Citation
Estrogen-central nervous system interactions in cardiovascular control and parturition

Material Information

Title:
Estrogen-central nervous system interactions in cardiovascular control and parturition
Creator:
Purinton, Scott Christopher, 1970-
Publication Date:

Subjects

Subjects / Keywords:
Carotid sinus ( jstor )
Estrogens ( jstor )
Fetus ( jstor )
Hypotension ( jstor )
Hypothalamus ( jstor )
Parturition ( jstor )
Placebos ( jstor )
Plasmas ( jstor )
Secretion ( jstor )
Sheep ( jstor )

Record Information

Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
30160776 ( ALEPH )
51638613 ( OCLC )

Downloads

This item has the following downloads:


Full Text












ESTROGEN-CENTRAL NERVOUS SYSTEM INTERACTIONS IN
CARDIOVASCULAR CONTROL AND PARTURITION














By

SCOTT CHRISTOPHER PURINTON


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1999























To the following, I dedicate this dissertation.
Without them, none of this would have been possible.


To my family: Thank you for your support and encouragement
To LeighAnn: Thank you for believing in and standing by me

To David M. Bush: Thank you for your friendship
Wish you could have been here













ACKNOWLEDGMENTS


First and foremost I would like to thank Dr. Charles E. Wood, chair of my

supervisory committee, for his guidance, wisdom, and friendship. Dr. Wood has

certainly been a true mentor to me as a scientist, as well as a role model to anyone

wishing to balance a fulfilling life with a successful career in science. For this and

everything else he has done for me, I am very grateful.

I would like to thank the other members of my supervisory committee, Drs.

Maureen Keller-Wood, Pushpa Kalra, and James Simpkins for their guidance and advice.

A special thanks goes to Dr. Maureen Keller-Wood for her extensive knowledge and for

being an indispensable resource. Also, a special thanks goes to Sherry McDaniel for her

assistance with the surgery and care of the animals.

Last but certainly not least, I would like to extend my gratitude to my family for

their never-ending love and support. They have always stood by me in whatever tasks I

set forth to accomplish. I especially would like to thank Dr. LeighAnn Stubley, my best

friend and fiancee. Without LeighAnn's love and support, I can truly say that none of

this would have been possible. She has taught me more about myself than I could ever

have learned from graduate school. For this I will be forever indebted to her.














TABLE OF CONTENTS

page

ACKNOW LEDGM ENTS..................................................................................................... iii

ABSTRACT ............................................................................................................................. vi

CHAPTERS

1 INTRODUCTION ..............................................................................................................1

General Background and Significance.............................................................................. 1
Specific Aims and Hypotheses .......................................................................................... 4
Experimental Protocol........................................................................................................ 5
Experimental M ethods ....................................................................................................... 7

2 LITERATURE REVIEW ......................................................................................... 10

Control of Parturition....................................................................................................... 10
The Hypothalamus-Pituitary-Adrenal Axis............................................................. 13
The Regulation of ACTH Secretion.........................................................................6......
The Development of the Fetal HPA Axis ....................................................................... 24
The Importance of Gonadal Steroids in Parturition and Cardiovascular Control........27
Cardiovascular Reflex Responsiveness.......................................................................... 33

3 GENERAL MATERIALS AND METHODS ........................................................39

Surgical Preparation of Fetal Sheep................................................................................. 39
In Vivo Experimental Procedures ...................................................................................42
Peptide Assays.................................................................................................................. 44
Steroid Assays.................................................................................................................. 45
Estrone Sulfatase Activity................................................................................................ 45
W western Blotting.........................................................................................4.. ..........47
Immunohistochemical Techniques............................................................................. 48

4 HORMONAL RESPONSIVENESS IN AN ESTRADIOL, HYPOTENSIVE,
CAROTID SINUS DENERVATED OVINE MODEL.......................................... 50

Introduction................................................................................................................ 50
M ethods and M aterials.................................................................. ...................52








Results.............................................................................................. .................58
D iscussion.................................................................... 60

5 NEURONAL ACTIVATION IN AN ESTRADIOL, HYPOTENSIVE,
CAROTID SINUS DENERVATED OVINE MODEL.......................................... 69

Introduction.......................... 69
M ethods and M aterials.............................................................................................. 70
Results ............................................................................ ........................................... 72
D discussion ......................................................................................................................... 76

6 ONTOGENY OF ESTROGEN SULFATASE AND ESTROGEN
SULFOTRANSFERASE IN BRAIN REGIONS IMPORTANT FOR
HYPOTHALAMUS-PITUITARY-ADRENAL AXIS CONTROL ....................95

Introduction...............................................................95
M ethods and M aterials.............................................................................................. 97
Results.......................................................................................................... ... 100
Discussion............................................................................................... 102

7 SUMMARY AND CONCLUSIONS..................................................................... 121

REFEREN CES .................................................................................................................... 138

BIOGRAPHICAL SKETCH........................................................................................ 158














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ESTROGEN-CENTRAL NERVOUS SYSTEM INTERACTIONS IN
CARDIOVASCULAR CONTROL AND PARTURITION

By

Scott Christopher Purinton

May 1999

Chairman: Charles E. Wood
Major Department: Physiology

In the fetal sheep, parturition is triggered by an increase in the activity of

the fetal hypothalamus-pituitary-adrenal (HPA) axis. Parturition can be delayed by

destruction of the pituitary or stimulated by infusions ofadrenocorticotropin (ACTH) or

glucocorticoids. The last days of gestation are marked by an increase in the activity of

the fetal hypothalamus as seen by elevated levels of fetal plasma ACTH. Estrogen has

been shown to trigger this preparturient increase in ACTH. I hypothesized that

estrogen's actions on fetal cardiovascular reflex responsiveness to hypotension will be

measurable in intact fetuses but not in baroreceptor/chemoreceptor denervated fetuses.

Research has previously demonstrated that denervation attenuates the reflex hormonal

and hemodynamic responses to moderate reduction in arterial blood pressure. I found

that interruption of this afferent pathway eliminated the effect of estrogen on reflex

cardiovascular responsiveness. This was shown by measuring hormone responsiveness








(ACTH, vasopressin, and cortisol) to hypotension by brachiocephalic occlusion. I further

hypothesized that estrogen's actions within the fetal central nervous system are centered

within the nucleus of the tractus solitarius (NTS), the paraventricular nucleus (PVN), or

within the components of cardiovascular regulatory centers receiving input from

baroreceptors or chemoreceptors. I used immunohistological techniques to identify the

neuroanatomical regions which are activated by hypotension and, subsequently, those

areas modified by estrogen's action and baroreceptor/chemoreceptor denervation. The

use of these techniques allows for the measurement of c-fos expression, an early response

gene which can be used as a marker of neuronal activity. I found that estradiol implanted

animals had more c-fos abundance, and hence more c-fos staining in relevant brain areas

(NTS, PVN, etc.) as compared to control animals. Furthermore, it was revealed that c-fos

staining was negligible in denervated animals. Finally, c-fos staining was significantly

elevated in all hypotensive animals as compared to normotensive animals. These results

suggest that estrogen works within the ovine fetal baroreceptor/chemoreceptor afferent

pathway in brain regions relevant for HPA axis control in order to augment ACTH

secretion in response to hypotension as well as play a role in the mechanism of

parturition.














CHAPTER 1
INTRODUCTION


General Background and Significance

There is a wealth of information concerning the influence of estrogen on the

cardiovascular system of the adult. The use of high dose estrogen contraceptives has

stimulated a great deal of interest in the role that exogenous estrogens play in the

pathogenesis of hypertension and stroke. However, it should be noted that little work has

been conducted regarding the influence of either endogenous or exogenous estrogen on

the control of cardiovascular function by the central nervous system. Furthermore, with

the exception of the studies conducted in Dr. Charles E. Wood's laboratory at the

University of Florida, Gainesville, Florida, there are no data concerning the influence of

estrogen on cardiovascular function in the fetus.

Increases in plasma estrogen concentration are thought to be an important

component of the maternal adaptation to pregnancy. During gestation, the growing fetus

and enlarging uterus exhibit an increasing demand for oxygen. For this reason, the utero-

placental blood flow constitutes an ever-increasing proportion of maternal cardiac output

(Rudolph, 1974). The increased flow demands are supported by an increase in maternal

blood volume and an increase in vascular compliance (Longo, 1983). This typically

produces a physiological condition in which maternal cardiac output and blood volume are

higher than in the nonpregnant state; however, maternal arterial pressure and central









venous pressure are lower (Keller-Wood, 1994; Longo, 1983). Though this response is

not entirely consistent among mammalian species, it has been proposed that estrogen plays

an important role in this phenomenon (Ueda, 1986). No studies have yet been conducted

that investigate the effects of estrogen on cardiovascular reflex responsiveness. By doing

such studies in fetal sheep, it is possible to investigate the mechanism behind parturition,

because an increase in the activity of the hypothalamus-pituitary-adrenal (HPA) axis has

been shown to trigger parturition (Liggins et al., 1973).

Both androgen and estrogen receptors can be found in the anterior pituitary as well

as in various regions of the brain, including the hypothalamus and the brainstem.

Receptors, and the mRNA encoding for these receptors, can be found in the various

structures, including the arcuate nucleus and the preoptic nucleus (Simerly et al., 1990). In

addition to the structures known for control of the hypothalamus-pituitary-gonadal axis

and for reproductive behavior, androgen and estrogen receptors are found in regions

better known for control of the hypothalamus-pituitary-adrenal axis, including the
/
paraventricular nucleus (PVN) and hippocampus (Lehman et al., 1993; Pomerantz and

Sholl, 1987). Estrogen receptors have been localized in the magnocellular portion of the

PVN, and may therefore influence either oxytocin, vasopressin (AVP), or both (Lehman et

al., 1993; Simerly et al., 1990). Both androgen and estrogen receptors are also found at

sites which are known to be relay centers on afferent pathways [the nucleus of the tractus

solitarius or (NTS)] mediating adrenocorticotropic hormone (ACTH) responses to

stresses such as hypoxia, hypercapnia, and hypotension (Simerly et al., 1990). Finally,

estrogen receptors have been localized in GABA-ergic cells, suggesting the possible








interaction of estrogen and ACTH secretion via this neuronal system within the brain

(Herbison et al., 1993).

The focus of the experiments is the neuroendocrine (ACTH, AVP) responsiveness

to hypotension. Of particular interest to me at the present time is the relationship between

neuroendocrine mechanisms controlling AVP and ACTH and how they regulate the

cardiovascular system of the fetus as well as trigger parturition. These two hormonal

systems are inextricably linked. In the fetus (as in the adult), afferent baroreceptor

pathways are shared by both endocrine systems. It is likely that the central pathways

carrying afferent information from the NTS to the hypothalamus are also mostly shared by

the two systems. AVP is synthesized in the PVN of the hypothalamus and can be found in

both magnocellular and parvocellular neurons (projecting to the posterior pituitary and

median eminence, respectively). Magnocellular AVP is secreted into the bloodstream by

the posterior pituitary. Parvocellular AVP is secreted into the hypothalamo-hypophyseal

portal system and acts at the anterior pituitary to induce the release of ACTH. ACTH, via

stimulation of cortisol, act to maintain vascular reactivity, promote plasma protein

biosynthesis, and indirectly alter fetal blood pressure and distribution of combined

ventricular output. Parvocellular neurons, in addition to AVP, synthesize and release

corticotropin releasing hormone (CRH) which also stimulates the release of ACTH from

the anterior pituitary. Parvocellular CRH neurons project axons to various parts of the

brain in addition to the median eminence. Most notably, these neurons project to the

cardiovascular control centers in the hypothalamus and medulla. When released from

these neurons, CRH stimulates increases in sympathetic efferent tone: measured in the

cardiovascular system as an increase in blood pressure and heart rate and a redistribution








of cardiac output (Lenz et al., 1987). Accordingly, it has been suggested that CRH is the

transmitter which coordinates all of the endocrine and cardiovascular responses to stress,

including hypotension (Lenz et al., 1987).

Preliminary experiments suggest that estrogen augments ACTH response to

hypotension, but that it does not alter AVP responses. Furthermore, estrogen augments

the ACTH response to cardiovascular stimuli, but not to purely endocrine stimuli.

Because of this difference between ACTH and AVP and between cardiovascular and non-

cardiovascular stimuli, I hypothesized that estrogen acts within the central nervous system

to affect the parvocellular CRH biosynthesis and secretion. Thus, it is proposed that

estrogen affects either CRH neurons or the ascending pathways which stimulate CRH

neurons, as well as affecting the activity of the descending (toward cardiovascular

regulatory centers) CRH pathways.

Specific Aims and Hypotheses

In the following experiments, a combination of in vivo (whole animal) and in vitro

(immunohistochemistry) techniques was employed.

Aim 1 The actions of estrogen on fetal cardiovascular reflex responsiveness to

hypotension will be measurable in intact fetuses but not in baro- and chemo-denervated

fetuses. Research has previously demonstrated that the combined baro- and chemo-

denervation attenuates (approximately 50%) the reflex hormonal and hemodynamic

responses to moderate (50 %) reduction in arterial blood pressure. I proposed that the

interruption of the afferent pathways would eliminate the effect of estrogen on the reflex

cardiovascular responsiveness. If so, it could concluded that estrogen acts on, within, or

requires input from, the afferent baroreceptor and chemoreceptor pathways.






5

Aim 2 The actions of estrogen within the fetal central nervous system are centered within

the nucleus of the tractus solitarius, the parvocellular neurons of the paraventricular

nucleus, or within the components of the cardiovascular regulatory centers receiving input

from baroreceptors or chemoreceptors. I used immunohistological techniques to identify

the neuroanatomical regions which were activated by hypotension and, subsequently,

those areas modified by estrogen's action and baro- and chemo-denervation. The use of

these techniques allows for the measurement of c-fos expression, the protein product of

the early response gene which can be used as a marker of neuronal activity. I proposed

that estradiol implanted animals will have more Fos activity, and hence more c-fos staining

in relevant brain areas (NTS, PVN, etc.) as compare to control animals. Furthermore, it is

expected that c-fos staining will be negligible in denervated animals. Finally, c-fos staining

should be significantly elevated in all hypotensive animals as compared to normotensive

animals.

In addition to the c-fos immunohistochenmical studies, the action of estrogen

sulfatase and estrogen sulfotransferase was investigated. Since the concentration of

estrogen sulfate precedes the increase in HPA axis activity (Nathanielsz et al., 1982), and

since conjugated estrogens circulate in much higher concentrations than unconjugated

estrogens (Carnegie and Robertson, 1978; Tsang, 1974), I hypothesized that these

enzymes would be present in brain areas important for HPA axis control. If true, a local

mechanism would be in place for conversion of biologically inactive to active estrogen.

Experimental Protocol

Design A total of 40 pregnant ewes were studied (5 per experimental group). Animals

were randomly assigned to the following groups:









1. placebo implant, intact (sham-denervated),normotensive fetuses

2. placebo implant, carotid sinus denervated, normotensive fetuses

3. placebo implant, intact (sham-denervated) fetuses subjected to hypotension

4. placebo implant, carotid sinus denervated fetuses subjected to hypotension

5. estradiol implant (5 mg/21 day release), intact (sham-denervated), normotensive

fetuses

6. estradiol implant (5 mg/21 day release), carotid sinus denervated, normotensive

fetuses

7. estradiol implant (5 mg/21 day release), intact (sham-denervated) fetuses

subjected to hypotension

8. estradiol implant (5 mg/21 day release), carotid sinus denervated fetuses

subjected to hypotension

Fetal sheep were chronically prepared with vascular catheters and implants on

around day 115 and allowed post-surgical recovery for 5 days. Fetuses were subjected to

experiments on day 1203, during which time each fetus was subjected to a 10 min period

of hypotension produced by brachiocephalic occlusion (no occlusion for control animals).

Fetal arterial blood samples (5 ml) were drawn at 0 min, +10 min, +20 min, and +60 min

relative to the initiation of hypotension. An additional 1 ml blood sample was drawn

anaerobically for the measurement of fetal blood gases and hematocrit at 0 min, +10 min,

and +20 min time points. Immediately after drawing the +60 min blood sample, the ewes

and fetuses were euthanized using an overdose of sodium pentobarbital. In each

experimental group, five fetuses will be studied, then prepared for immunohistochemistry

as detailed below. Immunohistochemistry was performed for the detection of c-fos. A










subset of sections was double-stained for ACTH and c-fos (pituitary), AVP and c-fos

(hypothalamus), and CRH and c-fos (hypothalamus).

Analysis The neuroanatomical areas which express c-fos in response to hypotension

(NTS, anterior hypothalamus, PVN, and pituitary) were identified. Immunohistochemistry

of c-fos was analyzed in a semi-quantitative manner (counting c-fos positive cells in the

relative regions). The number of cells expressing immunoreactive c-fos in each

experimental group at each site was be compared using two- and three-way ANOVA. I

further classified neuronal activation by counting cells which express/co-express c-fos and

ACTH (anterior pituitary), c-fos and CRH and c-fos and AVP (PVN).

Experimental Methods

Aim 1 Prior to surgery, food was withheld from the pregnant ewe (2-6 years old of mixed

breed, mostly Columbia-Ramboillet and Suffolk) for 24 hours. Before and during surgery,

the ewe was anesthetized with halothane (0.5-2.0%/) in oxygen. Fetal catheters were

routed to one flank of the ewe and held in place with a synthetic pocket and an expandable

bandage.

After exposing the uterus and the fetal hindlimbs via a midline incision, fetal

catheters were placed in the femoral arteries where their tips are advanced to the

subdiaphragmatic aorta. An estradiol or control implant was placed subcutaneously at this

time. An amniotic fluid catheter was sewn to the hindlimb before returning the fetus to the

amniotic cavity. Catheterization of fetal vessels is routine in Dr. Wood's laboratory

(Keller-Wood and Wood, 1991; Raff and Wood, 1991; Raff and Wood, 1992; Wood et

al., 1990).










For carotid sinus denervated fetuses, the common carotid artery was exposed and

stripped of nerves and connective tissue between the carotid-occipital arterial junction and

the carotid-lingual arterial junction. The occipital artery was ligated and divided and the

lingual artery was stripped of all nerves and connective tissue. Before closure of the fetus,

catheters were passed into the lingual arteries and advanced approximately 1 cm into the

carotid artery toward the heart. This entire procedure is conducted bilaterally. Dr.

Wood's laboratory has had extensive experience with this procedure and has published

descriptions of this technique (Wood, 1989, 1995).

Hypotension was achieved via brachiocephalic occlusion. A 10 minute period of

hypotension is necessary for activation of the HPA axis. Access to the artery was gained

through the second intercostal space on the left side of the chest. The occluder was tested

and deflated before the ribs, skin, and uterus were sutured closed.

Throughout the experiments, intravascular and amniotic pressures as well as heart

rate were measured utilizing a polygraph. Monitoring blood pressure assured that I

achieved a consistent and appropriate (50%) state of hypotension via brachiocephalic

occlusion. Blood pressure measurements were analyzed utilizing a computer program in

order to help quantify the level of estrogen augmentation of reflex responsiveness. Blood

gases and hematocrits were measured immediately after completion of each experiment.

Blood samples were processed (centrifuged, etc., to obtain plasma) and frozen until

hormone levels were measured via radioimmunoassay or enzyme immunoassay techniques

used routinely in Dr. Wood's laboratory. Plasma hormone concentrations (ACTH, AVP,

cortisol, and estradiol) were analyzed by 2- and 3-way ANOVA.










Aim 2 In preparation for immunohistochemistry, the head of the fetus was perfused with

heparinized saline followed by 4% paraformaldehyde. The fetal hypothalamus and

brainstem were dissected and processed accordingly (alcohol dehydrated and embedded in

paraffin). Tissues were sectioned (6-10 micron) using a Zeiss rotary microtome and

mounted on subbed slides. Staining for ACTH, AVP, and CRH was performed using

antisera produced in Dr. Wood's laboratory. Staining for c-fos was done using

commercially available antibodies from Oncogene Sciences. Staining for estrogen

sulfatase and estrogen sulfotransferase were done using custom made antibodies from

Alpha Diagnostic. Staining was visualized using a Histostain-SP kit (Zymed streptavidin-

biotin system).














CHAPTER 2
LITERATURE REVIEW


Control of Parturition

Though it has been known for some time that the fetus controls parturition in the

sheep, the precise mechanism has yet to be fully understood. However, much is known

about the major endocrine axis that initiates this process as well as controls blood pressure

in the fetus. These processes are controlled by the hypothalamus-pituitary-adrenal (HPA)

axis. One of the first pieces of evidence that linked the fetal HPA axis to the control of

parturition was reported by Binns et al. (1991). This was a natural phenomenon that

occurred when sheep ate a particular plant on day 14 of gestation. This plant, Veratrwnum

californicum, delayed the birth of the fetus indefinitely until caesarian section or death of

the fetusor ewe. The fetus had a number of birth defects, including cyclopia and a

dysfunctional hypothalamus-pituitary (HP) axis due to dislocation of the pituitary from its

normal position. It was realized later that this malformed, dysfunctional axis was

responsible for delayed parturition. This observation and others led Liggins (1973) to

conclude that the fetus controls parturition in the sheep. Liggins has credited Hippocrates

with first suggesting this idea when he wrote that the fetus pushes its way out of the

womb when the nutrition supplied by the mother is no longer sufficient for further growth.

Malpus (1933) put forth a more modem observation suggesting a fetal role in the timing

of birth when he reported the association of fetal ancephally and prolonged gestation in








women. These ideas laid the groundwork for much of the ongoing research to determine

the precise mechanism of parturition.

Though many have contributed to this idea of a fetal role in the initiation of

parturition, no one has contributed to this field of research more than Liggins. Liggins

was the first to hypothesize and directly test that the fetal pituitary is intricately involved in

this process (Liggins et al., 1967). This was done by first hypophysectomizing fetal sheep

by surgical electrocoagulation. In fetuses where 70% or more of the pituitary was ablated,

gestation was significantly prolonged and delivery was achieved only after caesarian

section. Fetal adrenalectomy was also found to prolong gestation (Drost and Holm,

1968). Disconnection of the hypothalamus from the pituitary in the fetal sheep between

108-112 days gestation delays birth by at least eight days (Antolovich et al., 1990).

Following hypophysectomy infusion of adrenocorticotropin (ACTH) or glucocorticoid still

induces parturition (Kendall et al., 1977). Further studies from this group also

demonstrate the necessity of the HP axis in maturation of pituitary corticotropes (Kendall
s
et al., 1977). After disconnection of the hypothalamus from the pituitary, fetuses were

infused with saline or cortisol. In the cortisol-infused fetuses the proportion of fetal-type

corticotropins was significantly lower than in the saline-infused fetuses however the

number of adult-type corticotropins did not change. A direct effect of cortisol on pituitary

corticotropin maturation requires the presence of complete HP axis (Antolovich et al.,

1992). More specifically, following destruction of the fetal paraventricular nucleus (PVN)

of the hypothalamus, parturition was delayed (McDonald and Nathanielsz, 1991).

Therefore, the signal for parturition may either be sent to the PVN which receives input

from the nucleus of the tractus solitarius (NTS), amygdala, or hippocampus, or possibly be









derived in corticotrophin releasing hormone (CRH) and arginine vasopressin (AVP)

producing neurons in the PVN.

Whereby disruption of the fetal HPA axis causes a delay in the timing of birth,

stimulation of this endocrine axis can result in premature parturition. The involvement of

the fetal adrenal cortex in the initiation of parturition in sheep was suggested after in utero

plasma concentration of corticosteroid revealed dramatic increases in these hormones

prior to birth (Bassett and Thorbumrn, 1969). Before this conclusion was made a number

of studies aimed at investigating the role of the HPA axis in the birth process were

performed in the fetal sheep by Liggins (1968). ACTH infused into fetal sheep induced

parturition within four to seven days along with producing adrenal hypertrophy. Cortisol

infusion into the fetus induced parturition within five days. The same doses of ACTH or

cortisol infused into ewes did not induce parturition. Although estradiol at 2 mg/24 hr had

no effect, infusion of cortisol at 25 mg/24 hr plus estradiol resulted in delivery after four

days (Liggins, 1968). Further studies by Liggins (1969) showed that it was glucocorticoid

activity (not mineralocorticoid) that was important for the initiation of parturition,

evidence of this effect was shown by the inability of deoxycorticosterone or corticosterone

to induce parturition. Dexamethasone infused at rates of 0.06 4.0 mg/24 hr in the fetus

and 4 mg/24 hr in the ewe was ineffective in producing premature delivery.

Since the earlier experiments of Liggins, many other studies have been performed

which confirm the previous results. Parturition was induced by continuous intravenous

cortisol infusion at 130 days gestation (Thomas et al., 1978). Activation of fetal adrenal

function by pulsatile ACTH administration in 125-127 day fetal sheep induced labor and

delivery in four to five days, resulting in four to six fold elevation in fetal plasma cortisol








concentrations (Lye et al., 1983). Furthermore, it was shown that hypophysectomized

fetuses that were administered dexamethasone or ACTH infusions exogenously would

undergo parturition (Kendall et al., 1977).

The Hypothalamus-Pituitary-Adrenal Axis

The HPA axis integrates a variety of neuroendocrine inputs to regulate the

synthesis and secretion of the adrenocorticosteroids which are required for the

maintenance of life. These steroid hormones exert effects to minimize any disturbance in

homeostasis. The critical role of adrenocorticosteroids can clearly be observed after

adrenalectomy or during hypoadrenocorticism either induced by drug or due to a disease

state. Without adrenal corticosteroids disrupted electrolyte balance or carbohydrate

metabolism leads to circulatory collapse of hypoglycemic coma and death. Physical,

emotional, and chemical stresses such as pain, trauma, hypoxia, acute hypoglycemia, cold

exposure, and vasopressin administration have all been shown to stimulate ACTH and

cortisol secretion (Gannet al., 1981).

The Hypothalamus The hypothalamus contains several nuclei of neuronal cells. The PVN

of the hypothalamus, located bilaterally on the ventricle, contains specialized

neurosecretory cells which synthesize CRH and AVP. The relatively large-celled (hence,

termed magnocellular) neurons in the PVN contain AVP and project to the posterior

pituitary (Lechan, 1987; Reichlin, 1992). In addition to the large projection to the

posterior pituitary, it has been established that small-celled (parvocellular) neurons in the

PVN contain CRH and AVP and project to the median eminence and are involved in the

regulation of ACTH release (Sawchenko and Swanson, 1980). The increase in plasma

ACTH concentration during stress is mediated by CRH as well as AVP from the










hypothalamus. These two factors are released into the hypothalamo-pituitary portal

circulation and diffused into the anterior pituitary to act on corticotropes to stimulate the

release of ACTH.

The Pituitary The pituitary is divided into the anterior lobe, the intermediate lobe, and the

neural or posterior lobe. Vessels of the hypophyseal-pituitary portal system deliver blood

from the median eminence of the hypothalamus to the anterior pituitary. This system

delivers hormones released from hypothalamic neuronal axons in the median eminence to

the anterior pituitary. CRH and AVP diffuse into the anterior pituitary and bind to their

receptors on specialized cells called corticotropes, which represent 15-20% of

adenohypophyseal cells. Once the releasing hormone binds to its receptor in the

corticotrope, a single mRNA the directs the synthesis of the large precursor molecule

called proopiomelanocorticotropin (POMC). POMC is then processed to produce the

smaller biologically active fragment ACTH. ACTH is then released into the systemic
/
circulation where it acts on adrenocortical cells to stimulate synthesis and secretion of

glucocorticoids (Reichlin, 1985).

The Adrenal Cortex The adrenal glands are endocrinologically complex organs that are

composed of two distinct endocrine tissues derived from different embryologic sources.

The outer zone is called the adrenal cortex and constitutes 80-90% of the gland. The

cortex is the source of the steroid hormones. The smaller inner zone is the adrenal

medulla which is the major source of circulating catecholamines. The adrenal cortex is

highly vascularized and receives its main arterial supply from branches of the inferior

phrenic artery, the renal arteries, and the aorta. There are three major groups of hormones

produced by the adrenal cortex: the mineralocorticoids, the glucocorticoids, and the sex










steroids. Histologically the adult cortex is composed of three zones: an outer zona

glomerulosa, a zona fasciculata, and an inner zona reticularis. The primary product of the

zona glomerulosa is the mineralocorticoid aldosterone. The zona fasciculata and

reticularis produce cortisol and androgens as their primary products (Pescovitz et al.,

1990). ACTH stimulates the secretion of glucocorticoids, mineralocorticoids, and

androgenic steroids from the adrenal cortex. ACTH binds to the receptors on the adrenal

cortex and provokes steroidogenesis through stimulation of cAMP production. cAMP

activates protein kinase A, which catalyzes the phosphorylation of a variety of proteins

thereby producing cholesterol. ACTH also stimulates synthesis of new adrenal proteins

and this increases adrenal weight. Glucocorticoids exert negative feedback at the

pituitary, hypothalamus, and other neural sites (Keller-Wood and Dallman, 1984).

Cortisol is carried in blood bound to transcortin (59%) and albumin (19%), while

about 22% is free in ovine plasma (Patterson and Hills, 1976). The basal production rate

of cortisol is 600 ,g/hr and the metabolic clearance rate of cortisol is about 51 L/hr in

sheep (Panaretto, 1974). The liver and the kidney are the principle organs involved in

clearing the steroid hormones from the circulation. Although most tissues can metabolize

steroids, the liver is the primary site of steroid hormone metabolism and the kidney is the

primary site of steroid hormone excretion. The plasma half-life of cortisol is 60-100

minutes in the human (Pescovitz et al., 1990).

Cortisol produces a number of diverse physiological actions to maintain

homeostasis. As the term homeostasis implies, an excess or deficiency ofglucocorticoids

affects every tissue of the body. Glucocorticoids are essential for survival (Baxter, 1972;

Gann et al., 1981; Pescovitz et al., 1990). The term glucocorticoid refers to the glucose










regulating properties of these hormones. However, glucocorticoids have multiple effects

that include important roles in carbohydrate, lipid, and protein metabolism (Baxter, 1972).

These increase blood glucose by increasing gluconeogensis in the liver and kidneys,

increasing hepatic glycogenesis and decreasing glucose uptake in tissues. The

glucocorticoids increase lipolysis and proteolysis. The glucocorticoids also have

stimulatory effects on cardiovascular function by increasing cardiac output and increasing

vascular response to catecholamines. At high concentrations, the glucocorticoids inhibit

most immunologic and inflammatory responses. Although these effects may have

beneficial aspects, they may also be detrimental to the host by inducing a state of

immunosuppression that will predispose the host to infection (Parrillo and Fauci, 1979).

The glucocorticoids also influence growth, development, bone metabolism, and central

nervous system activity.

The Regulation of ACTH Secretion
i
Corticotropin Releasing Hormone (CRH) Since 1955 it has been known that the

hypothalamus contains substances that acted at the pituitary gland to increase ACTH

secretion in vitro (Guillemin and Rosenberg, 1955; Saffi-an and Schally, 1955). In 1981,

Vale et al. (1981) characterized a 41 amino acid peptide from sheep hypothalamus that

stimulated ACTH secretion from corticotrophins and published the primary structure of

ovine CRH. Ovine CRH was further characterized when the cDNA was cloned and

sequenced (Furutani, 1983). At this time, a similarity was discovered between the

precursor proteins for CRH, AVP, and ACTH which implies a common evolutionary

beginning. A 20 kD immunoreactive form of CRH was identified from rat hypothalamus










and is close to the value for ovine and human pre-pro-CRH based on their cDNA

sequences (Lauber et al., 1984).

CRH increases ACTH secretion from the anterior pituitary gland by binding to

high affinity receptors (Wynn et al., 1983) located on the corticotrophs (Leroux and

Pelletier, 1984). Activation of the receptor complex increases adenylate cyclase activity

(Perrin et al., 1986) and cAMP which results in an increase in ACTH secretion. In

cultured rat pituitary cells CRH can enhance the rates of ACTH synthesis as well as

release (Vale et al., 1983). ACTH release can be modulated by down-regulation of CRH

receptors in the anterior pituitary. There is evidence that CRH (Wynn et al., 1988), AVP

(Hauger and Aguilera, 1993), and glucocorticoids (Haugher et al., 1987; Schwartz et al.,

1986; Wynn et al., 1985) can all act to regulate CRH receptor number. Therefore, an

alteration in CRH receptor number, receptor activity, receptor coupling, or even

corticotrophin number can affect the ability of CRH to stimulate ACTH secretion.
/
Arginine Vasopressin (AVP) The other major regulator of ACTH secretion of

hypothalamic origin is AVP. Classically, AVP is known as antidiuretic hormone (ADH)

for its role in renal regulation of fluid balance. An increase in plasma osmolality is the

most potent stimulus to AVP secretion in that very small increases in osmolality cause an

almost immediate secretary response from the posterior pituitary. AVP binds to receptors

on the basal-lateral membrane of the cortical and medullary collecting ducts of the

nephron. Binding to these receptors (V,) results in activation of adenylate cyclase which

subsequently increases cAMP. This second messenger is then thought to facilitate an

increase in protein channels found in the luminal membrane, thereby increasing the

diffusion of water out of the nephron and concentrating urine while retaining fluid










(Vander, 1985). In addition to AVP's role in modulating fluid reabsorption in the renal

system, AVP is also a potent vasoconstrictor of the cardiovascular system (binding to V,

receptors). Significant decreases in blood volume (15-20%) produce large increases in

plasma vasopressin. Decreases in blood volume are sensed as decreased stretch of the

arterial baroreceptors located in the carotid sinus as well as the receptors in the left atrium

and AVP secretion is reflexively stimulated (Berne and Levy, 1986).

AVP is one of two hormones secreted from the posterior pituitary gland (oxytocin

is the other). The posterior pituitary also called the neurohypophysis, is comprised of

axons and axon terminals which account for 42% of its total volume (Nordmann, 1977).

These axons project from magnocellular neurons of the supraoptic nucleus (SON) and

PVN from the hypothalamus. AVP and oxytocin are structurally similar hormones with

very different functions. AVP has actions on the renal and circulatory systems while

oxytocin causes milk ejection and uterine contractions. AVP and oxytocin are produced

in different neurons of the same nuclei and are stored in secretary vesicles or granules with

their appropriate neurophysin (Silverman and Zimmerman, 1975).

AVP is produced from a large precursor protein containing not only AVP but also

neurophysin and a glycopeptide signal sequence (Sachs et al., 1969). The precursor

protein molecule is packaged in granules with the enzymes needed for processing AVP to

its final form. As the granules move down the axons, post-translational processing of the

precursor molecule occurs within the granules. When the granules reach the axon

terminals, the nerve is depolarized and the granules are exocytosed and the contents of the

granules are released (Brownstein et al., 1980). Magnocellular neurons containing AVP










project fibers to the median eminence and therefore maybe important in the regulation of

ACTH secretion (Holmes et al., 1986).

AVP is a potent modulator of pituitary ACTH secretion. In fact, in sheep, AVP is

a more potent stimulator of ACTH than CRH (Familari et al., 1989; Liu et al., 1990).

AVP binds to receptors on the anterior pituitary corticotroph to increase plasma ACTH

secretion. These receptors are different from the pressor receptors (subtype V1) or anti-

diuretic receptors (subtype V2) that are found in the periphery. Data from two different

groups (Baertschei and Friedli, 1985; Jard et al., 1986) suggest that a subtype (classified

as Vib or V3) distinct from the peripheral receptors, exists in the brain with protein kinase

C as its second messenger.

AVP can effect anterior pituitary secretion in two ways: either by AVP secretion

from axons which terminate in the median eminence or by AVP secretion from the

posterior pituitary. Evidence for both possibilities exist. AVP-containing neurons of the

SON and PVN are known to project to the external zone of the median eminence

(Hoffminann et al., 1991). A further distinction has been made in that AVP-containing

parvocellular neurons of the PVN were found to innervate the external zone of the median

eminence. The magnocellular neurons of the PVN pass through the internal zone of the

median eminence to the neurohypophysis, but also contribute to the AVP found in the

median eminence (Holmes et al., 1986). AVP secretion from the posterior pituitary gland

has also been implicated in plasma ACTH secretion. In studies performed in dogs,

neurohypophysectomy attenuated the plasma ACTH response to hypotension. After

restoration of plasma AVP levels to those observed in the intact animal, plasma ACTH










levels were almost completely returned to normal (Raffet al., 1988). The action of AVP

appears to be through a direct effect at the pituitary gland.

Synergism of CRH and AVP The synergistic activity of CRF and AVP secretion has been

well documented. In support of this is an overwhelming amount of anatomical data for

the interaction between these two hormones. CRH and AVP are found in the same

neurosecretory vesicles in the median eminence (Whitnall et al., 1985). In normal rats,

staining of the median eminence by immunohistochemistry revealed co-localization of

AVP in 50% of CRH axons (Whitnall et al., 1987). Repeated stress (immobilization in

rats) increases the co-localization of AVP in CRH nerve terminals in the median eminence

(de Goeij et al., 1991). Following adrenalectomy in rats, CRH immunostaining increases

in parvocellular neurons of the PVN and the amount of co-localization with AVP

increases (Sawchenko et al., 1984). The increase in CRH and AVP immunoreactivity

following adrenalectomy is prevented by intracerebroventricular injection of

dexamethasone (Sawchenko, 1987).

From in vitro studies performed in cultured pituitary cells, CRH is considered to be

the more potent secretagogue for ACTH secretion in rats while AVP appears to be a more

potent stimulus to ACTH release in sheep (Familari et al., 1989). However, the action of

each is potentiated when administered together in cultured pituitary cells (Gillies et al.,

1982), adult freely moving rats (Rivier and Vale, 1983a) and in fetal sheep (Brooks and

White, 1990). In a study performed in conscious sheep, stress-induced ACTH secretion

(audiovisual and insulin-induced hypoglycemia) was accompanied by increases in

hypothalamic CRH and AVP secretion (Familari et al., 1989). What is most interesting is

that CRH:AVP molar ratio was altered with the stress. Portal plasma AVP was increased










above that of CRH, increasing the ratio of AVP to CRH. Since CRH and AVP can be

found in the same neurosecretory vesicles, this suggests that differential regulation of each

individual hormone also occurs. A synergistic effect of CRH and AVP on ACTH

secretion was also observed as seen by a lack of 1:1 concordance between hypothalamic

AVP/CRH secretion and pituitary ACTH secretion. This effect may be due to secretion of

other hypothalamic factors that increase ACTH secretion.

Other ACTH Secretagogues The control of adrenocorticotropin secretion is a complex

process involving numerous factors (neurotransmitters and neuropeptides) that augment

ACTH secretion. Rat pituitary corticotrophs in culture release ACTH in response to

epminephrine and norepinephrine acting on a l-adrenergic receptors (Giguere et al., 1981).

Epinephrine has also been identified in portal plasma suggesting a physiological role in the

control of anterior pituitary function (Johnson et al., 1983). Neuropeptide Y (NPY)

injected centrally in fetal and adult sheep increases plasma ACTH concentrations but does

not stimulate the pituitary directly (Brooks et al., 1994). This suggests that NPY acts

centrally to increase the activity of the HPA axis. Evidence for NPY stimulation of CRH

secretion supports this conclusion (Haas and George, 1987). Endogenous opiates are

capable of stimulating the HPA axis in late gestational fetal sheep but do not tonically

stimulate the axis in a regulatory manner (Brooks and Challis, 1988). Serotonin stimulates

ACTH secretion in humans, demonstrates by pharmacologically increasing serotonin with

fenfluramine (Lewis and Sherman, 1984). However, there are conflicting data as to the

site of action of serotonin (hypothalamus or pituitary). In rats, CRH secretion is

decreased after elimination of endogenous hypothalamic catecholamines suggesting a role

for central catecholinergic neurons in the control of ACTH release (Guillaume et al.,










1987). Angiotensin II also increases plasma ACTH secretion by induction ofCRH (Rivier

and Vale, 1983). Prostaglandin E2 alone does not increase ACTH secretion but enhances

the ability of AVP to stimulate ACTH secretion with no effect on CRH (Brooks and

Gibson, 1992). In addition to stimulating the activity of the HPA axis, factors from the

brain also inhibit the axis. The dopaminergic system in the amygdaloid central nucleus has

been found to inhibit ACTH secretion by action on the anterior and lateral hypothalamus

(Beaulieu et al., 1987). Atrial Natriuretic Peptide (ANP) has also been shown to alter

ACTH secretion. Brain ANP is secreted into the hypophyseal portal vessels from the

hypothalamus and physiological concentrations inhibit ACTH release from pituitary cells

in vitro (Dayanithi and Antoni, 1989; Lim et al., 1990; Sheward et al., 1991). In vivo

immunoneutralization of ANP significantly increases ACTH release but has no effect on

release during ether stress (Fink et al., 1991). These results suggest a role for ANP as a

mediator in the regulation of ACTH secretion.
/
Negative Feedback Control of ACTH Secretion Glucocorticoids are very versatile

steroids and necessary for survival in a number of species including sheep and primates.

Binding of the glucocorticoid to its receptor promotes binding to and transcription of

DNA, production of mRNA for synthesis of enzymes, and eventually alteration of cell

function. Glucocorticoids act in the body to increase plasma glucose concentrations by

increasing hepatic glycogenesis and gluconeogenesis. They also increase protein

catabolism and in the periphery, glucocorticoids exert actions that counter the effects of

insulin. Glucocorticoids are also necessary for vascular reactivity. Without them, the

vascular smooth muscle becomes unresponsive to epinephrine and norepinephrine, the

capillaries expand and their walls become permeable to proteins in the plasma. Finally,










glucocorticoids are released in response to stress and noxious stimuli. In animals that lack

normal secretion of glucocorticoids, exposure to a stress can be life-threatening (Ganong,

1985).

Secretion of glucocorticoids is regulated by adrenocorticotropin hormone from the

pituitary. When a stimulus of ACTH secretion from the hypothalamus reaches the

pituitary gland, ACTH is secreted into the general circulation, binds to its receptor at the

adrenal gland and stimulates cortisol secretion. Cortisol then acts at target organs to

increase plasma glucose levels, etc. However, cortisol also acts at the levels of the brain

to reduce ACTH secretion. Cortisol acts at the hypothalamus and pituitary gland

negatively to inhibit further ACTH secretion. This is called cortisol negative feedback

inhibition of ACTH secretion.

The mechanism of action of cortisol to reduce ACTH secretion has been studied at

length in many species and by many investigators. A study by Canny et al. (1989)
t
performed in sheep, examined both hypothalamic and pituitary sites. of action for

glucocorticoids. Measurements of hypophyseal portal concentrations of AVP and CRH

and systemic measurements of ACTH and cortisol concentrations were made before and

after dexamethasone infusion with different stimuli to ACTH secretion. The data suggest

that glucocorticoid act in a site-specific manner to inhibit ACTH secretion (Canny et al.,

1989). During audio-visual stress, hypothalamic CRH and AVP secretion were unaltered

but ACTH secretion was inhibited suggesting a pituitary site of action of dexamethasone.

In response to hypoglycemia, dexamethasone inhibited both the hypothalamic and pituitary

responses to the stress. Studies in rats have shown that glucocorticoids inhibit CRH

secretion as well as CRH synthesis (Sato et al., 1975) suggesting a mechanism for a










hypothalamic site of action. On the other hand, glucocorticoids may interfere with CRH

activation of second-messenger systems (cAMP) at the pituitary and therefore prevent

stimulation of ACTH secretion (Bilezikjian and Vale, 1983).

In the fetus, cortisol has the ability to inhibit plasma ACTH secretion in response

to a hypotensive stimulus by approximately 90 days gestation (Hargrave and Rose, 1985).

High concentrations of glucocorticoids near the fetal PVN prevent increased ACTH

secretion in response to hypotension and hypoxemia (McDonald et al., 1990). Between

117 and 131 days gestation, fetal sheep are extremely sensitive to negative feedback

effects of cortisol. This was demonstrated by infusions of cortisol that caused less than 2

ng/ml increases in plasma cortisol concentrations but which completely inhibited the

normal ACTH response to hypotension (Wood, 1986). This knowledge predicts the

existence of a normal feedback response in the fetus, which is the case in late gestational

fetal sheep. However, in near-term fetal sheep, cortisol negative feedback regulation of
/
ACTH secretion becomes ineffective. In experiments in which infusions of cortisol

increased plasma cortisol concentrations to approximately 60 ng/ml, fetal plasma ACTH

secretion was still not suppressed (Wood, 1987; 1988). This mechanism of this reduction

of glucocorticoid negative feedback efficacy is not fully understood at present.

The Development of the Fetal HPA Axis

Development of the ovine fetal HPA axis begins during the first third of gestation

(term being 145-148 days gestation) with the formation of the pituitary and adrenal

glands. The fetal pituitary gland can be detected as early as 31 days gestation and

differentiation of the anterior pituitary can be seen at about 40 days (Perry et al., 1982).

Staining of cells in the anterior pituitary indicate the presence of ACTH immunoreactivity










by as early as 50 days gestations and by 60 days gestation, processing of ACTH from

POMC can be detected in the intermediate lobe of the pituitary of the fetal sheep

(Mulvogue et al., 1986).

The fetal adrenal can be identified by approximately 28 days gestation in the sheep

(Wintour et al., 1977). By 40-50 days gestation, in vivo experiments demonstrate that the

fetal adrenal readily secretes cortisol in response to ACTH (Wintour et al., 1975;

Glickman and Challis, 1980). Between 90-120 days gestation, the cells of the zona

fasciculata (which are responsible for cortisol synthesis) are relatively immature (Robinson

et al., 1979) and approximately 90% of fetal plasma cortisol is derived from the maternal

circulation (Hennessy et al., 1982). After 120 days gestation, fetal adrenal sensitivity to

plasma ACTH increases (Liggins et al., 1973; Rose et al., 1982), the proportion of cortisol

that is of fetal origin increases (Hennessy et al., 1982), and the correlation between fetal

ACTH secretion and fetal cortisol secretion becomes significant (Hennessy et al., 1982).

Adrenal weight also increases as a function ofgestational age (Comline and Silver, 1961).

Early studies involving fetal hypophysectomy (Liggins et al., 1967) and

adrenalectomy (Droust and Holm, 1968) with prolongation of pregnancy suggested a link

between the adrenal steroid production and parturition. If this is the case, fetal plasma

cortisol secretion should be altered as gestation nears and ends. Before about 130 days

gestation, fetal plasma corticosterone levels are low but several days before, parturition

plasma levels increase and peak at birth (Bassett and Thorburn, 1969). Nathanielsz et al.

(1972) found that fetal plasma cortisol concentrations began to increase about three to

four days before parturition and then steadily declined in the newborn lamb. A more

elaborate study of cortisol secretion was performed by Magyar et al. (1980) in which










exponential curves were fit to the data to more accurately describe the increase in fetal

plasma cortisol concentrations. This analysis revealed fetal plasma cortisol concentrations

increasing exponentially about 10-15 days prior to parturition. Cortisol is secreted in

response to ACTH binding to receptors at the adrenal gland. In vivo experiments by

Brown et al., (1978) demonstrated that not only did glucocorticoids increase with

development, but the ability of the fetal adrenal to secrete cortisol in response to ACTH

was observed around 120-129 days gestation. The increase in cortisol secretion that

occurs near the end of gestation is in part due to an increase in adrenal sensitivity to

stimulation by ACTH (Madill and Bassett, 1973) but also possibly due to increased plasma

ACTH secretion from the fetus.

Jones et al. (1977) presented some of the first data examining the changes in fetal

plasma ACTH concentration during development. They demonstrated that fetal plasma

ACTH concentrations increase prior to parturition. However, they concluded that the
/
increase occurred after the increase in fetal plasma cortisol and therefore is probably not

the reason for the changes in fetal plasma concentrations. Experiments performed later by

other investigators revealed that the fetal plasma ACTH concentrations increase much

sooner than previously described by Jones et al. (Maclssac et al., 1985; Wintour, 1984;

Norman et al., 1985). In fact, fetal plasma ACTH concentrations increase during the last

30 days of gestation, well before the increase in plasma cortisol concentrations which

occurred approximately 120 days gestation.

The results of increasing plasma ACTH concentrations in vivo when taken

together with the in vitro pituitary ACTH secretion studies support the presence of an

additional factor involved in the process of parturition. Basal output of ACTH from fetal










sheep pituitaries in culture does not increase as a function ofgestational age (Durand et

al., 1986). Pituitary ACTH secretion in culture does not increase during the last week of

gestation at a time when in vivo circulating plasma ACTH concentrations are increasing

exponentially (McMillen and Merei, 1993). These data suggest that the preparturient

increase in ACTH secretion is dependent on some other factor to stimulate secretion and

not a function of basal pituitary output. In addition, McMillen and Merei (1993) also

found no change in responsiveness of the fetal corticotroph to CRH as a function of

gestational age. However, prior exposure of the fetal pituitary to cortisol increased its

responsiveness to CRH. There appears to be a signal, possibly from the hypothalamus,

that increases the activity of the fetal pituitary and therefore the fetal adrenal and is the

trigger to parturition in the sheep.

The Importance of Gonadal Steroids in Parturition and Cardiovascular Control

In addition to the increase in fetal plasma ACTH and cortisol concentrations that

occur at the end of gestation, the fetal plasma estrogen and androgen concentrations also

increase. Plasma estrone concentrations appear to increase over the last four days of

gestation while estradiol may be increasing over the last eight days in the fetus and

amniotic fluid (Challis and Patrick, 1981; Findlay and Cox, 1970). In a paper by Challis

and Patrick, fetal plasma estrone concentrations increase from 40 pg/ml at 14 days prior to

birth to near 400 pg/ml the day before birth. Fetal plasma estradiol concentrations

increased from 20 pg/ml at 14 days prior to birth to 80 pg/ml on the day before

parturition. Plasma estradiol concentrations in the ewe increase over the last two days of

pregnancy in the ewe (Robertson and Smeaton, 1973), with a ten-fold increase on the day

before parturition, from 20 to 40 pg/ml up to 411 pg/ml (Challis, 1971). At the time










plasma estrogens increase, there is a decrease in plasma progesterone (Bedford et al.,

1972). This is possibly due to a conversion of estrogen to progesterone, with plasma

progesterone acting as a reservoir for plasma estrogen production. Since progesterone is

also a precursor for androgen production, one would expect to see increases in plasma

concentrations of these steroids as well. Plasma androstenedione and testosterone

concentrations do indeed increase in the late gestational fetus in a manner similar to

estrogen concentrations (Pomerantz and Nalbandov, 1975; Yu et al., 1983).

This increase in plasma estrogen and androgen concentrations is a result of the

actions ofcortisol on the placenta (Anderson et al., 1975; Steele et al., 1976). Although

the placenta has aromatase activity the plasma concentrations of estrogens and androgens

are very low throughout gestation (Mann et al., 1975). Cortisol acts at the placenta to

induce an enzyme, cytochrome P450c7, which has 17a-hydroxylase and 17,20 lyase

activities. Upon induction, P450c17 facilitates the production of estrogens and androgens
/
from progesterone, thus increasing plasma concentrations of these hormones.

Gap junctions in the myometrium, which are very important for synchronized

uterine contractions, are believed to be formed at the time of progesterone withdrawal

(Garfield et al., 1977). It has been proposed that as a result of decreased progesterone

and increased estrogen, there is an augmentation in synthesis of proteins associated with

gap junctions (Garfield et al., 1978; Garfield, 1984, 1977). These gap junction proteins

are inserted into the plasma membrane of the myometrium and aggregate to form gap

junctions. Gap junctions are necessary for the uterus to contract in a unified, coordinated

fashion in order to expel the fetus. Prostaglandins and oxytocin, as well as estradiol, act in

concert to stimulate uterine contractions in the process of labor (Lye et al., 1983).










Tissue estrogen concentrations in sheep also increase towards term or after

ACTH-induced labor, especially in the myometrium (Power and Challis, 1987). The ovine

placenta, through sulfatase and aromatase activities, converts estradiol and estrone sulfate

to 173-Estradiol and estrone, the more potent estrogen for the target organ, the

myometrium (Rossier and Pierrepoint, 1974).

It has long been known that female rats have greater activity of the HPA axis than

male rats. Studies in adult animals demonstrate that female rats have increased

corticosterone secretion following ACTH administration and greater adrenal

responsiveness to tropic stimulation (Kitay, 1961). As the major difference between the

sexes is gonad and steroid production, experiments involving gonadectomy and

replacement of gonadal steroids were performed. After gonadectomy, testosterone

depressed ACTH content and steroid clearance in male rats but increased adrenal

responsiveness to ACTH. In female rats, estradiol had a consistent stimulatory effect on

ACTH secretion (Kitay, 1963). In female rats, plasma ACTH and corticosterone

responses to restraint stress were enhanced during proestrus, when estradiol

concentrations were highest (Kitay, 1963). In ovariectomized (ovx) rats replaced with

estradiol, this effect can be restored (Viau and Meaney, 1991). Following ovx, there was

a decreased capacity of the pituitary to synthesize ACTH and a decreased responsiveness

to stimulation by hypothalamic extracts (Coyne and Kitay, 1969). In ovx rats, estradiol

implants into the area of the anterior pituitary, arcuate nucleus, and lateral mammilary

bodies in rats facilitated pituitary-adrenal activity, suggesting a central nervous system

effect (Richard, 1965). However, estrogen stimulation of corticosterone secretion in ovx

rats, may be due in part to a direct effect on the adrenal cortex (Kitay et al., 1965). In ovx










rats, plasma ACTH and corticosterone responses to foot shock and ether vapor stress

were lower than in estrogen-replaced ovx rats (Burgess and Handa, 1992). From these

results, it was concluded that the increased activity in the pituitary-adrenal axis was due to

an impairment of the glucocorticoid negative feedback system.

The mechanism of action of estrogen stimulation on HPA axis activity is not fully

understood. Based on the present data, estrogen may interact with a number of systems

impinging on the HPA axis. Estrogen uptake has been demonstrated in corticotrophins

isolated from anterior pituitary cells from adrenalectomized rats (Keefer, 1981) suggesting

a possible direct effect of estrogen on the pituitary. Estradiol has also been shown to

concentrate in tyrosine hydroxylase containing neurons in the arcuate and periventricular

nuclei of the rat (Sar, 1984). In another study, estradiol concentrating cells have been

found in the amygdaloid central nucleus (Beaulieu et al., 1987). Since this dopaminergic

system can inhibit ACTH secretion, this may be important in modulating HPA axis

activity. In rats given 100lg estradiol exogenously for two weeks, there was an increase

in AVP in the SON and PVN of the hypothalamus without any changes in pituitary ACTH

and AVP content or basal plasma ACTH or AVP concentrations (Hashimoto et al., 1981).

In another study, plasma AVP concentrations were found to be greatest when estrogen

concentrations were highest. If the rats were ovx, plasma AVP concentrations decreased

but were restored when estrogen was replaced (Skowsky et al., 1979). These data

suggest a possible role of hypothalamic releasing factors in the mediation of estrogen

stimulation of HPA axis activity.

The results of this dissertation demonstrate that there is significant estrogen

sulfatase activity in ovine fetal hypothalamus, hippocampus, and brainstem, and that there










are statistically significant ontogenetic changes in activity of this enzyme in the

hippocampus. Also shown is the presence of estrogen sulfotransferase in the fetal

hypothalamus and brainstem. It has previously been demonstrated that estrogens in fetal

plasma increase both basal- and stimulated- fetal plasma ACTH secretion. The present

results suggest a mechanism by which the most abundant form of estrogen in ovine fetal

plasma, estrone sulfate, might be made available to areas within the fetal brain known to

be involved in the control of the fetal HPA axis.

Mathew and Balasubramanian (1982) and Lakshmi and Balasubramanian (1979)

have previously demonstrated estrogen sulfatase and sulfotransferase activity in adult

sheep brain tissue. Other investigators have demonstrated these enzymatic activities in

adult brain tissue from rats (Connolly and Resko, 1989; Kawano and Aikawa, 1987), mice

(Hobkirk, 1987), non-human primates (Lakshmi and Balasubramanian, 1981), and human

beings (Platia, 1984). Hobkirk and coworkers demonstrated that enzyme activities are
/
transiently increased postnatally in the brain of the mouse (1987). While the development

of brain estrogen sulfatase and sulfotransferase activity have not been studied in sheep, the

development of activities in mice suggests the possibility that this might be an important

developmental process in the perinatal period.

Using a histochemical technique, Kawano and Aikawa found that sulfatase activity

is highest in pineal gland, choroid plexus, and pars distalis of the pituitary in adult rats

(1987). I investigated the activity in hypothalamus, brainstem, and hippocampus because

these areas are known to contain nuclei involved in integration, afferent signal relay, or

negative feedback inhibition within the HPA axis (Grizzle et al., 1974; Keller-Wood and

Dallman, 1984; Maran, 1978; Ward, 1978). The presence of activity in any of these areas










could be important for the deconjugation of sulfated estrogens in the blood perfusing the

brain. Rosenfeld et al in 1980 reported that the majority of estrogen produced by the

ovine placenta is sulfoconjugated and thus protected since sulfatase in not present. My

data suggest otherwise given that sulfoconjugates in the fetal compartment may have

specific regional roles. The effect of estrogen on both basal- and hypotension stimulated-

concentrations of ACTH could be the result of an action of estrogen on the PVN in the

hypothalamus, an action on the hippocampus (which mediates some of the negative

feedback actions of corticosteroids on ACTH secretion), an action on the NTS (which

relays neural traffic from visceral afferents), or an action on any part of the pathways

leading from the NTS to the PVN (e.g., the RVLM). Estrogen receptors have been

demonstrated in the NTS and hippocampus (Lehman, 1993). While estrogen receptors

within the hypothalamus are most concentrated in the arcuate nucleus, estrogen receptors

have been demonstrated in the PVN (Lehman, 1993; Simerly, 1990). The results of the

present experiments identify the cellular location of the sulfatase activity which is

consistent with these centers for HPA axis control. I found widespread staining

throughout nuclei and fiber tracts of the hypothalamus and brainstem. Neuronal staining

was much more concentrated than fiber tract staining, however both were observable.

While estrogen sulfatase may be responsible for decongugating estrone sulfate

locally within the fetal to increase HPA axis activity directly, the role of estrogen

sulfotransferase is probably more indiscrete. Naturally one such role of the enzyme is to

maintain high levels of circulating conjugated estrogens that cannot be readily degraded.

A less obvious role of estrogen sulfotransferase might be to conjugate cortisol so that










inhibition of HPA axis negative feedback is achieved. This, concomitant with local

activation of estrogens via estrogen sulfatase, would increase ACTH release.

Cardiovascular Reflex Responsiveness

Arterial baroreceptors are mechano-receptors located in the walls of large systemic

arteries including the aortic arch, brachiocephalic artery, and carotid sinuses (Boss and

Green, 1956; Green, 1954). Afferent signals from the arch of the aorta are transmitted

through the left and right aortic depressor nerves to the vagus nerve and ultimately to the

nucleus of the tractus solitarius in the medullary area of the brain stem (Boss and Green,

1956; Nakayma, 1965; Nonindez, 1935). Sensory input from the carotid sinus region

travels to the nucleus of the tractus solitarius as well, but via the sinus (Hering's) and

glossopharyngeal nerves.

Barosensitive nerve endings are found in areas of the arteries with large quantities

of elastic tissue (Muratori, 1967). Approximately 40% of the tissue comprising the walls

of the aortic arch is an elastin-collagen mixture (Bader, 1963) which is almost free of

smooth muscle (Gregoreva, 1962). Similarly, the carotid sinus is thinner (Adams, 1958;

Addison, 1944; Rees, 1968; Rees and Jepson, 1970), contains less smooth muscle

(Addison, 1944; Bagshaw and Fischer, 1971; Muratori, 1967; Rees and Jepson, 1970),

and shows a higher elastin content than other areas of the carotid artery (Addison, 1944;

Rees and Jepson, 1970).

Although their name implies a pressure-sensitive quality, baroreceptors are stretch

receptors which respond to deformation of the vessel wall in which they are located

(Hauss et al., 1949; Angell-James, 1971). There is evidence which shows that the degree

of wall deformation determines the electrical activity of the carotid sinus and aortic arch










baroreceptors. Hauss et al. (1949) demonstrated that the reflex fall of blood pressure

produced by an increase in carotid sinus pressure is abolished if the stretching of the

carotid artery is prevented by a plaster cast applied to the outside of the sinus region.

Additionally, Angell-James (1971) reported that increased baroreceptor activity produced

by elevation of intrathorasic pressure could be prevented by simultaneously increasing the

extramural pressure by the same amount. Experiments in man have shown that changing

pressure in a chamber surrounding the neck results in reflex changes in heart rate and

blood pressure and provide further evidence that altered transmural pressure is a stimulus

for baroreceptor activation (Bevegard and Shepard, 1966; Emrnestine and Parry, 1957).

Koch (1931) was the first to demonstrate that when carotid sinus pressure was

changed in a stepwise manner, mean arterial pressure exhibited an inverse sigmoidal

response to the change in intrasinus pressure. Bronk and Stella (1932) who observed that

the impulse frequency in Herring's nerve exhibited a positive sigmoidal, relationship to

changes in sinus pressure later substantiated these findings. These early findings identified

that baroreceptors can be characterized as having a threshold pressure range for which the

discharge rate increases with a rise in mean arterial pressure, as an asymptotic saturation

pressure beyond which there is little increase in baroreceptor activity (Koushanpour,

1991).

The aortic and carotid baroreceptors exhibit different threshold and saturation

characteristics. Carotid sinus baroreceptors are silent at arterial pressures between 0 and

60 mmHg, but above 60 mmHg, they respond progressively and reach maximum discharge

capacity at approximately 180 mmHg (Koushanpour, 1991). Aortic baroreceptors

respond in a manner similar to that of the carotids except that they exhibit a threshold










pressure approximately 30 mmHg higher (Koushanpour, 1991). Therefore, in the normal

operating range of approximately 100 mmHg (80 to 180 mmHg), slight changes in

pressure elicit strong baroreceptor-mediated autonomic reflexes to return arterial pressure

to within homeostatic limits.

Blood pressure control in the fetus is similar with some differences. Decreases in

blood pressure increase the secretion of ACTH, cortisol, AVP, and rein (Robillard et al.,

1979; Rose et al., 1981; Wood 1989). As in the adult, the magnitude of the responses are

proportional to the magnitude of the decrease in arterial pressure (Wood et al., 1982), and

responses are attenuated by sinoaortic denervation (Wood, 1989). Chemoreceptors have

a critical role in maintaining blood pressure as well by monitoring the levels of oxygen,

carbon dioxide, and hydrogen ions in the blood.

Effective baroreceptor function is necessary to respond to transient alterations in

arterial pressure (Brown, 1980). In the face of increasing pressure, baroreceptor-

generated signals ascend afferent pathways and enter the nucleus tractus solitarius where

secondary signals inhibit the medullary vasoconstrictor center and excite the vagal center

stimulating vasodilation and decreased myocardial ionotropic and chronotropic response

(Brown, 1980). These actions lead to lowered peripheral resistance, cardiac output, and

ultimately, lower blood pressure. Conversely, a sudden fall in arterial pressure leads to

reflex actions which increase cardiac output and systemic resistance to raise blood

pressure.

The bradycardic response to baroreceptor stimulation in humans is mediated

through vagal cholinergic mechanisms. Several investigators (Eckberg et al., 1971;

Pickering et al., 1972; Simon et al., 1977; Takeshita et al., 1979) have demonstrated that










elongation of the R-R interval which accompanied a rise in arterial pressure following

administration ofphenylephrine was not reduced by propranolol (Jose and Taylor, 1969),

but was abolished by atropine. Others have reported similar observations in response to

stimulation of the carotid baroreceptors by neck suction (Eckberg, 1977; Eckberg et al.,

1976).

In contrast, there is a lack of consensus regarding the autonomic mechanisms

mediating the tachycardic response to arterial baroreceptor unloading. It has been

observed that the early tachycardia observed after administration of vasodilators was

unaffected by propranolol, but abolished by atropine, suggesting a predominant vagal

mediation of this response (Leon et al., 1970; Mancia et al., 1979; Mroczek et al., 1976;

Pickering et al., 1972). Contrary to these reports, others have demonstrated that the

increase in heart rate produced during infusions of nitroglycerin was reduced by atropine,

but could only be abolished by combined administration of atropine and a beta-adrenergic
/
blocker (Goldstein et al., 1975; Robinson et al., 1966). In addition, it has been

demonstrated that during lower body negative pressure, tachycardia was diminished 52%

by propranolol with the remaining response abolished by atropine (Bjurstedt et al., 1977).

Therefore, it appears there is a significant vagal component to the cardioacceleration

which accompanies baroreceptor unloading. However, an increase of sympathetic cardiac

influence may contribute to the more sustained component ofbaroreflex-mediated

tachycardia (Mancia and Mark, 1983). Taken together, these results suggest redundancy

in mechanisms by which the autonomic nervous system mediates baroreflex-induced

tachycardia.










The baroreceptor system markedly reduces daily variation in arterial pressure.

This phenomenon is readily demonstrated in sinoaortic denervated animals who exhibit

elevated blood pressure (Cowley et al., 1973). Controversy exists regarding the

persistence of this hypertension with some authors arguing it eventually subsides (Guyton

et al., 1974) while others suggest elevated blood pressure persists (Scher and Ito, 1978;

Alexander, 1979; Touw et al., 1979; Werber and Fink, 1979).

Studies involving the measurement of substances released from the hypothalamus,

the pituitary or the adrenal require the evaluation of blood pressure as blood pressure is

reflexively defended at least in part by hypothalamic, pituitary and adrenal responses. In

both the adult and the fetus, responses to hypotensive stimuli involve neural and hormonal

changes that are responsible for restoring blood pressure. In the adult, decreases in blood

pressure are detected by stretch receptors in the atria (Cryer and Gann, 1974) and by

baroreceptors in the high pressure circulatory system (Roseetal, 1981) and result in the
/*
increased release of ACTH, cortisol (Gann, 1979), renin and AVP (Claybaugh and Share,

1973). In the fetus the ACTH, cortisol, AVP and renin responses to hypotension are not

vagally mediated but instead are thought to involve changes in blood pH and central

chemoreceptors (Wood et al., 1989). ACTH is important in both adult and fetal animals

for inducing the appropriate adrenocortical responses to noxious stimuli such as

hypotension (Wood and Rudolph, 1983). Cortisol is essential for the restitution of blood

volume following hemorrhage and as a permissive substance for appropriate

vasoconstriction following hypotension in both the adult (Grimes et al., 1987; Pirkle et al.,

1976) and fetus (Brace, 1983). Vasopressin is responsible for vasoconstriction,






38



redistribution of blood flow and antidiuresis in the adult (Cowley et al., 1974) and fetus

(Iwamoto et al., 1979).













CHAPTER 3
GENERAL METHODS


Surgical Preparation of Fetal Sheep

The sheep used in this study were all pregnant ewes of 115 days gestation or later.

Animals were purchased from various suppliers (Institute of Food and Agricultural

Sciences, University of Florida, Gainesville, Fl; Tom Morris, MD) and were of various

breeds (Florida Native, Mixed Western, etc.). Prior to surgery, all animals were housed in

approved pens in the Health Science Center or the 34'h Street facility at the University of

Florida. Prior to experimentation, all animals were housed in Animal Resources at the

Health Science Center and were maintained under controlled lighting and temperature.

Pens were cleaned daily and ewes were given food and water ad libitum.

Aseptic fetal surgery was performed in Animal Resources or at the 34th Street

facility under general anesthesia with 0.5% 2.0% halothane. All ewes were between 115

and 125 days gestation at the time of surgery. Food and water were withheld from ewes

24 hours prior to surgery. Ewes were sheared close to the skin around the abdomen and

prepared for surgery with povidone iodine (Betadine, Purdue Fredrick Co., Norwalk,

CT). Animals were intubated and connected to a respirator to allow for constant

anesthesia. Heart rate, blood pressure, ventilatory 02 and CO2, respirations per minute,

and rectal temperature were all monitored at the time of surgery. Animals were closely

monitored from the time of intubation until recovery when the animal could stand on its








own effort. Ewes were allowed free access to food and water throughout the post-

operative period.

The uterus was exposed using a midline incision beginning at the umbilicus and

extending caudally approximately 10 cm. Once the hindlimbs were located, a small

incision was made in the uterus. Hindlimbs were delivered through the uterine incision

one at a time for the purpose of placing a polyvinyl chloride catheter (0.03" ID, 0.05" OD)

into each femoral artery. Later, these catheters would be used for blood sampling and

blood pressure recording. The tips of each femoral catheter were advanced to the

subdiaphragmatic aorta. At this time an estradiol implant (5 mg/21 days or 250 gig/day;

Innovative Research of America, Toledo OH) or placebo was inserted subcutaneously into

the area of the gluteus medius before suturing the incised hindlimbs. An amniotic catheter

made of polyvinyl chloride (0.05" ID, 0.09" OD) was sutured to the exterior of a hindlimb

for the purpose of antibiotic delivery as well as amniotic fluid pressure measurements.

Hindlimb and uterine incisions were closed using 2.0 silk suture. Hindlimb incisions were

closed using a simple continuous suture pattern. All uterine incisions were closed first

with a locking simple continuous pattern followed by umbrication of the uterus.

Using a technique similar to the one described above, catheters were placed in the

lingual arteries and advanced 1 cm into the carotid artery toward the heart. Upon closure

of the neck incision, lingual catheters were anchored to the chin of the fetus with 2.0 silk

suture. Lingual catheters were of the same material and size and femoral catheters.

Depending upon the experimental setup, carotid sinus denervation was employed

at this time. After exposing the common carotid artery, denervation was accomplished by

stripping all nerves and connective tissue between the carotid-occipital arterial junction








and the carotid-lingual arterial junction. The occipital artery was ligated, for this is the

only method by which all of the carotid sinus baroreceptor and chemoreceptor afferent

fibers are cut. The lingual artery was stripped rostrally of all nerves and connective tissue

for approximately 1 cm from the carotid-lingual junction. This denervation was conducted

bilaterally.

Before returning the head of the fetus to the uterus, an occluder was placed around

the brachiocephalic artery. These silastic occluders resemble miniature blood pressure

cuffs and were purchased from In Vivo Metric (Cat. # OC8, Healdsburg, CA). The left

forelimb of the fetus was delivered through a uterine incision. An incision was made into

the second intercostal space and the brachiocephalic artery was located. Once the

occluder was sewn in place, the incision under the left forearm was closed and the fetus

returned to the uterus. The uterine incision was closed once again using a locking simple

continuous suture pattern followed by an umbrication technique. 750 mg ampicillin

(Polyflex, Ft. Dodge Laboratories, Ft. Dodge, IA) was administered into the amniotic

cavity before closure of the maternal linea alba and skin.

All catheters were filled with heparin (1000 units/ml, Elkins-Sinn, Inc., Cherry Hill,

NJ) and closed with a sterile brad inserted into the end. Catheters and occluders were

flanked and exteriorized via a trochar. Catheters were held in place with an elastic

bandage. The linea alba was closed with #3 polyamide suture (Pitmann-Moore, France)

while the skin was closed with #1. 750 mg ampicillin was administered intramuscularly to

the ewe.

It should be noted that in ewes with twin pregnancies, both fetuses were surgically

manipulated the same way. At the time of experimentation however, only one of the








animals was made hypotensive while the other animal served as a control. All ewes were

treated with 750 mg ampicillin twice per day for five days post-operatively. In addition,

rectal temperatures were taken to monitor for infection. All ewes were monitored closely

for any indication of poor health.

In Vivo Experimental Procedures

All ewes were given five days to recover from surgery. On the day of

experimentation, catheters were removed from the elastic bandage and the distal ends

were cleaned with povidone iodine and alcohol. Each brad was removed and a sterile

blunt adapter with a three-way stopcock was inserted. This procedure was always done

for both femoral catheters and the amniotic catheter. Lingual catheters were only utilized

if the fetus was to be made hypotensive. All catheters to be used were flushed with

heparinized saline (2.0%/ v/v). One femoral catheter and the amniotic catheter were

attached to transducers (Statham P23Id, Statham Instruments, Oxnard, CA) for

measurement of fetal arterial and amniotic fluid pressure. As stated previously, if the fetus

was made hypotensive lingual pressure was also monitored to assure proper occlusion.

Arterial and amniotic fluid pressures were measured for the first 35 minutes of the

experiment using a Grass Model 7 recorder. The data were digitized and stored using an

IBM AT Microcomputer and a Keithley analog-to-digital converter on-line.

All experiments were performed between 120 and 135 days gestation to minimize

variation in hormone concentrations between experiments and animals. All animals were

studied in their pens utilizing six sections of PVC tubing to limit movement of the ewe.

Once catheters were removed from the elastic bandage ewes were not touched in order to

limit the amount of external stress placed upon the animals. Experiments lasted one hour










with blood samples taken at 0, 10, 20, and 60 minute time points. If the fetus was to be

made hypotensive to activate the HPA axis, the brachiocephalic occluder was inflated after

collection of the 0 time point sample for 10 minutes. The brachiocephalic occluder was

inflated via an infusion of saline through the silastic tubing. This, in turn, causes a

hypoperfusion of blood to the fetal brain, which activates the HPA axis. Five ml of blood

were taken at each time point and collected in chilled tubes containing Na4 EDTA (50 tg

EDTA/ml blood, Sigma Chemical Co., St. Louis, MO). An additional 1.5 ml of blood was

drawn anaerobically into syringes coated with heparin for measurement of fetal blood

gases using a Ciba-Corning 288 Blood Gas System. A small portion of this blood was

used to measure hematocrit using an IEC microhematocrit centrifuge. After sampling,

volume of the catheter was restored with 0.9% normal saline with 2.0% v/v heparin.

Blood samples (5 ml) were kept on ice until further analysis for hormone levels.

Samples were centrifuged at 3000 x g for 30 minutes at 4 C in a refrigerated centrifuge
i
(Sorvall RT 600B, DuPont, Newtown, CA). After centrifugation, the plasma was

transferred and aliquotted to polystyrene tubes and stored at -20 C until hormones were

assayed.

Upon conclusion of each experiment, ewes were sacrificed with an overdose of

sodium pentobarbital via the jugular vein. Fetuses were immediately removed for

perfusion of the brain. The chest cavity of each fetus was opened and the brachiocephalic

artery was located and cannulated. Either by means of a pump or by syringe, brains were

perfused first with one liter of phosphate buffered saline (pH 7.4, 2.0% v/v heparin)

followed by two liters of 4% paraformaldehyde. Brains and pituitary glands were

removed and stored in 4% paraformaldehyde until processing for immunohistochemistry.










Peptide Assays

Arginine Vasopressin (AVP) Plasma AVP concentrations were measured using an

antibody raised in rabbits (Raffet al., 1991). lodinated AVP was purchased from

Amersham and synthetic AVP from Sigma Chemical Co. AVP was first extracted from

0.5 ml plasma with 1.0 ml bentonite slurry (0.3% w/v in distilled water) and acidified with

0.05 ml 1 N HCI. Extracts were eluted with 1 ml acid:acetone (20% IN HCI:80%

acetone) with sonication. Samples were then evaporated to dryness and stored at -20 C

until assayed. Extracts were reconstituted with 0.25 ml assay buffer (0.05 M phosphate

buffer, pH 7.4 with 0.01 M EDTA (Sigma, #ED4SS) and 0.2% BSA w/v (Sigma, #A-

7638)). Extraction recovery was corrected by comparing samples to a standard curve

prepared from standard extracted with each set of samples.

Adrenocorticotropin (ACTH) Plasma ACTH concentrations were measured by

radioimmunoassay (RIA) as previously described (Bell et al., 1991) using an antibody
t
raised in rabbits developed in Dr. Wood's laboratory to human-ACTH (1-24). lodinated

ACTH (125 ACTH) was prepared using the chloramine-T method (Berson and Yalow,

1968) with human-ACTH (1-39)(Sigma Chemical Co., St. Louis, MO) and radioactive

sodium iodide (Amersham, Arlington Heights, IL). ). I125 ACTH was made fresh

approximately every six weeks. ACTH was first extracted from plasma before assaying.

ACTH (0.5 ml) was extracted on glass (35 mg/tube) (100-200 mesh glass. Corning

Glassworks, Comrning, NY) in 0.50 ml assay buffer (0.05 M phosphate buffer, pH 7.4 with

0.2% w/v silicic acid-extracted bovine serum albumin (BSA, Sigma Chemical Co.,

#9647)). The supernatant was aspirated and the glass washed with assay buffer. The

ACTH was eluted from the glass with 1 ml acid:acetone (1 volume 0.25 N HCI: 1 volume









acetone). The extracts were dried under vacuum (Savant Instruments, Farmingdale, NY)

and frozen at -20 C until assayed. Extracts were reconstituted overnight in 0.5 ml assay

buffer containing 0.5% v/v mercaptoethanol. Extraction recovery was corrected by

comparing samples to a standard curve prepared from standard extracted with each set of

samples.

Steroid Asays

Cortisol Plasma cortisol concentrations were measured as previously described (Wood et

al., 1993) using an antibody raised in rabbits and titrated cortisol purchased from

Amersham (#TRK-407) and cortisol standard from Sigma Chemical Co. Cortisol was

extracted from 20 Wi plasma in duplicate with 1 ml ethanol. Standard was prepared in

ethanol, and standards and samples were dried under vacuum with heat and immediately

reconstituted with 0.5 ml assay buffer (0.05 M phosphate buffer (using 0.06 M sodium

phosphate dibasic and 0.04 M sodium phosphate monobasic) pH 7.0 with 0.15 M NaCl,

0.1% w/v gelatin, and 0.1% w/v sodium azide).

Estradiol Plasma estradiol concentrations were measured utilizing an enzyme

immunoassay (EIA) kit from Oxford Biochemical Inc. (#EA70). 2 ml of plasma was

extracted with 16 ml ethyl ether. Extracts were dried under air and reconstituted to 200

4l with assay buffer (provided with kit). 50 u.l of extract was assayed for estradiol in

duplicate. The values obtained were divided by 10 to give ng/ml concentrations. The

antiserum in this particular EIA kit had a very low cross-reactivity with estrone (0.10%).

Estrone Sulfatase Activity

I studied fetuses (86-147 days gestation, term= 147 days), 4 lambs (3-4 weeks old),

and 4 adult nonpregnant ewes to determine estrone sulfatase activity. The sheep were










sacrificed using an intravenous overdose of sodium pentobarbital. Gestational ages of the

fetal sheep were calculated from known breeding dates. Whole brains were rapidly

removed, dissected into discrete regions, and quickly frozen on dry ice or in a slurry of dry

ice and acetone. All tissues were stored at -20C or -40C until studied.

Hypothalami, brainstems, and hippocampi were then processed to determine

estrone sulfatase activity. Each tissue sample was homogenized in medium 199 (Sigma,

St. Louis, MO) containing 25 mM HEPES. Homogenization was performed using a

Polytron homogenizer (Tekmar, Cincinnati, OH). The concentration of each tissue in the

homogenate was 0.5 g tissue in 5 mL medium.

Tissues were centrifuged at 1200 rpm for 5 min; supernatant was then collected

and assayed immediately. A sample of each homogenate was assayed for protein

concentration using the method of Bradford (1976) using a commercially-available assay

kit (Bio-Rad Laboratories, Hercules, CA). Homogenate (0.1 mL) was' aliquotted in

duplicate into borosilicate tubes (16 x 150 mm) containing 0.8 mL of a mixture of 3H-

[6,7]-estrone sulfate (DuPont-NEN, Wilmington, DE) and unlabeled estrone sulfate

(EiS04, Sigma, St. Louis, MO). All reactions were run at 37C. Reactions were

terminated by immediate cooling on ice, addition of 5 volumes of ethyl acetate:hexane

(3:2), and vigorous mixing for 30 seconds. The aqueous phase was frozen by submersion

of the reaction tube into a dry ice and acetone slurry. Subsequently, the organic phase

containing the 3H-estrone was decanted into 13x75 mm borosilicate glass tubes and dried

under a gentle stream of room air. Dried extracts were reconstituted in scintillant

(Cytoscint, ICN Corp., Costa Mesa, CA) and counted in a scintillation counter (LKB

Corp., Gaithersburg, MD).










