<%BANNER%>

Reduction in pre-tetinal neovascularization by ribozymes that cleave the A2b receptor mRNA

University of Florida Institutional Repository

PAGE 1

REDUCTION IN PRE-RETINAL NEOVASCULARIZATION BY RIBOZYMES THAT CLEAVE THE A2B RECEPTOR mRNA By AQEELA AFZAL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2003

PAGE 2

To my son, Faris Wasim.

PAGE 3

iii ACKNOWLEDGMENTS It is a pleasure to thank the many people who have made this dissertation possible. Many people have been a part of my gra duate education as friends, teachers and colleagues. My mentor, Maria Grant, has been al l of those. It is difficult to overstate my gratitude for her. She has in stilled in me the qua lities of being a good scientist. Her infectious enthusiasm for clinical research has been a major driving force during my career at the University of Flor ida. This dissertation is a sm all tribute to an exceptional woman from a student who is sti ll anxious to learn from her. My sincerest thanks are also due to L ynn Shaw. He patiently taught me all the techniques I needed to complete my work. He also spent countless hours editing and doing the graphics for this dissertation. His insightful comments were crucial for editing the many drafts into the final dissertation. My thanks are also due to Polyxenie E. Spoerri who taught me all th e tissue culture tech niques I needed to complete this dissertation. Thanks also to Sergio Ca ballero, Rehae Miller a nd past and present members of the Grant lab: Tom Ruzich, Nilanjana Sengupta, Christopher Beadle, Hao Pan I would like to thank my committee members: Dr. Don. A. Samuelson (Professor of Veterinary Medicine); Dr. Dennis. E. Brooks (Professor of Veterinary Medicine); Dr. John. B. Dame (Professor of Veterinary Medicine); Dr. Donald. A. Armstrong, Dr. Elizabeth C. Uhl (Clinical Assistant Professo r of Veterinary Medicine) and Dr. Harm J.

PAGE 4

iv Knot (Assistant Professor of Pharmacology and Therapeutics) for their guidance over the years. My son, Faris Wasim, has been a great s ource of inspiration. Being tired of not being able to fulfill his requests when I wanted to and missing him has been the best motivation for completing this dissertation. My husband, Wasim Asghar, has also shared this exciting journey with me. He has provided constant supp ort and encouragement throughout my graduate career. A very special thanks to the two people to whom I owe everything I am today, my parents, Mohammed Afzal and Mussarat Afzal Their unwavering faith and confidence in my ability and in me is what has shaped me to be the person I am today. Thank-you for everything. My thanks are also due to my sister, Aneela Afzal, and brother Yaseen Afzal, for their support and c ountless hours of babysitting. My family opened their hearts to me and my little one and ma de it possible for me to come to work knowing that he was in good hands. In addition, I would also like to thank the Department of Pharmacology and Therapeutics at the College of Medicine at th e University of Florida, and the College of Veterinary Medicine at the Un iversity of Florida for their financial support during my graduate career. Thanks also to the men and women who dona ted their eyes to our research. Their gift has made it possible for us to understand se veral eye diseases and prevent them in the future.

PAGE 5

v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF FIGURES.........................................................................................................viii LIST OF ABBREVIATIONS...........................................................................................xii ABSTRACT....................................................................................................................xvi i CHAPTER 1 BACKGROUND AND SIGNIFICANCE....................................................................1 Anatomy of the Eye......................................................................................................1 The Retina.............................................................................................................4 Blood Supply to the Retina....................................................................................8 The Blood Retinal Barrier.....................................................................................9 Retinopathies..............................................................................................................10 Age Related Macular Degeneration....................................................................10 Diabetic Retinopathy..................................................................................................11 Non-Proliferative Diabet ic Retinopathy (NPDR)...............................................11 Proliferative Diabetic Retinopathy (PDR)...........................................................15 Retinopathy of Prematurity.........................................................................................17 Treatment of Retinopathies.................................................................................19 Angiogenesis...............................................................................................................24 Extracellular matrix (ECM).........................................................................26 ECM degradation.........................................................................................26 Bound Factors......................................................................................................30 Integrins........................................................................................................30 EpH receptors...............................................................................................31 Vascular endothelial (VE) cadherins...........................................................32 Growth Factors...........................................................................................................33 Angiopoeitins......................................................................................................33 Vascular Endothelial Growth Factor...................................................................35 Fibroblast Growth Factor....................................................................................38 Platelet Derived Growth Factor...........................................................................39 Transforming Growth Factor...........................................................................39 Adenosine...................................................................................................................44 Adenosine and the Retina....................................................................................47

PAGE 6

vi Adenosine Receptors...........................................................................................49 Pharmacology of the A2B receptors..............................................................59 Distribution of the Adenosine Receptors............................................................60 Intracellular Pathways Regulated by A 2B Receptors .........................................62 Ribozymes..................................................................................................................64 Self Splicing Introns...................................................................................................64 Group I Introns....................................................................................................64 Group II Introns...................................................................................................67 RNase P RNA......................................................................................................67 Small Self Cleaving Ribozymes..........................................................................71 Hepatitis Delta Virus...........................................................................................71 Hairpin Ribozymes..............................................................................................71 Hammerhead Ribozymes.....................................................................................71 Experimental Aim.......................................................................................................77 2 METHODS AND MATERIALS...............................................................................85 Defining Location of the Target Sequence.................................................................85 Preparation of the Target Oligo-Nucleotide...............................................................86 Time Course of Cleavage Reactions fo r Mouse and Human Targets (Hammerhead Ribozymes)............................................................................................................86 Multiple Turnover Kinetics........................................................................................87 Cloning of the Hammerhead Ribozymes into the rAAV Expression Vector.............88 Sequencing of the Clones...........................................................................................88 Human Retinal Endothelial Ce ll (HREC) Tissue Culture..........................................89 LDL Uptake of the HREC..........................................................................................90 Transfection of HREC using DEAE-Dextran.............................................................91 Transfection Efficiency usi ng DEAE Dextran for HRECs.......................................92 Cell Migration Assay..................................................................................................92 Morphology of HEK Cells..........................................................................................93 Transfection using Lipofectamine on HEK 293 cells.................................................93 Transfection Efficiency for HEK Cells using Lipofectamine Reagent......................94 cAMP Assay on Transfected HEK 293 Cells.............................................................94 Total Retinal RNA Extraction for PCR......................................................................97 Real Time PCR...........................................................................................................97 Animals.......................................................................................................................9 8 Intraocular Injection into the Mouse Model of Oxygen Induced Retinopathy...........98 Statistical Analysis......................................................................................................99 3 RESULTS.................................................................................................................100 Determining Accessibility of the Target Site...........................................................100 Time Course of Ribozyme cleavage.........................................................................103 Multiple Turnover Kinetics......................................................................................106 Cloning of the Hammerhead Ribozyme into an rAAV Expression Vector..............109 Sequencing of the Clones.........................................................................................111 Cell Cultures.............................................................................................................111

PAGE 7

vii Transfection of HREC..............................................................................................114 Transfection Using Lipofectmaine on HEK Cells....................................................118 CAMP Assay on Transfected HEK Cells.................................................................121 Real Time PCR..................................................................................................125 Effect of A2B Ribozymes on Neovascularization in the ROP Mouse Model...........125 4 DISCUSSION...........................................................................................................131 Ribozymes As Tools To Study Gene Expression.....................................................132 Delivery Of The Ribozyme In vivo..........................................................................135 Promoter Considerations..........................................................................................139 Future Studies...........................................................................................................142 5 LIST OF REFERENCES..........................................................................................147 BIOGRAPHICAL SKETCH...........................................................................................165

PAGE 8

viii LIST OF FIGURES Figure page 1-1 Cross sectional view of the components of the eye...................................................2 1-2 The ten layers of the retina.........................................................................................6 1-3 A fundus shot of ARMD..........................................................................................12 1-4 Non-proliferative retinopathy...................................................................................14 1-5 New blood vessel growth around optic nerve in PDR.............................................16 1-6 ICROP definition of retinopathy..............................................................................18 1-7 The five stages of ROP.............................................................................................20 1-8 Laser treatment of the eye........................................................................................21 1-9 Cartoon showing cryotherapy applicati on to the anterior avascular retina..............23 1-10 The process of angiogenesis.....................................................................................25 1-11 PAs hydrolyze plasminogen to plasmin...................................................................28 1-12 Angiopoeitins are ligand for the Tie 1 and Tie 2 receptors......................................34 1-13 The vascular endothelial cell growth factor (VEGF R2) signaling pathway...........37 1-14 The FGF receptor and signaling pathway................................................................40 1-15 The PDGF receptor and signaling pathway.............................................................41 1-16 The TGFreceptor signaling pathway...................................................................43 1-17 Intracellular and extracellu lar production of adenosine...........................................46 118 Role of the high and low affinity adenosine receptors.............................................50 1-19 Homology of the A1 receptor for human and mouse...............................................53 1-20 Homology of the A2A receptor between human and mouse.....................................54

PAGE 9

ix 1-21 Homology of the A2B receptor between the human and the mouse.........................55 1-22 The A 2B receptor......................................................................................................57 1-23. The A2A receptor......................................................................................................58 1-24 The A 2B signaling pathway.....................................................................................63 1-25 The secondary stru cture group I introns...................................................................65 1-26 Splicing mechanism of the group I introns..............................................................66 1-27 Secondary structure of Group II introns...................................................................68 1-28 The splicing mechanism of the Group II introns......................................................69 1-29 Cleavage of the tRNA 5 leader sequence by Rnase P............................................70 1-30 Self-cleaving ribozymes resolve concatemers formed by rolling-circle replication into individual genomic molecules..........................................................................72 1-31 Structure of the hairpin ribozyme.............................................................................73 1-32 Structure of the hammerhead ribozyme...................................................................75 1-33 The hammerhead ribozyme cleaves it s substrate by a transesterification reaction.....................................................................................................................76 1-34 Cleavage of the A2B receptor by a ribozyme prevents translation of the protein.....79 1-35 Target sequences of the human and mouse A2B ribozymes 1 and 2.........................80 1-36 Hammerhead ribozymes for the A 2B Rz1 and Rz2................................................81 1-37 The p21Newhp Vector with the CMV e nhancer and beta actin promoter...............82 1-38 Time course for the ROP model...............................................................................84 3-1 Theoretical tertiary structures of the active A2B Rz1 generated by the mfold program..................................................................................................................101 3-2 Theoretical tertiary structures of the active A2B Rz2 generated by the mfold program..................................................................................................................102 3-3 Time course autoradiograph of a 10% polyacrylamide 8M urea gel showing products of cleavage of the A2B Rz2 on the mouse target......................................104 3-4 Time course analysis data......................................................................................105

PAGE 10

x 3-5 Time course of the ribozyme with increasing target concentrations......................107 3-6 Time course cleavage reaction with varying temperatures (37 C/25C) and magnesium concentrations of 20mM/1mM...........................................................108 3-7 Kinetic analysis of the ribozymes..........................................................................110 3-8 Sequence of the active and inactive versions of the A2B ribozymes at the site of insertion within the p21NewHp vector..................................................................112 3-9 Pebble stone morphology of the HREC.................................................................113 3-10. LDL uptake of HREC.............................................................................................115 3-11 The GFP plasmid. This plasmid was driven by a CMV enhancer and a chicken beta actin promoter.................................................................................................116 3-12 Transfection efficiency of the HREC.....................................................................117 3-13 Theory of migration assay......................................................................................119 3-14 Migration data for the ce lls transfected with the active and inactive versions of the A2B receptor and the vector control. 10% FBS/DMEM is the positive control and DMEM alone is the negative control...............................................................120 3-15 Transfection Efficiency of HEK cells....................................................................122 3-16 HEK cells transfection efficiency following passage 1.........................................123 3-17 cAMP accumulation in HEK cells tran sfected with the control, active A2B Rz2 and inactive A2B Rz 2....................................................................................................124 3-18 Real time RT-PCR results showing relative levels of the adenosine A2A and A2B receptor mRNAs isolated from HEK cells transfected with plasmid DNA...........126 3-19 The mice eyes were embedded in paraffi n and three hundred serial sections were done........................................................................................................................127 3-20 Injection with the control plasmid pr ior to exposure to high oxygen shows a high number of endothelial cell nuc lei surrounding blood vessel lumen.......................128 3-21 Injection with the active A 2B ribozyme prior to high oxygen exposure significantly reduced the pre-retina l neovascularization.............................................................129 3-22 Injection of the active an d inactive versions of the A2B Rz2 and the vector control in the ROP mouse model........................................................................................130 4-1 Entry of the AAV and transferrin into the cell.......................................................137

PAGE 11

xi 4-2 Diagram of the expression cassettes fusion protein and alkaline phosphatase (Alk Phos)..............................................................................................................141 4-3 A2B signaling pathway with theoretical downs tream effects, which have yet to be confirmed...............................................................................................................146

PAGE 12

xii LIST OF ABBREVIATIONS a-LDL Acetylated 1,1’-Dioctdycl3,3,3’,3’ tetra methyl indocarbocyanin perchlorate ABAM Antibiotic antimycotic mix A1 Adenosine receptor type 1 A2A Adenosine receptor type 2A A2B Adenosine receptor type 2B A2B Rz1 Adenosine receptor type 2 ribozyme 1 A2B Rz2 Adenosine receptor type 2 ribozyme 1 A2R Adenosine receptor type 2 A3 Adenosine receptor type 3 ADA Adenosise deaminase AK Adenosine kinase AMP Adenosine monophosphate ANG-1 Angiopoeitin 1 ANG-2 Angiopoeitin 2 ARMD Age Related Macular Degeneration ARNT Aryl hydrocarbon receptor nuclear translocator ARVO Association for Research in Vision and Ophthalmology ATP Adenosine triphosphate v3 Alpha V beta 3 integrin

PAGE 13

xiii v5 Integrin bFGF Basic fibroblast growth factor BSA Bovine serum albumin cAMP 3c, 5c-cyclic monophosphate CAT Chloramphenicol acetyltransferase CGS21680 A2A agonist. 2-{4[(2-car boxylethyl)-phenyl]ethylamine}-5’-Nethylcarboxamidoadenosine CHA Cyclohexyladenosine CHO Chinese hamster ovary cells CMV Cytomegalovirus DMEM Dubellco’s modifeid eagle medium DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DPSPX Non-seletive adenosine r eceptor antagonist. 1,3-dipropyl-8(psulfophenyl)xanthine DTT Dithiothreitol ECM Extracellular matrix EDTA Ethylenediamine tetraacetic acid EGS External guide sequence Eph receptor Ephrin receptor FAK Focal adhesion kinase FAT Focal adhesion targeting sequence FBS Fetal bovine serum Flt VEGF fms like tyrosine kinase GAGs Glycosaminoglycans

PAGE 14

xiv GC Guanosine cytosine content GCL Ganglion cell layer GFP Green fluorescent protein GPI Glycosylphosphatidylinositol HBSS Hanks balanced salt solution HDV Hepatitis delta virus HEK 293 Human embryonic kidney cells HIF Hypoxia inducible factor HIV Human immunodeficiency virus HRE Hypoxia response element HRECs Human retinal endothelial cells HSPGs Heparan sulfate proteoglycans IACUC Institution Animal Care and Use Committee. IB-MECA Selective A3 adenosine recep tor agonist. N6 (3-iodobenzyl)Ado-5’Nmethyl Uronamide ICROP International classificatio n of Retinopathy of Prematurity ILM Inner limiting membrane INL Inner nuclear layer IP3 Inositol triphosphate IPL Inner plexiform layer KDR VEGF kinase insert domain LAP Latency associated peptide MMP Metalloproteinases NAD Nicotinamide adenine dinucleotide

PAGE 15

xv NBTI Nitrobenzylthioinosine NECA N-ethylcarboxyamidoadenosine NFL Nerve fibre layer NPDR Non proliferative diabetic retinopathy 5’NT 5’ Nucleotodase OLM Outer limiting membrane ONL Outer nuclear layer OPL Outer plexiform layer. PAs Plasminogen activators PAI-1 Plasminogen activator inhibitor-1 PAI-2 Plasminogen activator inhibitor-2 PBS Phosphate buffered saline PDGF Platelet derived growth factor PKC Protein kinase C PLC Phospholipase C PDR Proliferative diabetic retinopathy rAAV Recombinant adeno associated virus RBCs Red blood cells ROP Retinopathy of Prematurity RNA Ribonucleic acid rRNA Ribosomal RNA RNasin Ribonuclease inhibitor RPE Retinal pigment epithelium

PAGE 16

xvi R-PIA Selective A1 receptor agonist. R-phenylisopropyl-adenosine SAH S-Adenosylhomocysteine TBS Tris buffered saline TGF Transforming growth factor Tie 1 and 2 Angiopoeitin receptors 1 and 2 TIMPS Tissue inhibitors of matrix metalloproteinases TNFTumor necrosis factor alpha tPA Tissue type plasminogen activator tRNA Transfer RNA TR Inverted terminal repeats uPA Urokinase type plasminogen inhibitor VE cadherin Vascular endothelial cadherins VEGF Vascular endothe lial growth factor VEGF-R1 Vascular endothelial growth factor-receptor 1 VEGF-R2 Vascular endothe lial growth-receptor 2 WBCs White blood cells XAC Xanthine amine cogener XDH Xanthine dehydrogenase XO Xanthine oxidase

PAGE 17

xvii Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy REDUCTION IN PRE-RETINAL NEOVASCULARIZATION BY RIBOZYMES THAT CLEAVE THE A 2B RECEPTOR MRNA By Aqeela Afzal May 2003 Chair: Dr. M.B. Grant Cochair: Dr. D. Samuelson Major Department: Veterinary Medical Sciences Tissue hypoxia and ischemia initiate events that lead to pre-retinal angiogenesis. Adenosine modulates a variety of cellular functions by interacti ng with specific cell surface G-protein coupled receptors (A1, A2A, A2B, A3) and is a potential mediator of angiogenesis. The A2B receptor has been implicated in the mediation of angiogenesis. The lack of a potent, selective A2B receptor inhibitor has hampered its characterization. Our goal was to design and characterize a hamme rhead ribozyme that would specifically cleave the A2B receptor mRNA and examine its eff ect on retinal angiogenesis. Active and inactive ribozymes specific for the mouse and human A2B receptor mRNAs were designed and cloned in expression plasmids. HEK 293 cells were tran sfected with these plasmids, and A2B mRNA levels were determined by quantitative RT-PCR. Human retinal endothelial cells (HREC) were also transfected, and cell migration was examined. The effects of these ribozymes on the levels of pre-retinal neovascularization were determined using a mouse model of oxyge n-induced retinopathy. We produced a

PAGE 18

xviii ribozyme with a Vmax of 10.8 pmole min-1 and a kcat of 36.1 min1. Transfection of HEK 293 cells with the plasmid expressing ribozyme resulted in a reduction of A2B mRNA levels by 45%. Transfection of HREC reduced NECA stimulated migration of the cells by 47%. Intraocular injection of the construc ts into the mouse model reduced pre-retinal neovascularization by 54%. Ou r results suggest that the A2B receptor ribozyme will provide a tool for the selective inhibition of this receptor, and provide further support for the role of the A2B receptor in retinal angiogenesis.

PAGE 19

1 CHAPTER 1 BACKGROUND AND SIGNIFICANCE The formation of blood vessels is a funda mental process that can be broken down into two basic pathways. The first is vasc ulogenesis, which is the formation of new blood vessels such as seen in embryogenesis. The second is angioge nesis, which is the formation of blood vessels from pre-existing blood vessels. Angiogenesis is common in both normal physiological processes (pre gnancy, menstruation, wound healing) and disease states (cancer, retinopath ies, psoriasis). The focus of this study is the process of angiogenesis in retinopathies, including di abetic retinopathy, the leading cause of blindness in adults, and reti nopathy of prematurity (ROP). Anatomy of the Eye The two eyes in humans are oriented to facilitate binocular single vision, which results from the forward position of the eyes and the chiasmal crossing from axons of ganglion cells. Axons from the right visual fi eld carry impulses to th e left optic tract and vice versa. The eye contains the elements that take in light and converts them to neural signals. For protection, the eye is loca ted within the bone and connective tissue framework of the orbit. The ey elids cover and protect the ante rior surface of the eye and contain glands, which produce a lubricating film (tears).1 The globe has three spaces within it: the anterior chamber, posterior chamber and the vitreous chamber. 1 (Figure 1-1)

PAGE 20

2 Figure 1-1. Cross sectional view of the components of the eye

PAGE 21

3 The anterior and posterior chambers contai n aqueous humour, which is produced by the ciliary body and provides nourishment for th e surrounding structures. The vitreous chamber is the largest space in the eye and lies adjacent to the inner retinal layer and contains the gel-like vitreous humor.1 The eye is made up of three layers: an out er fibrous layer, a middle vascular layer and an inner neural layer (retina).1 The outer fiber layer is a dense connective tissue that provides protection for structures within, ma intains the shape of the eye, and provides resistance to the pressure of the fluids inside the eye. Th e sclera is the opaque white of the eye, and the cornea is transparent and a llows light to enter the eye where the lens refracts it to bring light rays into focus on the retina.1 The middle layer of the eye is made up of three structures. The iris acts as a diaphragm to regulate the amount of light en tering the pupil. The ciliary body produces components of the aqueous humor and has musc les that control the shape of the lens during accomodation. The choroid is an an astomosing network of blood vessels with a dense capillary network.1 The principle functions of the choroid are to nourish the outer retina and to provide a pathway for the vessels that supply the anterior eye. The choroid is an egress for catabolites from the retina, which diffuse through Bruch’s membrane into the choriocapillaris. The suprachoroidal space provides a pathway for the posterior vessels and nerves that supply the anterior segment.1,2 The choroid also plays a role in the maintenance of intraocular pressure due to the high blood flow in its vessels. The choroid has the largest sized a nd the greatest number of vascul ar channels in the eye, and the amount of blood flowing through these ch annels at any time has an effect on the

PAGE 22

4 intraocular pressure. The choroid also provi des a regular smooth internal surface for the support of the retina. The smoothness of Bruc h’s membrane is important in maintaining the exact relationship between the retinal pigment epithelium (RPE) and the outer segments of the adjacent phot oreceptor rods and cones. 1,3 The Retina The retina is located between the choroi d and the vitreous, and extends from the circular edge of the optic disc, where the nerve fibers exit the eye, to the ora serrata and is continuous with the epithelia l layers of the ciliary body.1,4 The retina is a thin, delicate and transparent tissue that lines the inner eye. The neural retina is at tached loosely to the choroid through the pigment epithelium. Exte rnally, the RPE contacts the collagen and elastic tissue of Bruch’s membrane of the chor oid. Bruchs membrane is an elastic layer that stabilizes the RPE and the photoreceptors. Internally, the retina lies next to the vitreous. Anteriorly, the RPE gives rise to the ciliary body, a nd posteriorly, all the retinal layers terminate at the optic disc except the nerve fiber layer. The retina is thickest at the equator and thins at the ora serrata.1 The retina can be divided into the centra l retina and the peripheral retina. The central retina is thick and includes the macu la, fovea and foveola. The macula has a yellow appearance due to xanthophyl (a caroteno id), which is found in the ganglion cells. The peripheral retina includes the remainder of the retina from the macula to the temporal or nasal side. The ora serrata is the extreme periphery of the retina. It is the junction where the retina ends and gives rise to the te eth like processes that form the ciliary body. The peripheral retina ends at the ora serrata and forms the te eth like processes that form the base of the ciliary body.1