Enzyme activities at different developmental ages and in different tissues were

measured using a substrate concentration of 3 pM and 3H-estrone sulfate specific activity

of approximately 0.67 gCi/nmol. For this experiment, reactions were allowed to run for 5

min. Using these conditions, less than 20% of the substrate was converted to 3H-estrone.

Western Blotting

Hypothalami and brainstems were harvested from fetuses, lambs, and adults of

known gestational and postnatal ages. The number and ages of animals varied slightly

between hypothalami and brainstem but 11-12 fetuses, 3-4 lambs and 2 adults were used

per tissue type. These tissues were originally obtained and homogenized for other studies

(Saoud and Wood, 1996). Unfortunately, hippocampi from these animals were not

available. All tissue was homogenized in reducing buffer and boiled for 5 minutes. The

samples were centrifuged to remove particulate matter and supernatant was recovered.

Protein.concentrations were obtained utilizing the Bradford technique (1976). Western

blots were performed using a mini-Protean electrophoresis system (Bio-Rad, Hercules,

CA) on 10% pre-cast polyacrylamide gels purchased from Bio-Rad laboratories. Samples

were diluted so that an equal amount of protein was loaded per lane (20 jg for brainstem

and 40 jg for hypothalami). The protein was then transferred to a nitrocellulose

membrane and probed for either estrogen sulfatase or estrogen sulfotransferase using

custom-made rabbit polyclonal antibodies (Alpha Diagnostic, San Antonio, TX). The

peptide sequence used from the human sulfatase gene, amino acids 294-309, was NH2-

FSSKDFAGKSQHGVYGC-COOH (Simerly et al., 1990). The peptide sequence used

from the bovine sulfotransferase gene, amino acids 273-295, was NH2-

RERFEEHYQQQMKDC-COOH (Nash et al., 1988). Primary antibodies were diluted to










a concentration of 1:1000 in antibody diluent (1 % BSA in phosphate buffered saline with

0.05 % Tween 20). Visualization of the protein-antibody complex was accomplished

utilizing a chemiluminescence detection system (Renaissance, DuPont NEN, Boston, MA)

and analyzed by densitometry (Bio-Rad). Antibody specificity was confirmed by

preabsorption of the primary antibodies with peptides (1 .g/ml) also supplied by Alpha

Diagnostic. Developmental changes were calculated using multiple linear regression in

order to control for differences between gel running conditions (SigmaStat, Jandel

Scientific, San Rafael, CA).

Immunohistochemical Techniques

Fetal brains were perfusion fixed with 4% paraformaldehyde, dissected and cut

into gross tissue regions (hypothalamus, midbrain, pons, medulla, spinal cord, etc.).

Tissue was processed for embedding by dehydration with progressively increasing

concentrations of ethanol, followed by xylene. All tissue was embedded in paraffin and

cut into 10 pm sections using a Zeiss microtome. Sections were mounted on poly-L-

lysine slides, deparaffinized with xylene and rehydrated in decreasing concentrations of

ethanol. Immunohistochemistry and visualization were made possible utilizing a

Histostain-SP kit from Zymed and metal-enhanced DAB (Pierce, Rockford, IL). Sections

were stained for estrogen sulfatase, estrogen sulfotransferase, c-fos, ACTH, AVP, and

CRH (see Table 3.1). Primary antibodies were diluted in antibody diluent (1% BSA in

phosphate buffered saline with 0.01% Triton X-100). Specific staining was confirmed by

dilution tests, as staining decreased as primary antibodies were further diluted. Specific

staining was absent upon replacing primary antibodies with 10% normal goat serum. All








slides were dehydrated prior to mounting of coverslips with Permount (Fisher Scientific,

Pittsburgh, PA).