PAGE 23

5 Under the light microscope, ten layers of th e retina can be differentiated (Figure 12): RPE, rod and cone layer, outer lim iting membrane (OLM), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), ganglion cell layer (GCL), nerve fi bre layer (NFL) and the inner limiting membrane (ILM). The visual pathway cons ists of three interconnecting neurons and some receptor cells. The neurons include the bi polar cells, located within the retina; the ganglion cells, located in the inner retina, the axons of which go through the optic nerve to the chiasm and end in the lateral genicula te nucleus; and the third neuron is from the geniculate body to th e occipital cortex.1 The rods and cones are the sensory receptors. The outer segments have photopigments, which are excited by light, resul ting in a visual response. The cell bodies of the rods and cones lie in the ONL and the axons synapse with dendr ites of bipolar cells in the OPL. The dendrites of the bipolar cells extend to the OPL and synapse with axons of rods and cones; their axons extend to the IPL and synapse w ith dendrites of the ganglion cells from the NFL. The INL also has horizontal and amacrine cells. The horizontal and amacrine cells in this layer provide horizontal integration.1 The RPE is a single layer of uniform cells. It is located in the outer ci rcumference of the retina and extends from the edge of the optic disc to th e ora serrata. The cells of this layer are hexagonal shaped and carry a brown pigment. These cells may be multinucleated, especially in the ora serrata. The RPE provi des metabolites to the receptors and removes the outermost ends of external segments of the photoreceptors. If the RPE cells are damaged or diseased, these cells are not repl aced; instead, adjacent cell s slide laterally to fill the space of the necrotic cells. The RPE cells possess microvilli on

PAGE 24

6 Figure 1-2. The ten layers of the retina incl ude: retinal pigment epithelium, rod and cone layer, outer limiting membrane, outer nuc lear layer, outer plexiform layer, inner nuclear layer, inne r plexiform layer, gangli on cell layer, nerve fibre layer and the inner limiting membrane

PAGE 25

7 the apical surface that interdigitate with the photoreceptors. The RPE is critical to vitamin A metabolism and photoreceptor maintenance.1 The rod and cone layer lies external to th e OLM. It has a thick inner segment and thin outer segments joined by a slight c onstriction. The cell membrane is continuous between the constrictions. The outer segments have parallel processes, which are short in cones and long and thin in rods. 1 The OLM under a light microscope has a thin fenestrated membrane-like appearance. However, the OLM is not a basement membrane. Electron microscopy revealed it to be a zonula adherens between the photoreceptors and the Mller cells. The zonula adherens probably serve to keep th e highly elongated photoreceptors in place.1 The ONL has the cell bodies of the rods a nd the cones. The axons of the rods and cones synapse in the OPL with bipolar and horizontal cells.1 The OPL is a reticular structure, which is a transition zone be tween the receptors (neuroepithelial). The OPL is a layer of synaptic contacts between photoreceptors, bipolar cells and horizontal cells. The axons of the rods end here in spherules (oval shaped) and those of the cones end in pedicles (broad conica l swellings). The spherules are invaginated and synapse with bipolar dend rites or horizontal cells, and can make 2-4 contacts. The pedicles, on the other hand, c ontact many dendrites of horizontal cells and bipolar cells.1 The INL is a band of nuclei belonging to horizontal cells, bipolar cells, and amacrine cells. The Muller cells provide support and nutrition to the retina. They surround capillary walls and extend from the ILM to the extracellular membrane.1

PAGE 26

8 The IPL is a junction between the first or der neuron (bipolar cells) and the ganglion cell layer. This layer contains the nuclei of displaced ganglion or amacrine cells and processes of the Mller cells.1 The GCL contains the cell bodies of ganglion cells, which are thin in the nasal area and thicker near the macula. The axons of the ganglion cells run internally and then become parallel to the inner surface of the reti na to give rise to the NFL and the optic nerve fibres.1 The NFL has the axons of the ganglion cells and is thickest around the optic nerve.1 Branching processes of the Mller cel ls and a basal lamina like structure secreted by them forms the ILM. The macula is the center of the retina (area centralis) and is divided into the fovea (cone dominate d), parafovea (ganglion cell dominated) and the perifoveal retina (singl e layer of ganglion cells).1 Blood Supply to the Retina The retina has the highest rate of metabol ism of any tissue in the body and thus has a dual blood supply from the retinal and choroidal capillaries. If either of these sources is interrupted, ischemia develops and leads to loss of function. The out er retina is supplied by the choriocapillaris and the cen tral retinal artery supplies the remainder. The retinal artery is different from other arteries and does not have an in ternal elastic lamina but does have a prominently developed muscularis.1,5 The outer retinal layers r eceive their nutrition from th e choroidal capillary bed; metabolites diffuse through Bruch’s membrane a nd the RPE into the neural retina. The central retinal artery provides nutrients to the inner retinal layers. The artery enters the retina through the optic disc, us ually slightly nasal of center, and branches into a superior and inferior retinal artery, each of which furt her divides into nasal and temporal branches, and these vessels continue to bifurcate. The nasal branches run a relatively straight

PAGE 27

9 course toward the ora serrata, but the tem poral vessels arch around the macular area en route to the periphery. Two capillary networks exist within the retina. The deepest one lies in the inner nuclear layer ne ar the outer plexiform layer, and the superficial one is in the nerve fiber or ganglion ce ll layer. The OPL is avascu lar and thought to receive its nutrients from both retinal and choroidal vessels.1,5 Retinal arterial circulation is terminal; therefore there is no direct communication between the retina and other ve ssel systems. The junctions of endothelial cells in retinal vessels are tight or occluded. Thus, to enter or leave the retina, most substances require active transport across th e endothelial cells. The outer retina l layers receive their nutrition from the choroidal capillary bed; the central re tinal artery provides nutrients to the inner retinal layers. These vessels are distributed to the four quadrants of the retina.6 The retinal artery and vein to a particular quadrant supply most of the quadrant. If arterial supply to a retinal quadrant is interrupte d, infarction of that section o ccurs. The retinal capillaries supply the inner two thirds of the retina; the choroidal circ ulation supplies the remaining outer retina via regulated transport across the pigment epithelium.1,5 The Blood Retinal Barrier The epithelial portion of the blood-retinal barr ier is the retinal pigment epithelium. This barrier separates the c horoidal tissue fluid, which is similar to plasma, from the retinal tissue fluid. Tight junc tions that exist between the endothelial cells of the retinal vessels and similar tight junctions in the RP E maintain the blood retinal barrier. Thus, the retinal vessels are impermeable to the pa ssage of molecules gr eater than 20-30 kDa, and small molecules such as glucose and asco rbate are transported by facilitated diffusion through the RPE.1,5,7

PAGE 28

10 Vascular beds are situated to provide nourishment. To avoid problems with the presence of blood vessels in the outer retina, the outer layers of th e retina receive their nourishment from the choriocapillaris.1 Retinopathies The retina of the eye is uniquely situat ed to provide optimal vision. Blood supply to the retina is also strategically placed to avoid any hindrance of the visual pathway. Retinopathies (diseases affecting the retina) disrupt this balan ce and lead to loss of vision. Retinopathies affecting humans include: age related macular degeneration (ARMD), which primarily affects the aging population; diabetic retinopathy (DR), which primarily affects the working population; and retinopat hy of prematurity (ROP) which primarily affects the newborns. Age Related Macular Degeneration ARMD is a disease which affects the RPE and leads to blindness in the aged populations.8 There are two forms of ARMD: dry and wet.9-11 Dry ARMD is characterized by the presen ce of soft drusen and pigmentary abnormalities. Drusen is an amorphous acellu lar debris present within the basement membrane of the RPE. It is seen as ‘ye llow’ spots within the macula. Low amounts of drusen are a consequence of age; however, a larger amount present within the retina is indicative of ARMD.10 Drusen leads to mild vision loss and increases the risk of progression of the disease to the wet form of ARMD.8,9 The wet form of ARMD (also known as the exudative or the neovascular phase) is characterized by choroidal neovascularization, RPE detachment and disciform scarring. The wet form of ARMD leads to rapid visi on loss. The choroida l neovascularization

PAGE 29

11 (CNV) leads to the formation of immature bl ood vessels which result in leakage of serum and blood and loss of central vision.9,12 (Figure 1-3) Diabetic Retinopathy Diabetes Mellitus affects millions of peopl e worldwide and is the leading cause of blindness in working age adults.13,14,15 There are two forms of diabetes mellitus: Type I, which typically affects juveniles and is known as insulin dependent diabetes mellitus, and type II, which is the adult onset form of diabetes and is known as non-insulin dependent diabetes mellitus.15 Diabetes also leads to systemic complications such as kidney failure, hypertension and cardiovascular disease.16,13 DR is the most frequent diabetic complication. Eye problems due to diabetes can be asymptomatic and if left untreated can lead to serious visual loss. The longer a patient has diabetes, the more likely they are to develop diabetic retinopathy.13,16,17,15 Diabetic eye disease can be divided into two phases: background diabetic re tinopathy (non-proliferative phase) and proliferative diabetic retinopathy (PDR). Non-Proliferative Diabetic Retinopathy (NPDR) In NPDR small retinal blood vessels are damaged. NPDR is the result of two major processes which affect retinal blood vessels, vessel closure and abnormal vessel permeability.13 The vessels leak fluid (edema) and later blood (hemorrhage) into the retina. Macular edema is the most common cau se of reduced vision in patients with nonproliferative diabetic retinopathy and is seen as milkiness of the retina surrounded with exudates (yellow clumps). 16,15 These exudates are th e result of fat or protein leaking out of the vessels. Water is quickly reabsorbed into the vessels

PAGE 30

12 Figure 1-3. A. A fundus shot showing drusen (yellow). B. Wet form of ARMD showing blood leakage. (National Eye Institute)

PAGE 31

13 or tissue under the retina. However, the fatty material is absorbed very slowly and thus left behind surrounding the leakage site.16,18 (Figure 1-4) Vessel closure may be due to blood cell clumping, damaged endothelium, swelling of an abnormally permeable vessel wall or compression of the capillary by surrounding retinal swelling. Diabetic patients have cl osure/non-perfusion of capillaries, which leads to a decreased oxygen supply. In areas surr ounding the area of non-pe rfusion capillaries dilate to compensate for the decrea sed oxygen supply. Small focal dilations (microaneurysms) of retinal capillaries also develop due to weakened capillary walls, thus allowing for bulging. 13 When multiple areas of the retina have lost their blood supply, angiogenic factors are released wh ich stimulate proliferation of new blood vessels. These new blood vessels are small a nd fragile, therefore, cause bleeding and the formation of scar tissue within the retina. Sm all arterial closures follow capillary closure, and deprive larger regions of the retina of blood supply. This is seen as ‘cotton wool spots’ on the retina in fluorescein angiography.16 Blood vessels in the body ar e usually fenestrated allo wing fluid to pass through vessel walls. These openings are small enough to allow water and ions to pass through, while preventing the passage of blood cells and larger proteins. In contrast, retinal blood vessels have tight junctions be tween the endothelial cells of blood vessel. Therefore, all fluids and molecules exiting the vessels have to pass through the cell. This lack of fenestration helps to keep the retina thin a nd dehydrated for proper function. These tight junctions form the blood retinal barrier, whic h partitions the neur al retina from the circulation and protects the retina from circulating inflammatory cells.16 The tight junctions are formed by a number of protei ns such as: occludin and claudin. These

PAGE 32

14 Figure 1-4. A. Non-proliferative reti nopathy. Hemorrhage (arrowhead) short arrow microaneurysm, larger arrow exudates. B. Macular edema. (National Eye Institute)

PAGE 33

15 proteins limit the flow of fluid between endot helial cells. Diabetic patients have a lower amount of occludin at the tight junctions in the retinal e ndothelial cells and can leak fluid.16 Proliferative Diabetic Retinopathy (PDR). Proliferative diabetic retinopathy is th e stage of diabetes characterized by angiogenesis on the surface of the retina. 13 Patients can have NPDR for years before progressing to PDR. PDR is diagnosed by th e presence of prolif erating blood vessels within the retina or optic disc. These vesse ls grow on the retinal surface or into the vitreous cavity and take on a frond-li ke configuration as they grow.19 (Figure 1-5) The new blood vessels form due to the closure of re tinal capillaries, which leads to ischemia. As patches of the retina are deprived of oxygen and nutrients, vasoproliferative factors are released which diffuse into the vitreous cavity. These factors stimulate growth of new vessels throughout the retina.15 The new blood vessels are not located in the same location as the ischemically damaged retina and are very fragile and bleed into the vitreous. A small amount of blood may be removed in a few weeks and larger blood hemorrhages may take a few months. If dense blood from multiple recurrent hemorrhages occurs then vision may not be restored since the residual inflammatory debris and dead cells cannot be removed. Another complication of PDR is traction retinal det achment. New vessels grow and regress and lay down fibrous scar tissue, which contra cts and shrinks as it matures. If the neovascularization is on the surface of the reti na then contraction of the fibrous scar distorts the retina. However, if the vessels grow into the vitreous and contract, retinal detachment occurs which leads to blindness. 13,15

PAGE 34

16 Figure 1-5. New blood vessel growth around optic nerve in PDR (Top). Hemorrhage from new blood vessel growth (Bot tom). (National Eye Institute)

PAGE 35

17 Retinopathy of Prematurity Retinopathy of prematurity (ROP), also known as retrolental fibroplasia, is a potentially blinding condition affecting the retina of new borns. In the 1950s, it was associated with the use of high oxygen levels in neonatal units.20 Modern neonatal care has curbed the incidence of ROP, but because the survival rate of low-birth-weight infants is increasing, the exposure of surviv ing babies to high oxygen levels is also increasing and ROP is still a relevant clinical problem.21,22 ROP causes more blindness among children in the world than all other causes combined. It begins after removal from high oxygen conditions and may progress rapidly to blindness over a period of weeks.23 Active growth of the fetal eye occurs between the last 12 weeks of full term deliv ery (28-40 weeks of gestation) At 16 weeks of gestation, blood vessels gradually grow over the surface of the retina. Vessels reach the anterior edge of the retina and stop progressi ng at about 40 weeks of gestation.20,21,24,25 The international classification of RO P (ICROP) defines retinopathy by several distinct criteria: location, ex tent, stage, and plus disease.26 Location refers to the location of the damage to the retina relative to the opt ic nerve. Normally retinal vessels begin growth at the optic nerve and gradually move toward the edge of the retina. Vessels further from the optic nerve ar e more mature. To standard ize the location of ROP, the retina is divided into three zones: Zone I is centered on the optic disc and extends from the optic disc to twice the distance between th e disc and macula; Zone II is a concentric ring around zone I and extends to the nasal ora serrata (the edge of the retina on the side toward the nose); and zone III is the remain ing crescent of retina on the temporal side (side towards the temple) (Figure 1-6). The extent of ROP is described by the clock hours

PAGE 36

18 Figure 1-6. ICROP definition of retinopathy. The retina is divided into three zones: Zone I, Zone II and Zone III

PAGE 37

19 of the retina involved in the ROP. For example, if the ROP extends from 1:00 to 5:00, the extent of ROP is 4 clock hours.24,27 ROP is a progressive disease that begins with some mild changes in vessels and may progress on to more severe changes. The five stages of ROP describe the progression of the disease (Figure 1-7). Stag e 1 is characterized by a demarcation line between the normal retina (near the optic nerve) and vascularized retina. In stage 2, a ridge of scar tissue rises up from the retina due to growth of abnormal vessels. This ridge forms in place of the demarcation line. In st age 3, the vascular ridg e grows due to spread of abnormal vessels and extends into the vi treous. Stages 4 and 5 refer to retinal detachment; stage 4 refers to a partial retin al detachment caused by contraction of the ridge, thus pulling the retina away from the wall of the eye; and stage 5 refers to complete retinal detachment. Plus dis ease is a very severe form of ROP which is characterized by the abnormal growth of blood ve ssels near the optic nerve.24,28 Treatment of Retinopathies. Spot laser photocoagulation is used for the treatment of ROP.29 This uses an argon/diode laser to burn spots on the peripheral and middle por tions of the retina. When laser light hits blood or pigment, it is abso rbed as heat energy and produces a small burn. The laser treatment leads to a decrease in th e level of vasoprolifer ative factors produced by the ischemic retina. The avascular retina is treated using a small laser spot (Figure 18). The laser spot directly treats the re tina and the underlying tissue, thus reducing inflammation and results in less damage to othe r ocular structures. Destruction of small patches of the ischemic retina reduc es the oxygen demand and decreases the vasoproliferative factor produc tion. Laser treatment also th ins the pigmented tissue under

PAGE 38

20 Figure 1-7. The five stages of ROP (National Eye Institute)

PAGE 39

21 Figure 1-8 Laser treatment of the eye. The laser spot directly tr eats the retina and the underlying tissue. Laser treatment thin s the pigmented tissue under the retina and allows more oxygen to diffuse in from the vessels under the retina

PAGE 40

22 the retina thus allowi ng better oxygen diffusion in the retin a. Laser treatment increases oxygen supply, lowers the demand for oxygen and lowers the incidents for new vessels growth.30,28 Laser photocoagulation also causes less pain than other therapies. Currently laser treatment is the best option for the treatment of retinopathies.14,31,29,32 Cryotherapy is also one of the treatments available for the treatment of retinopathies.33,34 This technique involves placing a co ld probe on the sclera until an ice ball forms on the retinal surface. Multiple ap plications are done to cover the entire vascular area (Figure 1-9). This thins th e tissue under the retina (by destroying it) and allows easier oxygen diffusion through the retina.31 Due to the pain involved in cryotherapy, anesthesia has to be administer ed which is a risk factor for premature infants. If no anesthesia is administered, ca rdiac arrest follows. Another complication is hemorrhage due to excessive bleeding.31,33,35,36 If laser photocoagulation or cryotherapy is unsuccessful, a scleral buckle may be used.37 This involves surgery and is used if ther e is shallow retinal detachment due to the contraction of the ridge. A silicone band is tightly placed around the equator of the eye thus producing a slight inde ntation on the inside of the eye.38 This indentation relieves traction of the vitreous gel and allows the retina to flatten back onto the wall of the eye. The silicone band is then remove d a few months later to allow the eye to grow.14,31,39 If the scleral buckle is not sufficien t, vitrectomy may be performed. Small incisions are made into the eye, the vitreous removed and replaced by saline. This technique also has had limited success. Current available therapies for the different types of retinopathies have had limited success. The underlying cause of retinopathies is angiogenesis

PAGE 41

23 Figure 1-9. Cartoon showing cr yotherapy application to the anterior avascular retina A cold probe is placed on the sclera till an ice ball forms on the retinal surface. Multiple applications are done to cove r the entire vascular area. This treatment thins the tissue under the re tina and allows easier oxygen diffusion through the retina

PAGE 42

24 (abnormal blood vessel formation). Developmen t of other effective therapies for the diseases requires an understandi ng of the process of angiogenesis.28,33,40-42 Angiogenesis Vasulogenesis is the formation of new bl ood vessels. Precursor cells (angioblasts) differentiate into endothelia l cells which later link to fo rm blood vessels. Angiogenesis on the other hand is the spr outing of blood vessels from pre-existing blood vessels.43 The vasculature of the retina unde rgoes both vasculogenesis and angiogenesis. The superficial retinal vessels, which originate at the optic disc, are formed by the process of vasculogenesis and the process of angiogene sis later forms the capillary beds. The process of angiogenesis involves e ndothelial branching, sprouting, migration, proliferation and anastomosing with e ndothelial cells in existing vessels.43-45 (Figure 110) Vascular endothelial cells form a monolayer throughout the entire vasculature. They are polarized cells with an apical surface and a basal surface, which is surrounded by a basal lamina.46-49 Mural cells wrap around this struct ure and are contra ctile cells, which regulate vessel diameter and consequently blood flow.47,50 On large vessels they are multi layered and referred to as smooth muscle cells. On capillaries mural cells are sparse and usually referred to as pericytes.48,50 The extracellular matrix, bound factors and the soluble growth factors all play an importa nt role in the process of angiogenesis.

PAGE 43

25 Figure 1-10. The process of angiogenesis. The process of angiogenesis involves endothelial branching, sprouting, migrat ion and proliferation. Vascular endothelial cells form a monolayer throughout the entire vasculature. Pericytes wrap around these cells.

PAGE 44

26 Extracellular matrix (ECM). The ECM surrounds and provides mechanic al support for blood vessels. The activated endothelial cells cerate gaps in th e basement membrane, which allows them to sprout into the ECM. The ECM is com posed of two compartments: the interstitial matrix and the vascular basement membrane.51,52 The interstitial matrix consists of fi brillar collagen and glycoproteins (e.g. fibronectin, laminin). Fibr onectin attaches cells to a va riety of ECM components, and laminin anchors cell surfaces to the basal lami na. Collagen provides structural support, is synthesized by fibroblasts and is the most abundant protein comprising the ECM. There are 12 types of collagen and types I, II and I II are the most abundant types of collagen in the ECM.51,52 The vascular basement membrane li es between the endothelial cells and pericytes. It is composed of type IV collagen, which forms the basal lamina upon which the endothelium rests, and heparan sulfate proteoglycans.51 Proteoglycans are glycosaminoglycans ( GAGs) linked to proteins. Cell surface heparan sulfate proteoglycans (HSPGs) func tion as endothelial cell receptors that recognize the ECM. They are present in the basement membranes and cell surfaces. These proteoglycans modulate the response of e ndothelial cells to basic fibroblast growth factor (bFGF), vascular endot helial growth factor (VEGF) and other heparan binding angiogenic factors by sequestering these molecu les in the ECM. Heparatinases trigger the release of these growth factors from the ECM and make them available for angiogenic stimuli.51,53 ECM degradation Endothelial cells degrade the surroundi ng ECM by the release of plasminogen activators (PAs) and matrix metalloproteases (MMPs).54 The PAs hydrolyze plasminogen

PAGE 45

27 to plasmin, which is a general protease that can digest most protei ns. (Figure 1-11) It also converts latent collagenase into active collagensase wh ich can then degrade collagen type I, II and III. 54 There are two types of PAs: tissue type PA (tPA) and urokinase-type PA (uPA).55 Both PAs utilize the same substr ate, plasminogen, and both have two specific inhibitors, plasminogen activator i nhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2). PAI-1 is produced by endothelial cells to i nhibit PA activity to ensure a balanced degradation of the ECM. uPA and PAI-1 are also upregulated by angiogenic factors such as basi c fibroblast growth factor (b FGF) and vascular endothelial growth factor (VEGF)54-56 Matrix metalloproteases (MMPs) are zi nc dependent endopeptidases which are secreted as zymogens a nd proteolytically activated by other MMPs or plasmin.51 MMP expression may also be regulated by grow th factors such as VEGF, bFGF and TGF.51,56 MMPs degrade components of the ECM a nd are subdivided into: collagenases, stromelysins (cleave laminin and fibronectin), matrilysins, gelatinases (cleave collagen type IV), membrane type (MT) MMP and ot her MMPs. Endothelial cells, smooth muscle cells and fibroblasts produce collagenase 1 (MMP-1), stromelysin (MMP3), gelatinase A (MMP-2), gelatinase B (MMP-9), matr ilysin (MMP-7), and MT1-MMP (MMP-14, which has fibrinolytic activity).54,55

PAGE 46

28 Figure 1-11. PAs hydrolyze plasminogen to pl asmin. Plasmin subsequently activates matrix metalloproteases, which degrade the extra cellular matrix. PA=plasminogen activator; uPA=urokin ase type PA; tPA=tissue type PA; PAI=plasminogen activator inhibito r; MMP=matrix metalloproteases; TIMPs=tissue inhibitors of MMPs

PAGE 47

29 Endothelial cells also produce tissue inhi bitors of MMPs (TIMPs) that are specific inhibitors of MMPs and modulate the degrad ation of the ECM. TIMPs are secreted proteins, which inhibit MMPs in a 1:1 stoichio metry. They reversibly interact with the catalytic domain of the MMPs to inhibit their activity. TIMPs differ in their ability to interact with various MMPs. For exam ple, TIMP2 inhibits MT-MMP and TIMP3 inhibits MMP9. TIMPs also bind to the he paran sulfate proteogl ycans in the ECM and concentrate them to the specifi c regions within the tissue. 56 Angiostatin and endostatin are naturally occurring anti-angiogenic molecules, however, they are also produced by proteoly tic cleavage by MMPs from the pro-forms of plasminogen and collagen XVIII, respectively.57 The production of MMPs, however, is cell and tissue specific. For example, bFGF and VEGF upregulate in terstitial collagenase (MMP-1) and also increa se the formation of plasmin. Plasmin converts the inactive form of MMP-1 to the active form. Gelatinase A (MMP-2) is upregulated by calcium influx. It is responsible for the angiogenic switch a nd for the differentiation of the endothelial cells into tubes. MMPs promote capill ary tube formation, however, at high concentrations, they have an opposite effect. ECM degradation produces fragments, which have the opposite effect of the intact molecule. For example, hyaluronan, a GAG found in the ECM, has anti-angiogenic properties. However, when cleaved, it enha nces the action of a ngiogenic factors. Conversely, proteolytic degrad ation of fibronectin, plasmi nogen and collagen produces fragments, which have both anti-angiogenic and angioge nic activity. MMP2 undergoes proteolysis to produce PEX, which is the C-terminal non-catalytic domain of MMP2. PEX is anti-angiogenic and inhibits the gelatinolytic activity of MMP2.

PAGE 48

30 Bound Factors. Activated endothelial cells are anchorage dependent for survival. In addition to degradation of the ECM, the endothelial cells require bound fact ors to help in migration towards the ischemic stimulus. These bound factors include integrins, Eph receptor Eph/Ephrins complexes, and VE cadherins Integrins. Integrins provide the scaffolding for the cells to migrate upon and are used by the endothelial cells to recognize the ECM.58 Integrins play a role in regulating cell growth, differentiation and survival.59-63 Integrins are cellular receptors for ECM pr oteins and are expressed by all adhesive cells.64 Integrins are composed of and chain heterocomplexes, which are integral membrane glycoproteins. They have long extracellular domains, which are the ligand binding regions. Eighteen different subunits, and 8 subnits have been identified. These subunits can associate in 24 known co mbinations. A short transmembrane region follows the short intracel lular domains of both the and the subunits and the cytoplasmic tail of the beta subunit links the inte grins to cytoskeletal actin of the endothelial cell.62,63,65 Integrins are linked intracellulary to actin filaments by specific actin binding proteins, such as Talin, alpha actinin, vinicluin and paxillin.66 Focal adhesion kinase (FAK) is a protein with tyrosine kinase activit y and is composed of a large kinase domain flanked by an amino and carboxyl terminus. A region of the c-terminus, known as focal adhesion targeting sequence (FAT) recruits FAK to paxillin. Integrin mediated cell adhesion occurs when FAK is tyrosine phosphorylated.58

PAGE 49

31 v3 has a well-characterized ro le in angiogenesis. It me diates adhesion of cells to vitronection, fibronectin, von Willebra nd factor, osteopontin, tenascin and thrombospondin. Although the v3 integrin is minimally expressed on normal resting blood vessels, it is significantly upregulated in newly formed blood vessels within tumors, in healing wounds and in re sponse to certain growth factors. v3 expression is upregulated in endothelial ce lls exposed to angiogenic factors and those exposed to hypoxia. Integrins also help to target the activity of the MMPs, for example, v3 interacts with MMP-2 and also regulates sign aling via the vascular endothelial growth factor receptor –2 (VEGF-R2). Natural co mponents of the ECM, such as, endostatin, angiostatin, thrombospondin and tumastatin ar e all anti-angiogenic and exert their effect by binding to the v3 intergrin and disrupting the endot helial cell-ECM interaction. If this integrin is disrupted using an anti body (LM609) or a peptide antagonist (cyclic peptide 203, RGDfv), it results in the disrupt ion of angiogenesis pr ogression. VEGF and bFGF are capable of inducing the expression of v3 integrin of endothelial cells.67,68 EpH receptors. To discriminate cell partners from fibr oblasts or inflammatory cells, the Eph receptor is utilized. The Eph receptor is th e largest receptor of the receptor tyrosine kinase family (RTK) family.69,70 The receptors are divided based on ligand affinity into class A and class B. The extracellular domain of the Eph receptor consists of the ligand binding globular domain, cysteine rich region and 3 fibronec tin type II repeats. The cytoplasmic portion of the receptor consists of a juxtamembrane domain, and a carboxyl terminus. The ligands for these receptors are ephrins, which are also divided into subclass A and subclass B. Ephrins subclass A are anchored to the plasma membrane by

PAGE 50

32 glycosylphosphatidylinositol (GPI) anchor a nd ligands A1-A5 have been identified. Ephrins subclass B have a short transmembran e domain and a short cytoplasmic tail. Only three subclass B ephrins have been iden tified (B1-B3). Both the receptor and the ligands are membrane bound and therefore a signal is transduced in the receptor expressing cells and the ligand expressing cells.70,71 Prior to cell-cell contact, the Eph recepto r and ehprins ligand are loosely clustered at the cell surface. Following cell-cell cont act, the receptor and ligand heterodimerize and tetramerize. These receptors are capab le of bi-directional signaling (forward and reverse signaling). Eph A receptor enhances the adhesion of cells and the number of focal adhesion points and is known to be i nvolved in forward signaling. Eph receptor B is phosphorylated in the intracellular domain and is known to be capable of both forward and reverse signaling.72 Vascular endothelial (VE) cadherins Endothelial cells express at least th ree cadherins: N-, P and VE cadherin. Ncadherin is diffusely spread across the cell, P cadherin is present in trace amounts and VE cadherin is specifically localized to inter e ndothelial cell junctions. Beta catenine and plakoglobulin are anchored to the cadherin through actin and catinine. VE cadherin mediates contact inhibition of endothelial cells by decreasi ng the amount of proliferation and allows endothelial cell monolayers formation in the vessel wall. VEGF increases endothelial cell permeability by phosphorylation of a tyrosine residue of VE cadherin. This phosphorylation leads to dissociation of the VE cadherin and translocation of the beta catenine/plakoglobulin complex to the nucleus to regulate gene transcritption.73-75

PAGE 51

33 Growth Factors. The process of angiogenesis requires co-ordin ation of several growth factors, which play distinct roles in the pr ocess, examples include: angi opoeitins, vascular endothelial growth factor (VEGF), fibroblast growth fact or (FGF), platelet derived growth factor (PDGF) and transforming growth factor (TGF). Angiopoeitins Angiopoeitins are secreted ligands for the tw o Tie receptors: Tie 1 and Tie2 (Tek). Both receptors are endothelium specific,73 and have an extracellular domain composed of two immunoglobulin like folds a nd three fibronectin repeats. The cytoplasmic region has a tyrosine kinase domain interr upted by a short kinase insert. Angiopoeitin-1 and angipoeitin-2 are ligands for the Tie receptors. Both ligands can bind the receptors, however, onl y ANG1 can phosphorylate the receptor.76 ANG-2 inhibits Tie2, detaches peri cytes and loosens the matrix surrounding the vessel. ANG-1 does not initiate endothelial network organization it stabi lizes networks initiated by VEGF by enhancing the interaction betw een endothelial cells and pericytes Binding of ANG1 to the Tie 2 receptor in itiates cell survival through th e PI3 kinase, Akt pathway. Akt leads to the upregulation of survivi n, which is an apoptosis inhibitor. Phosphorylation of Tie2 leads to the phosphorylation of Dok. Dok then activates the Ras, Nck, and Crk pathway, which are involved in cell migration, proliferation and organization of the cytoskeleton. Molecules interacting with the Tie2 SH2 domains are Grb2, SHP2 which modulate cell growth, differentiation, migration and survival.76 (Figure 1-12)

PAGE 52

34 Figure 1-12. Angiopoeitins are ligand for th e Tie 1 and Tie 2 receptors. Binding of angiopoeitin 1 to the Tie 2 receptor lead s to endothelial cells proliferation, migration and cell survival. Angi opoeitin 2 inhibits Tie 2. ANG1=angiopoeitin 1; ANG-2=angiopoetin 2.

PAGE 53

35 Vascular Endothelial Growth Factor VEGF is a heparan binding potent endot helial cell mitogen. It promotes endothelial cell survival via activation of the phosphatidylinositol 3-ki nase (PI3K/Akt) pathway and inhibits apoptosis.77 VEGF undergoes alterna tive splicing to produce 5 known isoforms: VEGF 121, VEGF 145, VEGF 165, VEGF 189 and VEGF 206.73 The isoforms differ in storage in the ECM and their extracellular pathways.78 VEGF 121 and VEGF 165 are secreted extracellularly, whereas VEGF 189, VEGF 206 and possibly VEGF 165 are either cell or matrix associated due to their affinity for heparan sulfate. VEGF is a mitogen for endothelial cells and each isoform has varying effects during angiogenesis:73 VEGF 189 decreases lumen diameter, 121 and 165 increase lumen diameter and increases vessel length. VE GF 165 binds to the EC M and releases bFGF stored in the ECM, thus, bFGF and VEGF have a synergistic angiogenic effect. 78 There are three tyrosine kinase receptors for VEGF: VEGF R1 (Flt1), VEGF R2 (KDR/Flk-1) and VEGF R3 (Flt3).73 The receptors all have seven immunoglobulin like extracellular domains, a transmembrane domai n and an intracellular tyrosine kinase domain, which is interrupted by a kinase insert.78 VEGFR1 and VEGR2 transduce different signals to endothelia l cells. VEGFR1 promotes cel l migration and VEGFR2 is mitogenic for the endo thelial cells an d also promotes migration. 73 Hypoxia upregulates VEGFR1 and induces the expr ession of VEGF by endothe lial cells. The increased production of VEGF activates the VE GFR2 receptor phosphorylation and cell proliferation. 78 Ligand binding to VEGFR1 leads to the activation of the small adaptor proteins: Fyn, Yes and GAP. Ligand bi nding to VEGFR2, however, leads to phosphorylation of the SHP-1 and SHP-2 ad aptor proteins and PLC-gamma. PLC gamma hydrolyzes phosphatidyl inositol 4,5-bisphosphate (PIP2) to form inositol

PAGE 54

36 triphosphate (IP3) and diacylglycerol (DAG) The DAG remains associated with the plasma membrane and activates protein kina se C (PKC). PKC is a soluble cytosolic protein, which is activated by the increase in calcium concentration. Activation of PKC leads to cell proliferation and permeability. VEGFR2 also leads to the activation of the PI3 kinase/Akt pathway, which enhances cell survival. VEGFR2 plays a role in cell migration by recruiting FAK. The MAPK path way is also activated through Grb2, which is an SH2 adaptor protein. It has two SH 3 domains, which bind the guanine nucleotide exchange factor SOS. SOS then leads to th e activation of RAS. Activated RAS binds to the N-terminal of RAF which phosphorylat es MEK and phosphoryl ates MAP kinase.78 The activated MAPK pathway then leads to th e activation of the intranuclear proteins such as cyclin D which is important in the progression of the cell cycle from the G1 phase to the S phase.79 (Figure 1-13) VEGF also incr eases vascular permeability and allows leakage of plasma proteins, forma tion of the ECM and upregulates the production of uPA and tPA and PAI-1 by endothelial cells VEGF production is regulated by local oxygen concentrations. Hypoxia upregulates production of VEGF by binding to the hypoxia inducible factor (HIF).73 During retinal development astrocytes and neuronal precursors migrate away from existing blood pre-existing blood vessels. As the distance between the astrocytes and the pre-existing blood vessel increase s, the astrocytes sense a st ate of hypoxia. Astrocytes are more sensitive to hypoxia than neuronal cells and thus the astrocytes upregulate the production of VEGF, which lead s to angiogenesis. This upregulation of VEGF by the astrocytes creates a concentration gradie nt of VEGF. This stimulates blood

PAGE 55

37 Figure 1-13. The vascular endothelial cell growth factor (VEGF R2) signaling pathway. VEGFR2 activates several pathways a ll of which lead to angiogenesis.

PAGE 56

38 vessel formation towards the astrocytes, wh ich produce VEGF. In ROP babies are placed in a high oxygen incubator because th eir lungs are not fully developed. The hyperoxia inhibits the VEGF production by th e astrocytes thus causing newly formed blood vessels to regress. Once the babies are ta ken out of the incubato r all the cells of the retina sense hypoxia and upregul ate the production of VEGF. This leads to abnormal angiogenesis and unregulat ed blood vessel growth.78 Fibroblast Growth Factor Fibroblast growth factor (FGF ) is ubiquitously expresse d as either basic FGF or acidic FGF. FGF is either in the cytopl asm or bound to the ECM due to its intrinsic affinity for heparan.73 FGF binds to four related receptors, which are expressed on many cells. Ligand binding induces receptor dimeri zation. Endogenous hepara n sulfate in cells is required for the activation of FGF. The receptor for FGF has three immunoglobulin like folds; two intracellular tyrosine kinase domains, a short transmembrane region and a juxtamembrane domain, which is lo nger than any other receptor.80 The intracellular domain has two phosphorylation sites.81 Ligand binding to the FGF receptor induces tyrosine phosphorylation of an adaptor mol ecule, FRS2. The phosphorylated FRS2 then allows binding of a small adaptor molecule GR B2. GRB2 is involved in the activation of the GTP binding protein Ras. Since FRS2 doe s not have an SH2 domain, another adaptor molecule, SHP-2 associates with FR S2 alpha in the active FGF receptor.81 The importance of the association of this molecule with the FRS2 is not well defined. GRB2 exists with SOS, which catalyses the exch ange of GDP for GTP on Ras for activation. Therefore, SOS, facilitates the coupling of GRB2 to Ras.81 The activated MAPK then leads to the activation of the intranuclear pr oteins such as cyclin D which progresses the cell from the G1 phase to the S-phase.79 (Figure 1-14) This growth factor induces

PAGE 57

39 processes in endothelial cells and stimulates proliferation and migration of endothelial cells and pericytes, and production of PA by th e endothelial cells. bFGF plays a role in blood vessel remodeling by stimulating endothelia l cells to form tube like structures.73,82 Platelet Derived Growth Factor Platelet derived growth factor (PDGF) is a mitogen for smooth muscle cells73 and potent chemoattractant factor for smooth muscle cells, monocytes and fibroblasts. PDGF is a dimer consisting of two pol ypeptide chains: A and B. These chains combine to form 3 PDGF isoforms of PDGF AA or BB or heterodimers of PDGF AB.73,83 The PDGF receptor consists of a single transmembran e domain which has intrinsic kinase activity.83 The receptor is also a dimeric mixture of the alpha and beta subunits.73 Ligand binding induces receptor dimerization and trans phosphorylates tyrosine residues in the cytoplasmic domain of the receptor.83 Endothelial cells express the beta receptor and are stimulated by PDGF-BB.73,83 PDGF-BB acts through the MAPK/ERK pathway to stimulate c-jun/c-fos related genes in the nucleus to stimulate proliferation.83 PDGF also acts through the PI3kinase path way to activate PKB, which s timulates cell survival and proliferation. PDGF also plays a role in angiogenic chords formation and stimulates sprout formation. PDGF also mediates pr oliferation and migration of pericytes along angiogenic sprouts.73 (Figure 1-15) Transforming Growth FactorTransforming growth factor beta (TGF) is produced by almost all cells and thus its activation represents an important control mechanism.84 TGFis hydrolyzed

PAGE 58

40 Figure 1-14. The FGF receptor and signa ling pathway. Ligand binding to the FGF receptor leads to tyrosine phosphorylation of adaptor molecules and activation of the MAPK pathway. The MAPK pathway leads to endothelial cell proliferation, and migration.

PAGE 59

41 Figure 1-15. The PDGF receptor and signa ling pathway. The PDGF receptor acts through the MAPK pathway to stimulate proliferation and also stimulates endothelial cell survival thr ough the PI3-kinase/PKB pathway.

PAGE 60

42 intracellularly by a furin peptidase to produce the carboxyl terminal peptide.73 This peptide associates with the amino terminal to form the latency associated peptide (LAP). The LAP dimerizes to form the mature TGFwhich is then secreted in the inactive form.73 Plasmin activates the latent complex. TGFalso produces PaI-1 which inhibits plasminogen. Thus, showing that the action of TGFis self limiting.85 There are three different types of TGFreceptors designated, I, II and III. TGFbinds directly to the TGFII receptor. Binding of the II receptor is followed by the recruitment of the TGFI receptor. Both the receptors then form a stable complex and receptor II then phosphorylates receptor I which induces the signal cascade of the receptor.85 Once the TGFR2 is bound to the TGF1 receptor, the kinase activity of receptor 1 is activated. This leads to the recruitment and the accu mulation of the Smad proteins, which are then phosphorylated by th e receptor. The name SMAD is derived from the genes encoding them. The genes we re first identified in drosophila and C. elegans. The drosophila gene was named MAD (mother against decapentapleigic) and the gene from C. elegans was named SMA (small body size).86,87 (Figure 1-16) TGFis a bifunctional regulat or. At low levels, TGFstimulates angiogenesis, and at high levels it inhibits angiogenesis.85 TGFis found in the ECM, on endothelial cells and on pericytes. It supports the anchorage independe nt growth of fibroblasts.73 TGFalso controls cell adhesi on by regulating the production of ECM and integrins. Endothelial cell migration and formation of tube like structures are regulated by TGF. TGFalso upregulates the pr oduction of TIMPS, thus has anti-proteolytic activity. 73 TGFinhibits endothelial cell proliferation73 by blocking the effect of other mitogenic growth factors and enhances pericyte differentiation. It helps to form the vessel

PAGE 61

43 Figure 1-16. The TGFreceptor signaling pathway. Ligand binding to the TGFreceptor II leads to th e recruitment of TGFreceptor I. The activated receptor recruits the Smad proteins and stimulates angiogenesis at low levels and inhibits angiogenesis at high levels of TGF.

PAGE 62

44 wall by stimulating the production of the extrace llular matrix, strengthens the vessel wall and has matrix modulating effects and also stimulates tube assembly.73,87 Angiogenesis is a complex process, whic h involves the extra cellular matrix, bound factors and soluble factors. Of the soluble f actors, VEGF plays an important role in the early phases of angiogenesis. VEGF is an important mediator of compensatory angiogenesis and is a potent mitogen i nduced by hypoxia and nucleosides such as adenosine. 53,88,89 However, even though the angiogenesis process may solve the nourishment aspect of the outer retinal layers if the choriocapillaris was impaired, it would still cause vision impairment.90,90-94 Tissue hypoxia and ischemia initiate a se ries of events which lead to the development of collateral blood vessels, fo llowed by compensatory angiogenesis, which is detrimental and results in aberrant bl ood vessels that are friable and prone to bleeding.92,95 Mediators of compensatory angiogene sis include VEGF, which is a potent mitogen induced by hypoxia and nucleosides such as adenosine.95-97 Depending on the character of the ischemic stimulus, adenosin e plays two roles: as an intracellular signaling factor which promotes neovasc ularization following chronic hypoxia or ischemia, and as an endogenous protective fact or which is capable of protecting the retina from acute ischemia. Adenosine also upreg ulates VEGF in retinal endothelial cells. Therefore, adenosine may be a critical signal in the control of gene expression after retinal ischemia.91,98,99 Adenosine Adenosine is an endogenous nucleoside, which modulates many physiological processes such as cardiac myocyte contra ctility, modulation of neurosecretion and neurotransmission, cell growth and gene expression, regulatio n of intestinal tone and

PAGE 63

45 control of vascular tone.100 Adenosine serves as a signal to increase energy supply and demand by affecting cellular metabolic rate s and tissue perfusi on. Metabolites of adenosine may also have significant physiologica l and pathological effects. The level of adenosine available for these effects is dete rmined by a number of factors including the rate of production, transport and metabolism.100 Stimuli that mediate the local production of adenosine includ e hypoxia, ischemia and inflammation. The endothelium is a barrie r to adenosine, thus the adenosine formed within the lumen of the blood vessels may be derived from nucleotides released from platelets or endothelial cells. Ischemic pare nchymal cells or nucleotides derived from nerves or intestinal mast cells give rise to interstitial ad enosine. This adenosine may produce vasodilation via the A2A receptor on vascular smoot h muscle cells, which are especially accessible to the interstitial nucle oside. Adenosine may also be derived from adenine nucleotides from many cell t ypes by mechanisms which are not well understood.100,101 Since AMP is derived from the breakdown of ATP, adenosine formation is closely linked to the cellular energy state. Adenos ine may be formed intra or extracellularly. (Figure 1-17) The enzyme 5’ nucleotidase (5’N) cataylzes the metabolism of ATP to adenosine. S-adenosylhomocysteine hydrol ase also catalyses the break down of Sadenosylhomocysteine (SAH) into adenosin e. SAH contributes significantly to adenosine formation in the heart and ischemic conditions in the brain. Once formed, intracellular adenosine is transported out of the cell to exert e ffects on specific cell surface receptors. The transport of adenosine is bidirectional.