Primary antibody Vendor (Source) Dilution
Estrogen Sulfatase Alpha Diagnostics (same as 1:500
for Western Blots)__________
Estrogen Sulfotransferase Alpha Diagnostics (same as 1:500
for Western Blots)
c-fos Oncogene Biomedical Inc. 1:5000
~~~____~_______ (cat. PC38)
ACTH Wood Laboratory (same as 1:10,000
for RIA)
AVP Wood Laboratory (same as 1:20,000
~~____~________for RIA) ______I___
CRH Keller-Wood Laboratory 1:100,000


Table 3.1: Primary antibodies used in immunohistochemical experiments. All antibodies

were diluted in 1% BSA in phosphate buffered saline with 0.01% Triton X-100.













CHAPTER 4
HORMONAL RESPONSIVENESS IN AN ESTRADIOL, HYPOTENSIVE, CAROTID
SINUS DENERVATED OVINE MODEL


Introduction

In the fetal sheep, parturition is triggered by an increase in the activity of the fetal

HPA axis (Challis and Brooks, 1989; Liggins et al., 1973). Parturition can be delayed by

destruction of the pituitary (Liggins et al., 1966, 1967; Liggins and Kennedy, 1968) or

stimulated by infusions ofACTH (Liggins, 1968, 1969) or glucocorticoids (Jack et al.,

1975; Wood and Keller-Wood, 1991). The last few days of gestation are marked by an

increase in the activity of the fetal hypothalamus as seen by elevated levels of fetal plasma

ACTH. This increase in plasma ACTH causes a corresponding increase in plasma

cortisol.. Along with this increase in HPA axis activity is a decreased sensitivity of the axis

to cortisol negative feedback (Wood, 1988). It is well known that in the sheep, cortisol

acts at the placenta to increase the activity of an enzyme, cytochrome p450 (17-

hydroxylase and 17,20 lyase activities), which in turn, increases the ratio of estrogen to

progesterone (Anderson et al., 1975; Pomeranz and Nalbandov, 1975; Steele et al., 1976;

Yu et al., 1983). This cascade of events essentially increases the total amount of estrogen.

Estrogen is known to be an important factor in the initiation of parturition by

causing the uterus to contract (Liggins, 1974). 173-estradiol has been shown to increase

the activity of the HPA axis in sheep and rats. In a study by Saoud and Wood (1995),






51

estrogen was found to augment fetal plasma ACTH secretion in response to stress. Other

studies have shown similar results in adult animals (Viau and Meaney, 1991).

Understanding the mechanism of the increased fetal HPA axis at the end of

gestation is key to understanding the mechanism of spontaneous parturition in sheep.

These experiments were conducted to see if estradiol has it's stimulatory effect on HPA

axis activity through the afferent baroreceptor and chemoreceptor pathway. More

specifically, I hypothesized that estradiol's actions on fetal cardiovascular reflex

responsiveness to hypotension will be measurable in intact fetuses but not in baro- and

chemo- denervated fetuses. This augmentation of HPA axis activity will be assessed by

using a surgically manipulated ovine model. ACTH, AVP, estradiol, and cortisol levels

will be measured and compared across eight different treatment groups. These groups

include fetuses which are: (1) estradiol treated; (2) estradiol treated, hypotensive; (3)

estradiol treated, carotid sinus denervated; (4) estradiol treated, carotid sinus denervated,

hypotensive; (5) placebo treated; (6) placebo treated, hypotensive; (7) placebo treated,

carotid sinus denervated; (8) placebo treated, carotid sinus denervated, hypotensive.

Research has previously demonstrated that the combined baro- and chemo- denervation

attenuates (approximately 50%) the reflex hormonal and hemodynamic responses to

moderate (50 %) reduction in arterial blood pressure. I proposed that the interruption of

the afferent pathways would eliminate the effect of estrogen on the reflex cardiovascular

responsiveness. If so, it could concluded that estrogen acts on, within, or requires input

from, the afferent baroreceptor and chemoreceptor pathways.










Methods and Materials

Surgical Procedures Aseptic fetal surgery was performed in Animal Resources or at the

34th Street facility under general anesthesia with 0.5% 2.0% halothane. All ewes were

between 115 and 125 days gestation at the time of surgery. A total of 40 ewes were set

up and studied (n=5 per group). Food and water were withheld from ewes 24 hours prior

to surgery. Ewes were sheared close to the skin around the abdomen and prepared for

surgery with povidone iodine (Betadine, Purdue Fredrick Co., Norwalk, CT). Animals

were intubated and connected to a respirator to allow for constant anesthesia. Heart rate,

blood pressure, ventilatory 02 and CO2, respirations per minute, and rectal temperature

were all monitored at the time of surgery. Animals were closely monitored from the time

of intubation until recovery when the animal could stand on its own effort. Ewes were

allowed free access to food and water throughout the post-operative period.

The uterus was exposed using a midline incision beginning at the umbilicus and
f
extending caudally approximately 10 cm. Once the hindlimbs were.located, a small

incision was made in the uterus. Hindlimbs were delivered through the uterine incision

one at a time for the purpose of placing a polyvinyl chloride catheter (0.03" ID, 0.05" OD)

into each femoral artery. Later, these catheters would be used for blood sampling and

blood pressure recording. The tips of each femoral catheter were advanced to the

subdiaphragmatic aorta. At this time an estradiol implant (5 mg/21 days or 250 tg/day;

Innovative Research of America, Toledo OH) or placebo was inserted subcutaneously into

the area of the gluteous medius before suturing the incised hindlimbs. An amniotic

catheter made of polyvinyl chloride (0.05" ID, 0.09" OD) was sutured to the exterior of a

hindlimb for the purpose of antibiotic delivery as well as amniotic fluid pressure










measurements. Hindlimb and uterine incisions were closed using 2.0 silk suture. Hindlimb

incisions were closed using a simple continuous suture pattern. All uterine incisions were

closed first with a locking simple continuous pattern followed by umbrication of the

uterus.

Using a technique similar to the one described above, catheters were placed in the

lingual arteries and advanced approximately 1 cm into the carotid artery toward the heart.

Upon closure of the neck incision, lingual catheters were anchored to the chin of the fetus

with 2.0 silk suture. Lingual catheters were of the same material and size and femoral

catheters.

Depending upon the experimental setup, carotid sinus denervation was employed

at this time. After exposing the common carotid artery, denervation was accomplished by

stripping all nerves and connective tissue between the carotid-occipital arterial junction

and the carotid-lingual arterial junction. The occipital artery was ligated, for this is the

only method by which all of the carotid sinus baroreceptor and chemoreceptor afferent

fibers are cut. The lingual artery was stripped rostrally of all nerves and connective tissue

for approximately 1 cm from the carotid-lingual junction. This denervation was conducted

bilaterally.

Before returning the head of the fetus to the uterus, an occluder was placed around

the brachiocephalic artery. These silastic occluders resemble miniature blood pressure

cuffs and were purchased from In Vivo Metric (Cat. # OC8, Healdsburg, CA). The left

forelimb of the fetus was delivered through a uterine incision. An incision was made into

the second intercostal space and the brachiocephalic artery was located. Once the

occluder was sewn in place, the incision under the left forearm was closed and the fetus










returned to the uterus. The uterine incision was closed once again using a locking simple

continuous suture pattern followed by an umbrication technique. 750 mg ampicillin

(Polyflex, Ft. Dodge Laboratories, Ft. Dodge, IA) was administered into the amniotic

cavity before closure of the maternal linea alba and skin.

All catheters were filled with heparin (1000 units/ml, Elkins-Sinn, Inc., Cherry Hill,

NJ) and closed with a sterile brad inserted into the end. Catheters and occluders were

flanked and exteriorized via a trochar. Catheters were held in place with an elastic

bandage. The linea alba was closed with #3 polyamide suture (Pitmann-Moore, France)

while the skin was closed with #1. 750 mg ampicillin was administered intramuscularly to

the ewe.

It should be noted that in ewes with twin pregnancies, both fetuses were surgically

manipulated the same way. At the time of experimentation however, only one of the

animals was made hypotensive while the other animal served as a control. All ewes were

treated with 750 mg ampicillin twice per day for five days post-operatively. In addition,

rectal temperatures were taken to monitor for infection. All ewes were monitored closely

for any indication of poor health.

In Vivo Experimental Procedures All ewes were given five days to recovery from

surgery. On the day of experimentation, catheters were removed from the elastic bandage

and the distal ends were cleaned with povidone iodine and alcohol. Each brad was

removed and a sterile blunt adapter with a three-way stopcock was inserted. This

procedure was always done for both femoral catheters and the amniotic catheter. Lingual

catheters were only utilized if the fetus was to be made hypotensive. All catheters to be

used were flushed with heparinized saline (2.0% v/v). One femoral catheter and the










amniotic catheter were attached to transducers (Statham P23Id, Statham Instruments,

Oxnard, CA) for measurement of fetal arterial and amniotic fluid pressure. There were

eight different experimental groups in this study: (1) estradiol treated; (2) estradiol

treated, hypotensive; (3) estradiol treated, carotid sinus denervated; (4) estradiol treated,

carotid sinus denervated, hypotensive; (5) placebo treated; (6) placebo treated,

hypotensive; (7) placebo treated, carotid sinus denervated; (8) placebo treated, carotid

sinus denervated, hypotensive. As stated previously, if the fetus was made hypotensive

lingual pressure was also monitored to assure proper occlusion. Arterial and amniotic

fluid pressures were measured for the first 35 minutes of the experiment using a Grass

Model 7 recorder. The data were digitized and stored using an IBM AT Microcomputer

and a Keithley analog-to-digital converter on-line.

All experiments were performed between 120 and 135 days gestation to minimize

variation in hormone concentrations between experiments and animals.- All animals were

studied in their pens utilizing six sections of PVC tubing to limit movement of the ewe.

Once catheters were removed from the elastic bandage ewes were not touched in order to

limit the amount of external stress placed upon the animals. Experiments lasted one hour

with blood samples taken at 0, 10, 20, and 60 minute time points. If the fetus was to be

made hypotensive to activate the HPA axis, the brachiocephalic occluder was inflated after

collection of the 0 time point sample for 10 minutes. The brachiocephalic occluder was

inflated via an infusion of saline through the silastic tubing. This, in turn, causes a

hypoperfuision of blood to the fetal brain, which activates the HPA axis. Five ml of blood

were taken at each time point and collected in chilled tubes containing Na4 EDTA (50 jig

EDTA/ml blood, Sigma Chemical Co., St. Louis, MO). An additional 1.5 ml of blood was










drawn anaerobically into syringes coated with heparin for measurement of fetal blood

gases using a Ciba-Comrning 288 Blood Gas System. A small portion of this blood was

used to measure hematocrit using an IEC microhematocrit centrifuge. After sampling,

blood volume was restored with 0.9% normal saline with 2.0% v/v heparin.

Blood samples (5 ml) were kept on ice until further processing. Samples were

centrifuged at 3000 x g for 30 minutes at 4 C in a refrigerated centrifuge (Sorvall RT

600B, DuPont, Newtown, CA). After centrifugation, the plasma was transferred and

aliquotted to polystyrene tubes and stored at -20 C until hormones were assayed.

Upon conclusion of each experiment, ewes were sacrificed with an overdose of

sodium pentobarbital via the jugular vein. Fetuses were immediately removed for

perfusion of the brain. The chest cavity of each fetus was opened up and the

brachiocephalic artery was located and cannulated. Either by means of a pump or by

syringe, brains were perfused first with one liter of phosphate buffered' saline (pH 7.4,

2.0% v/v heparin) followed by two liters of 4% paraformaldehyde. Brains and pituitary

glands were removed and stored in 4% paraformaldehyde until processing for

immunohistochemistry (Chapter 5). The experimental design can be viewed pictorially in

Figure 4.1.

Estradiol Assay Plasma estradiol concentrations were measured utilizing an enzyme

immunoassay (EIA) kit from Oxford Biochemical Inc. (#EA70). 2 ml of plasma was

extracted with 16 ml ethyl ether. Extracts were dried under air and reconstituted to 200

ll with assay buffer (provided with kit). 50 ul of extract was assayed for estradiol in

duplicate. The values obtained were divided by 10 to give ng/ml concentrations. This

particular EIA kit had a very low cross-reactivity with estrone (0.10%).










Adrenocorticotropin (ACTH) Assay Plasma ACTH concentrations were measured by

radioimmunoassay (RIA) as previously described (Bell et al., 1991) using an antibody

raised in rabbits developed in Dr. Wood's laboratory to human-ACTH (1-24). lodinated

ACTH (I125 ACTH) was prepared using the chloramine-T method (Berson and Yalow,

1968) with human-ACTH (1-39)(Sigma Chemical Co., St. Louis, MO) and radioactive

sodium iodide (Amersham, Arlington Heights, IL).). I125 ACTH was made fresh

approximately every six weeks. ACTH was first extracted from plasma before assaying.

ACTH (0.5 ml) was extracted on glass (35 mg/tube) (100-200 mesh glass, Coming

Glassworks, Coming, NY) in 0.50 ml assay buffer (0.05 M phosphate buffer, pH 7.4 with

0.2% w/v silicic acid-extracted bovine serum albumin (BSA, Sigma Chemical Co.,

#9647)). The supernatant was aspirated and the glass washed with assay buffer. The

ACTH was eluted from the glass with 1 ml acid:acetone (1 volume 0.25 N HCI: 1 volume

acetone). The extracts were dried under vacuum (Savant Instruments,,Farmingdale, NY)
/
and frozen at -20 C until assayed. Extracts were reconstituted overnight in 0.5 ml assay

buffer containing 0.5% v/v mercaptoethanol. Extraction recovery was corrected by

comparing samples to a standard curve prepared from standard extracted with each set of

samples.

Arginine Vasopressin (AVP) Assay Plasma AVP concentrations were measured using an

antibody raised in rabbits (Raffet al., 1991). lodinated AVP was purchased from

Amersham and synthetic AVP from Sigma Chemical Co. AVP was first extracted from

0.5 ml plasma with 1.0 ml bentonite slurry (0.3% w/v in distilled water) and acidified with

0.05 ml 1 N HCI. Extracts were eluted with 1 ml acid:acetone (20% IN HC1:80%

acetone) with sonication. Samples were then evaporated to dryness and stored at -20 C










until assayed. Extracts were reconstituted with 0.25 ml assay buffer (0.05 M phosphate

buffer, pH 7.4 with 0.01 M EDTA (Sigma, #ED4SS) and 0.2% BSA w/v (Sigma, #A-

7638)). Extraction recovery was corrected by comparing samples to a standard curve

prepared from standard extracted with each set of samples.

Cortisol Assay Plasma cortisol concentrations were measured as previously described

(Wood et al., 1993) using an antibody raised in rabbits and titrated cortisol purchased

from Amersham (#TRK-407) and cortisol standard from Sigma Chemical Co. Cortisol

was extracted from 20 pl plasma in duplicate with 1 ml ethanol. Standard was prepared in

ethanol, and standards and samples were dried under vacuum with heat and immediately

reconstituted with 0.5 ml assay buffer (0.05 M phosphate buffer (using 0.06 M sodium

phosphate dibasic and 0.04 M sodium phosphate monobasic) pH 7.0 with 0.15 M NaCl,

0.1% w/v gelatin, and 0.1% w/v sodium azide).

Statistical Analyses ACTH, AVP, and cortisol levels were analyzed via three way

ANOVA. Estradiol treated fetuses were analyzed separately from placebo treated fetuses

by this means using hypotension/normotension, CSD/intact, and time as factors. Student

Newman-Keuls Test was employed as a multiple comparison procedure for statistically

significant groups within each grouping. Estradiol treated fetuses were compared to

placebo treated fetuses relative to time and treatment group by a t-test. Estradiol level

comparison between estradiol treated fetuses and placebo treated fetuses was made using

a t-test.

Results

Estradiol Assay Since my hypotheses depend upon the a difference in estradiol levels

between treatment groups, it seems only logical to discuss the results of the estradiol assay










first. There were a total of 40 animals studied in this experiment. Half(20) were

pretreated with an estradiol implant during surgery. These implants release estradiol at a

constant rate of 5 mg/21 day period or around 250 utg/day. Estradiol levels were

measured by EIA and statistical analyses showed that the estradiol treated fetuses were

significantly different than the placebo treated fetuses by a t-test (n=20 per group,

p<0.001). Result can be seen in Figure 4.2 which shows group means measured in units

of pg/ml +SEM.

Adrenocorticotropin (ACTH) Assay Plasma ACTH levels are shown in Figure 4.3.

Results are plotted as group means +SEM. Placebo treated fetuses and estradiol fetuses

were analyzed separately by three way ANOVA with hypotension / normotension, carotid

sinus denervation / intact, and time (0min., 10min., 20min., and 60min.) as experimental

factors. In both cases (placebo and estradiol treatment), groups were found to be

statistically different (n=5 per group, p<0.001). Specifically, ACTH levels in the
t
hypotensive animals at 10min. and 20min. as well as hypotensive, carotid sinus denervated

animals at 10min. and 20min. were found to differ significantly from the rest of the

treatment groups when further analyzed by Student Newman Keuls Comparison (n=5 per

group, p<0.001). This was true for both placebo treated fetuses and estradiol treated

fetuses. Also, all placebo treated fetuses differed significantly from their corresponding

estradiol treated fetuses by t-test (n=5 per group, p<0.01).

Arginine Vasopressin (AVP) Assay Plasma AVP levels are shown in Figure 4.4. Results

are plotted as group means +SEM. Placebo treated fetuses and estradiol fetuses were

analyzed separately by three way ANOVA with hypotension / normotension, carotid sinus

denervation / intact, and time (Omin., 10min., 20min., and 60min.) as experimental factors.










In both cases (placebo and estradiol treatment), groups were found to be statistically

different (n=5 per group, p<0.001). Specifically, AVP levels in the hypotensive animals at

10min. and 20min. as well as hypotensive, carotid sinus denervated animals at 10min. and

20min. were found to differ significantly from the rest of the treatment groups when

further analyzed by Student Newman Keuls Comparison (n=5 per group, p<0.001). This

was true for both placebo treated fetuses and estradiol treated fetuses. Also, all placebo

treated fetuses differed significantly from their corresponding estradiol treated fetuses by

t-test (n=5 per group, p<0.01).

Cortisol Assay Plasma cortisol levels are shown in Figure 4.5. Results are plotted as

group means SEM. Placebo treated fetuses and estradiol fetuses were analyzed

separately by three way ANOVA with hypotension / normotension, carotid sinus

denervation / intact, and time (Omin., 10min., 20min., and 60min.) as experimental factors.

In both cases (placebo and estradiol treatment), groups were found to be statistically
/
different (n=5 per group, p<0.001). Specifically, cortisol levels in the hypotensive animals

at 10min. and 20min. as well as hypotensive, carotid sinus denervated animals at 10min.

and 20min. were found to differ significantly from the rest of the treatment groups when

further analyzed by Student Newman Keuls Comparison (n=5 per group, p<0.001). This

was true for both placebo treated fetuses and estradiol treated fetuses. Also, all placebo

treated fetuses differed significantly from their corresponding estradiol treated fetuses by

t-test (n=5 per group, p<0.01).

Discussion

Understanding the mechanism of the increased fetal HPA axis at the end of

gestation is key to understanding the mechanism of spontaneous parturition in sheep.










These experiments were conducted to see if estradiol has it's stimulatory effect on HPA

axis activity through the afferent baroreceptor and chemoreceptor pathway. More

specifically, I hypothesized that estradiol's actions on fetal cardiovascular reflex

responsiveness to hypotension will be measurable in intact fetuses but not in baro- and

chemo- denervated fetuses. This augmentation of HPA axis activity was assessed by using

a surgically manipulated ovine model. ACTH, AVP, estradiol, and cortisol levels were

measured and compared across eight different treatment groups. These groups include

fetuses which are: (1) estradiol treated; (2) estradiol treated, hypotensive; (3) estradiol

treated, carotid sinus denervated; (4) estradiol treated, carotid sinus denervated,

hypotensive; (5) placebo treated; (6) placebo treated, hypotensive; (7) placebo treated,

carotid sinus denervated; (8) placebo treated, carotid sinus denervated, hypotensive.

This study has shown that that estradiol does have an effect on cardiovascular

responsiveness. Figures 4.3-4.5 show that pretreatment with estradiol increases even
I
basal ACTH, AVP, and cortisol levels. This effect of estradiol is seen best perhaps after a

ten minute period of hypotension. At this point, hormonal responsiveness has been greatly

augmented with regard to ACTH, AVP, and cortisol levels. Carotid sinus denervation,

essentially eliminates this augmented HPA axis activity in response to brachiocephalic

occlusion. Any hormone response leftover after denervation may be due in part to

incomplete denervation at the time of surgery. Of course another explanation lies with the

fact that other pathways or systems might be responsible for hormone secretion. Carotid

sinus denervation by itself, however, caused no HPA axis activity or augmentation,

deeming the surgical elimination of baroreceptor / chemoreceptor responsiveness an

appropriate and effective control. Research has previously demonstrated that the










combined baro- and chemo- denervation attenuates (approximately 50%) the reflex

hormonal and hemodynamic responses to moderate (50 %) reduction in arterial blood

pressure. This study supports this finding demonstrated by the fact that ACTH, AVP, and

cortisol levels at 10min. and 20min. time points in hypotensive, carotid sinus denervated

animals are approximately half their corresponding levels with hypotension alone. At the

very least, it can be concluded that estrogen acts on, within, or requires input from, the

afferent baroreceptor and chemoreceptor pathways.

This study utilized an artificial stimulus for HPA axis activation. This taken with

the fact that the animals in this study are at about 85% gestation makes direct comparison

to the time of parturition impossible. However, this study is useful in that it sheds light on

how an ovine fetus responds to bought of hypotension. Furthermore, the effect of

estradiol on cardiovascular responsiveness has been shown to greatly augment HPA axis

activity in response to such hypotension as controlled through the baroreceptor /

chemoreceptor pathway. These are important findings because although this ovine model

done not exactly mimic the process of parturition, important conclusions can be made. All

of the animals in this study are at least at 125 days gestation. Before around 120 days

gestation, the HPA axis is fairly unresponsive to stimuli. The estradiol implants used in

this study raised plasma estradiol levels to within a physiological range. Also, plasma

estradiol levels were comparable to time just prior to parturition, when estradiol levels are

thought to activate the HPA axis. For these reasons, it is evident that the model system

used in this study is appropriate for deciphering not only fetal responsiveness to

cardiovascular stimuli, but also the mechanism of parturition itself










Though the results of this study elude to an estradiol responsive baroreceptor /

chemoreceptor afferent pathway participation in cardiovascular reflex control and trigger

for parturition, they do not by themselves answer account for total control over these

processes. As stated before, research has previously demonstrated that the combined

baro- and chemo- denervation attenuates (approximately 50%) the reflex hormonal and

hemodynamic responses to moderate (50 %) reduction in arterial blood pressure. This

means that 50% of the mechanism / pathway responsible for cardiovascular reflex

responsiveness and parturition is unaccounted for. It is plausible that the remaining

compensatory mechanisms exist within the central nervous system itself Since the

cerebral circulation of the fetus exhibits autoregulation, one could hypothesize that

hypotension would cause reduced cerebral blood flow. This reduced flow may be

defended against by a local release ofvasoactive substances. Prostanoids represent one

such group of compounds that may if fact be responsible for this response. Although it

has not been quantified at this time, our lab has shown that estradiol treatment does in fact

increase prostaglandin E2 (PGE2) within areas of the brain that are responsible for HPA

axis control. Other studies have eluded to a role of thromboxane A2 (TxA2) in this

process. TxA2 acts within the area perfused by the cerebral vasculature to stimulate

ACTH secretion in the fetus (Wood et al., 1993), which suggests that local generation of

TxA2 would effectively stimulate the HPA axis. Whatever the mechanism, it is obvious

that further research needs to be done in order to fully understand the inner-workings of

this complex system.













Blood sampling time points






Omin. 1 Omin. 20min.


Hypotension
(Brachiocephalic Occlusion)


Euthanize and
perfuse brain
(Prepare for
Immunohistochemistry)


Figure 4.1: Experimental design for in vivo studies. 5ml blood samples are taken at
0min., lOmin., 20min., and 60min.(additional blood is taken at 0min., 10min., and
20min. for blood gas and hematocrit measurement). Hypotension via brachiocephalic
occlusion takes place between 0min. and 10min. Blood pressure and heart rate are
recorded for the first 35min. of the experiment. Animals are sacrificed at 60min. and
brains are extracted for immunohistochemistry.


60min.






































Estradiol treated
fetuses


Placebo treated
fetuses


Figure 4.2: Estradiol levels +SEM for fetuses treated with a 5 mg/21day estradiol
implant and fetuses treated with a placebo implant. The group means are
significantly different (n=20 per group, p<0.001).







1600 -

1400 -

1200 -

1000 -
-SD
S800 -

G 600-

400 -

200 -

0 -


1600 -

1400 -

1200 -

S1000 -

S800-

S600-
.<

400 -

200 -

0-


0 10 20 30 40 50 60


Estradiol treated fetuses


-Control
I IHypotensive
- CSD
-- Hypo/CSD


fI I


II


I~I


Time (minutes)



Figure 4.3: ACTH plasma levels plotted as group means +SEM. Top graph
shows placebo treated fetuses and bottom graph shows estradiol treated fetuses.
*,+ denotes statistical significance (n=5 per group, p<0.001). All estradiol groups
were significantly different from placebo groups relative to treatment and time
(n=5 per group, p<0.01).


111 i1l i I I r, i


III I







Placebo treated fetuses


- Control
' IHypotensive
- CSD
aHypo/CSD


in i I


+ *

jA ,HEr


.non


I I I I I
0 10 20 30 40 50 60
60
Estradiol treated fetuses Control
50 I Hypotensive
SCSD
----I Hypo/CSD
40 -

30 + *

20 -



': oii ____________
0 10 20 30 40 50 60
Time (minutes)


Figure 4.4: AVP plasma levels plotted as group means +SEM. Top graph
shows placebo treated fetuses and bottom graph shows estradiol treated fetuses.
*,+ denotes statistical significance (n=5 per group, p were significantly different from placebo groups relative to treatment and time
(n=5 per group, p<0.01).








Placebo treated fetuses


M Control
Zi Hypotensive
- CSD
Hypo/CSD


*
15
+
10 +

5

0
0 10 20


InI
60


0 10 20 30 40 50 60


Time (minutes)


Figure 4.5: Cortisol plasma levels plotted as group means +SEM. Top graph
shows placebo treated fetuses and bottom graph shows estradiol treated fetuses.
*,+ denotes statistical significance (n=5 per group, p<0.001). All estradiol groups
were significantly different from placebo groups relative to treatment and time
(n=5 per group, p<0.01).


30 -

25 -

20 -


I














CHAPTER 5
NEURONAL ACTIVATION IN AN ESTRADIOL, HYPOTENSIVE, CAROTID
SINUS DENERVATED OVINE MODEL


Introduction

Parturition in the sheep has been shown to be controlled by the fetal HPA axis

(Challis and Brooks, 1989; Liggins et al., 1973). The last few days of gestation are

marked by an increase in the activity of the fetal hypothalamus as seen by elevated levels

of fetal plasma ACTH. This increase in plasma ACTH causes a corresponding increase in

plasma cortisol. Along with this increase in HPA axis activity is a decreased sensitivity of

the axis to cortisol negative feedback (Wood, 1988). It is well known that in the sheep,

cortisol acts at the placenta to increase the activity of an enzyme, cytochrome p450 (17-

hydroxylase and 17,20 lyase activities), which in turn, increases the ratio of estrogen to

progesterone (Anderson et al., 1975; Pomeranz and Nalbandov, 1975; Steele et al., 1976;

Yu et al., 1983). This cascade of events essentially increases the total amount of estrogen.

Estrogen is known to be an important factor in the initiation of parturition by

causing the uterus to contract (Liggins, 1974). 1703-estradiol has been shown to increase

the activity of the HPA axis in sheep and rats. In a study by Saoud and Wood (1995),

estrogen was found to augment fetal plasma ACTH secretion in response to stress. Other

studies have shown similar results in adult animals (Viau and Meaney, 1991).

Understanding the mechanism of the increased fetal HPA axis at the end of

gestation is key to understanding the mechanism of spontaneous parturition in sheep.








These experiments were conducted to see at what point in the HPA axis that estradiol has

in augmenting ACTH secretion. More specifically, I hypothesized that neuronal activity

will be highest in areas important for HPA axis control in estradiol treated, hypotensive

animals (see Chapter 4). I hypothesized that the baroreceptor / chemoreceptor afferent

pathway is involved, thus, carotid sinus denervation will eliminate the augmented HPA

axis activity. Neuronal activity was assessed by measuring the level of c-fos, any early

response gene, in brain areas important for HPA axis control. This method has been used

in numerous studies to assess neuronal activation due to physiological stress (Hoffinan et

al., 1991; Shen et al., 1992; Chan et al., 1993).

Methods and Materials

Immunohistochemical Techniques Fetal ovine brains were perfusion fixed with 4%

paraformaldehyde, dissected and cut into gross tissue regions (hypothalamus, midbrain,

pons, medulla, spinal cord, etc.). These brains were the obtained from the experiments

discussed in Chapter 4. All animal were euthanized via an overdose of'sodium

pentobarbital one hour after the beginning of the experiment (50 minutes after a ten

minute hypotensive or corresponding normotensive period). There were a total of 24 fetal

brains used for the histological experiments (n=3 per group). The fetal ovine groups were

as followed: (1) estradiol treated; (2) estradiol treated, hypotensive; (3) estradiol treated,

carotid sinus denervated; (4) estradiol treated, carotid sinus denervated, hypotensive; (5)

placebo treated; (6) placebo treated, hypotensive; (7) placebo treated, carotid sinus

denervated; (8) placebo treated, carotid sinus denervated, hypotensive. Tissue was

processed for embedding by dehydration with progressively increasing concentrations of

ethanol, followed by xylene. All tissue was embedded in paraffin and cut into 10 pm










sections using a Zeiss microtome. Sections were mounted on poly-L-lysine slides,

deparaffinized with xylene and rehydrated in decreasing concentrations of ethanol.

Immunohistochemistry and visualization were made possible utilizing a Histostain-SP kit

from Zymed and metal-enhanced DAB (Pierce, Rockford, IL). Sections were stained for

c-fos, ACTH, AVP, and CRH (see Table 3.1). Primary antibodies were diluted in

antibody diluent (1% BSA in phosphate buffered saline with 0.01% Triton X-100).

Specific staining was confirmed by dilution tests, as staining decreased as primary

antibodies were further diluted. Specific staining was absent upon replacing primary

antibodies with 10%/ normal goat serum. All slides were dehydrated prior to mounting

coverslips with Permount (Fisher Scientific, Pittsburgh, PA).

Fetal brains regions important in HPA axis control were measured for c-fos

generation by means of Microcomputer Imaging Device (MCID) from Imagining Research

Inc. Cerebellum and cortex were also measured to verify if c-fos activity was specific or

just a general activation of the central nervous system. The following brain regions were

analyzed: (1) paraventricular nucleus (PVN), (2) nucleus of the tractus solitarius (NTS),

(3) rostral ventral lateral medulla (RVLM), (4) hippocampus, (5) cerebellum, (6) cortex,

and (7) pituitary gland. For each of the eight treatment groups (listed in the Materials and

Methods section), n=3. As paraffin blocks were cut on the microtome, the fifth section

was used once the region of interest was identified. This assured homogeneity among

fetal brains.

All densitometry was performed in a similar mannor. The region of interest was

outlined designating the scanned area. The computer would then count the number of

cells stained positive depending upon the assigned criteria. This value or target number










was multiplied by the mean target area (calculated by the computer) to establish the total

target area. Finally, the total target area was divided by the scanned area to establish the

proportion of positive stained cells in each brain section. Values were then analyzed via

three way Analysis of Variance (ANOVA) followed by a multiple comparison procedure.

The multiple comparison procedure employed was Student-Newman-Keuls Method. All

statistics were run using Sigma Stat.

Results

PVN C-fos staining was found to be significantly different among the eight treatment

groups by three way ANOVA (n-3 per group, p<0.001). The mean densitometry values

+SEM are plotted in Figure 5.1. A further analysis of the data revealed statistically

significant interactions between all three factors (placebo vs. estradiol, hypotensive vs.

normotensive, and carotid sinus denervated vs. intact). Student Newman Keuls Method

revealed-the following significant differences among treatment groups: '(1) mean values of

c-fos staining among the different levels of placebo treated fetuses vs. estradiol treated

fetuses was significantly different (n=3 per group, p<0.001), and (2) mean values of c-fos

staining among the different levels of hypotensive fetuses vs. normotensive fetuses was

significantly different (n=3 per group, p<0.001). Representative photomicrographs of the

PVN are shown in Figure 5.2. It can be seen visually that estradiol treated fetuses have

more c-fos generation in the PVN when compared to control fetuses (panels A and B).

Also shown is significantly more positive staining in estradiol treated, hypotensive fetuses

compared to placebo treated, hypotensive fetuses (panels C and D). As stated before

hypotensive animals also have more c-fos generation compared to normotensive animals

(panels A and C).










Carotid sinus denervation by itself did not cause significant c-fos generation. This

is show in Figure 5.3 which shows a representative estradiol treated fetus along with a

representative estradiol, carotid sinus denervated fetus. The region shown is the PVN and

visually, as well as statistically, there is no difference in the amount of staining. Figure 5.4

shows the effect of carotid sinus denervation on hypotension. The PVN of a

representative placebo, hypotensive fetus along with a representative placebo,

hypotensive, carotid sinus denervated fetus. Photomicrographs show that denervation

eliminates the effect of hypotension. These effects of carotid sinus denervation were true

of all brain regions studied.

Along with c-fos staining in the PVN, serial sections were also stained for AVP,

and CRF. Though statistics were not performed for the relative levels of these hormones,

the pattern of staining seemed to be consistent with that of c-fos. Not only was the

staining of AVP and CRF co-localized with c-fos, but also the levels of these hormones
t
seemed to increase with estradiol treatment as well as in hypotensive animals.

NTS C-fos staining was found to be significantly different among the eight treatment

groups by three way ANOVA (n-3 per group, p<0.001). The mean densitometry values

+SEM are plotted in Figure 5.5. A further analysis of the data revealed statistically

significant interactions between all three factors (placebo vs. estradiol, hypotensive vs.

normotensive, and carotid sinus denervated vs. intact). Student Newman Keuls Method

revealed the following significant differences among treatment groups: (1) mean values of

c-fos staining among the different levels of placebo treated fetuses vs. estradiol treated

fetuses was significantly different (n=3 per group, p<0.001), and (2) mean values of c-fos

staining among the different levels of hypotensive fetuses vs. normotensive fetuses was










significantly different (n=3 per group, p<0.001. Representative photomicrographs of the

NTS are shown in Figure 5.6. It can be seen visually that estradiol treated fetuses have

more c-fos generation in the NTS when compared to control fetuses (panels A and B).

Also shown is significantly more positive staining in estradiol treated, hypotensive fetuses

compared to placebo treated, hypotensive fetuses (panels C and D). As stated before

hypotensive animals also have more c-fos generation compared to normotensive animals

(panels A and C). These results coincide with those of the PVN.

RVLM C-fos staining was found to be significantly different among the eight treatment

groups by three way ANOVA (n-3 per group, p<0.001). The mean densitometry values

+SEM are plotted in Figure 5.7. A further analysis of the data revealed statistically

significant interactions between all three factors (placebo vs. estradiol, hypotensive vs.

normotensive, and carotid sinus denervated vs. intact). Student Newman Keuls Method

revealed, the following significant differences among treatment groups: (1) mean values of

c-fos staining among the different levels of placebo treated fetuses vs. estradiol treated

fetuses was significantly different (n=3 per group, p<0.001), (2) mean values of c-fos

staining among the different levels of hypotensive fetuses vs. normotensive fetuses was

significantly different (n=3 per group, p<0.001. Representative photomicrographs of the

RVLM are shown in Figure 5.8. It can be seen visually that estradiol treated fetuses have

more c-fos generation in the RVLM when compared to control fetuses (panels A and B).

Also shown is significantly more positive staining in estradiol treated, hypotensive fetuses

compared to placebo treated, hypotensive fetuses (panels C and D). As stated before

hypotensive animals also have more c-fos generation compared to normotensive animals

(panels A and C). These results coincide with those of the PVN and the NTS.











Hippocampus C-fos staining was found to be significantly different among the eight

treatment groups by three way ANOVA (n-3 per group, p<0.001). The mean

densitometry values +SEM are plotted in Figure 5.9. Note that the y axis is smaller than

that of the PVN, etc. because the level ofc-fos staining is lower in the hippocampus.

Student-Newman-Keuls Method revealed the that the mean values of c-fos staining among

the different levels ofhypotensive fetuses vs. normotensive fetuses was significantly

different (n=3 per group, p<0.001). However, mean values of c-fos staining among the

different levels of placebo treated fetuses vs. estradiol treated fetuses was not significantly

different (n=3 per group, p=0.407). Representative photomicrographs of the

hippocampus are shown in Figure 5.10. It can be seen visually that hypotensive fetuses

have more c-fos generation in the hippocampus when compared to normotensive fetuses

(panels A and B). These results resemble those of the PVN, NTS, and RVLM with regard

to the effect of hypotension, however, the absence of an estradiol effect is novel.
n
Cerebellum Cerbellum was analyzed as a peripheral tissue that does not integration in the

system being studied. As suspected, analysis by three way ANOVA did not yield any

differences among treatment groups (n=3 per group, p=0.721). Mean group values

+SEM of the densitometry analysis are shown in Figure 5.11. Note that the y axis is much

smaller than that of the PVN, etc. because of the absence of any significant staining.

Representative photomicrographs of fetal ovine cerebellum are shown in Figure 5.12.

Panel A shows a control animal while panel B shows a hypotensive animal. Visually, as

well as statistically, there is no difference. Though not shown pictorially, the same is true

of estradiol treated fetuses vs. placebo treated fetuses.










Cortex As the case with cerebellum, cortex was analyzed as a peripheral tissue that does

not integration in the system being studied. As suspected, analysis by three way ANOVA

did not yield any differences among treatment groups (n=3 per group, p=0.399). Mean

group values +SEM of the densitometry analysis are shown in Figure 5.13. Note that the

y axis is much smaller than that of the PVN, etc. because of the absence of any significant

staining. Representative photomicrographs of fetal ovine cortex are shown in Figure 5.14.

Panel A shows a control animal while panel B shows a hypotensive animal. Visually, as

well as statistically, there is no difference. Though not shown pictorially, the same is true

ofestradiol treated fetuses vs. placebo treated fetuses.

Pituitary gland Pituitary glands were stained for c-fos as well as ACTH and AVP.

Though statistics were not performed for the relative levels of these hormones, the pattern

of staining seemed to be consistent with that of the PVN, NTS, and RVLM. Not only was

the c-fos increased with estradiol treatment and hypotension, but the levels of ACTH and

AVP seemed to mimic the pattern and level of c-fos staining. ACTH was seen in the

anterior pituitary in and increased with both estradiol treatment and hypotension. AVP

was seen mostly in the posterior pituitary in areas of increased c-fos generation.

Discussion

Understanding the mechanism of the increased fetal HPA axis at the end of

gestation is key to understanding the mechanism of spontaneous parturition in sheep.

These experiments were conducted to see at what point in the HPA axis that estradiol has

in augmenting ACTH secretion. More specifically, I hypothesized that neuronal activity

will be highest in areas important for HPA axis control in estradiol treated, hypotensive

animals (see Chapter 4). I hypothesized that the baroreceptor / chemoreceptor afferent










pathway is involved, thus, carotid sinus denervation will eliminate the augmented HPA

axis activity. Neuronal activity was assessed by measuring the level of c-fos, any early

response gene, in brain areas important for HPA axis control. This method has been used

in numerous studies to assess neuronal activation due to physiological stress (Hoffmnan et

al., 1991; Shen et al., 1992; Chan et al., 1993).

The results of this study support to my original hypothesis. Estradiol augments

HPA axis activity through baroreceptor / chemoreceptor pathways and this augmentation

seems to be within brain regions important for HPA axis activity. Furthermore, the

elimination of increased neuronal activity in carotid sinus denervated animals shows that

the pathway involved is probably this central cardiovascular reflex pathway.

The immunohistochemistry results show that the PVN, NTS, and RVLM respond

similarly with regard to specific treatments. This finding comes as no surprise as all of

these areas are involved in HPA axis control. The PVN is the main collection of neuronal
f
cell bodies in the hypothalamus where CRH and AVP are synthesized and released to act

at the anterior pituitary to cause ACTH release (Lehman et al., 1993; Pomerantz and

Sholl, 1987). The NTS is the first synapse point in the pathway connecting the afferent

baroreceptors with the PVN. The RVLM is a cardiovascular regulatory center which

coordinates information from the periphery. It has been shown that all of these brain

regions have estrogen receptors, enabling them to respond to estradiol treatment prior to

brachiocephalic occlusion (Lehman et al., 1993; Simerly et al., 1990). Estradiol caused a

significant increase in neuronal activity in the PVN, NTS, and RVLM. In fetuses made

hypotensive via brachiocephalic occlusion, this effect was further augmented as shown by










statistical analyses. Carotid sinus denervation eliminated increases in c-fos activity in these

HPA axis regulatory centers.

An interesting finding of this study was the absence of an estradiol effect in the

hippocampus. Like the PVN, NTS, and RVLM, an increase in c-fos activity was seen in

hypotensive animals and this effect was eliminated with carotid sinus denervation.

However, basal levels as well as stressed levels of neuronal activity in estradiol treated

animals compared to placebo treated animals did not differ statistically. This reveals that

estradiol has no effect at the level of the hippocampus in augmenting HPA axis activity in

response to hypotension. This is interesting because there have been estrogen receptors

reported within the hippocampus (Lehman et al., 1993; Simerly et al., 1990). Perhaps the

answer to this discrepancy lies in fact that the hippocampus is a classic site of cortisol

negative feedback. Wood has further shown that the HPA axis is insensitive to inhibition

via cortisol negative feedback towards the end of gestation (1987). Possibly, the

hippocampus is unresponsive to an estradiol augmentation of ACTH secretion at this time.

Of course, this may be in the best interest of the fetus since an increase in neuronal activity

at the level of the hippocampus may lead to inhibition rather than stimulation of the axis.

It was certainly encouraging that in peripheral brain regions, an increase in

neuronal activity was observed in neither estradiol treated nor estradiol treated,

hypotensive fetuses. This lack of c-fos staining in the cortex and cerebellum indicate that

the response to treatment within the CNS was specific. All things being equal, only brain

regions important in HPA axis control seemed to respond to estradiol treatment and

brachiocephalic occlusion.










Pituitary glands were stained for c-fos as well as ACTH and AVP. Though

statistics were not performed for the relative levels of these hormones, the pattern of

staining seemed to be consistent with that of the PVN, NTS, and RVLM. Not only was

the c-fos increased with estradiol treatment and hypotension, but the levels of ACTH and

AVP seemed to mimic the pattern and level of c-fos staining. ACTH was seen in the

anterior pituitary in and increased with both estradiol treatment and hypotension. AVP

was seen mostly in the posterior pituitary in areas of increased c-fos generation. All of

this information fits with my hypothesis. One would expect to see an increase in staining

in these areas within the pituitary with both estradiol treatment and hypotension. There

are estrogen receptors within the pituitary and an increase in activity of the pituitary would

be logical during hypotension. It is possible that ACTH and AVP synthesis is increased

during estradiol treatment. This would also be true in the normal fetus towards the end of

gestation when estradiol is increasing. If these hormone levels are in fact increasing, The

fetus would be able to better respond to episodes of hypotension as well as trigger

parturition with an augmented ACTH and AVP response.

I have reported that within the PVN, NTS, RVLM, hippocampus, and pituitary of

fetal sheep, an in crease in c-fos activity is observed with estradiol treatment and

brachiocephalic occlusion. It may seem logical that rather than an increase in neuronal

activity, a decrease in c-fos staining at the level of the NTS would accompany

hypotension. Hypotension causes a decrease in the rate of firing of the baroreceptors, and

thus a decrease in signal at the NTS. Perhaps what is being observed here is a

chemoreceptor mediated effect. As the brachiocephalic artery is occluded, the blood

perfusing the head becomes acidotic and hypoxic. This is revealed through elevated levels











of hydrogen ions, elevated partial pressure of carbon dioxide (PCO2), and a decrease in the

partial pressure pressure of oxygen (PO,) in the blood. All of these factors were in fact

monitored throughout the duration of the experiment. I believe that the fetal PO2 level is

strictly monitored in the carotid artery and in a state of hypotension, the compensatory

response is an increase in HPA axis activity in order to re-establish adequate blood flow to

the brain. This will have the inevitable response of bringing blood gas levels back to a

homeostatic level.

It is important to realize that the HPA axis is not only integral in controlling blood

pressure, but it is also the endocrine axis of parturition. This is a fact which has been

proven many times without actual confirmation of a precise mechanism. This study sheds

light on the internal working of this system. I have shown that an intact afferent

baroreceptor / chemoreceptor pathway is necessary for an ovine fetus to respond to a state

of hypotension. I have further shown that estradiol augments this response through this
t
cardiovascular reflex pathway. More precisely, I have shown through the utilization of

immunohistochemistry, the brain areas involved in this process. This study used an

artificial stimulus to increase HPA axis activity, however, it is not disputed that the

pathway described is in fact involved in triggering parturition.












0.08







+,+









20.04



0.03



0.02



0.01









Figure 5.1: C-fos immunohistochemistry staining in the fetal ovine paraventricular
nucleus (Plac= placebo implant, E2= estradiol implant, Hypo= hypotensive, CSD=
carotid sinus denervated). There is a significant difference between Plac vs. E2
(*, p<0.001) and Control vs. Hypo (+, p<0.001).






82









A\ I ~; <"" *-*




,
























nucleus (A- Control; B- Estradiol treated; C- Hypotensive; D- Estradiol treated,
hypotensive). All photomicrographs are at a magnification of 40X.






83





A





I









B


4J
b i

It





















Figure 5.3: Photomicrographs of the fetal ovine PVN comparing c-fos staining in an
intact estradiol treated fetus (A) and an estradiol treated, carotid sinus denervated
fetus (B). A and B were not statistically different showing that denervation alone did
not cause c-fos generation.
















































Figure 5.4: Photomicrographs of the fetal ovine PVN comparing c-fos staining in a
hypotensive fetus (A) and a hypotensive, carotid sinus denervated fetus (B). A and B
were statistically different (p<001) showing that denervation diminished c-fos
generation in hypotensive animals.










0.08 -



0.07


0.06*+
0.06 -


0.05 -



0.04 -



0.03 -


0.02 -



0.01 -



0.00 -


0


V\CW


I ~ -T
~f~VO


Figure 5.5: C-fos immunohistochemistry staining in the fetal ovine nucleus
of the tractus solitarius (Plac= placebo implant, E2= estradiol implant, Hypo=
hypotensive, CSD=-- carotid sinus denervated). There is a significant difference
between Plac vs. E2 (*, p<0.001) and Control vs. Hypo (+, p<0.001).


V0\


















*0






q.


n




*b


B












em,





& 4
5'
-a


*1k


S.

4 .*


Figure 5.6: C-fos immunohistochemistry staining in the fetal ovine nucleus of the
tractus solitarius (A- Control; B- Estradiol treated; C- Hypotensive; D- Estradiol
treated, hypotensive). All photomicrographs are at a magnification of 100X.


I.
St
a-
S


A


Am










0.08



0.07



0.06


0.05 -



0.04 -



0.03 -


0.02 -



0.01 -



0.00-


CH ',


Figure 5.7: C-fos immunohistochemistry staining in the fetal ovine rostral
ventral lateral medulla (Plac= placebo implant, E2= estradiol implant, Hypo=
hypotensive, CSD= carotid sinus denervated). There is a significant difference
between Plac vs. E2 (*, p<0.001) and Control vs. Hypo (+, p<0.001).








































Figure 5.8: C-fos immunohistochemistry staining in the fetal ovine rostral ventral
lateral medulla (A- Control; B- Estradiol treated; C- Hypotensive; D- Estradiol treated,
hypotensive). All photomicrographs are at a magnification of 100X.


A B







C D.v ^-^
..*.., A'
"- *..
: i,-
A ,, I, D d "
aI I










0.040



0.035 -



0.030 -

CC

-. 0.025 -
S+
U +

o 0.020



0.015



0.010



0.005



0.000 -





Figure 5.9: C-fos immunohistochemistry staining in the fetal ovine hippocampus
(Plac= placebo implant, E2= estradiol implant, Hypo= hypotensive, CSD= carotid
sinus denervated). There is a significant difference between Control vs. Hypo
(+, p







90














i' .'. ;'. ?' >*', '*


*,. > .,. ,.
.... .. .








.,N .. ,





































Figure 5.10: C-fos immunohistochemistry staining in the fetal ovine hippocampus
(A- Control; B- Hypotensive). All photomicrographs are at a magnification of 40X.













0.0020


0.0018 -


0.0016 -


0.0014 -


0.0012
0













"\c *^*
C 0.0010


0.0008
0

0.0006


0.0004


0.0002


0.0000





Figure 5.11: C-fos immunohistochemistry staining in the fetal ovine cerebellum
(Plac= placebo implant, E2= estradiol implant, Hypo= hypotensive, CSD=
carotid sinus denervated).




















































Figure 5.12: C-fos immunohistochemistry staining in the fetal ovine cerebellum
(A- Control; B- Hypotensive). All photomicrographs are at a magnification of 40X.
A and B are not statistically different.














0.0014 -

0.0012 -

0.0010 -

0.0008 -

0.0006 -

0.0004 -

0.0002 -

0.0000 -


,s~# ~c~0\C5~'9


Figure 5.13: C-fos immunohistochemistry staining in the fetal ovine cortex
(Plac= placebo implant, E2= estradiol implant, Hypo= hypotensive, CSD=
carotid sinus denervated).


0.0020 -

0.0018 -

0.0016 -


IL
p 0


IL.1
I > --r




Full Text
ESTROGEN-CENTRAL NERVOUS SYSTEM INTERACTIONS IN
CARDIOVASCULAR CONTROL AND PARTURITION
By
SCOTT CHRISTOPHER PURINTON
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1999

To the following, I dedicate this dissertation.
Without them, none of this would have been possible.
To my family: Thank you for your support and encouragement
To Leigh Ann: Thank you for believing in and standing by me
To David M. Bush: Thank you for your friendship
Wish you could have been here

ACKNOWLEDGMENTS
First and foremost I would like to thank Dr. Charles E. Wood, chair of my
supervisory committee, for his guidance, wisdom, and friendship. Dr. Wood has
certainly been a true mentor to me as a scientist, as well as a role model to anyone
wishing to balance a fulfilling life with a successful career in science. For this and
everything else he has done for me, I am very grateful.
I would like to thank the other members of my supervisory committee, Drs.
Maureen Keller-Wood, Pushpa Kalra, and James Simpkins for their guidance and advice.
A special thanks goes to Dr. Maureen Keller-Wood for her extensive knowledge and for
being an indispensable resource. Also, a special thanks goes to Sherry McDaniel for her
assistance with the surgery and care of the animals.
Last but certainly not least, I would like to extend my gratitude to my family for
their never-ending love and support. They have always stood by me in whatever tasks I
set forth to accomplish. I especially would like to thank Dr. LeighAnn Stubley, my best
friend and fiancée. Without LeighAnn’s love and support, I can truly say that none of
this would have been possible. She has taught me more about myself than I could ever
have learned from graduate school. For this I will be forever indebted to her.
iii

TABLE OF CONTENTS
page
ACKNOWLEDGMENTS iii
ABSTRACT vi
CHAPTERS
1INTRODUCTION 1
General Background and Significance 1
Specific Aims and Hypotheses 4
Experimental Protocol 5
Experimental Methods 7
2LITERATURE REVIEW 10
Control of Parturition 10
The Hypothalamus-Pituitary-Adrenal Axis 13
The Regulation of ACTH Secretion 16
The Development of the Fetal HP A Axis 24
The Importance of Gonadal Steroids in Parturition and Cardiovascular Control 27
Cardiovascular Reflex Responsiveness 33
3GENERAL MATERIALS AND METHODS 39
Surgical Preparation of Fetal Sheep 39
In Vivo Experimental Procedures 42
Peptide Assays 44
Steroid Assays 45
Estrone Sulfatase Activity 45
Western Blotting 47
Immunohistochemical Techniques 48
4HORMONAL RESPONSIVENESS IN AN ESTRADIOL, HYPOTENSIVE,
CAROTID SINUS DENERVATED OVINE MODEL 50
Introduction 50
Methods and Materials 52
IV

Results 58
Discussion 60
5NEURONAL ACTIVATION IN AN ESTRADIOL, HYPOTENSIVE,
CAROTID SINUS DENERVATED OVINE MODEL 69
Introduction 69
Methods and Materials 70
Results 72
Discussion 76
6ONTOGENY OF ESTROGEN SULFATASE AND ESTROGEN
SULFOTRANSFERASE IN BRAIN REGIONS IMPORTANT FOR
HYPOTHALAMUS-PITUITARY-ADRENAL AXIS CONTROL ~ 95
Introduction 95
Methods and Materials 97
Results 100
Discussion 102
7SUMMARY AND CONCLUSIONS 121
REFERENCES J38
BIOGRAPHICAL SKETCH 158
v

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
ESTROGEN-CENTRAL NERVOUS SYSTEM INTERACTIONS IN
CARDIOVASCULAR CONTROL AND PARTURITION
By
Scott Christopher Purinton
May 1999
Chairman: Charles E. Wood
Major Department: Physiology
In the fetal sheep, parturition is triggered by an increase in the activity of
the fetal hypothalamus-pituitary-adrenal (HPA) axis. Parturition can be delayed by
destruction of the pituitary or stimulated by infusions of adrenocorticotropin (ACTFI) or
f
glucocorticoids. The last days of gestation are marked by an increase in the activity of
the fetal hypothalamus as seen by elevated levels of fetal plasma ACTH. Estrogen has
been shown to trigger this preparturient increase in ACTH. I hypothesized that
estrogen’s actions on fetal cardiovascular reflex responsiveness to hypotension will be
measurable in intact fetuses but not in baroreceptor/chemoreceptor denervated fetuses.
Research has previously demonstrated that denervation attenuates the reflex hormonal
and hemodynamic responses to moderate reduction in arterial blood pressure. I found
that interruption of this afferent pathway eliminated the effect of estrogen on reflex
cardiovascular responsiveness. This was shown by measuring hormone responsiveness
vi

(ACTH, vasopressin, and cortisol) to hypotension by brachiocephalic occlusion. I further
hypothesized that estrogen’s actions within the fetal central nervous system are centered
within the nucleus of the tractus solitarius (NTS), the paraventricular nucleus (PVN), or
within the components of cardiovascular regulatory centers receiving input from
baroreceptors or chemoreceptors. I used immunohistological techniques to identify the
neuroanatomical regions which are activated by hypotension and, subsequently, those
areas modified by estrogen’s action and baroreceptor/chemoreceptor denervation. The
use of these techniques allows for the measurement of c-fos expression, an early response
gene which can be used as a marker of neuronal activity. I found that estradiol implanted
animals had more c-fos abundance, and hence more c-fos staining in relevant brain areas
(NTS, PVN, etc.) as compared to control animals. Furthermore, it was revealed that c-fos
staining was negligible in denervated animals. Finally, c-fos staining was significantly
elevated in all hypotensive animals as compared to normotensive animals. These results
suggest that estrogen works within the ovine fetal baroreceptor/chemoreceptor afferent
pathway in brain regions relevant for HPA axis control in order to augment ACTH
secretion in response to hypotension as well as play a role in the mechanism of
parturition.
vi 1

CHAPTER 1
INTRODUCTION
General Background and Significance
There is a wealth of information concerning the influence of estrogen on the
cardiovascular system of the adult. The use of high dose estrogen contraceptives has
stimulated a great deal of interest in the role that exogenous estrogens play in the
pathogenesis of hypertension and stroke. However, it should be noted that little work has
been conducted regarding the influence of either endogenous or exogenous estrogen on
the control of cardiovascular function by the central nervous system. Furthermore, with
the exception of the studies conducted in Dr. Charles E. Wood’s laboratory at the
University of Florida, Gainesville, Florida, there are no data concerning the influence of
estrogen on cardiovascular function in the fetus.
Increases in plasma estrogen concentration are thought to be an important
component of the maternal adaptation to pregnancy. During gestation, the growing fetus
and enlarging uterus exhibit an increasing demand for oxygen. For this reason, the utero¬
placental blood flow constitutes an ever-increasing proportion of maternal cardiac output
(Rudolph, 1974). The increased flow demands are supported by an increase in maternal
blood volume and an increase in vascular compliance (Longo, 1983). This typically
produces a physiological condition in which maternal cardiac output and blood volume are
higher than in the nonpregnant state; however, maternal arterial pressure and central

2
venous pressure are lower (Keller-Wood, 1994; Longo, 1983). Though this response is
not entirely consistent among mammalian species, it has been proposed that estrogen plays
an important role in this phenomenon (Ueda, 1986). No studies have yet been conducted
that investigate the effects of estrogen on cardiovascular reflex responsiveness. By doing
such studies in fetal sheep, it is possible to investigate the mechanism behind parturition,
because an increase in the activity of the hypothalamus-pituitary-adrenal (HP A) axis has
been shown to trigger parturition (Liggins et al., 1973).
Both androgen and estrogen receptors can be found in the anterior pituitary as well
as in various regions of the brain, including the hypothalamus and the brainstem.
Receptors, and the mRNA encoding for these receptors, can be found in the various
structures, including the arcuate nucleus and the preoptic nucleus (Simerly et al., 1990). In
addition to the structures known for control of the hypothalamus-pituitary-gonadal axis
and for reproductive behavior, androgen and estrogen receptors are found in regions
better known for control of the hypothalamus-pituitary-adrenal axis, including the
t
paraventricular nucleus (PVN) and hippocampus (Lehman et al., 1993; Pomerantz and
Sholl, 1987). Estrogen receptors have been localized in the magnocellular portion of the
PVN, and may therefore influence either oxytocin, vasopressin (AVP), or both (Lehman et
al., 1993; Simerly et al., 1990). Both androgen and estrogen receptors are also found at
sites which are known to be relay centers on afferent pathways [the nucleus of the tractus
solitarius or (NTS)] mediating adrenocorticotropic hormone (ACTH) responses to
stresses such as hypoxia, hypercapnia, and hypotension (Simerly et al., 1990). Finally,
estrogen receptors have been localized in GABA-ergic cells, suggesting the possible

3
interaction of estrogen and ACTH secretion via this neuronal system within the brain
(Herbison et al., 1993).
The focus of the experiments is the neuroendocrine (ACTH, AVP) responsiveness
to hypotension. Of particular interest to me at the present time is the relationship between
neuroendocrine mechanisms controlling AVP and ACTH and how they regulate the
cardiovascular system of the fetus as well as trigger parturition. These two hormonal
systems are inextricably linked. In the fetus (as in the adult), afferent baroreceptor
pathways are shared by both endocrine systems. It is likely that the central pathways
carrying afferent information from the NTS to the hypothalamus are also mostly shared by
the two systems. AVP is synthesized in the PVN of the hypothalamus and can be found in
both magnocellular and parvocellular neurons (projecting to the posterior pituitary and
median eminence, respectively). Magnocellular AVP is secreted into the bloodstream by
the posterior pituitary. Parvocellular AVP is secreted into the hypothalamo-hypophyseal
portal system and acts at the anterior pituitary to induce the release of ACTH. ACTH, via
stimulation of cortisol, act to maintain vascular reactivity, promote plasma protein
biosynthesis, and indirectly alter fetal blood pressure and distribution of combined
ventricular output. Parvocellular neurons, in addition to AVP, synthesize and release
corticotropin releasing hormone (CRH) which also stimulates the release of ACTH from
the anterior pituitary. Parvocellular CRH neurons project axons to various parts of the
brain in addition to the median eminence. Most notably, these neurons project to the
cardiovascular control centers in the hypothalamus and medulla. When released from
these neurons, CRH stimulates increases in sympathetic efferent tone: measured in the
cardiovascular system as an increase in blood pressure and heart rate and a redistribution

4
of cardiac output (Lenz et al., 1987). Accordingly, it has been suggested that CRH is the
transmitter which coordinates all of the endocrine and cardiovascular responses to stress,
including hypotension (Lenz et al., 1987).
Preliminary experiments suggest that estrogen augments ACTH response to
hypotension, but that it does not alter AVP responses. Furthermore, estrogen augments
the ACTH response to cardiovascular stimuli, but not to purely endocrine stimuli.
Because of this difference between ACTH and AVP and between cardiovascular and non-
cardiovascular stimuli, I hypothesized that estrogen acts within the central nervous system
to affect the parvocellular CRH biosynthesis and secretion. Thus, it is proposed that
estrogen affects either CRH neurons or the ascending pathways which stimulate CRH
neurons, as well as affecting the activity of the descending (toward cardiovascular
regulatory centers) CRH pathways.
Specific Aims and Hypotheses
In the following experiments, a combination of in vivo (whole animal) and in vitro
r
(immunohistochemistry) techniques was employed.
Aim 1 The actions of estrogen on fetal cardiovascular reflex responsiveness to
hypotension will be measurable in intact fetuses but not in baro- and chemo-denervated
fetuses. Research has previously demonstrated that the combined baro- and chemo-
denervation attenuates (approximately 50%) the reflex hormonal and hemodynamic
responses to moderate (50 %) reduction in arterial blood pressure. I proposed that the
interruption of the afferent pathways would eliminate the effect of estrogen on the reflex
cardiovascular responsiveness. If so, it could concluded that estrogen acts on, within, or
requires input from, the afferent baroreceptor and chemoreceptor pathways.

5
Aim 2 The actions of estrogen within the fetal central nervous system are centered within
the nucleus of the tractus solitarius, the parvocellular neurons of the paraventricular
nucleus, or within the components of the cardiovascular regulatory centers receiving input
from baroreceptors or chemoreceptors. I used immunohistological techniques to identify
the neuroanatomical regions which were activated by hypotension and, subsequently,
those areas modified by estrogen’s action and baro- and chemo-denervation. The use of
these techniques allows for the measurement of c-fos expression, the protein product of
the early response gene which can be used as a marker of neuronal activity. I proposed
that estradiol implanted animals will have more Fos activity, and hence more c-fos staining
in relevant brain areas (NTS, PVN, etc.) as compare to control animals. Furthermore, it is
expected that c-fos staining will be negligible in denervated animals. Finally, c-fos staining
should be significantly elevated in all hypotensive animals as compared to normotensive
animals.
In addition to the c-fos immunohistochemical studies, the action of estrogen
/
sulfatase and estrogen sulfotransferase was investigated. Since the concentration of
estrogen sulfate precedes the increase in HPA axis activity (Nathanielsz et al., 1982), and
since conjugated estrogens circulate in much higher concentrations than unconjugated
estrogens (Carnegie and Robertson, 1978; Tsang, 1974), I hypothesized that these
enzymes would be present in brain areas important for HPA axis control. If true, a local
mechanism would be in place for conversion of biologically inactive to active estrogen.
Experimental Protocol
Design A total of 40 pregnant ewes were studied (5 per experimental group). Animals
were randomly assigned to the following groups:

6
1. placebo implant, intact (sham-denervated),normotensive fetuses
2. placebo implant, carotid sinus denervated, normotensive fetuses
3. placebo implant, intact (sham-denervated) fetuses subjected to hypotension
4. placebo implant, carotid sinus denervated fetuses subjected to hypotension
5. estradiol implant (5 mg/21 day release), intact (sham-denervated), normotensive
fetuses
6. estradiol implant (5 mg/21 day release), carotid sinus denervated, normotensive
fetuses
7. estradiol implant (5 mg/21 day release), intact (sham-denervated) fetuses
subjected to hypotension
8. estradiol implant (5 mg/21 day release), carotid sinus denervated fetuses
subjected to hypotension
Fetal sheep were chronically prepared with vascular catheters and implants on
around day 115 and allowed post-surgical recovery for 5 days. Fetuses were subjected to
experiments on day 120+3, during which time each fetus was subjected to a 10 min period
of hypotension produced by brachiocephalic occlusion (no occlusion for control animals).
Fetal arterial blood samples (5 ml) were drawn at 0 min, +10 min, +20 min, and +60 min
relative to the initiation of hypotension. An additional 1 ml blood sample was drawn
anaerobically for the measurement of fetal blood gases and hematocrit at 0 min, +10 min,
and +20 min time points. Immediately after drawing the +60 min blood sample, the ewes
and fetuses were euthanized using an overdose of sodium pentobarbital. In each
experimental group, five fetuses will be studied, then prepared for immunohistochemistry
as detailed below. Immunohistochemistry was performed for the detection of c-fos. A

7
subset of sections was double-stained for ACTH and c-fos (pituitary), AVP and c-fos
(hypothalamus), and CRH and c-fos (hypothalamus).
Analysis The neuroanatomical areas which express c-fos in response to hypotension
(NTS, anterior hypothalamus, PVN, and pituitary) were identified. Immunohistochemistry
of c-fos was analyzed in a semi-quantitative manner (counting c-fos positive cells in the
relative regions). The number of cells expressing immunoreactive c-fos in each
experimental group at each site was be compared using two- and three-way ANOVA. I
further classified neuronal activation by counting cells which express/co-express c-fos and