PAGE 64

46 Figure 1-17. Intracellular a nd extracellular production of adenosine. SAH hydrolase: Sadenosyl homocysteine hydrolase. 5’NT:5’nucleotidase. XDH:xanthine dehydrogenase. XO:xanthine oxidase

PAGE 65

47 Ecto 5’N catalyses the breakdown of 5’AM P to adenosine, thus giving rise to extracellular adenosine. The intracellula r and the extracellula r adenosine can be differentiated from each other using specific inhibitors of ecto 5’N.100,101 Adenosine deaminase (ADA) and adenosin e kinase (AK) catalyze the breakdown of adenosine in the cytoplasm. Both ADA a nd AKA are found in the cytoplasm. ADA is a 36 kDa protein which catalyzes the forma tion of inosine. ADA is heterogeneously distributed in tissues and hi ghest activity is during deve lopment. AK is a 38-56 kDa monomeric protein. It is al so widely distributed thr oughout the body. AK catalyses the phosphorylation of adenosine to 5’AMP. If the intracellular ade nosine is high, then AK is inhibited.100,101 Five types of adenosine transporters have been classified according to sensitivity to nitrobenzylthioinosone (NBTI), which is an aden osine transport inhibitor. Most of these transporters are sodium dependent and are bidirectional. Following degradation of adenosine, inosine leaves the intracellular environment and forms hypoxanthine. Xanthine dehydrogenase catalyses the oxi dation of hypoxanthine to xanthine and subsequently to uric acid. Conversion of xa nthine to uric acid also reduces NAD to NADH. Xanthine oxidase genetrates supe roxide and hydrogen peroxide, both of which are damaging to cells. Endothelial cells stim ulated by ischemia and reperfusion are key sources of xanthine oxidase formation and activity.100 Adenosine and the Retina Adenosine is heterogeneously distributed th roughout the retina of various species, such as rat, guinea pig, monkey, human and mouse.100 Adenosine immunoreactivity is found in the ganglion cell layer, the inner pl exiform layer and the i nner nuclear layer. 91,102 Under resting conditions, e ndogenous purines in the retin a are in the form of ATP

PAGE 66

48 (70%) and adenosine (2%). During devel opment, the retinal Mller cells provide glycosaminoglycan to the extracellular spaces for angioblasts which provides a scaffold for angioblast migration and organization. In developing and adult mammalian retina Mller cells express 5’ nucleotidase (5’N) ectoenzyme, a glycoprotein. This enzyme catalyzes the hydrolysis of purine nucle otide monophosphates, to the corresponding nucleoside. The 5’NT can metabolize a ll purine monophosphates, however, the major product is adenosine. Adenosine is an in tercellular communication molecule and is a modulator of synaptic transmission in the brai n and the retina, and is a local regulator of blood flow in several organs. In the retina, ad enosine is released in response to ischemia, thereby modulating the blood flow in the a dult and neonates. Adenosine is also chemotactic and a mitogen for endothelial ce lls, and enhances endothelial cell migration and tube formation.102 An increase in the 5’NT activit y in cerebral ischemia was shown by Braun et al.103 The pattern of 5’NT changes as the retina develops. In the early stages of development, the greatest act ivity of 5’NT is found in the inner Mller cell processes. When the inner retinal vasculature reaches completion (about 22 days of age), the inner retina activity of the enzyme decreases and th e activity in the outer retina increases (in both plexiform layers).102 Lutty et al showed that at days 1-5, an increased adenosine immunorectivity is found in the inner retina and the edge of the formed vasculature in the neonatal dog. An increase in the adenosine product shifted toward the ora serrat a as the vascular development progressed radially. On day 8 the 5’NT is increased in the inner retina, and on day 15 there is an increase in the adenos ine immunorecativity in the nerve fibre layer and the inner nuclear layer. When the radial progression of the inne r retinal vasculature

PAGE 67

49 is complete on day 22, the 5’NT and adenos ine are decreased throughout the nerve fibre layer and increased in the ganglion cell layer, the i nner nuclear layer and the photoreceptor inner segments.102 An increase in adenosine le vels at most ages was found to be proportional to an incr ease in the 5’NT activity. In summary, the 5’NT activity shifts from the nerve fibre layer to the i nner plexiform layer during development and the adenosine location is also shifted. Thus the Mller cells provide a glycosaminoglycan rich extracellular milieu for angioblast differentiation and also provide adenosine which is a stimulus for blood vessel formation.100,102 Adenosine Receptors Adenosine receptors have been impli cated in mast cell activation, asthma, regulation of cell growth, inte stinal function, neurosecretion modulation and vasodilation. Adenosine receptors modulate cAMP (adenosine 3c, 5c-cyclic monophosphate) intracellulary. Based on their ability to i nhibit or stimulate adenylyl cyclase, the adenosine receptors were initially divided into A1 and A2 subtypes. 100,104,105 The A2 receptor was further divided into 2 subtypes based on the finding of a high affinity A2 receptor in the rat striatum and a low affinity A2R in the brain 106 Both of these receptors activate adenylyl cyclase. The high affinity receptor was designated as A2A and the low affinity receptor was designated A2B.100,107 Adenosine activates four di fferent cell receptors: A1, A2A, A2B and A3. In most cell types, adenosine activates the A1 receptor to lower oxygen demand, and activates the A2 receptors to increase the oxygen supply. Thus the A1 and A2 receptors act to rectify imbalances between oxygen supply and demand.100,108(Figure 1-18)

PAGE 68

50 Figure 1-18. Role of the high and low affinity adenosine receptors. The A2 receptors increase oxygen supply. The A2A receptor leads to vasodilation and the A2B receptors lead to angiogenesis

PAGE 69

51 A1, A2B and A3 adenosine receptors are N-linked glycoproteins, which have sites for palmitoylation near the carboxyl terminus. Glycosylation has no effect on the affinity of ligands for these receptors, thus these si tes may be involved in targeting newly formed receptors to the cell surface. All receptors can be readily deglycosylated upon incubation with glycosidase.101 The molecular pharmacological a nd physiological relevance of the A1, A2A and A3 receptors is well known. However, the A2B receptor is not as well characterized due to a lack of selective pha rmacological probes and because this receptor has a low affinity for adenosine.100 The A1 receptor was initially cloned from rat, human, bovine and rabbit. The A1 receptor has seven transmembrane domains and is 326 amino acids in length and is about 36-37 kDa. Mutations in the H 274 and H 251 region result in loss of agonist and antagonist binding. Chimeric receptor construc ts reveal transmembrane domains 5, 6 and 7 to be important for bind ing. In the brain, the A1 receptors couple to Gi and Gs and inhibit the actions of adenosine. The A1 receptor decreases membrane potential (by increasing K+ and Clconductivity), lowers neurotransmitter release (e.g. glutamate and dopamine) and decreases calcium influx by stimulating calci um mobilization via the pertusis toxin sensitive pathway through the activat ion of PLC beta with G protein / subunit.101 All of these effects of the A1 receptor lead to a decrease in neuronal excitability and metabolism. Thus, the A1 receptor has a neuroprotective role in ischemic tissue.100 The A2A receptor was initially cloned from canine, rat and human and produced responses which are anti-inflammatory.101 It has seven transmembrane domains consisting of 410-412 amino acids and is about 45 kDa (comparable to A1). Mutations in

PAGE 70

52 H 274 and H 251 also lead to loss of agonist and antagonist binding. Adenosine relaxes vascular smooth muscle via the A2 receptor mediated mechanism and thus increases tissue perfusion. In the retina, the vasodilatory eff ects of adenosine are mediated by A2 linked to potassium ATP channels.100,101 Adenosine increases glyconeogenesis via the A2 receptor and thus promotes an increase in the supply and demand ratio for metabolic substrates in the retina. A2A decreases the superoxide release from activated neutrophils and inhibits platelet aggregation. These ar e all anti-inflammatory actions, the importance of which in retinal response to ischemia has not been established. Ideally, a drug that is an A2A agonist and an A2B antagonist is needed to fu rther understand the two receptors.100 The A2B receptor was initially cloned from rat hypothalamus109, human hippocampus110 and mouse mast cells 100 The receptor was found in these tissues by PCR with degenerate DNA oligonucleotides that recognized conserved regions of the G protein coupled receptors. The human, rat and mouse A2B receptors share 86-87% amino acid homology.109 The human A1 and the human A2A, and A2B receptors share 45% amino acid homology.100 Closely related species such as rat and mouse share 96% homology. The A1 receptors have 87% amino acid hom ology in various species (Figure 1-19) 111,112, the A2A receptors have 90% homology (Figure1-20). 113 while the A3 receptors differ significantly between species.111,112 Figure 1-21 shows the homology between the human and the mouse A2B receptor.100

PAGE 71

53 1 50 A1 human (1) VPAMPPSISAFQAAYIGIEVLIALVSVPGNVLVIWAVKVNQALRDATFCF A1 mouse (1) ---MPPYISAFQAAYIGIEVLIALVSVPGNVLVIWAVKVNQALRDATFCF 51 100 A1 human (51) IVSLAVADVAVGALVIPLAILINIGPQTYFHTCLMVACPVLILTQSSILA A1 mouse (48) IVSLAVADVAVGALVIPLAILINIGPQTYFHTCLMVACPVLILTQSSILA 101 150 A1 human (101) LLAIAVDRYLRVKIPLRYKMVVTPRRAAVAIAGCWILSFVVGLTPMFGWN A1 mouse (98) LLAIAVDRYLRVKIPLRYKTVVTQRRAAVAIAGCWILSLVVGLTPMFGWN 151 200 A1 human (151) NLSAVERAWAANGSMGEPVIKCEFEKVISMEYMVYFNFFVWVLPPLLLMV A1 mouse (148) NLSEVEQAWIANGSVGEPVIKCEFEKVISMEYMVYFNFFVWVLPPLLLMV 201 250 A1 human (201) LIYLEVFYLIRKQLNKKVSASSGDPQKYYGKELKIAKSLALILFLFALSW A1 mouse (198) LIYLEVFYLIRKQLNKKVSASSGDPQKYYGKELKIAKSLALILFLFALSW 251 300 A1 human (251) LPLHILNCITLFCPSCHKPSILTYIAIFLTHGNSAMNPIVYAFRIQKFRV A1 mouse (248) LPLHILNCITLFCPTCQKPSILIYIAIFLTHGNSAMNPIVYAFRIHKFRV 301 329 A1 human (301) TFLKIWNDHFRCQPAPPIDEDLPEERPDD A1 mouse (298) TFLKIWNDHFRCQPKPPIEEDIPEEKADD Figure 1-19. Homology of the A1 receptor for human and mous e. The yellow sequences indicate homology between the human and mouse A1 receptor sequences. The white sequences indicate non-homol ogous regions and the blue sequences indicate conserved sequences.

PAGE 72

54 1 50 A2A human (1) --MPIMGSSVYITVELAIAVLAILGNVLVCWAVWLNSNLQNVTNYFVVSL A2A mouse (1) VASPAMGSSVYIMVELAIAVLAILGNVLVCWAVWINSNLQNVTNFFVVSL 51 100 A2A human (49) AAADIAVGVLAIPFAITISTGFCAACHGCLFIACFVLVLTQSSIFSLLAI A2A mouse (51) AAADIAVGVLAIPFAITISTGFCAACHGCLFFACFVLVLTQSSIFSLLAI 101 150 A2A human (99) AIDRYIAIRIPLRYNGLVTGTRAKGIIAICWVLSFAIGLTPMLGWNNCGQ A2A mouse (101) AIDRYIAIRIPLRYNGLVTGMRAKGIIAICWVLSFAIGLTPMLGWNNCSQ 151 200 A2A human (149) PKEGKNHSQGCGEGQVACLFEDVVPMNYMVYFNFFACVLVPLLLMLGVYL A2A mouse (151) KDEN--STKTCGEGRVTCLFEDVVPMNYMVYYNFFAFVLLPLLLMLAIYL 201 250 A2A human (199) RIFLAARRQLKQMESQPLPGERARSTLQKEVHAAKSLAIIVGLFALCWLP A2A mouse (199) RIFLAARRQLKQMESQPLPGERTRSTLQKEVHAAKSLAIIVGLFALCWLP 251 300 A2A human (249) LHIINCFTFFCPDCSHAPLWLMYLAIVLSHTNSVVNPFIYAYRIREFRQT A2A mouse (249) LHIINCFTFFCSTCQHAPPWLMYLAIILSHSNSVVNPFIYAYRIREFRQT 301 350 A2A human (299) FRKIIRSHVLRQQEPFKAAGTSARVLAAHGSDGEQVSLRLNGHPPGVWAN A2A mouse (299) FRKIIRTHVLRRQEPFRAGGSSAWALAAHSTEGEQVSLRLNGHPLGVWAN 351 400 A2A human (349) GSAPHPERRPNGYALGLVSGGSAQESQGNTGLPDVELLSHELKGVCPEPP A2A mouse (349) GSAPHSGRRPNGYTLGPGGGGSTQGSPG-----DVELLTQEHQEGQ-EHP 401 423 A2A human (399) GLDDPLAQDGAGVS--------A2A mouse (393) GLGDHLAQGRVGTASWSSEFAPS Figure 1-20. Homology of the A2A receptor between human and mouse. The yellow sequences indicate homology betw een the human and mouse A2A receptor sequences. The white sequences in dicate non-homologous regions and the blue sequences indicate conserved sequences.

PAGE 73

55 1 50 A2B human (1) MLLETQDALYVALELVIAALSVAGNVLVCAAVGTANTLQTPTNYFLVSLA A2B mouse (1) MQLETQDALYVALELVIAALAVAGNVLVCAAVGASSALQTPTNYFLVSLA 51 100 A2B human (51) AADVAVGLFAIPFAITISLGFCTDFYGCLFLACFVLVLTQSSIFSLLAVA A2B mouse (51) TADVAVGLFAIPFAITISLGFCTDFHGCLFLACFVLVLTQSSIFSLLAVA 101 150 A2B human (101) VDRYLAICVPLRYKSLVTGTRARGVIAVLWVLAFGIGLTPFLGWNSKDSA A2B mouse (101) VDRYLAIRVPLRYKGLVTGTRARGIIAVLWVLAFGIGLTPFLGWNSKDSA 151 200 A2B human (151) TNNCTEPWDGTTNESCCLVKCLFENVVPMSYMVYFNFFGCVLPPLLIMLV A2B mouse (151) TSNCTELGDGIANKSCCPLTCLFENVVPMSYMVYFNFFGCVLPPLLIMLV 201 250 A2B human (201) IYIKIFLVACRQLQRTELMDHSRTTLQREIHAAKSLAMIVGIFALCWLPV A2B mouse (201) IYIKIFMVACKQLQSMELMDHSRTTLQREIHAAKSLAMIVGIFALCWLPV 251 300 A2B human (251) HAVNCVTLFQPAQGKNKPKWAMNMAILLSHANSVVNPIVYAYRNRDFRYT A2B mouse (251) HAINCITLFHPALAKDKPKWVMNVAILLSHANSVVNPIVYAYRNRDFRYS 301 333 A2B human (301) FHKIISRYLLCQADVKSGNGQAGVQPALGVGLA2B mouse (301) FHKIISRYVLCQAETKGGSGQAGAQSTLSLGLFigure 1-21. Homology of the A2B receptor between the human and the mouse. The yellow sequences indicate homology between the human and mouse A2B receptor sequences. The white sequences indicate non-homologous regions and the blue sequences i ndicate conserved sequences.

PAGE 74

56 The membrane structure of the A2B receptors is that of a typical G protein coupled receptor consisting of a 7 transmembrane dom ains connected via 3 extracellular and 3 intracellular loops. (Figure 1-22)100,110,114 Trans membrane domains have a high de gree of amino acid homology in different species. The human, mouse and rat A2B receptors have 2 potential N-glycosylation sites in the second extracellular loop.109 The human N-linked glycosylation sites are Asp 153 and 163 which are in the second extracellular loo p. Both of these sites are conserved in all of the A2B sequences of all specie s that have been cloned.100,115 The A2A intracellular and the third intracellu lar loop are involved in coupling A2A receptor to G proteins. 100,111 The third intracellular loop is a 15 peptide portion of the A2A receptor which has 57% amino acid homology with the A2B receptor and also determines the selective coupling with GS.100,116 Both A2A and A2B are coupled to Gs. The A2A and A1 receptors have 27% amino acid homology and the A1 is not coupled to Gs. Amino acids in the second intr acellular loop ma y modulate the A2A receptor coupling since lysine and glutamic acid are necessary for efficient A2A adenosine receptor Gs coupling.100,116 Analogous lysine and glutamic acid residues are also present in the A2B receptor. The major difference between the A2A and the A2B receptor is the long intracellular C-terminal tail of the A2A. (Figure 1-23) This long tail is not involved in Gs coupling to the receptor. Removal of the c-terminal tail of the A2A receptor does not inhibit stimulation of adenylyl cyclase wh en truncated receptor is expressed in CHO cell.100,111,116 Mutational studies of the A2A receptors have shown that the Thr 298 residue of the C-terminal tail of the A2A receptor is located close to the seventh membrane

PAGE 75

57 Figure 1-22. The A 2B receptor is a G protein coupled receptor consisting of a seven transmembrane domain connected via 3 ex tracellular and 3 intracellular loops flanked by an extracellular Nterminal and an intracellular C-terminal.

PAGE 76

58 Figure 1-23. The A2A receptor structure consists of 7 transmembrane domains connected via 3 extracellular and 3 intracellula r loops flanked by an extracellular Nterminal and a long intr acellular C-terminal.

PAGE 77

59 span and is essential for the deve lopment of rapid agonist mediated desensitization.100,111,117 The threonine residue is also present in the human A2B (Thr 300), however, its role in receptor desensitization has not been explored. A2B receptors can be coupled to other intracellular signaling pathways in addition to Gs and adenylyl cyclase.100 The A3 receptor was cloned from the human, rat and sheep. It is composed of 320 amino acids and has about 40-50% homology to the A1 and A2 receptors. It has low affinity for alkylxanthine antagonists such as theophylline and caffeine (which is a classic antagonist for A1 and A2). The non specific A3 antagonist IB-MECA inhibits adenyly l cylcase and increase PLC, calcium mobilization and decrease TNF-alpha. Higher concentrations of adenosine are required to activate the A3 receptors than are required to activate the A1 or the A2 receptors.100 Pharmacology of the A2B receptors Highly selective and potent agonists designed for A1, A2A, and A3 receptors are available and are important tools for the char acterization of adenosine receptors. The lack of a potent selective A2B antagonist hampers the characterization of its cellular functions. 95,100 The most potent agonist for A2B is NECA.100,118-120 At a concentration of 2 M, NECA produces half the maximal effect (EC50) for stimulation of adenylyl cyclase.120 NECA is non-selective and thus ac tivates other adenosine receptors with greater affinity. The EC50 for the A1 and A2A receptors is in the low nanomolar range and that of the A3 receptors is in the high nanom olar range. Therefore, the characterization of the A2B receptor depends on the use of compounds, which are potent

PAGE 78

60 selective agonists of other receptor subtypes. Therefore the A2B receptor is usually characterized by exclusion.100 CGS 21680121 is an A2A selective agonist that can differentiate A2A and A2B receptors.100,122-125 The A2A and the A2B receptors are both positively coupled to adenylyl cyclase and are activated by the non selec tive agonist NECA. CGS 21680, on the other hand, is ineffective on A2B receptors and as potent as NECA when activating A2A receptors.100,120-122,126-128 R-PIA is an A1 selective agonist and the A2B receptor has low affinity for it.100,119,120 The pharmacological characte rization of the adenosine receptors is based on apparent agonist potencies. Th is is not ideal as it depends on agonist binding to the receptor and multiple processes of signal transduction. Therefore, for receptor subtype identification, selective an tagonists are preferable 100,129 Highly selective A2B antagonists are not available. However, it is known that A2B has a low affinity for agonists, but a high affinity for antagonists. Enprofylline (3 -n-propylxanthine) is an anti-asthmatic drug, and is the most selective, but not potent, A2B antagonist known. Other potent but nonselective A2B receptor antagonists include 1,3-dipropyl-8 (p-sulfophenyl)xanthine (DPSPX), 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), xanthine amine cogener (XAC) and IPDX 95,119,120 Distribution of the Adenosine Receptors Initially the A2B receptor mRNA was found in the rat. The highest levels of the receptor was found in the cecum, bowel, bladder, followed by the spinal cord, lung, epidydimus, vas deferens and the pituitary.100,130 Subsequently more sensitive RT-PCR showed that the A2B receptors were present in all tissues of the rat, with the highest level in the proximal colon and the lowest level in the liver.131 Primary tissue cultures have

PAGE 79

61 different adenosine receptors present in the cell s. This may be because there are different populations of cells and each cell expresses a different type of adenosine receptor.100,132134 Studies on established cell lines also show ed multiple adenosine receptor subtypes on a single target.100,122,124,125 Also, studies on single cells s how the presence of one or more adenosine receptor subtype.135-137 Clonal cell lines also ha ve co-expression of the A2A and the A2B receptors.100,124 However, subsequent studies showed minute amounts of other receptors too! Therefore it is possible that adenosine selective antagonists are needed to better characterize the distribution of these rece ptors in cells. It is however, unclear why there is simultaneous expression of multiple adenosine receptors in a single cell. Both A1 and A2A receptors have a high affini ty for adenosine and need to be blocked before the effects of the A2B receptor can be seen 100,135,136,138 However, this is not always the case and may be a reason for discrepancies published in the literature. El fman et al showed that glial cells of ra t astrocytes have A1 and A2B adenosine receptors which stimulate cAMP.133,139-141 However, when the cells were st imulated by the non-selective agonist, NECA, cAMP accumulation was se en even though there are A1 receptors present. Therefore it may be that the importance of the A2B receptors is maximal where adenosine receptor levels are high, such as, in tissues with a high metabolic demand or conditions when oxygen is decreased. Both the A1 and the A2 receptors may modulate the response to lower the concentrations of adenosine.100 The widespread unique localization of ade nosine suggests that it is well positioned to serve as mediator of important physiologi cal and pathophysiologi cal processes in the retina. 91,100,142 In the retina, adenosine receptors are lo calized to the same retinal layers as endogenous adenosine. In the mouse a tritiated A1 agonist, cyclohexyladenosine (CHA)

PAGE 80

62 was used to localize the A1 to the inner retina (over the inner plexiform layer) and the A2 receptor was localized to the RPE (outer re tina) and the outer and inner segments of photoreceptors by using tritiated NECA.100,142 No A3 receptor has been found in the retina. The location of adenosine receptor mRNA transcripts generally correlated with the autoradiographic localization of the A1 receptors, but not the A2 receptors.100 Intracellular Pathways Regulated by A 2B Receptors Adenosine receptors activate a diverse cascade of intracellular signaling. The A1 and A3 receptors inhibit adenylyl cyclase and stimulate PLC by activation of pertussis toxin sensitive G proteins Gi and Go.143 Adenosine binding to the A2A and the A2B receptors couples them to Gs and adenylyl cyclase pos itively, however, the A2B receptor is also active in other signaling pathways. The A2B receptor coupled to Gs can also increase calcium transport into the cells by the cholera toxin sensitive pathway. This pathway is cAMP independent even though it is coupled to Gs. 144,100 The A2B receptor is also coupled to G q and leads to the activation of two distinct pathways. One of those pathways lead to the activat ion of the MAPK pathway and the other pathway activates the PI3 kinase/PkB pathway. (Figure 1-24) Angiogenesis is a complex process and is the underlying cause of several retinopathies. Currently avai lable treatments for retinopathie s are painful and have had limited success. Since adenosine exerts its a ngiogenic effects upstream of VEGF, it is an attractive target for inhibiting the process of angiogenesis. However a lack of selective and potent A2B antagonists requires the use of mo lecular techniques to target the A2B receptor. One such approach is the use of ri bozymes to target receptors at the molecular level.

PAGE 81

63 Figure 1-24. The A 2B signaling pathway. The A2B receptor couples to Gs and G q and leads to an increase in calcium transport and also leads to the activation of the MAPK pathway

PAGE 82

64 Ribozymes Ribozymes are catalytic RNA molecules that cleave other RNA molecules. Ribozyme is short for ribonucleotide en zyme, which, catalyze the hydrolysis and phosphopryl exchange at the phosphodiester li nkages between RNA bases resulting in cleavage of the substrate. 145,146 146,147 Ribozymes can be classified into 3 main groups based on function and size: self splicing introns, RNase P, small self cleaving ribozymes. Self Splicing Introns Group I Introns Self splicing introns can be divided into 2 classes: Group I intron and Group II introns, based on the conserved secondary st ructure and splicing mechanisms (Figure 125). Group I introns are found in a wide number of species, such as, eubacteria, bacteriphages, fungal mitochondria, pl ant chloroplasts and rRNA of lower eukaryotes.148,149,150 The splicing action c onsists of two consecutive transphotoesterification reactions. In a tr ansphotoesterification reaction, the number of phosphodiester bonds remain constant, however the position of the bonds changes. (Figure 1-26)150,151 Only the Tetrahymena large rRNA group I intron has been shown to function without a protein in vivo. All ot her known group I introns require a single protein co-factor to provide a scaffold th at helps position the introns in a catalytic conformation.152

PAGE 83

65 Figure 1-25. The secondar y structure group I introns.

PAGE 84

66 Figure 1-26. Splicing mechanism of the group I introns

PAGE 85

67 Group II Introns Group II introns are self sp licing introns found within nuclear pre-mRNA and in the pre-mRNA of organelles from fungi and plants.153 (Figure 1-27) High concentrations of magnesium and potassium ions are essential for their proper folding.153 Group II introns also require a complex of protei ns and small nuclear RNAs (SnRNA) for cleavage. These components form the sp liceocome. Group II spli cing occurs via two consecutive trans photoesterifi cation reactions similar to group I introns. The main difference in the splicing mechanism between the two introns is the nature of the hydroxyl group, which initiates the initial phot otransesterification reaction. In group I introns, the reaction is ini tiated by the 3’ hydroxyl group of the exogenous guanosine and in the group II introns, the reac tion is initiated by the 2’ hydr oxyl group of the internal adenosine.154 (Figure 1-28) RNase P RNA RNase P is an endoribonuclease which removes the 5’ leader sequence from precursor tRNAs. RNase P has an RNA and a pr otein unit, both of which are essential to its function. The RNA component is the ca talytic component of the complex. The protein subunit enhances the turnover rate of the reaction by acting as a scaffold for the RNA that forces the RNA into a catalytic conformation.155,156 RNase P can recognize and cleave 60 different tRNA substrates.157 RNase P recognizes the structure of the tRNA and only a minimal tRNA structure is required fo r the creation of the RNase P cleavage site ( Figure 1-29 ). 158,157

PAGE 86

68 Figure 1-27. Secondary st ructure of Group II introns.

PAGE 87

69 Figure 1-28. The splicing mech anism of the Group II introns.

PAGE 88

70 Figure 1-29. Cleavage of the tRNA 5’ leader sequence by Rnase P.

PAGE 89

71 Small Self Cleaving Ribozymes Small Self cleaving ribozymes are nucle olytic RNA’s and are found naturally. They are associated with viruses and sa tellite RNA and can catalyze RNA cleavage reactions in the absence of protein. 159 There are several types of small ribozymes, the most extensively studied ones include: he patitis delta virus (HDV), hammerhead and hairpin ribozymes. The hammer head and ha irpin ribozymes are derived from tobacco ring spot virus satellite RNA. Hepatitis Delta Virus Hepatitis delta virus (HDV) is a short single stranded RNA found in patients infected with human hepatitis B. It ha s a circular RNA genome, which encodes a ribozyme in both orientations. HDV replicat es through a rolling circle mechanism like other self cleaving ribozymes (Figure 1-30), and the ribozyme is required for the cleavage of the HDV genome into discrete units prior to packaging. 160,161 Hairpin Ribozymes The hairpin ribozyme was originally found in the tobacco ring spot virus satellite RNA. The hairpin ribozyme binds the substr ate and forms a structure with 4 helices and 2 loops (Figure 1-31). The arms of the hairpin ribozyme hybridi ze to the substrate molecule to from helix 1 (6 base pair) and helix 4 (4 base pair). Loop A has a BNGUC target sequence required for cleavage, where B is G, C or U, and N is any nucleotide.162 There are no conserved nucleoti des in any of the helices. 163,164 Hammerhead Ribozymes The catalytic domain of the hammerhead ribozyme was discovered by comparing self cleaving RNA sequences of a number of diff erent viroid infectious RNA molecules.

PAGE 90

72 Figure 1-30. Self-cleaving ribozymes resolve concatemers formed by rolling-circle replication into individual genomic molecules

PAGE 91

73 Figure 1-31. Structure of the ha irpin ribozyme. The arrow indi cates the site of cleavage. The hairpin ribozyme binds the substrate and forms a structure with 4 helices (1-4) and 2 loops (A and B).

PAGE 92

74 Hammerhead ribozymes are small, appr oximately 34 base RNA molecules and cleave RNA target in trans. The hammerh ead ribozymes bind substrate to form a structure, which consists of a stem and thr ee loops and a catalytic core with a conserved nine nucleotide sequence (Figure 1-32). A mu tation in any of the conserved nucleotides prevents RNA cleavage.165 The catalytic core of the hammerhead ri bozyme has two functions: it destabilizes the substrate strand by twisting it into a cleavable conformation and binds the metal cofactor needed for catalysis.166 The hammerhead ribozyme cleaves the substrate by a tranesterification reaction (Figure 1-33). The reacti on requires the presence of magnesium and water. The hydrated magnesi um ion has two functions, both mediated by water molecules. First, one molecule of water binds to one of the oxygen atoms of the phosphate group, holding it in the proper orie ntation for the enzymatic mechanism. Secondly, the environment of the active site lo wers the pKa of another water molecule so that it can donate a proton to the aqueous envi ronment. In the transition state, five oxygen atoms are arranged in a triangular bipyramid around the phosphorus atom. A bond is formed between the 2’ oxygen of cytosine 17 and the phosphorus atom. Simultaneously a bond is broken between th e phosphorous atom and the hydroxyl oxygen of the next nucleotide, adenine 1.1. This leaves the cytosine with a 2’-3’ cyclic phosphate group. The 5’nucleotide recovers a proton from the aqueous environment, completing a hydroxyl group. The reaction products diffuse away from the active site leaving the ribozyme free to bind a second substrate molecule and complete another reaction cycle The hammerhead ribozyme recognizes substrate sequences on either side of a NUX cleavage site, where N is any nucle otide and X is any nuc leotide except G.

PAGE 93

75 Figure 1-32. Structure of the hammerhead ribozyme. The hammerhead ribozyme binds substrate to form a structure, which c onsists of a stem and three loops and a catalytic core with a conserved nine nuc leotide sequence. Arrow indicates site of cleavage.

PAGE 94

76 A B C D Figure 1-33. The hammerhead ribozyme cleav es its substrate by a transesterification reaction. A A molecule of water binds to an oxygen of the phosphate group. B. Another water molecule donates a proton. A bond is formed between the 2’ oxygen of cytosine 17 and the phosphorous atom. C. A bond is broken between the phosphorous atom and th e hydroxyl oxygen of adenine 1.1. D Cytosine remains with a 2’3’ cyc lic phosphate group. The 5’ nucleotide recovers a proton to complete a hydr oxyl group. The reaction products then diffuse away from the active site leav ing the ribozyme fr ee to bind a second substrate molecule and complete another reaction.

PAGE 95

77 The ribozyme anneals to the substrate mR NA by means of two flanking arms which hybridize to form helices III and I. Cleavage oc curs at the 3’ end of the cleavage site. Not all cleavage sites demonstrate the same efficiency. Generally, GUC is the most efficient cleavage site, then CUC, UUC and AUC. The remainder of the cleavage sites are cleaved at least 10 times less efficientl y than the GUC site. The hammerhead and hairpin ribozymes are being examined as gene therapies for a number of different diseases because they are small and can be easily cloned and packaged into many of the existing viral vectors for delivery to target cells. The advantage of the hammerhead ribozyme is that it can recognize a greater num ber of cleavage sites than HDV or hairpin ribozymes.167,168 Experimental Aim Currently, the only available treatment for ROP is laser treatment of the retina, which has limited success. The aim of this pr oject is to design a hammerhead ribozyme that will specifically target and cleave the A2B receptor mRNA resulting in a reduction in expression of the A2B receptor protein and a reduc tion of cellular and physiological functions affected by this receptor. We are using a hammerhead ribozyme primarily as a tool to study the path ways that involve A2B. But this ribozyme can also be used as ‘proof of concept’ for conventio nal drugs targeting the A2B receptor and, finally there is a possibility that the hammerhead ribozyme itself could be used as a therapeutic agent. The goal of this project was to examine the effectiveness of ribozymes in the treatment of ROP. Previously we have s hown that proliferative blood vessels have an enhanced expression of the A2B receptor, therefore, there is justification to target this protein in controlling the disease. Ribozymes were designed to specifically cleave the mRNA of the A2B receptor to decrease the expression of the receptor protein. The

PAGE 96

78 underlying hypothesis was that the cleavage of the mRNA of the A2B receptor at the mRNA level would prevent translation of th e protein and subseque ntly progression of angiogenesis in ROP by preventing the growth of abnormal blood vessels. (Figure 1-34) The selected target site was a s hort region of the mRNA for the A2B receptor. The first step of the project was to design a ri bozyme to cleave the sequence of the A2B receptor in the mouse and the human. (Figure 1-35) Two hammerhead ribozymes were developed, each of which had an inactive version with a single base mutation. (Figure 1-36) The most efficient ribozyme was cloned into an rAAV construct (p21Newhp) for further analysis. (Figure 1-37) The s econd step of the project was to develop in vitro assays to examine the ability of the ribozyme to cleav e the mRNA. These assays were used to determine if the ribozymes would be effectiv e for reducing pre-retinal neovascularization in an oxygen-induced mouse model of retinopathy. To test the ribozyme in vivo, the A2B Rz2 was intravitreally injected to the mouse model. Several models for oxygen-indu ced retinopathy have been developed. Dembinska169 and Chowers170 both used a rat model. The rats were placed in alternating hypoxic and hyperoxic environments. The alterna ting environments lead to severe retinal complications, which were not representative of retinopathy in huma n babies. Since the timing and duration of the hypoxia was inadequate, it gave inconsistent results. In our study we used a mouse model developed by Louis Smith.171 An advantage of using the mouse retina is that in the newborn mouse the retinal vesse l development stage is the same as that of premature human babies. Also, normal retinal vascular development in mice occurs within two weeks of bi rth, thus illustra ting the evolution

PAGE 97

79 Figure 1-34. Cleavage of the A2B receptor by a ribozyme prevents translation of the protein.

PAGE 98

80 Figure 1-35. Target sequences of the human and mouse A2B ribozymes 1 and 2. Red indicates a difference in sequence be tween the human and mouse species.

PAGE 99

81 Figure 1-36. Hammerhead ribozymes for the A 2B Rz1 and Rz2. The target sequences are indicated in red. For each ribozym e an inactive version of the ribozyme was made with a C replaced by the G (arrow).

PAGE 100

82 Figure 1-37. A)The p21Newhp Vector with th e CMV enhancer and beta actin promoter. The hammer head ribozyme was cloned between the HindIII and SpeI sites. B) The hammerhead and the hairpin cleavage sites.

PAGE 101

83 of the vascular bed. Mouse retinal vessels develop from spindle-cell precursors, which are found in the superficial layer of the retina and the deeper retinal vessels develop later. The same developmental pattern is observed in the human retina. Thus the similarities between the mouse retinal vasculature and th at of the human premature babies makes it amenable for use in a model of ROP to better understand the human form of the disease. In the Smith model, seven day old mouse pups are placed in a 75% oxygen chamber for 5 days. Upon return to normal air, these mice developed retinal neovascularization. Five days following retu rn to normoxia (day 17), the animals are sacrificed and the eyes rem oved (Figure 1-38). Smith described two methods to quantify the neovascularization in the mice retina while minimizing the error and making the model applicable to retinopathy.171 First, Smith recommended fluorescein labeled dextran perfusion of the animals into the left ventri cle followed by flat mounting of the retinas. The perfusion technique delin eated the blood vessels, however, it was not permanent as the fluorescein is sensitive to light. Thus, perfusion was recommended for a quick survey of the blood vessels present in the retina following expos ure to hyperoxia. A second method that Smith recommended was counting the vascular nuclei in paraffin cross sections of the retina. Serial cross sections were stained with hematoxylin and eosin and the blood vessels were quantified by counting th e number of vascular cell nuclei on the vitreal side of the inner limiting membrane This method is more time consuming, however, it was reported by Smith to be mo re sensitive, reliable and reproducible.171 This model has also been used by our la b for other studies pertaining to retinal development. For example, Mino et al used this model to examine the effects of various adenosine antagonists on neovascularization. 95

PAGE 102

84 Figure 1-38. Time course for the ROP model. Seven day old pups are placed in a 75% oxygen chamber for 5 days. On day 12, the mice are returned to normal air and their eyes enucleated on day 17.

PAGE 103

85 CHAPTER 2 METHODS AND MATERIALS Defining Location of the Target Sequence It is important to define the region of target mRNA where the ribozyme will bind and cleave. For hammer h ead ribozymes the highest kcat (enzymatic rate of catalysis) has been observed for a GUC ribonucleotide sequence. Thus, it is important to locate all the GUC triplets within the target mRNA. To decide where in the mRNA to target the ribozyme, Genbank was used to obtain sequences for the human A2B receptor and for the mouse A2B receptor. The results were transferred into Vector NTI and all of the GUC sequences identified. All the sites containing the GUC triplet were considered to be potential targ et regions, however, additional criteria were used to narrow thes e potential sites. Target regions with six nucleotides on either side of the arms consis ting of a 50% GC content were selected since an ideal length for the target region is betw een 6-7 nucleotides. The presence of a U/A at the 3’ cleavage site also enhances the kcat ten fold. Therefore, a pplication of these criteria reduced the potential number of target sequences to those containing GUCU/GUCA regions. Selected target regions were examined using the RNA folding algorithum designed by Micheal Zucker.172 The folding program was used to fold 00-200 nucleotides on either side of the GUC target to determine whether the ribozyme binding location was acceptable. A blast search was also conducted to en sure that the ribozyme did not cleave another known mRNA sequence within th e known mouse or human sequences.

PAGE 104

86 Preparation of the Target Oligo-Nucleotide. The target oligonucleotide to be used in the cleavage reactions was radioactively labeled at the 5’end using T4 polynucleot ide kinase (New England Biochmeicals; Beverly, MA). The reactions were set up as follows: 2 l of the RNA oligo (10pmol/ l, 20pmol total) was added to a mixture contai ning 1ul of 10X polynucleotide kinase buffer (Promega, Madison, WI), 1ul RNASin (Pro mega, Madison, WI), 1ul 0.1M DTT (Sigma, St. Louis, MO), 3 l water, 1 l (gamma 32P) dATP (10uci) (ICN Santa Clara CA) and 1 l of polynucleotide kinase (5 units) (Sig ma, St. Louis, MO). The reaction was incubated at 37 C for 30 minutes. 90 l of TE (Fisher, Swanee, GA) was added to the reaction prior to extraction of the unincorporated nucleotides. A spin column (1ml syringe) was prepared with sterile glass wool and loaded with sephadex (Sigma, St. Louis, MO) saturated in water. The column was centrifuged at 1000 RPM for 5 minutes to remove any excess water and to further pack the sephadex. The 32P labeled target (100 l) was loaded on to the column. The column was sealed with parafilm and centrifuged again at 1000 RP M for 5 minutes. The labeled elute was collected in a 1.5ml Eppendorf tube (Fishe r, Swanee, GA) and was stored at -20 C. Time Course of Cleavage Reactions fo r Mouse and Human Targets (Hammerhead Ribozymes) To evaluate the time course of cleavage of the ribozyme for the target, a cleavage reaction was set up as follows: 13 l of 400mM Tris-HCL (Fisher, Swanee, GA), pH 7.47.5 was added to 1ul ribozyme (2pmol) and 88ul of water. The mixture was incubated at 65 C for 2 minutes and then left at room temperature for 10 minutes. 13 l of a 1:10 ratio of RNASEin:0.1M DTT was added to the reaction mixture along with 13 l of 200mM Magnesium chloride (20mM final) (S igma, St. Louis, MO). The reaction was

PAGE 105

87 then incubated at 37 C for 10 minutes. 1 l of the 32P labeled target (0.2 pmol) and 1 l of the cold target (20pmol total) were prem ixed and added to the reaction mixture at 37C. Time points were take n at 0, 1, 2, 3, 4, 5, 10, 15, and 30 minutes and at 1, 2, and 3 hours and overnight. For each time point, 10 l of the reaction mixture was removed from 37 C and added to a tube containing 10ul of formamide dye mix (90% formamide (Sigma, St. Louis, MO), 50mM ehtylenediam ine tetra acetic acid (EDTA) pH 8 (Fisher, Swanee, GA), 0.05% bromophenol blue (Sigma, St. Louis, MO), and 0.05% xylene cyanol (Sigma, St. Louis, MO). The sample s were initially placed on ice and then heat denatured at 90 C for 3 minutes. The denatured sa mples were cooled on ice before loading 6 l onto a 10% PAGE-8M urea gel to sepa rate the products. Bromophenol blue was run about 2/3 down the gel. The gels were analyzed on a molecular dynamics phosphoimager. Multiple Turnover Kinetics Multiple turnover kinetics were perfor med on the ribozymes. Reactions were performed in a final volume of 20 l. Ribozyme (0.3 picomol, 15nM final) in 40mM Tris –HCL (pH 7.5) was incubated at 65 C for 2 minutes and then incubated at 25 C for 10 minutes. DTT (20mM final) and magnesium chloride (20mM final) and 4 units of RNasin were added. The reac tions were incubated at 37 C for 10 minutes, and cleavage was initiated by the addition of increasing con centrations of the targ et oligonucleotide (0300 picomol; 0-1500 nM final). The reactions were incubated at 37 C for a fixed interval determined in the time course analysis of cleavage. A vari ation on this protocol was incubation at 25 C in 1mM MgCl2. Reactions were terminated by the addition of 20 l of formamide stop buffer and held on ice. The samples were then heat denatured at 95

PAGE 106

88 C for 2 minutes, placed on ice and th e reaction products separated on 10% polyacrylamide-8M urea gels. The gels were analyzed on a molecular dynamics phosphoimager. Cloning of the Hammerhead Ribozymes into the rAAV Expression Vector Two complimentary DNA oligonucleotides (Invitrogen, Carlsbad, CA) were annealed in order to produce a double stranded DNA fragment coding for each hammerhead ribozyme. All DNA oligonucleotid es were synthesized with 5’phosphate groups. The DNA oligonucleotides were designed to generate a cut Hi ndIII site at the 5’ end and a cut SpeI site at the 3’ end afte r annealing. The DNA oligonucleotides were incubated at 65 C for 2 minutes and annealed by slow cooling to room temperature for 30 minutes. The resulting double stranded DNA fragment was ligated into the HindIII and SpeI sites of the rAAV vector pTRUF-21 (UF vector Core, http://www.gtc.ufl.edu/gtc-home.htm). A self cleaving hairpin ribozyme has been cloned downstream of the inserted hammerhead ribozy mes into the SpeI and NsiI sites. This vector has the cytomegalovirus (CMV) beta -actin chimeric enhancer-promoter and results in the hairpin ribozyme cleaving ei ght bases downstream of the 3’end of the hammerhead ribozymes. The ligated plasmids were transformed into SURE electroporation competent cells (Stratagene, La Jolla, CA) in order to maintain the integrity of the inverted terminal repeat s. The ribozyme clones were verified by sequencing. Sequencing of the Clones Prior to sequencing the hammerhead inserts, the integrity of the inverted terminal repeats (TR’s) was verified by digestion w ith the restriction en donuclease SmaI. This

PAGE 107

89 digest also served to determine the appr oximate concentration of the plasmid. The hammerhead inserts were verified by sequencing using the Ladderman Dideoxy sequencing kit (TaKaRa Shuzo Co, Japan) ac cording to the manu facturers protocol. Human Retinal Endothelial Cell (HREC) Tissue Culture HREC were isolated from human donor eyes within 24 hours of death. The eyes were placed on a sterile ga uze pad (Johnson and Johnson Me dical Supplies, Arlington, Texas) in a laminar flow hood and washed with 5ml betadine (Courtesy, Shands Hospital, Gainesville, Fl). Sterile s calpels (No. 1, Feather Industr ies limited, Japan) and tweezers were used to dissect the eyes and remove th e neural retina from th e posterior portion of both eyes. The RPE layer was not included in the harvested retina. The retinae were placed on a 53 micron mesh nylon membrane (T etko Inc, Lab Pack, Kansas City, MO) and the remainder of the ocular components discarded. The retinas were washed with phosphate buffered saline (PBS) containing 2% antibiotic/antimycotic mix (ABAM) (Sigma, St. Louis, MO). A sterile wooden sp atula was used to grind the retina over the nylon membrane while washing. The remaining re tinae were aspirated into a sterile 10ml pipette and added to a 20ml Erlenmeyer flask containing 10ml of PBS with antibiotics. Approximately 1mg of collagenase (342 u/m g, Worthington Biomedical Corporation, Lakewood, NJ) was added to the flask and placed in a 37 C water bath for 15 minutes. The flask contents were mixed every 5 minut es to keep the collagenase dissolved. Following the 15 minute incubation in the 37 C water bath, 20ml of complete endothelial cell media was a dded to the flask. (250ml Dulbelco’s Modified Eagle Medium (DMEM) low glucose, 250 ml HAM’s F12, 10% fetal bovine serum, 15% endothelial cell growth supplement, 15% insu lin/transferring/selen ium, 2% L-glutamic

PAGE 108

90 acid, 2% antibiotic/antimycotic mix). The cells were washed twice with media and plated onto a T25 flask (Fisher, Springfiel d, NJ) coated with 1% gelatin (Sigma, St. Louis, MO). The cells were allowed to gr ow and attach for 48-72 hours before changing the media and adding fresh antibiotics. Once th e T25 flask was confluent, the cells were passed and split into T75 flasks using 5ml of trypsin EDTA solution for endothelial cells (Sigma, St. Louis, MO). For passing the cells the cells were washed twice with PBS and then 5ml of trypsin added. Th e flask was placed into the CO2 incubator for 45 seconds. The trypsin was neutralized usi ng 2x volume of the complete endothelial cell media. The cells were centrifuged at 1000 RPM in an Eppendorf CT 5810R. The pellet was resuspended in 6ml of complete endothelial cell media and plated in a T75 flask (Fisher, Swanee, GA) containing 15ml of complete endothelial ce ll growth media and fresh antibiotics. LDL Uptake of the HREC HREC were seeded in a 6 well tissue cultu re plate (Corning Incorporated, Corning, NY) and allowed to attach and grow to a bout 80-85% confluency. Morphology of the HRECs was observed and recorded using a Ca rl Zeiss Microscope (Zeiss, Goettingen, Germany). The HREC were washed twice with Hanks Balanced salt solution (HBSS) (Sigma, St. Louis, MO). 50 g/ml of Dil labeled (1,1’-di octdycl-3,3,3’,3’-tetramethylindocarbocyanin perchlorate) acetylated LDL (Molecular probes, Eugene, OR) was added to the cells overnight in serum free me dium. The cells were then washed with HBSS and examined by fluorescence microscopy for the uptake of the label. The cells were quantified as a percentage of labeled cells/total cells.