ACTH (anterior pituitary), c-fos and CRH and c-fos and AVP (PVN).
Experimental Methods
Aim 1 Prior to surgery, food was withheld from the pregnant ewe (2-6 years old of mixed
breed, mostly Columbia-Ramboillet and Suffolk) for 24 hours. Before and during surgery,
the ewe was anesthetized with halothane (0.5-2.0%) in oxygen. Fetal catheters were
f
routed to one flank of the ewe and held in place with a synthetic pocket and an expandable
bandage.
After exposing the uterus and the fetal hindlimbs via a midline incision, fetal
catheters were placed in the femoral arteries where their tips are advanced to the
subdiaphragmatic aorta. An estradiol or control implant was placed subcutaneously at this
time. An amniotic fluid catheter was sewn to the hindlimb before returning the fetus to the
amniotic cavity. Catheterization of fetal vessels is routine in Dr. Wood’s laboratory
(Keller-Wood and Wood, 1991; Raff and Wood, 1991; Raff and Wood, 1992; Wood et
al., 1990).

8
For carotid sinus denervated fetuses, the common carotid artery was exposed and
stripped of nerves and connective tissue between the carotid-occipital arterial junction and
the carotid-lingual arterial junction. The occipital artery was ligated and divided and the
lingual artery was stripped of all nerves and connective tissue. Before closure of the fetus,
catheters were passed into the lingual arteries and advanced approximately 1 cm into the
carotid artery toward the heart. This entire procedure is conducted bilaterally. Dr.
Wood’s laboratory has had extensive experience with this procedure and has published
descriptions of this technique (Wood, 1989, 1995).
Hypotension was achieved via brachiocephalic occlusion. A 10 minute period of
hypotension is necessary for activation of the HPA axis. Access to the artery was gained
through the second intercostal space on the left side of the chest. The occluder was tested
and deflated before the ribs, skin, and uterus were sutured closed.
Throughout the experiments, intravascular and amniotic pressures as well as heart
f
rate were measured utilizing a polygraph. Monitoring blood pressure assured that I
achieved a consistent and appropriate (50%) state of hypotension via brachiocephalic
occlusion. Blood pressure measurements were analyzed utilizing a computer program in
order to help quantify the level of estrogen augmentation of reflex responsiveness. Blood
gases and hematocrits were measured immediately after completion of each experiment.
Blood samples were processed (centrifuged, etc., to obtain plasma) and frozen until
hormone levels were measured via radioimmunoassay or enzyme immunoassay techniques
used routinely in Dr. Wood’s laboratory. Plasma hormone concentrations (ACTH, AVP,
cortisol, and estradiol) were analyzed by 2- and 3-way ANOVA.

9
Aim 2 In preparation for immunohistochemistry, the head of the fetus was perfused with
heparinized saline followed by 4% paraformaldehyde. The fetal hypothalamus and
brainstem were dissected and processed accordingly (alcohol dehydrated and embedded in
paraffin). Tissues were sectioned (6-10 micron) using a Zeiss rotary microtome and
mounted on subbed slides. Staining for ACTH, AVP, and CRH was performed using
antisera produced in Dr. Wood’s laboratory. Staining for c-fos was done using
commercially available antibodies from Oncogene Sciences. Staining for estrogen
sulfatase and estrogen sulfotransferase were done using custom made antibodies from
Alpha Diagnostic. Staining was visualized using a Histostain-SP kit (Zymed streptavidin-
biotin system).

CHAPTER 2
LITERATURE REVIEW
Control of Parturition
Though it has been known for some time that the fetus controls parturition in the
sheep, the precise mechanism has yet to be fully understood. However, much is known
about the major endocrine axis that initiates this process as well as controls blood pressure
in the fetus. These processes are controlled by the hypothalamus-pituitary-adrenal (HPA)
axis. One of the first pieces of evidence that linked the fetal HPA axis to the control of
parturition was reported by Binns et al. (1991). This was a natural phenomenon that
occurred when sheep ate a particular plant on day 14 of gestation. This plant, Veratrum
califomicum, delayed the birth of the fetus indefinitely until caesarian section or death of
the fetus or ewe. The fetus had a number of birth defects, including cyclopia and a
dysfunctional hypothalamus-pituitary (HP) axis due to dislocation of the pituitary from its
normal position. It was realized later that this malformed, dysfunctional axis was
responsible for delayed parturition. This observation and others led Liggins (1973) to
conclude that the fetus controls parturition in the sheep. Liggins has credited Hippocrates
with first suggesting this idea when he wrote that the fetus pushes its way out of the
womb when the nutrition supplied by the mother is no longer sufficient for further growth.
Malpus (1933) put forth a more modem observation suggesting a fetal role in the timing
of birth when he reported the association of fetal ancephally and prolonged gestation in
10

11
women. These ideas laid the groundwork for much of the ongoing research to determine
the precise mechanism of parturition.
Though many have contributed to this idea of a fetal role in the initiation of
parturition, no one has contributed to this field of research more than Liggins. Liggins
was the first to hypothesize and directly test that the fetal pituitary is intricately involved in
this process (Liggins et al., 1967). This was done by first hypophysectomizing fetal sheep
by surgical electrocoagulation. In fetuses where 70% or more of the pituitary was ablated,
gestation was significantly prolonged and delivery was achieved only after caesarian
section. Fetal adrenalectomy was also found to prolong gestation (Drost and Holm,
1968). Disconnection of the hypothalamus from the pituitary in the fetal sheep between
108-112 days gestation delays birth by at least eight days (Antolovich et al., 1990).
Following hypophysectomy infusion of adrenocorticotropin (ACTH) or glucocorticoid still
induces parturition (Kendall et al., 1977). Further studies from this group also
demonstrate the necessity of the HP axis in maturation of pituitary corticotropes (Kendall
et al., 1977). After disconnection of the hypothalamus from the pituitary, fetuses were
infused with saline or cortisol. In the cortisol-infused fetuses the proportion of fetal-type
corticotropins was significantly lower than in the saline-infused fetuses however the
number of adult-type corticotropins did not change. A direct effect of cortisol on pituitary
corticotropin maturation requires the presence of complete HP axis (Antolovich et al.,
1992). More specifically, following destruction of the fetal paraventricular nucleus (PVN)
of the hypothalamus, parturition was delayed (McDonald and Nathanielsz, 1991).
Therefore, the signal for parturition may either be sent to the PVN which receives input
from the nucleus of the tractus solitarius (NTS), amygdala, or hippocampus, or possibly be

12
derived in corticotrophin releasing hormone (CRH) and arginine vasopressin (AVP)
producing neurons in the PVN.
Whereby disruption of the fetal HP A axis causes a delay in the timing of birth,
stimulation of this endocrine axis can result in premature parturition. The involvement of
the fetal adrenal cortex in the initiation of parturition in sheep was suggested after in útero
plasma concentration of corticosteroid revealed dramatic increases in these hormones
prior to birth (Bassett and Thorbum, 1969). Before this conclusion was made a number
of studies aimed at investigating the role of the HPA axis in the birth process were
performed in the fetal sheep by Liggins (1968). ACTH infused into fetal sheep induced
parturition within four to seven days along with producing adrenal hypertrophy. Cortisol
infusion into the fetus induced parturition within five days. The same doses of ACTH or
cortisol infused into ewes did not induce parturition. Although estradiol at 2 mg/24 hr had
no effect, infusion of cortisol at 25 mg/24 hr plus estradiol resulted in delivery after four
days (Liggins, 1968). Further studies by Liggins (1969) showed that it was glucocorticoid
activity (not mineralocorticoid) that was important for the initiation of parturition,
evidence of this effect was shown by the inability of deoxycorticosterone or corticosterone
to induce parturition. Dexamethasone infused at rates of 0.06 - 4.0 mg/24 hr in the fetus
and 4 mg/24 hr in the ewe was ineffective in producing premature delivery.
Since the earlier experiments of Liggins, many other studies have been performed
which confirm the previous results. Parturition was induced by continuous intravenous
cortisol infusion at 130 days gestation (Thomas et al., 1978). Activation of fetal adrenal
function by pulsatile ACTH administration in 125-127 day fetal sheep induced labor and
delivery in four to five days, resulting in four to six fold elevation in fetal plasma cortisol

13
concentrations (Lye et al., 1983). Furthermore, it was shown that hypophysectomized
fetuses that were administered dexamethasone or ACTH infusions exogenously would
undergo parturition (Kendall et al., 1977).
The Hvpothalamus-Pituitarv-Adrenal Axis
The HPA axis integrates a variety of neuroendocrine inputs to regulate the
synthesis and secretion of the adrenocorticosteroids which are required for the
maintenance of life. These steroid hormones exert effects to minimize any disturbance in
homeostasis. The critical role of adrenocorticosteroids can clearly be observed after
adrenalectomy or during hypoadrenocorticism either induced by drug or due to a disease
state. Without adrenal corticosteroids disrupted electrolyte balance or carbohydrate
metabolism leads to circulatory collapse of hypoglycemic coma and death. Physical,
emotional, and chemical stresses such as pain, trauma, hypoxia, acute hypoglycemia, cold
exposure, and vasopressin administration have all been shown to stimulate ACTH and
cortisol secretion (Gann et al., 1981).
/
The Hypothalamus The hypothalamus contains several nuclei of neuronal cells. The PVN
of the hypothalamus, located bilaterally on the ventricle, contains specialized
neurosecretory cells which synthesize CRH and AVP. The relatively large-celled (hence,
termed magnocellular) neurons in the PVN contain AVP and project to the posterior
pituitary (Lechan, 1987; Reichlin, 1992). In addition to the large projection to the
posterior pituitary, it has been established that small-celled (parvocellular) neurons in the
PVN contain CRH and AVP and project to the median eminence and are involved in the
regulation of ACTH release (Sawchenko and Swanson, 1980). The increase in plasma
ACTH concentration during stress is mediated by CRH as well as AVP from the

14
hypothalamus. These two factors are released into the hypothalamo-pituitary portal
circulation and diffused into the anterior pituitary to act on corticotropes to stimulate the
release of ACTH.
The Pituitary The pituitary is divided into the anterior lobe, the intermediate lobe, and the
neural or posterior lobe. Vessels of the hypophyseal-pituitary portal system deliver blood
from the median eminence of the hypothalamus to the anterior pituitary. This system
delivers hormones released from hypothalamic neuronal axons in the median eminence to
the anterior pituitary. CRH and AVP diffuse into the anterior pituitary and bind to their
receptors on specialized cells called corticotropes, which represent 15-20% of
adenohypophyseal cells. Once the releasing hormone binds to its receptor in the
corticotrope, a single mRNA the directs the synthesis of the large precursor molecule
called proopiomelanocorticotropin (POMC). POMC is then processed to produce the
smaller biologically active fragment ACTH. ACTH is then released into the systemic
circulation where it acts on adrenocortical cells to stimulate synthesis and secretion of
glucocorticoids (Reichlin, 1985).
The Adrenal Cortex The adrenal glands are endocrinologically complex organs that are
composed of two distinct endocrine tissues derived from different embryologic sources.
The outer zone is called the adrenal cortex and constitutes 80-90% of the gland. The
cortex is the source of the steroid hormones. The smaller inner zone is the adrenal
medulla which is the major source of circulating catecholamines. The adrenal cortex is
highly vascularized and receives its main arterial supply from branches of the inferior
phrenic artery, the renal arteries, and the aorta. There are three major groups of hormones
produced by the adrenal cortex: the mineralocorticoids, the glucocorticoids, and the sex

15
steroids Histologically the adult cortex is composed of three zones: an outer zona
glomerulosa, a zona fasciculata, and an inner zona reticularis. The primary product of the
zona glomerulosa is the mineralocorticoid aldosterone. The zona fasciculata and
reticularis produce cortisol and androgens as their primary products (Pescovitz et al.,
1990). ACTH stimulates the secretion of glucocorticoids, mineralocorticoids, and
androgenic steroids from the adrenal cortex. ACTH binds to the receptors on the adrenal
cortex and provokes steroidogenesis through stimulation of cAMP production. cAMP
activates protein kinase A, which catalyzes the phosphorylation of a variety of proteins
thereby producing cholesterol. ACTH also stimulates synthesis of new adrenal proteins
and this increases adrenal weight. Glucocorticoids exert negative feedback at the
pituitary, hypothalamus, and other neural sites (Keller-Wood and Dallman, 1984).
Cortisol is carried in blood bound to transcortin (59%) and albumin (19%), while
about 22% is free in ovine plasma (Patterson and Hills, 1976). The basal production rate
f
of cortisol is 600 pg/hr and the metabolic clearance rate of cortisol is about 51 L/hr in
sheep (Panaretto, 1974). The liver and the kidney are the principle organs involved in
clearing the steroid hormones from the circulation. Although most tissues can metabolize
steroids, the liver is the primary site of steroid hormone metabolism and the kidney is the
primary site of steroid hormone excretion. The plasma half-life of cortisol is 60-100
minutes in the human (Pescovitz et al., 1990).
Cortisol produces a number of diverse physiological actions to maintain
homeostasis. As the term homeostasis implies, an excess or deficiency of glucocorticoids
affects every tissue of the body. Glucocorticoids are essential for survival (Baxter, 1972;
Gann et al., 1981; Pescovitz et al., 1990). The term glucocorticoid refers to the glucose

16
regulating properties of these hormones. However, glucocorticoids have multiple effects
that include important roles in carbohydrate, lipid, and protein metabolism (Baxter, 1972).
These increase blood glucose by increasing gluconeogensis in the liver and kidneys,
increasing hepatic glycogenesis and decreasing glucose uptake in tissues. The
glucocorticoids increase lipolysis and proteolysis. The glucocorticoids also have
stimulatory effects on cardiovascular function by increasing cardiac output and increasing
vascular response to catecholamines. At high concentrations, the glucocorticoids inhibit
most immunologic and inflammatory responses. Although these effects may have
beneficial aspects, they may also be detrimental to the host by inducing a state of
immunosuppression that will predispose the host to infection (Parrillo and Fauci, 1979).
The glucocorticoids also influence growth, development, bone metabolism, and central
nervous system activity.
The Regulation of ACTH Secretion
/
Corticotropin Releasing Hormone fCRJT) Since 1955 it has been known that the
hypothalamus contains substances that acted at the pituitary gland to increase ACTH
secretion in vitro (Guillemin and Rosenberg, 1955; Saffran and Schally, 1955). In 1981,
Vale et al. (1981) characterized a 41 amino acid peptide from sheep hypothalamus that
stimulated ACTH secretion from corticotrophins and published the primary structure of
ovine CRH. Ovine CRH was further characterized when the cDNA was cloned and
sequenced (Furutani, 1983). At this time, a similarity was discovered between the
precursor proteins for CRH, AVP, and ACTH which implies a common evolutionary
beginning. A 20 kD immunoreactive form of CRH was identified from rat hypothalamus

17
and is close to the value for ovine and human pre-pro-CRH based on their cDNA
sequences (Lauber et al., 1984).
CRH increases ACTH secretion from the anterior pituitary gland by binding to
high affinity receptors (Wynn et al., 1983) located on the corticotrophs (Leroux and
Pelletier, 1984). Activation of the receptor complex increases adenylate cyclase activity
(Perrin et al., 1986) and cAMP which results in an increase in ACTH secretion. In
cultured rat pituitary cells CRH can enhance the rates of ACTH synthesis as well as
release (Vale et al., 1983). ACTH release can be modulated by down-regulation of CRH
receptors in the anterior pituitary. There is evidence that CRH (Wynn et al., 1988), AVP
(Hauger and Aguilera, 1993), and glucocorticoids (Haugher et al., 1987; Schwartz et al.,
1986; Wynn et al., 1985) can all act to regulate CRH receptor number. Therefore, an
alteration in CRH receptor number, receptor activity, receptor coupling, or even
corticotrophin number can affect the ability of CRH to stimulate ACTH secretion.
/
Arginine Vasopressin (AVP) The other major regulator of ACTH secretion of
hypothalamic origin is AVP. Classically, AVP is known as antidiuretic hormone (ADH)
for its role in renal regulation of fluid balance. An increase in plasma osmolality is the
most potent stimulus to AVP secretion in that very small increases in osmolality cause an
almost immediate secretory response from the posterior pituitary. AVP binds to receptors
on the basal-lateral membrane of the cortical and medullary collecting ducts of the
nephron. Binding to these receptors (V2) results in activation of adenylate cyclase which
subsequently increases cAMP This second messenger is then thought to facilitate an
increase in protein channels found in the luminal membrane, thereby increasing the
diffusion of water out of the nephron and concentrating urine while retaining fluid

18
(Vander, 1985). In addition to A VP’s role in modulating fluid reabsorption in the renal
system, A VP is also a potent vasoconstrictor of the cardiovascular system (binding to Vi
receptors). Significant decreases in blood volume (15-20%) produce large increases in
plasma vasopressin. Decreases in blood volume are sensed as decreased stretch of the
arterial baroreceptors located in the carotid sinus as well as the receptors in the left atrium
and AVP secretion is reflexively stimulated (Berne and Levy, 1986).
AVP is one of two hormones secreted from the posterior pituitary gland (oxytocin
is the other). The posterior pituitary also called the neurohypophysis, is comprised of
axons and axon terminals which account for 42% of its total volume (Nordmann, 1977).
These axons project from magnocellular neurons of the supraoptic nucleus (SON) and
PVN from the hypothalamus. AVP and oxytocin are structurally similar hormones with
very different functions. AVP has actions on the renal and circulatory systems while
oxytocin causes milk ejection and uterine contractions. AVP and oxytocin are produced
r
in different neurons of the same nuclei and are stored in secretory vesicles or granules with
their appropriate neurophysin (Silverman and Zimmerman, 1975).
AVP is produced from a large precursor protein containing not only AVP but also
neurophysin and a glycopeptide signal sequence (Sachs et al., 1969). The precursor
protein molecule is packaged in granules with the enzymes needed for processing AVP to
its final form. As the granules move down the axons, post-translational processing of the
precursor molecule occurs within the granules. When the granules reach the axon
terminals, the nerve is depolarized and the granules are exocytosed and the contents of the
granules are released (Brownstein et al., 1980). Magnocellular neurons containing AVP

19
project fibers to the median eminence and therefore maybe important in the regulation of
ACTH secretion (Holmes et al., 1986).
AVP is a potent modulator of pituitary ACTH secretion. In fact, in sheep, AVP is
a more potent stimulator of ACTH than CRH (Familari et al., 1989; Liu et al., 1990).
AVP binds to receptors on the anterior pituitary corticotroph to increase plasma ACTH
secretion. These receptors are different from the pressor receptors (subtype Vi) or anti¬
diuretic receptors (subtype V2) that are found in the periphery. Data from two different
groups (Baertschei and Friedli, 1985; Jard et al., 1986) suggest that a subtype (classified
as Vib or V3) distinct from the peripheral receptors, exists in the brain with protein kinase
C as its second messenger.
AVP can effect anterior pituitary secretion in two ways: either by AVP secretion
from axons which terminate in the median eminence or by AVP secretion from the
posterior pituitary. Evidence for both possibilities exist. AVP-containing neurons of the
/
SON and PVN are known to project to the external zone of the median eminence
(Hoffmann et al., 1991). A further distinction has been made in that AVP-containing
parvocellular neurons of the PVN were found to innervate the external zone of the median
eminence. The magnocellular neurons of the PVN pass through the internal zone of the
median eminence to the neurohypophysis, but also contribute to the AVP found in the
median eminence (Holmes et al., 1986). AVP secretion from the posterior pituitary gland
has also been implicated in plasma ACTH secretion. In studies performed in dogs,
neurohypophysectomy attenuated the plasma ACTH response to hypotension. After
restoration of plasma AVP levels to those observed in the intact animal, plasma ACTH

20
levels were almost completely returned to normal (Raff et al., 1988). The action of AVP
appears to be through a direct effect at the pituitary gland.
Synergism of CRH and AVP The synergistic activity of CRF and AVP secretion has been
well documented. In support of this is an overwhelming amount of anatomical data for
the interaction between these two hormones. CRH and AVP are found in the same
neurosecretory vesicles in the median eminence (Whitnall et al., 1985). In normal rats,
staining of the median eminence by immunohistochemistry revealed co-localization of
AVP in 50% of CRH axons (Whitnall et al., 1987). Repeated stress (immobilization in
rats) increases the co-localization of AVP in CRH nerve terminals in the median eminence
(de Goeij et al., 1991). Following adrenalectomy in rats, CRH immunostaining increases
in parvocellular neurons of the PVN and the amount of co-localization with AVP
increases (Sawchenko et al., 1984). The increase in CRH and AVP immunoreactivity
following adrenalectomy is prevented by intracerebroventricular injection of
f
dexamethasone (Sawchenko, 1987).
From in vitro studies performed in cultured pituitary cells, CRH is considered to be
the more potent secretagogue for ACTH secretion in rats while AVP appears to be a more
potent stimulus to ACTH release in sheep (Familari et al., 1989). However, the action of
each is potentiated when administered together in cultured pituitary cells (Gillies et al.,
1982), adult freely moving rats (Rivier and Vale, 1983a) and in fetal sheep (Brooks and
White, 1990). In a study performed in conscious sheep, stress-induced ACTH secretion
(audiovisual and insulin-induced hypoglycemia) was accompanied by increases in
hypothalamic CRH and AVP secretion (Familari et al., 1989). What is most interesting is
that CRH:AVP molar ratio was altered with the stress. Portal plasma AVP was increased

21
above that of CRH, increasing the ratio of AVP to CRH. Since CRH and AVP can be
found in the same neurosecretory vesicles, this suggests that differential regulation of each
individual hormone also occurs. A synergistic effect of CRH and AVP on ACTH
secretion was also observed as seen by a lack of 1:1 concordance between hypothalamic
AVP/CRH secretion and pituitary ACTH secretion. This effect may be due to secretion of
other hypothalamic factors that increase ACTH secretion.
Other ACTH Secretagogues The control of adrenocorticotropin secretion is a complex
process involving numerous factors (neurotransmitters and neuropeptides) that augment
ACTH secretion. Rat pituitary corticotrophs in culture release ACTH in response to
epinephrine and norepinephrine acting on al-adrenergic receptors (Giguere et al., 1981).
Epinephrine has also been identified in portal plasma suggesting a physiological role in the
control of anterior pituitary function (Johnson et al., 1983). Neuropeptide Y (NPY)
injected centrally in fetal and adult sheep increases plasma ACTH concentrations but does
f
not stimulate the pituitary directly (Brooks et al., 1994). This suggests that NPY acts
centrally to increase the activity of the HPA axis. Evidence for NPY stimulation of CRH
secretion supports this conclusion (Haas and George, 1987). Endogenous opiates are
capable of stimulating the HPA axis in late gestational fetal sheep but do not tonically
stimulate the axis in a regulatory manner (Brooks and Challis, 1988). Serotonin stimulates
ACTH secretion in humans, demonstrates by pharmacologically increasing serotonin with
fenfluramine (Lewis and Sherman, 1984). However, there are conflicting data as to the
site of action of serotonin (hypothalamus or pituitary). In rats, CRH secretion is
decreased after elimination of endogenous hypothalamic catecholamines suggesting a role
for central catecholinergic neurons in the control of ACTH release (Guillaume et al.,

22
1987). Angiotensin II also increases plasma ACTH secretion by induction of CRH (Rivier
and Vale, 1983). Prostaglandin E2 alone does not increase ACTH secretion but enhances
the ability of AVP to stimulate ACTH secretion with no effect on CRH (Brooks and
Gibson, 1992). In addition to stimulating the activity of the HPA axis, factors from the
brain also inhibit the axis. The dopaminergic system in the amygdaloid central nucleus has
been found to inhibit ACTH secretion by action on the anterior and lateral hypothalamus
(Beaulieu et al., 1987). Atrial Natriuretic Peptide (ANP) has also been shown to alter
ACTH secretion. Brain ANP is secreted into the hypophyseal portal vessels from the
hypothalamus and physiological concentrations inhibit ACTH release from pituitary cells
in vitro (Dayanithi and Antoni, 1989; Lim et al., 1990; Sheward et al., 1991). In vivo
immunoneutralization of ANP significantly increases ACTH release but has no effect on
release during ether stress (Fink et al., 1991). These results suggest a role for ANP as a
mediator in the regulation of ACTH secretion.
/
Negative Feedback Control of ACTH Secretion Glucocorticoids are very versatile
steroids and necessary for survival in a number of species including sheep and primates.
Binding of the glucocorticoid to its receptor promotes binding to and transcription of
DNA, production of mRNA for synthesis of enzymes, and eventually alteration of cell
function. Glucocorticoids act in the body to increase plasma glucose concentrations by
increasing hepatic glycogenesis and gluconeogenesis. They also increase protein
catabolism and in the periphery, glucocorticoids exert actions that counter the effects of
insulin. Glucocorticoids are also necessary for vascular reactivity. Without them, the
vascular smooth muscle becomes unresponsive to epinephrine and norepinephrine, the
capillaries expand and their walls become permeable to proteins in the plasma. Finally,

23
glucocorticoids are released in response to stress and noxious stimuli. In animals that lack
normal secretion of glucocorticoids, exposure to a stress can be life-threatening (Ganong,
1985).
Secretion of glucocorticoids is regulated by adrenocorticotropin hormone from the
pituitary. When a stimulus of ACTH secretion from the hypothalamus reaches the
pituitary gland, ACTH is secreted into the general circulation, binds to its receptor at the
adrenal gland and stimulates cortisol secretion. Cortisol then acts at target organs to
increase plasma glucose levels, etc. However, cortisol also acts at the levels of the brain
to reduce ACTH secretion. Cortisol acts at the hypothalamus and pituitary gland
negatively to inhibit further ACTH secretion. This is called cortisol negative feedback
inhibition of ACTH secretion.
The mechanism of action of cortisol to reduce ACTH secretion has been studied at
length in many species and by many investigators. A study by Canny et al. (1989)
/
performed in sheep, examined both hypothalamic and pituitary sites, of action for
glucocorticoids. Measurements of hypophyseal portal concentrations of AVP and CRH
and systemic measurements of ACTH and cortisol concentrations were made before and
after dexamethasone infusion with different stimuli to ACTH secretion. The data suggest
that glucocorticoid act in a site-specific manner to inhibit ACTH secretion (Canny et al.,
1989). During audio-visual stress, hypothalamic CRH and AVP secretion were unaltered
but ACTH secretion was inhibited suggesting a pituitary site of action of dexamethasone.
In response to hypoglycemia, dexamethasone inhibited both the hypothalamic and pituitary
responses to the stress. Studies in rats have shown that glucocorticoids inhibit CRH
secretion as well as CRH synthesis (Sato et al., 1975) suggesting a mechanism for a

24
hypothalamic site of action. On the other hand, glucocorticoids may interfere with CRH
activation of second-messenger systems (cAMP) at the pituitary and therefore prevent
stimulation of ACTH secretion (Bilezikjian and Vale, 1983).
In the fetus, cortisol has the ability to inhibit plasma ACTH secretion in response
to a hypotensive stimulus by approximately 90 days gestation (Hargrave and Rose, 1985).
High concentrations of glucocorticoids near the fetal PVN prevent increased ACTH
secretion in response to hypotension and hypoxemia (McDonald et al., 1990). Between
117 and 131 days gestation, fetal sheep are extremely sensitive to negative feedback
effects of cortisol. This was demonstrated by infusions of cortisol that caused less than 2
ng/ml increases in plasma cortisol concentrations but which completely inhibited the
normal ACTH response to hypotension (Wood, 1986). This knowledge predicts the
existence of a normal feedback response in the fetus, which is the case in late gestational
fetal sheep. However, in near-term fetal sheep, cortisol negative feedback regulation of
ACTH secretion becomes ineffective. In experiments in which infusions of cortisol
increased plasma cortisol concentrations to approximately 60 ng/ml, fetal plasma ACTH
secretion was still not suppressed (Wood, 1987; 1988). This mechanism of this reduction
of glucocorticoid negative feedback efficacy is not fully understood at present.
The Development of the Fetal HP A Axis
Development of the ovine fetal HPA axis begins during the first third of gestation
(term being 145-148 days gestation) with the formation of the pituitary and adrenal
glands. The fetal pituitary gland can be detected as early as 31 days gestation and
differentiation of the anterior pituitary can be seen at about 40 days (Perry et al., 1982).
Staining of cells in the anterior pituitary indicate the presence of ACTH immunoreactivity

25
by as early as 50 days gestations and by 60 days gestation, processing of ACTH from
POMC can be detected in the intermediate lobe of the pituitary of the fetal sheep
(Mulvogue et al., 1986).
The fetal adrenal can be identified by approximately 28 days gestation in the sheep
(Wintour et al., 1977). By 40-50 days gestation, in vivo experiments demonstrate that the
fetal adrenal readily secretes cortisol in response to ACTH (Wintour et al., 1975;
Glickman and Challis, 1980). Between 90-120 days gestation, the cells of the zona
fasciculata (which are responsible for cortisol synthesis) are relatively immature (Robinson
et al., 1979) and approximately 90% of fetal plasma cortisol is derived from the maternal
circulation (Hennessy et al., 1982). After 120 days gestation, fetal adrenal sensitivity to
plasma ACTH increases (Liggins et al., 1973; Rose et al., 1982), the proportion of cortisol
that is of fetal origin increases (Hennessy et al., 1982), and the correlation between fetal
ACTH secretion and fetal cortisol secretion becomes significant (Hennessy et al., 1982).
/
Adrenal weight also increases as a function of gestational age (Comline and Silver, 1961).
Early studies involving fetal hypophysectomy (Liggins et al., 1967) and
adrenalectomy (Droust and Holm, 1968) with prolongation of pregnancy suggested a link
between the adrenal steroid production and parturition. If this is the case, fetal plasma
cortisol secretion should be altered as gestation nears and ends. Before about 130 days
gestation, fetal plasma corticosterone levels are low but several days before, parturition
plasma levels increase and peak at birth (Bassett and Thorbum, 1969). Nathanielsz et al.
(1972) found that fetal plasma cortisol concentrations began to increase about three to
four days before parturition and then steadily declined in the newborn lamb. A more
elaborate study of cortisol secretion was performed by Magyar et al. (1980) in which

26
exponential curves were fit to the data to more accurately describe the increase in fetal
plasma cortisol concentrations. This analysis revealed fetal plasma cortisol concentrations
increasing exponentially about 10-15 days prior to parturition. Cortisol is secreted in
response to ACTH binding to receptors at the adrenal gland. In vivo experiments by
Brown et al., (1978) demonstrated that not only did glucocorticoids increase with
development, but the ability of the fetal adrenal to secrete cortisol in response to ACTH
was observed around 120-129 days gestation. The increase in cortisol secretion that
occurs near the end of gestation is in part due to an increase in adrenal sensitivity to
stimulation by ACTH (Madill and Bassett, 1973) but also possibly due to increased plasma
ACTH secretion from the fetus.
Jones et al. (1977) presented some of the first data examining the changes in fetal
plasma ACTH concentration during development. They demonstrated that fetal plasma
ACTH concentrations increase prior to parturition. However, they concluded that the
/
increase occurred after the increase in fetal plasma cortisol and therefore is probably not
the reason for the changes in fetal plasma concentrations. Experiments performed later by
other investigators revealed that the fetal plasma ACTH concentrations increase much
sooner than previously described by Jones et al. (Maclssac et al., 1985; Wintour, 1984;
Norman et al., 1985). In fact, fetal plasma ACTH concentrations increase during the last
30 days of gestation, well before the increase in plasma cortisol concentrations which
occurred approximately 120 days gestation.
The results of increasing plasma ACTH concentrations in vivo when taken
together with the in vitro pituitary ACTH secretion studies support the presence of an
additional factor involved in the process of parturition. Basal output of ACTH from fetal

27
sheep pituitaries in culture does not increase as a function of gestational age (Durand et
al., 1986). Pituitary ACTH secretion in culture does not increase during the last week of
gestation at a time when in vivo circulating plasma ACTH concentrations are increasing
exponentially (McMillen and Merei, 1993). These data suggest that the preparturient
increase in ACTH secretion is dependent on some other factor to stimulate secretion and
not a function of basal pituitary output. In addition, McMillen and Merei (1993) also
found no change in responsiveness of the fetal corticotroph to CRH as a function of
gestational age. However, prior exposure of the fetal pituitary to cortisol increased its
responsiveness to CRH. There appears to be a signal, possibly from the hypothalamus,
that increases the activity of the fetal pituitary and therefore the fetal adrenal and is the
trigger to parturition in the sheep.
The Importance of Gonadal Steroids in Parturition and Cardiovascular Control
In addition to the increase in fetal plasma ACTH and cortisol concentrations that
/
occur at the end of gestation, the fetal plasma estrogen and androgen concentrations also
increase. Plasma estrone concentrations appear to increase over the last four days of
gestation while estradiol may be increasing over the last eight days in the fetus and
amniotic fluid (Challis and Patrick, 1981; Findlay and Cox, 1970). In a paper by Challis
and Patrick, fetal plasma estrone concentrations increase from 40 pg/ml at 14 days prior to
birth to near 400 pg/ml the day before birth. Fetal plasma estradiol concentrations
increased from 20 pg/ml at 14 days prior to birth to 80 pg/ml on the day before
parturition. Plasma estradiol concentrations in the ewe increase over the last two days of
pregnancy in the ewe (Robertson and Smeaton, 1973), with a ten-fold increase on the day
before parturition, from 20 to 40 pg/ml up to 411 pg/ml (Challis, 1971). At the time

28
plasma estrogens increase, there is a decrease in plasma progesterone (Bedford et al.,
1972). This is possibly due to a conversion of estrogen to progesterone, with plasma
progesterone acting as a reservoir for plasma estrogen production. Since progesterone is
also a precursor for androgen production, one would expect to see increases in plasma
concentrations of these steroids as well. Plasma androstenedione and testosterone
concentrations do indeed increase in the late gestational fetus in a manner similar to
estrogen concentrations (Pomerantz and Nalbandov, 1975; Yu et al., 1983).
This increase in plasma estrogen and androgen concentrations is a result of the
actions of cortisol on the placenta (Anderson et al., 1975; Steele et al., 1976). Although
the placenta has aromatase activity the plasma concentrations of estrogens and androgens
are very low throughout gestation (Mann et al., 1975). Cortisol acts at the placenta to
induce an enzyme, cytochrome P450Cn, which has 17a-hydroxylase and 17,20 lyase
activities. Upon induction, P450Ci7 facilitates the production of estrogens and androgens
/
from progesterone, thus increasing plasma concentrations of these hormones.
Gap junctions in the myometrium, which are very important for synchronized
uterine contractions, are believed to be formed at the time of progesterone withdrawal
(Garfield et al., 1977). It has been proposed that as a result of decreased progesterone
and increased estrogen, there is an augmentation in synthesis of proteins associated with
gap junctions (Garfield et al., 1978; Garfield, 1984, 1977). These gap junction proteins
are inserted into the plasma membrane of the myometrium and aggregate to form gap
junctions. Gap junctions are necessary for the uterus to contract in a unified, coordinated
fashion in order to expel the fetus. Prostaglandins and oxytocin, as well as estradiol, act in
concert to stimulate uterine contractions in the process of labor (Lye et al., 1983).

29
Tissue estrogen concentrations in sheep also increase towards term or after
ACTH-induced labor, especially in the myometrium (Power and Challis, 1987). The ovine
placenta, through sulfatase and aromatase activities, converts estradiol and estrone sulfate
to 173-Estradiol and estrone, the more potent estrogen for the target organ, the
myometrium (Rossier and Pierrepoint, 1974).
It has long been known that female rats have greater activity of the HPA axis than
male rats. Studies in adult animals demonstrate that female rats have increased
corticosterone secretion following ACTH administration and greater adrenal
responsiveness to tropic stimulation (Kitay, 1961). As the major difference between the
sexes is gonad and steroid production, experiments involving gonadectomy and
replacement of gonadal steroids were performed. After gonadectomy, testosterone
depressed ACTH content and steroid clearance in male rats but increased adrenal
responsiveness to ACTH. In female rats, estradiol had a consistent stimulatory effect on
ACTH secretion (Kitay, 1963). In female rats, plasma ACTH and corticosterone
responses to restraint stress were enhanced during proestrus, when estradiol
concentrations were highest (Kitay, 1963). In ovariectomized (ovx) rats replaced with
estradiol, this effect can be restored (Viau and Meaney, 1991). Following ovx, there was
a decreased capacity of the pituitary to synthesize ACTH and a decreased responsiveness
to stimulation by hypothalamic extracts (Coyne and Kitay, 1969). In ovx rats, estradiol
implants into the area of the anterior pituitary, arcuate nucleus, and lateral mammilary
bodies in rats facilitated pituitary-adrenal activity, suggesting a central nervous system
effect (Richard, 1965). However, estrogen stimulation of corticosterone secretion in ovx
rats, may be due in part to a direct effect on the adrenal cortex (Kitay et al., 1965). In ovx

30
rats, plasma ACTH and corticosterone responses to foot shock and ether vapor stress
were lower than in estrogen-replaced ovx rats (Burgess and Handa, 1992). From these
results, it was concluded that the increased activity in the pituitary-adrenal axis was due to
an impairment of the glucocorticoid negative feedback system.
The mechanism of action of estrogen stimulation on HPA axis activity is not fully
understood. Based on the present data, estrogen may interact with a number of systems
impinging on the HPA axis. Estrogen uptake has been demonstrated in corticotrophins
isolated from anterior pituitary cells from adrenalectomized rats (Keefer, 1981) suggesting
a possible direct effect of estrogen on the pituitary. Estradiol has also been shown to
concentrate in tyrosine hydroxylase containing neurons in the arcuate and periventricular
nuclei of the rat (Sar, 1984). In another study, estradiol concentrating cells have been
found in the amygdaloid central nucleus (Beaulieu et al., 1987). Since this dopaminergic
system can inhibit ACTH secretion, this may be important in modulating HPA axis
f
activity. In rats given 100|ig estradiol exogenously for two weeks, there was an increase
in AVP in the SON and PVN of the hypothalamus without any changes in pituitary ACTH
and AVP content or basal plasma ACTH or AVP concentrations (Hashimoto et al., 1981).
In another study, plasma AVP concentrations were found to be greatest when estrogen
concentrations were highest. If the rats were ovx, plasma AVP concentrations decreased
but were restored when estrogen was replaced (Skowsky et al., 1979). These data
suggest a possible role of hypothalamic releasing factors in the mediation of estrogen
stimulation of HPA axis activity.
The results of this dissertation demonstrate that there is significant estrogen
sulfatase activity in ovine fetal hypothalamus, hippocampus, and brainstem, and that there

31
are statistically significant ontogenetic changes in activity of this enzyme in the
hippocampus. Also shown is the presence of estrogen sulfotransferase in the fetal
hypothalamus and brainstem. It has previously been demonstrated that estrogens in fetal
plasma increase both basal- and stimulated- fetal plasma ACTH secretion. The present
results suggest a mechanism by which the most abundant form of estrogen in ovine fetal
plasma, estrone sulfate, might be made available to areas within the fetal brain known to
be involved in the control of the fetal HPA axis.
Mathew and Balasubramanian (1982) and Lakshmi and Balasubramanian (1979)
have previously demonstrated estrogen sulfatase and sulfotransferase activity in adult
sheep brain tissue. Other investigators have demonstrated these enzymatic activities in
adult brain tissue from rats (Connolly and Resko, 1989; Kawano and Aikawa, 1987), mice
(Hobkirk, 1987), non-human primates (Lakshmi and Balasubramanian, 1981), and human
beings (Platia, 1984). Hobkirk and coworkers demonstrated that enzyme activities are
/
transiently increased postnatally in the brain of the mouse (1987). While the development
of brain estrogen sulfatase and sulfotransferase activity have not been studied in sheep, the
development of activities in mice suggests the possibility that this might be an important
developmental process in the perinatal period.
Using a histochemical technique, Kawano and Aikawa found that sulfatase activity
is highest in pineal gland, choroid plexus, and pars distalis of the pituitary in adult rats
(1987). I investigated the activity in hypothalamus, brainstem, and hippocampus because
these areas are known to contain nuclei involved in integration, afferent signal relay, or
negative feedback inhibition within the HPA axis (Grizzle et al., 1974; Keller-Wood and
Dallman, 1984; Maran, 1978, Ward, 1978). The presence of activity in any of these areas

32
could be important for the deconjugation of sulfated estrogens in the blood perfusing the
brain. Rosenfeld et al in 1980 reported that the majority of estrogen produced by the
ovine placenta is sulfoconjugated and thus protected since sulfatase in not present. My
data suggest otherwise given that sulfoconjugates in the fetal compartment may have
specific regional roles. The effect of estrogen on both basal- and hypotension stimulated-
concentrations of ACTH could be the result of an action of estrogen on the PVN in the
hypothalamus, an action on the hippocampus (which mediates some of the negative
feedback actions of corticosteroids on ACTH secretion), an action on the NTS (which
relays neural traffic from visceral afiferents), or an action on any part of the pathways
leading from the NTS to the PVN (e.g., the RVLM). Estrogen receptors have been
demonstrated in the NTS and hippocampus (Lehman, 1993). While estrogen receptors
within the hypothalamus are most concentrated in the arcuate nucleus, estrogen receptors
have been demonstrated in the PVN (Lehman, 1993; Simerly, 1990). The results of the
present experiments identify the cellular location of the sulfatase activity which is
consistent with these centers for HPA axis control. I found widespread staining
throughout nuclei and fiber tracts of the hypothalamus and brainstem. Neuronal staining
was much more concentrated than fiber tract staining, however both were observable.
While estrogen sulfatase may be responsible for decongugating estrone sulfate
locally within the fetal to increase HPA axis activity directly, the role of estrogen
sulfotransferase is probably more indiscrete. Naturally one such role of the enzyme is to
maintain high levels of circulating conjugated estrogens that cannot be readily degraded.
A less obvious role of estrogen sulfotransferase might be to conjugate cortisol so that

33
inhibition of HPA axis negative feedback is achieved. This, concomitant with local
activation of estrogens via estrogen sulfatase, would increase ACTH release.
Cardiovascular Reflex Responsiveness
Arterial baroreceptors are mechano-receptors located in the walls of large systemic
arteries including the aortic arch, brachiocephalic artery, and carotid sinuses (Boss and
Green, 1956; Green, 1954). Afferent signals from the arch of the aorta are transmitted
through the left and right aortic depressor nerves to the vagus nerve and ultimately to the
nucleus of the tractus solitarius in the medullary area of the brain stem (Boss and Green,
1956; Nakayma, 1965; Nonindez, 1935). Sensory input from the carotid sinus region
travels to the nucleus of the tractus solitarius as well, but via the sinus (Hering’s) and
glossopharyngeal nerves.
Barosensitive nerve endings are found in areas of the arteries with large quantities
of elastic tissue (Muratori, 1967). Approximately 40% of the tissue comprising the walls
/
of the aortic arch is an elastin-collagen mixture (Bader, 1963) which is almost free of
smooth muscle (Gregoreva, 1962). Similarly, the carotid sinus is thinner (Adams, 1958;
Addison, 1944; Rees, 1968; Rees and Jepson, 1970), contains less smooth muscle
(Addison, 1944; Bagshaw and Fischer, 1971; Muratori, 1967; Rees and Jepson, 1970),
and shows a higher elastin content than other areas of the carotid artery (Addison, 1944;
Rees and Jepson, 1970).
Although their name implies a pressure-sensitive quality, baroreceptors are stretch
receptors which respond to deformation of the vessel wall in which they are located
(Hauss et al., 1949; Angell-James, 1971). There is evidence which shows that the degree
of wall deformation determines the electrical activity of the carotid sinus and aortic arch

34
baroreceptors. Hauss et al. (1949) demonstrated that the reflex fall of blood pressure
produced by an increase in carotid sinus pressure is abolished if the stretching of the
carotid artery is prevented by a plaster cast applied to the outside of the sinus region.
Additionally, Angell-James (1971) reported that increased baroreceptor activity produced
by elevation of intrathorasic pressure could be prevented by simultaneously increasing the
extramural pressure by the same amount. Experiments in man have shown that changing
pressure in a chamber surrounding the neck results in reflex changes in heart rate and
blood pressure and provide further evidence that altered transmural pressure is a stimulus
for baroreceptor activation (Bevegard and Shepard, 1966; Ernestine and Parry, 1957).
Koch (1931) was the first to demonstrate that when carotid sinus pressure was
changed in a stepwise mannor, mean arterial pressure exhibited an inverse sigmoidal
response to the change in intrasinus pressure. Bronk and Stella (1932) who observed that
the impulse frequency in Herring’s nerve exhibited a positive sigmoidal' relationship to
changes in sinus pressure later substantiated these findings. These early findings identified
that baroreceptors can be characterized as having a threshold pressure range for which the
discharge rate increases with a rise in mean arterial pressure, as an asymptotic saturation
pressure beyond which there is little increase in baroreceptor activity (Koushanpour,
1991).
The aortic and carotid baroreceptors exhibit different threshold and saturation
characteristics. Carotid sinus baroreceptors are silent at arterial pressures between 0 and
60 mmHg, but above 60 mmHg, they respond progressively and reach maximum discharge
capacity at approximately 180 mmHg (Koushanpour, 1991). Aortic baroreceptors
respond in a manner similar to that of the carotids except that they exhibit a threshold

35
pressure approximately 30 mmHg higher (Koushanpour, 1991). Therefore, in the normal
operating range of approximately 100 mmHg (80 to 180 mmHg), slight changes in
pressure elicit strong baroreceptor-mediated autonomic reflexes to return arterial pressure
to within homeostatic limits.
Blood pressure control in the fetus is similar with some differences. Decreases in
blood pressure increase the secretion of ACTH, cortisol, AVP, and renin (Robillard et al.,
1979; Rose et al., 1981; Wood 1989). As in the adult, the magnitude of the responses are
proportional to the magnitude of the decrease in arterial pressure (Wood et al., 1982), and
responses are attenuated by sinoaortic denervation (Wood, 1989). Chemoreceptors have
a critical role in maintaining blood pressure as well by monitoring the levels of oxygen,
carbon dioxide, and hydrogen ions in the blood.
Effective baroreceptor function is necessary to respond to transient alterations in
arterial pressure (Brown, 1980). In the face of increasing pressure, baroreceptor-
/
generated signals ascend afferent pathways and enter the nucleus tractus solitarius where
secondary signals inhibit the medullary vasoconstrictor center and excite the vagal center
stimulating vasodilation and decreased myocardial ionotropic and chronotropic response
(Brown, 1980). These actions lead to lowered peripheral resistance, cardiac output, and
ultimately, lower blood pressure. Conversely, a sudden fall in arterial pressure leads to
reflex actions which increase cardiac output and systemic resistance to raise blood
pressure.
The bradycardic response to baroreceptor stimulation in humans is mediated
through vagal cholinergic mechanisms. Several investigators (Eckberg et al., 1971;
Pickering et al., 1972; Simon et al., 1977; Takeshita et al., 1979) have demonstrated that

36
elongation of the R-R interval which accompanied a rise in arterial pressure following
administration of phenylephrine was not reduced by propranolol (Jose and Taylor, 1969),
but was abolished by atropine. Others have reported similar observations in response to
stimulation of the carotid baroreceptors by neck suction (Eckberg, 1977; Eckberg et al.,
1976).
In contrast, there is a lack of consensus regarding the autonomic mechanisms
mediating the tachycardic response to arterial baroreceptor unloading. It has been
observed that the early tachycardia observed after administration of vasodilators was
unaffected by propranolol, but abolished by atropine, suggesting a predominant vagal
mediation of this response (Leon et al., 1970; Mancia et al., 1979; Mroczek et al., 1976;
Pickering et al., 1972). Contrary to these reports, others have demonstrated that the
increase in heart rate produced during infusions of nitroglycerin was reduced by atropine,
but could only be abolished by combined administration of atropine and a beta-adrenergic
f
blocker (Goldstein et al., 1975; Robinson et al., 1966). In addition, it has been
demonstrated that during lower body negative pressure, tachycardia was diminished 52%
by propranolol with the remaining response abolished by atropine (Bjurstedt et al., 1977).
Therefore, it appears there is a significant vagal component to the cardioacceleration
which accompanies baroreceptor unloading. However, an increase of sympathetic cardiac
influence may contribute to the more sustained component of baroreflex-mediated
tachycardia (Mancia and Mark, 1983). Taken together, these results suggest redundancy
in mechanisms by which the autonomic nervous system mediates baroreflex-induced
tachycardia.

37
The baroreceptor system markedly reduces daily variation in arterial pressure.
This phenomenon is readily demonstrated in sinoaortic denervated animals who exhibit
elevated blood pressure (Cowley et al., 1973). Controversy exists regarding the
persistence of this hypertension with some authors arguing it eventually subsides (Guyton
et al., 1974) while others suggest elevated blood pressure persists (Scher and Ito, 1978;
Alexander, 1979; Touw et al., 1979; Werber and Fink, 1979).
Studies involving the measurement of substances released from the hypothalamus,
the pituitary or the adrenal require the evaluation of blood pressure as blood pressure is
reflexively defended at least in part by hypothalamic, pituitary and adrenal responses. In
both the adult and the fetus, responses to hypotensive stimuli involve neural and hormonal
changes that are responsible for restoring blood pressure. In the adult, decreases in blood
pressure are detected by stretch receptors in the atria (Cryer and Gann, 1974) and by
baroreceptors in the high pressure circulatory system (Roseetal, 1981) and result in the
/
increased release of ACTH, cortisol (Gann, 1979), renin and AVP (Claybaugh and Share,
1973). In the fetus the ACTH, cortisol, AVP and renin responses to hypotension are not
vagally mediated but instead are thought to involve changes in blood pH and central
chemoreceptors (Wood et al., 1989). ACTH is important in both adult and fetal animals
for inducing the appropriate adrenocortical responses to noxious stimuli such as
hypotension (Wood and Rudolph, 1983). Cortisol is essential for the restitution of blood
volume following hemorrhage and as a permissive substance for appropriate
vasoconstriction following hypotension in both the adult (Grimes et al., 1987; Pirkle et al.,
1976) and fetus (Brace, 1983). Vasopressin is responsible for vasoconstriction,

38
redistribution of blood flow and antidiuresis in the adult (Cowley et al., 1974) and fetus
(Iwamoto et al., 1979).

CHAPTER 3
GENERAL METHODS
Surgical Preparation of Fetal Sheep
The sheep used in this study were all pregnant ewes of 115 days gestation or later.
Animals were purchased from various suppliers (Institute of Food and Agricultural
Sciences, University of Florida, Gainesville, FI; Tom Morris, MD) and were of various
breeds (Florida Native, Mixed Western, etc.). Prior to surgery, all animals were housed in
approved pens in the Health Science Center or the 34th Street facility at the University of
Florida. Prior to experimentation, all animals were housed in Animal Resources at the
Health Science Center and were maintained under controlled lighting and temperature.
Pens were cleaned daily and ewes were given food and water ad libitum.
Aseptic fetal surgery was performed in Animal Resources or at the 34th Street
facility under general anesthesia with 0.5% - 2.0% halothane. All ewes were between 115
and 125 days gestation at the time of surgery. Food and water were withheld from ewes
24 hours prior to surgery. Ewes were sheared close to the skin around the abdomen and
prepared for surgery with povidone iodine (Betadine®, Purdue Fredrick Co., Norwalk,
CT). Animals were intubated and connected to a respirator to allow for constant
anesthesia. Heart rate, blood pressure, ventilatory 0; and C02, respirations per minute,
and rectal temperature were all monitored at the time of surgery. Animals were closely
monitored from the time of intubation until recovery when the animal could stand on its
39

40
own effort. Ewes were allowed free access to food and water throughout the post¬
operative period.
The uterus was exposed using a midline incision beginning at the umbilicus and
extending caudally approximately 10 cm. Once the hindlimbs were located, a small
incision was made in the uterus. Hindlimbs were delivered through the uterine incision
one at a time for the purpose of placing a polyvinyl chloride catheter (0.03” ED, 0.05” OD)
into each femoral artery. Later, these catheters would be used for blood sampling and
blood pressure recording. The tips of each femoral catheter were advanced to the
subdiaphragmatic aorta. At this time an estradiol implant (5 mg/21 days or 250 |ig/day;
Innovative Research of America, Toledo OH) or placebo was inserted subcutaneously into
the area of the gluteus medius before suturing the incised hindlimbs. An amniotic catheter
made of polyvinyl chloride (0.05” ID, 0.09” OD) was sutured to the exterior of a hindlimb
for the purpose of antibiotic delivery as well as amniotic fluid pressure measurements.
Hindlimb and uterine incisions were closed using 2.0 silk suture. Hindlimb incisions were
/
closed using a simple continuous suture pattern. All uterine incisions were closed first
with a locking simple continuous pattern followed by umbrication of the uterus.
Using a technique similar to the one described above, catheters were placed in the
lingual arteries and advanced 1 cm into the carotid artery toward the heart. Upon closure
of the neck incision, lingual catheters were anchored to the chin of the fetus with 2.0 silk
suture. Lingual catheters were of the same material and size and femoral catheters.
Depending upon the experimental setup, carotid sinus denervation was employed
at this time. After exposing the common carotid artery, denervation was accomplished by
stripping all nerves and connective tissue between the carotid-occipital arterial junction

41
and the carotid-lingual arterial junction. The occipital artery was ligated, for this is the
only method by which all of the carotid sinus baroreceptor and chemoreceptor afferent
fibers are cut. The lingual artery was stripped rostrally of all nerves and connective tissue
for approximately 1 cm from the carotid-lingual junction. This denervation was conducted
bilaterally.
Before returning the head of the fetus to the uterus, an occluder was placed around
the brachiocephalic artery. These silastic occluders resemble miniature blood pressure
cuffs and were purchased from In Vivo Metric (Cat. # OC8, Healdsburg, CA). The left
forelimb of the fetus was delivered through a uterine incision. An incision was made into
the second intercostal space and the brachiocephalic artery was located. Once the
occluder was sewn in place, the incision under the left forearm was closed and the fetus
returned to the uterus. The uterine incision was closed once again using a locking simple
continuous suture pattern followed by an umbrication technique. 750 mg ampicillin
(Polyflex®, Ft. Dodge Laboratories, Ft. Dodge, IA) was administered into the amniotic
/
cavity before closure of the maternal linea alba and skin.
All catheters were filled with heparin (1000 units/ml, Elkins-Sinn, Inc., Cherry Hill,
NJ) and closed with a sterile brad inserted into the end. Catheters and occluders were
flanked and exteriorized via a trochar Catheters were held in place with an elastic
bandage. The linea alba was closed with #3 polyamide suture (Pitmann-Moore, France)
while the skin was closed with #1. 750 mg ampicillin was administered intramuscularly to
the ewe.
It should be noted that in ewes with twin pregnancies, both fetuses were surgically
manipulated the same way. At the time of experimentation however, only one of the

42
animals was made hypotensive while the other animal served as a control. All ewes were
treated with 750 mg ampicillin twice per day for five days post-operatively. In addition,
rectal temperatures were taken to monitor for infection. All ewes were monitored closely
for any indication of poor health.
In Vivo Experimental Procedures
All ewes were given five days to recover from surgery. On the day of
experimentation, catheters were removed from the elastic bandage and the distal ends
were cleaned with povidone iodine and alcohol. Each brad was removed and a sterile
blunt adapter with a three-way stopcock was inserted. This procedure was always done
for both femoral catheters and the amniotic catheter. Lingual catheters were only utilized
if the fetus was to be made hypotensive. All catheters to be used were flushed with
heparinized saline (2.0% v/v). One femoral catheter and the amniotic catheter were
attached to transducers (Statham P23Id, Statham Instruments, Oxnard, CA) for
measurement of fetal arterial and amniotic fluid pressure. As stated previously, if the fetus
r
was made hypotensive lingual pressure was also monitored to assure proper occlusion.
Arterial and amniotic fluid pressures were measured for the first 35 minutes of the
experiment using a Grass Model 7 recorder. The data were digitized and stored using an
IBM AT Microcomputer and a Keithley analog-to-digital converter on-line.
All experiments were performed between 120 and 135 days gestation to minimize
variation in hormone concentrations between experiments and animals. All animals were
studied in their pens utilizing six sections of PVC tubing to limit movement of the ewe.
Once catheters were removed from the elastic bandage ewes were not touched in order to
limit the amount of external stress placed upon the animals. Experiments lasted one hour

43
with blood samples taken at 0, 10, 20, and 60 minute time points. If the fetus was to be
made hypotensive to activate the HPA axis, the brachiocephalic occluder was inflated after
collection of the 0 time point sample for 10 minutes. The brachiocephalic occluder was
inflated via an infusion of saline through the silastic tubing. This, in turn, causes a
hypoperfusion of blood to the fetal brain, which activates the HPA axis. Five ml of blood
were taken at each time point and collected in chilled tubes containing N&, EDTA (50 fig
EDTA/ml blood, Sigma Chemical Co., St. Louis, MO). An additional 1.5 ml of blood was
drawn anaerobically into syringes coated with heparin for measurement of fetal blood
gases using a Ciba-Coming 288 Blood Gas System. A small portion of this blood was
used to measure hematocrit using an IEC microhematocrit centrifuge. After sampling,
volume of the catheter was restored with 0.9% normal saline with 2.0% v/v heparin.
Blood samples (5 ml) were kept on ice until further analysis for hormone levels.
Samples were centrifuged at 3000 x g for 30 minutes at 4° C in a refrigerated centrifuge
/
(Sorvall RT 600B, DuPont, Newtown, CA). After centrifugation, the plasma was
transferred and aliquotted to polystyrene tubes and stored at -20° C until hormones were
assayed.
Upon conclusion of each experiment, ewes were sacrificed with an overdose of
sodium pentobarbital via the jugular vein. Fetuses were immediately removed for
perfusion of the brain. The chest cavity of each fetus was opened and the brachiocephalic
artery was located and cannulated. Either by means of a pump or by syringe, brains were
perfused first with one liter of phosphate buffered saline (pH 7.4, 2.0% v/v heparin)
followed by two liters of 4% paraformaldehyde. Brains and pituitary glands were
removed and stored in 4% paraformaldehyde until processing for immunohistochemistry.

44
Peptide Assays
Arginine Vasopressin (AVP) Plasma AVP concentrations were measured using an
antibody raised in rabbits (Raff et al., 1991). Iodinated AVP was purchased from
Amersham and synthetic AVP from Sigma Chemical Co. AVP was first extracted from
0.5 ml plasma with 1.0 ml bentonite slurry (0.3% w/v in distilled water) and acidified with
0.05 ml 1 N HC1. Extracts were eluted with 1 ml acid:acetone (20% IN HCl.80%
acetone) with sonication. Samples were then evaporated to dryness and stored at -20° C
until assayed. Extracts were reconstituted with 0.25 ml assay buffer (0.05 M phosphate
buffer, pH 7.4 with 0.01 M EDTA (Sigma, #ED4SS) and 0.2% BSA w/v (Sigma, #A-
7638)). Extraction recovery was corrected by comparing samples to a standard curve
prepared from standard extracted with each set of samples.
Adrenocorticotropin (ACTH) Plasma ACTH concentrations were measured by
radioimmunoassay (RIA) as previously described (Bell et al., 1991) using an antibody
raised in rabbits developed in Dr. Wood’s laboratory to human-ACTH (1-24). Iodinated
ACTH (I125 ACTH) was prepared using the chloramine-T method (Berson and Yalow,
1968) with human-ACTH (l-39)(Sigma Chemical Co., St. Louis, MO) and radioactive
sodium iodide (Amersham, Arlington Heights, IL). ). I125 ACTH was made fresh
approximately every six weeks. ACTH was first extracted from plasma before assaying.
ACTH (0.5 ml) was extracted on glass (35 mg/tube) (100-200 mesh glass, Coming
Glassworks, Coming, NY) in 0.50 ml assay buffer (0.05 M phosphate buffer, pH 7.4 with
0.2% w/v silicic acid-extracted bovine serum albumin (BSA, Sigma Chemical Co.,
#9647)). The supernatant was aspirated and the glass washed with assay buffer. The
ACTH was eluted from the glass with 1 ml acid:acetone (1 volume 0.25 N HC1.1 volume

45
acetone). The extracts were dried under vacuum (Savant Instruments, Farmingdale, NY)
and frozen at -20° C until assayed Extracts were reconstituted overnight in 0.5 ml assay
buffer containing 0.5% v/v mercaptoethanol. Extraction recovery was corrected by
comparing samples to a standard curve prepared from standard extracted with each set of
samples.
Steroid Assays
Cortisol Plasma cortisol concentrations were measured as previously described (Wood et
al., 1993) using an antibody raised in rabbits and titrated cortisol purchased from
Amersham (#TRK-407) and cortisol standard from Sigma Chemical Co. Cortisol was
extracted from 20 pi plasma in duplicate with 1 ml ethanol. Standard was prepared in
ethanol, and standards and samples were dried under vacuum with heat and immediately
reconstituted with 0.5 ml assay buffer (0.05 M phosphate buffer (using 0.06 M sodium
phosphate dibasic and 0.04 M sodium phosphate monobasic) pH 7.0 with 0.15 M NaCl,
0.1% w/v gelatin, and 0.1% w/v sodium azide).
Estradiol Plasma estradiol concentrations were measured utilizing an enzyme
immunoassay (EIA) kit from Oxford Biochemical Inc. (#EA70). 2 ml of plasma was
extracted with 16 ml ethyl ether. Extracts were dried under air and reconstituted to 200
M-l with assay buffer (provided with kit). 50 pi of extract was assayed for estradiol in
duplicate. The values obtained were divided by 10 to give ng/ml concentrations. The
antiserum in this particular EIA kit had a very low cross-reactivity with estrone (0.10%).
Estrone Sulfatase Activity
I studied fetuses (86-147 days gestation, term=147 days), 4 lambs (3-4 weeks old),
and 4 adult nonpregnant ewes to determine estrone sulfatase activity. The sheep were

46
sacrificed using an intravenous overdose of sodium pentobarbital. Gestational ages of the
fetal sheep were calculated from known breeding dates. Whole brains were rapidly
removed, dissected into discrete regions, and quickly frozen on dry ice or in a slurry of dry
ice and acetone. All tissues were stored at -20°C or -40°C until studied.
Hypothalami, brainstems, and hippocampi were then processed to determine
estrone sulfatase activity. Each tissue sample was homogenized in medium 199 (Sigma®,
St. Louis, MO) containing 25 mM HEPES. Homogenization was performed using a
Polytron homogenizer (Tekmar, Cincinnati, OH). The concentration of each tissue in the
homogenate was 0.5 g tissue in 5 mL medium.
Tissues were centrifuged at 1200 rpm for 5 min; supernatant was then collected
and assayed immediately. A sample of each homogenate was assayed for protein
concentration using the method of Bradford (1976) using a commercially-available assay
kit (Bio-Rad Laboratories, Hercules, CA). Homogenate (0.1 mL) was aliquotted in
r
duplicate into borosilicate tubes (16 x 150 mm) containing 0.8 mL of a mixture of 3H-
[6,7]-estrone sulfate (DuPont-NEN, Wilmington, DE) and unlabeled estrone sulfate
(E1SO4, Sigma®, St. Louis, MO). All reactions were run at 37°C. Reactions were
terminated by immediate cooling on ice, addition of 5 volumes of ethyl acetate:hexane
(3:2), and vigorous mixing for 30 seconds. The aqueous phase was frozen by submersion
of the reaction tube into a dry ice and acetone slurry. Subsequently, the organic phase
containing the 3H-estrone was decanted into 13x75 mm borosilicate glass tubes and dried
under a gentle stream of room air. Dried extracts were reconstituted in scintillant
(Cytoscint®, ICN Corp., Costa Mesa, CA) and counted in a scintillation counter (LKB
Corp., Gaithersburg, MD).

47
Enzyme activities at different developmental ages and in different tissues were
measured using a substrate concentration of 3 pM and 3H-estrone sulfate specific activity
of approximately 0.67 pCi/nmol. For this experiment, reactions were allowed to run for 5
min. Using these conditions, less than 20% of the substrate was converted to 3H-estrone.
Western Blotting
Hypothalami and brainstems were harvested from fetuses, lambs, and adults of
known gestational and postnatal ages. The number and ages of animals varied slightly
between hypothalami and brainstem but 11-12 fetuses, 3-4 lambs and 2 adults were used
per tissue type. These tissues were originally obtained and homogenized for other studies
(Saoud and Wood, 1996). Unfortunately, hippocampi from these animals were not
available. All tissue was homogenized in reducing buffer and boiled for 5 minutes. The
samples were centrifuged to remove particulate matter and supernatant was recovered.
Protein concentrations were obtained utilizing the Bradford technique (1976). Western
blots were performed using a mini-Protean electrophoresis system (Bio-Rad, Hercules,
CA) on 10% pre-cast polyacrylamide gels purchased from Bio-Rad laboratories. Samples
were diluted so that an equal amount of protein was loaded per lane (20 pg for brainstem
and 40 pg for hypothalami). The protein was then transferred to a nitrocellulose
membrane and probed for either estrogen sulfatase or estrogen sulfotransferase using
custom-made rabbit polyclonal antibodies (Alpha Diagnostic, San Antonio, TX). The
peptide sequence used from the human sulfatase gene, amino acids 294-309, was NH2-
FSSKDFAGKSQHGVYGC-COOH (Simerly et al., 1990). The peptide sequence used
from the bovine sulfotransferase gene, amino acids 273-295, was NH2-
RERFEEHYQQQMKDC-COOH (Nash et al., 1988). Primary antibodies were diluted to

48
a concentration of 1:1000 in antibody diluent (1 % BSA in phosphate buffered saline with
0.05 % Tween 20). Visualization of the protein-antibody complex was accomplished