PAGE 109

91 Transfection of HREC using DEAE-Dextran HREC were transfected at 70% confluency on an 150 mm tissue culture dish (BioRad, Hercules, CA). The cells were washed twice with PBS (Biowhittaker, Walkersville, Maryland) and 10.5ml of 10% Nuserum (B io-Rad, Bedford, MA) was added to the culture dish. (10% Nuserum has all the ingr edients of endothelial cell complete media except for the fetal bovine serum). 10 g of DNA in Tris buffered saline (TBS) (BioRad, Hercules, CA) (total volume of 108 l) was prepared in an eppendorf tube. 216 ul of DEAE dextran (diethylami noethyl-dextran) (Sigma, St. Louis, MO) (10mg/ml) were added dropwise to the tube while co nstantly mixing on a vortex mixer. 324 l of this mixture was added to the 150mm tissue cultur e dish containing 10.5ml of 10% Nuserum. The mixture was added dropwise across the pl ate and the plate swirled to distribute completely. Immediately, 8.1 l of chloroquine (100 mM) (Sigma, St. Louis, MO) was added to the plate and swirled. Chloroquine prevents the lysosm es from releasing DNAses that would destroy the DNA. The plates were incubated at 37 C for 4 hours in a CO2 incubator and the plates were mixed ever y 15 minutes to ensure even distribution of the plasmid. After incubation, the cells were treated with 15ml of 10% dimethly sulfoxide (DMSO) (Sigma, St. Louis, MO) in PBS for 1 minute. The cells were then washed twice with PBS. 20ml of complete e ndothelial cell media was added to the plates and the plates held in a CO2 incubator at 37 C. Media with fresh antibiotics was replaced after 24 hours. The cells were then allowed to grow for about 48 hours following replacement of media before harv esting the cells for further analysis.

PAGE 110

92 Transfection Efficiency using DEAE Dextran for HREC’s To determine the efficiency of HREC transfection, HREC we re transfected as described above, using a plasmid expressing GFP (PTRUF11) obtained from the Vector Core at the University of Florida. The level of GFP expression was observed using a Carl Zeiss fluorescent microscope (Zeiss, Goettingen, Germany). Cell Migration Assay HREC were grown in 150mm tissue culture dishes and transfected using the DEAE dextran method. The HREC were washed twice with PBS. 7ml of trypsin was added to the culture dish and incubated at 37 C in a CO2 incubator for 45 seconds. The cells were observed during this period to ensure maximal detachment of the cells. The trypsin was neutralized using 14ml of complete endothelial cell media containing fetal bovine serum. The cells were then centrifuged at 1000 RPM for 5 minute. The supernatant was discarded and the cells washed three times with basal media. The number of cells was determined with a hemacytometer (Hausser Scientific Horsham, PA). The cells were suspended in 50 l of PBS to a final concentration of 10,000 cell/ l. 30ul of the resuspended cells were loaded into the wells of a Chemotaxis Chamber (Neuro Probe Inc, Gaithersburg, MD ). The wells were covered with a 12uM porous polycarbonate membrane (Neuro Probe Inc, Gaithersburg, MD) pre-coated with 10% bovine collagen. The chemotaxis chamber was then sealed and placed upside down in a 37 C CO2 incubator. This allowed the cells to attach to the lower side of the 12 M porous membrane. After 4 hours, the chamber was removed, and the cells stimulated with 1, 10 or 100 M concentrations of NECA (dissolv ed in endothelial cell basal media (serum free). 50ul of the positive control (c omplete endothelial cell media), negative

PAGE 111

93 control (endothelial cell basa l media), or NECA concentratio ns were added to the top of the chamber. The chamber was then incubated at 37 C in a CO2 incubator for 12 hours, to allow the attached cells to migrate thr ough the porous membrane. The chamber was disassembled and the porous membrane removed from the chamber. The dull side of the membrane (c ontaining all the attached cells) was scraped using a cell scraper. The membrane was then stained using a diff Quik R stain set (Dade Behring Inc, Newark, DE) and mounted onto a gl ass slide. The migrated cell nuclei per well were counted per high power field. Morphology of HEK Cells HEK 293 cells were obtained from Amer ican Type Culture Collection (ATCC) (Manassas, VA) and plated onto a 150 mm tissu e culture dish. The cells were allowed to attach and grow to about 80% confluency. To ensure a homogenous population of HEK cells, the morphology of the HEK cells was r ecorded using a Zeiss microscope (Zeiss, Goettingen, Germany). Transfection using Lipofectamine on HEK 293 cells HEK 293 cells (ATCC, Manassas, VA) were seeded onto a 150mm tissue culture dish and allowed to attach and grow to 85-90% confluency. The HEK cells were fed with 1X high glucose Dubellco’s modified Eagle medium (Gibco, Carlsbad, CA) containing 2% fetal bovine serum and 2% ABAM Prior to transfection, the media was removed and 20.1 ml of high glucose DMEM w ith 2% FBS was added to the cultured cells. The antibiotics were not added fo r the duration of th e transfection assay. 33.5g of DNA was dissolved in 2,010 l of optimem I reduced serum media (Gibco, Carlsbad, CA) (final volume). 134ul of lipofectamine 2000 reagent (Invitrogen,

PAGE 112

94 Carlsbad, CA) was also diluted in 2,010 l of optimem (final volume) and incubated at room temperature for 5 minutes. The d iluted DNA was then added to the diluted lipofectamine reagent and incubated at room temperature for a further 20 minutes. The entire mixture was then added to the cultu red HEK 293 cells and gently swirled. The cells were incubated at 37 C in a CO2 incubator. After 24 hours, the media was changed and the antibiotics replaced. Transfection Efficiency for HEK Cells using Lipofectamine Reagent The HEK cells were transfected as ab ove with the plasmid pTRUF-11 which expresses GFP. GFP expression was determin ed using a fluorescence microscope at: 6hr, 24 hr, 48hr, 72 hr, 96hr, P1 and P2. cAMP Assay on Transfected HEK 293 Cells HEK cells transfected with the plasmids expressing either the active or inactive ribozymes, or the control plasmid were ha rvested for the cAMP assay. A Bradford protein assay (Bio-Rad) was used to determin e the amount of protei n in the transfected cells. A stock solution of 1mg/ml of BSA (S igma, St. Louis, MO) was used to prepare the standard curve. 100ul final volum e of protein standards containing 50 g, 100 g, 200 g and 300 g of BSA were prepared in water. The unknown samples were diluted 1:10 with water to a final volume of 100 l. 2ml of protein assay dye reagent (Bio-Rad, Hercules, CA) diluted 1:5 with water wa s added to all the standards and unknown samples. The absorbance of standards and samples at 595nm was determined using a Beckman spectrophotometer. The concentration of protei n in the unknown samples was determined by comparison to the linear regressi on plot of the standard curve. Based on the protein concentration in the unknown samp les, the unknowns were diluted to a final

PAGE 113

95 concentration of 1.5mg/ml with Hanks balanc ed salt solution (HBSS) (Gibco, Carlsbad, CA.). Unknown samples were diluted immediat ely prior to the addi tion of the adenosine agonist. For each transfected dish, a total of 16 Eppendorf tubes were set up and the cells stimulated with NECA. Basal levels were determined in duplicate in the absence of NECA. Seven concentrations of NECA were prepared from a 1x10-2 stock solution (1x10-4, 5x10-5, 1x10-5, 5x10-6, 1x10-6, 1x10-7, 1x10-8 (final concentrations in HBSS). Each concentration of NECA was done in duplicate. Tubes cont ained 150ul of HBSS and 50ul of cells (1.5mg/ml). Cells were pre-warmed in a 37 C water bath for 5 minutes. Following the incubation, 50ul of a phosphodiestera se inhibitor (10 M roliprom (Sigma, RBI, St. Louis, MO) (100 mM final concentration ) was added to all the tubes including the basal tubes. 5ul of the NECA concentra tions were added in duplicate to all 16 tubes. No NECA was added to the basal tubes. The tubes were incubated for a further 10 minutes at 36 C water bath. To end the reacti on, all tubes were placed in a boiling water bath for minutes. The tubes were cooled to room temperature and centrifuged at 2000 rpm for 2 minutes. The s upernatants were then assayed for cAMP content. A cAMP standard curve ranging from 1 nm ol to 25 pmol was prepared using a stock solution of 1x10-3 M cAMP (Sigma, St Louis, MO). Each unknown was assayed in duplicate. Additional tubes used to dete rmine total binding and non-specific binding were also included. The resultant cAMP sta ndard curve ranged from the non-specific to the total binding and was a sigmoidal curve. The samples were expected to fall within the linear range of the sigmoidal curve.

PAGE 114

96 For samples, 50 l of the supernatant was added to a glass test tube. To the nonspecific tube, 50 l of the highest cAMP concentration was added. 50 l of (125I) ScAMP (adenosine 3’5’ cyclic phos phoric acid 2’) -succinyl (125I) iodotyrosine methyl ester, courtesy Dr. John Shyrock, Dept of Pharm acology and Therapeutics, University of Florida, Gainesville) was added to all the tubes including the cAMP standard curve. 50 l of the cAMP antibody (Accurate Chemical Company, Westburg, NY) was also added to all tubes including the standard curve. The tubes were then covered with parafilm and incubated at 4 C overnight for at least 12 hours. Next, 75 l of a hydroxyapatite solution (Biome dical Research and Development Laboratories), diluted 1:3 with water, was added to the tubes. The cAMP and the antibody bound to the hydroxyapatitie. The t ubes were then washed with cold 10mM Tris-HCl pH 7.0 using a cell ha rvester (Brandell, Gathersburg, Maryland). A glass fiber filter, grade No.32 (pore size 2.3 um) (Schleicher and Schuell, Keena, NH) was used to trap the complexes bound to the hydroxyapatite. The individual filters were then placed in a Beckman G5500 counter which measured the 125I emissions in a one minute window. The results were analyzed with the statisti cal analysis and Graphics software, Prism (Graphpad software, San Diego, CA) was used. The unknown sample concentrations were determined from the standard curve as pmole per sample of cAMP accumulated. The cAMP per mg of protein was determined. Basal values were subtracted from the NECA stimulated tubes to obt ain a net response of NECA. Based on these values, a dose response graph was plotted. Maximum binding was determined by the (125I) ScAMP bound in the absence of additional non-radi oactive cAMP. Non-specific binding was

PAGE 115

97 determined by the addition of 500 pmoles cA MP and was subtracted from all the other readings before plotting the standard curve. By subtracting non specific binding from the total binding, specific binding for the assay wa s determined. The percent inhibition of (125 I) ScAMP binding to the antibody by additi onal cAMP (1nmol to 25 pmoles) was graphed. As the cAMP c oncentraion increased, less 125 I was detected. An EC 50 for the effect of NECA was also calcu lated. EC 50 is the effective concentration that displays a 50% response in cAMP production. Total Retinal RNA Extraction for PCR Total retinal RNA was isolated from HEK 293 cells using TRizol LS reagent (Invitrogen, Carlsbad, CA) followi ng the manufacturers protocol. Real Time PCR The cDNA was synthesized using either 2 or 4 g of total RNA and TaqMan R reverse transcription reagents (PE App lied Biosystems, Foster City, CA) in 100 l RT reactions. TaqMan R real time PCR analysis was app lied using 1ul cDNA per reaction and SYBR R Green PCR core reagents on AB I prism sequence detection system 5700 (PE Applied Biosystem, Foster City, CA). In each experiment, a standard curve for each primer pair was obtained using a serial dilution of total RNA samples prepared from cells that over expressed A2B adenosine receptor. At the end of the PCR cycle, a dissociation curve was generated to ensure the amplific ation of a single product and the threshold cycle time (ct values) for each gene was determined. Relative mRNA levels were calculated based on the ct values and normalized to a house-keeping gene: cyclophilin (100%).

PAGE 116

98 Animals All animals were treated in accordance w ith the IACUC of the University of Florida and the ARVO statement for the use of animals in ophthalmic and vision research. All animal protocols were approve d by the IACUC at the University of Florida prior to experimentation. C5BL6/J pregna nt mice on gestation day 14 were obtained from Jackson Laboratory (Bar harbor, ME). The mice were housed in the University of Florida Health Science Animal Resources facili ties. A Total of 24 animals were used (8 animals per plasmid) Animals were sacrificed by injecting a lethal dose of ketamine followed by spinal dislocation. Intraocular Injection into the Mouse Model of Oxygen Induced Retinopathy One day following birth, the mouse pups r eceived a 0.5ul intrav itreal injection of plasmid (2mg/ml) OD (right eye). In th e neonatal mouse model of oxygen induced retinopathy, 7 day old mice were placed with their nursing dams in a 75% oxygen atmosphere for 5 days. The oxygen chamber was monitored with an oxygen sensor within the closed chamber. The chamber oxyge n level was maintained for 5 days at 75%. After the fifth day, the oxygen chamber was sl owly returned to 21% oxygen over a period of one hour. The animals were removed from the oxygen chamber and placed in clean bedding. Upon return to normal air, these mice developed retinal neovascularization, with peak development occurring 5 days after their return to normoxia. After the fifth day following return to normoxia (day 17), the animals were sacrificed and the eyes removed and fixed in 4% paraformaldehyde and embedded in paraffin. Three hundred serial sections (6 m) were cut sagitally through the co rnea parallel to the optic disc. Every thirtieth section was placed on slid es and stained with hematoxylin and eosin (H&E). This resulted in te n sections from each eye being scored in a masked fashion

PAGE 117

99 using light microscopy by c ounting endothelial cell nucl ei extending beyond the inner limiting membrane into the vitreous. The e fficacy of treatment with each plasmid was then calculated as the percent average nuclei pe r section in the injected eye versus the uninjected control. Statistical Analysis. Student T-test was used to evaluate the da ta generated for all the experiments. A p value of less than 0.05 was considered to be significant.

PAGE 118

100 CHAPTER 3 RESULTS The expression of the A2B receptor upregulates VEGF expression which in turn leads to angiogenesis. The hypothesis underl ying this research project was that a decrease in the A2B receptor expression may prevent or decrease the severity of the angiogenesis. In this study, two differen t ribozymes were desi gned to cleave the A2B receptor mRNA. Two corresponding inactive versions of the ribozyme were also made. Determining Accessibility of the Target Site Success of ribozyme therapy depends on the id entification of an RNA target site. The m-fold program analysis was used to determine if the chos en target would be accessible to the ribozyme for binding. Figure 31 shows the theoretical tertiary structure of the active A2B Rz1 and Figure 3-2 shows the active A2B Rz2 mRNA under cellular conditions. Ribozymes generally fold into one of four secondary structure types. Type A, B, C and D. Type A structures have the highest activity based on the delta G values. The targeting arms in the type A structure do not interact to form a secondary structure and thus are readily available for target bindi ng. Types B and C compete with the type A structure and reduce the activity of the ri bozyme. However, these structures are catalytically active in vitro and should thus be effective in vivo. Ribozymes that form

PAGE 119

101 Figure 3-1. Theoretical tertiary structures of the active A2B Rz1 generated by the mfold program.

PAGE 120

102 Figure 3-2. Theoretical tertiary structures of the active A2B Rz2 generated by the mfold program.

PAGE 121

103 structures which are significantly more stable than the type A, such as type D, can completely inhibit the ribozymes catalytic ac tivity and should not be considered for in vitro and in vivo studies. The m-fold analysis showed that the A2B Rz1 could fold into two structures, types A and C, with dG’s of -7.6 and 7.0 kcal/moles respectively. The A2B Rz2 analysis by mfold produced three structures types A, B and C with dG’s of -7.6, -7.0 and -6.9 kcal/mol respectively. Based on this analysis both of the A2B ribozymes should be effective in vitro, and, therefore we deci ded to test both ribozymes. Time Course of Ribozyme cleavage Time course of cleavage analysis was done for the A2B Rz1 and the A2B Rz 2 as described in the Methods section. Figur e 3-3 is an autoradiograph from a 10% polyacrylamide-8M urea gel used to sepa rate the products of cleavage of the A2B Rz2 on the mouse target for reactions performed at 37 C and at 20 mM MgCl2. The autoradiograph shows an increase in the 5’cleaved product over tim e and a corresponding decrease in the target. Significant produc t accumulation was found at 1 minute after the addition of the target to the ribozyme. Fi gure 3-4 shows the graphical representation of the data in Figure 3-3 in addition to the data for the A2B Rz1 on the mouse target. The A2B Rz2 appears to have the hi gher rate of cleavage. Kinetic analysis on the hammerhead riboz ymes is performed at the time point where no more than 15% of the target has been cleaved. We select this point because the ribozyme cleavage rate is at a maximum and is linear at this point and the ribozyme is saturated by the target. Figur e 42 shows that this time point is less than one minute for the A2B Rz2. Since we do our kinetic analysis manually, as opposed to using a flow

PAGE 122

104 Figure 3-3. Time course autoradiograph of a 10% polyacrylamide 8M urea gel showing products of cleavage of the A2B Rz2 on the mouse target. The autoradiogarph shows an increase in the 5’ cleava ge product over time and a corresponding decrease in target.

PAGE 123

105 0.0 0.2 0.4 0.6 0.8 1.0 020406080100120 Time (minutes)Fraction of Target Cleaved A2B Rz1 A2B Rz2 Figure 3-4 Time course analys is data. Graphical representation of the cleavage for the A2B Rz1 and A2B Rz2 appears to have a higher rate of cleavag e with 15% of the target being cleaved in less than one minute.

PAGE 124

106 cytometer, we decided to reduce the rate of cleavage of both ribozymes in order to find a time point where manual kinetic analysis could be easily completed. Time course reactions were performed with increasing concentrations of the target (1:10, 1:40, 1:66, 1:100; Rz:Tar). (Figure 3-5) Increasing the target concentrations also did not slow the ribozyme down enough to asse ss the time point at which 15% of the cleavage occurs. Next the temperature and Mg Cl2 concentration were varied for the time course of cleavage reactions. Figure 3-6 shows the results of this analysis for the A2B Rz2 which yielded a time point of about 1 minute where the amount of target was reduced by 15%. Based on this type of analys is we did kinetic analysis on the A2B Rz1 at 37 C at 20mM MgCl2 at a time point of 6 minutes, and on the A2B Rz2 at 25 C at 1 mM MgCl2 at a time point of 1 minute. Multiple Turnover Kinetics To ensure cleavage of the RNA substr ate in vivo, it is important to design ribozymes with the highest possible catalytic activity and therefore this turnover number (kcat) and the Michaelis constant (Km) of the ribozymes were determined. This analysis relies on several assumptions: first, measuremen t of the initial rate of the reaction ensures that changes in the formation of the product and depletion of substr ate do not affect the rate of reaction. Conventionally, kinetic m easurements are made when no more than 15% of the substrate is converted to the products. A second assumption is that the concentration of the ribozyme is lower than the Km. A low ratio of the ribozyme to target ensures that the

PAGE 125

107 0 0.2 0.4 0.6 0.8 1 0102030405060 Time (minutes)Fraction of Target Cleaved 1:10 (Rz:Tar) 1:40 (Rz:Tar) 1:66(Rz:Tar) 1:100 (Rz:Tar) Figure 3-5. Time course of the ribozyme with increasing target concentrations.

PAGE 126

108 Figure 3-6. Time course cleavage reaction wi th varying temperatur es (37 C/25C) and magnesium concentrations of 20mM/1mM. 0.00 0.20 0.40 0.60 0.80 1.00 0246810 Time (minutes)Fraction of Target Cleaved 37/20 25/1

PAGE 127

109 velocity of the reaction is pr oportional to the concentration of the ribozyme thus allowing the turnover rate of the reaction to be determine d. The initial rate (v0) is determined by dividing the concentration of the reaction product over time. A liner regression curve is then plotted of 1/v vs 1/s and the kinetic para meters (Figure 3-7) can thus be determined from the equation for the line generated from the data (1/VMAX=abs/y at X=0, 1Km= abs/X at y=0). Each analysis was performe d a minimum of 3 times. The reactions were done at 37C and terminated at 6 minutes for the A2B Rz1 and at 1 minute for the A2B Rz2. At 37 C and 20 mM MgCl2, the A2B Rz1 has a Vmax of 27.3 nM/min, km of 8.3 M and a kcat of 1.8/min. Under the same conditions, the A2B Rz2 had a Vmax of 515 nM/min, Km of 4.3 M and a kcat of 36.1/min. At 25 C and 1mM MgCl2, the A2B Rz2 had a Vmax of 16.9 M/min, a km of 14.4 M, and a kcat of 1.1/min. At 37 C and 20 mM MgCl2, the A2B Rz2 had the highest catalytic activity of the two ribozymes and based on these results we dropped the A2B Rz1 and only cloned the A2B Rz2 for further testing. Cloning of the Hammerhead Ribozyme into an rAAV Expression Vector Ribozymes injected directly into an in vivo model are suscepti ble to endonuclease attack. Modification of the ribozyme by a dding phosphorothioate groups would protect the ribozyme from the endonucleases. Howe ver, modifications such as these are expensive and potentially toxic. Therefore, the A2B Rz2 was cloned into an rAAV vector, p21Newhp. These vectors are suitable for injectio n of the plasmid directly into an in vivo model and for the HREC transfection. This ve ctor allows us the option to package into AAV for subsequent experiment s in vitro and in vivo.

PAGE 128

110 Figure 3-7. Kinetic anal ysis of the ribozymes.

PAGE 129

111 Sequencing of the Clones Once the A2B Rz2 was cloned into the rAAV vect or, it was sequenced to confirm the sequences of the active A2B Rz2 ribozyme, inactive Rz2 and the p21Newhp vector alone. Figure 3-8 shows the sequencing resu lts. The C/G base difference between the active and the inactive ribozyme are indicated. Cell Cultures Human retinal endothelial cells (HREC) were grown and assesed for purity of culture. A special mesh pore nylon membrane was used to isolate these cells. This membrane retains the retinal endothelial cells but allows passage of the retinal components. Morphologically, the HREC usually have a ‘pebble stone’ morphology (Figure 3-9). The pebble stone morphology is not always easily distinguishable. If the culture flask is coated with 1% gelatin, it help s the cells to adhere to the culture flask and display their morphological charac teristics. They have a rounded nucleus which fills up most of the cell and a scanty cytoplasm. It is not always possibl e to have a 100% pure culture of the retinal endothel ial cells, and the presence of some pericytes is not unusual. To confirm the purity of the HREC the abil ity of the endothelial cells to take up acetylated LDL was assesed. The human LDL complex delivers cholesterol to the cells by receptor mediated endocytosis. The complex consists of a core of ester and triglycerides surrounded by a thic k shell of phospholip ids, unesterified cholesterol and an apoprotein B unit. Once internalized, LDL disso ciates from its receptors and appears in lysosomes. The lysine residues of LDL’ s apoprotein B can be acetylated to form acetylated LDL. Once acetylated, the LDL comp lex no longer binds to the LDL receptor

PAGE 130

112 Figure 3-8. Sequence of the active and inactive versions of the A2B ribozymes at the site of insertion within the p 21NewHp vector. The G to C change in the catalytic core at position 15 in the upper panel is also noted on the 6% polyacrylamide8M urea gel in the lower panel. The yellow sequences indicate the HindIII and SpeI sites. The blue sequences indicate the target. 1 56 10 20 30 40 (1) A A G C T T G G C A T A C T G A T G A G C C G T T C G C G G C G A A A C A A T G A C T A G Tp21A2Bactive(1) A A G C T T G G C A T A C T C A T G A G C C G T T C G C G G C G A A A C A A T G A C T A G Tp21A2Binactive(1) A A G C T T G C A T G C C T G C A G A C T A G Tp21NewHp(1) A A G C T T G G C A T A C T A T G A G C C G T T C G C G G C G A A A C A A T G A C T A G TConsensus(1) p21A2Bactive p21A2Binactivep21NewHp

PAGE 131

113 Figure 3-9. Pebble stone morphology of the HREC

PAGE 132

114 and is taken up by scavenger re ceptors specific for modified LDL. Endothelial cells and macrophages have these scavenger recep tors. The acetylated LDL complexes accumulate within the cells diffusely in the cytoplasm (Figure 3-10). To asses the purity of the culture, the number of cells that take up the LDL are expressed as a percentage of the total cells. Transfection of HREC The HREC were transfected with the ribozyme constructs to asses the ribozymes ability to cleave the target mRNA. HREC we re initially transfected with a rAAV vector PTRUF11. This plasmid is similar to th e p21NewHp, and, it expresses GFP downstream of a beta-actin CMV promoter (Figure 3-11). HR EC were transfected with this plasmid to determine the transfection efficiency using th e DEAE dextran protocol. The cells were transfected and observed under a fluorescent microscope for expression of GFP in the HREC over time. The number of cells expressi ng GFP increased with time as expected. However, the maximal expression of GFP wa s not seen until 3 w eeks post transfection. (Figure 3-12). Once the efficiency of the protocol was established, HREC were transfected with the active and inactive versions of the A2B Rz2, and the vector c ontrol (p21Newhp) using the DEAE dextran protocol. Following transf ection, the cells were allowed to grow for 72 hours. After 72 hours, the cells were not able to sustain normal morphology or characteristics of HRECs. Thus, the cells were harvested at 72 hours post transfection for further analysis.

PAGE 133

115 Figure 3-10. LDL uptake of HREC.

PAGE 134

116 Figure 3-11. The GFP plasmid. This plas mid was driven by a CMV enhancer and a chicken beta actin promoter pTR-UF117200 bp ApR ColE1 ori f1(+) origin TR TR GFPh neoR PYF441 enhancer CMV ie enhancer Intron SV40 poly(A) bGH poly(A) HSV-tk Chiken b-actin promot e Exon1 Chicken -actin p romote r

PAGE 135

117 Figure 3-12. Transfection efficiency of the HREC. A)24hrs. B)48hrs. C)72hrs. D)96hrs. E)3 weeks and F)Passage 1 post transfection.

PAGE 136

118 A migration assay was used to assess the levels of the A2B receptor in the transfected HRECs. For a migration assay, ce lls are placed in a chemotaxis chamber and covered with a porous membrane and allowed to attach to the lower side of this membrane. A stimulus is provided to the ce lls. The cells respond to the stimulus and migrate through the pores of the membrane. For this experiment, we placed the transfected cells into the wells of the chemot axis chamber and stimulated the cells with increasing concentrations of NECA. NE CA is an adenosine analogue and cells transfected with the active A2B Rz2 were not expected to re spond to NECA as readily as the inactive transfected, or the vector transf ected cells. The cells transfected with the active A2B Rz2 are expected to have the A2B receptor mRNA cleaved and thus have a decreased dose-dependent response to NECA th an the other cells. (Figure 3-13) Cells transfected with the pl asmid coding for active A2B Rz 2 reduced migration of cells by an average of 39% when compared to the p21NewHp control at increasi ng concentrations of NECA (10 and 100ng/ml), and cells transfected with plasmid coding for the inactive A2B Rz2 reduced migration of cells by an average of 15%. (Figure 3-14) Transfection Using Lipofectmaine on HEK Cells Human embryonic kidney cells (HEK) 293 cells were also transfected with the same plasmids for further in vitro analysis. Since these cells were transfected using a different protocol than the HREC the transf ection efficiency had to be determined for these cells also. To assess the transfection efficiency, the HEK cells were transfected with the GFP plasmid and the cells observed for expr ession of GFP under the fluorescence

PAGE 137

119 Figure 3-13. Theory of migration assay. Cells are plated into a well and covered with a porous membrane. Cells migrate through the membrane in response to a stimulus (eg. NECA). When A2B Rz2 transfected cells ar e placed in the well, fewer cells respond to the same stimulus.

PAGE 138

120 0 20 40 60 80 100 120 140 160 020406080100 NECA ( M)Average number of cells Active Inactive Control 10% FBS/DMEM DMEM Figure 3-14. Migration data fo r the cells transfected with th e active and inactive versions of the A2B receptor and the vector control. 10% FBS/DMEM is the positive control and DMEM alone is the negative control

PAGE 139

121 microscope. In this case, expression of th e GFP plasmid was observed within 24 hours of transfection. The number of ce lls expressing the GFP increased with time. (Figure 3-15) After 96 hours post-transfection, the cells were passaged to assess the level of expression. The HEK cells continued to e xpress GFP following passage P 1. (Figure 3-16) The level of expression of GFP also continued to increas e with time. Ninety six hours after the first passage, the cells were passaged again to determine if the GFP expression was maintained with repeated passaging. The HEK cells showed a considerably diffe rent pattern of expression of the same GFP plasmid used for the tran sfection of the HRECs. Th ere are a number of possible explanations for this observation. First and foremost, the HEK cells were obtained from an established cell line and the purity of th eir culture guaranteed. The HEK cells are much smaller than the HRECs and the transfection protocol of the HEK cells was completely different from that of the HRECs. CAMP Assay on Transfected HEK Cells Once the transfection efficiency of the HEK cells was established, the A2B RZ2 plasmids were used to transfect the cells. Seventy two hours post-transfection, the cells were harvested for a cAMP assay. The transf ected cells were stimulated with NECA, the adenosine analogue. Adenosine binding to receptors leads to pr oduction of cAMP. Figure 3-17 shows the results of the cAMP assay. There is a significant reduction of cAMP production in the HEK cells transfected with the active A2B Rz2. However, this effect was not reproducible.

PAGE 140

122 Figure 3-15. Transfection Efficiency of HE K cells. A)6hrs, B)24hr s, C)48hrs, D)72hrs and E)96hrs post transfection.

PAGE 141

123 Figure 3-16. HEK cells transfection effici ency following passage 1. A)24hrs, B)48hrs, C)72hrs, and D)96hrs following passage 1.

PAGE 142

124 0 100 200 300 400 500 600 700 ControlActiveInactivecAMP accumulation (pmole/mg) ** Figure 3-17. cAMP accumulation in HEK cells transfected with the control, active A2B Rz2 and inactive A2B Rz 2.

PAGE 143

125 Real Time PCR Figure 3-18 shows the results of TaqMan real time RT-PCR on mRNA isolated from HEK cells transfected with the active and inactive versions of the A2B Rz2, and with the control plasmid. The active A2B Rz2 reduces mRNA levels by 43%. The inactive A2B ribozyme shows no significant reduction in mRNA levels as expected for the catalytically inert ribozyme. The leve l of mRNA detected were normalized to cyclophilin. Effect of A2B Ribozymes on Neovascularization in the ROP Mouse Model Active and inactive A2B ribozymes and the cloning vect or p21NewHp were injected intra-ocularly on post natal day one in th e right eye of mouse pups. There was no injection in the left eyes (which served as controls), and the pups and their dams were taken through the oxygen-induced model of retinopathy. On day 17 the mice were euthanized and their eyes enucleated and fi xed in 4% paraformaldehyde. The eyes were embedded in paraffin and three hundred serial, 6uM sections were done Every thirtieth section was placed on a slide and stained with hematoxylin and eosin. (Figure 3-19). The extent of angiogenesis was determined by counting the pre-retinal endothelial cell nuclei surrounding a blood vesse l lumen. The cloning vector p21NewHp showed marked angiogenesis. (Figure 3-20) The active A2B Rz2 reduced the average number of nuclei per section on average by 55% (Figure 3-21) The inactive A2B Rz2 reduced the average number of nuclei on aver age by 5%. (Figure 3-22)

PAGE 144

126 0 0.2 0.4 0.6 0.8 1 1.2 p21 NewHp (control) p21A2BRz2 (active) p21A2BRz2i (inactive)Normalized RNA Level (Cyclophilin is 100%) A2A A2B Figure 3-18. Real time RT-PCR results showi ng relative levels of the adenosine A2A and A2B receptor mRNAs isolated from HEK ce lls transfected with plasmid DNA.

PAGE 145

127 Figure 3-19. The mice eyes were embedded in paraffin and three hundr ed serial sections were done. Every thirtieth section was placed on a slide and stained with hematoxylin and eosin. This figure s hows a representative mouse eye that was stained. Arrows indicate the cornea, sclera and the lens of the mouse eye.

PAGE 146

128 Figure 3-20. Injection with the control plas mid prior to exposure to high oxygen shows a high number of endothelial cell nuclei surrounding blood vessel lumen. This indicates that the control plasmid di d not reduce the amount of pre-retinal angiogenic vessels.

PAGE 147

129 Figure 3-21. Injection w ith the active A 2B ribozyme prior to high oxygen exposure significantly reduced the preretinal neovascularization.

PAGE 148

130 Figure 3-22. Injection of the activ e and inactive versions of the A2B Rz2 and the vector control in the ROP mouse model

PAGE 149

131 CHAPTER 4 DISCUSSION The overall goal of this project was to develop a hammerhead ribozyme targeted against the adenosine A2B receptor that would cleave the A2B mRNA and reduce the expression of this receptor in cell cult ure and in a mouse model of oxygen-induced retinopathy. The value of this ribozyme is that it allows us to study the involvement of the A2B receptor in the complex physiological path way of angiogenesis. Secondary to this is the potential of using this ribozyme as a therapy in the treatment of pathologies that produce abnormal neovascularization such as retinopathies and tumorogenesis. Our results demonstrate that we have developed a hammerhead ribozyme that specifically cleaves the mouse and human adenosine A2B receptor mRNA. We have demonstrated that this ribozyme reduces the expression and function of the A2B receptor in HREC and HEK cells and we have shown that this ribozyme reduces abnormal retinal neovascularization in the mouse model of oxygen-induced retinopathy. The ability of the active ribozyme to inhibit the expression of the A2B mRNA in HEK cells was clearly shown. Reduction in A2B mRNA signal in cells transfected with the active ribozyme was 43% compared to cell s transfected with the control plasmid. The chemotactic migration of HRECs acro ss a porous membrane toward solutions containing increasing concentrations of NE CA is dependent on the presence of A2B receptors on the cell surface. The reduction of this migration in HREC transfected with a plasmid expressing the active A2B ribozyme suggests that cell surface levels of the A2B receptor have been reduced due to inhibition of expression of the A2B mRNA by the

PAGE 150

132 ribozyme. A reduction of 39% in the numbe r of migrating cells was found in cells expressing the active ribozyme. Our result s show that approximately 60% of this reduction in expression is due to cleavage of the message while the remaining inhibition results from an antisense effect of ribozyme binding to the target. These results also show that ribozyme cleavage of the A2B mRNA reduces expression of the protein in cultured cells to a level that significantly inhibits the cellular function of the A2B receptor. Therefore, inhibition of the A2B receptor as it affects othe r components of this pathway can be quantitatively examined. In addition this ribozyme inhibits pre-re tinal neovascularization in vivo in a mouse model of oxygen-induced retinopathy, where a re duction in pre-retina l neovascularization of 55% in eyes injected with the plasmi d expressing the active ribozyme was achieved and only a small portion of this reduction, ap proximately 5%, is due to an anti-sense effect. These results suggest that the reduc tion in neovascularizati on is the result of ribozyme inhibition of the expression of the A2B receptor and demonstrate that this ribozyme will also be useful for in vivo studies of A2B receptor function including studies on retinopathies. Ribozymes As Tools To Study Gene Expression There are a number of reports on the ab ility of the hammerhead ribozymes to control expression of specifi c genes in cell culture. For example, a hammerhead ribozyme designed to cleave mRNA encoding CHa Ras mutation inhibited formation of foci of transformed cells by 50%.173 Hammerhead ribozymes have also been used to target anticancer therapies. For example, hammerhead ribozymes were used to target mRNA of a BCR/ABL fusion protein, which is responsible for chronic myelogenous leukemia. The hammerhead ribozyme elim inated expression of the protein in K562

PAGE 151

133 cells.174 Hammerhead ribozymes targeted to surviv in, which is expressed in carcinoma cells were also able to re duce survivin mRNA by 74%.175 In another study, a hammerhead ribozyme was used to target bc l-2 mRNA in tumors, which overexpress the protein. The ribozyme was tested in a lympho ma cell line and showed a decrease in the bcl-2 mRNA and protein following transfection with the ribozyme.176 In yet another study, ribozymes were designed as HIV therap ies by a number of groups. A hammerhead ribozyme expressed in Hum7 cells decreas ed the hepatitis B viral production by 83%.177 Naturally occurring RNAs have distin ct advantages over DNA. RNA has a 2’OH group associated with the sugar moiety of RNA which participates dir ectly in a chemical reaction which enhances the reactivity of the adjacent 3’OH group. The 2’OH group is also a hydrogen donor and acceptor, thus re ndering RNA more versatile then DNA in tertiary structure formation.178 DNA is predominantly a base paired duplex of complementary strands and RNA is folded from a single strand. RNA, can thus form extensive secondary structures due to pairing of imperfect complementary sequences in the RNA strand. Single stranded regions punc tuate through this s econdary structure leading to a variety of RNA conformations. RNA also has a uridine base instead of a thymidine allowing it to form unique secondary st ructures. For example, a uridine turn in tRNA and hammerhead ribozymes causes an abrupt turn in direction of the polynucleotide backbone and allows formation of complex tertiary interactions. The uridine turn is stabilized by hydrogen bonding and Van Der Waals interactions between uridine and the surr ounding nucleotides. Antisense therapy has been used by a number of investig ators to inhibit angiogenesis. For example, Robinson et al used an antisense oligonucelotide against the

PAGE 152

134 mouse VEGF to inhibit re tinal neovascularization.179 The oligos were injected into the mouse eye intravitreally and then exposed to hypoxia. Treatment with the anti-sense oligo’s resulted in a 25% decr ease in angiogenic vessel growt h. This study was able to confirm the importance of VEGF in the angi ogenic pathway, however, the antisense. olinucleotides were not succe ssful at potently inhibiting th e growth of abnormal vessels. 179 Another study used an o ligonucleotide targetting the human and rat VEGF forms and were only able to lower the incidence of choroidal neovascularization by 30%. Antisense therapy is effective when the targeted mRNA is not abundant. Antisense nucleotides are complementary DNA or RNA sequences whic h hybridize to specific mRNA. These nucleotides are specific to the target, easy to design and synthesize. However, antisense nucleotides used for therapy of disease have a poor uptake, are unstable. The nucleotides are also sensitive to degradation by exogenous and endogenous nucleases. Antisense nucleotides are toxic to cells because a higher concentration of nucleotides is required to significantly affect the expression of a gene. The stability of anti sense oligonucleotides is enhanced by phosphorothioate bonds, wh ich are toxic to mammalian cells. 180,181 An advantage of using ribozymes instead of antisense oligonucleotides is their small size making them inexpensive and easy to produce. Their small size allows them to be inserted into gene encoding ribozymes into viral vectors used for their delivery to cells in vivo. Trans acting ribozymes inactivate or m odify multiple mRNA molecules, which is a distinct advantage over anti-sense o ligonucleotides, which act stoichiometrically. Ribozymes also do not require host cellular machinery to degrade the substrate. Therefore trans-acting ribozymes inactivate targ et RNA more efficiently than anti-sense oligonucleotides. In comparison to the conve ntional antisense RNAs, ribozymes provide

PAGE 153

135 the potential of turnover, with a single molecu le being able to inactivate multiple target RNAs. 181 Ribozymes and protein enzymes share simila rities in their mode of action. Both, ribozymes and protein enzymes require a comp lex secondary and tertiary structure for catalysis.166,182,183 They also share similar mechanisms of acid base catalysis to carry out a reaction184 and stabilize a transitio n state between substrate and product formation. Catalysis is driven by intrinsic binding energy resulting from interactions between enzyme and its substrate at sites different from the catalytic core.185 Ribozymes can recognize RNA substrates via base pairing, thus their specificity is easy to manipulate. Ribozymes also do not evoke an immune re sponse to the same degree as (foreign) proteins evoke. RNA is a natural component of the cell and has a low half-life, thus low toxicity. Proteins, on the ot her hand require the host ce ll machinery to degrade the substrate. Delivery Of The Ribozyme In vivo The plasmid used for the expression of the A2B Rz2 has inverted terminal repeats (ITRs) at either ends of the ribozyme. ITRs contain the sequences, which are required for replication, packaging and inte gration of the plasmid into an rAAV vector. AAV is a single stranded, encapsulated DNA virus. AAV contains ITRs, which flank the AAV genome. This region of the genome is replaced with the gene of inte rest to generate the rAAV. 186 rAAV has been successfully used to deliv er transgenes into the eyes. While the A2B ribozyme was cloned into this vector for potential packaging into AAV, we have demonstrated a significant response of the Active A2B Rz2 naked plasmid and thus did not proceed to packing the ribozyme into an rAAV virus to enhance the effect.

PAGE 154

136 There are several advantages to using plasmid DNA versus rAAV. The plasmid DNA is easier to propagate and is usually of higher quality. 187 Plasmids can also carry larger DNA sequences as compared to limited 5kb capacity of rAAVs.188 Plasmid DNA can also be repeatedly administered du e to its low immunogenicity and toxicity. 189,190 Another method for the delivery of the ribozyme involves packaging the plasmid into a liposomal mixture that would bind to cell surface receptors and enhance uptake of the plasmid. 191,192 Liposomes are microscopic vesse ls composed of an aqueous compartment surrounded by a lipid layer. The transferrin receptor can be used as a vehicle to carry liposome/DNA complex into the cell. Transferrin binds to iron extracellulary and subsequently binds to the transferrin receptor on the cell surface. The receptor and transferrin are taken up by endocytos is into the cell. The iron is released and the transferrin and the receptor are recycled out of the cell. The transferrin helps the uptake of plasmid DNA by the cell. (Figure 41) Transferrin complexes can also be used to deliver cancer chemotherapeutic drugs to the tumor directly. For example, doxorubin, a cytotoxic drug was complexed with transferrin in liposomes and delivered to the site of tumor development. In another study, li posomal mediated delivery of the alpha interferon to murine bladder tumor cell line MBT2 was shown to increase the uptake of alpha interferon and enhance proliferative activ ity. Liposomal mediat ed delivery is safe, non-immunogenic, can be re-administered wi thout harm and an unlimited size of DNA can be delivered. However, a major drawb ack using the liposome /transferrin route of delivery is the poor transfection e fficiency of liposomal vectors. 193

PAGE 155

137 Figure 4-1. Entry of the AAV into the cell involves binding of the virus particle to heparan receptors on the cell surface follo wed by uptake of the virus into the cell and eventual release of viral DNA. In the transferrin cycle, transferrin after binding to the cell, it binds to th e transferrin receptor on the cell surface and is taken into the cell where the ir on is released and transferrin and its receptor are recycled out of the cell.

PAGE 156

138 Our lab has also tested the delivery of liposomes versus the naked DNA in an ROP model. A GFP plasmid was packed into a liposome with transferrin and suspended in HEPES buffer. Naked DNA resuspended in HEPES buffer was also used. Both formulations were tested in an ROP model of retinopathy in the mice. The mice were sacrificed and their eyes flat mounted to observe the expressi on of GFP. Results showed that the naked plasmid DNA injection had higher levels of GFP expression, thus supporting the results that naked DNA plasmid injection is sufficient. Use of the ribozyme in tissue cultures has shown a decrease in the expression of the A2B receptor in HREC. However, the efficiency of the transfection might be improved with increasing the amounts of the ribozyme. The transfection efficiency was determined by the transfection efficiency of a similar pl asmid containing a GFP expression site. GFP is a unique fluorophore, which forms intracellulary. It does not require additional cofactors and the emitted fluorescent intensity is proportional to the GFP expression levels within the cells. Thus GFP intensity can be measured at a single cell level. The GFP plasmid is also driven by a CMV promoter and thus would be taken up by any type of cell present in the culture. Using this GFP plasmid it was shown that the cells expressed GFP for up to three passages in the HEK ce lls and up to two passages in the HREC. However, this plasmid was not exactly the same as the one used to carry the ribozyme. To further enhance the chance of transfecting more cells, it would be better to place GFP upstream of the ribozyme and th en transfect the cells. The tissue culture can then be monitored for cells that expre ss the GFP. Any cells expres sing the GFP can be analyzed by flow cytometry which, can simultaneously measure and analyze multiple physical characteristics of cells as they flow in a fluid stream thr ough a beam of light. Properties

PAGE 157

139 it can measure include cell size, granularity a nd relative fluorescence. The cells can also be separated by a fluorescence activated cell so rter (FACS) and subsequently grown as pure culture. The pure culture would allow be tter characterization of the effects of the ribozyme. Sorenson et al 194 transfected H9 cells with a GFP marker located upstream of a retroviral vector. They were able to sort cells into two distinct populations by measuring the expression and the intensity of GFP expression. The sorted cells were viable for culture following sorting and the presence of GFP did not affect the cells. Promoter Considerations A CMV promoter was used to drive the expression of the plasmid, which carried the ribozyme. The CMV promoter is used for mammalian promoter strength to enhance the level of transient transgene expression in a majority of mammalian cells. The promoter is ubiquitous and affects most cells types. To better understand the pathophysiology of retinal angiogenesis, the CMV/beta actin promoter needs to be replaced with a proliferating endothelial cell specific promoter which is under the control of cell cycle gene switch. Such a promoter would only be expre ssed in proliferating endothelial cells in animals undergoing angi ogenesis. A promoter has been designed with a cdc6 cell cycle promoter and endot helin elements. Cdc6 is expressed in proliferating cells195,196 and the expression of endotheli n on mainly endothelial cells makes it an attractive targ et for endothelial cells.197,198,199 The cdc6 promoter was inserted into a plasmid and a mouse multimerized endothelin enhancer was inserted upstream of the cdc6 promoter. A five-fold increase in the expression of activity for the endothelin enhancer/cdc6 promoter was obser ved in diving cells ve rsus the non-diving endothelial cells. However, th is promoter was not found to be sufficient for adequately expressing ribozymes. Therefore a GAL4 DN A binding protein was used. The trans-

PAGE 158

140 activating protein was fused to the NF-kB p65 transactivation domain. Expression of the fusion protein was driven by a CMV enhancer promoter (Figure 4-2). To test the specificity of the endothelin/cdc6 promoter fo r endothelial proliferating cells, mice were implanted with SCCVII tumor cells. The tumo r was allowed to develop for a few days following and IV injection w ith liposome/plasmid complexes. The tumor and lung tissues were harvested at 18 hours and 4 days post injection and assayed for CAT expression. At 18hours post injection, the tumo r did not have any Cat activity present, and the lung tissue had detectable levels of CAT. However, at 4 days post injection, the tumor had ten times the activity of CAT and the lung tissue had less amounts. These results indicated that the CAT expressi ng promoter was repressed in non-dividing endothelial cells of the lung but was very high in the dividing cells of the tumor. It was however, interesting to note that there was no expression of CAT following 18hours of injection into the tumor. It is possible that endothelial cells need time to pass through at least one round of cell divisi on for the promoter to become activated. Experiments are currently underway to clone the A2B Rz2 downstream of this promoter. Thus far the distribution of the A2B receptors in different tissues has been based on the characterization of the receptors based on agonist binding. Antagonists would have been more preferable to determine the loca lization of these receptors, however, none are available. The A2B receptor has also been shown to be present in fibroblasts, which are present at sites of angiogenesis. Th erefore, the widespread pattern of A2B receptor distribution, based on agonist bind ing studies, may be misleading.200 Cloning of the A2B

PAGE 159

141 Figure 4-2. Diagram of the expression casse ttes fusion protein and alkaline phosphatase (Alk Phos). UT12 refers to the consen sus untranslated region of the message. IVS8 is a consensus splice site for mRNA processing. HGHpA is the human growth hormone poly A sequence. +1 is the transcriptional start site.