utilizing a chemiluminescence detection system (Renaissance, DuPont NEN, Boston, MA)
and analyzed by densitometry (Bio-Rad). Antibody specificity was confirmed by
preabsorption of the primary antibodies with peptides (1 pg/ml) also supplied by Alpha
Diagnostic. Developmental changes were calculated using multiple linear regression in
order to control for differences between gel running conditions (SigmaStat, Jandel
Scientific, San Rafael, CA).
Immunohistochemical Techniques
Fetal brains were perfusion fixed with 4% paraformaldehyde, dissected and cut
into gross tissue regions (hypothalamus, midbrain, pons, medulla, spinal cord, etc.).
Tissue was processed for embedding by dehydration with progressively increasing
concentrations of ethanol, followed by xylene. All tissue was embedded in paraffin and
cut into 10 pm sections using a Zeiss microtome. Sections were mounted on poly-L-
lysine slides, deparaffinized with xylene and rehydrated in decreasing concentrations of
ethanol. Immunohistochemistry and visualization were made possible utilizing a
Histostain-SP kit from Zymed and metal-enhanced DAB (Pierce, Rockford, EL). Sections
were stained for estrogen sulfatase, estrogen sulfotransferase, c-fos, ACTH, AVP, and
CRH (see Table 3.1). Primary antibodies were diluted in antibody diluent (1% BSA in
phosphate buffered saline with 0.01% Triton X-100). Specific staining was confirmed by
dilution tests, as staining decreased as primary antibodies were further diluted. Specific
staining was absent upon replacing primary antibodies with 10% normal goat serum. All

49
slides were dehydrated prior to mounting of coverslips with Permount (Fisher Scientific,
Pittsburgh, PA).
Primary antibody
Vendor (Source)
Dilution
Estrogen Sulfatase
Alpha Diagnostics (same as
for Western Blots)
1:500
Estrogen Sulfotransferase
Alpha Diagnostics (same as
for Western Blots)
1:500
c-fos
Oncogene Biomedical Inc.
(cat. PC38)
1:5000
ACTH
Wood Laboratory (same as
for RIA)
1:10,000
AVP
Wood Laboratory (same as
for RIA)
1:20,000
CRH
Keller-Wood Laboratory
1:100,000
Table 3.1: Primary antibodies used in immunohistochemical experiments. All antibodies
were diluted in 1% BSA in phosphate buffered saline with 0.01% Triton X-100.

CHAPTER 4
HORMONAL RESPONSIVENESS IN AN ESTRADIOL, HYPOTENSIVE, CAROTID
SINUS DENERVATED OVINE MODEL
Introduction
In the fetal sheep, parturition is triggered by an increase in the activity of the fetal
HPA axis (Challis and Brooks, 1989; Liggins et al., 1973). Parturition can be delayed by
destruction of the pituitary (Liggins et al., 1966, 1967; Liggins and Kennedy, 1968) or
stimulated by infusions of ACTH (Liggins, 1968, 1969) or glucocorticoids (Jack et al.,
1975; Wood and Keller-Wood, 1991). The last few days of gestation are marked by an
increase in the activity of the fetal hypothalamus as seen by elevated levels of fetal plasma
ACTH. This increase in plasma ACTH causes a corresponding increase in plasma
cortisol.. Along with this increase in HPA axis activity is a decreased sensitivity of the axis
to cortisol negative feedback (Wood, 1988). It is well known that in the sheep, cortisol
acts at the placenta to increase the activity of an enzyme, cytochrome p450 (17-
hydroxylase and 17,20 lyase activities), which in turn, increases the ratio of estrogen to
progesterone (Anderson et al., 1975; Pomeranz and Nalbandov, 1975; Steele et al., 1976;
Yu et al., 1983). This cascade of events essentially increases the total amount of estrogen.
Estrogen is known to be an important factor in the initiation of parturition by
causing the uterus to contract (Liggins, 1974). 17(3-estradiol has been shown to increase
the activity of the HPA axis in sheep and rats. In a study by Saoud and Wood (1995),
50

51
estrogen was found to augment fetal plasma ACTH secretion in response to stress. Other
studies have shown similar results in adult animals (Viau and Meaney, 1991).
Understanding the mechanism of the increased fetal HPA axis at the end of
gestation is key to understanding the mechanism of spontaneous parturition in sheep.
These experiments were conducted to see if estradiol has it’s stimulatory effect on HPA
axis activity through the afferent baroreceptor and chemoreceptor pathway. More
specifically, I hypothesized that estradiol’s actions on fetal cardiovascular reflex
responsiveness to hypotension will be measurable in intact fetuses but not in baro- and
chemo- denervated fetuses. This augmentation of HPA axis activity will be assessed by
using a surgically manipulated ovine model. ACTH, AVP, estradiol, and cortisol levels
will be measured and compared across eight different treatment groups. These groups
include fetuses which are: (1) estradiol treated; (2) estradiol treated, hypotensive; (3)
estradiol treated, carotid sinus denervated; (4) estradiol treated, carotid sinus denervated,
hypotensive; (5) placebo treated; (6) placebo treated, hypotensive; (7) placebo treated,
/
carotid sinus denervated; (8) placebo treated, carotid sinus denervated, hypotensive.
Research has previously demonstrated that the combined baro- and chemo- denervation
attenuates (approximately 50%) the reflex hormonal and hemodynamic responses to
moderate (50 %) reduction in arterial blood pressure. I proposed that the interruption of
the afferent pathways would eliminate the effect of estrogen on the reflex cardiovascular
responsiveness. If so, it could concluded that estrogen acts on, within, or requires input
from, the afferent baroreceptor and chemoreceptor pathways.

52
Methods and Materials
Surgical Procedures Aseptic fetal surgery was performed in Animal Resources or at the
34th Street facility under general anesthesia with 0.5% - 2.0% halothane. All ewes were
between 115 and 125 days gestation at the time of surgery. A total of 40 ewes were set
up and studied (n=5 per group). Food and water were withheld from ewes 24 hours prior
to surgery. Ewes were sheared close to the skin around the abdomen and prepared for
surgery with povidone iodine (Betadine®, Purdue Fredrick Co., Norwalk, CT). Animals
were intubated and connected to a respirator to allow for constant anesthesia. Heart rate,
blood pressure, ventilatory 02 and C02, respirations per minute, and rectal temperature
were all monitored at the time of surgery. Animals were closely monitored from the time
of intubation until recovery when the animal could stand on its own effort. Ewes were
allowed free access to food and water throughout the post-operative period.
The uterus was exposed using a midline incision beginning at the umbilicus and
/
extending caudally approximately 10 cm. Once the hindlimbs were located, a small
incision was made in the uterus. Hindlimbs were delivered through the uterine incision
one at a time for the purpose of placing a polyvinyl chloride catheter (0.03” ID, 0.05” OD)
into each femoral artery. Later, these catheters would be used for blood sampling and
blood pressure recording. The tips of each femoral catheter were advanced to the
subdiaphragmatic aorta. At this time an estradiol implant (5 mg/21 days or 250 ng/day;
Innovative Research of America, Toledo OH) or placebo was inserted subcutaneously into
the area of the gluteous medius before suturing the incised hindlimbs. An amniotic
catheter made of polyvinyl chloride (0.05” ID, 0.09” OD) was sutured to the exterior of a
hindlimb for the purpose of antibiotic delivery as well as amniotic fluid pressure

53
measurements. Hindlimb and uterine incisions were closed using 2.0 silk suture. Hindlimb
incisions were closed using a simple continuous suture pattern. All uterine incisions were
closed first with a locking simple continuous pattern followed by umbrication of the
uterus.
Using a technique similar to the one described above, catheters were placed in the
lingual arteries and advanced approximately 1 cm into the carotid artery toward the heart.
Upon closure of the neck incision, lingual catheters were anchored to the chin of the fetus
with 2.0 silk suture. Lingual catheters were of the same material and size and femoral
catheters.
Depending upon the experimental setup, carotid sinus denervation was employed
at this time. After exposing the common carotid artery, denervation was accomplished by
stripping all nerves and connective tissue between the carotid-occipital arterial junction
and the carotid-lingual arterial junction. The occipital artery was ligated, for this is the
/
only method by which all of the carotid sinus baroreceptor and chemoreceptor afferent
fibers are cut. The lingual artery was stripped rostrally of all nerves and connective tissue
for approximately 1 cm from the carotid-lingual junction. This denervation was conducted
bilaterally.
Before returning the head of the fetus to the uterus, an occluder was placed around
the brachiocephalic artery. These silastic occluders resemble miniature blood pressure
cuffs and were purchased from In Vivo Metric (Cat. # OC8, Healdsburg, CA). The left
forelimb of the fetus was delivered through a uterine incision. An incision was made into
the second intercostal space and the brachiocephalic artery was located. Once the
occluder was sewn in place, the incision under the left forearm was closed and the fetus

54
returned to the uterus. The uterine incision was closed once again using a locking simple
continuous suture pattern followed by an umbrication technique. 750 mg ampicillin
(Polyflex®, Ft. Dodge Laboratories, Ft. Dodge, IA) was administered into the amniotic
cavity before closure of the maternal linea alba and skin.
All catheters were filled with heparin (1000 units/ml, Elkins-Sinn, Inc., Cherry Hill,
NJ) and closed with a sterile brad inserted into the end. Catheters and occluders were
flanked and exteriorized via a trochar. Catheters were held in place with an elastic
bandage. The linea alba was closed with #3 polyamide suture (Pitmann-Moore, France)
while the skin was closed with #1. 750 mg ampicillin was administered intramuscularly to
the ewe.
It should be noted that in ewes with twin pregnancies, both fetuses were surgically
manipulated the same way. At the time of experimentation however, only one of the
animals was made hypotensive while the other animal served as a control. All ewes were
treated with 750 mg ampicillin twice per day for five days post-operatively. In addition,
rectal temperatures were taken to monitor for infection. All ewes were monitored closely
for any indication of poor health.
In Vivo Experimental Procedures All ewes were given five days to recovery from
surgery. On the day of experimentation, catheters were removed from the elastic bandage
and the distal ends were cleaned with povidone iodine and alcohol. Each brad was
removed and a sterile blunt adapter with a three-way stopcock was inserted. This
procedure was always done for both femoral catheters and the amniotic catheter. Lingual
catheters were only utilized if the fetus was to be made hypotensive. All catheters to be
used were flushed with heparinized saline (2.0% v/v). One femoral catheter and the

55
amniotic catheter were attached to transducers (Statham P23Id, Statham Instruments,
Oxnard, CA) for measurement of fetal arterial and amniotic fluid pressure. There were
eight different experimental groups in this study: (1) estradiol treated; (2) estradiol
treated, hypotensive; (3) estradiol treated, carotid sinus denervated; (4) estradiol treated,
carotid sinus denervated, hypotensive; (5) placebo treated; (6) placebo treated,
hypotensive; (7) placebo treated, carotid sinus denervated; (8) placebo treated, carotid
sinus denervated, hypotensive. As stated previously, if the fetus was made hypotensive
lingual pressure was also monitored to assure proper occlusion. Arterial and amniotic
fluid pressures were measured for the first 35 minutes of the experiment using a Grass
Model 7 recorder. The data were digitized and stored using an IBM AT Microcomputer
and a Keithley analog-to-digital converter on-line.
All experiments were performed between 120 and 135 days gestation to minimize
variation in hormone concentrations between experiments and animals.- All animals were
f
studied in their pens utilizing six sections of PVC tubing to limit movement of the ewe.
Once catheters were removed from the elastic bandage ewes were not touched in order to
limit the amount of external stress placed upon the animals. Experiments lasted one hour
with blood samples taken at 0, 10, 20, and 60 minute time points. If the fetus was to be
made hypotensive to activate the HPA axis, the brachiocephalic occluder was inflated after
collection of the 0 time point sample for 10 minutes. The brachiocephalic occluder was
inflated via an infusion of saline through the silastic tubing. This, in turn, causes a
hypoperfusion of blood to the fetal brain, which activates the HPA axis. Five ml of blood
were taken at each time point and collected in chilled tubes containing Na< EDTA (50 p.g
EDTA/ml blood, Sigma Chemical Co., St. Louis, MO). An additional 1.5 ml of blood was

56
drawn anaerobically into syringes coated with heparin for measurement of fetal blood
gases using a Ciba-Coming 288 Blood Gas System. A small portion of this blood was
used to measure hematocrit using an IEC microhematocrit centrifuge. After sampling,
blood volume was restored with 0.9% normal saline with 2.0% v/v heparin.
Blood samples (5 ml) were kept on ice until further processing. Samples were
centrifuged at 3000 x g for 30 minutes at 4° C in a refrigerated centrifuge (Sorvall RT
600B, DuPont, Newtown, CA). After centrifugation, the plasma was transferred and
aliquotted to polystyrene tubes and stored at -20° C until hormones were assayed.
Upon conclusion of each experiment, ewes were sacrificed with an overdose of
sodium pentobarbital via the jugular vein. Fetuses were immediately removed for
perfusion of the brain. The chest cavity of each fetus was opened up and the
brachiocephalic artery was located and cannulated. Either by means of a pump or by
syringe,, brains were perfused first with one liter of phosphate buffered saline (pH 7.4,
f
2.0% v/v heparin) followed by two liters of 4% paraformaldehyde. Brains and pituitary
glands were removed and stored in 4% paraformaldehyde until processing for
immunohistochemistry (Chapter 5). The experimental design can be viewed pictorially in
Figure 4.1.
Estradiol Assay Plasma estradiol concentrations were measured utilizing an enzyme
immunoassay (EIA) kit from Oxford Biochemical Inc. (#EA70). 2 ml of plasma was
extracted with 16 ml ethyl ether. Extracts were dried under air and reconstituted to 200
pi with assay buffer (provided with kit). 50 pi of extract was assayed for estradiol in
duplicate. The values obtained were divided by 10 to give ng/ml concentrations. This
particular EIA kit had a very low cross-reactivity with estrone (0.10%).

57
Adrenocorticotropin (ACTH) Assay Plasma ACTH concentrations were measured by
radioimmunoassay (RIA) as previously described (Bell et al., 1991) using an antibody
raised in rabbits developed in Dr. Wood’s laboratory to human-ACTH (1-24). Iodinated
ACTH (I125 ACTH) was prepared using the chloramine-T method (Berson and Yalow,
1968) with human-ACTH (l-39)(Sigma Chemical Co., St. Louis, MO) and radioactive
sodium iodide (Amersham, Arlington Heights, IL). ). I125 ACTH was made fresh
approximately every six weeks. ACTH was first extracted from plasma before assaying.
ACTH (0.5 ml) was extracted on glass (35 mg/tube) (100-200 mesh glass, Coming
Glassworks, Coming, NY) in 0.50 ml assay buffer (0.05 M phosphate buffer, pH 7.4 with
0.2% w/v silicic acid-extracted bovine serum albumin (BSA, Sigma Chemical Co.,
#9647)). The supernatant was aspirated and the glass washed with assay buffer. The
ACTH was eluted from the glass with 1 ml acid:acetone (1 volume 0.25 N HC1:1 volume
acetone). The extracts were dried under vacuum (Savant Instruments,'Farmingdale, NY)
f
and frozen at -20° C until assayed. Extracts were reconstituted overnight in 0.5 ml assay
buffer containing 0.5% v/v mercaptoethanol. Extraction recovery was corrected by
comparing samples to a standard curve prepared from standard extracted with each set of
samples.
Arginine Vasopressin (AVP) Assay Plasma AVP concentrations were measured using an
antibody raised in rabbits (Raff et al., 1991). Iodinated AVP was purchased from
Amersham and synthetic AVP from Sigma Chemical Co. AVP was first extracted from
0.5 ml plasma with 1.0 ml bentonite slurry (0.3% w/v in distilled water) and acidified with
0.05 ml 1 N HC1. Extracts were eluted with 1 ml acid:acetone (20% IN HC1:80%
acetone) with sonication. Samples were then evaporated to dryness and stored at -20° C

58
until assayed. Extracts were reconstituted with 0.25 ml assay buffer (0.05 M phosphate
buffer, pH 7.4 with 0.01 M EDTA (Sigma, #ED4SS) and 0.2% BSA w/v (Sigma, #A-
7638)). Extraction recovery was corrected by comparing samples to a standard curve
prepared from standard extracted with each set of samples.
Cortisol Assay Plasma cortisol concentrations were measured as previously described
(Wood et al., 1993) using an antibody raised in rabbits and titrated cortisol purchased
from Amersham (#TRK-407) and cortisol standard from Sigma Chemical Co. Cortisol
was extracted from 20 pi plasma in duplicate with 1 ml ethanol. Standard was prepared in
ethanol, and standards and samples were dried under vacuum with heat and immediately
reconstituted with 0.5 ml assay buffer (0.05 M phosphate buffer (using 0.06 M sodium
phosphate dibasic and 0.04 M sodium phosphate monobasic) pH 7.0 with 0.15 M NaCl,
0.1% w/v gelatin, and 0.1% w/v sodium azide).
Statistical Analyses ACTH, AVP, and cortisol levels were analyzed via three way
/
ANOVA. Estradiol treated fetuses were analyzed separately from placebo treated fetuses
by this means using hypotension/normotension, CSD/intact, and time as factors. Student
Newman-Keuls Test was employed as a multiple comparison procedure for statistically
significant groups within each grouping. Estradiol treated fetuses were compared to
placebo treated fetuses relative to time and treatment group by a t-test. Estradiol level
comparison between estradiol treated fetuses and placebo treated fetuses was made using
a t-test.
Results
Estradiol Assay Since my hypotheses depend upon the a difference in estradiol levels
between treatment groups, it seems only logical to discuss the results of the estradiol assay

59
first. There were a total of 40 animals studied in this experiment. Half (20) were
pretreated with an estradiol implant during surgery. These implants release estradiol at a
constant rate of 5 mg/21 day period or around 250 pg/day. Estradiol levels were
measured by EIA and statistical analyses showed that the estradiol treated fetuses were
significantly different than the placebo treated fetuses by a t-test (n=20 per group,
p<0.001). Result can be seen in Figure 4.2 which shows group means measured in units
of pg/ml +SEM.
Adrenocorticotropin (ACTH) Assay Plasma ACTH levels are shown in Figure 4.3.
Results are plotted as group means +SEM. Placebo treated fetuses and estradiol fetuses
were analyzed separately by three way ANOVA with hypotension / normotension, carotid
sinus denervation / intact, and time (Omin., lOmin., 20min., and 60min.) as experimental
factors. In both cases (placebo and estradiol treatment), groups were found to be
statistically different (n=5 per group, p<0.001). Specifically, ACTH levels in the
r
hypotensive animals at 1 Omin. and 20min. as well as hypotensive, carotid sinus denervated
animals at lOmin. and 20min. were found to differ significantly from the rest of the
treatment groups when further analyzed by Student Newman Keuls Comparison (n=5 per
group, p<0.001). This was true for both placebo treated fetuses and estradiol treated
fetuses. Also, all placebo treated fetuses differed significantly from their corresponding
estradiol treated fetuses by t-test (n=5 per group, p<0.01).
Arginine Vasopressin (AVP) Assay Plasma AVP levels are shown in Figure 4.4. Results
are plotted as group means +SEM. Placebo treated fetuses and estradiol fetuses were
analyzed separately by three way ANOVA with hypotension / normotension, carotid sinus
denervation / intact, and time (Omin., lOmin., 20min., and 60min.) as experimental factors.

60
In both cases (placebo and estradiol treatment), groups were found to be statistically
different (n=5 per group, p<0.001). Specifically, AVP levels in the hypotensive animals at
lOmin. and 20min. as well as hypotensive, carotid sinus denervated animals at lOmin. and
20min. were found to differ significantly from the rest of the treatment groups when
further analyzed by Student Newman Keuls Comparison (n=5 per group, p<0.001). This
was true for both placebo treated fetuses and estradiol treated fetuses. Also, all placebo
treated fetuses differed significantly from their corresponding estradiol treated fetuses by
t-test (n=5 per group, p<0.01).
Cortisol Assay Plasma cortisol levels are shown in Figure 4.5. Results are plotted as
group means +SEM. Placebo treated fetuses and estradiol fetuses were analyzed
separately by three way ANOVA with hypotension / normotension, carotid sinus
denervation / intact, and time (Omin., lOmin., 20min., and 60min.) as experimental factors.
In both cases (placebo and estradiol treatment), groups were found to be statistically
/
different (n=5 per group, p<0.001). Specifically, cortisol levels in the hypotensive animals
at lOmin. and 20min. as well as hypotensive, carotid sinus denervated animals at lOmin.
and 20min. were found to differ significantly from the rest of the treatment groups when
further analyzed by Student Newman Keuls Comparison (n=5 per group, p<0.001). This
was true for both placebo treated fetuses and estradiol treated fetuses. Also, all placebo
treated fetuses differed significantly from their corresponding estradiol treated fetuses by
t-test (n=5 per group, p<0.01).
Discussion
Understanding the mechanism of the increased fetal HPA axis at the end of
gestation is key to understanding the mechanism of spontaneous parturition in sheep.

61
These experiments were conducted to see if estradiol has it’s stimulatory effect on HPA
axis activity through the afferent baroreceptor and chemoreceptor pathway. More
specifically, I hypothesized that estradiol’s actions on fetal cardiovascular reflex
responsiveness to hypotension will be measurable in intact fetuses but not in baro- and
chemo- denervated fetuses. This augmentation of HPA axis activity was assessed by using
a surgically manipulated ovine model. ACTH, AVP, estradiol, and cortisol levels were
measured and compared across eight different treatment groups. These groups include
fetuses which are: (1) estradiol treated; (2) estradiol treated, hypotensive; (3) estradiol
treated, carotid sinus denervated; (4) estradiol treated, carotid sinus denervated,
hypotensive; (5) placebo treated; (6) placebo treated, hypotensive; (7) placebo treated,
carotid sinus denervated; (8) placebo treated, carotid sinus denervated, hypotensive.
This study has shown that that estradiol does have an effect on cardiovascular
responsiveness. Figures 4.3-4 5 show that pretreatment with estradiol increases even
/
basal ACTH, AVP, and cortisol levels. This effect of estradiol is seen best perhaps after a
ten minute period of hypotension. At this point, hormonal responsiveness has been greatly
augmented with regard to ACTH, AVP, and cortisol levels. Carotid sinus denervation,
essentially eliminates this augmented HPA axis activity in response to brachiocephalic
occlusion. Any hormone response leftover after denervation may be due in part to
incomplete denervation at the time of surgery. Of course another explanation lies with the
fact that other pathways or systems might be responsible for hormone secretion. Carotid
sinus denervation by itself, however, caused no HPA axis activity or augmentation,
deeming the surgical elimination of baroreceptor / chemoreceptor responsiveness an
appropriate and effective control. Research has previously demonstrated that the

62
combined baro- and chemo- denervation attenuates (approximately 50%) the reflex
hormonal and hemodynamic responses to moderate (50 %) reduction in arterial blood
pressure. This study supports this finding demonstrated by the fact that ACTH, AVP, and
cortisol levels at lOmin. and 20min. time points in hypotensive, carotid sinus denervated
animals are approximately half their corresponding levels with hypotension alone. At the
very least, it can be concluded that estrogen acts on, within, or requires input from, the
afferent baroreceptor and chemoreceptor pathways.
This study utilized an artificial stimulus for HPA axis activation. This taken with
the fact that the animals in this study are at about 85% gestation makes direct comparison
to the time of parturition impossible. However, this study is useful in that it sheds light on
how an ovine fetus responds to boughts of hypotension. Furthermore, the effect of
estradiol on cardiovascular responsiveness has been shown to greatly augment HPA axis
activity jn response to such hypotension as controlled through the baroreceptor /
/
chemoreceptor pathway. These are important findings because although this ovine model
done not exactly mimic the process of parturition, important conclusions can be made. All
of the animals in this study are at least at 125 days gestation. Before around 120 days
gestation, the HPA axis is fairly unresponsive to stimuli. The estradiol implants used in
this study raised plasma estradiol levels to within a physiological range. Also, plasma
estradiol levels were comparable to time just prior to parturition, when estradiol levels are
thought to activate the HPA axis. For these reasons, it is evident that the model system
used in this study is appropriate for deciphering not only fetal responsiveness to
cardiovascular stimuli, but also the mechanism of parturition itself.

63
Though the results of this study elude to an estradiol responsive baroreceptor /
chemoreceptor afferent pathway participation in cardiovascular reflex control and trigger
for parturition, they do not by themselves answer account for total control over these
processes. As stated before, research has previously demonstrated that the combined
baro- and chemo- denervation attenuates (approximately 50%) the reflex hormonal and
hemodynamic responses to moderate (50 %) reduction in arterial blood pressure. This
means that 50% of the mechanism / pathway responsible for cardiovascular reflex
responsiveness and parturition is unaccounted for. It is plausible that the remaining
compensatory mechanisms exist within the central nervous system itself. Since the
cerebral circulation of the fetus exhibits autoregulation, one could hypothesize that
hypotension would cause reduced cerebral blood flow. This reduced flow may be
defended against by a local release of vasoactive substances. Prostanoids represent one
such group of compounds that may if fact be responsible for this response. Although it
t
has not been quantified at this time, our lab has shown that estradiol treatment does in fact
increase prostaglandin E2 (PGE2) within areas of the brain that are responsible for HPA
axis control. Other studies have eluded to a role of thromboxane A2 (TxA2) in this
process. TxA2 acts within the area perfused by the cerebral vasculature to stimulate
ACTH secretion in the fetus (Wood et al., 1993), which suggests that local generation of
TxA2 would effectively stimulate the HPA axis. Whatever the mechanism, it is obvious
that further research needs to be done in order to fully understand the inner-workings of
this complex system.

64
Blood sampling time points
III I
Omin.
1 Omin. 20
min.
60
min.
Hypotension
(Brachiocephalic Occlusion)
I
Euthanize and
perfuse brain
(Prepare for
Immunohistochemistry)
Figure 4.1: Experimental design for in vivo studies. 5ml blood samples are taken at
Omin., lOmin., 20min., and 60min.(additional blood is taken at Omin., lOmin., and
20min. for blood gas and hematocrit measurement). Hypotension via brachiocephalic
occlusion takes place between Omin. and lOmin. Blood pressure and heart rate are
recorded for the first 35min. of the experiment. Animals are sacrificed at 60min. and
brains are extracted for immunohistochemistry.

65
00
&
-a
c3
i~
i-i
c/i
w
Estradiol treated
fetuses
Placebo treated
fetuses
Figure 4.2: Estradiol levels +SEM for fetuses treated with a 5 mg/21 day estradiol
implant and fetuses treated with a placebo implant. The group means are
significantly different (n=20 per group, pO.OOl).

ACTH (pg/ml) ACTH (pg/ml)
Time (minutes)
Figure 4.3: ACTH plasma levels plotted as group means +SEM. Top graph
shows placebo treated fetuses and bottom graph shows estradiol treated fetuses.
*,+ denotes statistical significance (n=5 per group, p<0.001). All estradiol groups
were significantly different from placebo groups relative to treatment and time
(n=5 per group, p<0.01).
66

AVP (pg/ml) AVP (pg/ml)
67
Time (minutes)
Figure 4.4: AVP plasma levels plotted as group means +SEM. Top graph
shows placebo treated fetuses and bottom graph shows estradiol treated fetuses.
*,+ denotes statistical significance (n=5 per group, p<0.001). All estradiol groups
were significantly different from placebo groups relative to treatment and time
(n=5 per group, p<0.01).

Cortisol (ng/ml) Cortisol (ng/ml)
68
30
25
20
15
10
5
0
0 10 20 30 40 50 60
Estradiol treated fetuses
+ *
T I
Control
Hypotensive
CSD
Hypo/CSD
Time (minutes)
Figure 4.5: Cortisol plasma levels plotted as group means+SEM. Top graph
shows placebo treated fetuses and bottom graph shows estradiol treated fetuses.
*,+ denotes statistical significance (n=5 per group, p<0.001). All estradiol groups
were significantly different from placebo groups relative to treatment and time
(n=5 per group, p<0.01).

CHAPTER 5
NEURONAL ACTIVATION IN AN ESTRADIOL, HYPOTENSIVE, CAROTID
SINUS DENERVATED OVINE MODEL
Introduction
Parturition in the sheep has been shown to be controlled by the fetal HPA axis
(Challis and Brooks, 1989; Liggins et al., 1973). The last few days of gestation are
marked by an increase in the activity of the fetal hypothalamus as seen by elevated levels
of fetal plasma ACTH. This increase in plasma ACTH causes a corresponding increase in
plasma cortisol. Along with this increase in HPA axis activity is a decreased sensitivity of
the axis to cortisol negative feedback (Wood, 1988). It is well known that in the sheep,
cortisol acts at the placenta to increase the activity of an enzyme, cytochrome p450 (17-
hydroxylase and 17,20 lyase activities), which in turn, increases the ratio of estrogen to
/
progesterone (Anderson et al., 1975; Pomeranz and Nalbandov, 1975; Steele et al., 1976;
Yu et al., 1983). This cascade of events essentially increases the total amount of estrogen.
Estrogen is known to be an important factor in the initiation of parturition by
causing the uterus to contract (Liggins, 1974). 17P-estradiol has been shown to increase
the activity of the HPA axis in sheep and rats. In a study by Saoud and Wood (1995),
estrogen was found to augment fetal plasma ACTH secretion in response to stress. Other
studies have shown similar results in adult animals (Viau and Meaney, 1991).
Understanding the mechanism of the increased fetal HPA axis at the end of
gestation is key to understanding the mechanism of spontaneous parturition in sheep.
69

70
These experiments were conducted to see at what point in the HPA axis that estradiol has
in augmenting ACTH secretion. More specifically, I hypothesized that neuronal activity
will be highest in areas important for HPA axis control in estradiol treated, hypotensive
animals (see Chapter 4). I hypothesized that the baroreceptor / chemoreceptor afferent
pathway is involved, thus, carotid sinus denervation will eliminate the augmented HPA
axis activity. Neuronal activity was assessed by measuring the level of c-fos, any early
response gene, in brain areas important for HPA axis control. This method has been used
in numerous studies to assess neuronal activation due to physiological stress (Hoffman et
al., 1991; Shen et al., 1992; Chan et al., 1993).
Methods and Materials
Immunohistochemical Techniques Fetal ovine brains were perfusion fixed with 4%
paraformaldehyde, dissected and cut into gross tissue regions (hypothalamus, midbrain,
pons, medulla, spinal cord, etc.). These brains were the obtained from the experiments
discussed in Chapter 4. All animal were euthanized via an overdose of sodium
r
pentobarbital one hour after the beginning of the experiment (50 minutes after a ten
minute hypotensive or corresponding normotensive period). There were a total of 24 fetal
brains used for the histological experiments (n=3 per group). The fetal ovine groups were
as followed. (1) estradiol treated; (2) estradiol treated, hypotensive; (3) estradiol treated,
carotid sinus denervated; (4) estradiol treated, carotid sinus denervated, hypotensive; (5)
placebo treated; (6) placebo treated, hypotensive; (7) placebo treated, carotid sinus
denervated; (8) placebo treated, carotid sinus denervated, hypotensive. Tissue was
processed for embedding by dehydration with progressively increasing concentrations of
ethanol, followed by xylene. All tissue was embedded in paraffin and cut into 10 jam

71
sections using a Zeiss microtome. Sections were mounted on poly-L-lysine slides,
deparaffinized with xylene and rehydrated in decreasing concentrations of ethanol.
Immunohistochemistry and visualization were made possible utilizing a Histostain-SP kit
from Zymed and metal-enhanced DAB (Pierce, Rockford, IL). Sections were stained for
c-fos, ACTH, AVP, and CRH (see Table 3.1). Primary antibodies were diluted in
antibody diluent (1% BSA in phosphate buffered saline with 0.01% Triton X-100).
Specific staining was confirmed by dilution tests, as staining decreased as primary
antibodies were further diluted. Specific staining was absent upon replacing primary
antibodies with 10% normal goat serum. All slides were dehydrated prior to mounting
coverslips with Permount (Fisher Scientific, Pittsburgh, PA).
Fetal brains regions important in HPA axis control were measured for c-fos
generation by means of Microcomputer Imaging Device (MCID) from Imagining Research
Inc. Cerebellum and cortex were also measured to verify if c-fos activity was specific or
f
just a general activation of the central nervous system. The following brain regions were
analyzed: (1) paraventricular nucleus (PVN), (2) nucleus of the tractus solitarius (NTS),
(3) rostral ventral lateral medulla (RVLM), (4) hippocampus, (5) cerebellum, (6) cortex,
and (7) pituitary gland. For each of the eight treatment groups (listed in the Materials and
Methods section), n=3. As paraffin blocks were cut on the microtome, the fifth section
was used once the region of interest was identified. This assured homogeneity among
fetal brains.
All densitometry was performed in a similar mannor. The region of interest was
outlined designating the scanned area. The computer would then count the number of
cells stained positive depending upon the assigned criteria. This value or target number

72
was multiplied by the mean target area (calculated by the computer) to establish the total
target area. Finally, the total target area was divided by the scanned area to establish the
proportion of positive stained cells in each brain section. Values were then analyzed via
three way Analysis of Variance (ANOVA) followed by a multiple comparison procedure.
The multiple comparison procedure employed was Student-Newman-Keuls Method. All
statistics were run using Sigma Stat.
Results
PVN C-fos staining was found to be significantly different among the eight treatment
groups by three way ANOVA (n-3 per group, p<0.001). The mean densitometry values
+SEM are plotted in Figure 5.1. A further analysis of the data revealed statistically
significant interactions between all three factors (placebo vs. estradiol, hypotensive vs.
normotensive, and carotid sinus denervated vs. intact). Student Newman Keuls Method
revealed.the following significant differences among treatment groups: (1) mean values of
f
c-fos staining among the different levels of placebo treated fetuses vs. estradiol treated
fetuses was significantly different (n=3 per group, p<0.001), and (2) mean values of c-fos
staining among the different levels of hypotensive fetuses vs. normotensive fetuses was
significantly different (n=3 per group, p<0.001). Representative photomicrographs of the
PVN are shown in Figure 5.2. It can be seen visually that estradiol treated fetuses have
more c-fos generation in the PVN when compared to control fetuses (panels A and B).
Also shown is significantly more positive staining in estradiol treated, hypotensive fetuses
compared to placebo treated, hypotensive fetuses (panels C and D). As stated before
hypotensive animals also have more c-fos generation compared to normotensive animals
(panels A and C).

73
Carotid sinus denervation by itself did not cause significant c-fos generation. This
is show in Figure 5.3 which shows a representative estradiol treated fetus along with a
representative estradiol, carotid sinus denervated fetus. The region shown is the PVN and
visually, as well as statistically, there is no difference in the amount of staining. Figure 5.4
shows the effect of carotid sinus denervation on hypotension. The PVN of a
representative placebo, hypotensive fetus along with a representative placebo,
hypotensive, carotid sinus denervated fetus. Photomicrographs show that denervation
eliminates the effect of hypotension. These effects of carotid sinus denervation were true
of all brain regions studied.
Along with c-fos staining in the PVN, serial sections were also stained for AVP,
and CRF. Though statistics were not performed for the relative levels of these hormones,
the pattern of staining seemed to be consistent with that of c-fos. Not only was the
staining of AVP and CRF co-localized with c-fos, but also the levels of these hormones
/
seemed to increase with estradiol treatment as well as in hypotensive animals.
NTS C-fos staining was found to be significantly different among the eight treatment
groups by three way ANOVA (n-3 per group, p<0.001). The mean densitometry values
+SEM are plotted in Figure 5.5. A further analysis of the data revealed statistically
significant interactions between all three factors (placebo vs. estradiol, hypotensive vs.
normotensive, and carotid sinus denervated vs. intact). Student Newman Keuls Method
revealed the following significant differences among treatment groups. (1) mean values of
c-fos staining among the different levels of placebo treated fetuses vs. estradiol treated
fetuses was significantly different (n=3 per group, p<0.001), and (2) mean values of c-fos
staining among the different levels of hypotensive fetuses vs. normotensive fetuses was

74
significantly different (n=3 per group, p<0.001. Representative photomicrographs of the
NTS are shown in Figure 5.6. It can be seen visually that estradiol treated fetuses have
more c-fos generation in the NTS when compared to control fetuses (panels A and B).
Also shown is significantly more positive staining in estradiol treated, hypotensive fetuses
compared to placebo treated, hypotensive fetuses (panels C and D). As stated before
hypotensive animals also have more c-fos generation compared to normotensive animals
(panels A and C). These results coincide with those of the PVN.
RVLM C-fos staining was found to be significantly different among the eight treatment
groups by three way ANOVA (n-3 per group, p<0.001). The mean densitometry values
+SEM are plotted in Figure 5.7. A further analysis of the data revealed statistically
significant interactions between all three factors (placebo vs. estradiol, hypotensive vs.
normotensive, and carotid sinus denervated vs. intact). Student Newman Keuls Method
revealed the following significant differences among treatment groups: (1) mean values of
/
c-fos staining among the different levels of placebo treated fetuses vs. estradiol treated
fetuses was significantly different (n=3 per group, p<0.001), (2) mean values of c-fos
staining among the different levels of hypotensive fetuses vs. normotensive fetuses was
significantly different (n=3 per group, p<0.001. Representative photomicrographs of the
RVLM are shown in Figure 5.8. It can be seen visually that estradiol treated fetuses have
more c-fos generation in the RVLM when compared to control fetuses (panels A and B).
Also shown is significantly more positive staining in estradiol treated, hypotensive fetuses
compared to placebo treated, hypotensive fetuses (panels C and D). As stated before
hypotensive animals also have more c-fos generation compared to normotensive animals
(panels A and C). These results coincide with those of the PVN and the NTS.

75
Hippocampus C-fos staining was found to be significantly different among the eight
treatment groups by three way ANOVA (n-3 per group, p<0.001). The mean
densitometry values +SEM are plotted in Figure 5.9. Note that the y axis is smaller than
that of the PVN, etc. because the level of c-fos staining is lower in the hippocampus .
Student-Newman-Keuls Method revealed the that the mean values of c-fos staining among
the different levels of hypotensive fetuses vs. normotensive fetuses was significantly
different (n=3 per group, p<0.001). However, mean values of c-fos staining among the
different levels of placebo treated fetuses vs. estradiol treated fetuses was not significantly
different (n=3 per group, p=0.407). Representative photomicrographs of the
hippocampus are shown in Figure 5.10. It can be seen visually that hypotensive fetuses
have more c-fos generation in the hippocampus when compared to normotensive fetuses
(panels A and B). These results resemble those of the PVN, NTS, and RVLM with regard
to the effect of hypotension, however, the absence of an estradiol effect is novel.
/
Cerebellum Cerbellum was analyzed as a peripheral tissue that does not integration in the
system being studied. As suspected, analysis by three way ANOVA did not yield any
differences among treatment groups (n=3 per group, p=0.721). Mean group values
+SEM of the densitometry analysis are shown in Figure 5.11. Note that the y axis is much
smaller than that of the PVN, etc. because of the absence of any significant staining.
Representative photomicrographs of fetal ovine cerebellum are shown in Figure 5.12.
Panel A shows a control animal while panel B shows a hypotensive animal. Visually, as
well as statistically, there is no difference. Though not shown pictorially, the same is true
of estradiol treated fetuses vs. placebo treated fetuses.

76
Cortex As the case with cerebellum, cortex was analyzed as a peripheral tissue that does
not integration in the system being studied. As suspected, analysis by three way ANOVA
did not yield any differences among treatment groups (n=3 per group, p=0.399). Mean
group values +SEM of the densitometry analysis are shown in Figure 5.13. Note that the
y axis is much smaller than that of the PVN, etc. because of the absence of any significant
staining. Representative photomicrographs of fetal ovine cortex are shown in Figure 5.14.
Panel A shows a control animal while panel B shows a hypotensive animal. Visually, as
well as statistically, there is no difference. Though not shown pictorially, the same is true
of estradiol treated fetuses vs. placebo treated fetuses.
Pituitary gland Pituitary glands were stained for c-fos as well as ACTH and AVP.
Though statistics were not performed for the relative levels of these hormones, the pattern
of staining seemed to be consistent with that of the PVN, NTS, and RVLM. Not only was
the c-fos increased with estradiol treatment and hypotension, but the levels of ACTH and
r
AVP seemed to mimic the pattern and level of c-fos staining. ACTH was seen in the
anterior pituitary in and increased with both estradiol treatment and hypotension. AVP
was seen mostly in the posterior pituitary in areas of increased c-fos generation.
Discussion
Understanding the mechanism of the increased fetal HPA axis at the end of
gestation is key to understanding the mechanism of spontaneous parturition in sheep.
These experiments were conducted to see at what point in the HPA axis that estradiol has
in augmenting ACTH secretion. More specifically, I hypothesized that neuronal activity
will be highest in areas important for HPA axis control in estradiol treated, hypotensive
animals (see Chapter 4). I hypothesized that the baroreceptor / chemoreceptor afferent

77
pathway is involved, thus, carotid sinus denervation will eliminate the augmented HPA
axis activity. Neuronal activity was assessed by measuring the level of c-fos, any early
response gene, in brain areas important for HPA axis control. This method has been used
in numerous studies to assess neuronal activation due to physiological stress (Hoffman et
al., 1991; Shen et al., 1992; Chan et al., 1993).
The results of this study support to my original hypothesis. Estradiol augments
HPA axis activity through baroreceptor / chemoreceptor pathways and this augmentation
seems to be within brain regions important for HPA axis activity. Furthermore, the
elimination of increased neuronal activity in carotid sinus denervated animals shows that
the pathway involved is probably this central cardiovascular reflex pathway.
The immunohistochemistry results show that the PVN, NTS, and RVLM respond
similarly with regard to specific treatments. This finding comes as no surprise as all of
these areas are involved in HPA axis control. The PVN is the main collection of neuronal
r
cell bodies in the hypothalamus where CRH and AVP are synthesized and released to act
at the anterior pituitary to cause ACTH release (Lehman et al., 1993; Pomerantz and
Sholl, 1987). The NTS is the first synapse point in the pathway connecting the afferent
baroreceptors with the PVN. The RVLM is a cardiovascular regulatory center which
coordinates information from the periphery. It has been shown that all of these brain
regions have estrogen receptors, enabling them to respond to estradiol treatment prior to
brachiocephalic occlusion (Lehman et al., 1993; Simerly et al., 1990). Estradiol caused a
significant increase in neuronal activity in the PVN, NTS, and RVLM. In fetuses made
hypotensive via brachiocephalic occlusion, this effect was further augmented as shown by

78
statistical analyses Carotid sinus denervation eliminated increases in c-fos activity in these
HPA axis regulatory centers.
An interesting finding of this study was the absence of an estradiol effect in the
hippocampus. Like the PVN, NTS, and RVLM, an increase in c-fos activity was seen in
hypotensive animals and this effect was eliminated with carotid sinus denervation.
However, basal levels as well as stressed levels of neuronal activity in estradiol treated
animals compared to placebo treated animals did not differ statistically. This reveals that
estradiol has no effect at the level of the hippocampus in augmenting HPA axis activity in
response to hypotension. This is interesting because there have been estrogen receptors
reported within the hippocampus (Lehman et al., 1993; Simerly et al., 1990). Perhaps the
answer to this discrepancy lies in fact that the hippocampus is a classic site of cortisol
negative feedback. Wood has further shown that the HPA axis is insensitive to inhibition
via cortisol negative feedback towards the end of gestation (1987). Possibly, the
/
hippocampus is unresponsive to an estradiol augmentation of ACTH secretion at this time.
Of course, this may be in the best interest of the fetus since an increase in neuronal activity
at the level of the hippocampus may lead to inhibition rather than stimulation of the axis.
It was certainly encouraging that in peripheral brain regions, an increase in
neuronal activity was observed in neither estradiol treated nor estradiol treated,
hypotensive fetuses. This lack of c-fos staining in the cortex and cerebellum indicate that
the response to treatment within the CNS was specific. All things being equal, only brain
regions important in HPA axis control seemed to respond to estradiol treatment and
brachiocephalic occlusion.

79
Pituitary glands were stained for c-fos as well as ACTH and AVP Though
statistics were not performed for the relative levels of these hormones, the pattern of
staining seemed to be consistent with that of the PVN, NTS, and RVLM. Not only was
the c-fos increased with estradiol treatment and hypotension, but the levels of ACTH and
AVP seemed to mimic the pattern and level of c-fos staining. ACTH was seen in the
anterior pituitary in and increased with both estradiol treatment and hypotension. AVP
was seen mostly in the posterior pituitary in areas of increased c-fos generation. All of
this information fits with my hypothesis. One would expect to see an increase in staining
in these areas within the pituitary with both estradiol treatment and hypotension. There
are estrogen receptors within the pituitary and an increase in activity of the pituitary would
be logical during hypotension. It is possible that ACTH and AVP synthesis is increased
during estradiol treatment. This would also be true in the normal fetus towards the end of
gestation when estradiol is increasing. If these hormone levels are in fact increasing, The
r
fetus would be able to better respond to episodes of hypotension as well as trigger
parturition with an augmented ACTH and AVP response.
I have reported that within the PVN, NTS, RVLM, hippocampus, and pituitary of
fetal sheep, an in crease in c-fos activity is observed with estradiol treatment and
brachiocephalic occlusion. It may seem logical that rather than an increase in neuronal
activity, a decrease in c-fos staining at the level of the NTS would accompany
hypotension. Hypotension causes a decrease in the rate of firing of the baroreceptors, and
thus a decrease in signal at the NTS. Perhaps what is being observed here is a
chemoreceptor mediated effect. As the brachiocephalic artery is occluded, the blood
perfusing the head becomes acidotic and hypoxic. This is revealed through elevated levels

80
of hydrogen ions, elevated partial pressure of carbon dioxide (PC02), and a decrease in the
partial pressure pressure of oxygen (P02) in the blood. All of these factors were in fact
monitored throughout the duration of the experiment. I believe that the fetal P02 level is
strictly monitored in the carotid artery and in a state of hypotension, the compensatory
response is an increase in HPA axis activity in order to re-establish adequate blood flow to
the brain. This will have the inevitable response of bringing blood gas levels back to a
homeostatic level.
It is important to realize that the HPA axis is not only integral in controlling blood
pressure, but it is also the endocrine axis of parturition. This is a fact which has been
proven many times without actual confirmation of a precise mechanism. This study sheds
light on the internal working of this system. I have shown that an intact afferent
baroreceptor / chemoreceptor pathway is necessary for an ovine fetus to respond to a state
of hypotension. I have further shown that estradiol augments this response through this
t
cardiovascular reflex pathway. More precisely, I have shown through the utilization of
immunohistochemistry, the brain areas involved in this process. This study used an
artificial stimulus to increase HPA axis activity, however, it is not disputed that the
pathway described is in fact involved in triggering parturition.

Total Target Area / Scanned Area
81
Figure 5.1: C-fos immunohistochemistry staining in the fetal ovine paraventricular
nucleus (Plac= placebo implant, E2= estradiol implant, Hypo= hypotensive, CSD=
carotid sinus denervated). There is a significant difference between Plac vs. E2
(*, p<0.001) and Control vs. Hypo (+, p<0.001).

82
Figure 5.2: C-fos immunohistochemistry staining in the fetal ovine paraventricular
nucleus (A- Control; B- Estradiol treated; C- Hypotensive; D- Estradiol treated,
hypotensive). All photomicrographs are at a magnification of 40X.

83
Figure 5.3: Photomicrographs of the fetal ovine PVN comparing c-fos staining in an
intact estradiol treated fetus (A) and an estradiol treated, carotid sinus denervated
fetus (B). A and B were not statistically different showing that denervation alone did
not cause c-fos generation.

84
Figure 5.4: Photomicrographs of the fetal ovine PVN comparing c-fos staining in a
hypotensive fetus (A) and a hypotensive, carotid sinus denervated fetus (B). A and B
were statistically different (p<.001) showing that denervation diminished c-fos
generation in hypotensive animals.

Total Target Area / Scanned Area
85
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
Figure 5.5: C-fos immunohistochemistry staining in the fetal ovine nucleus
of the tractus solitarius (Plac= placebo implant, E2= estradiol implant, Hypo=
hypotensive, CSD= carotid sinus denervated). There is a significant difference
between Plac vs. E2 (*, p<0.001) and Control vs. Hypo (+, p<0.001).

Figure 5.6: C-fos immunohistochemistry staining in the fetal ovine nucleus of the
tractus solitarius (A- Control; B- Estradiol treated; C- Hypotensive; D- Estradiol
treated, hypotensive). All photomicrographs are at a magnification of 100X.

Total Target Area / Scanned Area
87
Figure 5.7: C-fos immunohistochemistry staining in the fetal ovine rostral
ventral lateral medulla (Plac= placebo implant, E2= estradiol implant, Hypo=
hypotensive, CSD= carotid sinus denervated). There is a significant difference
between Plac vs. E2 (*, p<0.001) and Control vs. Hypo (+, p<0.001).

88
Figure 5.8: C-fos immunohistochemistry staining in the fetal ovine rostral ventral
lateral medulla (A- Control; B- Estradiol treated; C- Hypotensive; D- Estradiol treated,
hypotensive). All photomicrographs are at a magnification of 100X.

Total Target Area / Scanned Area
89
0.040
0.035
0.030
0.025
0.020
0.015
0.010
0.005
0.000
Figure 5.9: C-fos immunohistochemistry staining in the fetal ovine hippocampus
(Plac= placebo implant, E2= estradiol implant, Hypo= hypotensive, CSD= carotid
sinus denervated). There is a significant difference between Control vs. Hypo
(+, p<0.001).

90
Figure 5.10: C-fos immunohistochemistry staining in the fetal ovine hippocampus
(A- Control; B- Hypotensive). All photomicrographs are at a magnification of 40X.

Total Target Area / Scanned Area
91
0.0020
0.0018
0.0016
0.0014
0.0012
0.0010
0.0008
0.0006
0.0004
0.0002
0.0000
Figure 5.11: C-fos immunohistochemistry staining in the fetal ovine cerebellum
(Plac= placebo implant, E2= estradiol implant, Hypo= hypotensive, CSD=
carotid sinus denervated).

92
Figure 5.12: C-fos immunohistochemistry staining in the fetal ovine cerebellum
(A- Control; B- Hypotensive). All photomicrographs are at a magnification of 40X.
A and B are not statistically different.

Total Target Area / Scanned Area
93
0.0020
0.0018
0.0016
0.0014
0.0012
0.0010
0.0008
0.0006
0.0004
0.0002
0.0000
Figure 5.13: C-fos immunohistochemistry staining in the fetal ovine cortex
(Plac= placebo implant, E2= estradiol implant, Hypo= hypotensive, CSD=
carotid sinus denervated).

94
Figure 5.14: C-fos immunohistochemistry staining in the fetal ovine cortex
(A- Control, B- Hypotensive). All photomicrographs are at a magnification of 40X.
A and B are not statistically different.

CHAPTER 6
ONTOGENY OF ESTROGEN SULFATASE AND ESTROGEN
SULFOTRANSFERASE IN BRAIN REGIONS IMPORTANT FOR
HYPOTHALAMUS-PITUITARY-ADRENAL AXIS CONTROL
Introduction
In ovine pregnancy, developmental changes in the synthesis of estrogens and
progesterone are important for the initiation, maintenance, and spontaneous termination of
the pregnancy. My interests have focused on the mechanisms controlling spontaneous
parturition. In the ovine fetus, parturition is initiated by an increase in the activity of the
fetal hypothalamus-pituitary-adrenal axis (Liggins et al., 1973). The resultant increase in
fetal plasma cortisol induces the activity of cytochrome P450cn in the placenta (Mason et
al., 1989). This enzyme has both 17-hydroxylase and 17,20 lyase activities; induction of
this enzyme allows proportionately more estrogen and proportionately less progesterone
biosynthesis. The increase in the so-called "estrogen-to-progesterone" (E/P) ratio in
plasma and locally (within the uterine tissues) allows increased uterine contractility
(Liggins et al., 1973). It is the increased uterine tone which initiates labor and delivery of
the fetus.
It has recently been demonstrated that physiological increases in fetal plasma
estrogen concentrations greatly augment fetal ACTH secretion (Saoud and Wood, 1996).
This effect of estrogen can be demonstrated on both basal and hypotension stimulated fetal
ACTH secretion. While estrogen has a potentially important effect on fetal ACTH
95

96
secretion, it is well-known that fetal plasma concentrations of unconjugated estrogens
increase only after the beginning of the increase in fetal plasma ACTH and cortisol
(Nathanielsz et al., 1982; Strott et al., 1974). Conjugated estrogens, mostly estrone
sulfate, circulate in high concentrations compared to unconjugated estrogens (Carnegie
and Robertson, 1978; Tsang, 1974). The concentration of estrone sulfate increases before
the increase in fetal HPA axis activity (Nathanielsz et al., 1982). However, conjugated
steroids cannot bind to nuclear receptors unless deconjugated (Hobkirk, 1985). It is
possible that estrone sulfate is converted to estrone locally within the fetal brain, and that
the circulating estrone sulfate acts as a reservoir for estrone acting within the fetal brain.
Conjugation and deconjugation of estrone are accomplished via the enzymes estrogen
sulfotransferase and estrogen sulfatase, respectively.
Estrogen sulfotransferase and sulfatase activity have been reported in brain tissue
from adult sheep (Lakshmi and Balasubramanian, 1979), rats (Connolly and Resko, 1989;
Kawano and Aikawa, 1987), primates (Lakshmi and Balasubramanian, 1979), and human
r
beings (Platia et al., 1984). Activity of these enzymes has not been investigated in fetal
sheep. I hypothesized that estrogen sulfotransferase and sulfatase activity could be
demonstrated in the hypothalamus, brainstem and hippocampus of fetal sheep, and that the
activity changes as a function of fetal gestational age. I further hypothesized that these
enzymes would be present throughout the final trimester of fetal development, as well as
in the postnatal animal, in brain regions relevant for HPA axis control. The experiments
conducted were designed to test these hypotheses.

97
Methods and Materials
Enzyme Activity I studied fetuses (86-147 days gestation, term=147 days), 4 lambs (3-4
weeks old), and 4 adult nonpregnant ewes to determine estrone sulfatase activity. The
sheep were sacrificed using an intravenous overdose of sodium pentobarbital. Gestational
ages of the fetal sheep were calculated from known breeding dates. Whole brains were
rapidly removed, dissected into discrete regions, and quickly frozen on dry ice or in a
slurry of dry ice and acetone. All tissues were stored at -20°C or -40°C until studied.
Hypothalami, brainstems, and hippocampi were then processed to determine
estrone sulfatase activity. Each tissue sample was homogenized in medium 199 (Sigma®,
St. Louis, MO) containing 25 mM HEPES. Homogenization was performed using a
Polytron homogenizer (Tekmar, Cincinnati, OH). The concentration of each tissue in the
homogenate was 0.5 g tissue in 5 mL medium.
Tissues were centrifuged at 1200 rpm for 5 min; supernatant was then collected
and assayed immediately. A sample of each homogenate was assayed for protein
concentration using the method of Bradford (1976) using a commercially-available assay
kit (Bio-Rad Laboratories, Hercules, CA). Homogenate (0.1 mL) was aliquotted in
duplicate into borosilicate tubes (16 x 150 mm) containing 0.8 mL of a mixture of 3H-
[6,7]-estrone sulfate (DuPont-NEN, Wilmington, DE) and unlabeled estrone sulfate
(E1SO4 , Sigma®, St. Louis, MO). All reactions were run at 37°C. Reactions were
terminated by immediate cooling on ice, addition of 5 volumes of ethyl acetate:hexane
(3:2), and vigorous mixing for 30 seconds. The aqueous phase was frozen by submersion
of the reaction tube into a dry ice and acetone slurry. Subsequently, the organic phase
containing the 3H-estrone was decanted into 13x75 mm borosilicate glass tubes and dried

98
under a gentle stream of room air. Dried extracts were reconstituted in scintillant
(Cytoscint®, ICN Corp., Costa Mesa, CA) and counted in a scintillation counter (LKB
Corp., Gaithersburg, MD).
Enzyme activities at different developmental ages and in different tissues were
measured using a substrate concentration of 3 pM and 3H-estrone sulfate specific activity
of approximately 0.67 pCi/nmol. For this experiment, reactions were allowed to run for 5
min. Using these conditions, less than 20% of the substrate was converted to 3H-estrone.
Comparison of relative activities at different developmental ages was achieved
using one-way analysis of variance (ANOVA). A posteriori comparison of individual
mean values was performed using Newman-Keuls multiple range test (Winer, 1971).
Comparison of two means was performed using Student's t-test (Zar, 1984). All statistical
computations were performed using SigmaStat (Jandel Scientific, San Rafael, CA).
Western Blotting Hypothalami and brainstems were harvested from fetuses, lambs, and
/â– 
adults of known gestational and postnatal ages. The number and ages of animals varied
slightly between hypothalami and brainstem but 11-12 fetuses, 3-4 lambs and 2 adults
were used per tissue type. These tissues were originally obtained and homogenized for
other studies (Saoud and Wood, 1996). Unfortunately, hippocampi from these animals
were not available. All tissue was homogenized in reducing buffer and boiled for 5
minutes. The samples were centrifuged to remove particulate matter and supernatant was
recovered. Protein concentrations were obtained utilizing the Bradford technique (1976).
Western blots were performed using a mini-Protean electrophoresis system (Bio-Rad,
Hercules, CA) on 10% pre-cast polyacrylamide gels purchased from Bio-Rad laboratories.
Samples were diluted so that an equal amount of protein was loaded per lane (20 pg for

99
brainstem and 40 jig for hypothalami). The protein was then transferred to a
nitrocellulose membrane and probed for either estrogen sulfatase or estrogen
sulfotransferase using custom-made rabbit polyclonal antibodies (Alpha Diagnostic, San
Antonio, TX). The peptide sequence used from the human sulfatase gene, amino acids
294-309, was NH2-FSSKDFAGKSQHGVYGC-COOH (Simerly et al., 1990). The
peptide sequence used from the bovine sulfotransferase gene, amino acids 273-295, was
NH2-RERFEEHYQQQMKDC-COOH (Nash et al., 1988). Primary antibodies were
diluted to a concentration of 1:1000 in antibody diluent (1 % BSA in phosphate buffered
saline with 0.05 % Tween 20). Visualization of the protein-antibody complex was
accomplished utilizing a chemiluminescence detection system (Renaissance, DuPont NEN,
Boston, MA) and analyzed by densitometry (Bio-Rad). Antibody specificity was
confirmed by preabsorption of the primary antibodies with peptides (1 pg/ml) also
supplied by Alpha Diagnostic. Developmental changes were calculated, using multiple
linear regression in order to control for differences between gel running conditions
(SigmaStat, Jandel Scientific, San Rafael, CA).
Immunohistochemistrv Fetal brains were perfusion fixed with 4% paraformaldehyde,
dissected and cut into gross tissue regions (hypothalamus, midbrain, pons, medulla, spinal
cord, etc.). Tissue was processed for embedding by dehydration with progressively
increasing concentrations of ethanol, followed by xylene. All tissue was embedded in
paraffin and cut into 10 |am sections using a Zeiss microtome. Sections were mounted on
poly-L-lysine slides, deparaffinized with xylene and rehydrated in decreasing
concentrations of ethanol. Immunohistochemistry and visualization were made possible
utilizing a Histostain-SP kit from Zymed and metal-enhanced DAB (Pierce, Rockford, IL).

100
Sections were stained for estrogen sulfatase and estrogen sulfotransferase using the same
custom made antibody used for western blotting. Primary antibodies were diluted to a
concentration of 1 500 in antibody diluent (1% BSA in phosphate buffered saline with
0.01% Triton X-100). Specific staining was confirmed by dilution tests, as staining
decreased as primary antibodies were further diluted. Specific staining was absent upon
replacing primary antibodies with 10% normal goat serum. All slides were dehydrated
prior to mounting of coverslips with Permount (Fisher Scientific, Pittsburgh, PA).
Results
Enzyme Activity Estrogen sulfatase activity was measurable in ovine fetal, neonatal, and
adult hypothalamus, hippocampus, and brainstem (Figure 6.1). The activity in the
hippocampus was significantly increased in late-gestation fetuses compared to younger
fetuses, lambs, and adult ewes as tested by ANOVA and Newman-Keuls multiple range
test (n=3-4 per group; p<0.01). The activity in hypothalamus appeared to decrease in
/
more mature animals, but this was not statistically significant possibly due in part to
insufficient numbers in each group. The activity in brainstem was highly variable, and
overall did not change as a function of age. High variability may have distorted the
statistical analysis of estrogen sulfatase within the brainstem.
As shown in Figure 6.2, the activity of the estrogen sulfatase within these brain
regions is very high, even when compared to ovine myometrium. When compared by
Student's t-test, the activity in ovine adult hypothalamus was significantly different than
the activity in ovine myometrium (n=3 per group; p<0.001).
Western Blotting In performing Western blots, tissues were distributed among gels so
that each gel contained a subset of the developmental ages studied. For brainstem, 11

101
fetuses, 3 lambs and 2 adults were used. The developmental ages were as follows: 90-110
days (n=3), 119-129 days (n=4), 134-138 days (n=3), 140-147 days (n=2), lambs 1-21
days (n=3) and adult ewes (n=2).
Western blots revealed three distinct bands at 66, 45, and 30 kD for estrogen
sulfatase. The 66 kD band is shown in Figure 6.3 and corresponds to the correct
molecular weight of 65,492 Daltons for estrogen sulfatase (Stein et al., 1989). As shown
by Figure 6.3, there are slight molecular weight variations of the 66 kD band for ovine
brainstem. Preabsorption of the enzyme revealed the 66 kD band to be specific, while the
lower molecular weight bands were shown to be nonspecific. Multiple linear regression
did not reveal the 66 kD band to vary between groups. Figure 6.4 (top panel) shows
group means ±SEM measured in relative optical density across developmental age for the
66 kD band present in brainstem.
Estrogen sulfotransferase revealed two distinct bands at 35 and 30 kD for
/
brainstem. Figure 6.5 shows the 35 kD band corresponding to the correct molecular
weight of estrogen sulfotransferase of 34,640 Daltons (Nash et al., 1988). This specificity
of molecular weight was confirmed by preabsorption of the primary antibody. Multiple
linear regression did not reveal any of the bands to be significantly different among
developmental age. Figure 6.6 (top panel) shows group means ±SEM measured in optical
density units for the 35 kD estrogen sulfotransferase band present in brainstem.
For hypothalamic tissue, 12 fetuses, 4 lambs and 2 adults were used. The
developmental ages were as follows: 86-110 days (n=2), 119-129 days (n=4), 134-138
days (n=3), 140-147 days (n=3), lambs 1-21 days (n=4) and adults (n=2).

102
Western blots again revealed three distinct bands at 66, 45, and 30 kD for estrogen
sulfatase. Again, preabsorption of the enzyme revealed only the 66 kD band to be
specific. Western blots showing the 66 kD band for ovine hypothalamus, as well as
brainstem, are shown in Figure 6.3. The 66 kD band was not statistically different
between groups by multiple linear regression. Figure 6.4 (bottom panel) shows group
means ±SEM measured in relative optical density across developmental age for the 66 kD
band present in hypothalami.
Estrogen sulfotransferase revealed three distinct bands at 45, 35, and 30 kD for
hypothalami. Preabsorption of the enzyme revealed only the 35 kD to be specific. Multiple
linear regression did not reveal any of the bands to be significantly different among
developmental age. Figure 6.6 (bottom panel) shows group means ±SEM measured in optical
density units for the 35 kD estrogen sulfotransferase band present in hypothalami.
Immunohistochemistry Specific staining for estrogen sulfatase and sulfotransferase were
/
widespread throughout the hypothalamus and brainstem of all developmental and post-
developmental groups. Immunohistochemical results from regions important for HPA axis
control are shown in Figures 6.7-6.13. Specific neuronal staining was seen in the
paraventricular nucleus or PVN of the hypothalamus (Figures 6.7 and 6.11), the nucleus
of the tractus solitarius or NTS of the medulla (Figures 6.8 and 6.12), the rostral ventral
lateral medulla or RVLM (Figures 6.9 and 6.13), and the raphe nucleus (Figure 6.10).
Discussion
The results of this study demonstrate that there is significant estrogen sulfatase
activity in ovine fetal hypothalamus, hippocampus, and brainstem, and that there are
statistically significant ontogenetic changes in activity of this enzyme in the hippocampus.

103
Also shown is the presence of estrogen sulfotransferase in the fetal hypothalamus and
brainstem. It has previously been demonstrated that estrogens in fetal plasma increase
both basal- and stimulated- fetal plasma ACTH secretion. The present results suggest a
mechanism by which the most abundant form of estrogen in ovine fetal plasma, estrone
sulfate, might be made available to areas within the fetal brain known to be involved in the
control of the fetal HPA axis.
Mathew and Balasubramanian (1982) and Lakshmi and Balasubramanian (1979)
have previously demonstrated estrogen sulfatase and sulfotransferase activity in adult
sheep brain tissue. Other investigators have demonstrated these enzymatic activities in
adult brain tissue from rats (Connolly and Resko, 1989; Kawano and Aikawa, 1987), mice
(Hobkirk, 1987), non-human primates (Lakshmi and Balasubramanian, 1981), and human
beings (Platia, 1984). Hobkirk and coworkers demonstrated that enzyme activities are
transiently increased postnatally in the brain of the mouse (1987). While the development
/
of brain estrogen sulfatase and sulfotransferase activity have not been studied in sheep, the
development of activities in mice suggests the possibility that this might be an important
developmental process in the perinatal period.
In this present study, I found an unequal distribution of estrogen sulfatase activity
in the brain regions studied, and we found that the developmental changes in activities
were not identical among the regions. Among the areas that I studied, I found highest
activity in the hippocampus and lower but still substantial activity in the hypothalamus and
brainstem. Western blotting in the hypothalamus and brainstem confirmed the enzyme
activity results. Thus, estrogen sulfatase was present throughout development in both
hypothalami and brainstem, but it did not change significantly between groups. The

104
presence of multiple bands (66, 45, and 30 kD) is not surprising since the primary antibody
used was polyclonal The 66 kD band best represents the enzyme which has a molecular
weight of 65,492 Daltons (Stein et al., 1989) and was the only band that was specific as
tested by preabsorption. This band was shown to be non-significant across developmental
age (Figure 6.4), which agrees with the results of the enzyme kinetics. The 66 kD band
for brainstem exhibited slight variations in molecular weight as shown by Figure 6.3.
Though no previous evidence exists in the literature for such an observation, a probable
explanation may exist through post-translational modification. That is, events such as
phosphorylation or glycosylation could be responsible for slight variations in molecular
weight.
Using a histochemical technique, Kawano and Aikawa found that sulfatase activity
is highest in pineal gland, choroid plexus, and pars distalis of the pituitary in adult rats
(1987). I investigated the activity in hypothalamus, brainstem, and hippocampus because
r
these areas are known to contain nuclei involved in integration, afferent signal relay, or
negative feedback inhibition within the HP A axis (Grizzle et al., 1974; Keller-wood and
Dallman, 1984; Maran, 1978; Ward, 1978). The presence of activity in any of these areas
could be important for the deconjugation of sulfated estrogens in the blood perfusing the
brain. Rosenfeld et al in 1980 reported that the majority of estrogen produced by the
ovine placenta is sulfoconjugated and thus protected since sulfatase in not present. My
data suggest otherwise given that sulfoconjugates in the fetal compartment may have
specific regional roles. The effect of estrogen on both basal- and hypotension stimulated-
concentrations of ACTH could be the result of an action of estrogen on the PVN in the
hypothalamus, an action on the hippocampus (which mediates some of the negative

105
feedback actions of corticosteroids on ACTH secretion), an action on the NTS (which
relays neural traffic from visceral afferents), or an action on any part of the pathways
leading from the NTS to the PVN (e g., the RVLM). Estrogen receptors have been
demonstrated in the NTS and hippocampus (Lehman, 1993). While estrogen receptors
within the hypothalamus are most concentrated in the arcuate nucleus, estrogen receptors
have been demonstrated in the PVN (Lehman, 1993; Simerly, 1990). The results of the
present experiments identify the cellular location of the sulfatase activity which is
consistent with these centers for HPA axis control. I found widespread staining
throughout nuclei and fiber tracts of the hypothalamus and brainstem. Neuronal staining
was much more concentrated than fiber tract staining, however both were observable.
Specifically, we found intense neuronal staining in the PVN (Figure 6.7), the NTS (Figure
6.8), the RVLM (Figure 6.9), and the dorsal raphe nucleus (Figure 6.10).
While estrogen sulfatase may be responsible for decongugating estrone sulfate
/
locally within the fetal to increase HPA axis activity directly, the role of estrogen
sulfotransferase is probably more indiscrete. Naturally one such role of the enzyme is to
maintain high levels of circulating conjugated estrogens that cannot be readily degraded.
A less obvious role of estrogen sulfotransferase might be to conjugate cortisol so that
inhibition of HPA axis negative feedback is achieved. This, concomitant with local
activation of estrogens via estrogen sulfatase, would increase ACTH release.
The presence of estrogen sulfotransferase was found throughout development in
both ovine fetal hypothalami and brainstem, but it did not change significantly between
groups. Western blotting revealed the presence of multiple bands (45, 35, and 30 kD) is
not surprising since the primary antibody used was polyclonal. The 35 kD band best

106
represents the enzyme which has a molecular weight of 34,640 Daltons (Nash et al., 1988)
and was the only band that was specific as tested by preabsorption. This band was shown
to be non-significant across developmental age (Figure 6.6), which agrees with the results
of the enzyme kinetics. The 35 kD band for hypothalamus exhibited slight variations in
molecular weight as shown by Figure 6.5. Though no previous evidence exists in the
literature for such an observation, a probable explanation may exist through post-
translational modification. That is, events such as phosphorylation or glycosylation could
be responsible for slight variations in molecular weight.
Immunohistochemistry revealed the cellular location of estrogen sulfotransferase in
centers important for HPA axis control. As was the case for estrogen sulfatase, I found
widespread staining of estrogen sulfotransferase throughout nuclei and fiber tracts of the
hypothalamus and brainstem. Neuronal staining was much more concentrated than fiber
tract staining, however both were observable. Specifically, I found intense neuronal
/
staining in the PVN (Figure 6.11), the NTS (Figure 6.12), and the RVLM (Figure 6.13).
I propose that parturition in the sheep, and possibly in other species, involves an
interaction among several variables whose net result is the activation of the HPA axis. In
sheep, increases in fetal plasma cortisol concentration induce placental synthesis of
estrogens. In non-human primates and humans, increases in fetal plasma ACTH stimulate
fetal adrenal secretion of dehydroepiandrosterone which is then converted to estrogen by
the placenta. It has recently been reported that physiological increases in fetal plasma
estrogen concentrations stimulate fetal ACTH secretion (Saoud and Wood, 1996), and
that physiological increases in fetal plasma androgen concentrations decrease the
sensitivity of the fetal hypothalamo-pituitary unit to negative feedback inhibition by

107
cortisol (Saoud and Wood, 1996). Therefore, the increases in fetal plasma estrogen and
androgen concentrations, themselves in part a function of fetal HPA axis activity, further
augment fetal ACTH secretion. I hypothesize that parturition results from the onset of an
hypothalamic "drive" to ACTH secretion, with interaction between the adrenal cortex,
placenta, and hypothalamus producing a positive feedback cycle which ultimately
concludes with the separation of placenta from the fetal HPA axis (parturition). This
study suggests that the influence of estrogens on HPA axis activity could be expressed
earlier than would be predicted on the basis of changes in plasma concentrations of
unconjugated forms.

108
220
220
c
a>
200 )
o
k_
140 4
Q-
120
I
o
o
T—
C
E
80 A
60
o
E
40
Q_
20
0
&
&
A
V
Brainstem
\l
>
&
&
*
/
Figure 6.1: Estrone sulfatase activity in ovine hippocampus (top panel), hypothalamus
(middle panel) and brainstem (bottom panel). For all reactions, velocity is expressed as
pmol estrone formed per minute per mg protein. Substrate concentration in all reactions
was 3 pM. Data are represented as means ±SEM (n=3-4 per group).

109
Sulfatase Activity
(pmol / min /10 mg tissue)
14
12
10
8
6
4
2
0
Figure 6.2: Estrone sulfatase activity in ovine myometrium (n=3) and hypothalamus
(n=3). The activity is expressed as pmol estrone formed per minute per 10 mg tissue wet
weight. Substrate concentration in all reactions was 3 pM

110
Figure 6.3: Western blots showing the 66 kD band of estrogen sulfatase for ovine
hypothalami and brainstems. The number below each band designates developmental
age in number of days (L=lamb, A=adult ewe).

Ill
— 90-110 119-129 134-138 140-147 lambs adults
s
86-110 119-129 134-138 140-147 lambs adults
Developmental Age
Figure 6.4: Estrogen sulfatase (66 kD band) in ovine brainstem (top panel) and in ovine
hypothalamus (bottom panel). Bars represent means +SEM of designated age groups
from western blot analyses plotted as relative optical density units (n= 2-4 per group).

112
Hypothalamus
8 1 1 1 1 1 L L A
6 2 2 3 4 4
5 6 5 1 4
Hypothalamus
9 1 1 1 1 1 L L A
0 2 2 3 3 4
5 6 5 7 1
Brainstem
—
;f|«
9
1
1
1
1
1
L
A
2
1
2
3
3
4
0
6
7
8
1
Brainstem
m
—
, -
1
1
1
1
1
L
L
A
1
2
2
3
4
0
5
8
5
1
Figure 6.5: Western blots showing the 35 kD band of estrogen sulfotransferase
for ovine hypothalami and brainstems. The number below each band designates
developmental age in number of days (L=lamb, A=adult ewe).

113
86-110 119-129 134-138 140-147 lambs
adults
~i— —i— —i-
86-110 119-129 134-138 140-147 lambs
adults
Developmental Age
Figure 6.6: Estrogen sulfotransferase (35 kD band) in ovine brainstem (top panel) and in
ovine hypothalamus (bottom panel). Bars represent means +SEM of designated age
groups from western blot analyses plotted as relative optical density units (n=2-4 per
group).

114
Figure 6.7: Photomicrographs of neuronal estrogen sulfatase staining in the
fetal ovine PVN (A- 40X, B- 200X).

115
Figure 6.8: Photomicrographs of neuronal estrogen sulfatase staining in
the fetal ovine NTS (A- 100X, B- 200X).

Figure 6.9: Photomicrograph of neuronal estrogen sulfatase staining in
the fetal ovine RVLM (200X).

117
Figure 6.10: Photomicrograph of estrogen sulfatase staining in the fetal
ovine raphe nucleus (40X).

118
Figure 6.11: Photomicrographs of neuronal estrogen sulfotransferase in the fetal
ovine PVN (A- 40X, B- 200X).

119
Figure 6.12: Photomicrographs of neuronal
the fetal ovine NTS (A- 100X, B- 200X).
estrogen sulfotransferase staining in

120
Figure 6.13: Photomicrograph of neuronal estrogen sulfotransferase in the fetal
ovine RVLM (200X).

CHAPTER 7
SUMMARY AND CONCLUSIONS
This goal of this dissertation was to shed light on the mechanism of parturition as
well as reveal how an ovine fetus defends itself against hypotension. This was
accomplished by using a combination of in vivo (whole animal) and in vitro
(immunohistochemistry) techniques. In trying to accomplish this aim, two main
hypotheses were developed.
Hypothesis 1 The actions of estrogen on fetal cardiovascular reflex responsiveness to
hypotension will be measurable in intact fetuses but not in baro- and chemo-denervated
fetuses. Research has previously demonstrated that the combined baro- and chemo-
denervation attenuates (approximately 50%) the reflex hormonal and hemodynamic
responses to moderate (50 %) reduction in arterial blood pressure. I proposed that the
interruption of the afferent pathways will eliminate the effect of estrogen on the reflex
cardiovascular responsiveness. If so, it could concluded that estrogen acts on, within, or
requires input from, the afferent baroreceptor and chemoreceptor pathways.
Hypothesis 2 The actions of estrogen within the fetal central nervous system are centered
within the nucleus of the tractus solitarius, the parvocellular neurons of the paraventricular
nucleus, or within the components of the cardiovascular regulatory centers receiving input
from baroreceptors or chemoreceptors. I used immunohistological techniques to identify
the neuroanatomical regions which are activated by hypotension and, subsequently, those
121

122
areas modified by estrogen’s action and baro- and chemo- denervation. The use of these
techniques allows for the measurement of c-fos expression, the protein product of the
early response gene which can be used as a marker of neuronal activity. I proposed that
estradiol implanted animals will have more Fos activity, and hence more c-fos staining in
relevant brain areas (NTS, PVN, etc.) as compared to control animals. Furthermore, it is
expected that c-fos staining would be negligible in denervated animals. Finally, c-fos
staining should be significantly elevated in all hypotensive animals as compared to
normotensive animals.
In addition to the c-fos immunohistochemical studies, the action of estrogen
sulfatase and estrogen sulfotransferase was investigated. Since the increase in
concentration of estrogen sulfate precedes the increase in HPA axis activity (Nathanielsz
et al., 1982), and since conjugated estrogens circulate in much higher concentrations than
unconjugated estrogens (Carnegie and Robertson, 1978; Tsang, 1974), I hypothesized that
these enzymes would be present in brain areas important for HPA axis control. If true, a
/
local mechanism would be in place for conversion of biologically inactive to active
estrogen.
A total of 40 pregnant ewes were studied (5 per experimental group). Animals
were randomly assigned to the following groups:
1. placebo implant, intact (sham-denervated), normotensive fetuses
2. placebo implant, carotid sinus denervated, normotensive fetuses
3. placebo implant, intact (sham-denervated) fetuses subjected to hypotension
4. placebo implant, carotid sinus denervated fetuses subjected to hypotension

123
5. estradiol implant (5 mg/21 day release), intact (sham-denervated), normotensive
fetuses
6. estradiol implant (5 mg/21 day release), carotid sinus denervated, normotensive
fetuses
7. estradiol implant (5 mg/21 day release), intact (sham-denervated) fetuses
subjected to hypotension
8. estradiol implant (5 mg/21 day release), carotid sinus denervated fetuses
subjected to hypotension
Understanding the mechanism of the increased fetal HPA axis at the end of
gestation is key to understanding the mechanism of spontaneous parturition in sheep. The
in vivo experiments were conducted to see if estradiol has it’s stimulatory effect on HPA
axis activity through the afferent baroreceptor and chemoreceptor pathway. More
specifically, I hypothesized that the actions of estrogen on the fetal cardiovascular reflex
responsiveness to hypotension will be measurable in intact fetuses but riot in baro- and
chemo- denervated fetuses. This augmentation of HPA axis activity was assessed by using
a surgically manipulated ovine model. ACTH, AVP, estradiol, and cortisol levels were
measured and compared across treatment groups.
This study has shown that estradiol does have an effect on cardiovascular
responsiveness. Pretreatment with estradiol increases basal ACTH, AVP, and cortisol
levels. This effect of estradiol is seen best perhaps after a ten minute period of
hypotension. At this point, hormonal responsiveness has been greatly augmented with
regard to ACTH, AVP, and cortisol levels. Carotid sinus denervation, attenuates this
augmented HPA axis activity in response to brachiocephalic occlusion. Carotid sinus

124
denervation by itself, however, caused no HPA axis activity or augmentation, deeming the
surgical elimination of baroreceptor / chemoreceptor responsiveness an appropriate and
effective control. Research has previously demonstrated that the combined baro- and
chemo- denervation attenuates (approximately 50%) the reflex hormonal and
hemodynamic responses to moderate (50 %) reduction in arterial blood pressure. This
study supports this finding demonstrated by the fact that ACTH, AVP, and cortisol levels
at lOmin. and 20min. time points in hypotensive, carotid sinus denervated animals are
approximately half their corresponding levels with hypotension alone. At the very least, it
can be concluded that estrogen acts on, within, or requires input from, the afferent
baroreceptor and chemoreceptor pathways. Figure 7.1 shows a schematic of the
interaction between the HPA axis and the baroreceptor / chemoreceptor afferent pathway.
This study utilized an artificial stimulus for HPA axis activation. This taken with
the fact that the animals in this study are at about 85% gestation makes direct comparison
to the time of parturition impossible. However, this study is useful in that it sheds light on
/
how an ovine fetus responds to periods of hypotension. Furthermore, the effect of
estradiol on cardiovascular responsiveness has been shown to greatly augment HPA axis
activity in response to such hypotension as controlled through the baroreceptor /
chemoreceptor pathway. These are important findings because although this ovine model
done not exactly mimic the process of parturition, important conclusions can be made. All
of the animals in this study are at least at 125 days gestation. Before around 120 days
gestation, the HPA axis is fairly unresponsive to stimuli. The estradiol implants used in
this study raised plasma estradiol levels to within a physiological range. Also, plasma
estradiol levels were comparable to time just prior to parturition, when estradiol levels are

125
thought to activate the HPA axis. For these reasons, it is evident that the model system
used in this study is appropriate for deciphering not only fetal responsiveness to
cardiovascular stimuli, but also the mechanism of parturition itself.
Though the results of this study reveal an estradiol responsive baroreceptor /
chemoreceptor afferent pathway participation in cardiovascular reflex control and trigger
for parturition, they do not by themselves account for total control over these processes.
As stated before, research has previously demonstrated that the combined baro- and
chemo- denervation attenuates (approximately 50%) the reflex hormonal and
hemodynamic responses to moderate (50 %) reduction in arterial blood pressure. This
means that 50% of the mechanism / pathway responsible for cardiovascular reflex
responsiveness and parturition is unaccounted for. It is plausible that the remaining
compensatory mechanisms exist within the central nervous system itself. Since the
cerebral circulation of the fetus exhibits autoregulation, one could hypothesize that
/
hypotension would cause reduced cerebral blood flow. This reduced flow may be
defended against by a local release of vasoactive substances. Prostanoids represent one
such group of compounds that may if fact be responsible for this response. Although it
has not been quantified at this time, our lab has shown that estradiol treatment does in fact
increase prostaglandin E2 (PGE2) within areas of the brain that are responsible for HPA
axis control. Other studies have pointed to a role of thromboxane A2 (TxA2) in this
process. TxA2 acts within the area perfused by the cerebral vasculature to stimulate
ACTH secretion in the fetus (Wood et al., 1993), which suggests that local generation of
TxA2 would effectively stimulate the HPA axis. Whatever the mechanism, it is obvious

126
that further research needs to be done in order to fully understand the workings of this
complex system.
My second hypothesis explores the fetal brains themselves to show exactly what
areas are responsible for the HPA axis augmentation displayed through the in vivo
experimentation. Immunohistochemical experiments were conducted to see at what point
in the HPA axis that estradiol acts to augment ACTH secretion. More specifically, I
hypothesized that neuronal activity would be highest in areas important for HPA axis
control in estradiol treated, hypotensive animals. I hypothesized that the baroreceptor /
chemoreceptor afferent pathway is involved, thus, carotid sinus denervation will eliminate
the augmented HPA axis activity. Neuronal activity was assessed by measuring the level
of c-fos, an early response gene, in brain areas important for HPA axis control. This
method has been used in numerous studies to assess neuronal activation due to
physiological stress (Hoffman et al., 1991; Shen et al., 1992; Chan et al., 1993).
r
The results of this particular study certainly lend support to my original hypothesis.
That is, estradiol augments HPA axis activity through baroreceptor / chemoreceptor
pathways and this augmentation seems to be in brain regions important for HPA axis
activity. Furthermore, the elimination of increased neuronal activity in carotid sinus
denervated animals shows that the pathway involved is probably this central
cardiovascular reflex pathway.
The immunohistochemistry results show that the PVN, NTS, and RVLM respond
similarly with regard to specific treatments. This finding comes as no surprise as all of
these areas are involved in HPA axis control. The PVN is the main collection of neuronal
cell bodies in the hypothalamus where CRH and AVP are synthesized and released to act

127
at the anterior pituitary to cause ACTH release (Lehman et al., 1993; Pomerantz and
Sholl, 1987). The NTS is the first synapse point in the pathway connecting the afferent
baroreceptors with the PVN. The RVLM is a cardiovascular regulatory center which
coordinates information from the periphery. It has been shown that all of these brain
regions have estrogen receptors, enabling them to respond to estradiol treatment prior to
brachiocephalic occlusion (Lehman et al., 1993; Simerly et al., 1990). Estradiol caused a
significant increase in neuronal activity in the PVN, NTS, and RVLM. In fetuses made
hypotensive via brachiocephalic occlusion, this effect was further augmented as shown by
statistical analyses. Carotid sinus denervation eliminated increases in c-fos activity in these
HPA axis regulatory centers.
An interesting finding of this study was the absence of an estradiol effect in the
hippocampus. Like the PVN, NTS, and RVLM, an increase in c-fos activity was seen in
hypotensive animals and this effect was eliminated with carotid sinus denervation.
However, basal levels as well as stressed levels of neuronal activity in estradiol treated
animals compared to placebo treated animals did not differ statistically. This reveals that
estradiol has no effect at the level of the hippocampus in augmenting HPA axis activity in
response to hypotension. This is interesting because there have been estrogen receptors
reported within the hippocampus (Lehman et al., 1993; Simerly et al., 1990). Perhaps the
answer to this discrepancy lies in fact that the hippocampus is a classic site of cortisol
negative feedback. Wood has further shown that the HPA axis is insensitive to inhibition
via cortisol negative feedback towards the end of gestation (1987). Possibly, the
hippocampus is unresponsive to an estradiol augmentation of ACTH secretion at this time.

128
Of course, this may be in the best interest of the fetus since an increase in neuronal activity
at the level of the hippocampus may lead to inhibition rather than stimulation of the axis.
It was certainly encouraging that in peripheral brain regions, an increase in
neuronal activity was observed in neither estradiol treated nor estradiol treated,
hypotensive fetuses. This lack of c-fos staining in the cortex and cerebellum indicate that
the response to treatment within the CNS was specific. All things being equal, only brain
regions important in HPA axis control seemed to respond to estradiol treatment and
brachiocephalic occlusion.
Pituitary glands were stained for c-fos as well as ACTH and AVP. Though
statistics were not performed for the relative levels of these hormones, the pattern of
staining seemed to be consistent with that of the PVN, NTS, and RVLM. Not only was
the c-fos increased with estradiol treatment and hypotension, but the levels of ACTH and
AVP seemed to mimic the pattern and level of c-fos staining. ACTH was seen in the
f
anterior pituitary in and increased with both estradiol treatment and hypotension. AVP
was seen mostly in the posterior pituitary in areas of increased c-fos generation. All of
this information fits with my hypothesis. One would expect to see an increase in staining
in these areas within the pituitary with both estradiol treatment and hypotension. There
are estrogen receptors within the pituitary and an increase in activity of the pituitary would
be logical during hypotension. It is possible that ACTH and AVP synthesis is increased
during estradiol treatment. This would also be true in the normal fetus towards the end of
gestation when estradiol is increasing. If these hormone levels are in fact increasing, The
fetus would be able to better respond to episodes of hypotension as well as trigger
parturition with an augmented ACTH and AVP response.

129
I have reported that within the PVN, NTS, RVLM, hippocampus, and pituitary of
fetal sheep, an increase in c-fos activity is observed with estradiol treatment and
brachiocephalic occlusion. It may seem logical that rather than an increase in neuronal
activity, a decrease in c-fos staining at the level of the NTS would accompany
hypotension. Hypotension causes a decrease in the rate of firing of the baroreceptors, and
thus a decrease in signal at the NTS. Perhaps what is being observed here is a
chemoreceptor mediated effect. As the brachiocephalic artery is occluded, the blood
perfusing the head becomes acidotic and hypoxic. This is revealed through elevated levels
of hydrogen ions, elevated PC02, and a decrease in P02 in the blood. All of these factors
were in fact monitored throughout the duration of the experiment. I believe that the fetal
P02 level is strictly monitored in the carotid artery and in a state of hypotension, the
compensatory response is an increase in HPA axis activity in order to re-establish
adequate blood flow to the brain. This will have the inevitable response of bringing blood
gas levels back to a homeostatic level. In doing so, blood pressure will be restored to
homeostatic levels.
Is important to realize that the HPA axis is not only integral in controlling blood
pressure, but it is also the endocrine axis of parturition. This is a fact which has been
proven many times without actual confirmation of a precise mechanism. This study sheds
light on the internal working of this system. I have shown that an intact afferent
baroreceptor/chemoreceptor pathway is necessary for an ovine fetus to respond to a state
of hypotension. I have further shown that estradiol augments this response through this
cardiovascular reflex pathway. More precisely, I have shown through the utilization of
immunohistochemistry, the brain areas involved in this process. Figure 7.2 outlines some

130
of the brain areas modified by estradiol, thus allowing for an augmented HPA axis
response. This study used an artificial stimulus to increase HPA axis activity, however, it
is not disputed that the pathway described is in fact involved in triggering parturition.
It has recently been demonstrated that physiological increases in fetal plasma
estrogen concentrations greatly augment fetal ACTH secretion (Saoud and Wood, 1996).
This effect of estrogen can be demonstrated on both basal and hypotension stimulated fetal
ACTH secretion. While estrogen has a potentially important effect on fetal ACTH
secretion, it is well-known that fetal plasma concentrations of unconjugated estrogens
increase only after the beginning of the increase in fetal plasma ACTH and cortisol
(Nathanielsz et al., 1982; Strott et al., 1974). Conjugated estrogens, mostly estrone
sulfate, circulate in high concentrations compared to unconjugated estrogens (Carnegie
and Robertson, 1978; Tsang, 1974). The concentration of estrone sulfate increases before
the increase in fetal HPA axis activity (Nathanielsz et al., 1982). However, conjugated
steroids cannot bind to nuclear receptors unless deconjugated (Hobkirk, 1985). It is
possible that estrone sulfate is converted to estrone locally within the fetal brain, and that
the circulating estrone sulfate acts as a reservoir for estrone acting within the fetal brain.
Conjugation and deconjugation of estrone are accomplished via the enzymes estrogen
sulfotransferase and estrogen sulfatase, respectively.
Once again, immunohistochemical techniques, this time accompanied by Western
blotting and enzyme activity assays, were employed to decipher this discrepancy. The
results of this study demonstrated that there is significant estrogen sulfatase activity in
ovine fetal hypothalamus, hippocampus, and brainstem, and that there are statistically
significant ontogenetic changes in activity of this enzyme in the hippocampus. Also shown

131
is the presence of estrogen sulfotransferase in the fetal hypothalamus and brainstem. It has
previously been demonstrated that estrogens in fetal plasma increase both basal- and
stimulated- fetal plasma ACTH secretion. The present results suggest a mechanism by
which the most abundant form of estrogen in ovine fetal plasma, estrone sulfate, might be
made available to areas within the fetal brain known to be involved in the control of the
fetal HPA axis.
I found an unequal distribution of estrogen sulfatase activity in the brain regions
studied, and we found that the developmental changes in activities were not identical
among the regions. Among the areas that I studied, I found highest activity in the
hippocampus and lower but still substantial activity in the hypothalamus and brainstem.
Western blotting in the hypothalamus and brainstem confirmed the enzyme activity results.
Thus, estrogen sulfatase was present throughout development in both hypothalami and
brainstem, but it did not change significantly between groups.
/
Using a histochemical technique, Kawano and Aikawa found that sulfatase activity
is highest in pineal gland, choroid plexus, and pars distalis of the pituitary in adult rats
(1987). I investigated the activity in hypothalamus, brainstem, and hippocampus because
these areas are known to contain nuclei involved in integration, afferent signal relay, or
negative feedback inhibition within the HPA axis (Grizzle et al., 1974; Keller-Wood and
Dallman, 1984; Maran, 1978; Ward, 1978). The presence of activity in any of these areas
could be important for the deconjugation of sulfated estrogens in the blood perfusing the
brain. Rosenfeld et al in 1980 reported that the majority of estrogen produced by the
ovine placenta is sulfoconjugated and thus protected since sulfatase in not present. My
data suggest otherwise given that sulfoconjugates in the fetal compartment may have

132
specific regional roles. The effect of estrogen on both basal- and hypotension stimulated-
concentrations of ACTH could be the result of an action of estrogen on the PVN in the
hypothalamus, an action on the hippocampus (which mediates some of the negative
feedback actions of corticosteroids on ACTH secretion), an action on the NTS (which
relays neural traffic from visceral afferents), or an action on any part of the pathways
leading from the NTS to the PVN (e g., the RVLM). Estrogen receptors have been
demonstrated in the NTS and hippocampus (Lehman, 1993). While estrogen receptors
within the hypothalamus are most concentrated in the arcuate nucleus, estrogen receptors
have been demonstrated in the PVN (Lehman, 1993; Simerly, 1990). The results of the
present experiments identify the cellular location of the sulfatase activity which is
consistent with these centers for HPA axis control. I found widespread staining
throughout nuclei and fiber tracts of the hypothalamus and brainstem. Neuronal staining
was much more concentrated than fiber tract staining, however both were observable.
/
Specifically, we found intense neuronal staining in the PVN, NTS, RVLM, and the dorsal
raphe nucleus.
While estrogen sulfatase may be responsible for decongugating estrone sulfate
locally within the fetal to increase HPA axis activity directly, the role of estrogen
sulfotransferase is probably more indiscrete. Naturally one such role of the enzyme is to
maintain high levels of circulating conjugated estrogens that cannot be readily degraded.
A less obvious role of estrogen sulfotransferase might be to conjugate cortisol so that
inhibition of HPA axis negative feedback is achieved. This, concomitant with local
activation of estrogens via estrogen sulfatase, would increase ACTH release.

133
The presence of estrogen sulfotransferase was found throughout development in
both ovine fetal hypothalami and brainstem, but it did not change significantly between
groups. Immunohistochemistry further revealed the cellular location of estrogen
sulfotransferase in centers important for HPA axis control. As was the case for estrogen
sulfatase, I found widespread staining of estrogen sulfotransferase throughout nuclei and
fiber tracts of the hypothalamus and brainstem. Neuronal staining was much more
concentrated than fiber tract staining, however both were observable. Specifically, I found
intense neuronal staining in the PVN, NTS, and the RVLM. I propose that parturition in
the sheep, and possibly in other species, involves an interaction among several variables
whose net result is the activation of the HPA axis. In sheep, increases in fetal plasma
cortisol concentration induce placental synthesis of estrogens. In non-human primates and
humans, increases in fetal plasma ACTH stimulate fetal adrenal secretion of
dehydroepiandrosterone which is then converted to estrogen by the placenta. It has
f
recently been reported that physiological increases in fetal plasma estrogen concentrations
stimulate fetal ACTH secretion (Saoud and Wood, 1996), and that physiological increases
in fetal plasma androgen concentrations decrease the sensitivity of the fetal hypothalamo-
pituitary unit to negative feedback inhibition by cortisol (Saoud and Wood, 1996).
Therefore, the increases in fetal plasma estrogen and androgen concentrations, themselves
in part a function of fetal HPA axis activity, further augment fetal ACTH secretion. I
hypothesize that parturition results from the onset of an hypothalamic "drive" to ACTH
secretion, with interaction between adrenal, placenta, and hypothalamus producing a
positive feedback cycle which ultimately concludes with the separation of placenta from
the fetal HPA axis (parturition). This study suggests that the influence of estrogens on

134
HPA axis activity could be expressed earlier than would be predicted on the basis of
changes in plasma concentrations of unconjugated forms.
Preterm labor is still a major concern in this day and age. Despite efforts to inform
the public about proper nutrition and the need to seek prenatal care, a significant number
of infants are being bom prematurely. Approximately 5-10% of all deliveries in North
America and the United Kingdom occur prematurely, before 37 weeks gestation. This
figure can be higher or lower depending upon the developmental status of the country and
the socio-economic status of the mother. Complications due to premature delivery and
immaturity of the neonate contribute significantly to the life or death of these infants
(Creasy, 1980; Hall, 1985).
The findings of my research offer insight to the problem of preterm labor by
providing useful information that may be used to pharmacologically manipulate the
system. By deciphering the exact pathway involved in the process of parturition, one
/
could theoretically manipulate it in order to accelerate or decelerate the process in
question. The areas within central nervous system for this type of manipulation are
numerous given that there exists many HPA axis control centers. By using an estrogen
receptor antagonist in the PVN, NTS, or RVLM, the timing of parturition might be
delayed. Of course, there may exist complications in that the antagonist may bind to other
sites which may be problematic if inhibited. Another possibility is the use of estrogen
sulfatase inhibitors / degraders of estrogen sulfotransferase to alter the timing of birth. An
important aspect of this research is the usefulness of it to aid or even cure a health concern
that plagues the general public. In this case, the problem is infant mortality due to preterm

135
labor as well as a variety of problems that arise due to premature parturition for living
offspring (i.e. surfactant production).
The purpose of the preceding experiments was to understand some aspects of
parturition that had not yet been investigated. Through experimentation, I have been able
to answer some important questions that will inevitably aid in the problem of premature
birth. There are still, however, many questions about this phenomenon that have to be
answered. Perhaps it is better yet to say that the questions themselves need to pondered
and asked. The real power of research is both in the progress gained through the actual
scientific experimentation, as well as in the questions and ideas the research generates.
We still have much to learn about the process of parturition. The research performed in
this dissertation is but one piece of the puzzle. Still lingering in the future are some of the
most important answers concerning this area of research. What is the signal that increases
the drive to the HPA axis? Obviously estrogens play a role, but what is the signal for
f
increasing the levels of the estrogens themselves? Is it purely maternal, purely fetal, or is
it a combination of the two? Perhaps in investigating these areas, the most important
questions yet will be generated and maybe answered. Perhaps the only real constant in
research is that the more we learn about the inner-workings of organisms, the more we
realize just how little is known.

136
Nucleus of the
Tractus Solitarius
Hypothalamus
(Paraventricular Nucleus)
Cortisol
Pituitary <
Baro/Chemoreceptors
(Carotid sinus and Aortic arch)
ACTH
â–¼
Adrenal Cortex
Figure 7.1: Schematic of the baroreceptor/chemoreceptor afferent pathway and the HP A
axis. This system monitors blood pressure on a minute to minute basis. Hypotension
will be sensed at the level of the baroreceptors as a decrease in the rate of firing. This
will activate the HPA axis in order to return blood pressure to a homeostatic level.

137
Hippocampus
Figure 7.2: Baroreceptor reflex pathway showing possible sites of estradiol action (*)
as measured by an increase in c-fos generation. Hypotension (brachiocephalic
occlusion) activates the HPA axis due to a decrease in the rate of firing of the
baroreceptors. Pretreatment with estradiol allows for an augmented hormonal response
in the intact fetus. Carotid sinus denervation eliminates this enhanced response.

REFERENCES
Adams, W.E. The Comparative Morphology of the Carotid Body and Carotid Sinus.
Springfield, EL: Thomas, 1958
Addison, W.H.F. The extent of carotid pressoreceptor area in the cat as indicated by its
special elastic tissue wall. Anat. Record. 88:418-419, 1944.
Alexander, N. Plasma volumes and hematocrits in rats with chronic sinoaortic denervation
hypertension. Am. J. Physiol. 236:H92-H95, 1979.
Anderson, A.B.M., Flint, A.P., and Turnbull, A.C.. Mechanism of activation of
glucocorticoids in induction of ovine parturition: Effect on placental steroid
metabolism. J. Endocrinol. 66:61-70, 1975.
Angell-James, J. The effects of changes of extramural “intrathoracic” pressure on aortic
arch baroreceptors. Physiol. Lond. 214:89-103, 1971.
Antolovich, G.C., Clarke, I.J., McMillen, I.C., Perry, R.A., Robinson, P.M., Silver, M.,
and Young, R. Hypothalamo-pituitary disconnection in the fetal sheep.
Neuroendocrinol. 51:1-9, 1990.
Antolovich, G.C., McMillen, I.C., Robinson, P.M., Silver, M., Youngs, I.R., and Perry,
R.A. Effect of cortisol infusion on the pituitary-adrenal axis of the
hypothalamopituitary disconnected fetal sheep. Neuroendocrinol. 56:312-319,
1992.
Bader, H. The anatomy and physiology of the vascular wall. In: Handbook of
Physiology. Circulation, Section Two, Volume Two. Hamilton, W.F. and Dow, P.,
eds. Washington D C.: American Physiological Society, 1963.
Baertschi, A.J., and Friedli, M. A novel type of vasopressin receptor on anterior pituitary
corticotrophs? Endocrinol. 116:499-502, 1985.
Bagshaw, R.J., and Fischer, J.M. Morphology of the carotid sinus in the dog. J. Appl.
Physiol. 31:198-202, 1971.
Bassett, J.M., and Thorbum, G.D. Foetal plasma corticosteroids and the initiation of
parturition in the sheep. J. Endocrinol. 44:285, 1969.
138

139
Baxter, J.E. Glucocorticoid hormone action. Pharmacol. Ther.2:605,1972.
Beaulieu, S., Paolo, T.D., Cote, J., and Barden, N. Participation of the central
amygdaloid nucleus in the response of adrenocorticotropin secretion to
immobilization stress: Opposing roles of the noradrenergic and dopaminergic
systems. Neuroendocrinol. 45:37-46, 1987.
Bedfored, C.A., Challis, J.R.G., Harrison, F.A., and Heap, R.B. The role of oestrogens
and progesterone in the onset of parturition in various species. J. Reprod. Fert.
Suppl. 16:1-23, 1972.
Bell, ME., Wood, C.E., and Keller-Wood, M. Influence of reproductive state on
pituitary-adrenal activity in the ewe. Dorn. Anim. Endocrinol. 8: 245-254, 1991.
Berne, R.M., and Levy, M.N. Cardiovascular Physiology. 5th ed. St. Louis, MO: Mosby,
1986.
Bevegard, B.S. and Shepherd, J.T. Circulatory effects of stimulating the carotid stretch
receptors in man at rest and during exercise. J. Clin. Invest. 45:132-142, 1966.
Bilezikjian, L.M. and Vale, W.W. Glucocorticoids inhibit corticotropin-releasing factor-
induced production of adenosine 3’,5’-monophosphate in cultured anterior
pituitary cells. Endocrinol. 113:657-662, 1983.
Binns, W., Shupe, J.L., Keeler, R.F., and James, L.F. Chronologic evaluation of
teratogenicity in sheep fed Veratrum califomicum. J. Am. Vet. Med Assoc.
147:839-842, 1991.
/
Bjurstedt, H., Rosenhamer, G., and Tyden G. Cardiovascular responses to changes in
carotid sinus tranmural pressure in man. Acta. Physiol. Scand. 94:497-505, 1977.
Boss, J., and Green, J.H. The histology of the common carotid baroreceptor areas of the
cat. Circ. Res. 4:12-17, 1956.
Brace, R.A. Fetal blood volume responses to acute fetal hemorrhage. Circ. Res. 52:730-
734, 1983.
Bradford, M.M. A refined and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Analytical
Biochem. 72: 248-254, 1976.
Bronk, D.W., and Stella, G. Afferent impulses in the carotid sinus nerve I. The
relationship of the discharge from single end organs to arterial blood pressure.
J.Cell Comp. Physiol. 1:113-120, 1932.

Brooks, A N., and Challis, J.R.G. Exogeneous opioid regulation of the hypothalamic-
pituitary-adrenal axis in fetal sheep. J. Endocrinol. 119:389-395, 1988.
140
Brooks, A N., and Gibson, F. Prostaglandin E2 enhances AVP-stimulated but not CRF-
stimulated ACTH secretion from cultured fetal sheep pituitary cells. J.
Endocrinol. 132:33-38, 1992.
Brooks, A.N., Howe, D C., Porter, D.W.F., and Naylor, A M. Neuropeptide-Y stimulates
pituitary -adrenal activity in fetal and adult sheep. J. Neuroendocrinol. 6:161-166,
1994.
Brooks, A.N.,and White, A. Activation of pituitary-adrenal function in fetal sheep by
corticotropin-releasing factor and arginine vasopressin. J. Endocrinol. 124:27-35,
1990.
Brown, A.M. Receptors under pressure. Circ. Res. 46:1-10, 1980.
Brown, E.H., Coghlan, J.P., Hardy, K.J., and Wintour, E M. Aldosterone, corticosterone,
cortisol, 11-deoxycortisol and 11-deoxycorticosterone concentration in the blood
of chronically cannulated ovine foetuses: Effect of ACTH. Acta. Endocrinol.
88:364-374, 1978.
Brownstein, M.J., Russell, J.T., and Gainer, H. Synthesis, transport, and release of
posterior pituitary hormones. Science. 207:373-378, 1980.
Burgess, L.H., and Handa, R.J. Chronic estrogen-induced alterations in
adrenocorticotropin and corticosterone secretion, and glucocorticoid receptor-
mediated function in female rats. Endocrinol. 131:1261-1269, 1992.
Canny, B.J., Funder, J.W., and Clarke, I.J. Clucocorticoids regulate ovine hypophysiol
portal levels of corticotropin-releasing factor and arginine vasopressin in a stress-
specific manner. Endocrinol. 125:2532-2539, 1989.
Carnegie, J.A., and Robertson, H.A. Conjugated and unconjugated estrogens in fetal and
maternal fluids of the pregnant ewe: A possible role for estrone sulfate during early
pregnancy. Biol.Reprod. 19:202-211, 1978.
Challis, J.R.G. Sharp increase in free circulating oestrogens immediately before
parturition in sheep. Nature. 229:208, 1971.
Challis, J.R.G., and Brooks, A.N. Maturation and activation of hypothalamic-pituitary-
adrenal function in fetal sheep. Endocrine Rev. 10:182-204, 1989.

141
Challis, J R.G., and Patrick, J.E. Fetal and maternal estrogen concentrations throughout
pregnancy in the sheep. Can. J. Physiol. Pharmacol. 59:970-978, 1981.
Chan, R.K.W., Brown, E.R., Ericsson, A., Kovacs, K.J., and Sawchenko, P.E. A
comparison of two immediate-early genes, c-fos and NGFI-B, as markers for
functional activation in stress-related neuroendocrine circuitry. J. Neurosci.
13:5126-5136, 1993.
Claybaugh, J.R., and Share, L. Vasopressin, renin and cardiovascular responses to
continuous slow hemorrhage. Am. J. Physiol. 224:529-523, 1973.
Comline, R.S. and Silver, M. The release of adrenaline and nor-adrenaline from the
adrenal glands of the foetal sheep. J. Physiol. Lond. 210:41,1961.
Connolly, P.B., and Resko, J.A. Estrone sulfatase activity in rat brain and pituitary:
effects of gonadectomy and the estrous cycle. J.Steroid Biochemistry. 33: 1013-
1018, 1989.
Cowley, A.W., Liard, J.F., and Guyton, A.C. Role of the baroreceptor reflex in daily
control of arterial pressure and other variables in dogs. Circ. Res. 32:564-576,
1973.
Cowley, Jr., A.W., Monos, E., and Guyton, A.C. Interaction of vasopressin and the
varoreceptor reflex system in the regulation of arterial blood pressure in the dog.
Circ. Res. 34:505-514, 1974.
Coyne, M.D., and Kitay, J.I. Effect of ovariectomy on pituitary secretion of ACTH.
Endocrinol. 85:1097-1102, 1969.
Coyne, M.D., and Kitay, J.I. Effect of orchidectomy on pituitary secretion of ACTH.
Endocrinol. 89:1024-1028, 1971.
Creasy, R.K. Prevention of preterm labor. In: Premature Labor, Mead Johnson
Symposium on Perinatal and Developmental Medicine. 15:37. 1980.
Cryer, G.L., and Gann, D.S. Right atrial receptors mediate the adrenocortical response to
small hemorrhage. Am. J. Physiol. 227:325-328, 1974.
Dayanithi, G.A., and Antoni, F A. Atriopeptins are potent inhibitors of ACTH secretion
by rat anterior pituirary cells in vitro: Involvement of the atrial natriuretic factor
receptor domain of membrane bound guanylyl cyclase. J. Endocrinol. 125:39-44,
1989.

142
De Goeij, D.C.E., Kvetnansky, R., Whitnall, M.H., Jezova, D., Berkenbosch, F. and
Tilders, F.J. H. Repeated stress-induced activation of corticotropin-releasing
factor aneurons enhances vasopressin stores and colocalization with corticotropin-
releasing factor in the median eminence of rats. Neuroendocrinol. 53:150-159,
1991.
Drost, M., and Holm, L.M. Prolonged gestation in ewes after foetal adrenalectomy. J.
Endocrinol. 40:293-296, 1968.
Durand, P., Cathiard, A M., Dacheux, F., Annman, E., and Saez, J.M. In vitro stimulation
and inhibition of adrenocorticotropin release by pituitary cells from ovine fetuses
and lambs. Endocrinol. 118:1387-1394,1986.
Eckberg, D.L. Adaptation of the human carotid baroreceptor cardiac reflex. J. Physiol.
Lond. 269:561-589, 1977.
Eckberg, D.L., Abboud, F.M., and Mark, A.L. Modulation of carotid baroreflex
responsiveness in man: Effects of posture and propranolol. J. Appl. Physiol.
32:R215-R220, 1992.
Eckberg, D.L., Drabinski, M., and Vraunwald, E. Defective parasympathetic control in
patients with heart disease. NEJM. 285:877-883, 1971.
Emsting, J., and Parry, D.J. Some observations on the effects of stimulating the stretch
receptors in the carotid artery of man. J. Physiol. Lond. 135:45P-46P, 1957.
Familari, M., Smith, A.I., Smith, R., and Funder, J.W. Arginine vasopressin is a much
more potent stimulus to ACTH release from ovine anterior pituitary cells than
ovine corticotropin-releasing factor. Neuroendocrinol. 50:152-157, 1989.
Findlay, J.K., and Cox, R.I. Oestrogens in the plasma of the sheep foetus. J.
Endorcrinol. 46:281-282, 1970.
Fink, G., Dow, R.C., Casley, D., Johnston, D.I., Llim, A T., Copolov, D.L., Bennie, J.,
Carroll, S., and Dick, H. Atrial natruiretic peptide is a physiological inhibitor of
ACTH release. Evidence from immunoneurtralization in vivo. J. Endocrinol.
13 TR9-R12, 1991.
Furutani, Y., Morimoto, Y., Shibahara, S., Noda, M., Takahashi, H., Hirose, T., Asai, M.,
Inayama, S. Hayashida, H., Miyata, T., and Numa, S. Cloning and sequence
analysis ofcDNA for ovine corticotropiin-releasing factor precursor. Nature.
301:537-540, 1983.
Gann, D.S. Cortisol secretion after hemorrhage: Multiple mechanisms. Nephron. 23:119-
124, 1979.

143
Gann, D.S., Dallman, M.F., and Engeland, W.C. Reflex control and modulation of ACTH
and corticosteroids. Endocrine Physiology III. International Review of
Physiology, McCann, S.M., ed. Baltimore: University Park Press. 24:157-199,
1981.
Ganong, W.F. Review of Medical Physiology. 14th ed., Norwalk, CT: Lange Medical
Publications, 1985.
Garfield, R.E. Myometrial ultrastructure and uterine contractility. In: Uterine
Contractility. Boltari, S., Thomas, J.P., Volaer, A., and Vokaer, R. eds. Masson,
NY: Asmus. 1984.
Garfield, R.E., Sims, S., and Daniel, E.E. Gap junctions: Their presence and necessity in
myometrium during parturition. Science. 198:958-960,1977.
Garfield, R.E., Sims, S., Kannan, M.S., and Daniel, E.E. The possible role of gap
junctions in activiation of the myometrium during parturition. Am. J. Physiol.
235:C168-C179, 1978.
Giguere, V., Cote, J., and Labrie, F. Characteristics of the a-adrenergic stimulation of
adrenocorticotropin secretion in rat anterior pituitary cells. Endocrinol. 109:757-
762, 1981.
Gillies, G.E., Linton, E. A., and Lowry, P.J. Corticotropin releasing activity of the new
CRF is potentiated several times by vasopressin. Nature. 299:355-357, 1982.
Glickman, J.A., and Challis, J.R.G. The changing response pattern of sheep fetal adrenal
cells throughout the course of gestation. Endocrinol. 106:1371-1376, 1980.
Goldstein, R E., Beiser, G.D., Stampfer, M., and Epstein, S.E. Impairment of the
autonomically mediated heart rate control in patients with cardiac dysfunction.
Circ. Res. 36:571-578, 1975.
Green, J.H. Further barorecepor areas associated with the common carotid artery of the
cat. J. Physiol. Lond. 123:4IP, 1954.
Gregoreva, T.A. The Innervation of Blood Vessels. New York: Pergamon, 1962.
Grimes, J.M., Buss, L.A., and Brace, R.A. Blood volume restituition after hemorrhage in
adult sheep. Am. J. Physiol. 253:R541-R544, 1987.
Grizzle, W.E., Dallman, M.F., Schramm, L.P., and Gann, D.S. Inhibitory and facilitatory
hypothalamic areas mediation ACTH release in the cat. Endocrinol. 95: 1450-
1461, 1974.

144
Guillaume, V., Conte-Devolx, B., Szafarczyk, A., Malaval, F., Pares-Herbute, N., Grino,
M., Alonso, G., Assenmacher, I., and Oliver, C. The corticotropin-releasing factor
release in rat hypophysial portal blood is mediated by brain catecholamines.
Neuroendocrinol. 46:143-146, 1987.
Guillemin, R., and Rosenberg, B. Humoral hypothalamic control of anterior pituitary: A
study with combined tissue cultures. Endocrinol. 57:599-607,1955.
Guyton, A.C., Coleman, T.G., Cowley, A.W., Manning, R.D., Norman, R.A., and
Ferguson, J.D. A systems analysis approach to understanding long-range arterial
blood pressure control and hypertension. Circ. Res. 35:159-176, 1974.
Haas, D.A., and George, S.R. Neuropeptide-Y administration acutely increases
hypothalamic corticotropin-releasing factor immunoreactivity: Lack of effect in
other brain regions. Life Sci. 41:2725-2731, 1987.
Hall, M.H. Incidence and distribution of preterm labour. In: Preterm Labour and its
Consequences, Proceedings of the 13th Study Group, Royal College of
Obstetricians and Gynaecologists. Beard, R.W., and Sharp, F. eds. London:
RCOG, 1985.
Handa, R.J., Nunley, K.M, Lorens, S.A., Louie, J.P., McGivem, R.F., and Bollnow., M.R.
Androgen regulation of adrenocorticotropin and corticosterone secretion in the
male rat following novelty and foot shock stressors. Physiol. Behav. 55:117-124,
1994.
Hargrave, B.Y., and Rose, J.C. By 94 days of gestation plasma cortisol increases block
ACTH response to hypotension in lamb fetuses. Am. J. Physiol. 249:E350-E354,
1985.
Hashimoto, K., Ohno, N., Aoki, Y., Kageyama, I, Takahara, J., and Ofuji, T. A different
distribution of corticotropin releasing factor and arginine vasopressin contents in
the hypothalamic nuclei after estrogen administration. Acta. Med. Okayama.
35:37-43, 1981.
Hauger, R.L., and Auilera, G. Regulation of pituitary corticotropin releasing hormone
(CRH) receptors by CRF: Interaction with vasopressin. Endocrinol. 133:1708-
1714, 1993.
Hauger, R.L., Millan, M.A., Catt, K.J., and Aguilera, G. Differential regulation of brain
and pituitary corticotropin-releasing factor receptors by corticosterone.
Endocrinol. 120:1527-1533,1987.

145
Hauss, W.H, Kreuziger, H., and Asteroth, H. Uber die reizung der pressorezeptoren im
sinus caroticus beim hund. A Kreislaufforsch. 38:28-33, 1949.
Hennessy, D P., Coghlan, J.P., Hardy, K.J., Scoggins, BA., and Wintour, E.M. The
origin of cortisol in the blood of fetal sheep. J. Endocrinol. 95:71-79, 1982a.
Hennessy, D.P., Coghlan, J.P., Hardy, K.J., Scoggins, B.A., and Wintour, E.M.
Development of the pituitary-adrenal axis in chronically cannulated ovine fetuses.
J. Develop. Physiol. 4:339-352, 1982b.
Herbison, A. E., Robinson, J.E., and Skinner, D C. Distribution of estrogen
receptor-immunoreactive cells in the preoptic area of the ewe: co-localization with
glutamic acid decarboxylase but not luteinizing hormone-releasing hormone.
Neuroendocrinol. 57: 751-759, 1993.
Hobkirk, R. Steroid sulfotransferases and steroid sulfate sulfatases: characteristics and
biological roles. Can.J.Biochem.CellBiol. 63: 1127-1144, 1985.
Hobkirk, R., Fletcher, J.M., and Choi, H.Y. Estrone sulfate sulfohydrolase in the
developing brain and pituitary of rat, mouse and guinea pig. J. Steroid Biochem.
26: 413-416, 1987.
Hoñman, G.E., McDonald, T., Shedwick, R., and Nathanielsz, P.W. Activation of cFos
in ovine fetal corticotropin-releasing hormone neurons at the time of parturition.
Endocrinol. 129:3227-3233, 1991.
Holmes, M.C., Antoni, F.A., Aguilera, G., and Catt, K.J. Magnocellular axons in passage
through the median eminence release vasopressin. Nature. 319:326-329, 1986.
Iwamoto, H.S., Rudolph, A M., Keil, L.C., and Heymann, M.A. Hemodynamic responses
of the sheep fetus to vasopressin infusion. Circ. Res. 44:430-436, 1979.
Jard, S., Gailard, R.C., Guillon, G., Marie, J., Schoenenberg, P., Muller, A.F., Manning,
M., and Sawyer, W.H. Vasopressin antagonists allow demonstration of a novel
type of vasopressin receptor in the rat adenohypophysis. Mol. Pharmacol.
30:171-177, 1986.
Johnston, C.A., Gibbs, D M., and Negro-Vilar, A. High concentrations of epinephrine
derived from a central source and of 5-hydroxyindole-3-acetic acid in hypophysial
portal plasma. Endocrinol. 113:819-821,1983.
Jones, C.T., Boddy, K., and Robinson, J.S. Changes in the concentration of
adrenocorticotrophin and corticosteroid in the plasma of foetal sheep in the latte
half of pregnancy and during labour. J. Endocrinol. 72:293-300,1977.

146
Jose, A D ., and Taylor, R.R. Autonomic blockade by propranolol and atropine to study
intrinsic myocardial function in man. J. Clin. Invest. 48:2029-2031, 1969.
Kawano, J, and Aikawa, E. Regional distribution of arylsulphatase and estrone-sulfate
sulfatase activities in rat brain and hypophysis. Brain Res. 409: 391-394, 1987.
Keefer, D A. Quantification of in vivo 3H-estrogen uptake by individual anterior pituitary
cell types of male rat: A combined autoradiographic-immunocytochemical
technique. J. Histochem. Cytochem. 29:167-174, 1981.
Keller-Wood, M, and Dallman, M.F. Corticosteroid inhibition of ACTH secretion.
Endocrine Rev. 5: 1-22, 1984.
Keller-Wood, M. Vasopressin responses to hyperosmolality and hypotension during ovine
pregnancy. Am. J. Physiol. 266: R188-R193, 1994.
Keller-Wood, M., and Wood, C.E. Does the ovine placenta secrete ACTH under
normoxic or hypoxic conditions? Am. J. Physiol. 260: R389-R395, 1991.
Kendall, J.Z., Challis, J.R.G., Hart, I.C., Jones, C.T., Mitchell, M.S., Ritchie, J.W.K.,
Robinson, J.S., and Thorbum, G.D. Steroid and prostaglandin concentrations in
the plasma of pregnant ewes during infusion of adrenocorticotrophin or
dezamethasone to intact or hypophysectomized foetuses. J. Endocrinol. 75:59-
71, 1977.
Kerrigan, J.R., Krieg, Jr., R.J., and Rogol, A.D. Exogenous androgen does not alter
hypothalamic proopiomelanocortin gene transcript levels in the sexually immature
male rat. Neuroendocrinol. 56:264-270,1992.
Kitay, J.I. Sex differences in adrenal cortical secretion in the rat. Endocrinol. 68:818-
824, 1961.
Kitay, J.I. Pituitary-adrenal function in the rat after gonadectomy and gonadal hormone
replacement. Endocrinol. 73:253-260, 1963.
Kitay, J.I., Coyne, M.D., Newsom, W., and Nelson, R. Relation of the ovary to adrenal
corticosterone production and adrenal enzyme activity in the rat. Endocrinol.
77:902-908, 1965.
Koch, E. Die reflektorische selbsteuerung des kreislaufes. In: Ergebnisse der
Kreislaufforschung. Kirsch, B. ed. Dresden: Steinkopff, 1931.
Koushanpour, E. Baroreceptor discharge behavior and resetting. In: Baroreceptor
Reflexes. Persson, P.B., and Kirchheim, H R. eds. New York: Springer-Verlag,
1991.

147
Lakshmi, S., and A S. Balasubramanian. Studies on the chaotropically solubilized
arylsulfatase C and estrone sulfatase of sheep brain. Biochem.Biophys.Acta 567:
184-195, 1979.
Lakshmi, S., and Balasubramanian, A S. The distribution of estrone sulphatase,
dehydroepiandrosterone sulphatase, and arylsulphatase C in the primate (Macaca
radiata) brain and pituitary. J.Neurochem. 37: 358-362, 1981.
Lauber, M., Clavreul, C., Vaudry, H., and Cohen, P. Immunological detection of pro-
corticotropin releasing factor (CRF) in rat hypothalamus and pancreatic extracts.
Evidence for in vitro conversion into CRF. FEBS Lett. 173:222-226, 1984.
Lechan, R.M. Neuroendocrinology of pituitary hormone regulation. Endocrinol. Metab.
Clin. N. Am. 16:475-501, 1987.
Lehman, M. N., Ebling, F.J.P., Moenter, S.M., and Karsch, F.J. Distribution of estrogen
receptor-immunoreactive cells in the sheep brain. Endocrinol. 133: 876-886,
1993.
Lenz, H. J., Raedler, A., Greten, H., and Brown, M R. CRF initiates biological actions
within the brain that are observed in response to stress. Am. J. Physiol. 252:
R34-R39, 1987.
Leon, D.F., Shaver, J.A., and Leonard, J.J. Reflex heart rate control in man. Am. Heart
J. 80:729-739, 1970.
Leranth,' C., Antoni, F.A., and Palkovits, M. Ultrastructural demonstration of ovine CRF-
like immunoreactivity (oCRF-LI) in the rat hypothalamus: Processes of
magnocellular neurons establish membrane specializations with parvocellular
neurons containing oCRF-LI. Reg. Peptides. 6:179-188, 1983.
Leroux, P., and Pelletier, G. Radioautographic study of binding and internalization of
corticotropin-releasing factor by rat anterior pituitary corticotrophs. Endocrinol.
114:14-21, 1984.
Lewis, D.A., and Sherman, B.M. Serotonergic stimulation of adrenocorticotropin
secretion in man. J. Clin. Endocrinol. Metab. 58:458-462, 1984.
Liggins, G.C. Premature parturition after infusion of corticotropin or cortisol into foetal
lambs. J. Endocrinol. 42:323-329, 1968.
Liggins, G.C. Premature delivery of foetal lambs infused with glucocorticoids. J.
Endocrinol. 45:515-523, 1969.

148
Liggins, G.C., Fairclough, R J ., Grieves, S.A., Kendall, J.Z., and Knox, B.S. The
mechanism of initiation of parturition in the ewe Rec.Prog.Horm.Res. 29: 111-
159, 1973.
Liggins, G.C., Holm, L.W. and Kennedy, P C. Prolonged pregnancy following surgical
lesions of the foetal lamb pituitary. J. Reprod. Fértil. 12:419, 1966.
Liggins, G.C. and Kennedy, P C. Effects of electrocoagulation of the foetal lamb
hypophysis on growth and development. J. Endocrinol. 40:371-381, 1968.
Liggins, G.C., Kennedy, PC., and Holm, L.W. Failure of initiation of parturition after
electrocoagulation of the pituitary of the fetal lamb. Am. J. Obstet. Gynecol.
98:1080-1086, 1967.
Lim, A.T., Sheward, W.J., Copolov, D., Windmil, D., and Fink, G. J. Endocrinol. 2:15-
18, 1990.
Liu, J.P., Robinson, J., Funder, J.W., and Engler, D. The biosynthesis and secretion of
adrenocorticotropin by the ovine anterior pituitary is predominately regulated by
arginine vasopressin (AVP): Evidence that protein kinase C mediates the action of
A VP. J.Biol.Chem. 265:14136-14142,1990.
Longo, L. D. Maternal blood volume and cardiac output during pregnancy: a hypothesis
of endocrinologic control. Am. J. Physiol. R720-R729, 1983.
Lye, S.J., Sprague, C.L., Mitchell, B.F., and Challis, J.R.G. Activation of ovine fetal
adrenal function by pulsatile or continuous administration of adrenocorticotropin-
(1-24)1. Effects on fetal plasma corticosteroids. Endocrinol. 113:770-776, 1983.
Maclsaac, R.J., Bell, R.J., McDougall, J.G., Tregear, G.W., Wang, X. And Wintour, E.M.
Development of the hypothalamic-pituitary axis in the ovine fetus: Ontogeny of
action of ovine corticotropin-releasing factor. J. Dev. Physiol. 7:329, 1985.
Madill, D. and Bassett, J.M. Coerticosteroid release by adrenal tissue from foetal and
newborn lambs in response to corticotrophin stimulation in a perfusion system in
vitro. J. Endocrinol. 58:75-87, 1973.
Magyar, D M., Fridshal, D., Eisner, C.W., Glatz, T., Eliot, J., Klein, A.H., Lowe, K.C.,
Buster, J.E., and Nathanielsz, P.W. Time-trend analysis of plasma cortisol
concentrations in the fetal sheep in relation to partuirition. Endocrinol. 107:155-
159, 1980.
Malpas, P. Postmaturity and malformations of the foetus. J. Obstet. Gynecol. Brit.
Cwlth. 40:1046-1053, 1933.

149
Manda, G., Bonazzi, O., Pozzoni, L., Ferrari, A., Gardumi, M, Gregorini, L., and
Perondi, R. Baroreceptor control of artioventricular conduction in man. Circ.
Res. 44:752-758, 1979.
Manda, G., and Mark, A L. Arterial baroreflexes in human. In: Handbook of
Physiology: The Cardiovascular System. Peripheral Circulation and Organ Blood
Flow, Part Two, Volume Three. Shepherd, J.T., and Abboud, F.M., eds.,
Bethesda, MD.: American Physiological Society, 1983.
Mann, M R., Curet, L.B., and Colas, A.E. Aromatizing activity of placental microsomal
fractions from ewes in late gestation. J. Endocrinol. 65:117-125, 1975.
Maran, J.W., Carlson, D.E., Grizzle, W.E., Ward, D.G., and Gann, D.S. Organization of
the medial hypothalamus for control of adrenocorticotropin in the cat.
Endocrinol. 103: 957-970, 1978.
Mason, J.I., France, J.T., Magness, R.R., Murry, B.A., and Rosenfeld, C.R. Ovine
placental steroid 17 alpha-hydroxylase/C-17,20 lyase, aromatase, and sulphatase in
dexamethasone-induced and natural parturition. J.Endocrinol. 122:351-359,
1989.
Mathew, J. and Balasubramanian, A.S. Arylsulphatase C and estrone sulphatase of sheep
hypothalamus, preoptic area, and midbrain: separation by hydrophobic interaction
chromatography and evidence for differences in their lipid environment.
J.Neurochem. 39: 1205-1209, 1982.
McDonald, T.J., Hoffmann, G.E., Myers, D.A., and Nathanielsz, P.W. Hypothalamic
glucocorticoid implants prevent fetal ovine adrenocorticotropin secretion in
response to stress. Endocrinol. 127:2862-2868, 1990.
McDonald, T.J. and Nathanielsz, P.W. Bilateral destruction of the fetal paraventricular
nuclei prolongs gestation in shepp. Am. J. Obstet. Gynecol. 165:764-770, 1991.
McMillen, I.C. and Merei, J.J. Effect of gestational age, CRF, and cortisol on ACTH-
secretion from slices of fetal sheep pituitaries in an in vitro perfusion system.
Neuroendocrinol. 58:564-569, 1993.
Mroczek, W.J., Lee, W.R., David, M.E., and Finnerty, F A. Vasodialator administration
in the presence of beta-adrenergic blockade. Circulation. 53:985-988, 1976.
Mulvogue, H.M., McMillen, I.C., Robinson, P.M., and Perry, R.A. Immunocytochemical
localization of proyMSH, ACTH, and Pendorphin/plipotrophin in the fetal sheep
pituitary: An ontogenetic study. J. Dev. Physiol. 8:355-368, 1986.

150
Muratori, G. Histological observations on the structure of the carotid sinus in man and
mammals. In: Baroreceptors and Hypertension. Kedzi, P. ed. Oxford: Peragon
Press, 1967.
Nakayama, S. The circulatory and respiratory reflexes from the subclavian and
brachiocephalic artery. Jap. J. Physiol. 15:290-293, 1965.
Nash, A.R., Glenn, W.K., Moore, S.S., Kerr, J., Thompson, A.R., and Thompson, E.O.P.
Oestrogen sulfotransferase: molecular cloning and sequencing of cDNA for the
bovine placental enzyme. Aust. J. Biol. Sci. 41: 507-516, 1988.
Nathanielsz, P.W., Comlin, R.S., Silver, M., and Paisey, R.B. Cortisol metabolism in the
fetal and neonatal sheep. J. Reprod. Fert. Suppl. 16:39-59, 1972.
Nathanielsz, P.W., Eisner, C., Magyar, D., Fridshal, D., Freeman, A., and Buster, J.E.
Time trend analysis of plasma unconjugated and sulfoconjugated estrone and 3
beta-delta 5-steroids in fetal and maternal sheep plasma in relation to spontaneous
parturition at term. Endocrinol. 110: 1402-1407, 1982.
Nonindez, J.F. The aortic (depressor) nerve and its associated epitheloid body, the
glomus aorticum. Am. J. Anat. 57:259-301, 1935.
Nordmann, J. J. Ultrastructural morphometry of the rat neurohypophysis. J. Anat.
123:213-218, 1977.
Norman, L.J., Lye, S.J., Wlodek, M.E., and Challis, J.R.G. Changes in pituitary responses
to synthetic ovine corticotropin releasing factor in fetal sheep. Can. J. Physiol.
Pharmacol. 63:1398-1403, 1985.
Panaretto, B .A. Relationship of visceral blood flow to cortisol metabolism in cold-stressed
sheep. J. Endocrinol. 60:235-245, 1974.
Parrillo, J.E., and Fauci, A.S. Mechanisms of glucocorticoid action on immune processes.
Annu. Rev. Pharmacol. Toxicol. 19:179-201, 1979.
Patterson, J.Y.F., and Hills, F. The binding of cortisol by ovine plasma proteins. J.
Endocrinol. 37:261-268, 1976.
Perrin, M.H., Haas, Y., Rivier, J.E., and Vale, W.W. Corticotropin-releasing factor
binding to the anterior pituitary receptor is modulated by divalent cations and
guanyl nucleotides. Endocrinol. 118:1171 -1179, 1986.
Perry, R.A., Robinson, P.M., and Ryan, G.B. Ultrastructure of the pars intermedia of the
developing sheep hypophysis. Cell. Tis. Res. 224:369-381,1982.

151
Pescovitz, O.H., Cutler, G.B., and Loriaux, D L. Adrenocortical insufficiency In Becker,
KL ed. Principles and Practice of Endocrinology and Metabolism. Philadelphia:
Lippincott Co., 1990.
Pickering, T.G., Gribbin, B , Strange-Petersen, E., Cunningham, D J.C., and Sleight, P.
Effects of autonomic blockade on the baroreflex in man at rest and during exercise.
Circ. Res. 30:177-185, 1972.
Pirlde, Jr., J.C., and Gann, D.S. Restitution of blood volume after hemorrhage: Role of
the adrenal cortex. Am. J. Physiol. 230:1683-1687, 1976.
Platia, M.P., Fencl, M., Elkind-Hirsch, K.E., Canick, J.A., and Tulchinsky, D. Estrone
sulfatase activity in the human brain and estrone sulfate levels in the normal
menstrual cycle. J.SteroidBiochem. 21: 237-241, 1984.
Pomerantz, D.K., and Nalbandov, A.V. Androgen level in the sheep fetus during
gestation. Proc. Soc. Exp. Biol. Med. 149:413-416, 1975.
Pomerantz, S. M., and Sholl, S.A. Analysis of sex and regional differences in androgen
receptors in fetal rhesus monkey brain. Develop. Brain Res. 36:
151-156, 1987.
Power, S.G.A., and Challis, J.R.G. Tissue-specific concentration changes of estrone and
estradiol during spontaneous and ACTH-induced parturition in sheep. Can. J.
Physiol. Pharmacol. 65:130-135, 1987.
Raff, H., Kane, C., and Wood, C.E. Vasopressin responses to hypoxia and hypercapnia in
late-gestation fetal sheep. Am. J. Physiol. 260: R1077-R1081, 1991.
Raff, H., Merrill, D C., Skelton, M.M., Brownfield, M S., and Cowley, Jr., A.W. Control
of adrenocorticotropin secretion and adrenocortical sensitivity in
neurohypophysectomized conscious dogs: effects of acute and chronic vasopressin
replacement. Endocrinol. 122:1410-1418, 1988.
Raff, H., and Wood, C.E. Effect of age and blood pressure on the heart rate, vasopressin,
and renin response to hypoxia in fetal sheep. Am. J. Physiol. 263: R880-R884,
1992.
Rees, P.M. Electron microscopical observations on the architecture of the carotid sinus
walls, with special reference to the sinus portion. J. Anat. 103:35-47, 1968.
Rees, P.M., and Jepson, P. Measurement of arterial geometry and wall composition in the
carotid sinus baroreceptor area. Circ. Res. 26:461-467, 1970.

152
Reeves, W.B , Andreoli, T.E. The posterior pituitary and water metabolism. Williams
Textbook of Endocrinology Wilson, W.D and Foster, D.W. eds. Philadelphia:
Saunders, 1992.
Reichlin, S. Neuroendocrinology. Williams Textbook of Endocrinology. Wilson, W.D.
and Foster, D.W. eds. Philadelphia: Saunders, 1992.
Richard, R. Estrogen effects on pituitary-adrenal function via the hypothalamus and
hypophysis. Neuroendocrinol. 1:322-332, 1965.
Rivier, C., and Vale, W. Effect of antiotensin II on ACTH release in vivo: Role of
corticotropin-releasing factor. Reg. Peptides. 7:253-258, 1983a.
Rivier, C., and Vale, W. Interaction of corticotropin-releasing factor and arginine
vasopressin on adrenocorticotopin secretion in vivo. Endocrinol. 113:939-942,
1983b.
Robertson, H.A., and Smeaton, T.C. The concentration of unconjugated oestrone,
oestradiol-17a and oestradiol-173 in the maternal plasma of the pregnant ewe in
relation to the initiation of parturition and lactation. J. Reprod Fert. 35:461-468,
1973.
Robillard, J.E., Weitzman, R.E., Tisher, D.A., and Smith, F.G. The dynamics of
vasopressin release and blood volume regulation during fetal hemorrhage in the
lamb fetus. Pediatr. Res. 13:606-610, 1979.
Robinson, B.F., Epstein, S.E., Beiser, G.D., and Braunwald, E. Control of heart rate by
theautonomic nervous system. Studies in man on the interrelations between
baroreceptor mechanisms and exercise. Circ.Res. 19:400-411, 1966.
Robinson, P.M., Rowe, E.J., and Wintour, E.M. The histogenesis of the adrenal cortex in
the foetal sheep. Acta. Endocrinol. 91:134-149, 1979.
Rose, J.C., Meis, P., and Morris, M. Ontongeny of endocrine (ACTH, vasopressin,
cortisol) responses to hypotension in lamb fetuses. Am. J. Physiol. 240:E656-
E661, 1981.
Rose, J.C., Meis, P.J., Urban, R.B., and Greiss, Jr., F.C. In vivo evidence for increased
adrenal sensitivity to adrenocorticotropin-(l-24) in the lamb fetus late in gestation.
Endocrinol. 111:80-85, 1982.
Rose, J.C. Morris, M., and Meis, P.J. Hemorrhage in newborn lambs: Effects on arterial
blood pressure, ACTH, cortisol, and vasopressin. Am. J. Physiol. 240.E585-
E590,1981.

153
Rosenfeld, C.R., Worley, R.J., Milewich, L , Grant, N.F., and Parker, Jr, R. Ovine
Fetoplacental sulfoconjugation and aromatization of dehydroepiandrosterone.
Endocrinol. 106: 1971-1979, 1980.
Rossier, G., and Pierrepoint, C.G. Oestrogen metabolism in sheep myometrium. J.
Reprod. Fert. 37:43-49, 1974.
Rudolph, A. M. Congenital diseases of the heart. Chicago: Year Book Medical
Publishers, 1974.
Sachs, H., Fawcett, Y., Takabatake, Y., and Portanova, R. Biosynthesis and release of
vasopressin and neurophysin. Rec. Prog. Horm. Res. 25:447-491, 1969.
Saffran, M., and Schally, A.V. The release of corticotrophin by anterior pituitary tissue in
vitro. Can. J. Biochem. Physiol. 33:408-415,1955.
Saoud, C.J., and Wood, C.E. Ontogeny and molecular weight of immunoreactive arginine
vasopressin and corticotropin-releasing factor in the ovine fetal hypothalamus.
Peptides 17: 55-61, 1996.
Saoud, C.J., and Wood, C.E. Modulation of Ovine Fetal Adrenocorticotropin Secretion
by Androstenedione and 173-Estradiol. Am.J.Physiol. 272: R1128-R1134, 1997.
Sar, M. Estradiol is concentrated in tyrosine hydroxylase-containing neurons of the
hypothalamus. Science. 223:928-940, 1984.
/
Sato, T., Sato, M., Shinsako, J., and Dallman, M.F. Corticosterone-induced changes in
hypothalamic corticotropin-releasing factor (CRF) content after stress.
Endocrinol. 97:265-274, 1975.
Sawchenko, P.E. Evidence for a local site of action for glucocorticoids in inhibiting CRF
and vasopressin expression in the paraventricular nucleus. Brain Res. 403:213-
224, 1987.
Sawchenko, P.E., and Swanson, L.W. The organization of noradrenergic pathways from
the brainstem to the paraventricular and supraoptic nuclei in the rat. Brain Res.
4:275-325, 1980.
Sawchenko, P.E., Swanson, L.W., and Vale, W.W. Co-expression of corticotropin¬
releasing factor and vasopressin immunoreactivity in parvocellular neurosecretory
neurons of the adrenalectomized rat. Neurobiol. 81:1883-1887,1984.
Scher, A M. and Ito, C.S. Regulation of arterial blood pressure by aortic baroreceptors in
the unanesthetized dog. Circ. Res. 42:230-236, 1978.

154
Schwartz, J., Billestrup, N., Perrin, M., Rivier, J., and Vale, W. Identification of
corticotropin-releasing factor (CRF) target cells and effects of dexamethasone on
binding in anterior pituitary using a fluorescent analog of CRF. Endocrinol.
119:2376-2382, 1986.
Shen, E., Dun, S.L., Ren, C., Bennett-Clarke, C., and Dun, N.J. Hypotension
preferentially induces c-fos immunoreactivity in supraoptic vasopressin neurons.
Brain Res. 593:136-139, 1992.
Sheward, W.J., Lim, A., Alder, B., Copolov, D, Dow, R.C., and Fink, G. Hypothalamic
release of atrial natruiretic factor and beta endorphin into rat hypophysial portal
plasma: Relationship to oestrous cycle and effect of hypophysectomy. J.
Endocrinol. 131:113-125, 1991.
Silverman, A.J., and Zimmerman, E.A. Ultrastructural immunocytochemical localization
of neurophysin and vasopressin in the median eminence and posterior pituitary of
the guinea pig. Cell Tiss. Res. 159:291-301,1975.
Simerly, R.B., Chang, C., Muramatsu, M., and Swanson, L.W. Distribution of androgen
and estrogen receptor mRNA-containing cells in the rat brain: an in situ
hybridization study. J.Comp.Neurol. 294: 76-95, 1990.
Simon, A.C.H., Safar, ME., Weiss, Y.A., London, J.M., and Milliez, P.L. Varoreflex
sensitivity and cardiopulmonary blood volume in normotensive and hypertensive
patients. Br. Heart J. 39:799-805, 1977.
Skowsky, W.R., Swan, L., and Smith, P. Effects of sex steroid hormones on arginine
vasopressin in intact and castrated male and female rats. Endocrinol. 104:105-
108, 1979.
Spiess, J., Rivier, J., Rivier, C., and Vale, W. Primary structure of corticotropin-releasing
factor from ovine hypothalamus. Proc. Nat. Acad Sci. USA 78:6517-6521, 1981.
Steele, PA., Flint, A.P.F., and Turnbull, A C. Acitivty of steroid C-17,20 lyase in the
ovine placenta: Effect of exposure to foetal glucocorticoid. J. Endocrinol.
69:239-246, 1976.
Stein, C., Hille, A., Seidel, J., Rijnbout, S., Waheed, A., Schmidt, B., Geuze, H., and von
Figura, K. Cloning and expression of human steroid-sulfatase. Membrane
topology, glycosylation, and subcellular distribution in BHK-21. J.Biol.Chem
264: 13865-13872, 1989.
Strott, C.A., Sundel, H., and Stahlman, M.T. Maternal and fetal plasma progesterone,
cortisol, testosterone, and 17 beta-estradiol in preparturient sheep: response to
fetal ACTH infusion. Endocrinol. 95: 1327-1339, 1974.

155
Takeshita, A., Mark, A.L., Eckberg, D.L., and Abboud, F.M. Effect of central venous
pressure on arterial baroreflex control of heart rate Am. J. Physiol. 236 (Heart
Circ. Physiol.)5:H42-H47, 1979.
Telegdy, G., Schreiberg, G., and Endroczi, E. Effect of oestrogens implanted in to the
hypothalamus on the activity of the pituitary-adrenocortical system. Physiol.
25:229-234, 1963.
Thomas, A.L., Krane, E.J., and Nathanielsz, P.W. Changes in the fetal thyroid axis after
induction of premature partuirition by low dose continuous intravascular cortisol
infusion to the fetal sheep at 130 days gestation. Endocrinol. 103:17-23, 1978.
Touw, K., Fink, G., Haywood, J.R., Shaffer, R.A., and Brody, M.J. Elevated neurogenic
vasoconstrictor tone is the mechanism of hypertension in rats with aortic
varoreceptor reafferentation. Fed. Proc. 38:1232, 1979.
Tsang, C .P .W. Changes in plasma levels of estrone sulfate and estrone in the pregnant
ewe around parturition. Steroids 23: 855-868, 1974.
Ueda, S., Fortune, V., Bull, B.S., Valenzuela, G.J., and Longo, L.D. Estrogen effects on
plasma volume, arterial blood pressure interstitial space, plasma proteins, and
blood viscosity in sheep. Am. J. Obstet. Gynecol. July: 195-200, 1986.
Vale, W., Spiess, J., Rivier, C., and Rivier, J. Characterization of a 41-residue ovine
hypothalamic peptide that stimulates secretion of corticotropin and 3-endorphin.
Science. 213:1392-1397, 1981.
r
Vale, W. Vaughan, J., Smith, M., Yamamoto, G, Rivier, F., and Rivier, C. Effects of
synthetic ovine corticotropin-releasing factor, glucocorticoids, catecholamines,
neurohypophysial peptides, and other substances on cultured corticotropic cells.
Endocrinol. 113:1121-1131,1983.
Vander, A.J. Renal Physiology. 3rd ed. New York: McGraw, 1985.
Viau, V., and Meaney, M.J. Variations in the hypothalamic-pituitary-adrenal response to
stress during the estrous cycle in the rat. Endocrinol. 129:2503-2511, 1991.
Ward, D.G., Bolton, M.G., and Gann, D.S. Inhibitory and facilitatory areas of the ventral
midbrain mediating release of corticotropin in the cat. Endocrinol. 102: 1147-
1154, 1978.
Werber, A.H., and Fink, G.D. Aortic varoreceptor denervation in the rat as a model for
vorderline hypertension in the human. Fed. Proc. 38:1232, 1979.

156
Whitnall, M.H., Mezey, E., and Gainer, H. Co-localization of corticotropin-releasing
factor and vasopressin in median eminence neurosecretory vesicles. Nature
317:247-250, 1985.
Whitnall, M.H., Smyth, D., and Gainer, H. Vasopressin coexists in half of the
corticotropin-releasing factor axons present in the external zone of the median
eminence in normal rats. Neuroendocrinol. 45:420-424,1987.
Winer, B.J. Statistical Principles in Experimental Design. New York. McGraw-Hill, 1971.
Wintour, E.M. Developmental aspects of the hypothalamic-pituitary-adrenal axis. J. Dev.
Physiol. 6:291-299, 1984.
Wintour, E.M., Brown, E.H., Denton, D.A., Hardy, K.J., McDougall, J.G., Oddie, C.J.,
and Whipp, G.T. The ontogeny and regulation of corticosteroid secretion by the
ovine foetal adrenal: in vivo and in vitro studies. Acta. Endocrinol. 79:301-316,
1975.
Wintour, E.M., Brown, E.H., Denton, D A. Hardy, K.J., McDougall, J.G., Robinson,
P.M., Rowe, E.J., and Whipp. G.T. In vitro and in vivo adrenal cortical steroid
production by fetal sheep: Effect of antiotensin II, sodium deficiency, ACTH. Res.
Steroids 3:301-316, 1977.
Wood, C.E. Sensitivity of cortisol-induced inhibition of ACTH and renin in fetal sheep.
Am. J. Physiol. 252:R795-R802, 1986.
r
Wood, C.E. Does a decrease in cortisol negative feedback efficacy preded ovine
parturition? Am. J. Physiol. 252:R624-R627, 1987.
Wood, C.E., Insensitivity of near-term fetal sheep to cortisol: Possible relation to the
control of parturition. Endocrinol. 122:1565-1572, 1988.
Wood, C. E. Sinoaortic denervation attenuates the reflex responses to hypotension in
fetal sheep. Am. J. Physiol. 256: R1103-R1110, 1989.
Wood, C. E. Baroreflex and chemoreflex control of fetal hormone secretion.
Reprod. Fértil. Dev. 7: 479-489, 1995.
Wood, C.E., Chen, H.G., and Bell, M E. Role of vagosympathetic fibers in the control of
adrenocorticotropic hormone, vasopressin, and renin responses to hemorrhage in
fetal sheep. Circ. Res. 64:515-523, 1989.
Wood, C.E., Cudd, T.A., Kane, C., and Engelke, K. Fetal ACTH and blood pressure
responses to thromboxane mimetic U46619. Am. J. Physiol. 265:R858-R862,
1993.

157
Wood, C. E., Kane, C., and Raff, H. Peripheral chemoreceptor control of fetal renin
responses to hypoxia and hypercapnia. Circ. Res. 67: 722-732, 1990.
Wood, C.E., Keil, L.C., and Rudolph, A M. Hormonal and hemodynamic responses to
vena caval obstruction in fetal sheep. Am. J. Physiol. 243:E278-E286, 1982.
Wood, C.E., and Keller-Wood, M. Induction of parturition by cortisol: effects on
negative feedback sensitivity and plasma CRF. J. Dev. Physiol. 16:287-292,
1991.
Wood, C.E., and Rudolph, A M. Carotid vascular control of ACTH secretion in lambs.
Am. J. Physiol. 244:E555-E559, 1983.
Wynn, P.C., Aguilera, G., Morell, J., and Catt, K.J. Properties and regulation of high-
affinity pituitary receptors for corticotropin-releasing factor. Biochem. Biophys.
Res. Comm. 110:602-608,1983.
Wynn, PC., Harwood, J.P., Catt, K.J., and Auilera, G. Regulation of corticotropin¬
releasing factor (CRF) receptors in the rat pituitary gland: Effects of
adrenalectomy on CRF receptors and corticotroph responses. Endocrinol.
116:1653-1659, 1985.
Wynn, P.C., Harwood, J.P., Catt, K.J., and Aguilera, G. Corticotropin-releasing factor
(CRF) induces desensitization of the rat pituitary CRF receptor-adenylate cyclase
complex. Endocrinol. 122:351-358, 1988.
Yu, H.K., Cabalum, T., Jansen, C.A., Buster, J.E., and Nathanielsz, P.W.
Androstenedione, testosterone, and estradiol concentrations in fetal and maternal
plasma in late pregnancy in the sheep. Endocrinol. 113:2216-2220, 1983.
Zar, J.H. Biostatistical analysis. Englewood Cliffs, NJ: Prentice-Hall, 1984.

BIOGRAPHICAL SKETCH
Scott Christopher Purinton was bom November 14, 1970, in Saco, Maine. His
family moved to Orange Park, Florida, where Purinton attended Orange Park High
School and graduated salutatorian in June 1989. Purinton received his undergraduate
education from Furman University in Greenville, South Carolina He earned his B.S.
degree in the field of biology in May 1993. For the two years following graduation,
Purinton worked as a laboratory technician for Charles E. Wood at the University of
Florida, College of Medicine, in the Department of Physiology. This experience led him
to enter a doctoral program under the tutelage of Dr. Wood in August 1995, to study
cardiovascular and endocrine physiology. He was awarded his Ph.D. from the University
of Florida in May 1999. Following graduation, Purinton will attend medical school and
marry his best friend, Dr. LeighAnn Stubley.
158

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Charles E. Wood, Chair
Professor of Physiology
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
'¿lljAJUi '
Maureen Keller-Wood
Associate Professor of Pharmacodynamics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Pushpa S. Kalra
Professor of Physiology
• I certify that I have read this study and that in my opinion it conforms to
accepfable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy. *
Jgfpes W. Simpkins
rofessor of Pharmacodynamics
This dissertation was submitted to the Graduate Faculty of the College of
Medicine and to the Graduate School and was accepted as partial fulfillment of the
requirements for the degree of Doctor of Philosophy.
May 1999
CyC
c '
Dean, College of Medicine
Dean, Graduate School

UNIVERSITY OF FLORIDA



xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID ENA7TSSP2_1R1CZ8 INGEST_TIME 2014-06-24T20:53:25Z PACKAGE AA00022317_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES



PAGE 1

(6752*(1&(175$/ 1(59286 6<67(0 ,17(5$&7,216 ,1 &$5',29$6&8/$5 &21752/ $1' 3$5785,7,21 %\ 6&277 &+5,6723+(5 385,1721 $ ',66(57$7,21 35(6(17(' 72 7+( *5$'8$7( 6&+22/ 2) 7+( 81,9(56,7< 2) )/25,'$ ,1 3$57,$/ )8/),//0(17 2) 7+( 5(48,5(0(176 )25 7+( '(*5(( 2) '2&725 2) 3+,/2623+< 81,9(56,7< 2) )/25,'$

PAGE 2

7R WKH IROORZLQJ GHGLFDWH WKLV GLVVHUWDWLRQ :LWKRXW WKHP QRQH RI WKLV ZRXOG KDYH EHHQ SRVVLEOH 7R P\ IDPLO\ 7KDQN \RX IRU \RXU VXSSRUW DQG HQFRXUDJHPHQW 7R /HLJK $QQ 7KDQN \RX IRU EHOLHYLQJ LQ DQG VWDQGLQJ E\ PH 7R 'DYLG 0 %XVK 7KDQN \RX IRU \RXU IULHQGVKLS :LVK \RX FRXOG KDYH EHHQ KHUH

PAGE 3

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fV ORYH DQG VXSSRUW FDQ WUXO\ VD\ WKDW QRQH RI WKLV ZRXOG KDYH EHHQ SRVVLEOH 6KH KDV WDXJKW PH PRUH DERXW P\VHOI WKDQ FRXOG HYHU KDYH OHDUQHG IURP JUDGXDWH VFKRRO )RU WKLV ZLOO EH IRUHYHU LQGHEWHG WR KHU LLL

PAGE 4

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

PAGE 5

5HVXOWV 'LVFXVVLRQ 1(8521$/ $&7,9$7,21 ,1 $1 (675$',2/ +<327(16,9( &$527,' 6,186 '(1(59$7(' 29,1( 02'(/ ,QWURGXFWLRQ 0HWKRGV DQG 0DWHULDOV 5HVXOWV 'LVFXVVLRQ 2172*(1< 2) (6752*(1 68/)$7$6( $1' (6752*(1 68/)275$16)(5$6( ,1 %5$,1 5(*,216 ,03257$17 )25 +<327+$/$0863,78,7$5<$'5(1$/ $;,6 &21752/ a ,QWURGXFWLRQ 0HWKRGV DQG 0DWHULDOV 5HVXOWV 'LVFXVVLRQ 6800$5< $1' &21&/86,216 5()(5(1&(6 %,2*5$3+,&$/ 6.(7&+ Y

PAGE 6

$EVWUDFW RI 'LVVHUWDWLRQ 3UHVHQWHG WR WKH *UDGXDWH 6FKRRO RI WKH 8QLYHUVLW\ RI )ORULGD LQ 3DUWLDO )XOILOOPHQW RI WKH 5HTXLUHPHQWV IRU WKH 'HJUHH RI 'RFWRU RI 3KLORVRSK\ (6752*(1&(175$/ 1(59286 6<67(0 ,17(5$&7,216 ,1 &$5',29$6&8/$5 &21752/ $1' 3$5785,7,21 %\ 6FRWW &KULVWRSKHU 3XULQWRQ 0D\ &KDLUPDQ &KDUOHV ( :RRG 0DMRU 'HSDUWPHQW 3K\VLRORJ\ ,Q WKH IHWDO VKHHS SDUWXULWLRQ LV WULJJHUHG E\ DQ LQFUHDVH LQ WKH DFWLYLW\ RI WKH IHWDO K\SRWKDODPXVSLWXLWDU\DGUHQDO +3$f D[LV 3DUWXULWLRQ FDQ EH GHOD\HG E\ GHVWUXFWLRQ RI WKH SLWXLWDU\ RU VWLPXODWHG E\ LQIXVLRQV RI DGUHQRFRUWLFRWURSLQ $&7),f RU I JOXFRFRUWLFRLGV 7KH ODVW GD\V RI JHVWDWLRQ DUH PDUNHG E\ DQ LQFUHDVH LQ WKH DFWLYLW\ RI WKH IHWDO K\SRWKDODPXV DV VHHQ E\ HOHYDWHG OHYHOV RI IHWDO SODVPD $&7+ (VWURJHQ KDV EHHQ VKRZQ WR WULJJHU WKLV SUHSDUWXULHQW LQFUHDVH LQ $&7+ K\SRWKHVL]HG WKDW HVWURJHQf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

PAGE 7

$&7+ YDVRSUHVVLQ DQG FRUWLVROf WR K\SRWHQVLRQ E\ EUDFKLRFHSKDOLF RFFOXVLRQ IXUWKHU K\SRWKHVL]HG WKDW HVWURJHQfV DFWLRQV ZLWKLQ WKH IHWDO FHQWUDO QHUYRXV V\VWHP DUH FHQWHUHG ZLWKLQ WKH QXFOHXV RI WKH WUDFWXV VROLWDULXV 176f WKH SDUDYHQWULFXODU QXFOHXV 391f RU ZLWKLQ WKH FRPSRQHQWV RI FDUGLRYDVFXODU UHJXODWRU\ FHQWHUV UHFHLYLQJ LQSXW IURP EDURUHFHSWRUV RU FKHPRUHFHSWRUV XVHG LPPXQRKLVWRORJLFDO WHFKQLTXHV WR LGHQWLI\ WKH QHXURDQDWRPLFDO UHJLRQV ZKLFK DUH DFWLYDWHG E\ K\SRWHQVLRQ DQG VXEVHTXHQWO\ WKRVH DUHDV PRGLILHG E\ HVWURJHQfV DFWLRQ DQG EDURUHFHSWRUFKHPRUHFHSWRU GHQHUYDWLRQ 7KH XVH RI WKHVH WHFKQLTXHV DOORZV IRU WKH PHDVXUHPHQW RI FIRV H[SUHVVLRQ DQ HDUO\ UHVSRQVH JHQH ZKLFK FDQ EH XVHG DV D PDUNHU RI QHXURQDO DFWLYLW\ IRXQG WKDW HVWUDGLRO LPSODQWHG DQLPDOV KDG PRUH FIRV DEXQGDQFH DQG KHQFH PRUH FIRV VWDLQLQJ LQ UHOHYDQW EUDLQ DUHDV 176 391 HWFf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

PAGE 8

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fV ODERUDWRU\ DW WKH 8QLYHUVLW\ RI )ORULGD *DLQHVYLOOH )ORULGD WKHUH DUH QR GDWD FRQFHUQLQJ WKH LQIOXHQFH RI HVWURJHQ RQ FDUGLRYDVFXODU IXQFWLRQ LQ WKH IHWXV ,QFUHDVHV LQ SODVPD HVWURJHQ FRQFHQWUDWLRQ DUH WKRXJKW WR EH DQ LPSRUWDQW FRPSRQHQW RI WKH PDWHUQDO DGDSWDWLRQ WR SUHJQDQF\ 'XULQJ JHVWDWLRQ WKH JURZLQJ IHWXV DQG HQODUJLQJ XWHUXV H[KLELW DQ LQFUHDVLQJ GHPDQG IRU R[\JHQ )RU WKLV UHDVRQ WKH XWHURn SODFHQWDO EORRG IORZ FRQVWLWXWHV DQ HYHULQFUHDVLQJ SURSRUWLRQ RI PDWHUQDO FDUGLDF RXWSXW 5XGROSK f 7KH LQFUHDVHG IORZ GHPDQGV DUH VXSSRUWHG E\ DQ LQFUHDVH LQ PDWHUQDO EORRG YROXPH DQG DQ LQFUHDVH LQ YDVFXODU FRPSOLDQFH /RQJR f 7KLV W\SLFDOO\ SURGXFHV D SK\VLRORJLFDO FRQGLWLRQ LQ ZKLFK PDWHUQDO FDUGLDF RXWSXW DQG EORRG YROXPH DUH KLJKHU WKDQ LQ WKH QRQSUHJQDQW VWDWH KRZHYHU PDWHUQDO DUWHULDO SUHVVXUH DQG FHQWUDO

PAGE 9

YHQRXV SUHVVXUH DUH ORZHU .HOOHU:RRG /RQJR f 7KRXJK WKLV UHVSRQVH LV QRW HQWLUHO\ FRQVLVWHQW DPRQJ PDPPDOLDQ VSHFLHV LW KDV EHHQ SURSRVHG WKDW HVWURJHQ SOD\V DQ LPSRUWDQW UROH LQ WKLV SKHQRPHQRQ 8HGD f 1R VWXGLHV KDYH \HW EHHQ FRQGXFWHG WKDW LQYHVWLJDWH WKH HIIHFWV RI HVWURJHQ RQ FDUGLRYDVFXODU UHIOH[ UHVSRQVLYHQHVV %\ GRLQJ VXFK VWXGLHV LQ IHWDO VKHHS LW LV SRVVLEOH WR LQYHVWLJDWH WKH PHFKDQLVP EHKLQG SDUWXULWLRQ EHFDXVH DQ LQFUHDVH LQ WKH DFWLYLW\ RI WKH K\SRWKDODPXVSLWXLWDU\DGUHQDO +3 $f D[LV KDV EHHQ VKRZQ WR WULJJHU SDUWXULWLRQ /LJJLQV HW DO f %RWK DQGURJHQ DQG HVWURJHQ UHFHSWRUV FDQ EH IRXQG LQ WKH DQWHULRU SLWXLWDU\ DV ZHOO DV LQ YDULRXV UHJLRQV RI WKH EUDLQ LQFOXGLQJ WKH K\SRWKDODPXV DQG WKH EUDLQVWHP 5HFHSWRUV DQG WKH P51$ HQFRGLQJ IRU WKHVH UHFHSWRUV FDQ EH IRXQG LQ WKH YDULRXV VWUXFWXUHV LQFOXGLQJ WKH DUFXDWH QXFOHXV DQG WKH SUHRSWLF QXFOHXV 6LPHUO\ HW DO f ,Q DGGLWLRQ WR WKH VWUXFWXUHV NQRZQ IRU FRQWURO RI WKH K\SRWKDODPXVSLWXLWDU\JRQDGDO D[LV DQG IRU UHSURGXFWLYH EHKDYLRU DQGURJHQ DQG HVWURJHQ UHFHSWRUV DUH IRXQG LQ UHJLRQV EHWWHU NQRZQ IRU FRQWURO RI WKH K\SRWKDODPXVSLWXLWDU\DGUHQDO D[LV LQFOXGLQJ WKH W SDUDYHQWULFXODU QXFOHXV 391f DQG KLSSRFDPSXV /HKPDQ HW DO 3RPHUDQW] DQG 6KROO f (VWURJHQ UHFHSWRUV KDYH EHHQ ORFDOL]HG LQ WKH PDJQRFHOOXODU SRUWLRQ RI WKH 391 DQG PD\ WKHUHIRUH LQIOXHQFH HLWKHU R[\WRFLQ YDVRSUHVVLQ $93f RU ERWK /HKPDQ HW DO 6LPHUO\ HW DO f %RWK DQGURJHQ DQG HVWURJHQ UHFHSWRUV DUH DOVR IRXQG DW VLWHV ZKLFK DUH NQRZQ WR EH UHOD\ FHQWHUV RQ DIIHUHQW SDWKZD\V >WKH QXFOHXV RI WKH WUDFWXV VROLWDULXV RU 176f@ PHGLDWLQJ DGUHQRFRUWLFRWURSLF KRUPRQH $&7+f UHVSRQVHV WR VWUHVVHV VXFK DV K\SR[LD K\SHUFDSQLD DQG K\SRWHQVLRQ 6LPHUO\ HW DO f )LQDOO\ HVWURJHQ UHFHSWRUV KDYH EHHQ ORFDOL]HG LQ *$%$HUJLF FHOOV VXJJHVWLQJ WKH SRVVLEOH

PAGE 10

LQWHUDFWLRQ RI HVWURJHQ DQG $&7+ VHFUHWLRQ YLD WKLV QHXURQDO V\VWHP ZLWKLQ WKH EUDLQ +HUELVRQ HW DO f 7KH IRFXV RI WKH H[SHULPHQWV LV WKH QHXURHQGRFULQH $&7+ $93f UHVSRQVLYHQHVV WR K\SRWHQVLRQ 2I SDUWLFXODU LQWHUHVW WR PH DW WKH SUHVHQW WLPH LV WKH UHODWLRQVKLS EHWZHHQ QHXURHQGRFULQH PHFKDQLVPV FRQWUROOLQJ $93 DQG $&7+ DQG KRZ WKH\ UHJXODWH WKH FDUGLRYDVFXODU V\VWHP RI WKH IHWXV DV ZHOO DV WULJJHU SDUWXULWLRQ 7KHVH WZR KRUPRQDO V\VWHPV DUH LQH[WULFDEO\ OLQNHG ,Q WKH IHWXV DV LQ WKH DGXOWf DIIHUHQW EDURUHFHSWRU SDWKZD\V DUH VKDUHG E\ ERWK HQGRFULQH V\VWHPV ,W LV OLNHO\ WKDW WKH FHQWUDO SDWKZD\V FDUU\LQJ DIIHUHQW LQIRUPDWLRQ IURP WKH 176 WR WKH K\SRWKDODPXV DUH DOVR PRVWO\ VKDUHG E\ WKH WZR V\VWHPV $93 LV V\QWKHVL]HG LQ WKH 391 RI WKH K\SRWKDODPXV DQG FDQ EH IRXQG LQ ERWK PDJQRFHOOXODU DQG SDUYRFHOOXODU QHXURQV SURMHFWLQJ WR WKH SRVWHULRU SLWXLWDU\ DQG PHGLDQ HPLQHQFH UHVSHFWLYHO\f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f ZKLFK DOVR VWLPXODWHV WKH UHOHDVH RI $&7+ IURP WKH DQWHULRU SLWXLWDU\ 3DUYRFHOOXODU &5+ QHXURQV SURMHFW D[RQV WR YDULRXV SDUWV RI WKH EUDLQ LQ DGGLWLRQ WR WKH PHGLDQ HPLQHQFH 0RVW QRWDEO\ WKHVH QHXURQV SURMHFW WR WKH FDUGLRYDVFXODU FRQWURO FHQWHUV LQ WKH K\SRWKDODPXV DQG PHGXOOD :KHQ UHOHDVHG IURP WKHVH QHXURQV &5+ VWLPXODWHV LQFUHDVHV LQ V\PSDWKHWLF HIIHUHQW WRQH PHDVXUHG LQ WKH FDUGLRYDVFXODU V\VWHP DV DQ LQFUHDVH LQ EORRG SUHVVXUH DQG KHDUW UDWH DQG D UHGLVWULEXWLRQ

PAGE 11

RI FDUGLDF RXWSXW /HQ] HW DO f $FFRUGLQJO\ LW KDV EHHQ VXJJHVWHG WKDW &5+ LV WKH WUDQVPLWWHU ZKLFK FRRUGLQDWHV DOO RI WKH HQGRFULQH DQG FDUGLRYDVFXODU UHVSRQVHV WR VWUHVV LQFOXGLQJ K\SRWHQVLRQ /HQ] HW DO f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f &5+ SDWKZD\V 6SHFLILF $LPV DQG +\SRWKHVHV ,Q WKH IROORZLQJ H[SHULPHQWV D FRPELQDWLRQ RI LQ YLYR ZKROH DQLPDOf DQG LQ YLWUR I LPPXQRKLVWRFKHPLVWU\f WHFKQLTXHV ZDV HPSOR\HG $LP 7KH DFWLRQV RI HVWURJHQ RQ IHWDO FDUGLRYDVFXODU UHIOH[ UHVSRQVLYHQHVV WR K\SRWHQVLRQ ZLOO EH PHDVXUDEOH LQ LQWDFW IHWXVHV EXW QRW LQ EDUR DQG FKHPRGHQHUYDWHG IHWXVHV 5HVHDUFK KDV SUHYLRXVO\ GHPRQVWUDWHG WKDW WKH FRPELQHG EDUR DQG FKHPR GHQHUYDWLRQ DWWHQXDWHV DSSUR[LPDWHO\ bf WKH UHIOH[ KRUPRQDO DQG KHPRG\QDPLF UHVSRQVHV WR PRGHUDWH bf UHGXFWLRQ LQ DUWHULDO EORRG SUHVVXUH SURSRVHG WKDW WKH LQWHUUXSWLRQ RI WKH DIIHUHQW SDWKZD\V ZRXOG HOLPLQDWH WKH HIIHFW RI HVWURJHQ RQ WKH UHIOH[ FDUGLRYDVFXODU UHVSRQVLYHQHVV ,I VR LW FRXOG FRQFOXGHG WKDW HVWURJHQ DFWV RQ ZLWKLQ RU UHTXLUHV LQSXW IURP WKH DIIHUHQW EDURUHFHSWRU DQG FKHPRUHFHSWRU SDWKZD\V

PAGE 12

$LP 7KH DFWLRQV RI HVWURJHQ ZLWKLQ WKH IHWDO FHQWUDO QHUYRXV V\VWHP DUH FHQWHUHG ZLWKLQ WKH QXFOHXV RI WKH WUDFWXV VROLWDULXV WKH SDUYRFHOOXODU QHXURQV RI WKH SDUDYHQWULFXODU QXFOHXV RU ZLWKLQ WKH FRPSRQHQWV RI WKH FDUGLRYDVFXODU UHJXODWRU\ FHQWHUV UHFHLYLQJ LQSXW IURP EDURUHFHSWRUV RU FKHPRUHFHSWRUV XVHG LPPXQRKLVWRORJLFDO WHFKQLTXHV WR LGHQWLI\ WKH QHXURDQDWRPLFDO UHJLRQV ZKLFK ZHUH DFWLYDWHG E\ K\SRWHQVLRQ DQG VXEVHTXHQWO\ WKRVH DUHDV PRGLILHG E\ HVWURJHQfV DFWLRQ DQG EDUR DQG FKHPRGHQHUYDWLRQ 7KH XVH RI WKHVH WHFKQLTXHV DOORZV IRU WKH PHDVXUHPHQW RI FIRV H[SUHVVLRQ WKH SURWHLQ SURGXFW RI WKH HDUO\ UHVSRQVH JHQH ZKLFK FDQ EH XVHG DV D PDUNHU RI QHXURQDO DFWLYLW\ SURSRVHG WKDW HVWUDGLRO LPSODQWHG DQLPDOV ZLOO KDYH PRUH )RV DFWLYLW\ DQG KHQFH PRUH FIRV VWDLQLQJ LQ UHOHYDQW EUDLQ DUHDV 176 391 HWFf DV FRPSDUH WR FRQWURO DQLPDOV )XUWKHUPRUH LW LV H[SHFWHG WKDW FIRV VWDLQLQJ ZLOO EH QHJOLJLEOH LQ GHQHUYDWHG DQLPDOV )LQDOO\ FIRV VWDLQLQJ VKRXOG EH VLJQLILFDQWO\ HOHYDWHG LQ DOO K\SRWHQVLYH DQLPDOV DV FRPSDUHG WR QRUPRWHQVLYH DQLPDOV ,Q DGGLWLRQ WR WKH FIRV LPPXQRKLVWRFKHPLFDO VWXGLHV WKH DFWLRQ RI HVWURJHQ VXOIDWDVH DQG HVWURJHQ VXOIRWUDQVIHUDVH ZDV LQYHVWLJDWHG 6LQFH WKH FRQFHQWUDWLRQ RI HVWURJHQ VXOIDWH SUHFHGHV WKH LQFUHDVH LQ +3$ D[LV DFWLYLW\ 1DWKDQLHOV] HW DO f DQG VLQFH FRQMXJDWHG HVWURJHQV FLUFXODWH LQ PXFK KLJKHU FRQFHQWUDWLRQV WKDQ XQFRQMXJDWHG HVWURJHQV &DUQHJLH DQG 5REHUWVRQ 7VDQJ f K\SRWKHVL]HG WKDW WKHVH HQ]\PHV ZRXOG EH SUHVHQW LQ EUDLQ DUHDV LPSRUWDQW IRU +3$ D[LV FRQWURO ,I WUXH D ORFDO PHFKDQLVP ZRXOG EH LQ SODFH IRU FRQYHUVLRQ RI ELRORJLFDOO\ LQDFWLYH WR DFWLYH HVWURJHQ ([SHULPHQWDO 3URWRFRO 'HVLJQ $ WRWDO RI SUHJQDQW HZHV ZHUH VWXGLHG SHU H[SHULPHQWDO JURXSf $QLPDOV ZHUH UDQGRPO\ DVVLJQHG WR WKH IROORZLQJ JURXSV

PAGE 13

SODFHER LPSODQW LQWDFW VKDPGHQHUYDWHGfQRUPRWHQVLYH IHWXVHV SODFHER LPSODQW FDURWLG VLQXV GHQHUYDWHG QRUPRWHQVLYH IHWXVHV SODFHER LPSODQW LQWDFW VKDPGHQHUYDWHGf IHWXVHV VXEMHFWHG WR K\SRWHQVLRQ SODFHER LPSODQW FDURWLG VLQXV GHQHUYDWHG IHWXVHV VXEMHFWHG WR K\SRWHQVLRQ HVWUDGLRO LPSODQW PJ GD\ UHOHDVHf LQWDFW VKDPGHQHUYDWHGf QRUPRWHQVLYH IHWXVHV HVWUDGLRO LPSODQW PJ GD\ UHOHDVHf FDURWLG VLQXV GHQHUYDWHG QRUPRWHQVLYH IHWXVHV HVWUDGLRO LPSODQW PJ GD\ UHOHDVHf LQWDFW VKDPGHQHUYDWHGf IHWXVHV VXEMHFWHG WR K\SRWHQVLRQ HVWUDGLRO LPSODQW PJ GD\ UHOHDVHf FDURWLG VLQXV GHQHUYDWHG IHWXVHV VXEMHFWHG WR K\SRWHQVLRQ )HWDO VKHHS ZHUH FKURQLFDOO\ SUHSDUHG ZLWK YDVFXODU FDWKHWHUV DQG LPSODQWV RQ DURXQG GD\ DQG DOORZHG SRVWVXUJLFDO UHFRYHU\ IRU GD\V )HWXVHV ZHUH VXEMHFWHG WR H[SHULPHQWV RQ GD\ GXULQJ ZKLFK WLPH HDFK IHWXV ZDV VXEMHFWHG WR D PLQ SHULRG RI K\SRWHQVLRQ SURGXFHG E\ EUDFKLRFHSKDOLF RFFOXVLRQ QR RFFOXVLRQ IRU FRQWURO DQLPDOVf )HWDO DUWHULDO EORRG VDPSOHV POf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

PAGE 14

VXEVHW RI VHFWLRQV ZDV GRXEOHVWDLQHG IRU $&7+ DQG FIRV SLWXLWDU\f $93 DQG FIRV K\SRWKDODPXVf DQG &5+ DQG FIRV K\SRWKDODPXVf $QDO\VLV 7KH QHXURDQDWRPLFDO DUHDV ZKLFK H[SUHVV FIRV LQ UHVSRQVH WR K\SRWHQVLRQ 176 DQWHULRU K\SRWKDODPXV 391 DQG SLWXLWDU\f ZHUH LGHQWLILHG ,PPXQRKLVWRFKHPLVWU\ RI FIRV ZDV DQDO\]HG LQ D VHPLTXDQWLWDWLYH PDQQHU FRXQWLQJ FIRV SRVLWLYH FHOOV LQ WKH UHODWLYH UHJLRQVf 7KH QXPEHU RI FHOOV H[SUHVVLQJ LPPXQRUHDFWLYH FIRV LQ HDFK H[SHULPHQWDO JURXS DW HDFK VLWH ZDV EH FRPSDUHG XVLQJ WZR DQG WKUHHZD\ $129$ IXUWKHU FODVVLILHG QHXURQDO DFWLYDWLRQ E\ FRXQWLQJ FHOOV ZKLFK H[SUHVVFRH[SUHVV FIRV DQG $&7+ DQWHULRU SLWXLWDU\f FIRV DQG &5+ DQG FIRV DQG $93 391f ([SHULPHQWDO 0HWKRGV $LP 3ULRU WR VXUJHU\ IRRG ZDV ZLWKKHOG IURP WKH SUHJQDQW HZH \HDUV ROG RI PL[HG EUHHG PRVWO\ &ROXPELD5DPERLOOHW DQG 6XIIRONf IRU KRXUV %HIRUH DQG GXULQJ VXUJHU\ WKH HZH ZDV DQHVWKHWL]HG ZLWK KDORWKDQH bf LQ R[\JHQ )HWDO FDWKHWHUV ZHUH URXWHG WR RQH IODQN RI WKH HZH DQG KHOG LQ SODFH ZLWK D V\QWKHWLF SRFNHW DQG DQ H[SDQGDEOH EDQGDJH $IWHU H[SRVLQJ WKH XWHUXV DQG WKH IHWDO KLQGOLPEV YLD D PLGOLQH LQFLVLRQ IHWDO FDWKHWHUV ZHUH SODFHG LQ WKH IHPRUDO DUWHULHV ZKHUH WKHLU WLSV DUH DGYDQFHG WR WKH VXEGLDSKUDJPDWLF DRUWD $Q HVWUDGLRO RU FRQWURO LPSODQW ZDV SODFHG VXEFXWDQHRXVO\ DW WKLV WLPH $Q DPQLRWLF IOXLG FDWKHWHU ZDV VHZQ WR WKH KLQGOLPE EHIRUH UHWXUQLQJ WKH IHWXV WR WKH DPQLRWLF FDYLW\ &DWKHWHUL]DWLRQ RI IHWDO YHVVHOV LV URXWLQH LQ 'U :RRGfV ODERUDWRU\ .HOOHU:RRG DQG :RRG 5DII DQG :RRG 5DII DQG :RRG :RRG HW DO f

PAGE 15

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fV ODERUDWRU\ KDV KDG H[WHQVLYH H[SHULHQFH ZLWK WKLV SURFHGXUH DQG KDV SXEOLVKHG GHVFULSWLRQV RI WKLV WHFKQLTXH :RRG f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bf VWDWH RI K\SRWHQVLRQ YLD EUDFKLRFHSKDOLF RFFOXVLRQ %ORRG SUHVVXUH PHDVXUHPHQWV ZHUH DQDO\]HG XWLOL]LQJ D FRPSXWHU SURJUDP LQ RUGHU WR KHOS TXDQWLI\ WKH OHYHO RI HVWURJHQ DXJPHQWDWLRQ RI UHIOH[ UHVSRQVLYHQHVV %ORRG JDVHV DQG KHPDWRFULWV ZHUH PHDVXUHG LPPHGLDWHO\ DIWHU FRPSOHWLRQ RI HDFK H[SHULPHQW %ORRG VDPSOHV ZHUH SURFHVVHG FHQWULIXJHG HWF WR REWDLQ SODVPDf DQG IUR]HQ XQWLO KRUPRQH OHYHOV ZHUH PHDVXUHG YLD UDGLRLPPXQRDVVD\ RU HQ]\PH LPPXQRDVVD\ WHFKQLTXHV XVHG URXWLQHO\ LQ 'U :RRGfV ODERUDWRU\ 3ODVPD KRUPRQH FRQFHQWUDWLRQV $&7+ $93 FRUWLVRO DQG HVWUDGLROf ZHUH DQDO\]HG E\ DQG ZD\ $129$

PAGE 16

$LP ,Q SUHSDUDWLRQ IRU LPPXQRKLVWRFKHPLVWU\ WKH KHDG RI WKH IHWXV ZDV SHUIXVHG ZLWK KHSDULQL]HG VDOLQH IROORZHG E\ b SDUDIRUPDOGHK\GH 7KH IHWDO K\SRWKDODPXV DQG EUDLQVWHP ZHUH GLVVHFWHG DQG SURFHVVHG DFFRUGLQJO\ DOFRKRO GHK\GUDWHG DQG HPEHGGHG LQ SDUDIILQf 7LVVXHV ZHUH VHFWLRQHG PLFURQf XVLQJ D =HLVV URWDU\ PLFURWRPH DQG PRXQWHG RQ VXEEHG VOLGHV 6WDLQLQJ IRU $&7+ $93 DQG &5+ ZDV SHUIRUPHG XVLQJ DQWLVHUD SURGXFHG LQ 'U :RRGfV ODERUDWRU\ 6WDLQLQJ IRU FIRV ZDV GRQH XVLQJ FRPPHUFLDOO\ DYDLODEOH DQWLERGLHV IURP 2QFRJHQH 6FLHQFHV 6WDLQLQJ IRU HVWURJHQ VXOIDWDVH DQG HVWURJHQ VXOIRWUDQVIHUDVH ZHUH GRQH XVLQJ FXVWRP PDGH DQWLERGLHV IURP $OSKD 'LDJQRVWLF 6WDLQLQJ ZDV YLVXDOL]HG XVLQJ D +LVWRVWDLQ63 NLW =\PHG VWUHSWDYLGLQ ELRWLQ V\VWHPf

PAGE 17

&+$37(5 /,7(5$785( 5(9,(: &RQWURO RI 3DUWXULWLRQ 7KRXJK LW KDV EHHQ NQRZQ IRU VRPH WLPH WKDW WKH IHWXV FRQWUROV SDUWXULWLRQ LQ WKH VKHHS WKH SUHFLVH PHFKDQLVP KDV \HW WR EH IXOO\ XQGHUVWRRG +RZHYHU PXFK LV NQRZQ DERXW WKH PDMRU HQGRFULQH D[LV WKDW LQLWLDWHV WKLV SURFHVV DV ZHOO DV FRQWUROV EORRG SUHVVXUH LQ WKH IHWXV 7KHVH SURFHVVHV DUH FRQWUROOHG E\ WKH K\SRWKDODPXVSLWXLWDU\DGUHQDO +3$f D[LV 2QH RI WKH ILUVW SLHFHV RI HYLGHQFH WKDW OLQNHG WKH IHWDO +3$ D[LV WR WKH FRQWURO RI SDUWXULWLRQ ZDV UHSRUWHG E\ %LQQV HW DO f 7KLV ZDV D QDWXUDO SKHQRPHQRQ WKDW RFFXUUHG ZKHQ VKHHS DWH D SDUWLFXODU SODQW RQ GD\ RI JHVWDWLRQ 7KLV SODQW 9HUDWUXP FDOLIRPLFXP GHOD\HG WKH ELUWK RI WKH IHWXV LQGHILQLWHO\ XQWLO FDHVDULDQ VHFWLRQ RU GHDWK RI WKH IHWXV RU HZH 7KH IHWXV KDG D QXPEHU RI ELUWK GHIHFWV LQFOXGLQJ F\FORSLD DQG D G\VIXQFWLRQDO K\SRWKDODPXVSLWXLWDU\ +3f D[LV GXH WR GLVORFDWLRQ RI WKH SLWXLWDU\ IURP LWV QRUPDO SRVLWLRQ ,W ZDV UHDOL]HG ODWHU WKDW WKLV PDOIRUPHG G\VIXQFWLRQDO D[LV ZDV UHVSRQVLEOH IRU GHOD\HG SDUWXULWLRQ 7KLV REVHUYDWLRQ DQG RWKHUV OHG /LJJLQV f WR FRQFOXGH WKDW WKH IHWXV FRQWUROV SDUWXULWLRQ LQ WKH VKHHS /LJJLQV KDV FUHGLWHG +LSSRFUDWHV ZLWK ILUVW VXJJHVWLQJ WKLV LGHD ZKHQ KH ZURWH WKDW WKH IHWXV SXVKHV LWV ZD\ RXW RI WKH ZRPE ZKHQ WKH QXWULWLRQ VXSSOLHG E\ WKH PRWKHU LV QR ORQJHU VXIILFLHQW IRU IXUWKHU JURZWK 0DOSXV f SXW IRUWK D PRUH PRGHP REVHUYDWLRQ VXJJHVWLQJ D IHWDO UROH LQ WKH WLPLQJ RI ELUWK ZKHQ KH UHSRUWHG WKH DVVRFLDWLRQ RI IHWDO DQFHSKDOO\ DQG SURORQJHG JHVWDWLRQ LQ

PAGE 18

ZRPHQ 7KHVH LGHDV ODLG WKH JURXQGZRUN IRU PXFK RI WKH RQJRLQJ UHVHDUFK WR GHWHUPLQH WKH SUHFLVH PHFKDQLVP RI SDUWXULWLRQ 7KRXJK PDQ\ KDYH FRQWULEXWHG WR WKLV LGHD RI D IHWDO UROH LQ WKH LQLWLDWLRQ RI SDUWXULWLRQ QR RQH KDV FRQWULEXWHG WR WKLV ILHOG RI UHVHDUFK PRUH WKDQ /LJJLQV /LJJLQV ZDV WKH ILUVW WR K\SRWKHVL]H DQG GLUHFWO\ WHVW WKDW WKH IHWDO SLWXLWDU\ LV LQWULFDWHO\ LQYROYHG LQ WKLV SURFHVV /LJJLQV HW DO f 7KLV ZDV GRQH E\ ILUVW K\SRSK\VHFWRPL]LQJ IHWDO VKHHS E\ VXUJLFDO HOHFWURFRDJXODWLRQ ,Q IHWXVHV ZKHUH b RU PRUH RI WKH SLWXLWDU\ ZDV DEODWHG JHVWDWLRQ ZDV VLJQLILFDQWO\ SURORQJHG DQG GHOLYHU\ ZDV DFKLHYHG RQO\ DIWHU FDHVDULDQ VHFWLRQ )HWDO DGUHQDOHFWRP\ ZDV DOVR IRXQG WR SURORQJ JHVWDWLRQ 'URVW DQG +ROP f 'LVFRQQHFWLRQ RI WKH K\SRWKDODPXV IURP WKH SLWXLWDU\ LQ WKH IHWDO VKHHS EHWZHHQ GD\V JHVWDWLRQ GHOD\V ELUWK E\ DW OHDVW HLJKW GD\V $QWRORYLFK HW DO f )ROORZLQJ K\SRSK\VHFWRP\ LQIXVLRQ RI DGUHQRFRUWLFRWURSLQ $&7+f RU JOXFRFRUWLFRLG VWLOO LQGXFHV SDUWXULWLRQ .HQGDOO HW DO f )XUWKHU VWXGLHV IURP WKLV JURXS DOVR GHPRQVWUDWH WKH QHFHVVLW\ RI WKH +3 D[LV LQ PDWXUDWLRQ RI SLWXLWDU\ FRUWLFRWURSHV .HQGDOO HW DO f $IWHU GLVFRQQHFWLRQ RI WKH K\SRWKDODPXV IURP WKH SLWXLWDU\ IHWXVHV ZHUH LQIXVHG ZLWK VDOLQH RU FRUWLVRO ,Q WKH FRUWLVROLQIXVHG IHWXVHV WKH SURSRUWLRQ RI IHWDOW\SH FRUWLFRWURSLQV ZDV VLJQLILFDQWO\ ORZHU WKDQ LQ WKH VDOLQHLQIXVHG IHWXVHV KRZHYHU WKH QXPEHU RI DGXOWW\SH FRUWLFRWURSLQV GLG QRW FKDQJH $ GLUHFW HIIHFW RI FRUWLVRO RQ SLWXLWDU\ FRUWLFRWURSLQ PDWXUDWLRQ UHTXLUHV WKH SUHVHQFH RI FRPSOHWH +3 D[LV $QWRORYLFK HW DO f 0RUH VSHFLILFDOO\ IROORZLQJ GHVWUXFWLRQ RI WKH IHWDO SDUDYHQWULFXODU QXFOHXV 391f RI WKH K\SRWKDODPXV SDUWXULWLRQ ZDV GHOD\HG 0F'RQDOG DQG 1DWKDQLHOV] f 7KHUHIRUH WKH VLJQDO IRU SDUWXULWLRQ PD\ HLWKHU EH VHQW WR WKH 391 ZKLFK UHFHLYHV LQSXW IURP WKH QXFOHXV RI WKH WUDFWXV VROLWDULXV 176f DP\JGDOD RU KLSSRFDPSXV RU SRVVLEO\ EH

PAGE 19

GHULYHG LQ FRUWLFRWURSKLQ UHOHDVLQJ KRUPRQH &5+f DQG DUJLQLQH YDVRSUHVVLQ $93f SURGXFLQJ QHXURQV LQ WKH 391 :KHUHE\ GLVUXSWLRQ RI WKH IHWDO +3 $ D[LV FDXVHV D GHOD\ LQ WKH WLPLQJ RI ELUWK VWLPXODWLRQ RI WKLV HQGRFULQH D[LV FDQ UHVXOW LQ SUHPDWXUH SDUWXULWLRQ 7KH LQYROYHPHQW RI WKH IHWDO DGUHQDO FRUWH[ LQ WKH LQLWLDWLRQ RI SDUWXULWLRQ LQ VKHHS ZDV VXJJHVWHG DIWHU LQ WHUR SODVPD FRQFHQWUDWLRQ RI FRUWLFRVWHURLG UHYHDOHG GUDPDWLF LQFUHDVHV LQ WKHVH KRUPRQHV SULRU WR ELUWK %DVVHWW DQG 7KRUEXP f %HIRUH WKLV FRQFOXVLRQ ZDV PDGH D QXPEHU RI VWXGLHV DLPHG DW LQYHVWLJDWLQJ WKH UROH RI WKH +3$ D[LV LQ WKH ELUWK SURFHVV ZHUH SHUIRUPHG LQ WKH IHWDO VKHHS E\ /LJJLQV f $&7+ LQIXVHG LQWR IHWDO VKHHS LQGXFHG SDUWXULWLRQ ZLWKLQ IRXU WR VHYHQ GD\V DORQJ ZLWK SURGXFLQJ DGUHQDO K\SHUWURSK\ &RUWLVRO LQIXVLRQ LQWR WKH IHWXV LQGXFHG SDUWXULWLRQ ZLWKLQ ILYH GD\V 7KH VDPH GRVHV RI $&7+ RU FRUWLVRO LQIXVHG LQWR HZHV GLG QRW LQGXFH SDUWXULWLRQ $OWKRXJK HVWUDGLRO DW PJ KU KDG QR HIIHFW LQIXVLRQ RI FRUWLVRO DW PJ KU SOXV HVWUDGLRO UHVXOWHG LQ GHOLYHU\ DIWHU IRXU GD\V /LJJLQV f )XUWKHU VWXGLHV E\ /LJJLQV f VKRZHG WKDW LW ZDV JOXFRFRUWLFRLG DFWLYLW\ QRW PLQHUDORFRUWLFRLGf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f $FWLYDWLRQ RI IHWDO DGUHQDO IXQFWLRQ E\ SXOVDWLOH $&7+ DGPLQLVWUDWLRQ LQ GD\ IHWDO VKHHS LQGXFHG ODERU DQG GHOLYHU\ LQ IRXU WR ILYH GD\V UHVXOWLQJ LQ IRXU WR VL[ IROG HOHYDWLRQ LQ IHWDO SODVPD FRUWLVRO

PAGE 20

FRQFHQWUDWLRQV /\H HW DO f )XUWKHUPRUH LW ZDV VKRZQ WKDW K\SRSK\VHFWRPL]HG IHWXVHV WKDW ZHUH DGPLQLVWHUHG GH[DPHWKDVRQH RU $&7+ LQIXVLRQV H[RJHQRXVO\ ZRXOG XQGHUJR SDUWXULWLRQ .HQGDOO HW DO f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f 7KH +\SRWKDODPXV 7KH K\SRWKDODPXV FRQWDLQV VHYHUDO QXFOHL RI QHXURQDO FHOOV 7KH 391 RI WKH K\SRWKDODPXV ORFDWHG ELODWHUDOO\ RQ WKH YHQWULFOH FRQWDLQV VSHFLDOL]HG QHXURVHFUHWRU\ FHOOV ZKLFK V\QWKHVL]H &5+ DQG $93 7KH UHODWLYHO\ ODUJHFHOOHG KHQFH WHUPHG PDJQRFHOOXODUf QHXURQV LQ WKH 391 FRQWDLQ $93 DQG SURMHFW WR WKH SRVWHULRU SLWXLWDU\ /HFKDQ 5HLFKOLQ f ,Q DGGLWLRQ WR WKH ODUJH SURMHFWLRQ WR WKH SRVWHULRU SLWXLWDU\ LW KDV EHHQ HVWDEOLVKHG WKDW VPDOOFHOOHG SDUYRFHOOXODUf QHXURQV LQ WKH 391 FRQWDLQ &5+ DQG $93 DQG SURMHFW WR WKH PHGLDQ HPLQHQFH DQG DUH LQYROYHG LQ WKH UHJXODWLRQ RI $&7+ UHOHDVH 6DZFKHQNR DQG 6ZDQVRQ f 7KH LQFUHDVH LQ SODVPD $&7+ FRQFHQWUDWLRQ GXULQJ VWUHVV LV PHGLDWHG E\ &5+ DV ZHOO DV $93 IURP WKH

PAGE 21

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b RI DGHQRK\SRSK\VHDO FHOOV 2QFH WKH UHOHDVLQJ KRUPRQH ELQGV WR LWV UHFHSWRU LQ WKH FRUWLFRWURSH D VLQJOH P51$ WKH GLUHFWV WKH V\QWKHVLV RI WKH ODUJH SUHFXUVRU PROHFXOH FDOOHG SURRSLRPHODQRFRUWLFRWURSLQ 320&f 320& LV WKHQ SURFHVVHG WR SURGXFH WKH VPDOOHU ELRORJLFDOO\ DFWLYH IUDJPHQW $&7+ $&7+ LV WKHQ UHOHDVHG LQWR WKH V\VWHPLF FLUFXODWLRQ ZKHUH LW DFWV RQ DGUHQRFRUWLFDO FHOOV WR VWLPXODWH V\QWKHVLV DQG VHFUHWLRQ RI JOXFRFRUWLFRLGV 5HLFKOLQ f 7KH $GUHQDO &RUWH[ 7KH DGUHQDO JODQGV DUH HQGRFULQRORJLFDOO\ FRPSOH[ RUJDQV WKDW DUH FRPSRVHG RI WZR GLVWLQFW HQGRFULQH WLVVXHV GHULYHG IURP GLIIHUHQW HPEU\RORJLF VRXUFHV 7KH RXWHU ]RQH LV FDOOHG WKH DGUHQDO FRUWH[ DQG FRQVWLWXWHV b RI WKH JODQG 7KH FRUWH[ LV WKH VRXUFH RI WKH VWHURLG KRUPRQHV 7KH VPDOOHU LQQHU ]RQH LV WKH DGUHQDO PHGXOOD ZKLFK LV WKH PDMRU VRXUFH RI FLUFXODWLQJ FDWHFKRODPLQHV 7KH DGUHQDO FRUWH[ LV KLJKO\ YDVFXODUL]HG DQG UHFHLYHV LWV PDLQ DUWHULDO VXSSO\ IURP EUDQFKHV RI WKH LQIHULRU SKUHQLF DUWHU\ WKH UHQDO DUWHULHV DQG WKH DRUWD 7KHUH DUH WKUHH PDMRU JURXSV RI KRUPRQHV SURGXFHG E\ WKH DGUHQDO FRUWH[ WKH PLQHUDORFRUWLFRLGV WKH JOXFRFRUWLFRLGV DQG WKH VH[

PAGE 22

VWHURLGV +LVWRORJLFDOO\ WKH DGXOW FRUWH[ LV FRPSRVHG RI WKUHH ]RQHV DQ RXWHU ]RQD JORPHUXORVD D ]RQD IDVFLFXODWD DQG DQ LQQHU ]RQD UHWLFXODULV 7KH SULPDU\ SURGXFW RI WKH ]RQD JORPHUXORVD LV WKH PLQHUDORFRUWLFRLG DOGRVWHURQH 7KH ]RQD IDVFLFXODWD DQG UHWLFXODULV SURGXFH FRUWLVRO DQG DQGURJHQV DV WKHLU SULPDU\ SURGXFWV 3HVFRYLW] HW DO f $&7+ VWLPXODWHV WKH VHFUHWLRQ RI JOXFRFRUWLFRLGV PLQHUDORFRUWLFRLGV DQG DQGURJHQLF VWHURLGV IURP WKH DGUHQDO FRUWH[ $&7+ ELQGV WR WKH UHFHSWRUV RQ WKH DGUHQDO FRUWH[ DQG SURYRNHV VWHURLGRJHQHVLV WKURXJK VWLPXODWLRQ RI F$03 SURGXFWLRQ F$03 DFWLYDWHV SURWHLQ NLQDVH $ ZKLFK FDWDO\]HV WKH SKRVSKRU\ODWLRQ RI D YDULHW\ RI SURWHLQV WKHUHE\ SURGXFLQJ FKROHVWHURO $&7+ DOVR VWLPXODWHV V\QWKHVLV RI QHZ DGUHQDO SURWHLQV DQG WKLV LQFUHDVHV DGUHQDO ZHLJKW *OXFRFRUWLFRLGV H[HUW QHJDWLYH IHHGEDFN DW WKH SLWXLWDU\ K\SRWKDODPXV DQG RWKHU QHXUDO VLWHV .HOOHU:RRG DQG 'DOOPDQ f &RUWLVRO LV FDUULHG LQ EORRG ERXQG WR WUDQVFRUWLQ bf DQG DOEXPLQ bf ZKLOH DERXW b LV IUHH LQ RYLQH SODVPD 3DWWHUVRQ DQG +LOOV f 7KH EDVDO SURGXFWLRQ UDWH I RI FRUWLVRO LV SJKU DQG WKH PHWDEROLF FOHDUDQFH UDWH RI FRUWLVRO LV DERXW /KU LQ VKHHS 3DQDUHWWR f 7KH OLYHU DQG WKH NLGQH\ DUH WKH SULQFLSOH RUJDQV LQYROYHG LQ FOHDULQJ WKH VWHURLG KRUPRQHV IURP WKH FLUFXODWLRQ $OWKRXJK PRVW WLVVXHV FDQ PHWDEROL]H VWHURLGV WKH OLYHU LV WKH SULPDU\ VLWH RI VWHURLG KRUPRQH PHWDEROLVP DQG WKH NLGQH\ LV WKH SULPDU\ VLWH RI VWHURLG KRUPRQH H[FUHWLRQ 7KH SODVPD KDOIOLIH RI FRUWLVRO LV PLQXWHV LQ WKH KXPDQ 3HVFRYLW] HW DO f &RUWLVRO SURGXFHV D QXPEHU RI GLYHUVH SK\VLRORJLFDO DFWLRQV WR PDLQWDLQ KRPHRVWDVLV $V WKH WHUP KRPHRVWDVLV LPSOLHV DQ H[FHVV RU GHILFLHQF\ RI JOXFRFRUWLFRLGV DIIHFWV HYHU\ WLVVXH RI WKH ERG\ *OXFRFRUWLFRLGV DUH HVVHQWLDO IRU VXUYLYDO %D[WHU *DQQ HW DO 3HVFRYLW] HW DO f 7KH WHUP JOXFRFRUWLFRLG UHIHUV WR WKH JOXFRVH

PAGE 23

UHJXODWLQJ SURSHUWLHV RI WKHVH KRUPRQHV +RZHYHU JOXFRFRUWLFRLGV KDYH PXOWLSOH HIIHFWV WKDW LQFOXGH LPSRUWDQW UROHV LQ FDUERK\GUDWH OLSLG DQG SURWHLQ PHWDEROLVP %D[WHU f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f 7KH JOXFRFRUWLFRLGV DOVR LQIOXHQFH JURZWK GHYHORSPHQW ERQH PHWDEROLVP DQG FHQWUDO QHUYRXV V\VWHP DFWLYLW\ 7KH 5HJXODWLRQ RI $&7+ 6HFUHWLRQ &RUWLFRWURSLQ 5HOHDVLQJ +RUPRQH I&5-7f 6LQFH LW KDV EHHQ NQRZQ WKDW WKH K\SRWKDODPXV FRQWDLQV VXEVWDQFHV WKDW DFWHG DW WKH SLWXLWDU\ JODQG WR LQFUHDVH $&7+ VHFUHWLRQ LQ YLWUR *XLOOHPLQ DQG 5RVHQEHUJ 6DIIUDQ DQG 6FKDOO\ f ,Q 9DOH HW DO f FKDUDFWHUL]HG D DPLQR DFLG SHSWLGH IURP VKHHS K\SRWKDODPXV WKDW VWLPXODWHG $&7+ VHFUHWLRQ IURP FRUWLFRWURSKLQV DQG SXEOLVKHG WKH SULPDU\ VWUXFWXUH RI RYLQH &5+ 2YLQH &5+ ZDV IXUWKHU FKDUDFWHUL]HG ZKHQ WKH F'1$ ZDV FORQHG DQG VHTXHQFHG )XUXWDQL f $W WKLV WLPH D VLPLODULW\ ZDV GLVFRYHUHG EHWZHHQ WKH SUHFXUVRU SURWHLQV IRU &5+ $93 DQG $&7+ ZKLFK LPSOLHV D FRPPRQ HYROXWLRQDU\ EHJLQQLQJ $ N' LPPXQRUHDFWLYH IRUP RI &5+ ZDV LGHQWLILHG IURP UDW K\SRWKDODPXV

PAGE 24

DQG LV FORVH WR WKH YDOXH IRU RYLQH DQG KXPDQ SUHSUR&5+ EDVHG RQ WKHLU F'1$ VHTXHQFHV /DXEHU HW DO f &5+ LQFUHDVHV $&7+ VHFUHWLRQ IURP WKH DQWHULRU SLWXLWDU\ JODQG E\ ELQGLQJ WR KLJK DIILQLW\ UHFHSWRUV :\QQ HW DO f ORFDWHG RQ WKH FRUWLFRWURSKV /HURX[ DQG 3HOOHWLHU f $FWLYDWLRQ RI WKH UHFHSWRU FRPSOH[ LQFUHDVHV DGHQ\ODWH F\FODVH DFWLYLW\ 3HUULQ HW DO f DQG F$03 ZKLFK UHVXOWV LQ DQ LQFUHDVH LQ $&7+ VHFUHWLRQ ,Q FXOWXUHG UDW SLWXLWDU\ FHOOV &5+ FDQ HQKDQFH WKH UDWHV RI $&7+ V\QWKHVLV DV ZHOO DV UHOHDVH 9DOH HW DO f $&7+ UHOHDVH FDQ EH PRGXODWHG E\ GRZQUHJXODWLRQ RI &5+ UHFHSWRUV LQ WKH DQWHULRU SLWXLWDU\ 7KHUH LV HYLGHQFH WKDW &5+ :\QQ HW DO f $93 +DXJHU DQG $JXLOHUD f DQG JOXFRFRUWLFRLGV +DXJKHU HW DO 6FKZDUW] HW DO :\QQ HW DO f FDQ DOO DFW WR UHJXODWH &5+ UHFHSWRU QXPEHU 7KHUHIRUH DQ DOWHUDWLRQ LQ &5+ UHFHSWRU QXPEHU UHFHSWRU DFWLYLW\ UHFHSWRU FRXSOLQJ RU HYHQ FRUWLFRWURSKLQ QXPEHU FDQ DIIHFW WKH DELOLW\ RI &5+ WR VWLPXODWH $&7+ VHFUHWLRQ $UJLQLQH 9DVRSUHVVLQ $93f 7KH RWKHU PDMRU UHJXODWRU RI $&7+ VHFUHWLRQ RI K\SRWKDODPLF RULJLQ LV $93 &ODVVLFDOO\ $93 LV NQRZQ DV DQWLGLXUHWLF KRUPRQH $'+f IRU LWV UROH LQ UHQDO UHJXODWLRQ RI IOXLG EDODQFH $Q LQFUHDVH LQ SODVPD RVPRODOLW\ LV WKH PRVW SRWHQW VWLPXOXV WR $93 VHFUHWLRQ LQ WKDW YHU\ VPDOO LQFUHDVHV LQ RVPRODOLW\ FDXVH DQ DOPRVW LPPHGLDWH VHFUHWRU\ UHVSRQVH IURP WKH SRVWHULRU SLWXLWDU\ $93 ELQGV WR UHFHSWRUV RQ WKH EDVDOODWHUDO PHPEUDQH RI WKH FRUWLFDO DQG PHGXOODU\ FROOHFWLQJ GXFWV RI WKH QHSKURQ %LQGLQJ WR WKHVH UHFHSWRUV 9f UHVXOWV LQ DFWLYDWLRQ RI DGHQ\ODWH F\FODVH ZKLFK VXEVHTXHQWO\ LQFUHDVHV F$03 7KLV VHFRQG PHVVHQJHU LV WKHQ WKRXJKW WR IDFLOLWDWH DQ LQFUHDVH LQ SURWHLQ FKDQQHOV IRXQG LQ WKH OXPLQDO PHPEUDQH WKHUHE\ LQFUHDVLQJ WKH GLIIXVLRQ RI ZDWHU RXW RI WKH QHSKURQ DQG FRQFHQWUDWLQJ XULQH ZKLOH UHWDLQLQJ IOXLG

PAGE 25

9DQGHU f ,Q DGGLWLRQ WR $ 93fV UROH LQ PRGXODWLQJ IOXLG UHDEVRUSWLRQ LQ WKH UHQDO V\VWHP $ 93 LV DOVR D SRWHQW YDVRFRQVWULFWRU RI WKH FDUGLRYDVFXODU V\VWHP ELQGLQJ WR 9L UHFHSWRUVf 6LJQLILFDQW GHFUHDVHV LQ EORRG YROXPH bf SURGXFH ODUJH LQFUHDVHV LQ SODVPD YDVRSUHVVLQ 'HFUHDVHV LQ EORRG YROXPH DUH VHQVHG DV GHFUHDVHG VWUHWFK RI WKH DUWHULDO EDURUHFHSWRUV ORFDWHG LQ WKH FDURWLG VLQXV DV ZHOO DV WKH UHFHSWRUV LQ WKH OHIW DWULXP DQG $93 VHFUHWLRQ LV UHIOH[LYHO\ VWLPXODWHG %HUQH DQG /HY\ f $93 LV RQH RI WZR KRUPRQHV VHFUHWHG IURP WKH SRVWHULRU SLWXLWDU\ JODQG R[\WRFLQ LV WKH RWKHUf 7KH SRVWHULRU SLWXLWDU\ DOVR FDOOHG WKH QHXURK\SRSK\VLV LV FRPSULVHG RI D[RQV DQG D[RQ WHUPLQDOV ZKLFK DFFRXQW IRU b RI LWV WRWDO YROXPH 1RUGPDQQ f 7KHVH D[RQV SURMHFW IURP PDJQRFHOOXODU QHXURQV RI WKH VXSUDRSWLF QXFOHXV 621f DQG 391 IURP WKH K\SRWKDODPXV $93 DQG R[\WRFLQ DUH VWUXFWXUDOO\ VLPLODU KRUPRQHV ZLWK YHU\ GLIIHUHQW IXQFWLRQV $93 KDV DFWLRQV RQ WKH UHQDO DQG FLUFXODWRU\ V\VWHPV ZKLOH R[\WRFLQ FDXVHV PLON HMHFWLRQ DQG XWHULQH FRQWUDFWLRQV $93 DQG R[\WRFLQ DUH SURGXFHG LQ GLIIHUHQW QHXURQV RI WKH VDPH QXFOHL DQG DUH VWRUHG LQ VHFUHWRU\ YHVLFOHV RU JUDQXOHV ZLWK WKHLU DSSURSULDWH QHXURSK\VLQ 6LOYHUPDQ DQG =LPPHUPDQ f $93 LV SURGXFHG IURP D ODUJH SUHFXUVRU SURWHLQ FRQWDLQLQJ QRW RQO\ $93 EXW DOVR QHXURSK\VLQ DQG D JO\FRSHSWLGH VLJQDO VHTXHQFH 6DFKV HW DO f 7KH SUHFXUVRU SURWHLQ PROHFXOH LV SDFNDJHG LQ JUDQXOHV ZLWK WKH HQ]\PHV QHHGHG IRU SURFHVVLQJ $93 WR LWV ILQDO IRUP $V WKH JUDQXOHV PRYH GRZQ WKH D[RQV SRVWWUDQVODWLRQDO SURFHVVLQJ RI WKH SUHFXUVRU PROHFXOH RFFXUV ZLWKLQ WKH JUDQXOHV :KHQ WKH JUDQXOHV UHDFK WKH D[RQ WHUPLQDOV WKH QHUYH LV GHSRODUL]HG DQG WKH JUDQXOHV DUH H[RF\WRVHG DQG WKH FRQWHQWV RI WKH JUDQXOHV DUH UHOHDVHG %URZQVWHLQ HW DO f 0DJQRFHOOXODU QHXURQV FRQWDLQLQJ $93

PAGE 26

SURMHFW ILEHUV WR WKH PHGLDQ HPLQHQFH DQG WKHUHIRUH PD\EH LPSRUWDQW LQ WKH UHJXODWLRQ RI $&7+ VHFUHWLRQ +ROPHV HW DO f $93 LV D SRWHQW PRGXODWRU RI SLWXLWDU\ $&7+ VHFUHWLRQ ,Q IDFW LQ VKHHS $93 LV D PRUH SRWHQW VWLPXODWRU RI $&7+ WKDQ &5+ )DPLODUL HW DO /LX HW DO f $93 ELQGV WR UHFHSWRUV RQ WKH DQWHULRU SLWXLWDU\ FRUWLFRWURSK WR LQFUHDVH SODVPD $&7+ VHFUHWLRQ 7KHVH UHFHSWRUV DUH GLIIHUHQW IURP WKH SUHVVRU UHFHSWRUV VXEW\SH 9Lf RU DQWLn GLXUHWLF UHFHSWRUV VXEW\SH 9f WKDW DUH IRXQG LQ WKH SHULSKHU\ 'DWD IURP WZR GLIIHUHQW JURXSV %DHUWVFKHL DQG )ULHGOL -DUG HW DO f VXJJHVW WKDW D VXEW\SH FODVVLILHG DV 9LE RU 9f GLVWLQFW IURP WKH SHULSKHUDO UHFHSWRUV H[LVWV LQ WKH EUDLQ ZLWK SURWHLQ NLQDVH & DV LWV VHFRQG PHVVHQJHU $93 FDQ HIIHFW DQWHULRU SLWXLWDU\ VHFUHWLRQ LQ WZR ZD\V HLWKHU E\ $93 VHFUHWLRQ IURP D[RQV ZKLFK WHUPLQDWH LQ WKH PHGLDQ HPLQHQFH RU E\ $93 VHFUHWLRQ IURP WKH SRVWHULRU SLWXLWDU\ (YLGHQFH IRU ERWK SRVVLELOLWLHV H[LVW $93FRQWDLQLQJ QHXURQV RI WKH 621 DQG 391 DUH NQRZQ WR SURMHFW WR WKH H[WHUQDO ]RQH RI WKH PHGLDQ HPLQHQFH +RIIPDQQ HW DO f $ IXUWKHU GLVWLQFWLRQ KDV EHHQ PDGH LQ WKDW $93FRQWDLQLQJ SDUYRFHOOXODU QHXURQV RI WKH 391 ZHUH IRXQG WR LQQHUYDWH WKH H[WHUQDO ]RQH RI WKH PHGLDQ HPLQHQFH 7KH PDJQRFHOOXODU QHXURQV RI WKH 391 SDVV WKURXJK WKH LQWHUQDO ]RQH RI WKH PHGLDQ HPLQHQFH WR WKH QHXURK\SRSK\VLV EXW DOVR FRQWULEXWH WR WKH $93 IRXQG LQ WKH PHGLDQ HPLQHQFH +ROPHV HW DO f $93 VHFUHWLRQ IURP WKH SRVWHULRU SLWXLWDU\ JODQG KDV DOVR EHHQ LPSOLFDWHG LQ SODVPD $&7+ VHFUHWLRQ ,Q VWXGLHV SHUIRUPHG LQ GRJV QHXURK\SRSK\VHFWRP\ DWWHQXDWHG WKH SODVPD $&7+ UHVSRQVH WR K\SRWHQVLRQ $IWHU UHVWRUDWLRQ RI SODVPD $93 OHYHOV WR WKRVH REVHUYHG LQ WKH LQWDFW DQLPDO SODVPD $&7+

PAGE 27

OHYHOV ZHUH DOPRVW FRPSOHWHO\ UHWXUQHG WR QRUPDO 5DII HW DO f 7KH DFWLRQ RI $93 DSSHDUV WR EH WKURXJK D GLUHFW HIIHFW DW WKH SLWXLWDU\ JODQG 6\QHUJLVP RI &5+ DQG $93 7KH V\QHUJLVWLF DFWLYLW\ RI &5) DQG $93 VHFUHWLRQ KDV EHHQ ZHOO GRFXPHQWHG ,Q VXSSRUW RI WKLV LV DQ RYHUZKHOPLQJ DPRXQW RI DQDWRPLFDO GDWD IRU WKH LQWHUDFWLRQ EHWZHHQ WKHVH WZR KRUPRQHV &5+ DQG $93 DUH IRXQG LQ WKH VDPH QHXURVHFUHWRU\ YHVLFOHV LQ WKH PHGLDQ HPLQHQFH :KLWQDOO HW DO f ,Q QRUPDO UDWV VWDLQLQJ RI WKH PHGLDQ HPLQHQFH E\ LPPXQRKLVWRFKHPLVWU\ UHYHDOHG FRORFDOL]DWLRQ RI $93 LQ b RI &5+ D[RQV :KLWQDOO HW DO f 5HSHDWHG VWUHVV LPPRELOL]DWLRQ LQ UDWVf LQFUHDVHV WKH FRORFDOL]DWLRQ RI $93 LQ &5+ QHUYH WHUPLQDOV LQ WKH PHGLDQ HPLQHQFH GH *RHLM HW DO f )ROORZLQJ DGUHQDOHFWRP\ LQ UDWV &5+ LPPXQRVWDLQLQJ LQFUHDVHV LQ SDUYRFHOOXODU QHXURQV RI WKH 391 DQG WKH DPRXQW RI FRORFDOL]DWLRQ ZLWK $93 LQFUHDVHV 6DZFKHQNR HW DO f 7KH LQFUHDVH LQ &5+ DQG $93 LPPXQRUHDFWLYLW\ IROORZLQJ DGUHQDOHFWRP\ LV SUHYHQWHG E\ LQWUDFHUHEURYHQWULFXODU LQMHFWLRQ RI I GH[DPHWKDVRQH 6DZFKHQNR f )URP LQ YLWUR VWXGLHV SHUIRUPHG LQ FXOWXUHG SLWXLWDU\ FHOOV &5+ LV FRQVLGHUHG WR EH WKH PRUH SRWHQW VHFUHWDJRJXH IRU $&7+ VHFUHWLRQ LQ UDWV ZKLOH $93 DSSHDUV WR EH D PRUH SRWHQW VWLPXOXV WR $&7+ UHOHDVH LQ VKHHS )DPLODUL HW DO f +RZHYHU WKH DFWLRQ RI HDFK LV SRWHQWLDWHG ZKHQ DGPLQLVWHUHG WRJHWKHU LQ FXOWXUHG SLWXLWDU\ FHOOV *LOOLHV HW DO f DGXOW IUHHO\ PRYLQJ UDWV 5LYLHU DQG 9DOH Df DQG LQ IHWDO VKHHS %URRNV DQG :KLWH f ,Q D VWXG\ SHUIRUPHG LQ FRQVFLRXV VKHHS VWUHVVLQGXFHG $&7+ VHFUHWLRQ DXGLRYLVXDO DQG LQVXOLQLQGXFHG K\SRJO\FHPLDf ZDV DFFRPSDQLHG E\ LQFUHDVHV LQ K\SRWKDODPLF &5+ DQG $93 VHFUHWLRQ )DPLODUL HW DO f :KDW LV PRVW LQWHUHVWLQJ LV WKDW &5+$93 PRODU UDWLR ZDV DOWHUHG ZLWK WKH VWUHVV 3RUWDO SODVPD $93 ZDV LQFUHDVHG

PAGE 28

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f WKDW DXJPHQW $&7+ VHFUHWLRQ 5DW SLWXLWDU\ FRUWLFRWURSKV LQ FXOWXUH UHOHDVH $&7+ LQ UHVSRQVH WR HSLQHSKULQH DQG QRUHSLQHSKULQH DFWLQJ RQ DODGUHQHUJLF UHFHSWRUV *LJXHUH HW DO f (SLQHSKULQH KDV DOVR EHHQ LGHQWLILHG LQ SRUWDO SODVPD VXJJHVWLQJ D SK\VLRORJLFDO UROH LQ WKH FRQWURO RI DQWHULRU SLWXLWDU\ IXQFWLRQ -RKQVRQ HW DO f 1HXURSHSWLGH < 13
PAGE 29

f $QJLRWHQVLQ ,, DOVR LQFUHDVHV SODVPD $&7+ VHFUHWLRQ E\ LQGXFWLRQ RI &5+ 5LYLHU DQG 9DOH f 3URVWDJODQGLQ ( DORQH GRHV QRW LQFUHDVH $&7+ VHFUHWLRQ EXW HQKDQFHV WKH DELOLW\ RI $93 WR VWLPXODWH $&7+ VHFUHWLRQ ZLWK QR HIIHFW RQ &5+ %URRNV DQG *LEVRQ f ,Q DGGLWLRQ WR VWLPXODWLQJ WKH DFWLYLW\ RI WKH +3$ D[LV IDFWRUV IURP WKH EUDLQ DOVR LQKLELW WKH D[LV 7KH GRSDPLQHUJLF V\VWHP LQ WKH DP\JGDORLG FHQWUDO QXFOHXV KDV EHHQ IRXQG WR LQKLELW $&7+ VHFUHWLRQ E\ DFWLRQ RQ WKH DQWHULRU DQG ODWHUDO K\SRWKDODPXV %HDXOLHX HW DO f $WULDO 1DWULXUHWLF 3HSWLGH $13f KDV DOVR EHHQ VKRZQ WR DOWHU $&7+ VHFUHWLRQ %UDLQ $13 LV VHFUHWHG LQWR WKH K\SRSK\VHDO SRUWDO YHVVHOV IURP WKH K\SRWKDODPXV DQG SK\VLRORJLFDO FRQFHQWUDWLRQV LQKLELW $&7+ UHOHDVH IURP SLWXLWDU\ FHOOV LQ YLWUR 'D\DQLWKL DQG $QWRQL /LP HW DO 6KHZDUG HW DO f ,Q YLYR LPPXQRQHXWUDOL]DWLRQ RI $13 VLJQLILFDQWO\ LQFUHDVHV $&7+ UHOHDVH EXW KDV QR HIIHFW RQ UHOHDVH GXULQJ HWKHU VWUHVV )LQN HW DO f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

PAGE 30

JOXFRFRUWLFRLGV DUH UHOHDVHG LQ UHVSRQVH WR VWUHVV DQG QR[LRXV VWLPXOL ,Q DQLPDOV WKDW ODFN QRUPDO VHFUHWLRQ RI JOXFRFRUWLFRLGV H[SRVXUH WR D VWUHVV FDQ EH OLIHWKUHDWHQLQJ *DQRQJ f 6HFUHWLRQ RI JOXFRFRUWLFRLGV LV UHJXODWHG E\ DGUHQRFRUWLFRWURSLQ KRUPRQH IURP WKH SLWXLWDU\ :KHQ D VWLPXOXV RI $&7+ VHFUHWLRQ IURP WKH K\SRWKDODPXV UHDFKHV WKH SLWXLWDU\ JODQG $&7+ LV VHFUHWHG LQWR WKH JHQHUDO FLUFXODWLRQ ELQGV WR LWV UHFHSWRU DW WKH DGUHQDO JODQG DQG VWLPXODWHV FRUWLVRO VHFUHWLRQ &RUWLVRO WKHQ DFWV DW WDUJHW RUJDQV WR LQFUHDVH SODVPD JOXFRVH OHYHOV HWF +RZHYHU FRUWLVRO DOVR DFWV DW WKH OHYHOV RI WKH EUDLQ WR UHGXFH $&7+ VHFUHWLRQ &RUWLVRO DFWV DW WKH K\SRWKDODPXV DQG SLWXLWDU\ JODQG QHJDWLYHO\ WR LQKLELW IXUWKHU $&7+ VHFUHWLRQ 7KLV LV FDOOHG FRUWLVRO QHJDWLYH IHHGEDFN LQKLELWLRQ RI $&7+ VHFUHWLRQ 7KH PHFKDQLVP RI DFWLRQ RI FRUWLVRO WR UHGXFH $&7+ VHFUHWLRQ KDV EHHQ VWXGLHG DW OHQJWK LQ PDQ\ VSHFLHV DQG E\ PDQ\ LQYHVWLJDWRUV $ VWXG\ E\ &DQQ\ HW DO f U SHUIRUPHG LQ VKHHS H[DPLQHG ERWK K\SRWKDODPLF DQG SLWXLWDU\ VLWHV RI DFWLRQ IRU JOXFRFRUWLFRLGV 0HDVXUHPHQWV RI K\SRSK\VHDO SRUWDO FRQFHQWUDWLRQV RI $93 DQG &5+ DQG V\VWHPLF PHDVXUHPHQWV RI $&7+ DQG FRUWLVRO FRQFHQWUDWLRQV ZHUH PDGH EHIRUH DQG DIWHU GH[DPHWKDVRQH LQIXVLRQ ZLWK GLIIHUHQW VWLPXOL WR $&7+ VHFUHWLRQ 7KH GDWD VXJJHVW WKDW JOXFRFRUWLFRLG DFW LQ D VLWHVSHFLILF PDQQHU WR LQKLELW $&7+ VHFUHWLRQ &DQQ\ HW DO f 'XULQJ DXGLRYLVXDO VWUHVV K\SRWKDODPLF &5+ DQG $93 VHFUHWLRQ ZHUH XQDOWHUHG EXW $&7+ VHFUHWLRQ ZDV LQKLELWHG VXJJHVWLQJ D SLWXLWDU\ VLWH RI DFWLRQ RI GH[DPHWKDVRQH ,Q UHVSRQVH WR K\SRJO\FHPLD GH[DPHWKDVRQH LQKLELWHG ERWK WKH K\SRWKDODPLF DQG SLWXLWDU\ UHVSRQVHV WR WKH VWUHVV 6WXGLHV LQ UDWV KDYH VKRZQ WKDW JOXFRFRUWLFRLGV LQKLELW &5+ VHFUHWLRQ DV ZHOO DV &5+ V\QWKHVLV 6DWR HW DO f VXJJHVWLQJ D PHFKDQLVP IRU D

PAGE 31

K\SRWKDODPLF VLWH RI DFWLRQ 2Q WKH RWKHU KDQG JOXFRFRUWLFRLGV PD\ LQWHUIHUH ZLWK &5+ DFWLYDWLRQ RI VHFRQGPHVVHQJHU V\VWHPV F$03f DW WKH SLWXLWDU\ DQG WKHUHIRUH SUHYHQW VWLPXODWLRQ RI $&7+ VHFUHWLRQ %LOH]LNMLDQ DQG 9DOH f ,Q WKH IHWXV FRUWLVRO KDV WKH DELOLW\ WR LQKLELW SODVPD $&7+ VHFUHWLRQ LQ UHVSRQVH WR D K\SRWHQVLYH VWLPXOXV E\ DSSUR[LPDWHO\ GD\V JHVWDWLRQ +DUJUDYH DQG 5RVH f +LJK FRQFHQWUDWLRQV RI JOXFRFRUWLFRLGV QHDU WKH IHWDO 391 SUHYHQW LQFUHDVHG $&7+ VHFUHWLRQ LQ UHVSRQVH WR K\SRWHQVLRQ DQG K\SR[HPLD 0F'RQDOG HW DO f %HWZHHQ DQG GD\V JHVWDWLRQ IHWDO VKHHS DUH H[WUHPHO\ VHQVLWLYH WR QHJDWLYH IHHGEDFN HIIHFWV RI FRUWLVRO 7KLV ZDV GHPRQVWUDWHG E\ LQIXVLRQV RI FRUWLVRO WKDW FDXVHG OHVV WKDQ QJPO LQFUHDVHV LQ SODVPD FRUWLVRO FRQFHQWUDWLRQV EXW ZKLFK FRPSOHWHO\ LQKLELWHG WKH QRUPDO $&7+ UHVSRQVH WR K\SRWHQVLRQ :RRG f 7KLV NQRZOHGJH SUHGLFWV WKH H[LVWHQFH RI D QRUPDO IHHGEDFN UHVSRQVH LQ WKH IHWXV ZKLFK LV WKH FDVH LQ ODWH JHVWDWLRQDO IHWDO VKHHS +RZHYHU LQ QHDUWHUP IHWDO VKHHS FRUWLVRO QHJDWLYH IHHGEDFN UHJXODWLRQ RI $&7+ VHFUHWLRQ EHFRPHV LQHIIHFWLYH ,Q H[SHULPHQWV LQ ZKLFK LQIXVLRQV RI FRUWLVRO LQFUHDVHG SODVPD FRUWLVRO FRQFHQWUDWLRQV WR DSSUR[LPDWHO\ QJPO IHWDO SODVPD $&7+ VHFUHWLRQ ZDV VWLOO QRW VXSSUHVVHG :RRG f 7KLV PHFKDQLVP RI WKLV UHGXFWLRQ RI JOXFRFRUWLFRLG QHJDWLYH IHHGEDFN HIILFDF\ LV QRW IXOO\ XQGHUVWRRG DW SUHVHQW 7KH 'HYHORSPHQW RI WKH )HWDO +3 $ $[LV 'HYHORSPHQW RI WKH RYLQH IHWDO +3$ D[LV EHJLQV GXULQJ WKH ILUVW WKLUG RI JHVWDWLRQ WHUP EHLQJ GD\V JHVWDWLRQf ZLWK WKH IRUPDWLRQ RI WKH SLWXLWDU\ DQG DGUHQDO JODQGV 7KH IHWDO SLWXLWDU\ JODQG FDQ EH GHWHFWHG DV HDUO\ DV GD\V JHVWDWLRQ DQG GLIIHUHQWLDWLRQ RI WKH DQWHULRU SLWXLWDU\ FDQ EH VHHQ DW DERXW GD\V 3HUU\ HW DO f 6WDLQLQJ RI FHOOV LQ WKH DQWHULRU SLWXLWDU\ LQGLFDWH WKH SUHVHQFH RI $&7+ LPPXQRUHDFWLYLW\

PAGE 32

E\ DV HDUO\ DV GD\V JHVWDWLRQV DQG E\ GD\V JHVWDWLRQ SURFHVVLQJ RI $&7+ IURP 320& FDQ EH GHWHFWHG LQ WKH LQWHUPHGLDWH OREH RI WKH SLWXLWDU\ RI WKH IHWDO VKHHS 0XOYRJXH HW DO f 7KH IHWDO DGUHQDO FDQ EH LGHQWLILHG E\ DSSUR[LPDWHO\ GD\V JHVWDWLRQ LQ WKH VKHHS :LQWRXU HW DO f %\ GD\V JHVWDWLRQ LQ YLYR H[SHULPHQWV GHPRQVWUDWH WKDW WKH IHWDO DGUHQDO UHDGLO\ VHFUHWHV FRUWLVRO LQ UHVSRQVH WR $&7+ :LQWRXU HW DO *OLFNPDQ DQG &KDOOLV f %HWZHHQ GD\V JHVWDWLRQ WKH FHOOV RI WKH ]RQD IDVFLFXODWD ZKLFK DUH UHVSRQVLEOH IRU FRUWLVRO V\QWKHVLVf DUH UHODWLYHO\ LPPDWXUH 5RELQVRQ HW DO f DQG DSSUR[LPDWHO\ b RI IHWDO SODVPD FRUWLVRO LV GHULYHG IURP WKH PDWHUQDO FLUFXODWLRQ +HQQHVV\ HW DO f $IWHU GD\V JHVWDWLRQ IHWDO DGUHQDO VHQVLWLYLW\ WR SODVPD $&7+ LQFUHDVHV /LJJLQV HW DO 5RVH HW DO f WKH SURSRUWLRQ RI FRUWLVRO WKDW LV RI IHWDO RULJLQ LQFUHDVHV +HQQHVV\ HW DO f DQG WKH FRUUHODWLRQ EHWZHHQ IHWDO $&7+ VHFUHWLRQ DQG IHWDO FRUWLVRO VHFUHWLRQ EHFRPHV VLJQLILFDQW +HQQHVV\ HW DO f $GUHQDO ZHLJKW DOVR LQFUHDVHV DV D IXQFWLRQ RI JHVWDWLRQDO DJH &RPOLQH DQG 6LOYHU f (DUO\ VWXGLHV LQYROYLQJ IHWDO K\SRSK\VHFWRP\ /LJJLQV HW DO f DQG DGUHQDOHFWRP\ 'URXVW DQG +ROP f ZLWK SURORQJDWLRQ RI SUHJQDQF\ VXJJHVWHG D OLQN EHWZHHQ WKH DGUHQDO VWHURLG SURGXFWLRQ DQG SDUWXULWLRQ ,I WKLV LV WKH FDVH IHWDO SODVPD FRUWLVRO VHFUHWLRQ VKRXOG EH DOWHUHG DV JHVWDWLRQ QHDUV DQG HQGV %HIRUH DERXW GD\V JHVWDWLRQ IHWDO SODVPD FRUWLFRVWHURQH OHYHOV DUH ORZ EXW VHYHUDO GD\V EHIRUH SDUWXULWLRQ SODVPD OHYHOV LQFUHDVH DQG SHDN DW ELUWK %DVVHWW DQG 7KRUEXP f 1DWKDQLHOV] HW DO f IRXQG WKDW IHWDO SODVPD FRUWLVRO FRQFHQWUDWLRQV EHJDQ WR LQFUHDVH DERXW WKUHH WR IRXU GD\V EHIRUH SDUWXULWLRQ DQG WKHQ VWHDGLO\ GHFOLQHG LQ WKH QHZERUQ ODPE $ PRUH HODERUDWH VWXG\ RI FRUWLVRO VHFUHWLRQ ZDV SHUIRUPHG E\ 0DJ\DU HW DO f LQ ZKLFK

PAGE 33

H[SRQHQWLDO FXUYHV ZHUH ILW WR WKH GDWD WR PRUH DFFXUDWHO\ GHVFULEH WKH LQFUHDVH LQ IHWDO SODVPD FRUWLVRO FRQFHQWUDWLRQV 7KLV DQDO\VLV UHYHDOHG IHWDO SODVPD FRUWLVRO FRQFHQWUDWLRQV LQFUHDVLQJ H[SRQHQWLDOO\ DERXW GD\V SULRU WR SDUWXULWLRQ &RUWLVRO LV VHFUHWHG LQ UHVSRQVH WR $&7+ ELQGLQJ WR UHFHSWRUV DW WKH DGUHQDO JODQG ,Q YLYR H[SHULPHQWV E\ %URZQ HW DO f GHPRQVWUDWHG WKDW QRW RQO\ GLG JOXFRFRUWLFRLGV LQFUHDVH ZLWK GHYHORSPHQW EXW WKH DELOLW\ RI WKH IHWDO DGUHQDO WR VHFUHWH FRUWLVRO LQ UHVSRQVH WR $&7+ ZDV REVHUYHG DURXQG GD\V JHVWDWLRQ 7KH LQFUHDVH LQ FRUWLVRO VHFUHWLRQ WKDW RFFXUV QHDU WKH HQG RI JHVWDWLRQ LV LQ SDUW GXH WR DQ LQFUHDVH LQ DGUHQDO VHQVLWLYLW\ WR VWLPXODWLRQ E\ $&7+ 0DGLOO DQG %DVVHWW f EXW DOVR SRVVLEO\ GXH WR LQFUHDVHG SODVPD $&7+ VHFUHWLRQ IURP WKH IHWXV -RQHV HW DO f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f ,Q IDFW IHWDO SODVPD $&7+ FRQFHQWUDWLRQV LQFUHDVH GXULQJ WKH ODVW GD\V RI JHVWDWLRQ ZHOO EHIRUH WKH LQFUHDVH LQ SODVPD FRUWLVRO FRQFHQWUDWLRQV ZKLFK RFFXUUHG DSSUR[LPDWHO\ GD\V JHVWDWLRQ 7KH UHVXOWV RI LQFUHDVLQJ SODVPD $&7+ FRQFHQWUDWLRQV LQ YLYR ZKHQ WDNHQ WRJHWKHU ZLWK WKH LQ YLWUR SLWXLWDU\ $&7+ VHFUHWLRQ VWXGLHV VXSSRUW WKH SUHVHQFH RI DQ DGGLWLRQDO IDFWRU LQYROYHG LQ WKH SURFHVV RI SDUWXULWLRQ %DVDO RXWSXW RI $&7+ IURP IHWDO

PAGE 34

VKHHS SLWXLWDULHV LQ FXOWXUH GRHV QRW LQFUHDVH DV D IXQFWLRQ RI JHVWDWLRQDO DJH 'XUDQG HW DO f 3LWXLWDU\ $&7+ VHFUHWLRQ LQ FXOWXUH GRHV QRW LQFUHDVH GXULQJ WKH ODVW ZHHN RI JHVWDWLRQ DW D WLPH ZKHQ LQ YLYR FLUFXODWLQJ SODVPD $&7+ FRQFHQWUDWLRQV DUH LQFUHDVLQJ H[SRQHQWLDOO\ 0F0LOOHQ DQG 0HUHL f 7KHVH GDWD VXJJHVW WKDW WKH SUHSDUWXULHQW LQFUHDVH LQ $&7+ VHFUHWLRQ LV GHSHQGHQW RQ VRPH RWKHU IDFWRU WR VWLPXODWH VHFUHWLRQ DQG QRW D IXQFWLRQ RI EDVDO SLWXLWDU\ RXWSXW ,Q DGGLWLRQ 0F0LOOHQ DQG 0HUHL f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f ,Q D SDSHU E\ &KDOOLV DQG 3DWULFN IHWDO SODVPD HVWURQH FRQFHQWUDWLRQV LQFUHDVH IURP SJPO DW GD\V SULRU WR ELUWK WR QHDU SJPO WKH GD\ EHIRUH ELUWK )HWDO SODVPD HVWUDGLRO FRQFHQWUDWLRQV LQFUHDVHG IURP SJPO DW GD\V SULRU WR ELUWK WR SJPO RQ WKH GD\ EHIRUH SDUWXULWLRQ 3ODVPD HVWUDGLRO FRQFHQWUDWLRQV LQ WKH HZH LQFUHDVH RYHU WKH ODVW WZR GD\V RI SUHJQDQF\ LQ WKH HZH 5REHUWVRQ DQG 6PHDWRQ f ZLWK D WHQIROG LQFUHDVH RQ WKH GD\ EHIRUH SDUWXULWLRQ IURP WR SJPO XS WR SJPO &KDOOLV f $W WKH WLPH

PAGE 35

SODVPD HVWURJHQV LQFUHDVH WKHUH LV D GHFUHDVH LQ SODVPD SURJHVWHURQH %HGIRUG HW DO f 7KLV LV SRVVLEO\ GXH WR D FRQYHUVLRQ RI HVWURJHQ WR SURJHVWHURQH ZLWK SODVPD SURJHVWHURQH DFWLQJ DV D UHVHUYRLU IRU SODVPD HVWURJHQ SURGXFWLRQ 6LQFH SURJHVWHURQH LV DOVR D SUHFXUVRU IRU DQGURJHQ SURGXFWLRQ RQH ZRXOG H[SHFW WR VHH LQFUHDVHV LQ SODVPD FRQFHQWUDWLRQV RI WKHVH VWHURLGV DV ZHOO 3ODVPD DQGURVWHQHGLRQH DQG WHVWRVWHURQH FRQFHQWUDWLRQV GR LQGHHG LQFUHDVH LQ WKH ODWH JHVWDWLRQDO IHWXV LQ D PDQQHU VLPLODU WR HVWURJHQ FRQFHQWUDWLRQV 3RPHUDQW] DQG 1DOEDQGRY
PAGE 36

7LVVXH HVWURJHQ FRQFHQWUDWLRQV LQ VKHHS DOVR LQFUHDVH WRZDUGV WHUP RU DIWHU $&7+LQGXFHG ODERU HVSHFLDOO\ LQ WKH P\RPHWULXP 3RZHU DQG &KDOOLV f 7KH RYLQH SODFHQWD WKURXJK VXOIDWDVH DQG DURPDWDVH DFWLYLWLHV FRQYHUWV HVWUDGLRO DQG HVWURQH VXOIDWH WR (VWUDGLRO DQG HVWURQH WKH PRUH SRWHQW HVWURJHQ IRU WKH WDUJHW RUJDQ WKH P\RPHWULXP 5RVVLHU DQG 3LHUUHSRLQW f ,W KDV ORQJ EHHQ NQRZQ WKDW IHPDOH UDWV KDYH JUHDWHU DFWLYLW\ RI WKH +3$ D[LV WKDQ PDOH UDWV 6WXGLHV LQ DGXOW DQLPDOV GHPRQVWUDWH WKDW IHPDOH UDWV KDYH LQFUHDVHG FRUWLFRVWHURQH VHFUHWLRQ IROORZLQJ $&7+ DGPLQLVWUDWLRQ DQG JUHDWHU DGUHQDO UHVSRQVLYHQHVV WR WURSLF VWLPXODWLRQ .LWD\ f $V WKH PDMRU GLIIHUHQFH EHWZHHQ WKH VH[HV LV JRQDG DQG VWHURLG SURGXFWLRQ H[SHULPHQWV LQYROYLQJ JRQDGHFWRP\ DQG UHSODFHPHQW RI JRQDGDO VWHURLGV ZHUH SHUIRUPHG $IWHU JRQDGHFWRP\ WHVWRVWHURQH GHSUHVVHG $&7+ FRQWHQW DQG VWHURLG FOHDUDQFH LQ PDOH UDWV EXW LQFUHDVHG DGUHQDO UHVSRQVLYHQHVV WR $&7+ ,Q IHPDOH UDWV HVWUDGLRO KDG D FRQVLVWHQW VWLPXODWRU\ HIIHFW RQ $&7+ VHFUHWLRQ .LWD\ f ,Q IHPDOH UDWV SODVPD $&7+ DQG FRUWLFRVWHURQH UHVSRQVHV WR UHVWUDLQW VWUHVV ZHUH HQKDQFHG GXULQJ SURHVWUXV ZKHQ HVWUDGLRO FRQFHQWUDWLRQV ZHUH KLJKHVW .LWD\ f ,Q RYDULHFWRPL]HG RY[f UDWV UHSODFHG ZLWK HVWUDGLRO WKLV HIIHFW FDQ EH UHVWRUHG 9LDX DQG 0HDQH\ f )ROORZLQJ RY[ WKHUH ZDV D GHFUHDVHG FDSDFLW\ RI WKH SLWXLWDU\ WR V\QWKHVL]H $&7+ DQG D GHFUHDVHG UHVSRQVLYHQHVV WR VWLPXODWLRQ E\ K\SRWKDODPLF H[WUDFWV &R\QH DQG .LWD\ f ,Q RY[ UDWV HVWUDGLRO LPSODQWV LQWR WKH DUHD RI WKH DQWHULRU SLWXLWDU\ DUFXDWH QXFOHXV DQG ODWHUDO PDPPLODU\ ERGLHV LQ UDWV IDFLOLWDWHG SLWXLWDU\DGUHQDO DFWLYLW\ VXJJHVWLQJ D FHQWUDO QHUYRXV V\VWHP HIIHFW 5LFKDUG f +RZHYHU HVWURJHQ VWLPXODWLRQ RI FRUWLFRVWHURQH VHFUHWLRQ LQ RY[ UDWV PD\ EH GXH LQ SDUW WR D GLUHFW HIIHFW RQ WKH DGUHQDO FRUWH[ .LWD\ HW DO f ,Q RY[

PAGE 37

UDWV SODVPD $&7+ DQG FRUWLFRVWHURQH UHVSRQVHV WR IRRW VKRFN DQG HWKHU YDSRU VWUHVV ZHUH ORZHU WKDQ LQ HVWURJHQUHSODFHG RY[ UDWV %XUJHVV DQG +DQGD f )URP WKHVH UHVXOWV LW ZDV FRQFOXGHG WKDW WKH LQFUHDVHG DFWLYLW\ LQ WKH SLWXLWDU\DGUHQDO D[LV ZDV GXH WR DQ LPSDLUPHQW RI WKH JOXFRFRUWLFRLG QHJDWLYH IHHGEDFN V\VWHP 7KH PHFKDQLVP RI DFWLRQ RI HVWURJHQ VWLPXODWLRQ RQ +3$ D[LV DFWLYLW\ LV QRW IXOO\ XQGHUVWRRG %DVHG RQ WKH SUHVHQW GDWD HVWURJHQ PD\ LQWHUDFW ZLWK D QXPEHU RI V\VWHPV LPSLQJLQJ RQ WKH +3$ D[LV (VWURJHQ XSWDNH KDV EHHQ GHPRQVWUDWHG LQ FRUWLFRWURSKLQV LVRODWHG IURP DQWHULRU SLWXLWDU\ FHOOV IURP DGUHQDOHFWRPL]HG UDWV .HHIHU f VXJJHVWLQJ D SRVVLEOH GLUHFW HIIHFW RI HVWURJHQ RQ WKH SLWXLWDU\ (VWUDGLRO KDV DOVR EHHQ VKRZQ WR FRQFHQWUDWH LQ W\URVLQH K\GUR[\ODVH FRQWDLQLQJ QHXURQV LQ WKH DUFXDWH DQG SHULYHQWULFXODU QXFOHL RI WKH UDW 6DU f ,Q DQRWKHU VWXG\ HVWUDGLRO FRQFHQWUDWLQJ FHOOV KDYH EHHQ IRXQG LQ WKH DP\JGDORLG FHQWUDO QXFOHXV %HDXOLHX HW DO f 6LQFH WKLV GRSDPLQHUJLF V\VWHP FDQ LQKLELW $&7+ VHFUHWLRQ WKLV PD\ EH LPSRUWDQW LQ PRGXODWLQJ +3$ D[LV I DFWLYLW\ ,Q UDWV JLYHQ _LJ HVWUDGLRO H[RJHQRXVO\ IRU WZR ZHHNV WKHUH ZDV DQ LQFUHDVH LQ $93 LQ WKH 621 DQG 391 RI WKH K\SRWKDODPXV ZLWKRXW DQ\ FKDQJHV LQ SLWXLWDU\ $&7+ DQG $93 FRQWHQW RU EDVDO SODVPD $&7+ RU $93 FRQFHQWUDWLRQV +DVKLPRWR HW DO f ,Q DQRWKHU VWXG\ SODVPD $93 FRQFHQWUDWLRQV ZHUH IRXQG WR EH JUHDWHVW ZKHQ HVWURJHQ FRQFHQWUDWLRQV ZHUH KLJKHVW ,I WKH UDWV ZHUH RY[ SODVPD $93 FRQFHQWUDWLRQV GHFUHDVHG EXW ZHUH UHVWRUHG ZKHQ HVWURJHQ ZDV UHSODFHG 6NRZVN\ HW DO f 7KHVH GDWD VXJJHVW D SRVVLEOH UROH RI K\SRWKDODPLF UHOHDVLQJ IDFWRUV LQ WKH PHGLDWLRQ RI HVWURJHQ VWLPXODWLRQ RI +3$ D[LV DFWLYLW\ 7KH UHVXOWV RI WKLV GLVVHUWDWLRQ GHPRQVWUDWH WKDW WKHUH LV VLJQLILFDQW HVWURJHQ VXOIDWDVH DFWLYLW\ LQ RYLQH IHWDO K\SRWKDODPXV KLSSRFDPSXV DQG EUDLQVWHP DQG WKDW WKHUH

PAGE 38

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f DQG /DNVKPL DQG %DODVXEUDPDQLDQ f KDYH SUHYLRXVO\ GHPRQVWUDWHG HVWURJHQ VXOIDWDVH DQG VXOIRWUDQVIHUDVH DFWLYLW\ LQ DGXOW VKHHS EUDLQ WLVVXH 2WKHU LQYHVWLJDWRUV KDYH GHPRQVWUDWHG WKHVH HQ]\PDWLF DFWLYLWLHV LQ DGXOW EUDLQ WLVVXH IURP UDWV &RQQROO\ DQG 5HVNR .DZDQR DQG $LNDZD f PLFH +RENLUN f QRQKXPDQ SULPDWHV /DNVKPL DQG %DODVXEUDPDQLDQ f DQG KXPDQ EHLQJV 3ODWLD f +RENLUN DQG FRZRUNHUV GHPRQVWUDWHG WKDW HQ]\PH DFWLYLWLHV DUH WUDQVLHQWO\ LQFUHDVHG SRVWQDWDOO\ LQ WKH EUDLQ RI WKH PRXVH f :KLOH WKH GHYHORSPHQW RI EUDLQ HVWURJHQ VXOIDWDVH DQG VXOIRWUDQVIHUDVH DFWLYLW\ KDYH QRW EHHQ VWXGLHG LQ VKHHS WKH GHYHORSPHQW RI DFWLYLWLHV LQ PLFH VXJJHVWV WKH SRVVLELOLW\ WKDW WKLV PLJKW EH DQ LPSRUWDQW GHYHORSPHQWDO SURFHVV LQ WKH SHULQDWDO SHULRG 8VLQJ D KLVWRFKHPLFDO WHFKQLTXH .DZDQR DQG $LNDZD IRXQG WKDW VXOIDWDVH DFWLYLW\ LV KLJKHVW LQ SLQHDO JODQG FKRURLG SOH[XV DQG SDUV GLVWDOLV RI WKH SLWXLWDU\ LQ DGXOW UDWV f LQYHVWLJDWHG WKH DFWLYLW\ LQ K\SRWKDODPXV EUDLQVWHP DQG KLSSRFDPSXV EHFDXVH WKHVH DUHDV DUH NQRZQ WR FRQWDLQ QXFOHL LQYROYHG LQ LQWHJUDWLRQ DIIHUHQW VLJQDO UHOD\ RU QHJDWLYH IHHGEDFN LQKLELWLRQ ZLWKLQ WKH +3$ D[LV *UL]]OH HW DO .HOOHU:RRG DQG 'DOOPDQ 0DUDQ :DUG f 7KH SUHVHQFH RI DFWLYLW\ LQ DQ\ RI WKHVH DUHDV

PAGE 39

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f DQ DFWLRQ RQ WKH 176 ZKLFK UHOD\V QHXUDO WUDIILF IURP YLVFHUDO DILIHUHQWVf RU DQ DFWLRQ RQ DQ\ SDUW RI WKH SDWKZD\V OHDGLQJ IURP WKH 176 WR WKH 391 HJ WKH 59/0f (VWURJHQ UHFHSWRUV KDYH EHHQ GHPRQVWUDWHG LQ WKH 176 DQG KLSSRFDPSXV /HKPDQ f :KLOH HVWURJHQ UHFHSWRUV ZLWKLQ WKH K\SRWKDODPXV DUH PRVW FRQFHQWUDWHG LQ WKH DUFXDWH QXFOHXV HVWURJHQ UHFHSWRUV KDYH EHHQ GHPRQVWUDWHG LQ WKH 391 /HKPDQ 6LPHUO\ f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

PAGE 40

LQKLELWLRQ RI +3$ D[LV QHJDWLYH IHHGEDFN LV DFKLHYHG 7KLV FRQFRPLWDQW ZLWK ORFDO DFWLYDWLRQ RI HVWURJHQV YLD HVWURJHQ VXOIDWDVH ZRXOG LQFUHDVH $&7+ UHOHDVH &DUGLRYDVFXODU 5HIOH[ 5HVSRQVLYHQHVV $UWHULDO EDURUHFHSWRUV DUH PHFKDQRUHFHSWRUV ORFDWHG LQ WKH ZDOOV RI ODUJH V\VWHPLF DUWHULHV LQFOXGLQJ WKH DRUWLF DUFK EUDFKLRFHSKDOLF DUWHU\ DQG FDURWLG VLQXVHV %RVV DQG *UHHQ *UHHQ f $IIHUHQW VLJQDOV IURP WKH DUFK RI WKH DRUWD DUH WUDQVPLWWHG WKURXJK WKH OHIW DQG ULJKW DRUWLF GHSUHVVRU QHUYHV WR WKH YDJXV QHUYH DQG XOWLPDWHO\ WR WKH QXFOHXV RI WKH WUDFWXV VROLWDULXV LQ WKH PHGXOODU\ DUHD RI WKH EUDLQ VWHP %RVV DQG *UHHQ 1DND\PD 1RQLQGH] f 6HQVRU\ LQSXW IURP WKH FDURWLG VLQXV UHJLRQ WUDYHOV WR WKH QXFOHXV RI WKH WUDFWXV VROLWDULXV DV ZHOO EXW YLD WKH VLQXV +HULQJfVf DQG JORVVRSKDU\QJHDO QHUYHV %DURVHQVLWLYH QHUYH HQGLQJV DUH IRXQG LQ DUHDV RI WKH DUWHULHV ZLWK ODUJH TXDQWLWLHV RI HODVWLF WLVVXH 0XUDWRUL f $SSUR[LPDWHO\ b RI WKH WLVVXH FRPSULVLQJ WKH ZDOOV RI WKH DRUWLF DUFK LV DQ HODVWLQFROODJHQ PL[WXUH %DGHU f ZKLFK LV DOPRVW IUHH RI VPRRWK PXVFOH *UHJRUHYD f 6LPLODUO\ WKH FDURWLG VLQXV LV WKLQQHU $GDPV $GGLVRQ 5HHV 5HHV DQG -HSVRQ f FRQWDLQV OHVV VPRRWK PXVFOH $GGLVRQ %DJVKDZ DQG )LVFKHU 0XUDWRUL 5HHV DQG -HSVRQ f DQG VKRZV D KLJKHU HODVWLQ FRQWHQW WKDQ RWKHU DUHDV RI WKH FDURWLG DUWHU\ $GGLVRQ 5HHV DQG -HSVRQ f $OWKRXJK WKHLU QDPH LPSOLHV D SUHVVXUHVHQVLWLYH TXDOLW\ EDURUHFHSWRUV DUH VWUHWFK UHFHSWRUV ZKLFK UHVSRQG WR GHIRUPDWLRQ RI WKH YHVVHO ZDOO LQ ZKLFK WKH\ DUH ORFDWHG +DXVV HW DO $QJHOO-DPHV f 7KHUH LV HYLGHQFH ZKLFK VKRZV WKDW WKH GHJUHH RI ZDOO GHIRUPDWLRQ GHWHUPLQHV WKH HOHFWULFDO DFWLYLW\ RI WKH FDURWLG VLQXV DQG DRUWLF DUFK

PAGE 41

EDURUHFHSWRUV +DXVV HW DO f GHPRQVWUDWHG WKDW WKH UHIOH[ IDOO RI EORRG SUHVVXUH SURGXFHG E\ DQ LQFUHDVH LQ FDURWLG VLQXV SUHVVXUH LV DEROLVKHG LI WKH VWUHWFKLQJ RI WKH FDURWLG DUWHU\ LV SUHYHQWHG E\ D SODVWHU FDVW DSSOLHG WR WKH RXWVLGH RI WKH VLQXV UHJLRQ $GGLWLRQDOO\ $QJHOO-DPHV f UHSRUWHG WKDW LQFUHDVHG EDURUHFHSWRU DFWLYLW\ SURGXFHG E\ HOHYDWLRQ RI LQWUDWKRUDVLF SUHVVXUH FRXOG EH SUHYHQWHG E\ VLPXOWDQHRXVO\ LQFUHDVLQJ WKH H[WUDPXUDO SUHVVXUH E\ WKH VDPH DPRXQW ([SHULPHQWV LQ PDQ KDYH VKRZQ WKDW FKDQJLQJ SUHVVXUH LQ D FKDPEHU VXUURXQGLQJ WKH QHFN UHVXOWV LQ UHIOH[ FKDQJHV LQ KHDUW UDWH DQG EORRG SUHVVXUH DQG SURYLGH IXUWKHU HYLGHQFH WKDW DOWHUHG WUDQVPXUDO SUHVVXUH LV D VWLPXOXV IRU EDURUHFHSWRU DFWLYDWLRQ %HYHJDUG DQG 6KHSDUG (UQHVWLQH DQG 3DUU\ f .RFK f ZDV WKH ILUVW WR GHPRQVWUDWH WKDW ZKHQ FDURWLG VLQXV SUHVVXUH ZDV FKDQJHG LQ D VWHSZLVH PDQQRU PHDQ DUWHULDO SUHVVXUH H[KLELWHG DQ LQYHUVH VLJPRLGDO UHVSRQVH WR WKH FKDQJH LQ LQWUDVLQXV SUHVVXUH %URQN DQG 6WHOOD f ZKR REVHUYHG WKDW WKH LPSXOVH IUHTXHQF\ LQ +HUULQJfV QHUYH H[KLELWHG D SRVLWLYH VLJPRLGDOn UHODWLRQVKLS WR FKDQJHV LQ VLQXV SUHVVXUH ODWHU VXEVWDQWLDWHG WKHVH ILQGLQJV 7KHVH HDUO\ ILQGLQJV LGHQWLILHG WKDW EDURUHFHSWRUV FDQ EH FKDUDFWHUL]HG DV KDYLQJ D WKUHVKROG SUHVVXUH UDQJH IRU ZKLFK WKH GLVFKDUJH UDWH LQFUHDVHV ZLWK D ULVH LQ PHDQ DUWHULDO SUHVVXUH DV DQ DV\PSWRWLF VDWXUDWLRQ SUHVVXUH EH\RQG ZKLFK WKHUH LV OLWWOH LQFUHDVH LQ EDURUHFHSWRU DFWLYLW\ .RXVKDQSRXU f 7KH DRUWLF DQG FDURWLG EDURUHFHSWRUV H[KLELW GLIIHUHQW WKUHVKROG DQG VDWXUDWLRQ FKDUDFWHULVWLFV &DURWLG VLQXV EDURUHFHSWRUV DUH VLOHQW DW DUWHULDO SUHVVXUHV EHWZHHQ DQG PP+J EXW DERYH PP+J WKH\ UHVSRQG SURJUHVVLYHO\ DQG UHDFK PD[LPXP GLVFKDUJH FDSDFLW\ DW DSSUR[LPDWHO\ PP+J .RXVKDQSRXU f $RUWLF EDURUHFHSWRUV UHVSRQG LQ D PDQQHU VLPLODU WR WKDW RI WKH FDURWLGV H[FHSW WKDW WKH\ H[KLELW D WKUHVKROG

PAGE 42

SUHVVXUH DSSUR[LPDWHO\ PP+J KLJKHU .RXVKDQSRXU f 7KHUHIRUH LQ WKH QRUPDO RSHUDWLQJ UDQJH RI DSSUR[LPDWHO\ PP+J WR PP+Jf VOLJKW FKDQJHV LQ SUHVVXUH HOLFLW VWURQJ EDURUHFHSWRUPHGLDWHG DXWRQRPLF UHIOH[HV WR UHWXUQ DUWHULDO SUHVVXUH WR ZLWKLQ KRPHRVWDWLF OLPLWV %ORRG SUHVVXUH FRQWURO LQ WKH IHWXV LV VLPLODU ZLWK VRPH GLIIHUHQFHV 'HFUHDVHV LQ EORRG SUHVVXUH LQFUHDVH WKH VHFUHWLRQ RI $&7+ FRUWLVRO $93 DQG UHQLQ 5RELOODUG HW DO 5RVH HW DO :RRG f $V LQ WKH DGXOW WKH PDJQLWXGH RI WKH UHVSRQVHV DUH SURSRUWLRQDO WR WKH PDJQLWXGH RI WKH GHFUHDVH LQ DUWHULDO SUHVVXUH :RRG HW DO f DQG UHVSRQVHV DUH DWWHQXDWHG E\ VLQRDRUWLF GHQHUYDWLRQ :RRG f &KHPRUHFHSWRUV KDYH D FULWLFDO UROH LQ PDLQWDLQLQJ EORRG SUHVVXUH DV ZHOO E\ PRQLWRULQJ WKH OHYHOV RI R[\JHQ FDUERQ GLR[LGH DQG K\GURJHQ LRQV LQ WKH EORRG (IIHFWLYH EDURUHFHSWRU IXQFWLRQ LV QHFHVVDU\ WR UHVSRQG WR WUDQVLHQW DOWHUDWLRQV LQ DUWHULDO SUHVVXUH %URZQ f ,Q WKH IDFH RI LQFUHDVLQJ SUHVVXUH EDURUHFHSWRU JHQHUDWHG VLJQDOV DVFHQG DIIHUHQW SDWKZD\V DQG HQWHU WKH QXFOHXV WUDFWXV VROLWDULXV ZKHUH VHFRQGDU\ VLJQDOV LQKLELW WKH PHGXOODU\ YDVRFRQVWULFWRU FHQWHU DQG H[FLWH WKH YDJDO FHQWHU VWLPXODWLQJ YDVRGLODWLRQ DQG GHFUHDVHG P\RFDUGLDO LRQRWURSLF DQG FKURQRWURSLF UHVSRQVH %URZQ f 7KHVH DFWLRQV OHDG WR ORZHUHG SHULSKHUDO UHVLVWDQFH FDUGLDF RXWSXW DQG XOWLPDWHO\ ORZHU EORRG SUHVVXUH &RQYHUVHO\ D VXGGHQ IDOO LQ DUWHULDO SUHVVXUH OHDGV WR UHIOH[ DFWLRQV ZKLFK LQFUHDVH FDUGLDF RXWSXW DQG V\VWHPLF UHVLVWDQFH WR UDLVH EORRG SUHVVXUH 7KH EUDG\FDUGLF UHVSRQVH WR EDURUHFHSWRU VWLPXODWLRQ LQ KXPDQV LV PHGLDWHG WKURXJK YDJDO FKROLQHUJLF PHFKDQLVPV 6HYHUDO LQYHVWLJDWRUV (FNEHUJ HW DO 3LFNHULQJ HW DO 6LPRQ HW DO 7DNHVKLWD HW DO f KDYH GHPRQVWUDWHG WKDW

PAGE 43

HORQJDWLRQ RI WKH 55 LQWHUYDO ZKLFK DFFRPSDQLHG D ULVH LQ DUWHULDO SUHVVXUH IROORZLQJ DGPLQLVWUDWLRQ RI SKHQ\OHSKULQH ZDV QRW UHGXFHG E\ SURSUDQRORO -RVH DQG 7D\ORU f EXW ZDV DEROLVKHG E\ DWURSLQH 2WKHUV KDYH UHSRUWHG VLPLODU REVHUYDWLRQV LQ UHVSRQVH WR VWLPXODWLRQ RI WKH FDURWLG EDURUHFHSWRUV E\ QHFN VXFWLRQ (FNEHUJ (FNEHUJ HW DO f ,Q FRQWUDVW WKHUH LV D ODFN RI FRQVHQVXV UHJDUGLQJ WKH DXWRQRPLF PHFKDQLVPV PHGLDWLQJ WKH WDFK\FDUGLF UHVSRQVH WR DUWHULDO EDURUHFHSWRU XQORDGLQJ ,W KDV EHHQ REVHUYHG WKDW WKH HDUO\ WDFK\FDUGLD REVHUYHG DIWHU DGPLQLVWUDWLRQ RI YDVRGLODWRUV ZDV XQDIIHFWHG E\ SURSUDQRORO EXW DEROLVKHG E\ DWURSLQH VXJJHVWLQJ D SUHGRPLQDQW YDJDO PHGLDWLRQ RI WKLV UHVSRQVH /HRQ HW DO 0DQFLD HW DO 0URF]HN HW DO 3LFNHULQJ HW DO f &RQWUDU\ WR WKHVH UHSRUWV RWKHUV KDYH GHPRQVWUDWHG WKDW WKH LQFUHDVH LQ KHDUW UDWH SURGXFHG GXULQJ LQIXVLRQV RI QLWURJO\FHULQ ZDV UHGXFHG E\ DWURSLQH EXW FRXOG RQO\ EH DEROLVKHG E\ FRPELQHG DGPLQLVWUDWLRQ RI DWURSLQH DQG D EHWDDGUHQHUJLF I EORFNHU *ROGVWHLQ HW DO 5RELQVRQ HW DO f ,Q DGGLWLRQ LW KDV EHHQ GHPRQVWUDWHG WKDW GXULQJ ORZHU ERG\ QHJDWLYH SUHVVXUH WDFK\FDUGLD ZDV GLPLQLVKHG b E\ SURSUDQRORO ZLWK WKH UHPDLQLQJ UHVSRQVH DEROLVKHG E\ DWURSLQH %MXUVWHGW HW DO f 7KHUHIRUH LW DSSHDUV WKHUH LV D VLJQLILFDQW YDJDO FRPSRQHQW WR WKH FDUGLRDFFHOHUDWLRQ ZKLFK DFFRPSDQLHV EDURUHFHSWRU XQORDGLQJ +RZHYHU DQ LQFUHDVH RI V\PSDWKHWLF FDUGLDF LQIOXHQFH PD\ FRQWULEXWH WR WKH PRUH VXVWDLQHG FRPSRQHQW RI EDURUHIOH[PHGLDWHG WDFK\FDUGLD 0DQFLD DQG 0DUN f 7DNHQ WRJHWKHU WKHVH UHVXOWV VXJJHVW UHGXQGDQF\ LQ PHFKDQLVPV E\ ZKLFK WKH DXWRQRPLF QHUYRXV V\VWHP PHGLDWHV EDURUHIOH[LQGXFHG WDFK\FDUGLD

PAGE 44

7KH EDURUHFHSWRU V\VWHP PDUNHGO\ UHGXFHV GDLO\ YDULDWLRQ LQ DUWHULDO SUHVVXUH 7KLV SKHQRPHQRQ LV UHDGLO\ GHPRQVWUDWHG LQ VLQRDRUWLF GHQHUYDWHG DQLPDOV ZKR H[KLELW HOHYDWHG EORRG SUHVVXUH &RZOH\ HW DO f &RQWURYHUV\ H[LVWV UHJDUGLQJ WKH SHUVLVWHQFH RI WKLV K\SHUWHQVLRQ ZLWK VRPH DXWKRUV DUJXLQJ LW HYHQWXDOO\ VXEVLGHV *X\WRQ HW DO f ZKLOH RWKHUV VXJJHVW HOHYDWHG EORRG SUHVVXUH SHUVLVWV 6FKHU DQG ,WR $OH[DQGHU 7RXZ HW DO :HUEHU DQG )LQN f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f DQG E\ EDURUHFHSWRUV LQ WKH KLJK SUHVVXUH FLUFXODWRU\ V\VWHP 5RVHHWDO f DQG UHVXOW LQ WKH LQFUHDVHG UHOHDVH RI $&7+ FRUWLVRO *DQQ f UHQLQ DQG $93 &OD\EDXJK DQG 6KDUH f ,Q WKH IHWXV WKH $&7+ FRUWLVRO $93 DQG UHQLQ UHVSRQVHV WR K\SRWHQVLRQ DUH QRW YDJDOO\ PHGLDWHG EXW LQVWHDG DUH WKRXJKW WR LQYROYH FKDQJHV LQ EORRG S+ DQG FHQWUDO FKHPRUHFHSWRUV :RRG HW DO f $&7+ LV LPSRUWDQW LQ ERWK DGXOW DQG IHWDO DQLPDOV IRU LQGXFLQJ WKH DSSURSULDWH DGUHQRFRUWLFDO UHVSRQVHV WR QR[LRXV VWLPXOL VXFK DV K\SRWHQVLRQ :RRG DQG 5XGROSK f &RUWLVRO LV HVVHQWLDO IRU WKH UHVWLWXWLRQ RI EORRG YROXPH IROORZLQJ KHPRUUKDJH DQG DV D SHUPLVVLYH VXEVWDQFH IRU DSSURSULDWH YDVRFRQVWULFWLRQ IROORZLQJ K\SRWHQVLRQ LQ ERWK WKH DGXOW *ULPHV HW DO 3LUNOH HW DO f DQG IHWXV %UDFH f 9DVRSUHVVLQ LV UHVSRQVLEOH IRU YDVRFRQVWULFWLRQ

PAGE 45

UHGLVWULEXWLRQ RI EORRG IORZ DQG DQWLGLXUHVLV LQ WKH DGXOW &RZOH\ HW DO f DQG IHWXV ,ZDPRWR HW DO f

PAGE 46

&+$37(5 *(1(5$/ 0(7+2'6 6XUJLFDO 3UHSDUDWLRQ RI )HWDO 6KHHS 7KH VKHHS XVHG LQ WKLV VWXG\ ZHUH DOO SUHJQDQW HZHV RI GD\V JHVWDWLRQ RU ODWHU $QLPDOV ZHUH SXUFKDVHG IURP YDULRXV VXSSOLHUV ,QVWLWXWH RI )RRG DQG $JULFXOWXUDO 6FLHQFHV 8QLYHUVLW\ RI )ORULGD *DLQHVYLOOH ), 7RP 0RUULV 0'f DQG ZHUH RI YDULRXV EUHHGV )ORULGD 1DWLYH 0L[HG :HVWHUQ HWFf 3ULRU WR VXUJHU\ DOO DQLPDOV ZHUH KRXVHG LQ DSSURYHG SHQV LQ WKH +HDOWK 6FLHQFH &HQWHU RU WKH WK 6WUHHW IDFLOLW\ DW WKH 8QLYHUVLW\ RI )ORULGD 3ULRU WR H[SHULPHQWDWLRQ DOO DQLPDOV ZHUH KRXVHG LQ $QLPDO 5HVRXUFHV DW WKH +HDOWK 6FLHQFH &HQWHU DQG ZHUH PDLQWDLQHG XQGHU FRQWUROOHG OLJKWLQJ DQG WHPSHUDWXUH 3HQV ZHUH FOHDQHG GDLO\ DQG HZHV ZHUH JLYHQ IRRG DQG ZDWHU DG OLELWXP $VHSWLF IHWDO VXUJHU\ ZDV SHUIRUPHG LQ $QLPDO 5HVRXUFHV RU DW WKH WK 6WUHHW IDFLOLW\ XQGHU JHQHUDO DQHVWKHVLD ZLWK b b KDORWKDQH $OO HZHV ZHUH EHWZHHQ DQG GD\V JHVWDWLRQ DW WKH WLPH RI VXUJHU\ )RRG DQG ZDWHU ZHUH ZLWKKHOG IURP HZHV KRXUV SULRU WR VXUJHU\ (ZHV ZHUH VKHDUHG FORVH WR WKH VNLQ DURXQG WKH DEGRPHQ DQG SUHSDUHG IRU VXUJHU\ ZLWK SRYLGRQH LRGLQH %HWDGLQHp 3XUGXH )UHGULFN &R 1RUZDON &7f $QLPDOV ZHUH LQWXEDWHG DQG FRQQHFWHG WR D UHVSLUDWRU WR DOORZ IRU FRQVWDQW DQHVWKHVLD +HDUW UDWH EORRG SUHVVXUH YHQWLODWRU\ DQG & UHVSLUDWLRQV SHU PLQXWH DQG UHFWDO WHPSHUDWXUH ZHUH DOO PRQLWRUHG DW WKH WLPH RI VXUJHU\ $QLPDOV ZHUH FORVHO\ PRQLWRUHG IURP WKH WLPH RI LQWXEDWLRQ XQWLO UHFRYHU\ ZKHQ WKH DQLPDO FRXOG VWDQG RQ LWV

PAGE 47

RZQ HIIRUW (ZHV ZHUH DOORZHG IUHH DFFHVV WR IRRG DQG ZDWHU WKURXJKRXW WKH SRVWn RSHUDWLYH SHULRG 7KH XWHUXV ZDV H[SRVHG XVLQJ D PLGOLQH LQFLVLRQ EHJLQQLQJ DW WKH XPELOLFXV DQG H[WHQGLQJ FDXGDOO\ DSSUR[LPDWHO\ FP 2QFH WKH KLQGOLPEV ZHUH ORFDWHG D VPDOO LQFLVLRQ ZDV PDGH LQ WKH XWHUXV +LQGOLPEV ZHUH GHOLYHUHG WKURXJK WKH XWHULQH LQFLVLRQ RQH DW D WLPH IRU WKH SXUSRVH RI SODFLQJ D SRO\YLQ\O FKORULGH FDWKHWHU f (' f 2'f LQWR HDFK IHPRUDO DUWHU\ /DWHU WKHVH FDWKHWHUV ZRXOG EH XVHG IRU EORRG VDPSOLQJ DQG EORRG SUHVVXUH UHFRUGLQJ 7KH WLSV RI HDFK IHPRUDO FDWKHWHU ZHUH DGYDQFHG WR WKH VXEGLDSKUDJPDWLF DRUWD $W WKLV WLPH DQ HVWUDGLRO LPSODQW PJ GD\V RU _LJGD\ ,QQRYDWLYH 5HVHDUFK RI $PHULFD 7ROHGR 2+f RU SODFHER ZDV LQVHUWHG VXEFXWDQHRXVO\ LQWR WKH DUHD RI WKH JOXWHXV PHGLXV EHIRUH VXWXULQJ WKH LQFLVHG KLQGOLPEV $Q DPQLRWLF FDWKHWHU PDGH RI SRO\YLQ\O FKORULGH f ,' f 2'f ZDV VXWXUHG WR WKH H[WHULRU RI D KLQGOLPE IRU WKH SXUSRVH RI DQWLELRWLF GHOLYHU\ DV ZHOO DV DPQLRWLF IOXLG SUHVVXUH PHDVXUHPHQWV +LQGOLPE DQG XWHULQH LQFLVLRQV ZHUH FORVHG XVLQJ VLON VXWXUH +LQGOLPE LQFLVLRQV ZHUH FORVHG XVLQJ D VLPSOH FRQWLQXRXV VXWXUH SDWWHUQ $OO XWHULQH LQFLVLRQV ZHUH FORVHG ILUVW ZLWK D ORFNLQJ VLPSOH FRQWLQXRXV SDWWHUQ IROORZHG E\ XPEULFDWLRQ RI WKH XWHUXV 8VLQJ D WHFKQLTXH VLPLODU WR WKH RQH GHVFULEHG DERYH FDWKHWHUV ZHUH SODFHG LQ WKH OLQJXDO DUWHULHV DQG DGYDQFHG FP LQWR WKH FDURWLG DUWHU\ WRZDUG WKH KHDUW 8SRQ FORVXUH RI WKH QHFN LQFLVLRQ OLQJXDO FDWKHWHUV ZHUH DQFKRUHG WR WKH FKLQ RI WKH IHWXV ZLWK VLON VXWXUH /LQJXDO FDWKHWHUV ZHUH RI WKH VDPH PDWHULDO DQG VL]H DQG IHPRUDO FDWKHWHUV 'HSHQGLQJ XSRQ WKH H[SHULPHQWDO VHWXS FDURWLG VLQXV GHQHUYDWLRQ ZDV HPSOR\HG DW WKLV WLPH $IWHU H[SRVLQJ WKH FRPPRQ FDURWLG DUWHU\ GHQHUYDWLRQ ZDV DFFRPSOLVKHG E\ VWULSSLQJ DOO QHUYHV DQG FRQQHFWLYH WLVVXH EHWZHHQ WKH FDURWLGRFFLSLWDO DUWHULDO MXQFWLRQ

PAGE 48

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f 7KH OHIW IRUHOLPE RI WKH IHWXV ZDV GHOLYHUHG WKURXJK D XWHULQH LQFLVLRQ $Q LQFLVLRQ ZDV PDGH LQWR WKH VHFRQG LQWHUFRVWDO VSDFH DQG WKH EUDFKLRFHSKDOLF DUWHU\ ZDV ORFDWHG 2QFH WKH RFFOXGHU ZDV VHZQ LQ SODFH WKH LQFLVLRQ XQGHU WKH OHIW IRUHDUP ZDV FORVHG DQG WKH IHWXV UHWXUQHG WR WKH XWHUXV 7KH XWHULQH LQFLVLRQ ZDV FORVHG RQFH DJDLQ XVLQJ D ORFNLQJ VLPSOH FRQWLQXRXV VXWXUH SDWWHUQ IROORZHG E\ DQ XPEULFDWLRQ WHFKQLTXH PJ DPSLFLOOLQ 3RO\IOH[p )W 'RGJH /DERUDWRULHV )W 'RGJH ,$f ZDV DGPLQLVWHUHG LQWR WKH DPQLRWLF FDYLW\ EHIRUH FORVXUH RI WKH PDWHUQDO OLQHD DOED DQG VNLQ $OO FDWKHWHUV ZHUH ILOOHG ZLWK KHSDULQ XQLWVPO (ONLQV6LQQ ,QF &KHUU\ +LOO 1-f DQG FORVHG ZLWK D VWHULOH EUDG LQVHUWHG LQWR WKH HQG &DWKHWHUV DQG RFFOXGHUV ZHUH IODQNHG DQG H[WHULRUL]HG YLD D WURFKDU &DWKHWHUV ZHUH KHOG LQ SODFH ZLWK DQ HODVWLF EDQGDJH 7KH OLQHD DOED ZDV FORVHG ZLWK SRO\DPLGH VXWXUH 3LWPDQQ0RRUH )UDQFHf ZKLOH WKH VNLQ ZDV FORVHG ZLWK PJ DPSLFLOOLQ ZDV DGPLQLVWHUHG LQWUDPXVFXODUO\ WR WKH HZH ,W VKRXOG EH QRWHG WKDW LQ HZHV ZLWK WZLQ SUHJQDQFLHV ERWK IHWXVHV ZHUH VXUJLFDOO\ PDQLSXODWHG WKH VDPH ZD\ $W WKH WLPH RI H[SHULPHQWDWLRQ KRZHYHU RQO\ RQH RI WKH

PAGE 49

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b YYf 2QH IHPRUDO FDWKHWHU DQG WKH DPQLRWLF FDWKHWHU ZHUH DWWDFKHG WR WUDQVGXFHUV 6WDWKDP 3,G 6WDWKDP ,QVWUXPHQWV 2[QDUG &$f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

PAGE 50

ZLWK EORRG VDPSOHV WDNHQ DW DQG PLQXWH WLPH SRLQWV ,I WKH IHWXV ZDV WR EH PDGH K\SRWHQVLYH WR DFWLYDWH WKH +3$ D[LV WKH EUDFKLRFHSKDOLF RFFOXGHU ZDV LQIODWHG DIWHU FROOHFWLRQ RI WKH WLPH SRLQW VDPSOH IRU PLQXWHV 7KH EUDFKLRFHSKDOLF RFFOXGHU ZDV LQIODWHG YLD DQ LQIXVLRQ RI VDOLQH WKURXJK WKH VLODVWLF WXELQJ 7KLV LQ WXUQ FDXVHV D K\SRSHUIXVLRQ RI EORRG WR WKH IHWDO EUDLQ ZKLFK DFWLYDWHV WKH +3$ D[LV )LYH PO RI EORRG ZHUH WDNHQ DW HDFK WLPH SRLQW DQG FROOHFWHG LQ FKLOOHG WXEHV FRQWDLQLQJ 1t ('7$ ILJ ('7$PO EORRG 6LJPD &KHPLFDO &R 6W /RXLV 02f $Q DGGLWLRQDO PO RI EORRG ZDV GUDZQ DQDHURELFDOO\ LQWR V\ULQJHV FRDWHG ZLWK KHSDULQ IRU PHDVXUHPHQW RI IHWDO EORRG JDVHV XVLQJ D &LED&RPLQJ %ORRG *DV 6\VWHP $ VPDOO SRUWLRQ RI WKLV EORRG ZDV XVHG WR PHDVXUH KHPDWRFULW XVLQJ DQ ,(& PLFURKHPDWRFULW FHQWULIXJH $IWHU VDPSOLQJ YROXPH RI WKH FDWKHWHU ZDV UHVWRUHG ZLWK b QRUPDO VDOLQH ZLWK b YY KHSDULQ %ORRG VDPSOHV POf ZHUH NHSW RQ LFH XQWLO IXUWKHU DQDO\VLV IRU KRUPRQH OHYHOV 6DPSOHV ZHUH FHQWULIXJHG DW [ J IRU PLQXWHV DW r & LQ D UHIULJHUDWHG FHQWULIXJH 6RUYDOO 57 % 'X3RQW 1HZWRZQ &$f $IWHU FHQWULIXJDWLRQ WKH SODVPD ZDV WUDQVIHUUHG DQG DOLTXRWWHG WR SRO\VW\UHQH WXEHV DQG VWRUHG DW r & XQWLO KRUPRQHV ZHUH DVVD\HG 8SRQ FRQFOXVLRQ RI HDFK H[SHULPHQW HZHV ZHUH VDFULILFHG ZLWK DQ RYHUGRVH RI VRGLXP SHQWREDUELWDO YLD WKH MXJXODU YHLQ )HWXVHV ZHUH LPPHGLDWHO\ UHPRYHG IRU SHUIXVLRQ RI WKH EUDLQ 7KH FKHVW FDYLW\ RI HDFK IHWXV ZDV RSHQHG DQG WKH EUDFKLRFHSKDOLF DUWHU\ ZDV ORFDWHG DQG FDQQXODWHG (LWKHU E\ PHDQV RI D SXPS RU E\ V\ULQJH EUDLQV ZHUH SHUIXVHG ILUVW ZLWK RQH OLWHU RI SKRVSKDWH EXIIHUHG VDOLQH S+ b YY KHSDULQf IROORZHG E\ WZR OLWHUV RI b SDUDIRUPDOGHK\GH %UDLQV DQG SLWXLWDU\ JODQGV ZHUH UHPRYHG DQG VWRUHG LQ b SDUDIRUPDOGHK\GH XQWLO SURFHVVLQJ IRU LPPXQRKLVWRFKHPLVWU\

PAGE 51

3HSWLGH $VVD\V $UJLQLQH 9DVRSUHVVLQ $93f 3ODVPD $93 FRQFHQWUDWLRQV ZHUH PHDVXUHG XVLQJ DQ DQWLERG\ UDLVHG LQ UDEELWV 5DII HW DO f ,RGLQDWHG $93 ZDV SXUFKDVHG IURP $PHUVKDP DQG V\QWKHWLF $93 IURP 6LJPD &KHPLFDO &R $93 ZDV ILUVW H[WUDFWHG IURP PO SODVPD ZLWK PO EHQWRQLWH VOXUU\ b ZY LQ GLVWLOOHG ZDWHUf DQG DFLGLILHG ZLWK PO 1 +& ([WUDFWV ZHUH HOXWHG ZLWK PO DFLGDFHWRQH b ,1 +&b DFHWRQHf ZLWK VRQLFDWLRQ 6DPSOHV ZHUH WKHQ HYDSRUDWHG WR GU\QHVV DQG VWRUHG DW r & XQWLO DVVD\HG ([WUDFWV ZHUH UHFRQVWLWXWHG ZLWK PO DVVD\ EXIIHU 0 SKRVSKDWH EXIIHU S+ ZLWK 0 ('7$ 6LJPD ('66f DQG b %6$ ZY 6LJPD $ ff ([WUDFWLRQ UHFRYHU\ ZDV FRUUHFWHG E\ FRPSDULQJ VDPSOHV WR D VWDQGDUG FXUYH SUHSDUHG IURP VWDQGDUG H[WUDFWHG ZLWK HDFK VHW RI VDPSOHV $GUHQRFRUWLFRWURSLQ $&7+f 3ODVPD $&7+ FRQFHQWUDWLRQV ZHUH PHDVXUHG E\ UDGLRLPPXQRDVVD\ 5,$f DV SUHYLRXVO\ GHVFULEHG %HOO HW DO f XVLQJ DQ DQWLERG\ UDLVHG LQ UDEELWV GHYHORSHG LQ 'U :RRGfV ODERUDWRU\ WR KXPDQ$&7+ f ,RGLQDWHG $&7+ $&7+f ZDV SUHSDUHG XVLQJ WKH FKORUDPLQH7 PHWKRG %HUVRQ DQG
PAGE 52

DFHWRQHf 7KH H[WUDFWV ZHUH GULHG XQGHU YDFXXP 6DYDQW ,QVWUXPHQWV )DUPLQJGDOH 1
PAGE 53

VDFULILFHG XVLQJ DQ LQWUDYHQRXV RYHUGRVH RI VRGLXP SHQWREDUELWDO *HVWDWLRQDO DJHV RI WKH IHWDO VKHHS ZHUH FDOFXODWHG IURP NQRZQ EUHHGLQJ GDWHV :KROH EUDLQV ZHUH UDSLGO\ UHPRYHG GLVVHFWHG LQWR GLVFUHWH UHJLRQV DQG TXLFNO\ IUR]HQ RQ GU\ LFH RU LQ D VOXUU\ RI GU\ LFH DQG DFHWRQH $OO WLVVXHV ZHUH VWRUHG DW r& RU r& XQWLO VWXGLHG +\SRWKDODPL EUDLQVWHPV DQG KLSSRFDPSL ZHUH WKHQ SURFHVVHG WR GHWHUPLQH HVWURQH VXOIDWDVH DFWLYLW\ (DFK WLVVXH VDPSOH ZDV KRPRJHQL]HG LQ PHGLXP 6LJPDp 6W /RXLV 02f FRQWDLQLQJ P0 +(3(6 +RPRJHQL]DWLRQ ZDV SHUIRUPHG XVLQJ D 3RO\WURQ KRPRJHQL]HU 7HNPDU &LQFLQQDWL 2+f 7KH FRQFHQWUDWLRQ RI HDFK WLVVXH LQ WKH KRPRJHQDWH ZDV J WLVVXH LQ P/ PHGLXP 7LVVXHV ZHUH FHQWULIXJHG DW USP IRU PLQ VXSHUQDWDQW ZDV WKHQ FROOHFWHG DQG DVVD\HG LPPHGLDWHO\ $ VDPSOH RI HDFK KRPRJHQDWH ZDV DVVD\HG IRU SURWHLQ FRQFHQWUDWLRQ XVLQJ WKH PHWKRG RI %UDGIRUG f XVLQJ D FRPPHUFLDOO\DYDLODEOH DVVD\ NLW %LR5DG /DERUDWRULHV +HUFXOHV &$f +RPRJHQDWH P/f ZDV DOLTXRWWHG LQ U GXSOLFDWH LQWR ERURVLOLFDWH WXEHV [ PPf FRQWDLQLQJ P/ RI D PL[WXUH RI + >@HVWURQH VXOIDWH 'X3RQW1(1 :LOPLQJWRQ '(f DQG XQODEHOHG HVWURQH VXOIDWH (62 6LJPDp 6W /RXLV 02f $OO UHDFWLRQV ZHUH UXQ DW r& 5HDFWLRQV ZHUH WHUPLQDWHG E\ LPPHGLDWH FRROLQJ RQ LFH DGGLWLRQ RI YROXPHV RI HWK\O DFHWDWH KH[DQH f DQG YLJRURXV PL[LQJ IRU VHFRQGV 7KH DTXHRXV SKDVH ZDV IUR]HQ E\ VXEPHUVLRQ RI WKH UHDFWLRQ WXEH LQWR D GU\ LFH DQG DFHWRQH VOXUU\ 6XEVHTXHQWO\ WKH RUJDQLF SKDVH FRQWDLQLQJ WKH +HVWURQH ZDV GHFDQWHG LQWR [ PP ERURVLOLFDWH JODVV WXEHV DQG GULHG XQGHU D JHQWOH VWUHDP RI URRP DLU 'ULHG H[WUDFWV ZHUH UHFRQVWLWXWHG LQ VFLQWLOODQW &\WRVFLQWp ,&1 &RUS &RVWD 0HVD &$f DQG FRXQWHG LQ D VFLQWLOODWLRQ FRXQWHU /.% &RUS *DLWKHUVEXUJ 0'f

PAGE 54

(Q]\PH DFWLYLWLHV DW GLIIHUHQW GHYHORSPHQWDO DJHV DQG LQ GLIIHUHQW WLVVXHV ZHUH PHDVXUHG XVLQJ D VXEVWUDWH FRQFHQWUDWLRQ RI S0 DQG +HVWURQH VXOIDWH VSHFLILF DFWLYLW\ RI DSSUR[LPDWHO\ S&LQPRO )RU WKLV H[SHULPHQW UHDFWLRQV ZHUH DOORZHG WR UXQ IRU PLQ 8VLQJ WKHVH FRQGLWLRQV OHVV WKDQ b RI WKH VXEVWUDWH ZDV FRQYHUWHG WR +HVWURQH :HVWHUQ %ORWWLQJ +\SRWKDODPL DQG EUDLQVWHPV ZHUH KDUYHVWHG IURP IHWXVHV ODPEV DQG DGXOWV RI NQRZQ JHVWDWLRQDO DQG SRVWQDWDO DJHV 7KH QXPEHU DQG DJHV RI DQLPDOV YDULHG VOLJKWO\ EHWZHHQ K\SRWKDODPL DQG EUDLQVWHP EXW IHWXVHV ODPEV DQG DGXOWV ZHUH XVHG SHU WLVVXH W\SH 7KHVH WLVVXHV ZHUH RULJLQDOO\ REWDLQHG DQG KRPRJHQL]HG IRU RWKHU VWXGLHV 6DRXG DQG :RRG f 8QIRUWXQDWHO\ KLSSRFDPSL IURP WKHVH DQLPDOV ZHUH QRW DYDLODEOH $OO WLVVXH ZDV KRPRJHQL]HG LQ UHGXFLQJ EXIIHU DQG ERLOHG IRU PLQXWHV 7KH VDPSOHV ZHUH FHQWULIXJHG WR UHPRYH SDUWLFXODWH PDWWHU DQG VXSHUQDWDQW ZDV UHFRYHUHG 3URWHLQ FRQFHQWUDWLRQV ZHUH REWDLQHG XWLOL]LQJ WKH %UDGIRUG WHFKQLTXH f :HVWHUQ U EORWV ZHUH SHUIRUPHG XVLQJ D PLQL3URWHDQ HOHFWURSKRUHVLV V\VWHP %LR5DG +HUFXOHV &$f RQ b SUHFDVW SRO\DFU\ODPLGH JHOV SXUFKDVHG IURP %LR5DG ODERUDWRULHV 6DPSOHV ZHUH GLOXWHG VR WKDW DQ HTXDO DPRXQW RI SURWHLQ ZDV ORDGHG SHU ODQH SJ IRU EUDLQVWHP DQG SJ IRU K\SRWKDODPLf 7KH SURWHLQ ZDV WKHQ WUDQVIHUUHG WR D QLWURFHOOXORVH PHPEUDQH DQG SUREHG IRU HLWKHU HVWURJHQ VXOIDWDVH RU HVWURJHQ VXOIRWUDQVIHUDVH XVLQJ FXVWRPPDGH UDEELW SRO\FORQDO DQWLERGLHV $OSKD 'LDJQRVWLF 6DQ $QWRQLR 7;f 7KH SHSWLGH VHTXHQFH XVHG IURP WKH KXPDQ VXOIDWDVH JHQH DPLQR DFLGV ZDV 1+ )66.')$*.64+*9<*&&22+ 6LPHUO\ HW DO f 7KH SHSWLGH VHTXHQFH XVHG IURP WKH ERYLQH VXOIRWUDQVIHUDVH JHQH DPLQR DFLGV ZDV 1+ 5(5)((+<4440.'&&22+ 1DVK HW DO f 3ULPDU\ DQWLERGLHV ZHUH GLOXWHG WR

PAGE 55

D FRQFHQWUDWLRQ RI LQ DQWLERG\ GLOXHQW b %6$ LQ SKRVSKDWH EXIIHUHG VDOLQH ZLWK b 7ZHHQ f 9LVXDOL]DWLRQ RI WKH SURWHLQDQWLERG\ FRPSOH[ ZDV DFFRPSOLVKHG XWLOL]LQJ D FKHPLOXPLQHVFHQFH GHWHFWLRQ V\VWHP 5HQDLVVDQFH 'X3RQW 1(1 %RVWRQ 0$f DQG DQDO\]HG E\ GHQVLWRPHWU\ %LR5DGf $QWLERG\ VSHFLILFLW\ ZDV FRQILUPHG E\ SUHDEVRUSWLRQ RI WKH SULPDU\ DQWLERGLHV ZLWK SHSWLGHV SJPOf DOVR VXSSOLHG E\ $OSKD 'LDJQRVWLF 'HYHORSPHQWDO FKDQJHV ZHUH FDOFXODWHG XVLQJ PXOWLSOH OLQHDU UHJUHVVLRQ LQ RUGHU WR FRQWURO IRU GLIIHUHQFHV EHWZHHQ JHO UXQQLQJ FRQGLWLRQV 6LJPD6WDW -DQGHO 6FLHQWLILF 6DQ 5DIDHO &$f ,PPXQRKLVWRFKHPLFDO 7HFKQLTXHV )HWDO EUDLQV ZHUH SHUIXVLRQ IL[HG ZLWK b SDUDIRUPDOGHK\GH GLVVHFWHG DQG FXW LQWR JURVV WLVVXH UHJLRQV K\SRWKDODPXV PLGEUDLQ SRQV PHGXOOD VSLQDO FRUG HWFf 7LVVXH ZDV SURFHVVHG IRU HPEHGGLQJ E\ GHK\GUDWLRQ ZLWK SURJUHVVLYHO\ LQFUHDVLQJ FRQFHQWUDWLRQV RI HWKDQRO IROORZHG E\ [\OHQH $OO WLVVXH ZDV HPEHGGHG LQ SDUDIILQ DQG FXW LQWR SP VHFWLRQV XVLQJ D =HLVV PLFURWRPH 6HFWLRQV ZHUH PRXQWHG RQ SRO\/ O\VLQH VOLGHV GHSDUDIILQL]HG ZLWK [\OHQH DQG UHK\GUDWHG LQ GHFUHDVLQJ FRQFHQWUDWLRQV RI HWKDQRO ,PPXQRKLVWRFKHPLVWU\ DQG YLVXDOL]DWLRQ ZHUH PDGH SRVVLEOH XWLOL]LQJ D +LVWRVWDLQ63 NLW IURP =\PHG DQG PHWDOHQKDQFHG '$% 3LHUFH 5RFNIRUG (/f 6HFWLRQV ZHUH VWDLQHG IRU HVWURJHQ VXOIDWDVH HVWURJHQ VXOIRWUDQVIHUDVH FIRV $&7+ $93 DQG &5+ VHH 7DEOH f 3ULPDU\ DQWLERGLHV ZHUH GLOXWHG LQ DQWLERG\ GLOXHQW b %6$ LQ SKRVSKDWH EXIIHUHG VDOLQH ZLWK b 7ULWRQ ;f 6SHFLILF VWDLQLQJ ZDV FRQILUPHG E\ GLOXWLRQ WHVWV DV VWDLQLQJ GHFUHDVHG DV SULPDU\ DQWLERGLHV ZHUH IXUWKHU GLOXWHG 6SHFLILF VWDLQLQJ ZDV DEVHQW XSRQ UHSODFLQJ SULPDU\ DQWLERGLHV ZLWK b QRUPDO JRDW VHUXP $OO

PAGE 56

VOLGHV ZHUH GHK\GUDWHG SULRU WR PRXQWLQJ RI FRYHUVOLSV ZLWK 3HUPRXQW )LVKHU 6FLHQWLILF 3LWWVEXUJK 3$f 3ULPDU\ DQWLERG\ 9HQGRU 6RXUFHf 'LOXWLRQ (VWURJHQ 6XOIDWDVH $OSKD 'LDJQRVWLFV VDPH DV IRU :HVWHUQ %ORWVf (VWURJHQ 6XOIRWUDQVIHUDVH $OSKD 'LDJQRVWLFV VDPH DV IRU :HVWHUQ %ORWVf FIRV 2QFRJHQH %LRPHGLFDO ,QF FDW 3&f $&7+ :RRG /DERUDWRU\ VDPH DV IRU 5,$f $93 :RRG /DERUDWRU\ VDPH DV IRU 5,$f &5+ .HOOHU:RRG /DERUDWRU\ 7DEOH 3ULPDU\ DQWLERGLHV XVHG LQ LPPXQRKLVWRFKHPLFDO H[SHULPHQWV $OO DQWLERGLHV ZHUH GLOXWHG LQ b %6$ LQ SKRVSKDWH EXIIHUHG VDOLQH ZLWK b 7ULWRQ ;

PAGE 57

&+$37(5 +25021$/ 5(63216,9(1(66 ,1 $1 (675$',2/ +<327(16,9( &$527,' 6,186 '(1(59$7(' 29,1( 02'(/ ,QWURGXFWLRQ ,Q WKH IHWDO VKHHS SDUWXULWLRQ LV WULJJHUHG E\ DQ LQFUHDVH LQ WKH DFWLYLW\ RI WKH IHWDO +3$ D[LV &KDOOLV DQG %URRNV /LJJLQV HW DO f 3DUWXULWLRQ FDQ EH GHOD\HG E\ GHVWUXFWLRQ RI WKH SLWXLWDU\ /LJJLQV HW DO /LJJLQV DQG .HQQHG\ f RU VWLPXODWHG E\ LQIXVLRQV RI $&7+ /LJJLQV f RU JOXFRFRUWLFRLGV -DFN HW DO :RRG DQG .HOOHU:RRG f 7KH ODVW IHZ GD\V RI JHVWDWLRQ DUH PDUNHG E\ DQ LQFUHDVH LQ WKH DFWLYLW\ RI WKH IHWDO K\SRWKDODPXV DV VHHQ E\ HOHYDWHG OHYHOV RI IHWDO SODVPD $&7+ 7KLV LQFUHDVH LQ SODVPD $&7+ FDXVHV D FRUUHVSRQGLQJ LQFUHDVH LQ SODVPD FRUWLVRO $ORQJ ZLWK WKLV LQFUHDVH LQ +3$ D[LV DFWLYLW\ LV D GHFUHDVHG VHQVLWLYLW\ RI WKH D[LV WR FRUWLVRO QHJDWLYH IHHGEDFN :RRG f ,W LV ZHOO NQRZQ WKDW LQ WKH VKHHS FRUWLVRO DFWV DW WKH SODFHQWD WR LQFUHDVH WKH DFWLYLW\ RI DQ HQ]\PH F\WRFKURPH S K\GUR[\ODVH DQG O\DVH DFWLYLWLHVf ZKLFK LQ WXUQ LQFUHDVHV WKH UDWLR RI HVWURJHQ WR SURJHVWHURQH $QGHUVRQ HW DO 3RPHUDQ] DQG 1DOEDQGRY 6WHHOH HW DO
PAGE 58

HVWURJHQ ZDV IRXQG WR DXJPHQW IHWDO SODVPD $&7+ VHFUHWLRQ LQ UHVSRQVH WR VWUHVV 2WKHU VWXGLHV KDYH VKRZQ VLPLODU UHVXOWV LQ DGXOW DQLPDOV 9LDX DQG 0HDQH\ f 8QGHUVWDQGLQJ WKH PHFKDQLVP RI WKH LQFUHDVHG IHWDO +3$ D[LV DW WKH HQG RI JHVWDWLRQ LV NH\ WR XQGHUVWDQGLQJ WKH PHFKDQLVP RI VSRQWDQHRXV SDUWXULWLRQ LQ VKHHS 7KHVH H[SHULPHQWV ZHUH FRQGXFWHG WR VHH LI HVWUDGLRO KDV LWfV VWLPXODWRU\ HIIHFW RQ +3$ D[LV DFWLYLW\ WKURXJK WKH DIIHUHQW EDURUHFHSWRU DQG FKHPRUHFHSWRU SDWKZD\ 0RUH VSHFLILFDOO\ K\SRWKHVL]HG WKDW HVWUDGLROfV DFWLRQV RQ IHWDO FDUGLRYDVFXODU UHIOH[ UHVSRQVLYHQHVV WR K\SRWHQVLRQ ZLOO EH PHDVXUDEOH LQ LQWDFW IHWXVHV EXW QRW LQ EDUR DQG FKHPR GHQHUYDWHG IHWXVHV 7KLV DXJPHQWDWLRQ RI +3$ D[LV DFWLYLW\ ZLOO EH DVVHVVHG E\ XVLQJ D VXUJLFDOO\ PDQLSXODWHG RYLQH PRGHO $&7+ $93 HVWUDGLRO DQG FRUWLVRO OHYHOV ZLOO EH PHDVXUHG DQG FRPSDUHG DFURVV HLJKW GLIIHUHQW WUHDWPHQW JURXSV 7KHVH JURXSV LQFOXGH IHWXVHV ZKLFK DUH f HVWUDGLRO WUHDWHG f HVWUDGLRO WUHDWHG K\SRWHQVLYH f HVWUDGLRO WUHDWHG FDURWLG VLQXV GHQHUYDWHG f HVWUDGLRO WUHDWHG FDURWLG VLQXV GHQHUYDWHG K\SRWHQVLYH f SODFHER WUHDWHG f SODFHER WUHDWHG K\SRWHQVLYH f SODFHER WUHDWHG FDURWLG VLQXV GHQHUYDWHG f SODFHER WUHDWHG FDURWLG VLQXV GHQHUYDWHG K\SRWHQVLYH 5HVHDUFK KDV SUHYLRXVO\ GHPRQVWUDWHG WKDW WKH FRPELQHG EDUR DQG FKHPR GHQHUYDWLRQ DWWHQXDWHV DSSUR[LPDWHO\ bf WKH UHIOH[ KRUPRQDO DQG KHPRG\QDPLF UHVSRQVHV WR PRGHUDWH bf UHGXFWLRQ LQ DUWHULDO EORRG SUHVVXUH SURSRVHG WKDW WKH LQWHUUXSWLRQ RI WKH DIIHUHQW SDWKZD\V ZRXOG HOLPLQDWH WKH HIIHFW RI HVWURJHQ RQ WKH UHIOH[ FDUGLRYDVFXODU UHVSRQVLYHQHVV ,I VR LW FRXOG FRQFOXGHG WKDW HVWURJHQ DFWV RQ ZLWKLQ RU UHTXLUHV LQSXW IURP WKH DIIHUHQW EDURUHFHSWRU DQG FKHPRUHFHSWRU SDWKZD\V

PAGE 59

0HWKRGV DQG 0DWHULDOV 6XUJLFDO 3URFHGXUHV $VHSWLF IHWDO VXUJHU\ ZDV SHUIRUPHG LQ $QLPDO 5HVRXUFHV RU DW WKH WK 6WUHHW IDFLOLW\ XQGHU JHQHUDO DQHVWKHVLD ZLWK b b KDORWKDQH $OO HZHV ZHUH EHWZHHQ DQG GD\V JHVWDWLRQ DW WKH WLPH RI VXUJHU\ $ WRWDO RI HZHV ZHUH VHW XS DQG VWXGLHG Q SHU JURXSf )RRG DQG ZDWHU ZHUH ZLWKKHOG IURP HZHV KRXUV SULRU WR VXUJHU\ (ZHV ZHUH VKHDUHG FORVH WR WKH VNLQ DURXQG WKH DEGRPHQ DQG SUHSDUHG IRU VXUJHU\ ZLWK SRYLGRQH LRGLQH %HWDGLQHp 3XUGXH )UHGULFN &R 1RUZDON &7f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f ,' f 2'f LQWR HDFK IHPRUDO DUWHU\ /DWHU WKHVH FDWKHWHUV ZRXOG EH XVHG IRU EORRG VDPSOLQJ DQG EORRG SUHVVXUH UHFRUGLQJ 7KH WLSV RI HDFK IHPRUDO FDWKHWHU ZHUH DGYDQFHG WR WKH VXEGLDSKUDJPDWLF DRUWD $W WKLV WLPH DQ HVWUDGLRO LPSODQW PJ GD\V RU QJGD\ ,QQRYDWLYH 5HVHDUFK RI $PHULFD 7ROHGR 2+f RU SODFHER ZDV LQVHUWHG VXEFXWDQHRXVO\ LQWR WKH DUHD RI WKH JOXWHRXV PHGLXV EHIRUH VXWXULQJ WKH LQFLVHG KLQGOLPEV $Q DPQLRWLF FDWKHWHU PDGH RI SRO\YLQ\O FKORULGH f ,' f 2'f ZDV VXWXUHG WR WKH H[WHULRU RI D KLQGOLPE IRU WKH SXUSRVH RI DQWLELRWLF GHOLYHU\ DV ZHOO DV DPQLRWLF IOXLG SUHVVXUH

PAGE 60

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f 7KH OHIW IRUHOLPE RI WKH IHWXV ZDV GHOLYHUHG WKURXJK D XWHULQH LQFLVLRQ $Q LQFLVLRQ ZDV PDGH LQWR WKH VHFRQG LQWHUFRVWDO VSDFH DQG WKH EUDFKLRFHSKDOLF DUWHU\ ZDV ORFDWHG 2QFH WKH RFFOXGHU ZDV VHZQ LQ SODFH WKH LQFLVLRQ XQGHU WKH OHIW IRUHDUP ZDV FORVHG DQG WKH IHWXV

PAGE 61

UHWXUQHG WR WKH XWHUXV 7KH XWHULQH LQFLVLRQ ZDV FORVHG RQFH DJDLQ XVLQJ D ORFNLQJ VLPSOH FRQWLQXRXV VXWXUH SDWWHUQ IROORZHG E\ DQ XPEULFDWLRQ WHFKQLTXH PJ DPSLFLOOLQ 3RO\IOH[p )W 'RGJH /DERUDWRULHV )W 'RGJH ,$f ZDV DGPLQLVWHUHG LQWR WKH DPQLRWLF FDYLW\ EHIRUH FORVXUH RI WKH PDWHUQDO OLQHD DOED DQG VNLQ $OO FDWKHWHUV ZHUH ILOOHG ZLWK KHSDULQ XQLWVPO (ONLQV6LQQ ,QF &KHUU\ +LOO 1-f DQG FORVHG ZLWK D VWHULOH EUDG LQVHUWHG LQWR WKH HQG &DWKHWHUV DQG RFFOXGHUV ZHUH IODQNHG DQG H[WHULRUL]HG YLD D WURFKDU &DWKHWHUV ZHUH KHOG LQ SODFH ZLWK DQ HODVWLF EDQGDJH 7KH OLQHD DOED ZDV FORVHG ZLWK SRO\DPLGH VXWXUH 3LWPDQQ0RRUH )UDQFHf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b YYf 2QH IHPRUDO FDWKHWHU DQG WKH

PAGE 62

DPQLRWLF FDWKHWHU ZHUH DWWDFKHG WR WUDQVGXFHUV 6WDWKDP 3,G 6WDWKDP ,QVWUXPHQWV 2[QDUG &$f IRU PHDVXUHPHQW RI IHWDO DUWHULDO DQG DPQLRWLF IOXLG SUHVVXUH 7KHUH ZHUH HLJKW GLIIHUHQW H[SHULPHQWDO JURXSV LQ WKLV VWXG\ f HVWUDGLRO WUHDWHG f HVWUDGLRO WUHDWHG K\SRWHQVLYH f HVWUDGLRO WUHDWHG FDURWLG VLQXV GHQHUYDWHG f HVWUDGLRO WUHDWHG FDURWLG VLQXV GHQHUYDWHG K\SRWHQVLYH f SODFHER WUHDWHG f SODFHER WUHDWHG K\SRWHQVLYH f SODFHER WUHDWHG FDURWLG VLQXV GHQHUYDWHG f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f $Q DGGLWLRQDO PO RI EORRG ZDV

PAGE 63

GUDZQ DQDHURELFDOO\ LQWR V\ULQJHV FRDWHG ZLWK KHSDULQ IRU PHDVXUHPHQW RI IHWDO EORRG JDVHV XVLQJ D &LED&RPLQJ %ORRG *DV 6\VWHP $ VPDOO SRUWLRQ RI WKLV EORRG ZDV XVHG WR PHDVXUH KHPDWRFULW XVLQJ DQ ,(& PLFURKHPDWRFULW FHQWULIXJH $IWHU VDPSOLQJ EORRG YROXPH ZDV UHVWRUHG ZLWK b QRUPDO VDOLQH ZLWK b YY KHSDULQ %ORRG VDPSOHV POf ZHUH NHSW RQ LFH XQWLO IXUWKHU SURFHVVLQJ 6DPSOHV ZHUH FHQWULIXJHG DW [ J IRU PLQXWHV DW r & LQ D UHIULJHUDWHG FHQWULIXJH 6RUYDOO 57 % 'X3RQW 1HZWRZQ &$f $IWHU FHQWULIXJDWLRQ WKH SODVPD ZDV WUDQVIHUUHG DQG DOLTXRWWHG WR SRO\VW\UHQH WXEHV DQG VWRUHG DW r & XQWLO KRUPRQHV ZHUH DVVD\HG 8SRQ FRQFOXVLRQ RI HDFK H[SHULPHQW HZHV ZHUH VDFULILFHG ZLWK DQ RYHUGRVH RI VRGLXP SHQWREDUELWDO YLD WKH MXJXODU YHLQ )HWXVHV ZHUH LPPHGLDWHO\ UHPRYHG IRU SHUIXVLRQ RI WKH EUDLQ 7KH FKHVW FDYLW\ RI HDFK IHWXV ZDV RSHQHG XS DQG WKH EUDFKLRFHSKDOLF DUWHU\ ZDV ORFDWHG DQG FDQQXODWHG (LWKHU E\ PHDQV RI D SXPS RU E\ V\ULQJH EUDLQV ZHUH SHUIXVHG ILUVW ZLWK RQH OLWHU RI SKRVSKDWH EXIIHUHG VDOLQH S+ I b YY KHSDULQf IROORZHG E\ WZR OLWHUV RI b SDUDIRUPDOGHK\GH %UDLQV DQG SLWXLWDU\ JODQGV ZHUH UHPRYHG DQG VWRUHG LQ b SDUDIRUPDOGHK\GH XQWLO SURFHVVLQJ IRU LPPXQRKLVWRFKHPLVWU\ &KDSWHU f 7KH H[SHULPHQWDO GHVLJQ FDQ EH YLHZHG SLFWRULDOO\ LQ )LJXUH (VWUDGLRO $VVD\ 3ODVPD HVWUDGLRO FRQFHQWUDWLRQV ZHUH PHDVXUHG XWLOL]LQJ DQ HQ]\PH LPPXQRDVVD\ (,$f NLW IURP 2[IRUG %LRFKHPLFDO ,QF ($f PO RI SODVPD ZDV H[WUDFWHG ZLWK PO HWK\O HWKHU ([WUDFWV ZHUH GULHG XQGHU DLU DQG UHFRQVWLWXWHG WR SL ZLWK DVVD\ EXIIHU SURYLGHG ZLWK NLWf SL RI H[WUDFW ZDV DVVD\HG IRU HVWUDGLRO LQ GXSOLFDWH 7KH YDOXHV REWDLQHG ZHUH GLYLGHG E\ WR JLYH QJPO FRQFHQWUDWLRQV 7KLV SDUWLFXODU (,$ NLW KDG D YHU\ ORZ FURVVUHDFWLYLW\ ZLWK HVWURQH bf

PAGE 64

$GUHQRFRUWLFRWURSLQ $&7+f $VVD\ 3ODVPD $&7+ FRQFHQWUDWLRQV ZHUH PHDVXUHG E\ UDGLRLPPXQRDVVD\ 5,$f DV SUHYLRXVO\ GHVFULEHG %HOO HW DO f XVLQJ DQ DQWLERG\ UDLVHG LQ UDEELWV GHYHORSHG LQ 'U :RRGfV ODERUDWRU\ WR KXPDQ$&7+ f ,RGLQDWHG $&7+ $&7+f ZDV SUHSDUHG XVLQJ WKH FKORUDPLQH7 PHWKRG %HUVRQ DQG
PAGE 65

XQWLO DVVD\HG ([WUDFWV ZHUH UHFRQVWLWXWHG ZLWK PO DVVD\ EXIIHU 0 SKRVSKDWH EXIIHU S+ ZLWK 0 ('7$ 6LJPD ('66f DQG b %6$ ZY 6LJPD $ ff ([WUDFWLRQ UHFRYHU\ ZDV FRUUHFWHG E\ FRPSDULQJ VDPSOHV WR D VWDQGDUG FXUYH SUHSDUHG IURP VWDQGDUG H[WUDFWHG ZLWK HDFK VHW RI VDPSOHV &RUWLVRO $VVD\ 3ODVPD FRUWLVRO FRQFHQWUDWLRQV ZHUH PHDVXUHG DV SUHYLRXVO\ GHVFULEHG :RRG HW DO f XVLQJ DQ DQWLERG\ UDLVHG LQ UDEELWV DQG WLWUDWHG FRUWLVRO SXUFKDVHG IURP $PHUVKDP 75.f DQG FRUWLVRO VWDQGDUG IURP 6LJPD &KHPLFDO &R &RUWLVRO ZDV H[WUDFWHG IURP SL SODVPD LQ GXSOLFDWH ZLWK PO HWKDQRO 6WDQGDUG ZDV SUHSDUHG LQ HWKDQRO DQG VWDQGDUGV DQG VDPSOHV ZHUH GULHG XQGHU YDFXXP ZLWK KHDW DQG LPPHGLDWHO\ UHFRQVWLWXWHG ZLWK PO DVVD\ EXIIHU 0 SKRVSKDWH EXIIHU XVLQJ 0 VRGLXP SKRVSKDWH GLEDVLF DQG 0 VRGLXP SKRVSKDWH PRQREDVLFf S+ ZLWK 0 1D&O b ZY JHODWLQ DQG b ZY VRGLXP D]LGHf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

PAGE 66

ILUVW 7KHUH ZHUH D WRWDO RI DQLPDOV VWXGLHG LQ WKLV H[SHULPHQW +DOI f ZHUH SUHWUHDWHG ZLWK DQ HVWUDGLRO LPSODQW GXULQJ VXUJHU\ 7KHVH LPSODQWV UHOHDVH HVWUDGLRO DW D FRQVWDQW UDWH RI PJ GD\ SHULRG RU DURXQG SJGD\ (VWUDGLRO OHYHOV ZHUH PHDVXUHG E\ (,$ DQG VWDWLVWLFDO DQDO\VHV VKRZHG WKDW WKH HVWUDGLRO WUHDWHG IHWXVHV ZHUH VLJQLILFDQWO\ GLIIHUHQW WKDQ WKH SODFHER WUHDWHG IHWXVHV E\ D WWHVW Q SHU JURXS Sf 5HVXOW FDQ EH VHHQ LQ )LJXUH ZKLFK VKRZV JURXS PHDQV PHDVXUHG LQ XQLWV RISJPO 6(0 $GUHQRFRUWLFRWURSLQ $&7+f $VVD\ 3ODVPD $&7+ OHYHOV DUH VKRZQ LQ )LJXUH 5HVXOWV DUH SORWWHG DV JURXS PHDQV 6(0 3ODFHER WUHDWHG IHWXVHV DQG HVWUDGLRO IHWXVHV ZHUH DQDO\]HG VHSDUDWHO\ E\ WKUHH ZD\ $129$ ZLWK K\SRWHQVLRQ QRUPRWHQVLRQ FDURWLG VLQXV GHQHUYDWLRQ LQWDFW DQG WLPH 2PLQ O2PLQ PLQ DQG PLQf DV H[SHULPHQWDO IDFWRUV ,Q ERWK FDVHV SODFHER DQG HVWUDGLRO WUHDWPHQWf JURXSV ZHUH IRXQG WR EH VWDWLVWLFDOO\ GLIIHUHQW Q SHU JURXS S222Of 6SHFLILFDOO\ $&7+ OHYHOV LQ WKH K\SRWHQVLYH DQLPDOV DW 2PLQ DQG PLQ DV ZHOO DV K\SRWHQVLYH FDURWLG VLQXV GHQHUYDWHG DQLPDOV DW O2PLQ DQG PLQ ZHUH IRXQG WR GLIIHU VLJQLILFDQWO\ IURP WKH UHVW RI WKH WUHDWPHQW JURXSV ZKHQ IXUWKHU DQDO\]HG E\ 6WXGHQW 1HZPDQ .HXOV &RPSDULVRQ Q SHU JURXS Sf 7KLV ZDV WUXH IRU ERWK SODFHER WUHDWHG IHWXVHV DQG HVWUDGLRO WUHDWHG IHWXVHV $OVR DOO SODFHER WUHDWHG IHWXVHV GLIIHUHG VLJQLILFDQWO\ IURP WKHLU FRUUHVSRQGLQJ HVWUDGLRO WUHDWHG IHWXVHV E\ WWHVW Q SHU JURXS Sf $UJLQLQH 9DVRSUHVVLQ $93f $VVD\ 3ODVPD $93 OHYHOV DUH VKRZQ LQ )LJXUH 5HVXOWV DUH SORWWHG DV JURXS PHDQV 6(0 3ODFHER WUHDWHG IHWXVHV DQG HVWUDGLRO IHWXVHV ZHUH DQDO\]HG VHSDUDWHO\ E\ WKUHH ZD\ $129$ ZLWK K\SRWHQVLRQ QRUPRWHQVLRQ FDURWLG VLQXV GHQHUYDWLRQ LQWDFW DQG WLPH 2PLQ O2PLQ PLQ DQG PLQf DV H[SHULPHQWDO IDFWRUV

PAGE 67

,Q ERWK FDVHV SODFHER DQG HVWUDGLRO WUHDWPHQWf JURXSV ZHUH IRXQG WR EH VWDWLVWLFDOO\ GLIIHUHQW Q SHU JURXS Sf 6SHFLILFDOO\ $93 OHYHOV LQ WKH K\SRWHQVLYH DQLPDOV DW O2PLQ DQG PLQ DV ZHOO DV K\SRWHQVLYH FDURWLG VLQXV GHQHUYDWHG DQLPDOV DW O2PLQ DQG PLQ ZHUH IRXQG WR GLIIHU VLJQLILFDQWO\ IURP WKH UHVW RI WKH WUHDWPHQW JURXSV ZKHQ IXUWKHU DQDO\]HG E\ 6WXGHQW 1HZPDQ .HXOV &RPSDULVRQ QA SHU JURXS Sf 7KLV ZDV WUXH IRU ERWK SODFHER WUHDWHG IHWXVHV DQG HVWUDGLRO WUHDWHG IHWXVHV $OVR DOO SODFHER WUHDWHG IHWXVHV GLIIHUHG VLJQLILFDQWO\ IURP WKHLU FRUUHVSRQGLQJ HVWUDGLRO WUHDWHG IHWXVHV E\ WWHVW Q SHU JURXS Sf &RUWLVRO $VVD\ 3ODVPD FRUWLVRO OHYHOV DUH VKRZQ LQ )LJXUH 5HVXOWV DUH SORWWHG DV JURXS PHDQV 6(0 3ODFHER WUHDWHG IHWXVHV DQG HVWUDGLRO IHWXVHV ZHUH DQDO\]HG VHSDUDWHO\ E\ WKUHH ZD\ $129$ ZLWK K\SRWHQVLRQ QRUPRWHQVLRQ FDURWLG VLQXV GHQHUYDWLRQ LQWDFW DQG WLPH 2PLQ O2PLQ PLQ DQG PLQf DV H[SHULPHQWDO IDFWRUV ,Q ERWK FDVHV SODFHER DQG HVWUDGLRO WUHDWPHQWf JURXSV ZHUH IRXQG WR EH VWDWLVWLFDOO\ GLIIHUHQW Q SHU JURXS Sf 6SHFLILFDOO\ FRUWLVRO OHYHOV LQ WKH K\SRWHQVLYH DQLPDOV DW O2PLQ DQG PLQ DV ZHOO DV K\SRWHQVLYH FDURWLG VLQXV GHQHUYDWHG DQLPDOV DW O2PLQ DQG PLQ ZHUH IRXQG WR GLIIHU VLJQLILFDQWO\ IURP WKH UHVW RI WKH WUHDWPHQW JURXSV ZKHQ IXUWKHU DQDO\]HG E\ 6WXGHQW 1HZPDQ .HXOV &RPSDULVRQ Q SHU JURXS Sf 7KLV ZDV WUXH IRU ERWK SODFHER WUHDWHG IHWXVHV DQG HVWUDGLRO WUHDWHG IHWXVHV $OVR DOO SODFHER WUHDWHG IHWXVHV GLIIHUHG VLJQLILFDQWO\ IURP WKHLU FRUUHVSRQGLQJ HVWUDGLRO WUHDWHG IHWXVHV E\ WWHVW Q SHU JURXS Sf 'LVFXVVLRQ 8QGHUVWDQGLQJ WKH PHFKDQLVP RI WKH LQFUHDVHG IHWDO +3$ D[LV DW WKH HQG RI JHVWDWLRQ LV NH\ WR XQGHUVWDQGLQJ WKH PHFKDQLVP RI VSRQWDQHRXV SDUWXULWLRQ LQ VKHHS

PAGE 68

7KHVH H[SHULPHQWV ZHUH FRQGXFWHG WR VHH LI HVWUDGLRO KDV LWfV VWLPXODWRU\ HIIHFW RQ +3$ D[LV DFWLYLW\ WKURXJK WKH DIIHUHQW EDURUHFHSWRU DQG FKHPRUHFHSWRU SDWKZD\ 0RUH VSHFLILFDOO\ K\SRWKHVL]HG WKDW HVWUDGLROfV DFWLRQV RQ IHWDO FDUGLRYDVFXODU UHIOH[ UHVSRQVLYHQHVV WR K\SRWHQVLRQ ZLOO EH PHDVXUDEOH LQ LQWDFW IHWXVHV EXW QRW LQ EDUR DQG FKHPR GHQHUYDWHG IHWXVHV 7KLV DXJPHQWDWLRQ RI +3$ D[LV DFWLYLW\ ZDV DVVHVVHG E\ XVLQJ D VXUJLFDOO\ PDQLSXODWHG RYLQH PRGHO $&7+ $93 HVWUDGLRO DQG FRUWLVRO OHYHOV ZHUH PHDVXUHG DQG FRPSDUHG DFURVV HLJKW GLIIHUHQW WUHDWPHQW JURXSV 7KHVH JURXSV LQFOXGH IHWXVHV ZKLFK DUH f HVWUDGLRO WUHDWHG f HVWUDGLRO WUHDWHG K\SRWHQVLYH f HVWUDGLRO WUHDWHG FDURWLG VLQXV GHQHUYDWHG f HVWUDGLRO WUHDWHG FDURWLG VLQXV GHQHUYDWHG K\SRWHQVLYH f SODFHER WUHDWHG f SODFHER WUHDWHG K\SRWHQVLYH f SODFHER WUHDWHG FDURWLG VLQXV GHQHUYDWHG f SODFHER WUHDWHG FDURWLG VLQXV GHQHUYDWHG K\SRWHQVLYH 7KLV VWXG\ KDV VKRZQ WKDW WKDW HVWUDGLRO GRHV KDYH DQ HIIHFW RQ FDUGLRYDVFXODU UHVSRQVLYHQHVV )LJXUHV VKRZ WKDW SUHWUHDWPHQW ZLWK HVWUDGLRO LQFUHDVHV HYHQ EDVDO $&7+ $93 DQG FRUWLVRO OHYHOV 7KLV HIIHFW RI HVWUDGLRO LV VHHQ EHVW SHUKDSV DIWHU D WHQ PLQXWH SHULRG RI K\SRWHQVLRQ $W WKLV SRLQW KRUPRQDO UHVSRQVLYHQHVV KDV EHHQ JUHDWO\ DXJPHQWHG ZLWK UHJDUG WR $&7+ $93 DQG FRUWLVRO OHYHOV &DURWLG VLQXV GHQHUYDWLRQ HVVHQWLDOO\ HOLPLQDWHV WKLV DXJPHQWHG +3$ D[LV DFWLYLW\ LQ UHVSRQVH WR EUDFKLRFHSKDOLF RFFOXVLRQ $Q\ KRUPRQH UHVSRQVH OHIWRYHU DIWHU GHQHUYDWLRQ PD\ EH GXH LQ SDUW WR LQFRPSOHWH GHQHUYDWLRQ DW WKH WLPH RI VXUJHU\ 2I FRXUVH DQRWKHU H[SODQDWLRQ OLHV ZLWK WKH IDFW WKDW RWKHU SDWKZD\V RU V\VWHPV PLJKW EH UHVSRQVLEOH IRU KRUPRQH VHFUHWLRQ &DURWLG VLQXV GHQHUYDWLRQ E\ LWVHOI KRZHYHU FDXVHG QR +3$ D[LV DFWLYLW\ RU DXJPHQWDWLRQ GHHPLQJ WKH VXUJLFDO HOLPLQDWLRQ RI EDURUHFHSWRU FKHPRUHFHSWRU UHVSRQVLYHQHVV DQ DSSURSULDWH DQG HIIHFWLYH FRQWURO 5HVHDUFK KDV SUHYLRXVO\ GHPRQVWUDWHG WKDW WKH

PAGE 69

FRPELQHG EDUR DQG FKHPR GHQHUYDWLRQ DWWHQXDWHV DSSUR[LPDWHO\ bf WKH UHIOH[ KRUPRQDO DQG KHPRG\QDPLF UHVSRQVHV WR PRGHUDWH bf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b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

PAGE 70

7KRXJK WKH UHVXOWV RI WKLV VWXG\ HOXGH WR DQ HVWUDGLRO UHVSRQVLYH EDURUHFHSWRU FKHPRUHFHSWRU DIIHUHQW SDWKZD\ SDUWLFLSDWLRQ LQ FDUGLRYDVFXODU UHIOH[ FRQWURO DQG WULJJHU IRU SDUWXULWLRQ WKH\ GR QRW E\ WKHPVHOYHV DQVZHU DFFRXQW IRU WRWDO FRQWURO RYHU WKHVH SURFHVVHV $V VWDWHG EHIRUH UHVHDUFK KDV SUHYLRXVO\ GHPRQVWUDWHG WKDW WKH FRPELQHG EDUR DQG FKHPR GHQHUYDWLRQ DWWHQXDWHV DSSUR[LPDWHO\ bf WKH UHIOH[ KRUPRQDO DQG KHPRG\QDPLF UHVSRQVHV WR PRGHUDWH bf UHGXFWLRQ LQ DUWHULDO EORRG SUHVVXUH 7KLV PHDQV WKDW b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f ZLWKLQ DUHDV RI WKH EUDLQ WKDW DUH UHVSRQVLEOH IRU +3$ D[LV FRQWURO 2WKHU VWXGLHV KDYH HOXGHG WR D UROH RI WKURPER[DQH $ 7[$f LQ WKLV SURFHVV 7[$ DFWV ZLWKLQ WKH DUHD SHUIXVHG E\ WKH FHUHEUDO YDVFXODWXUH WR VWLPXODWH $&7+ VHFUHWLRQ LQ WKH IHWXV :RRG HW DO f ZKLFK VXJJHVWV WKDW ORFDO JHQHUDWLRQ RI 7[$ ZRXOG HIIHFWLYHO\ VWLPXODWH WKH +3$ D[LV :KDWHYHU WKH PHFKDQLVP LW LV REYLRXV WKDW IXUWKHU UHVHDUFK QHHGV WR EH GRQH LQ RUGHU WR IXOO\ XQGHUVWDQG WKH LQQHUZRUNLQJV RI WKLV FRPSOH[ V\VWHP

PAGE 71

%ORRG VDPSOLQJ WLPH SRLQWV ,,, 2PLQ 2PLQ PLQ PLQ +\SRWHQVLRQ %UDFKLRFHSKDOLF 2FFOXVLRQf (XWKDQL]H DQG SHUIXVH EUDLQ 3UHSDUH IRU ,PPXQRKLVWRFKHPLVWU\f )LJXUH ([SHULPHQWDO GHVLJQ IRU LQ YLYR VWXGLHV PO EORRG VDPSOHV DUH WDNHQ DW 2PLQ O2PLQ PLQ DQG PLQDGGLWLRQDO EORRG LV WDNHQ DW 2PLQ O2PLQ DQG PLQ IRU EORRG JDV DQG KHPDWRFULW PHDVXUHPHQWf +\SRWHQVLRQ YLD EUDFKLRFHSKDOLF RFFOXVLRQ WDNHV SODFH EHWZHHQ 2PLQ DQG O2PLQ %ORRG SUHVVXUH DQG KHDUW UDWH DUH UHFRUGHG IRU WKH ILUVW PLQ RI WKH H[SHULPHQW $QLPDOV DUH VDFULILFHG DW PLQ DQG EUDLQV DUH H[WUDFWHG IRU LPPXQRKLVWRFKHPLVWU\

PAGE 72

t ‘R W LW FQ Z (VWUDGLRO WUHDWHG IHWXVHV 3ODFHER WUHDWHG IHWXVHV )LJXUH (VWUDGLRO OHYHOV 6(0 IRU IHWXVHV WUHDWHG ZLWK D PJ GD\ HVWUDGLRO LPSODQW DQG IHWXVHV WUHDWHG ZLWK D SODFHER LPSODQW 7KH JURXS PHDQV DUH VLJQLILFDQWO\ GLIIHUHQW Q SHU JURXS S222Of

PAGE 73

$&7+ SJPOf $&7+ SJPOf 7LPH PLQXWHVf )LJXUH $&7+ SODVPD OHYHOV SORWWHG DV JURXS PHDQV 6(0 7RS JUDSK VKRZV SODFHER WUHDWHG IHWXVHV DQG ERWWRP JUDSK VKRZV HVWUDGLRO WUHDWHG IHWXVHV r GHQRWHV VWDWLVWLFDO VLJQLILFDQFH Q SHU JURXS Sf $OO HVWUDGLRO JURXSV ZHUH VLJQLILFDQWO\ GLIIHUHQW IURP SODFHER JURXSV UHODWLYH WR WUHDWPHQW DQG WLPH Q SHU JURXS Sf

PAGE 74

$93 SJPOf $93 SJPOf 7LPH PLQXWHVf )LJXUH $93 SODVPD OHYHOV SORWWHG DV JURXS PHDQV 6(0 7RS JUDSK VKRZV SODFHER WUHDWHG IHWXVHV DQG ERWWRP JUDSK VKRZV HVWUDGLRO WUHDWHG IHWXVHV r GHQRWHV VWDWLVWLFDO VLJQLILFDQFH Q SHU JURXS Sf $OO HVWUDGLRO JURXSV ZHUH VLJQLILFDQWO\ GLIIHUHQW IURP SODFHER JURXSV UHODWLYH WR WUHDWPHQW DQG WLPH Q SHU JURXS Sf

PAGE 75

&RUWLVRO QJPOf &RUWLVRO QJPOf (VWUDGLRO WUHDWHG IHWXVHV r 7 &RQWURO +\SRWHQVLYH &6' +\SR&6' 7LPH PLQXWHVf )LJXUH &RUWLVRO SODVPD OHYHOV SORWWHG DV JURXS PHDQV6(0 7RS JUDSK VKRZV SODFHER WUHDWHG IHWXVHV DQG ERWWRP JUDSK VKRZV HVWUDGLRO WUHDWHG IHWXVHV r GHQRWHV VWDWLVWLFDO VLJQLILFDQFH Q SHU JURXS Sf $OO HVWUDGLRO JURXSV ZHUH VLJQLILFDQWO\ GLIIHUHQW IURP SODFHER JURXSV UHODWLYH WR WUHDWPHQW DQG WLPH Q SHU JURXS Sf

PAGE 76

&+$37(5 1(8521$/ $&7,9$7,21 ,1 $1 (675$',2/ +<327(16,9( &$527,' 6,186 '(1(59$7(' 29,1( 02'(/ ,QWURGXFWLRQ 3DUWXULWLRQ LQ WKH VKHHS KDV EHHQ VKRZQ WR EH FRQWUROOHG E\ WKH IHWDO +3$ D[LV &KDOOLV DQG %URRNV /LJJLQV HW DO f 7KH ODVW IHZ GD\V RI JHVWDWLRQ DUH PDUNHG E\ DQ LQFUHDVH LQ WKH DFWLYLW\ RI WKH IHWDO K\SRWKDODPXV DV VHHQ E\ HOHYDWHG OHYHOV RI IHWDO SODVPD $&7+ 7KLV LQFUHDVH LQ SODVPD $&7+ FDXVHV D FRUUHVSRQGLQJ LQFUHDVH LQ SODVPD FRUWLVRO $ORQJ ZLWK WKLV LQFUHDVH LQ +3$ D[LV DFWLYLW\ LV D GHFUHDVHG VHQVLWLYLW\ RI WKH D[LV WR FRUWLVRO QHJDWLYH IHHGEDFN :RRG f ,W LV ZHOO NQRZQ WKDW LQ WKH VKHHS FRUWLVRO DFWV DW WKH SODFHQWD WR LQFUHDVH WKH DFWLYLW\ RI DQ HQ]\PH F\WRFKURPH S K\GUR[\ODVH DQG O\DVH DFWLYLWLHVf ZKLFK LQ WXUQ LQFUHDVHV WKH UDWLR RI HVWURJHQ WR I SURJHVWHURQH $QGHUVRQ HW DO 3RPHUDQ] DQG 1DOEDQGRY 6WHHOH HW DO
PAGE 77

7KHVH H[SHULPHQWV ZHUH FRQGXFWHG WR VHH DW ZKDW SRLQW LQ WKH +3$ D[LV WKDW HVWUDGLRO KDV LQ DXJPHQWLQJ $&7+ VHFUHWLRQ 0RUH VSHFLILFDOO\ K\SRWKHVL]HG WKDW QHXURQDO DFWLYLW\ ZLOO EH KLJKHVW LQ DUHDV LPSRUWDQW IRU +3$ D[LV FRQWURO LQ HVWUDGLRO WUHDWHG K\SRWHQVLYH DQLPDOV VHH &KDSWHU f K\SRWKHVL]HG WKDW WKH EDURUHFHSWRU FKHPRUHFHSWRU DIIHUHQW SDWKZD\ LV LQYROYHG WKXV FDURWLG VLQXV GHQHUYDWLRQ ZLOO HOLPLQDWH WKH DXJPHQWHG +3$ D[LV DFWLYLW\ 1HXURQDO DFWLYLW\ ZDV DVVHVVHG E\ PHDVXULQJ WKH OHYHO RI FIRV DQ\ HDUO\ UHVSRQVH JHQH LQ EUDLQ DUHDV LPSRUWDQW IRU +3$ D[LV FRQWURO 7KLV PHWKRG KDV EHHQ XVHG LQ QXPHURXV VWXGLHV WR DVVHVV QHXURQDO DFWLYDWLRQ GXH WR SK\VLRORJLFDO VWUHVV +RIIPDQ HW DO 6KHQ HW DO &KDQ HW DO f 0HWKRGV DQG 0DWHULDOV ,PPXQRKLVWRFKHPLFDO 7HFKQLTXHV )HWDO RYLQH EUDLQV ZHUH SHUIXVLRQ IL[HG ZLWK b SDUDIRUPDOGHK\GH GLVVHFWHG DQG FXW LQWR JURVV WLVVXH UHJLRQV K\SRWKDODPXV PLGEUDLQ SRQV PHGXOOD VSLQDO FRUG HWFf 7KHVH EUDLQV ZHUH WKH REWDLQHG IURP WKH H[SHULPHQWV GLVFXVVHG LQ &KDSWHU $OO DQLPDO ZHUH HXWKDQL]HG YLD DQ RYHUGRVH RI VRGLXP U SHQWREDUELWDO RQH KRXU DIWHU WKH EHJLQQLQJ RI WKH H[SHULPHQW PLQXWHV DIWHU D WHQ PLQXWH K\SRWHQVLYH RU FRUUHVSRQGLQJ QRUPRWHQVLYH SHULRGf 7KHUH ZHUH D WRWDO RI IHWDO EUDLQV XVHG IRU WKH KLVWRORJLFDO H[SHULPHQWV Q SHU JURXSf 7KH IHWDO RYLQH JURXSV ZHUH DV IROORZHG f HVWUDGLRO WUHDWHG f HVWUDGLRO WUHDWHG K\SRWHQVLYH f HVWUDGLRO WUHDWHG FDURWLG VLQXV GHQHUYDWHG f HVWUDGLRO WUHDWHG FDURWLG VLQXV GHQHUYDWHG K\SRWHQVLYH f SODFHER WUHDWHG f SODFHER WUHDWHG K\SRWHQVLYH f SODFHER WUHDWHG FDURWLG VLQXV GHQHUYDWHG f SODFHER WUHDWHG FDURWLG VLQXV GHQHUYDWHG K\SRWHQVLYH 7LVVXH ZDV SURFHVVHG IRU HPEHGGLQJ E\ GHK\GUDWLRQ ZLWK SURJUHVVLYHO\ LQFUHDVLQJ FRQFHQWUDWLRQV RI HWKDQRO IROORZHG E\ [\OHQH $OO WLVVXH ZDV HPEHGGHG LQ SDUDIILQ DQG FXW LQWR MLP

PAGE 78

VHFWLRQV XVLQJ D =HLVV PLFURWRPH 6HFWLRQV ZHUH PRXQWHG RQ SRO\/O\VLQH VOLGHV GHSDUDIILQL]HG ZLWK [\OHQH DQG UHK\GUDWHG LQ GHFUHDVLQJ FRQFHQWUDWLRQV RI HWKDQRO ,PPXQRKLVWRFKHPLVWU\ DQG YLVXDOL]DWLRQ ZHUH PDGH SRVVLEOH XWLOL]LQJ D +LVWRVWDLQ63 NLW IURP =\PHG DQG PHWDOHQKDQFHG '$% 3LHUFH 5RFNIRUG ,/f 6HFWLRQV ZHUH VWDLQHG IRU FIRV $&7+ $93 DQG &5+ VHH 7DEOH f 3ULPDU\ DQWLERGLHV ZHUH GLOXWHG LQ DQWLERG\ GLOXHQW b %6$ LQ SKRVSKDWH EXIIHUHG VDOLQH ZLWK b 7ULWRQ ;f 6SHFLILF VWDLQLQJ ZDV FRQILUPHG E\ GLOXWLRQ WHVWV DV VWDLQLQJ GHFUHDVHG DV SULPDU\ DQWLERGLHV ZHUH IXUWKHU GLOXWHG 6SHFLILF VWDLQLQJ ZDV DEVHQW XSRQ UHSODFLQJ SULPDU\ DQWLERGLHV ZLWK b QRUPDO JRDW VHUXP $OO VOLGHV ZHUH GHK\GUDWHG SULRU WR PRXQWLQJ FRYHUVOLSV ZLWK 3HUPRXQW )LVKHU 6FLHQWLILF 3LWWVEXUJK 3$f )HWDO EUDLQV UHJLRQV LPSRUWDQW LQ +3$ D[LV FRQWURO ZHUH PHDVXUHG IRU FIRV JHQHUDWLRQ E\ PHDQV RI 0LFURFRPSXWHU ,PDJLQJ 'HYLFH 0&,'f IURP ,PDJLQLQJ 5HVHDUFK ,QF &HUHEHOOXP DQG FRUWH[ ZHUH DOVR PHDVXUHG WR YHULI\ LI FIRV DFWLYLW\ ZDV VSHFLILF RU I MXVW D JHQHUDO DFWLYDWLRQ RI WKH FHQWUDO QHUYRXV V\VWHP 7KH IROORZLQJ EUDLQ UHJLRQV ZHUH DQDO\]HG f SDUDYHQWULFXODU QXFOHXV 391f f QXFOHXV RI WKH WUDFWXV VROLWDULXV 176f f URVWUDO YHQWUDO ODWHUDO PHGXOOD 59/0f f KLSSRFDPSXV f FHUHEHOOXP f FRUWH[ DQG f SLWXLWDU\ JODQG )RU HDFK RI WKH HLJKW WUHDWPHQW JURXSV OLVWHG LQ WKH 0DWHULDOV DQG 0HWKRGV VHFWLRQf Q $V SDUDIILQ EORFNV ZHUH FXW RQ WKH PLFURWRPH WKH ILIWK VHFWLRQ ZDV XVHG RQFH WKH UHJLRQ RI LQWHUHVW ZDV LGHQWLILHG 7KLV DVVXUHG KRPRJHQHLW\ DPRQJ IHWDO EUDLQV $OO GHQVLWRPHWU\ ZDV SHUIRUPHG LQ D VLPLODU PDQQRU 7KH UHJLRQ RI LQWHUHVW ZDV RXWOLQHG GHVLJQDWLQJ WKH VFDQQHG DUHD 7KH FRPSXWHU ZRXOG WKHQ FRXQW WKH QXPEHU RI FHOOV VWDLQHG SRVLWLYH GHSHQGLQJ XSRQ WKH DVVLJQHG FULWHULD 7KLV YDOXH RU WDUJHW QXPEHU

PAGE 79

ZDV PXOWLSOLHG E\ WKH PHDQ WDUJHW DUHD FDOFXODWHG E\ WKH FRPSXWHUf WR HVWDEOLVK WKH WRWDO WDUJHW DUHD )LQDOO\ WKH WRWDO WDUJHW DUHD ZDV GLYLGHG E\ WKH VFDQQHG DUHD WR HVWDEOLVK WKH SURSRUWLRQ RI SRVLWLYH VWDLQHG FHOOV LQ HDFK EUDLQ VHFWLRQ 9DOXHV ZHUH WKHQ DQDO\]HG YLD WKUHH ZD\ $QDO\VLV RI 9DULDQFH $129$f IROORZHG E\ D PXOWLSOH FRPSDULVRQ SURFHGXUH 7KH PXOWLSOH FRPSDULVRQ SURFHGXUH HPSOR\HG ZDV 6WXGHQW1HZPDQ.HXOV 0HWKRG $OO VWDWLVWLFV ZHUH UXQ XVLQJ 6LJPD 6WDW 5HVXOWV 391 &IRV VWDLQLQJ ZDV IRXQG WR EH VLJQLILFDQWO\ GLIIHUHQW DPRQJ WKH HLJKW WUHDWPHQW JURXSV E\ WKUHH ZD\ $129$ Q SHU JURXS Sf 7KH PHDQ GHQVLWRPHWU\ YDOXHV 6(0 DUH SORWWHG LQ )LJXUH $ IXUWKHU DQDO\VLV RI WKH GDWD UHYHDOHG VWDWLVWLFDOO\ VLJQLILFDQW LQWHUDFWLRQV EHWZHHQ DOO WKUHH IDFWRUV SODFHER YV HVWUDGLRO K\SRWHQVLYH YV QRUPRWHQVLYH DQG FDURWLG VLQXV GHQHUYDWHG YV LQWDFWf 6WXGHQW 1HZPDQ .HXOV 0HWKRG UHYHDOHGWKH IROORZLQJ VLJQLILFDQW GLIIHUHQFHV DPRQJ WUHDWPHQW JURXSV f PHDQ YDOXHV RI I FIRV VWDLQLQJ DPRQJ WKH GLIIHUHQW OHYHOV RI SODFHER WUHDWHG IHWXVHV YV HVWUDGLRO WUHDWHG IHWXVHV ZDV VLJQLILFDQWO\ GLIIHUHQW Q SHU JURXS Sf DQG f PHDQ YDOXHV RI FIRV VWDLQLQJ DPRQJ WKH GLIIHUHQW OHYHOV RI K\SRWHQVLYH IHWXVHV YV QRUPRWHQVLYH IHWXVHV ZDV VLJQLILFDQWO\ GLIIHUHQW Q SHU JURXS Sf 5HSUHVHQWDWLYH SKRWRPLFURJUDSKV RI WKH 391 DUH VKRZQ LQ )LJXUH ,W FDQ EH VHHQ YLVXDOO\ WKDW HVWUDGLRO WUHDWHG IHWXVHV KDYH PRUH FIRV JHQHUDWLRQ LQ WKH 391 ZKHQ FRPSDUHG WR FRQWURO IHWXVHV SDQHOV $ DQG %f $OVR VKRZQ LV VLJQLILFDQWO\ PRUH SRVLWLYH VWDLQLQJ LQ HVWUDGLRO WUHDWHG K\SRWHQVLYH IHWXVHV FRPSDUHG WR SODFHER WUHDWHG K\SRWHQVLYH IHWXVHV SDQHOV & DQG 'f $V VWDWHG EHIRUH K\SRWHQVLYH DQLPDOV DOVR KDYH PRUH FIRV JHQHUDWLRQ FRPSDUHG WR QRUPRWHQVLYH DQLPDOV SDQHOV $ DQG &f

PAGE 80

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f 7KH PHDQ GHQVLWRPHWU\ YDOXHV 6(0 DUH SORWWHG LQ )LJXUH $ IXUWKHU DQDO\VLV RI WKH GDWD UHYHDOHG VWDWLVWLFDOO\ VLJQLILFDQW LQWHUDFWLRQV EHWZHHQ DOO WKUHH IDFWRUV SODFHER YV HVWUDGLRO K\SRWHQVLYH YV QRUPRWHQVLYH DQG FDURWLG VLQXV GHQHUYDWHG YV LQWDFWf 6WXGHQW 1HZPDQ .HXOV 0HWKRG UHYHDOHG WKH IROORZLQJ VLJQLILFDQW GLIIHUHQFHV DPRQJ WUHDWPHQW JURXSV f PHDQ YDOXHV RI FIRV VWDLQLQJ DPRQJ WKH GLIIHUHQW OHYHOV RI SODFHER WUHDWHG IHWXVHV YV HVWUDGLRO WUHDWHG IHWXVHV ZDV VLJQLILFDQWO\ GLIIHUHQW Q SHU JURXS Sf DQG f PHDQ YDOXHV RI FIRV VWDLQLQJ DPRQJ WKH GLIIHUHQW OHYHOV RI K\SRWHQVLYH IHWXVHV YV QRUPRWHQVLYH IHWXVHV ZDV

PAGE 81

VLJQLILFDQWO\ GLIIHUHQW Q SHU JURXS S222O 5HSUHVHQWDWLYH SKRWRPLFURJUDSKV RI WKH 176 DUH VKRZQ LQ )LJXUH ,W FDQ EH VHHQ YLVXDOO\ WKDW HVWUDGLRO WUHDWHG IHWXVHV KDYH PRUH FIRV JHQHUDWLRQ LQ WKH 176 ZKHQ FRPSDUHG WR FRQWURO IHWXVHV SDQHOV $ DQG %f $OVR VKRZQ LV VLJQLILFDQWO\ PRUH SRVLWLYH VWDLQLQJ LQ HVWUDGLRO WUHDWHG K\SRWHQVLYH IHWXVHV FRPSDUHG WR SODFHER WUHDWHG K\SRWHQVLYH IHWXVHV SDQHOV & DQG 'f $V VWDWHG EHIRUH K\SRWHQVLYH DQLPDOV DOVR KDYH PRUH FIRV JHQHUDWLRQ FRPSDUHG WR QRUPRWHQVLYH DQLPDOV SDQHOV $ DQG &f 7KHVH UHVXOWV FRLQFLGH ZLWK WKRVH RI WKH 391 59/0 &IRV VWDLQLQJ ZDV IRXQG WR EH VLJQLILFDQWO\ GLIIHUHQW DPRQJ WKH HLJKW WUHDWPHQW JURXSV E\ WKUHH ZD\ $129$ Q SHU JURXS Sf 7KH PHDQ GHQVLWRPHWU\ YDOXHV 6(0 DUH SORWWHG LQ )LJXUH $ IXUWKHU DQDO\VLV RI WKH GDWD UHYHDOHG VWDWLVWLFDOO\ VLJQLILFDQW LQWHUDFWLRQV EHWZHHQ DOO WKUHH IDFWRUV SODFHER YV HVWUDGLRO K\SRWHQVLYH YV QRUPRWHQVLYH DQG FDURWLG VLQXV GHQHUYDWHG YV LQWDFWf 6WXGHQW 1HZPDQ .HXOV 0HWKRG UHYHDOHGWKH IROORZLQJ VLJQLILFDQW GLIIHUHQFHV DPRQJ WUHDWPHQW JURXSV f PHDQ YDOXHV RI FIRV VWDLQLQJ DPRQJ WKH GLIIHUHQW OHYHOV RI SODFHER WUHDWHG IHWXVHV YV HVWUDGLRO WUHDWHG IHWXVHV ZDV VLJQLILFDQWO\ GLIIHUHQW Q SHU JURXS Sf f PHDQ YDOXHV RI FIRV VWDLQLQJ DPRQJ WKH GLIIHUHQW OHYHOV RI K\SRWHQVLYH IHWXVHV YV QRUPRWHQVLYH IHWXVHV ZDV VLJQLILFDQWO\ GLIIHUHQW Q SHU JURXS S 5HSUHVHQWDWLYH SKRWRPLFURJUDSKV RI WKH 59/0 DUH VKRZQ LQ )LJXUH ,W FDQ EH VHHQ YLVXDOO\ WKDW HVWUDGLRO WUHDWHG IHWXVHV KDYH PRUH FIRV JHQHUDWLRQ LQ WKH 59/0 ZKHQ FRPSDUHG WR FRQWURO IHWXVHV SDQHOV $ DQG %f $OVR VKRZQ LV VLJQLILFDQWO\ PRUH SRVLWLYH VWDLQLQJ LQ HVWUDGLRO WUHDWHG K\SRWHQVLYH IHWXVHV FRPSDUHG WR SODFHER WUHDWHG K\SRWHQVLYH IHWXVHV SDQHOV & DQG 'f $V VWDWHG EHIRUH K\SRWHQVLYH DQLPDOV DOVR KDYH PRUH FIRV JHQHUDWLRQ FRPSDUHG WR QRUPRWHQVLYH DQLPDOV SDQHOV $ DQG &f 7KHVH UHVXOWV FRLQFLGH ZLWK WKRVH RI WKH 391 DQG WKH 176

PAGE 82

+LSSRFDPSXV &IRV VWDLQLQJ ZDV IRXQG WR EH VLJQLILFDQWO\ GLIIHUHQW DPRQJ WKH HLJKW WUHDWPHQW JURXSV E\ WKUHH ZD\ $129$ Q SHU JURXS Sf 7KH PHDQ GHQVLWRPHWU\ YDOXHV 6(0 DUH SORWWHG LQ )LJXUH 1RWH WKDW WKH \ D[LV LV VPDOOHU WKDQ WKDW RI WKH 391 HWF EHFDXVH WKH OHYHO RI FIRV VWDLQLQJ LV ORZHU LQ WKH KLSSRFDPSXV 6WXGHQW1HZPDQ.HXOV 0HWKRG UHYHDOHG WKH WKDW WKH PHDQ YDOXHV RI FIRV VWDLQLQJ DPRQJ WKH GLIIHUHQW OHYHOV RI K\SRWHQVLYH IHWXVHV YV QRUPRWHQVLYH IHWXVHV ZDV VLJQLILFDQWO\ GLIIHUHQW Q SHU JURXS Sf +RZHYHU PHDQ YDOXHV RI FIRV VWDLQLQJ DPRQJ WKH GLIIHUHQW OHYHOV RI SODFHER WUHDWHG IHWXVHV YV HVWUDGLRO WUHDWHG IHWXVHV ZDV QRW VLJQLILFDQWO\ GLIIHUHQW Q SHU JURXS S f 5HSUHVHQWDWLYH SKRWRPLFURJUDSKV RI WKH KLSSRFDPSXV DUH VKRZQ LQ )LJXUH ,W FDQ EH VHHQ YLVXDOO\ WKDW K\SRWHQVLYH IHWXVHV KDYH PRUH FIRV JHQHUDWLRQ LQ WKH KLSSRFDPSXV ZKHQ FRPSDUHG WR QRUPRWHQVLYH IHWXVHV SDQHOV $ DQG %f 7KHVH UHVXOWV UHVHPEOH WKRVH RI WKH 391 176 DQG 59/0 ZLWK UHJDUG WR WKH HIIHFW RI K\SRWHQVLRQ KRZHYHU WKH DEVHQFH RI DQ HVWUDGLRO HIIHFW LV QRYHO &HUHEHOOXP &HUEHOOXP ZDV DQDO\]HG DV D SHULSKHUDO WLVVXH WKDW GRHV QRW LQWHJUDWLRQ LQ WKH V\VWHP EHLQJ VWXGLHG $V VXVSHFWHG DQDO\VLV E\ WKUHH ZD\ $129$ GLG QRW \LHOG DQ\ GLIIHUHQFHV DPRQJ WUHDWPHQW JURXSV Q SHU JURXS S f 0HDQ JURXS YDOXHV 6(0 RI WKH GHQVLWRPHWU\ DQDO\VLV DUH VKRZQ LQ )LJXUH 1RWH WKDW WKH \ D[LV LV PXFK VPDOOHU WKDQ WKDW RI WKH 391 HWF EHFDXVH RI WKH DEVHQFH RI DQ\ VLJQLILFDQW VWDLQLQJ 5HSUHVHQWDWLYH SKRWRPLFURJUDSKV RI IHWDO RYLQH FHUHEHOOXP DUH VKRZQ LQ )LJXUH 3DQHO $ VKRZV D FRQWURO DQLPDO ZKLOH SDQHO % VKRZV D K\SRWHQVLYH DQLPDO 9LVXDOO\ DV ZHOO DV VWDWLVWLFDOO\ WKHUH LV QR GLIIHUHQFH 7KRXJK QRW VKRZQ SLFWRULDOO\ WKH VDPH LV WUXH RI HVWUDGLRO WUHDWHG IHWXVHV YV SODFHER WUHDWHG IHWXVHV

PAGE 83

&RUWH[ $V WKH FDVH ZLWK FHUHEHOOXP FRUWH[ ZDV DQDO\]HG DV D SHULSKHUDO WLVVXH WKDW GRHV QRW LQWHJUDWLRQ LQ WKH V\VWHP EHLQJ VWXGLHG $V VXVSHFWHG DQDO\VLV E\ WKUHH ZD\ $129$ GLG QRW \LHOG DQ\ GLIIHUHQFHV DPRQJ WUHDWPHQW JURXSV Q SHU JURXS S f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f K\SRWKHVL]HG WKDW WKH EDURUHFHSWRU FKHPRUHFHSWRU DIIHUHQW

PAGE 84

SDWKZD\ LV LQYROYHG WKXV FDURWLG VLQXV GHQHUYDWLRQ ZLOO HOLPLQDWH WKH DXJPHQWHG +3$ D[LV DFWLYLW\ 1HXURQDO DFWLYLW\ ZDV DVVHVVHG E\ PHDVXULQJ WKH OHYHO RI FIRV DQ\ HDUO\ UHVSRQVH JHQH LQ EUDLQ DUHDV LPSRUWDQW IRU +3$ D[LV FRQWURO 7KLV PHWKRG KDV EHHQ XVHG LQ QXPHURXV VWXGLHV WR DVVHVV QHXURQDO DFWLYDWLRQ GXH WR SK\VLRORJLFDO VWUHVV +RIIPDQ HW DO 6KHQ HW DO &KDQ HW DO f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f 7KH 176 LV WKH ILUVW V\QDSVH SRLQW LQ WKH SDWKZD\ FRQQHFWLQJ WKH DIIHUHQW EDURUHFHSWRUV ZLWK WKH 391 7KH 59/0 LV D FDUGLRYDVFXODU UHJXODWRU\ FHQWHU ZKLFK FRRUGLQDWHV LQIRUPDWLRQ IURP WKH SHULSKHU\ ,W KDV EHHQ VKRZQ WKDW DOO RI WKHVH EUDLQ UHJLRQV KDYH HVWURJHQ UHFHSWRUV HQDEOLQJ WKHP WR UHVSRQG WR HVWUDGLRO WUHDWPHQW SULRU WR EUDFKLRFHSKDOLF RFFOXVLRQ /HKPDQ HW DO 6LPHUO\ HW DO f (VWUDGLRO FDXVHG D VLJQLILFDQW LQFUHDVH LQ QHXURQDO DFWLYLW\ LQ WKH 391 176 DQG 59/0 ,Q IHWXVHV PDGH K\SRWHQVLYH YLD EUDFKLRFHSKDOLF RFFOXVLRQ WKLV HIIHFW ZDV IXUWKHU DXJPHQWHG DV VKRZQ E\

PAGE 85

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f 3HUKDSV WKH DQVZHU WR WKLV GLVFUHSDQF\ OLHV LQ IDFW WKDW WKH KLSSRFDPSXV LV D FODVVLF VLWH RI FRUWLVRO QHJDWLYH IHHGEDFN :RRG KDV IXUWKHU VKRZQ WKDW WKH +3$ D[LV LV LQVHQVLWLYH WR LQKLELWLRQ YLD FRUWLVRO QHJDWLYH IHHGEDFN WRZDUGV WKH HQG RI JHVWDWLRQ f 3RVVLEO\ WKH KLSSRFDPSXV LV XQUHVSRQVLYH WR DQ HVWUDGLRO DXJPHQWDWLRQ RI $&7+ VHFUHWLRQ DW WKLV WLPH 2I FRXUVH WKLV PD\ EH LQ WKH EHVW LQWHUHVW RI WKH IHWXV VLQFH DQ LQFUHDVH LQ QHXURQDO DFWLYLW\ DW WKH OHYHO RI WKH KLSSRFDPSXV PD\ OHDG WR LQKLELWLRQ UDWKHU WKDQ VWLPXODWLRQ RI WKH D[LV ,W ZDV FHUWDLQO\ HQFRXUDJLQJ WKDW LQ SHULSKHUDO EUDLQ UHJLRQV DQ LQFUHDVH LQ QHXURQDO DFWLYLW\ ZDV REVHUYHG LQ QHLWKHU HVWUDGLRO WUHDWHG QRU HVWUDGLRO WUHDWHG K\SRWHQVLYH IHWXVHV 7KLV ODFN RI FIRV VWDLQLQJ LQ WKH FRUWH[ DQG FHUHEHOOXP LQGLFDWH WKDW WKH UHVSRQVH WR WUHDWPHQW ZLWKLQ WKH &16 ZDV VSHFLILF $OO WKLQJV EHLQJ HTXDO RQO\ EUDLQ UHJLRQV LPSRUWDQW LQ +3$ D[LV FRQWURO VHHPHG WR UHVSRQG WR HVWUDGLRO WUHDWPHQW DQG EUDFKLRFHSKDOLF RFFOXVLRQ

PAGE 86

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

PAGE 87

RI K\GURJHQ LRQV HOHYDWHG SDUWLDO SUHVVXUH RI FDUERQ GLR[LGH 3&2f DQG D GHFUHDVH LQ WKH SDUWLDO SUHVVXUH SUHVVXUH RI R[\JHQ 32f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

PAGE 88

7RWDO 7DUJHW $UHD 6FDQQHG $UHD )LJXUH &IRV LPPXQRKLVWRFKHPLVWU\ VWDLQLQJ LQ WKH IHWDO RYLQH SDUDYHQWULFXODU QXFOHXV 3ODF SODFHER LPSODQW ( HVWUDGLRO LPSODQW +\SR K\SRWHQVLYH &6' FDURWLG VLQXV GHQHUYDWHGf 7KHUH LV D VLJQLILFDQW GLIIHUHQFH EHWZHHQ 3ODF YV ( r Sf DQG &RQWURO YV +\SR Sf

PAGE 89

)LJXUH &IRV LPPXQRKLVWRFKHPLVWU\ VWDLQLQJ LQ WKH IHWDO RYLQH SDUDYHQWULFXODU QXFOHXV $ &RQWURO % (VWUDGLRO WUHDWHG & +\SRWHQVLYH (VWUDGLRO WUHDWHG K\SRWHQVLYHf $OO SKRWRPLFURJUDSKV DUH DW D PDJQLILFDWLRQ RI ;

PAGE 90

)LJXUH 3KRWRPLFURJUDSKV RI WKH IHWDO RYLQH 391 FRPSDULQJ FIRV VWDLQLQJ LQ DQ LQWDFW HVWUDGLRO WUHDWHG IHWXV $f DQG DQ HVWUDGLRO WUHDWHG FDURWLG VLQXV GHQHUYDWHG IHWXV %f $ DQG % ZHUH QRW VWDWLVWLFDOO\ GLIIHUHQW VKRZLQJ WKDW GHQHUYDWLRQ DORQH GLG QRW FDXVH FIRV JHQHUDWLRQ

PAGE 91

)LJXUH 3KRWRPLFURJUDSKV RI WKH IHWDO RYLQH 391 FRPSDULQJ FIRV VWDLQLQJ LQ D K\SRWHQVLYH IHWXV $f DQG D K\SRWHQVLYH FDURWLG VLQXV GHQHUYDWHG IHWXV %f $ DQG % ZHUH VWDWLVWLFDOO\ GLIIHUHQW Sf VKRZLQJ WKDW GHQHUYDWLRQ GLPLQLVKHG FIRV JHQHUDWLRQ LQ K\SRWHQVLYH DQLPDOV

PAGE 92

7RWDO 7DUJHW $UHD 6FDQQHG $UHD )LJXUH &IRV LPPXQRKLVWRFKHPLVWU\ VWDLQLQJ LQ WKH IHWDO RYLQH QXFOHXV RI WKH WUDFWXV VROLWDULXV 3ODF SODFHER LPSODQW ( HVWUDGLRO LPSODQW +\SR K\SRWHQVLYH &6' FDURWLG VLQXV GHQHUYDWHGf 7KHUH LV D VLJQLILFDQW GLIIHUHQFH EHWZHHQ 3ODF YV ( r Sf DQG &RQWURO YV +\SR Sf

PAGE 93

)LJXUH &IRV LPPXQRKLVWRFKHPLVWU\ VWDLQLQJ LQ WKH IHWDO RYLQH QXFOHXV RI WKH WUDFWXV VROLWDULXV $ &RQWURO % (VWUDGLRO WUHDWHG & +\SRWHQVLYH (VWUDGLRO WUHDWHG K\SRWHQVLYHf $OO SKRWRPLFURJUDSKV DUH DW D PDJQLILFDWLRQ RI ;

PAGE 94

7RWDO 7DUJHW $UHD 6FDQQHG $UHD )LJXUH &IRV LPPXQRKLVWRFKHPLVWL\ VWDLQLQJ LQ WKH IHWDO RYLQH URVWUDO YHQWUDO ODWHUDO PHGXOOD 3ODF SODFHER LPSODQW ( HVWUDGLRO LPSODQW +\SR K\SRWHQVLYH &6' FDURWLG VLQXV GHQHUYDWHGf 7KHUH LV D VLJQLILFDQW GLIIHUHQFH EHWZHHQ 3ODF YV ( r Sf DQG &RQWURO YV +\SR Sf

PAGE 95

)LJXUH &IRV LPPXQRKLVWRFKHPLVWU\ VWDLQLQJ LQ WKH IHWDO RYLQH URVWUDO YHQWUDO ODWHUDO PHGXOOD $ &RQWURO % (VWUDGLRO WUHDWHG & +\SRWHQVLYH (VWUDGLRO WUHDWHG K\SRWHQVLYHf $OO SKRWRPLFURJUDSKV DUH DW D PDJQLILFDWLRQ RI ;

PAGE 96

7RWDO 7DUJHW $UHD 6FDQQHG $UHD )LJXUH &IRV LPPXQRKLVWRFKHPLVWU\ VWDLQLQJ LQ WKH IHWDO RYLQH KLSSRFDPSXV 3ODF SODFHER LPSODQW ( HVWUDGLRO LPSODQW +\SR K\SRWHQVLYH &6' FDURWLG VLQXV GHQHUYDWHGf 7KHUH LV D VLJQLILFDQW GLIIHUHQFH EHWZHHQ &RQWURO YV +\SR Sf

PAGE 97

)LJXUH &IRV LPPXQRKLVWRFKHPLVWU\ VWDLQLQJ LQ WKH IHWDO RYLQH KLSSRFDPSXV $ &RQWURO % +\SRWHQVLYHf $OO SKRWRPLFURJUDSKV DUH DW D PDJQLILFDWLRQ RI ;

PAGE 98

7RWDO 7DUJHW $UHD 6FDQQHG $UHD )LJXUH &IRV LPPXQRKLVWRFKHPLVWU\ VWDLQLQJ LQ WKH IHWDO RYLQH FHUHEHOOXP 3ODF SODFHER LPSODQW ( HVWUDGLRO LPSODQW +\SR K\SRWHQVLYH &6' FDURWLG VLQXV GHQHUYDWHGf

PAGE 99

)LJXUH &IRV LPPXQRKLVWRFKHPLVWU\ VWDLQLQJ LQ WKH IHWDO RYLQH FHUHEHOOXP $ &RQWURO % +\SRWHQVLYHf $OO SKRWRPLFURJUDSKV DUH DW D PDJQLILFDWLRQ RI ; $ DQG % DUH QRW VWDWLVWLFDOO\ GLIIHUHQW

PAGE 100

7RWDO 7DUJHW $UHD 6FDQQHG $UHD )LJXUH &IRV LPPXQRKLVWRFKHPLVWU\ VWDLQLQJ LQ WKH IHWDO RYLQH FRUWH[ 3ODF SODFHER LPSODQW ( HVWUDGLRO LPSODQW +\SR K\SRWHQVLYH &6' FDURWLG VLQXV GHQHUYDWHGf

PAGE 101

)LJXUH &IRV LPPXQRKLVWRFKHPLVWU\ VWDLQLQJ LQ WKH IHWDO RYLQH FRUWH[ $ &RQWURO % +\SRWHQVLYHf $OO SKRWRPLFURJUDSKV DUH DW D PDJQLILFDWLRQ RI ; $ DQG % DUH QRW VWDWLVWLFDOO\ GLIIHUHQW

PAGE 102

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f 7KH UHVXOWDQW LQFUHDVH LQ IHWDO SODVPD FRUWLVRO LQGXFHV WKH DFWLYLW\ RI F\WRFKURPH 3FL LQ WKH SODFHQWD 0DVRQ HW DO f 7KLV HQ]\PH KDV ERWK K\GUR[\ODVH DQG O\DVH DFWLYLWLHV LQGXFWLRQ RI WKLV HQ]\PH DOORZV SURSRUWLRQDWHO\ PRUH HVWURJHQ DQG SURSRUWLRQDWHO\ OHVV SURJHVWHURQH ELRV\QWKHVLV 7KH LQFUHDVH LQ WKH VRFDOOHG HVWURJHQWRSURJHVWHURQH (3f UDWLR LQ SODVPD DQG ORFDOO\ ZLWKLQ WKH XWHULQH WLVVXHVf DOORZV LQFUHDVHG XWHULQH FRQWUDFWLOLW\ /LJJLQV HW DO f ,W LV WKH LQFUHDVHG XWHULQH WRQH ZKLFK LQLWLDWHV ODERU DQG GHOLYHU\ RI WKH IHWXV ,W KDV UHFHQWO\ EHHQ GHPRQVWUDWHG WKDW SK\VLRORJLFDO LQFUHDVHV LQ IHWDO SODVPD HVWURJHQ FRQFHQWUDWLRQV JUHDWO\ DXJPHQW IHWDO $&7+ VHFUHWLRQ 6DRXG DQG :RRG f 7KLV HIIHFW RI HVWURJHQ FDQ EH GHPRQVWUDWHG RQ ERWK EDVDO DQG K\SRWHQVLRQ VWLPXODWHG IHWDO $&7+ VHFUHWLRQ :KLOH HVWURJHQ KDV D SRWHQWLDOO\ LPSRUWDQW HIIHFW RQ IHWDO $&7+

PAGE 103

VHFUHWLRQ LW LV ZHOONQRZQ WKDW IHWDO SODVPD FRQFHQWUDWLRQV RI XQFRQMXJDWHG HVWURJHQV LQFUHDVH RQO\ DIWHU WKH EHJLQQLQJ RI WKH LQFUHDVH LQ IHWDO SODVPD $&7+ DQG FRUWLVRO 1DWKDQLHOV] HW DO 6WURWW HW DO f &RQMXJDWHG HVWURJHQV PRVWO\ HVWURQH VXOIDWH FLUFXODWH LQ KLJK FRQFHQWUDWLRQV FRPSDUHG WR XQFRQMXJDWHG HVWURJHQV &DUQHJLH DQG 5REHUWVRQ 7VDQJ f 7KH FRQFHQWUDWLRQ RI HVWURQH VXOIDWH LQFUHDVHV EHIRUH WKH LQFUHDVH LQ IHWDO +3$ D[LV DFWLYLW\ 1DWKDQLHOV] HW DO f +RZHYHU FRQMXJDWHG VWHURLGV FDQQRW ELQG WR QXFOHDU UHFHSWRUV XQOHVV GHFRQMXJDWHG +RENLUN f ,W LV SRVVLEOH WKDW HVWURQH VXOIDWH LV FRQYHUWHG WR HVWURQH ORFDOO\ ZLWKLQ WKH IHWDO EUDLQ DQG WKDW WKH FLUFXODWLQJ HVWURQH VXOIDWH DFWV DV D UHVHUYRLU IRU HVWURQH DFWLQJ ZLWKLQ WKH IHWDO EUDLQ &RQMXJDWLRQ DQG GHFRQMXJDWLRQ RI HVWURQH DUH DFFRPSOLVKHG YLD WKH HQ]\PHV HVWURJHQ VXOIRWUDQVIHUDVH DQG HVWURJHQ VXOIDWDVH UHVSHFWLYHO\ (VWURJHQ VXOIRWUDQVIHUDVH DQG VXOIDWDVH DFWLYLW\ KDYH EHHQ UHSRUWHG LQ EUDLQ WLVVXH IURP DGXOW VKHHS /DNVKPL DQG %DODVXEUDPDQLDQ f UDWV &RQQROO\ DQG 5HVNR .DZDQR DQG $LNDZD f SULPDWHV /DNVKPL DQG %DODVXEUDPDQLDQ f DQG KXPDQ EHLQJV 3ODWLD HW DO f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

PAGE 104

0HWKRGV DQG 0DWHULDOV (Q]\PH $FWLYLW\ VWXGLHG IHWXVHV GD\V JHVWDWLRQ WHUP GD\Vf ODPEV ZHHNV ROGf DQG DGXOW QRQSUHJQDQW HZHV WR GHWHUPLQH HVWURQH VXOIDWDVH DFWLYLW\ 7KH VKHHS ZHUH VDFULILFHG XVLQJ DQ LQWUDYHQRXV RYHUGRVH RI VRGLXP SHQWREDUELWDO *HVWDWLRQDO DJHV RI WKH IHWDO VKHHS ZHUH FDOFXODWHG IURP NQRZQ EUHHGLQJ GDWHV :KROH EUDLQV ZHUH UDSLGO\ UHPRYHG GLVVHFWHG LQWR GLVFUHWH UHJLRQV DQG TXLFNO\ IUR]HQ RQ GU\ LFH RU LQ D VOXUU\ RI GU\ LFH DQG DFHWRQH $OO WLVVXHV ZHUH VWRUHG DW r& RU r& XQWLO VWXGLHG +\SRWKDODPL EUDLQVWHPV DQG KLSSRFDPSL ZHUH WKHQ SURFHVVHG WR GHWHUPLQH HVWURQH VXOIDWDVH DFWLYLW\ (DFK WLVVXH VDPSOH ZDV KRPRJHQL]HG LQ PHGLXP 6LJPDp 6W /RXLV 02f FRQWDLQLQJ P0 +(3(6 +RPRJHQL]DWLRQ ZDV SHUIRUPHG XVLQJ D 3RO\WURQ KRPRJHQL]HU 7HNPDU &LQFLQQDWL 2+f 7KH FRQFHQWUDWLRQ RI HDFK WLVVXH LQ WKH KRPRJHQDWH ZDV J WLVVXH LQ P/ PHGLXP 7LVVXHV ZHUH FHQWULIXJHG DW USP IRU PLQ VXSHUQDWDQW ZDV WKHQ FROOHFWHG DQG DVVD\HG LPPHGLDWHO\ $ VDPSOH RI HDFK KRPRJHQDWH ZDV DVVD\HG IRU SURWHLQ FRQFHQWUDWLRQ XVLQJ WKH PHWKRG RI %UDGIRUG f XVLQJ D FRPPHUFLDOO\DYDLODEOH DVVD\ NLW %LR5DG /DERUDWRULHV +HUFXOHV &$f +RPRJHQDWH P/f ZDV DOLTXRWWHG LQ GXSOLFDWH LQWR ERURVLOLFDWH WXEHV [ PPf FRQWDLQLQJ P/ RI D PL[WXUH RI + >@HVWURQH VXOIDWH 'X3RQW1(1 :LOPLQJWRQ '(f DQG XQODEHOHG HVWURQH VXOIDWH (62 6LJPDp 6W /RXLV 02f $OO UHDFWLRQV ZHUH UXQ DW r& 5HDFWLRQV ZHUH WHUPLQDWHG E\ LPPHGLDWH FRROLQJ RQ LFH DGGLWLRQ RI YROXPHV RI HWK\O DFHWDWHKH[DQH f DQG YLJRURXV PL[LQJ IRU VHFRQGV 7KH DTXHRXV SKDVH ZDV IUR]HQ E\ VXEPHUVLRQ RI WKH UHDFWLRQ WXEH LQWR D GU\ LFH DQG DFHWRQH VOXUU\ 6XEVHTXHQWO\ WKH RUJDQLF SKDVH FRQWDLQLQJ WKH +HVWURQH ZDV GHFDQWHG LQWR [ PP ERURVLOLFDWH JODVV WXEHV DQG GULHG

PAGE 105

XQGHU D JHQWOH VWUHDP RI URRP DLU 'ULHG H[WUDFWV ZHUH UHFRQVWLWXWHG LQ VFLQWLOODQW &\WRVFLQWp ,&1 &RUS &RVWD 0HVD &$f DQG FRXQWHG LQ D VFLQWLOODWLRQ FRXQWHU /.% &RUS *DLWKHUVEXUJ 0'f (Q]\PH DFWLYLWLHV DW GLIIHUHQW GHYHORSPHQWDO DJHV DQG LQ GLIIHUHQW WLVVXHV ZHUH PHDVXUHG XVLQJ D VXEVWUDWH FRQFHQWUDWLRQ RI S0 DQG +HVWURQH VXOIDWH VSHFLILF DFWLYLW\ RI DSSUR[LPDWHO\ S&LQPRO )RU WKLV H[SHULPHQW UHDFWLRQV ZHUH DOORZHG WR UXQ IRU PLQ 8VLQJ WKHVH FRQGLWLRQV OHVV WKDQ b RI WKH VXEVWUDWH ZDV FRQYHUWHG WR +HVWURQH &RPSDULVRQ RI UHODWLYH DFWLYLWLHV DW GLIIHUHQW GHYHORSPHQWDO DJHV ZDV DFKLHYHG XVLQJ RQHZD\ DQDO\VLV RI YDULDQFH $129$f $ SRVWHULRUL FRPSDULVRQ RI LQGLYLGXDO PHDQ YDOXHV ZDV SHUIRUPHG XVLQJ 1HZPDQ.HXOV PXOWLSOH UDQJH WHVW :LQHU f &RPSDULVRQ RI WZR PHDQV ZDV SHUIRUPHG XVLQJ 6WXGHQWnV WWHVW =DU f $OO VWDWLVWLFDO FRPSXWDWLRQV ZHUH SHUIRUPHG XVLQJ 6LJPD6WDW -DQGHO 6FLHQWLILF 6DQ 5DIDHO &$f :HVWHUQ %ORWWLQJ +\SRWKDODPL DQG EUDLQVWHPV ZHUH KDUYHVWHG IURP IHWXVHV ODPEV DQG DGXOWV RI NQRZQ JHVWDWLRQDO DQG SRVWQDWDO DJHV 7KH QXPEHU DQG DJHV RI DQLPDOV YDULHG VOLJKWO\ EHWZHHQ K\SRWKDODPL DQG EUDLQVWHP EXW IHWXVHV ODPEV DQG DGXOWV ZHUH XVHG SHU WLVVXH W\SH 7KHVH WLVVXHV ZHUH RULJLQDOO\ REWDLQHG DQG KRPRJHQL]HG IRU RWKHU VWXGLHV 6DRXG DQG :RRG f 8QIRUWXQDWHO\ KLSSRFDPSL IURP WKHVH DQLPDOV ZHUH QRW DYDLODEOH $OO WLVVXH ZDV KRPRJHQL]HG LQ UHGXFLQJ EXIIHU DQG ERLOHG IRU PLQXWHV 7KH VDPSOHV ZHUH FHQWULIXJHG WR UHPRYH SDUWLFXODWH PDWWHU DQG VXSHUQDWDQW ZDV UHFRYHUHG 3URWHLQ FRQFHQWUDWLRQV ZHUH REWDLQHG XWLOL]LQJ WKH %UDGIRUG WHFKQLTXH f :HVWHUQ EORWV ZHUH SHUIRUPHG XVLQJ D PLQL3URWHDQ HOHFWURSKRUHVLV V\VWHP %LR5DG +HUFXOHV &$f RQ b SUHFDVW SRO\DFU\ODPLGH JHOV SXUFKDVHG IURP %LR5DG ODERUDWRULHV 6DPSOHV ZHUH GLOXWHG VR WKDW DQ HTXDO DPRXQW RI SURWHLQ ZDV ORDGHG SHU ODQH SJ IRU

PAGE 106

EUDLQVWHP DQG _LJ IRU K\SRWKDODPLf 7KH SURWHLQ ZDV WKHQ WUDQVIHUUHG WR D QLWURFHOOXORVH PHPEUDQH DQG SUREHG IRU HLWKHU HVWURJHQ VXOIDWDVH RU HVWURJHQ VXOIRWUDQVIHUDVH XVLQJ FXVWRPPDGH UDEELW SRO\FORQDO DQWLERGLHV $OSKD 'LDJQRVWLF 6DQ $QWRQLR 7;f 7KH SHSWLGH VHTXHQFH XVHG IURP WKH KXPDQ VXOIDWDVH JHQH DPLQR DFLGV ZDV 1+)66.')$*.64+*9<*&&22+ 6LPHUO\ HW DO f 7KH SHSWLGH VHTXHQFH XVHG IURP WKH ERYLQH VXOIRWUDQVIHUDVH JHQH DPLQR DFLGV ZDV 1+5(5)((+<4440.'&&22+ 1DVK HW DO f 3ULPDU\ DQWLERGLHV ZHUH GLOXWHG WR D FRQFHQWUDWLRQ RI LQ DQWLERG\ GLOXHQW b %6$ LQ SKRVSKDWH EXIIHUHG VDOLQH ZLWK b 7ZHHQ f 9LVXDOL]DWLRQ RI WKH SURWHLQDQWLERG\ FRPSOH[ ZDV DFFRPSOLVKHG XWLOL]LQJ D FKHPLOXPLQHVFHQFH GHWHFWLRQ V\VWHP 5HQDLVVDQFH 'X3RQW 1(1 %RVWRQ 0$f DQG DQDO\]HG E\ GHQVLWRPHWU\ %LR5DGf $QWLERG\ VSHFLILFLW\ ZDV FRQILUPHG E\ SUHDEVRUSWLRQ RI WKH SULPDU\ DQWLERGLHV ZLWK SHSWLGHV SJPOf DOVR VXSSOLHG E\ $OSKD 'LDJQRVWLF 'HYHORSPHQWDO FKDQJHV ZHUH FDOFXODWHG XVLQJ PXOWLSOH OLQHDU UHJUHVVLRQ LQ RUGHU WR FRQWURO IRU GLIIHUHQFHV EHWZHHQ JHO UXQQLQJ FRQGLWLRQV 6LJPD6WDW -DQGHO 6FLHQWLILF 6DQ 5DIDHO &$f ,PPXQRKLVWRFKHPLVWUY )HWDO EUDLQV ZHUH SHUIXVLRQ IL[HG ZLWK b SDUDIRUPDOGHK\GH GLVVHFWHG DQG FXW LQWR JURVV WLVVXH UHJLRQV K\SRWKDODPXV PLGEUDLQ SRQV PHGXOOD VSLQDO FRUG HWFf 7LVVXH ZDV SURFHVVHG IRU HPEHGGLQJ E\ GHK\GUDWLRQ ZLWK SURJUHVVLYHO\ LQFUHDVLQJ FRQFHQWUDWLRQV RI HWKDQRO IROORZHG E\ [\OHQH $OO WLVVXH ZDV HPEHGGHG LQ SDUDIILQ DQG FXW LQWR _DP VHFWLRQV XVLQJ D =HLVV PLFURWRPH 6HFWLRQV ZHUH PRXQWHG RQ SRO\/O\VLQH VOLGHV GHSDUDIILQL]HG ZLWK [\OHQH DQG UHK\GUDWHG LQ GHFUHDVLQJ FRQFHQWUDWLRQV RI HWKDQRO ,PPXQRKLVWRFKHPLVWU\ DQG YLVXDOL]DWLRQ ZHUH PDGH SRVVLEOH XWLOL]LQJ D +LVWRVWDLQ63 NLW IURP =\PHG DQG PHWDOHQKDQFHG '$% 3LHUFH 5RFNIRUG ,/f

PAGE 107

6HFWLRQV ZHUH VWDLQHG IRU HVWURJHQ VXOIDWDVH DQG HVWURJHQ VXOIRWUDQVIHUDVH XVLQJ WKH VDPH FXVWRP PDGH DQWLERG\ XVHG IRU ZHVWHUQ EORWWLQJ 3ULPDU\ DQWLERGLHV ZHUH GLOXWHG WR D FRQFHQWUDWLRQ RI LQ DQWLERG\ GLOXHQW b %6$ LQ SKRVSKDWH EXIIHUHG VDOLQH ZLWK b 7ULWRQ ;f 6SHFLILF VWDLQLQJ ZDV FRQILUPHG E\ GLOXWLRQ WHVWV DV VWDLQLQJ GHFUHDVHG DV SULPDU\ DQWLERGLHV ZHUH IXUWKHU GLOXWHG 6SHFLILF VWDLQLQJ ZDV DEVHQW XSRQ UHSODFLQJ SULPDU\ DQWLERGLHV ZLWK b QRUPDO JRDW VHUXP $OO VOLGHV ZHUH GHK\GUDWHG SULRU WR PRXQWLQJ RI FRYHUVOLSV ZLWK 3HUPRXQW )LVKHU 6FLHQWLILF 3LWWVEXUJK 3$f 5HVXOWV (Q]\PH $FWLYLW\ (VWURJHQ VXOIDWDVH DFWLYLW\ ZDV PHDVXUDEOH LQ RYLQH IHWDO QHRQDWDO DQG DGXOW K\SRWKDODPXV KLSSRFDPSXV DQG EUDLQVWHP )LJXUH f 7KH DFWLYLW\ LQ WKH KLSSRFDPSXV ZDV VLJQLILFDQWO\ LQFUHDVHG LQ ODWHJHVWDWLRQ IHWXVHV FRPSDUHG WR \RXQJHU IHWXVHV ODPEV DQG DGXOW HZHV DV WHVWHG E\ $129$ DQG 1HZPDQ.HXOV PXOWLSOH UDQJH WHVW Q SHU JURXS Sf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nV WWHVW WKH DFWLYLW\ LQ RYLQH DGXOW K\SRWKDODPXV ZDV VLJQLILFDQWO\ GLIIHUHQW WKDQ WKH DFWLYLW\ LQ RYLQH P\RPHWULXP Q SHU JURXS Sf :HVWHUQ %ORWWLQJ ,Q SHUIRUPLQJ :HVWHUQ EORWV WLVVXHV ZHUH GLVWULEXWHG DPRQJ JHOV VR WKDW HDFK JHO FRQWDLQHG D VXEVHW RI WKH GHYHORSPHQWDO DJHV VWXGLHG )RU EUDLQVWHP

PAGE 108

IHWXVHV ODPEV DQG DGXOWV ZHUH XVHG 7KH GHYHORSPHQWDO DJHV ZHUH DV IROORZV GD\V Q f GD\V Q f GD\V Q f GD\V Q f ODPEV GD\V Q f DQG DGXOW HZHV Q f :HVWHUQ EORWV UHYHDOHG WKUHH GLVWLQFW EDQGV DW DQG N' IRU HVWURJHQ VXOIDWDVH 7KH N' EDQG LV VKRZQ LQ )LJXUH DQG FRUUHVSRQGV WR WKH FRUUHFW PROHFXODU ZHLJKW RI 'DOWRQV IRU HVWURJHQ VXOIDWDVH 6WHLQ HW DO f $V VKRZQ E\ )LJXUH WKHUH DUH VOLJKW PROHFXODU ZHLJKW YDULDWLRQV RI WKH N' EDQG IRU RYLQH EUDLQVWHP 3UHDEVRUSWLRQ RI WKH HQ]\PH UHYHDOHG WKH N' EDQG WR EH VSHFLILF ZKLOH WKH ORZHU PROHFXODU ZHLJKW EDQGV ZHUH VKRZQ WR EH QRQVSHFLILF 0XOWLSOH OLQHDU UHJUHVVLRQ GLG QRW UHYHDO WKH N' EDQG WR YDU\ EHWZHHQ JURXSV )LJXUH WRS SDQHOf VKRZV JURXS PHDQV s6(0 PHDVXUHG LQ UHODWLYH RSWLFDO GHQVLW\ DFURVV GHYHORSPHQWDO DJH IRU WKH N' EDQG SUHVHQW LQ EUDLQVWHP (VWURJHQ VXOIRWUDQVIHUDVH UHYHDOHG WZR GLVWLQFW EDQGV DW DQG N' IRU EUDLQVWHP )LJXUH VKRZV WKH N' EDQG FRUUHVSRQGLQJ WR WKH FRUUHFW PROHFXODU ZHLJKW RI HVWURJHQ VXOIRWUDQVIHUDVH RI 'DOWRQV 1DVK HW DO f 7KLV VSHFLILFLW\ RI PROHFXODU ZHLJKW ZDV FRQILUPHG E\ SUHDEVRUSWLRQ RI WKH SULPDU\ DQWLERG\ 0XOWLSOH OLQHDU UHJUHVVLRQ GLG QRW UHYHDO DQ\ RI WKH EDQGV WR EH VLJQLILFDQWO\ GLIIHUHQW DPRQJ GHYHORSPHQWDO DJH )LJXUH WRS SDQHOf VKRZV JURXS PHDQV s6(0 PHDVXUHG LQ RSWLFDO GHQVLW\ XQLWV IRU WKH N' HVWURJHQ VXOIRWUDQVIHUDVH EDQG SUHVHQW LQ EUDLQVWHP )RU K\SRWKDODPLF WLVVXH IHWXVHV ODPEV DQG DGXOWV ZHUH XVHG 7KH GHYHORSPHQWDO DJHV ZHUH DV IROORZV GD\V Q f GD\V Q f GD\V Q f GD\V Q f ODPEV GD\V Q f DQG DGXOWV Q f

PAGE 109

:HVWHUQ EORWV DJDLQ UHYHDOHG WKUHH GLVWLQFW EDQGV DW DQG N' IRU HVWURJHQ VXOIDWDVH $JDLQ SUHDEVRUSWLRQ RI WKH HQ]\PH UHYHDOHG RQO\ WKH N' EDQG WR EH VSHFLILF :HVWHUQ EORWV VKRZLQJ WKH N' EDQG IRU RYLQH K\SRWKDODPXV DV ZHOO DV EUDLQVWHP DUH VKRZQ LQ )LJXUH 7KH N' EDQG ZDV QRW VWDWLVWLFDOO\ GLIIHUHQW EHWZHHQ JURXSV E\ PXOWLSOH OLQHDU UHJUHVVLRQ )LJXUH ERWWRP SDQHOf VKRZV JURXS PHDQV s6(0 PHDVXUHG LQ UHODWLYH RSWLFDO GHQVLW\ DFURVV GHYHORSPHQWDO DJH IRU WKH N' EDQG SUHVHQW LQ K\SRWKDODPL (VWURJHQ VXOIRWUDQVIHUDVH UHYHDOHG WKUHH GLVWLQFW EDQGV DW DQG N' IRU K\SRWKDODPL 3UHDEVRUSWLRQ RI WKH HQ]\PH UHYHDOHG RQO\ WKH N' WR EH VSHFLILF 0XOWLSOH OLQHDU UHJUHVVLRQ GLG QRW UHYHDO DQ\ RI WKH EDQGV WR EH VLJQLILFDQWO\ GLIIHUHQW DPRQJ GHYHORSPHQWDO DJH )LJXUH ERWWRP SDQHOf VKRZV JURXS PHDQV s6(0 PHDVXUHG LQ RSWLFDO GHQVLW\ XQLWV IRU WKH N' HVWURJHQ VXOIRWUDQVIHUDVH EDQG SUHVHQW LQ K\SRWKDODPL ,PPXQRKLVWRFKHPLVWU\ 6SHFLILF VWDLQLQJ IRU HVWURJHQ VXOIDWDVH DQG VXOIRWUDQVIHUDVH ZHUH ZLGHVSUHDG WKURXJKRXW WKH K\SRWKDODPXV DQG EUDLQVWHP RI DOO GHYHORSPHQWDO DQG SRVW GHYHORSPHQWDO JURXSV ,PPXQRKLVWRFKHPLFDO UHVXOWV IURP UHJLRQV LPSRUWDQW IRU +3$ D[LV FRQWURO DUH VKRZQ LQ )LJXUHV 6SHFLILF QHXURQDO VWDLQLQJ ZDV VHHQ LQ WKH SDUDYHQWULFXODU QXFOHXV RU 391 RI WKH K\SRWKDODPXV )LJXUHV DQG f WKH QXFOHXV RI WKH WUDFWXV VROLWDULXV RU 176 RI WKH PHGXOOD )LJXUHV DQG f WKH URVWUDO YHQWUDO ODWHUDO PHGXOOD RU 59/0 )LJXUHV DQG f DQG WKH UDSKH QXFOHXV )LJXUH f 'LVFXVVLRQ 7KH UHVXOWV RI WKLV VWXG\ GHPRQVWUDWH WKDW WKHUH LV VLJQLILFDQW HVWURJHQ VXOIDWDVH DFWLYLW\ LQ RYLQH IHWDO K\SRWKDODPXV KLSSRFDPSXV DQG EUDLQVWHP DQG WKDW WKHUH DUH VWDWLVWLFDOO\ VLJQLILFDQW RQWRJHQHWLF FKDQJHV LQ DFWLYLW\ RI WKLV HQ]\PH LQ WKH KLSSRFDPSXV

PAGE 110

$OVR VKRZQ LV WKH SUHVHQFH RI HVWURJHQ VXOIRWUDQVIHUDVH LQ WKH IHWDO K\SRWKDODPXV DQG EUDLQVWHP ,W KDV SUHYLRXVO\ EHHQ GHPRQVWUDWHG WKDW HVWURJHQV LQ IHWDO SODVPD LQFUHDVH ERWK EDVDO DQG VWLPXODWHG IHWDO SODVPD $&7+ VHFUHWLRQ 7KH SUHVHQW UHVXOWV VXJJHVW D PHFKDQLVP E\ ZKLFK WKH PRVW DEXQGDQW IRUP RI HVWURJHQ LQ RYLQH IHWDO SODVPD HVWURQH VXOIDWH PLJKW EH PDGH DYDLODEOH WR DUHDV ZLWKLQ WKH IHWDO EUDLQ NQRZQ WR EH LQYROYHG LQ WKH FRQWURO RI WKH IHWDO +3$ D[LV 0DWKHZ DQG %DODVXEUDPDQLDQ f DQG /DNVKPL DQG %DODVXEUDPDQLDQ f KDYH SUHYLRXVO\ GHPRQVWUDWHG HVWURJHQ VXOIDWDVH DQG VXOIRWUDQVIHUDVH DFWLYLW\ LQ DGXOW VKHHS EUDLQ WLVVXH 2WKHU LQYHVWLJDWRUV KDYH GHPRQVWUDWHG WKHVH HQ]\PDWLF DFWLYLWLHV LQ DGXOW EUDLQ WLVVXH IURP UDWV &RQQROO\ DQG 5HVNR .DZDQR DQG $LNDZD f PLFH +RENLUN f QRQKXPDQ SULPDWHV /DNVKPL DQG %DODVXEUDPDQLDQ f DQG KXPDQ EHLQJV 3ODWLD f +RENLUN DQG FRZRUNHUV GHPRQVWUDWHG WKDW HQ]\PH DFWLYLWLHV DUH WUDQVLHQWO\ LQFUHDVHG SRVWQDWDOO\ LQ WKH EUDLQ RI WKH PRXVH f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

PAGE 111

SUHVHQFH RI PXOWLSOH EDQGV DQG N'f LV QRW VXUSULVLQJ VLQFH WKH SULPDU\ DQWLERG\ XVHG ZDV SRO\FORQDO 7KH N' EDQG EHVW UHSUHVHQWV WKH HQ]\PH ZKLFK KDV D PROHFXODU ZHLJKW RI 'DOWRQV 6WHLQ HW DO f DQG ZDV WKH RQO\ EDQG WKDW ZDV VSHFLILF DV WHVWHG E\ SUHDEVRUSWLRQ 7KLV EDQG ZDV VKRZQ WR EH QRQVLJQLILFDQW DFURVV GHYHORSPHQWDO DJH )LJXUH f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f LQYHVWLJDWHG WKH DFWLYLW\ LQ K\SRWKDODPXV EUDLQVWHP DQG KLSSRFDPSXV EHFDXVH U WKHVH DUHDV DUH NQRZQ WR FRQWDLQ QXFOHL LQYROYHG LQ LQWHJUDWLRQ DIIHUHQW VLJQDO UHOD\ RU QHJDWLYH IHHGEDFN LQKLELWLRQ ZLWKLQ WKH +3 $ D[LV *UL]]OH HW DO .HOOHUZRRG DQG 'DOOPDQ 0DUDQ :DUG f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

PAGE 112

IHHGEDFN DFWLRQV RI FRUWLFRVWHURLGV RQ $&7+ VHFUHWLRQf DQ DFWLRQ RQ WKH 176 ZKLFK UHOD\V QHXUDO WUDIILF IURP YLVFHUDO DIIHUHQWVf RU DQ DFWLRQ RQ DQ\ SDUW RI WKH SDWKZD\V OHDGLQJ IURP WKH 176 WR WKH 391 H J WKH 59/0f (VWURJHQ UHFHSWRUV KDYH EHHQ GHPRQVWUDWHG LQ WKH 176 DQG KLSSRFDPSXV /HKPDQ f :KLOH HVWURJHQ UHFHSWRUV ZLWKLQ WKH K\SRWKDODPXV DUH PRVW FRQFHQWUDWHG LQ WKH DUFXDWH QXFOHXV HVWURJHQ UHFHSWRUV KDYH EHHQ GHPRQVWUDWHG LQ WKH 391 /HKPDQ 6LPHUO\ f 7KH UHVXOWV RI WKH SUHVHQW H[SHULPHQWV LGHQWLI\ WKH FHOOXODU ORFDWLRQ RI WKH VXOIDWDVH DFWLYLW\ ZKLFK LV FRQVLVWHQW ZLWK WKHVH FHQWHUV IRU +3$ D[LV FRQWURO IRXQG ZLGHVSUHDG VWDLQLQJ WKURXJKRXW QXFOHL DQG ILEHU WUDFWV RI WKH K\SRWKDODPXV DQG EUDLQVWHP 1HXURQDO VWDLQLQJ ZDV PXFK PRUH FRQFHQWUDWHG WKDQ ILEHU WUDFW VWDLQLQJ KRZHYHU ERWK ZHUH REVHUYDEOH 6SHFLILFDOO\ ZH IRXQG LQWHQVH QHXURQDO VWDLQLQJ LQ WKH 391 )LJXUH f WKH 176 )LJXUH f WKH 59/0 )LJXUH f DQG WKH GRUVDO UDSKH QXFOHXV )LJXUH f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f LV QRW VXUSULVLQJ VLQFH WKH SULPDU\ DQWLERG\ XVHG ZDV SRO\FORQDO 7KH N' EDQG EHVW

PAGE 113

UHSUHVHQWV WKH HQ]\PH ZKLFK KDV D PROHFXODU ZHLJKW RI 'DOWRQV 1DVK HW DO f DQG ZDV WKH RQO\ EDQG WKDW ZDV VSHFLILF DV WHVWHG E\ SUHDEVRUSWLRQ 7KLV EDQG ZDV VKRZQ WR EH QRQVLJQLILFDQW DFURVV GHYHORSPHQWDO DJH )LJXUH f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f WKH 176 )LJXUH f DQG WKH 59/0 )LJXUH f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f DQG WKDW SK\VLRORJLFDO LQFUHDVHV LQ IHWDO SODVPD DQGURJHQ FRQFHQWUDWLRQV GHFUHDVH WKH VHQVLWLYLW\ RI WKH IHWDO K\SRWKDODPRSLWXLWDU\ XQLW WR QHJDWLYH IHHGEDFN LQKLELWLRQ E\

PAGE 114

FRUWLVRO 6DRXG DQG :RRG f 7KHUHIRUH WKH LQFUHDVHV LQ IHWDO SODVPD HVWURJHQ DQG DQGURJHQ FRQFHQWUDWLRQV WKHPVHOYHV LQ SDUW D IXQFWLRQ RI IHWDO +3$ D[LV DFWLYLW\ IXUWKHU DXJPHQW IHWDO $&7+ VHFUHWLRQ K\SRWKHVL]H WKDW SDUWXULWLRQ UHVXOWV IURP WKH RQVHW RI DQ K\SRWKDODPLF GULYH WR $&7+ VHFUHWLRQ ZLWK LQWHUDFWLRQ EHWZHHQ WKH DGUHQDO FRUWH[ SODFHQWD DQG K\SRWKDODPXV SURGXFLQJ D SRVLWLYH IHHGEDFN F\FOH ZKLFK XOWLPDWHO\ FRQFOXGHV ZLWK WKH VHSDUDWLRQ RI SODFHQWD IURP WKH IHWDO +3$ D[LV SDUWXULWLRQf 7KLV VWXG\ VXJJHVWV WKDW WKH LQIOXHQFH RI HVWURJHQV RQ +3$ D[LV DFWLYLW\ FRXOG EH H[SUHVVHG HDUOLHU WKDQ ZRXOG EH SUHGLFWHG RQ WKH EDVLV RI FKDQJHV LQ SODVPD FRQFHQWUDWLRQV RI XQFRQMXJDWHG IRUPV

PAGE 115

F f 2 4 f§A R R & ( $ R e &/ t t \ $ 9 %UDLQVWHP ?O t r )LJXUH (VWURQH VXOIDWDVH DFWLYLW\ LQ RYLQH KLSSRFDPSXV WRS SDQHOf K\SRWKDODPXV PLGGOH SDQHOf DQG EUDLQVWHP ERWWRP SDQHOf )RU DOO UHDFWLRQV YHORFLW\ LV H[SUHVVHG DV SPRO HVWURQH IRUPHG SHU PLQXWH SHU PJ SURWHLQ 6XEVWUDWH FRQFHQWUDWLRQ LQ DOO UHDFWLRQV ZDV S0 'DWD DUH UHSUHVHQWHG DV PHDQV s6(0 Q SHU JURXSf

PAGE 116

6XOIDWDVH $FWLYLW\ SPRO PLQ PJ WLVVXHf )LJXUH (VWURQH VXOIDWDVH DFWLYLW\ LQ RYLQH P\RPHWULXP Q f DQG K\SRWKDODPXV Q f 7KH DFWLYLW\ LV H[SUHVVHG DV SPRO HVWURQH IRUPHG SHU PLQXWH SHU PJ WLVVXH ZHW ZHLJKW 6XEVWUDWH FRQFHQWUDWLRQ LQ DOO UHDFWLRQV ZDV S0

PAGE 117

)LJXUH :HVWHUQ EORWV VKRZLQJ WKH N' EDQG RI HVWURJHQ VXOIDWDVH IRU RYLQH K\SRWKDODPL DQG EUDLQVWHPV 7KH QXPEHU EHORZ HDFK EDQG GHVLJQDWHV GHYHORSPHQWDO DJH LQ QXPEHU RI GD\V / ODPE $ DGXOW HZHf

PAGE 118

5HODWLYH 2SWLFDO 'HQVLW\ 8QLWV ODPEV DGXOWV +\SRWKDODPXV ODPEV DGXOWV 'HYHORSPHQWDO $JH )LJXUH (VWURJHQ VXOIDWDVH N' EDQGf LQ RYLQH EUDLQVWHP WRS SDQHOf DQG LQ RYLQH K\SRWKDODPXV ERWWRP SDQHOf %DUV UHSUHVHQW PHDQV 6(0 RI GHVLJQDWHG DJH JURXSV IURP ZHVWHUQ EORW DQDO\VHV SORWWHG DV UHODWLYH RSWLFDO GHQVLW\ XQLWV Q SHU JURXSf

PAGE 119

+\SRWKDODPXV f§ f§ f§ P f§r f§ rr J L / / $ +\SRWKDODPXV f§ r f§ / / $ %UDLQVWHP f§ IHMMM n rre O / $ %UDLQVWHP f§ / / $ )LJXUH :HVWHUQ EORWV VKRZLQJ WKH N' EDQG RI HVWURJHQ VXOIRWUDQVIHUDVH IRU RYLQH K\SRWKDODPL DQG EUDLQVWHPV 7KH QXPEHU EHORZ HDFK EDQG GHVLJQDWHV GHYHORSPHQWDO DJH LQ QXPEHU RI GD\V / ODPE $ DGXOW HZHf

PAGE 120

ODPEV DGXOWV L U ODPEV DGXOWV 'HYHORSPHQWDO $JH )LJXUH (VWURJHQ VXOIRWUDQVIHUDVH N' EDQGf LQ RYLQH EUDLQVWHP WRS SDQHOf DQG LQ RYLQH K\SRWKDODPXV ERWWRP SDQHOf %DUV UHSUHVHQW PHDQV 6(0 RI GHVLJQDWHG DJH JURXSV IURP ZHVWHUQ EORW DQDO\VHV SORWWHG DV UHODWLYH RSWLFDO GHQVLW\ XQLWV Q SHU JURXSf

PAGE 121

)LJXUH 3KRWRPLFURJUDSKV RI QHXURQDO HVWURJHQ VXOIDWDVH VWDLQLQJ LQ WKH IHWDO RYLQH 391 $ ; % ;f

PAGE 122

)LJXUH 3KRWRPLFURJUDSKV RI QHXURQDO HVWURJHQ VXOIDWDVH VWDLQLQJ LQ WKH IHWDO RYLQH 176 $ ; % ;f

PAGE 123

)LJXUH 3KRWRPLFURJUDSK RI QHXURQDO HVWURJHQ VXOIDWDVH VWDLQLQJ LQ WKH IHWDO RYLQH 59/0 ;f

PAGE 124

)LJXUH 3KRWRPLFURJUDSK RI HVWURJHQ VXOIDWDVH VWDLQLQJ LQ WKH IHWDO RYLQH UDSKH QXFOHXV ;f

PAGE 125

)LJXUH 3KRWRPLFURJUDSKV RI QHXURQDO HVWURJHQ VXOIRWUDQVIHUDVH LQ WKH IHWDO RYLQH 391 $ ; % ;f

PAGE 126

)LJXUH 3KRWRPLFURJUDSKV RI QHXURQDO HVWURJHQ VXOIRWUDQVIHUDVH VWDLQLQJ LQ WKH IHWDO RYLQH 176 $ ; % ;f

PAGE 127

)LJXUH 3KRWRPLFURJUDSK RI QHXURQDO HVWURJHQ VXOIRWUDQVIHUDVH LQ WKH IHWDO RYLQH 59/0 ;f

PAGE 128

&+$37(5 6800$5< $1' &21&/86,216 7KLV JRDO RI WKLV GLVVHUWDWLRQ ZDV WR VKHG OLJKW RQ WKH PHFKDQLVP RI SDUWXULWLRQ DV ZHOO DV UHYHDO KRZ DQ RYLQH IHWXV GHIHQGV LWVHOI DJDLQVW K\SRWHQVLRQ 7KLV ZDV DFFRPSOLVKHG E\ XVLQJ D FRPELQDWLRQ RI LQ YLYR ZKROH DQLPDOf DQG LQ YLWUR LPPXQRKLVWRFKHPLVWU\f WHFKQLTXHV ,Q WU\LQJ WR DFFRPSOLVK WKLV DLP WZR PDLQ K\SRWKHVHV ZHUH GHYHORSHG +\SRWKHVLV 7KH DFWLRQV RI HVWURJHQ RQ IHWDO FDUGLRYDVFXODU UHIOH[ UHVSRQVLYHQHVV WR K\SRWHQVLRQ ZLOO EH PHDVXUDEOH LQ LQWDFW IHWXVHV EXW QRW LQ EDUR DQG FKHPRGHQHUYDWHG IHWXVHV 5HVHDUFK KDV SUHYLRXVO\ GHPRQVWUDWHG WKDW WKH FRPELQHG EDUR DQG FKHPR GHQHUYDWLRQ DWWHQXDWHV DSSUR[LPDWHO\ bf WKH UHIOH[ KRUPRQDO DQG KHPRG\QDPLF UHVSRQVHV WR PRGHUDWH bf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

PAGE 129

DUHDV PRGLILHG E\ HVWURJHQfV DFWLRQ DQG EDUR DQG FKHPR GHQHUYDWLRQ 7KH XVH RI WKHVH WHFKQLTXHV DOORZV IRU WKH PHDVXUHPHQW RI FIRV H[SUHVVLRQ WKH SURWHLQ SURGXFW RI WKH HDUO\ UHVSRQVH JHQH ZKLFK FDQ EH XVHG DV D PDUNHU RI QHXURQDO DFWLYLW\ SURSRVHG WKDW HVWUDGLRO LPSODQWHG DQLPDOV ZLOO KDYH PRUH )RV DFWLYLW\ DQG KHQFH PRUH FIRV VWDLQLQJ LQ UHOHYDQW EUDLQ DUHDV 176 391 HWFf DV FRPSDUHG WR FRQWURO DQLPDOV )XUWKHUPRUH LW LV H[SHFWHG WKDW FIRV VWDLQLQJ ZRXOG EH QHJOLJLEOH LQ GHQHUYDWHG DQLPDOV )LQDOO\ FIRV VWDLQLQJ VKRXOG EH VLJQLILFDQWO\ HOHYDWHG LQ DOO K\SRWHQVLYH DQLPDOV DV FRPSDUHG WR r QRUPRWHQVLYH DQLPDOV ,Q DGGLWLRQ WR WKH FIRV LPPXQRKLVWRFKHPLFDO VWXGLHV WKH DFWLRQ RI HVWURJHQ VXOIDWDVH DQG HVWURJHQ VXOIRWUDQVIHUDVH ZDV LQYHVWLJDWHG 6LQFH WKH LQFUHDVH LQ FRQFHQWUDWLRQ RI HVWURJHQ VXOIDWH SUHFHGHV WKH LQFUHDVH LQ +3$ D[LV DFWLYLW\ 1DWKDQLHOV] HW DO f DQG VLQFH FRQMXJDWHG HVWURJHQV FLUFXODWH LQ PXFK KLJKHU FRQFHQWUDWLRQV WKDQ XQFRQMXJDWHG HVWURJHQV &DUQHJLH DQG 5REHUWVRQ 7VDQJ f K\SRWKHVL]HG WKDW WKHVH HQ]\PHV ZRXOG EH SUHVHQW LQ EUDLQ DUHDV LPSRUWDQW IRU +3$ D[LV FRQWURO ,I WUXH D ORFDO PHFKDQLVP ZRXOG EH LQ SODFH IRU FRQYHUVLRQ RI ELRORJLFDOO\ LQDFWLYH WR DFWLYH HVWURJHQ $ WRWDO RI SUHJQDQW HZHV ZHUH VWXGLHG SHU H[SHULPHQWDO JURXSf $QLPDOV ZHUH UDQGRPO\ DVVLJQHG WR WKH IROORZLQJ JURXSV SODFHER LPSODQW LQWDFW VKDPGHQHUYDWHGf QRUPRWHQVLYH IHWXVHV SODFHER LPSODQW FDURWLG VLQXV GHQHUYDWHG QRUPRWHQVLYH IHWXVHV SODFHER LPSODQW LQWDFW VKDPGHQHUYDWHGf IHWXVHV VXEMHFWHG WR K\SRWHQVLRQ SODFHER LPSODQW FDURWLG VLQXV GHQHUYDWHG IHWXVHV VXEMHFWHG WR K\SRWHQVLRQ

PAGE 130

HVWUDGLRO LPSODQW PJ GD\ UHOHDVHf LQWDFW VKDPGHQHUYDWHGf QRUPRWHQVLYH IHWXVHV HVWUDGLRO LPSODQW PJ GD\ UHOHDVHf FDURWLG VLQXV GHQHUYDWHG QRUPRWHQVLYH IHWXVHV HVWUDGLRO LPSODQW PJ GD\ UHOHDVHf LQWDFW VKDPGHQHUYDWHGf IHWXVHV VXEMHFWHG WR K\SRWHQVLRQ HVWUDGLRO LPSODQW PJ GD\ UHOHDVHf FDURWLG VLQXV GHQHUYDWHG IHWXVHV VXEMHFWHG WR K\SRWHQVLRQ 8QGHUVWDQGLQJ WKH PHFKDQLVP RI WKH LQFUHDVHG IHWDO +3$ D[LV DW WKH HQG RI JHVWDWLRQ LV NH\ WR XQGHUVWDQGLQJ WKH PHFKDQLVP RI VSRQWDQHRXV SDUWXULWLRQ LQ VKHHS 7KH LQ YLYR H[SHULPHQWV ZHUH FRQGXFWHG WR VHH LI HVWUDGLRO KDV LWf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

PAGE 131

GHQHUYDWLRQ E\ LWVHOI KRZHYHU FDXVHG QR +3$ D[LV DFWLYLW\ RU DXJPHQWDWLRQ GHHPLQJ WKH VXUJLFDO HOLPLQDWLRQ RI EDURUHFHSWRU FKHPRUHFHSWRU UHVSRQVLYHQHVV DQ DSSURSULDWH DQG HIIHFWLYH FRQWURO 5HVHDUFK KDV SUHYLRXVO\ GHPRQVWUDWHG WKDW WKH FRPELQHG EDUR DQG FKHPR GHQHUYDWLRQ DWWHQXDWHV DSSUR[LPDWHO\ bf WKH UHIOH[ KRUPRQDO DQG KHPRG\QDPLF UHVSRQVHV WR PRGHUDWH bf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b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

PAGE 132

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bf WKH UHIOH[ KRUPRQDO DQG KHPRG\QDPLF UHVSRQVHV WR PRGHUDWH bf UHGXFWLRQ LQ DUWHULDO EORRG SUHVVXUH 7KLV PHDQV WKDW b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f ZLWKLQ DUHDV RI WKH EUDLQ WKDW DUH UHVSRQVLEOH IRU +3$ D[LV FRQWURO 2WKHU VWXGLHV KDYH SRLQWHG WR D UROH RI WKURPER[DQH $ 7[$f LQ WKLV SURFHVV 7[$ DFWV ZLWKLQ WKH DUHD SHUIXVHG E\ WKH FHUHEUDO YDVFXODWXUH WR VWLPXODWH $&7+ VHFUHWLRQ LQ WKH IHWXV :RRG HW DO f ZKLFK VXJJHVWV WKDW ORFDO JHQHUDWLRQ RI 7[$ ZRXOG HIIHFWLYHO\ VWLPXODWH WKH +3$ D[LV :KDWHYHU WKH PHFKDQLVP LW LV REYLRXV

PAGE 133

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f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

PAGE 134

DW WKH DQWHULRU SLWXLWDU\ WR FDXVH $&7+ UHOHDVH /HKPDQ HW DO 3RPHUDQW] DQG 6KROO f 7KH 176 LV WKH ILUVW V\QDSVH SRLQW LQ WKH SDWKZD\ FRQQHFWLQJ WKH DIIHUHQW EDURUHFHSWRUV ZLWK WKH 391 7KH 59/0 LV D FDUGLRYDVFXODU UHJXODWRU\ FHQWHU ZKLFK FRRUGLQDWHV LQIRUPDWLRQ IURP WKH SHULSKHU\ ,W KDV EHHQ VKRZQ WKDW DOO RI WKHVH EUDLQ UHJLRQV KDYH HVWURJHQ UHFHSWRUV HQDEOLQJ WKHP WR UHVSRQG WR HVWUDGLRO WUHDWPHQW SULRU WR EUDFKLRFHSKDOLF RFFOXVLRQ /HKPDQ HW DO 6LPHUO\ HW DO f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f 3HUKDSV WKH DQVZHU WR WKLV GLVFUHSDQF\ OLHV LQ IDFW WKDW WKH KLSSRFDPSXV LV D FODVVLF VLWH RI FRUWLVRO QHJDWLYH IHHGEDFN :RRG KDV IXUWKHU VKRZQ WKDW WKH +3$ D[LV LV LQVHQVLWLYH WR LQKLELWLRQ YLD FRUWLVRO QHJDWLYH IHHGEDFN WRZDUGV WKH HQG RI JHVWDWLRQ f 3RVVLEO\ WKH KLSSRFDPSXV LV XQUHVSRQVLYH WR DQ HVWUDGLRO DXJPHQWDWLRQ RI $&7+ VHFUHWLRQ DW WKLV WLPH

PAGE 135

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

PAGE 136

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

PAGE 137

RI WKH EUDLQ DUHDV PRGLILHG E\ HVWUDGLRO WKXV DOORZLQJ IRU DQ DXJPHQWHG +3$ D[LV UHVSRQVH 7KLV VWXG\ XVHG DQ DUWLILFLDO VWLPXOXV WR LQFUHDVH +3$ D[LV DFWLYLW\ KRZHYHU LW LV QRW GLVSXWHG WKDW WKH SDWKZD\ GHVFULEHG LV LQ IDFW LQYROYHG LQ WULJJHULQJ SDUWXULWLRQ ,W KDV UHFHQWO\ EHHQ GHPRQVWUDWHG WKDW SK\VLRORJLFDO LQFUHDVHV LQ IHWDO SODVPD HVWURJHQ FRQFHQWUDWLRQV JUHDWO\ DXJPHQW IHWDO $&7+ VHFUHWLRQ 6DRXG DQG :RRG f 7KLV HIIHFW RI HVWURJHQ FDQ EH GHPRQVWUDWHG RQ ERWK EDVDO DQG K\SRWHQVLRQ VWLPXODWHG IHWDO $&7+ VHFUHWLRQ :KLOH HVWURJHQ KDV D SRWHQWLDOO\ LPSRUWDQW HIIHFW RQ IHWDO $&7+ VHFUHWLRQ LW LV ZHOONQRZQ WKDW IHWDO SODVPD FRQFHQWUDWLRQV RI XQFRQMXJDWHG HVWURJHQV LQFUHDVH RQO\ DIWHU WKH EHJLQQLQJ RI WKH LQFUHDVH LQ IHWDO SODVPD $&7+ DQG FRUWLVRO 1DWKDQLHOV] HW DO 6WURWW HW DO f &RQMXJDWHG HVWURJHQV PRVWO\ HVWURQH VXOIDWH FLUFXODWH LQ KLJK FRQFHQWUDWLRQV FRPSDUHG WR XQFRQMXJDWHG HVWURJHQV &DUQHJLH DQG 5REHUWVRQ 7VDQJ f 7KH FRQFHQWUDWLRQ RI HVWURQH VXOIDWH LQFUHDVHV EHIRUH WKH LQFUHDVH LQ IHWDO +3$ D[LV DFWLYLW\ 1DWKDQLHOV] HW DO f +RZHYHU FRQMXJDWHG I VWHURLGV FDQQRW ELQG WR QXFOHDU UHFHSWRUV XQOHVV GHFRQMXJDWHG +RENLUN f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

PAGE 138

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f LQYHVWLJDWHG WKH DFWLYLW\ LQ K\SRWKDODPXV EUDLQVWHP DQG KLSSRFDPSXV EHFDXVH WKHVH DUHDV DUH NQRZQ WR FRQWDLQ QXFOHL LQYROYHG LQ LQWHJUDWLRQ DIIHUHQW VLJQDO UHOD\ RU QHJDWLYH IHHGEDFN LQKLELWLRQ ZLWKLQ WKH +3$ D[LV *UL]]OH HW DO .HOOHU:RRG DQG 'DOOPDQ 0DUDQ :DUG f 7KH SUHVHQFH RI DFWLYLW\ LQ DQ\ RI WKHVH DUHDV FRXOG EH LPSRUWDQW IRU WKH GHFRQMXJDWLRQ RI VXOIDWHG HVWURJHQV LQ WKH EORRG SHUIXVLQJ WKH EUDLQ 5RVHQIHOG HW DO LQ UHSRUWHG WKDW WKH PDMRULW\ RI HVWURJHQ SURGXFHG E\ WKH RYLQH SODFHQWD LV VXOIRFRQMXJDWHG DQG WKXV SURWHFWHG VLQFH VXOIDWDVH LQ QRW SUHVHQW 0\ GDWD VXJJHVW RWKHUZLVH JLYHQ WKDW VXOIRFRQMXJDWHV LQ WKH IHWDO FRPSDUWPHQW PD\ KDYH

PAGE 139

VSHFLILF UHJLRQDO UROHV 7KH HIIHFW RI HVWURJHQ RQ ERWK EDVDO DQG K\SRWHQVLRQ VWLPXODWHG FRQFHQWUDWLRQV RI $&7+ FRXOG EH WKH UHVXOW RI DQ DFWLRQ RI HVWURJHQ RQ WKH 391 LQ WKH K\SRWKDODPXV DQ DFWLRQ RQ WKH KLSSRFDPSXV ZKLFK PHGLDWHV VRPH RI WKH QHJDWLYH IHHGEDFN DFWLRQV RI FRUWLFRVWHURLGV RQ $&7+ VHFUHWLRQf DQ DFWLRQ RQ WKH 176 ZKLFK UHOD\V QHXUDO WUDIILF IURP YLVFHUDO DIIHUHQWVf RU DQ DFWLRQ RQ DQ\ SDUW RI WKH SDWKZD\V OHDGLQJ IURP WKH 176 WR WKH 391 H J WKH 59/0f (VWURJHQ UHFHSWRUV KDYH EHHQ GHPRQVWUDWHG LQ WKH 176 DQG KLSSRFDPSXV /HKPDQ f :KLOH HVWURJHQ UHFHSWRUV ZLWKLQ WKH K\SRWKDODPXV DUH PRVW FRQFHQWUDWHG LQ WKH DUFXDWH QXFOHXV HVWURJHQ UHFHSWRUV KDYH EHHQ GHPRQVWUDWHG LQ WKH 391 /HKPDQ 6LPHUO\ f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

PAGE 140

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f DQG WKDW SK\VLRORJLFDO LQFUHDVHV LQ IHWDO SODVPD DQGURJHQ FRQFHQWUDWLRQV GHFUHDVH WKH VHQVLWLYLW\ RI WKH IHWDO K\SRWKDODPR SLWXLWDU\ XQLW WR QHJDWLYH IHHGEDFN LQKLELWLRQ E\ FRUWLVRO 6DRXG DQG :RRG f 7KHUHIRUH WKH LQFUHDVHV LQ IHWDO SODVPD HVWURJHQ DQG DQGURJHQ FRQFHQWUDWLRQV WKHPVHOYHV LQ SDUW D IXQFWLRQ RI IHWDO +3$ D[LV DFWLYLW\ IXUWKHU DXJPHQW IHWDO $&7+ VHFUHWLRQ K\SRWKHVL]H WKDW SDUWXULWLRQ UHVXOWV IURP WKH RQVHW RI DQ K\SRWKDODPLF GULYH WR $&7+ VHFUHWLRQ ZLWK LQWHUDFWLRQ EHWZHHQ DGUHQDO SODFHQWD DQG K\SRWKDODPXV SURGXFLQJ D SRVLWLYH IHHGEDFN F\FOH ZKLFK XOWLPDWHO\ FRQFOXGHV ZLWK WKH VHSDUDWLRQ RI SODFHQWD IURP WKH IHWDO +3$ D[LV SDUWXULWLRQf 7KLV VWXG\ VXJJHVWV WKDW WKH LQIOXHQFH RI HVWURJHQV RQ

PAGE 141

+3$ D[LV DFWLYLW\ FRXOG EH H[SUHVVHG HDUOLHU WKDQ ZRXOG EH SUHGLFWHG RQ WKH EDVLV RI FKDQJHV LQ SODVPD FRQFHQWUDWLRQV RI XQFRQMXJDWHG IRUPV 3UHWHUP ODERU LV VWLOO D PDMRU FRQFHUQ LQ WKLV GD\ DQG DJH 'HVSLWH HIIRUWV WR LQIRUP WKH SXEOLF DERXW SURSHU QXWULWLRQ DQG WKH QHHG WR VHHN SUHQDWDO FDUH D VLJQLILFDQW QXPEHU RI LQIDQWV DUH EHLQJ ERP SUHPDWXUHO\ $SSUR[LPDWHO\ b RI DOO GHOLYHULHV LQ 1RUWK $PHULFD DQG WKH 8QLWHG .LQJGRP RFFXU SUHPDWXUHO\ EHIRUH ZHHNV JHVWDWLRQ 7KLV ILJXUH FDQ EH KLJKHU RU ORZHU GHSHQGLQJ XSRQ WKH GHYHORSPHQWDO VWDWXV RI WKH FRXQWU\ DQG WKH VRFLRHFRQRPLF VWDWXV RI WKH PRWKHU &RPSOLFDWLRQV GXH WR SUHPDWXUH GHOLYHU\ DQG LPPDWXULW\ RI WKH QHRQDWH FRQWULEXWH VLJQLILFDQWO\ WR WKH OLIH RU GHDWK RI WKHVH LQIDQWV &UHDV\ +DOO f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

PAGE 142

ODERU DV ZHOO DV D YDULHW\ RI SUREOHPV WKDW DULVH GXH WR SUHPDWXUH SDUWXULWLRQ IRU OLYLQJ RIIVSULQJ LH VXUIDFWDQW SURGXFWLRQf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

PAGE 143

1XFOHXV RI WKH 7UDFWXV 6ROLWDULXV +\SRWKDODPXV 3DUDYHQWULFXODU 1XFOHXVf &RUWLVRO 3LWXLWDU\ %DUR&KHPRUHFHSWRUV &DURWLG VLQXV DQG $RUWLF DUFKf $&7+ $GUHQDO &RUWH[ )LJXUH 6FKHPDWLF RI WKH EDURUHFHSWRUFKHPRUHFHSWRU DIIHUHQW SDWKZD\ DQG WKH +3 $ D[LV 7KLV V\VWHP PRQLWRUV EORRG SUHVVXUH RQ D PLQXWH WR PLQXWH EDVLV +\SRWHQVLRQ ZLOO EH VHQVHG DW WKH OHYHO RI WKH EDURUHFHSWRUV DV D GHFUHDVH LQ WKH UDWH RI ILULQJ 7KLV ZLOO DFWLYDWH WKH +3$ D[LV LQ RUGHU WR UHWXUQ EORRG SUHVVXUH WR D KRPHRVWDWLF OHYHO

PAGE 144

)LJXUH %DURUHFHSWRU UHIOH[ SDWKZD\ VKRZLQJ SRVVLEOH VLWHV RI HVWUDGLRO DFWLRQ rf DV PHDVXUHG E\ DQ LQFUHDVH LQ FIRV JHQHUDWLRQ +\SRWHQVLRQ EUDFKLRFHSKDOLF RFFOXVLRQf DFWLYDWHV WKH +3$ D[LV GXH WR D GHFUHDVH LQ WKH UDWH RI ILULQJ RI WKH EDURUHFHSWRUV 3UHWUHDWPHQW ZLWK HVWUDGLRO DOORZV IRU DQ DXJPHQWHG KRUPRQDO UHVSRQVH LQ WKH LQWDFW IHWXV &DURWLG VLQXV GHQHUYDWLRQ HOLPLQDWHV WKLV HQKDQFHG UHVSRQVH

PAGE 145

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fLQWUDWKRUDFLFf SUHVVXUH RQ DRUWLF DUFK EDURUHFHSWRUV 3K\VLRO /RQG $QWRORYLFK *& &ODUNH ,0F0LOOHQ ,& 3HUU\ 5$ 5RELQVRQ 30 6LOYHU 0 DQG
PAGE 146

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ff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

PAGE 147

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

PAGE 148

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

PAGE 149

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

PAGE 150

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
PAGE 151

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f UHFHSWRUV E\ &5) ,QWHUDFWLRQ ZLWK YDVRSUHVVLQ (QGRFULQRO +DXJHU 5/ 0LOODQ 0$ &DWW .DQG $JXLOHUD 'LIIHUHQWLDO UHJXODWLRQ RI EUDLQ DQG SLWXLWDU\ FRUWLFRWURSLQUHOHDVLQJ IDFWRU UHFHSWRUV E\ FRUWLFRVWHURQH (QGRFULQRO

PAGE 152

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

PAGE 153

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
PAGE 154

/DNVKPL 6 DQG $ 6 %DODVXEUDPDQLDQ 6WXGLHV RQ WKH FKDRWURSLFDOO\ VROXELOL]HG DU\OVXOIDWDVH & DQG HVWURQH VXOIDWDVH RI VKHHS EUDLQ %LRFKHP%LRSK\V$FWD /DNVKPL 6 DQG %DODVXEUDPDQLDQ $ 6 7KH GLVWULEXWLRQ RI HVWURQH VXOSKDWDVH GHK\GURHSLDQGURVWHURQH VXOSKDWDVH DQG DU\OVXOSKDWDVH & LQ WKH SULPDWH 0DFDFD UDGLDWDf EUDLQ DQG SLWXLWDU\ -1HXURFKHP /DXEHU 0 &ODYUHXO & 9DXGU\ + DQG &RKHQ 3 ,PPXQRORJLFDO GHWHFWLRQ RI SUR FRUWLFRWURSLQ UHOHDVLQJ IDFWRU &5)f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n & $QWRQL )$ DQG 3DONRYLWV 0 8OWUDVWUXFWXUDO GHPRQVWUDWLRQ RI RYLQH &5) OLNH LPPXQRUHDFWLYLW\ R&5)/,f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

PAGE 155

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f (YLGHQFH WKDW SURWHLQ NLQDVH & PHGLDWHV WKH DFWLRQ RI $ 93 -%LRO&KHP /RQJR / 0DWHUQDO EORRG YROXPH DQG FDUGLDF RXWSXW GXULQJ SUHJQDQF\ D K\SRWKHVLV RI HQGRFULQRORJLF FRQWURO $P 3K\VLRO 55 /\H 66SUDJXH &/ 0LWFKHOO %) DQG &KDOOLV -5* $FWLYDWLRQ RI RYLQH IHWDO DGUHQDO IXQFWLRQ E\ SXOVDWLOH RU FRQWLQXRXV DGPLQLVWUDWLRQ RI DGUHQRFRUWLFRWURSLQ f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

PAGE 156

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

PAGE 157

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f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

PAGE 158

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

PAGE 159

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f UHVSRQVHV WR K\SRWHQVLRQ LQ ODPE IHWXVHV $P 3K\VLRO ( ( 5RVH -& 0HLV 38UEDQ 5% DQG *UHLVV -U )& ,Q YLYR HYLGHQFH IRU LQFUHDVHG DGUHQDO VHQVLWLYLW\ WR DGUHQRFRUWLFRWURSLQOf LQ WKH ODPE IHWXV ODWH LQ JHVWDWLRQ (QGRFULQRO 5RVH -& 0RUULV 0 DQG 0HLV 3+HPRUUKDJH LQ QHZERUQ ODPEV (IIHFWV RQ DUWHULDO EORRG SUHVVXUH $&7+ FRUWLVRO DQG YDVRSUHVVLQ $P 3K\VLRO ( (

PAGE 160

5RVHQIHOG & 5 :RUOH\ 50LOHZLFK / *UDQW 1) DQG 3DUNHU -U 5 2YLQH )HWRSODFHQWDO VXOIRFRQMXJDWLRQ DQG DURPDWL]DWLRQ RI GHK\GURHSLDQGURVWHURQH (QGRFULQRO 5RVVLHU DQG 3LHUUHSRLQW &* 2HVWURJHQ PHWDEROLVP LQ VKHHS P\RPHWULXP 5HSURG )HUW 5XGROSK $ 0 &RQJHQLWDO GLVHDVHV RI WKH KHDUW &KLFDJR
PAGE 161

6FKZDUW] %LOOHVWUXS 1 3HUULQ 0 5LYLHU DQG 9DOH : ,GHQWLILFDWLRQ RI FRUWLFRWURSLQUHOHDVLQJ IDFWRU &5)f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

PAGE 162

7DNHVKLWD $ 0DUN $/ (FNEHUJ '/ DQG $EERXG )0 (IIHFW RI FHQWUDO YHQRXV SUHVVXUH RQ DUWHULDO EDURUHIOH[ FRQWURO RI KHDUW UDWH $P 3K\VLRO +HDUW &LUF 3K\VLROf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
PAGE 163

:KLWQDOO 0+ 0H]H\ ( DQG *DLQHU + &RORFDOL]DWLRQ RI FRUWLFRWURSLQUHOHDVLQJ IDFWRU DQG YDVRSUHVVLQ LQ PHGLDQ HPLQHQFH QHXURVHFUHWRU\ YHVLFOHV 1DWXUH :KLWQDOO 0+ 6P\WK DQG *DLQHU + 9DVRSUHVVLQ FRH[LVWV LQ KDOI RI WKH FRUWLFRWURSLQUHOHDVLQJ IDFWRU D[RQV SUHVHQW LQ WKH H[WHUQDO ]RQH RI WKH PHGLDQ HPLQHQFH LQ QRUPDO UDWV 1HXURHQGRFULQRO :LQHU %6WDWLVWLFDO 3ULQFLSOHV LQ ([SHULPHQWDO 'HVLJQ 1HZ
PAGE 164

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n UHOHDVLQJ IDFWRU &5)f UHFHSWRUV LQ WKH UDW SLWXLWDU\ JODQG (IIHFWV RI DGUHQDOHFWRP\ RQ &5) UHFHSWRUV DQG FRUWLFRWURSK UHVSRQVHV (QGRFULQRO :\QQ 3& +DUZRRG -3 &DWW .DQG $JXLOHUD &RUWLFRWURSLQUHOHDVLQJ IDFWRU &5)f LQGXFHV GHVHQVLWL]DWLRQ RI WKH UDW SLWXLWDU\ &5) UHFHSWRUDGHQ\ODWH F\FODVH FRPSOH[ (QGRFULQRO
PAGE 165

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

PAGE 166

, FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSWDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH LQ VFRSH DQG TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ &KDUOHV ( :RRG &KDLU 3URIHVVRU RI 3K\VLRORJ\ FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSWDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH LQ VFRSH DQG TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ nOO M$-8 L n 0DXUHHQ .HOOHU:RRG $VVRFLDWH 3URIHVVRU RI 3KDUPDFRG\QDPLFV FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSWDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH LQ VFRSH DQG TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ 3XVKSD 6 .DOUD 3URIHVVRU RI 3K\VLRORJ\ f FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSIDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH LQ VFRSH DQG TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ r -JISHV : 6LPSNLQV URIHVVRU RI 3KDUPDFRG\QDPLFV 7KLV GLVVHUWDWLRQ ZDV VXEPLWWHG WR WKH *UDGXDWH )DFXOW\ RI WKH &ROOHJH RI 0HGLFLQH DQG WR WKH *UDGXDWH 6FKRRO DQG ZDV DFFHSWHG DV SDUWLDO IXOILOOPHQW RI WKH UHTXLUHPHQWV IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ 0D\ &VW 'HDQ &ROOHJH RI 0HGLFLQH 'HDQ *UDGXDWH 6FKRRO

PAGE 167

81,9(56,7< 2) )/25,'$