PAGE 160

142 Rz2 into the cdc6/endothelin promoter woul d allow better localizat ion and distribution patterns of the A2B receptor. The purity of the HRECs was important for the success of this project and for determining the presence of contaminating cell s such as pericytes. The purity of the culture was assessed either based on the morphology of the cells or the uptake of acetylated LDL by scavenger receptors. Both of these techniques are reliable for assessing the purity of the cultu res. In addition, a functional assay, Matrigel, can also be performed. This assay involves plating the ce lls onto a commercially available Matrigel. Matrigel is a basement membrane composed of collagens, laminin and proteoglycans. It also contains matrix degrading enzymes, TI MPs and growth factors. Endothelial cells plated onto a Matrigel helps them to form tube like structures. The Matrigel assay is time consuming and would not have provided any additional information to help in determining the purity of the HRECs culture. Future Studies Transfection with the active A2B ribozyme showed a 43% decrease in the levels of the A2B receptor by real time PCR. VEGF is a potent contributor to the process of angiogenesis and it has alrea dy been shown that the A2B receptor works upstream of VEGF. Hypoxia has been shown to upregulat e the expression of the VEGF receptor and the production of VEGF. Therefore, cells tr ansfected with the act ive form of the A2B ribozyme should also show a decrease in the levels of VEGF receptor and VEGF production. Therefore, it woul d be important to assess how down regulation of the A2B receptor affects the subsequent growth factors, which also play an important role in angiogenesis.

PAGE 161

143 It also remains to be ascertained whether down-regulation of the A2B receptor affects the other adenosine receptors. It is possible that down-regulation of one of the receptors causes the other receptors to enha nce their effects to compensate for the deficiency. The data from the real-tim e PCR shows that the levels of the A2A receptor mRNA remain unaffected following transfection with the A2B Rz2. Even though the mRNA for the A2B receptor has been shown to be decreased, it is possible that the amount of protein being produced is unaffected. Therefore, it would be useful to measure the A2B receptor protein by wester n blotting to confirm that a reduction in the amount of mRNA corresponds to a reduction in the am ount of protein. Currently, however, a suitable antibody for western blotting purposes is unavailable for the A2B receptor. Since a suitable antibody is available for the A2A receptor, it would be interesting to see if transfection of HRECs with the A2B Rz2 affects the level of protein production of the A2A receptor. Our lab has also made ribozymes to othe r components of the angiogenesis pathway, for example, VEGF and the integrins. Thes e ribozymes are made similar to the methods described for the A2B ribozymes. Sufficient in vivo and in vitro testing of the ribozymes has also been carried out. It would be interes ting to see the effect of a combination of the ribozymes might have on the angiogenesis pathway. Theoreticall y, a combination of ribozymes targeting various aspects of the a ngiogenic pathway would have a more potent effect. However, it is al so possible that down regulati on of too many of the growth factors may also hinder the deve lopment of normal blood vessels too. Downstream signaling of the A2B receptors is not well established. For example, the A2B receptors in xenopus have been shown to stimulate PLC, which in turn can

PAGE 162

144 activate calcium dependent chloride conductance. 201 This effect has not been shown in the retinal endothelial cells. The A2B receptor also leads to th e activation of the MAPK pathway, which may stabilize HIF-1 which, in turn may lead to the mitogenic effects of VEGF.108 The MAPK pathway may also stab ilize hypoxia inducible factor (HIF-1 ) by preventing its proteasomal degradation. HI F-1 is a heterodimeric protein, which is activated by hypoxia and regulates the transcription of many genes. It consists of constitutive HIF-1 and the rate limiting HIF-1 .202-204 Under normoxia, HIF-1 is regulated by the removal of the HIF-1 subunit by ubiquination and proteasomal degradation. HIF-1 is the aryl hydrocarbon receptor nuclear translocator (ARNT) which heterodimerizes with the aryl hydrocarbon receptor. 204 Hypoxia inhibits the removal of the HIF-1 subunit by the proteasome thus preventing HIF-1 destruction. The prevention of proteasomal degradation of HIF-1 is poorly understood. However, it is thought that the hydroxylation of two proline residues in the HIF-1 subunit prevent its degradation by the proteasome.203 The stabilized HIF-1 translocates to the nucleus and dimerizes with the HIF-1 (ARNT). The stabilized HIF-1 / subunit subsequently binds to the hypoxia response element (HRE). The HRE is a gene promoter which upregulates the expression of VEGF.205 Hypoxia also leads to the stab ilization of the short lived VEGF mRNA directly, thereby augm enting the effect of HIF-1 alpha.206 Glycogen synthase kinase-3 (GSK-3) is an enzyme, which catalyzes the breakdown of glycogen synthase. It is a pro-apoptotic enzyme and is constitutively active in cells. Phosphorylation of GSK-3 inhibits its activity. 207 The PI3 kinase pathway has been shown to phosphorylate GSK-3 and hen ce inhibit its activity. The A2B receptor couples to G q and activates the PI-3 kinase pathwa y. However, it has not been shown yet

PAGE 163

145 whether the PI-3 kinase pathway leads to the phosphorylation of GSK-3 by the A2B receptor. 207 Coupling of the A2B receptor to G q also leads to the act ivation of the MAPK pathway. It is possible that th e MAPK pathway stabilizes HIF-1 by preventing its proteasomal degradation. 207 Therefore it may also be hypothesized that phosphorylation of GSK-3 and subsequent inhibi tion of its activity also stabilizes the HIF-1 subunit. This pathway would contri bute to the upregulation of VEGF and angiogenic blood vessel growth (Figure 4-3) It is important to be able to dissect the signaling pathway of the receptor to better understand the angiogenic pathway. The active A2B ribozyme in combination with inhibito rs of some of the common kinases may be an important tool in studying this pathway.

PAGE 164

146 Figure 4-3. A2B signaling pathway with theoretical downstream effects, which have yet to be confirmed

PAGE 165

147 LIST OF REFERENCES 1. Hogan M. Histology of theHuman Eye Philadelphia: WB Saunders Co; 1976. 2. Snell R, Lemp, MA. Clinical Anatomy of the Eye Chicago, IL: Blackwell Scientific Publications.; 1989. 3. Gelatt K. Veterinary Ophthalmology NY: Lippincott and Williams; 1996. 4. Forrester J. The eye. Basic Sciences inPpractice Philadelphia, PA: WB Saunders Co Ltd; 1996. 5. Wise G, Dollery CT, Henkind P. The Retinal Circulation. NY: Harper & Row Publishers; 1971. 6. Hart W. Adler's Physiology of the Eye. 9th. ed. NY: Blackwell Scientific publications.; 1992. 7. Rubin LL, Staddon JM. The cell bi ology of the blood-brain barrier. Annu Rev Neurosci 1999;22:11-28. 8. Hyman L, Neborsky R. Risk factors fo r age-related macular degeneration: an update. Curr Opin Ophthalmol 2002;13:171-5. 9. Stone EM, Sheffield VC, Hageman GS. Molecular genetics of age-related macular degeneration. Hum Mol Genet 2001;10:2285-92. 10. Gottlieb JL. Age-related macular degeneration. Jama 2002;288:2233-6. 11. Harvey PT. Common eye diseases of el derly people: identifying and treating causes of vision loss. Gerontology 2003;49:1-11. 12. Campochiaro PA. Retinal and choroidal neovascularization. J Cell Physiol 2000;184:301-10. 13. Cai J, Boulton M. The pathogenesis of di abetic retinopathy: ol d concepts and new questions. Eye 2002;16:242-60.

PAGE 166

148 14. Aiello LP. The potential role of PKC beta in diabetic retinopathy and macular edema. Surv Ophthalmol 2002;47 Suppl 2:S263-9. 15. Sulochana K, Ramakrishnan, S., Rajesh M., Coral, K., and Badrinath, SS. Diabetic retinoapthy:moelcular mechanism, present regime of treatment and future perspectives. Current Science 2001;80:133-142. 16. Gardner TW, Antonetti DA, Barber AJ LaNoue KF, Levison SW. Diabetic retinopathy: more than meets the eye. Surv Ophthalmol 2002;47 Suppl 2:S25362. 17. Rosenbaum JT. Sugar creates a sticky business: round up the usual suspects. Am J Pathol 2002;160:1547-50. 18. Gardner TW, Antonetti DA, Barber AJ Lieth E, Tarbell JA. The molecular structure and function of the inner bl ood-retinal barrier. Penn State Retina Research Group. Doc Ophthalmol 1999;97:229-37. 19. Miller JW, Adamis AP, Aiello LP. Vascul ar endothelial growth factor in ocular neovascularization and prolif erative diabetic retinopathy. Diabetes Metab Rev 1997;13:37-50. 20. Weinberger B, Laskin DL, Heck DE, La skin JD. Oxygen toxicity in premature infants. Toxicol Appl Pharmacol 2002;181:60-7. 21. McColm JR, Fleck BW. Retinopa thy of prematurity: causation. Semin Neonatol 2001;6:453-60. 22. Wesolowski E, Smith LE. Effect of light on oxygen-induced retinopathy in the mouse. Invest Ophthalmol Vis Sci 1994;35:112-9. 23. Hack M, Flannery DJ, Schluchter M, Cart ar L, Borawski E, Klein N. Outcomes in young adulthood for very-low-birth-weight infants. N Engl J Med 2002;346:14957. 24. Wheatley CM, Dickinson JL, Mackey DA, Craig JE, Sale MM. Retinopathy of prematurity: recent advances in our understanding. Br J Ophthalmol 2002;86:696-700. 25. Kotecha S. Oxygen therapy for infants with chronic lung disease. Arch Dis Child Fetal Neonatal Ed. 2002;87:F11-4.

PAGE 167

149 26. An international classification of reti nopathy of prematurity. The Committee for the Classification of Retinopathy of Prematurity. Arch Ophthalmol 1984;102:1130-4. 27. Katz X, Kychenthal A, Dorta P. Zone I retinopathy of prematurity. J Aapos 2000;4:373-6. 28. Ellis A. Phot essay: regression of seve re retinopathy of prem aturity after laser treatment. Arch Ophthalmol 2002;120:1404-5. 29. Banach MJ, Berinstein DM. Laser th erapy for retinopathy of prematurity. Curr Opin Ophthalmol 2001;12:164-70. 30. Weinberger B, Laskin DL, Heck DE, La skin JD. Oxygen toxicity in premature infants. Toxicol Appl Pharmacol 2002;181:60-7. 31. Fielder AR, Reynolds JD. Retinopathy of prematurity: clinical aspects. Semin Neonatol 2001;6:461-75. 32. Mintz-Hittner HA, Kretzer FL. The rationale for cryothe rapy with a prophylactic scleral buckle for Zone I thresh old retinopathy of prematurity. Doc Ophthalmol 1990;74:263-8. 33. Connolly B, McNamara JA, Regillo CD, Vander JF, Tasman W. A comparison of laser photocoagulation with cryotherapy for threshold retinopathy of prematurity at 10 years: part 2 refractive outcome. Ophthalmology 2002;109:936-41. 34. Whitfill CR, Drack AV. Avoidance and tr eatment of retinopathy of prematurity. Semin Pediatr Surg 2000;9:103-5. 35. Mintz-Hittner HA, Prager TC, Kretzer FL. Visual acuity correlates with severity of retinopathy of prematurity in untreated infants wei ghing 750 g or less at birth. Arch Ophthalmol 1992;110:1087-91. 36. Kretzer FL, Mehta RS, Brown ES, Mi ntz-Hittner HA. The pathogenesis of retinopathy of prematurity as it relates to surgical treatment. Doc Ophthalmol 1990;74:205-11. 37. Hinz BJ, de Juan E, Jr., Repka MX. Sc leral buckling surgery for active stage 4A retinopathy of prematurity. Ophthalmology 1998;105:1827-30. 38. Chuang Y. Scleral buckeling for stage 4 ROP. Ophthalmic Surg Lasers 2002;31.

PAGE 168

150 39. McPherson AR, Hittner HM, Lemos R. Retinal detachment in young premature infants with acute retrolental fibroplasia. Thirty-two new cases. Ophthalmology 1982;89:1160-9. 40. Aggarwal R, Agarwal R, Deorari AK, Paul VK. Retinopathy of prematurity. Indian J Pediatr 2002;69:83-6. 41. Fielder AR, Reynolds JD. Retinopathy of prematurity: clinical aspects. Semin Neonatol 2001;6:461-75. 42. Agarwal R AR, Deorari AK, Paul VK. Retinopathy of prematurity. Indian J Pediatr 2002;69:83-6. 43. Bhushan M, Young HS, Brenchley PE, Griffiths CE. Recent advances in cutaneous angiogenesis. Br J Dermatol 2002;147:418-25. 44. Bussolino F, Mantovani A, Persico G. Molecular mechanisms of blood vessel formation. Trends Biochem Sci 1997;22:251-6. 45. Liekens S, De Clercq E, Neyts J. Angiogenesis: regulators and clinical applications. Biochem Pharmacol 2001;61:253-70. 46. Jekunen A, Kairemo K. Inhibition of a ngiogenesis at endothelial cell level. Microsc Res Tech 2003;60:85-97. 47. Fajardo LF. The complexity of endothelial cells. A review. Am J Clin Pathol 1989;92:241-50. 48. Hirschi KK, D'Amore PA. Pericytes in the microvasculature. Cardiovasc Res 1996;32:687-98. 49. Vestweber D. Molecular mechanisms th at control endothelial cell contacts. J Pathol 2000;190:281-91. 50. Diaz-Flores L, Gutierrez R, Varela H, Rancel N, Valladares F. Microvascular pericytes: a review of their morphological and func tional charac teristics. Histol Histopathol 1991;6:269-86. 51. Alexander JS, Elrod JW. Extracellular ma trix, junctional integrity and matrix metalloproteinase interactions in endothelial permeability regulation. J Anat 2002;200:561-74.

PAGE 169

151 52. Ingber D. Extracellular matrix and ce ll shape: potential control points for inhibition of angiogenesis. J Cell Biochem 1991;47:236-41. 53. Nicosia RF, Villaschi S. Autoregulation of angiogenesis by cells of the vessel wall. Int Rev Cytol 1999;185:1-43. 54. Mignatti P, Rifkin DB. Plasminogen activ ators and matrix metalloproteinases in angiogenesis. Enzyme Protein 1996;49:117-37. 55. Mignatti P, Rifkin DB. Plasmi nogen activators and angiogenesis. Curr Top Microbiol Immunol 1996;213 ( Pt 1):33-50. 56. Sternlicht MD, Werb Z. How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol 2001;17:463-516. 57. Cao Y, Ji RW, Davidson D, Schaller J, Marti D, Sohndel S, McCance SG, O'Reilly MS, Llinas M, Folkman J. Kringle domains of human angiostatin. Characterization of the anti-prolif erative activity on endothelial cells. J Biol Chem 1996;271:29461-7. 58. Juliano RL. Signal transduction by cell ad hesion receptors and the cytoskeleton: functions of integrins, cadherins, se lectins, and immunoglobulin-superfamily members. Annu Rev Pharmacol Toxicol 2002;42:283-323. 59. Shattil SJ, Ginsberg MH. Integrin signaling in va scular biology. J Clin Invest 1997;100:S91-5. 60. Plow EF, Haas TA, Zhang L, Loftus J, Smith JW. Ligand binding to integrins. J Biol Chem 2000;275:21785-8. 61. Harris ES, McIntyre TM, Prescott SM, Zimmerman GA. The leukocyte integrins. J Biol Chem 2000;275:23409-12. 62. Mizejewski GJ. Role of integrins in cancer: survey of expression patterns. Proc Soc Exp Biol Med 1999;222:124-38. 63. Uhm JH, Gladson CL, Rao JS. The role of integrins in the malignant phenotype of gliomas. Front Biosci 1999;4:D188-99. 64. Boudreau NJ, Jones PL. Extracellular matrix and integrin signalling: the shape of things to come. Biochem J 1999;339 ( Pt 3):481-8.

PAGE 170

152 65. Coppolino MG, Dedhar S. Bi-directional si gnal transduction by integrin receptors. Int J Biochem Cell Biol 2000;32:171-88. 66. Calderwood DA, Shattil SJ, Ginsberg MH. Integrins and actin filaments: reciprocal regulation of cell adhesion and signaling. J Biol Chem 2000;275:22607-10. 67. Senger DR, Ledbetter SR, Claffey KP Papadopoulos-Sergiou A, Peruzzi CA, Detmar M. Stimulation of endothelial cell migration by vasc ular permeability factor/vascular endothelial growth fa ctor through cooperative mechanisms involving the alphavbeta3 integr in, osteopontin, and thrombin. Am J Pathol 1996;149:293-305. 68. Eliceiri BP, Cheresh DA. The role of alphav integrins during angiogenesis: insights into potential mechanisms of action and clinical development. J Clin Invest 1999;103:1227-30. 69. Cheng N, Brantley DM, Chen J. The ephrins and Eph receptors in angiogenesis. Cytokine Growth Factor Rev 2002;13:75-85. 70. Gale NW, Yancopoulos GD. Ephrins and their receptors: a repulsive topic? Cell Tissue Res 1997;290:227-41. 71. Holmberg J, Frisen J. Ephrins are not only unattractive. Trends Neurosci 2002;25:239-43. 72. Kalo MS, Pasquale EB. Signal transfer by Eph receptors. Cell Tissue Res 1999;298:1-9. 73. Papetti M, Herman IM. Mechanisms of normal and tumor-derived angiogenesis. Am J Physiol Cell Physiol 2002;282:C947-70. 74. Dora KA. Cell-cell communi cation in the vessel wall. Vasc Med 2001;6:43-50. 75. Dejana E, Corada M, Lampugnani MG Endothelial cell-t o-cell junctions. Faseb J 1995;9:910-8. 76. Loughna S, Sato TN. A combinatorial role of angiopoietin-1 and orphan receptor TIE1 pathways in establishing vasc ular polarity during angiogenesis. Mol Cell 2001;7:233-9. 77. Chavakis E, Dimmeler S. Regulation of endothelial cell survival and apoptosis during angiogenesis. Arterioscler Thromb Vasc Biol 2002;22:887-93.

PAGE 171

153 78. Ortega N, Hutchings H, Plouet J. Signal relays in the VEGF system. Front Biosci 1999;4:D141-52. 79. Suhardja A, Hoffman H. Role of gr owth factors and their receptors in proliferation of microva scular endothelial cells. Microsc Res Tech 2003;60:70-5. 80. Dow JK, deVere White RW. Fibroblast growth factor 2: its structure and property, paracrine function, tumor angioge nesis, and prostate-related mitogenic and oncogenic functions. Urology 2000;55:800-6. 81. Klint P, Claesson-Welsh L. Signal tran sduction by fibroblast growth factor receptors. Front Biosci 1999;4:D165-77. 82. Gabrilove JL. Angiogenic growth factors: autocrine and para crine regulation of survival in hematologic malignancies. Oncologist 2001;6 Suppl 5:4-7. 83. Stice LL, Vaziri C, Faller DV. Regulat ion of platelet-derived growth factor signaling by activated p21Ras. Front Biosci 1999;4:D72-86. 84. Campochiaro PA, Chang M, Ohsato M, Vinores SA, Nie Z, Hjelmeland L, Mansukhani A, Basilico C, Zack DJ. Retinal degeneration in transgenic mice with photoreceptor-specific expression of a domin ant-negative fibroblast growth factor receptor. J Neurosci 1996;16:1679-88. 85. Pepper MS. Transforming growth factor-b eta: vasculogenesis, angiogenesis, and vessel wall integrity. Cytokine Growth Factor Rev 1997;8:21-43. 86. Itoh S, Itoh F, Goumans MJ, Ten Dijke P. Signaling of transforming growth factor-beta family member s through Smad proteins. Eur J Biochem 2000;267:6954-67. 87. Attisano L, Wrana JL. Signal transd uction by the TGF-beta superfamily. Science 2002;296:1646-7. 88. Burridge K, Chrzanowska Wodnicka M. Focal adhesions, contractility, and signaling. Ann Rev Cell Dev Biol 1996;12:463-518. 89. Aplin AE, Howe A, Alahari SK, Juli ano RL. Signal transduction and signal modulation by cell adhesion receptors: the role of integrins, cadherins, immunoglobulin-cell adhesion mo lecules, and selectins. Pharmacol Rev 1998;50:197-263.

PAGE 172

154 90. Hanahan D. Signaling vascular morphogenesis and maintenance. Science 1997;277:48-50. 91. Ghiardi GJ, Gidday JM, Roth S. The purine nucleoside adenosine in retinal ischemia-reperfusion injury. Vision Res 1999;39:2519-35. 92. Takagi H, King GL, Robinson GS, Ferrara N, Aiello LP. Adenosine mediates hypoxic induction of vascular endothelial gr owth factor in retinal pericytes and endothelial cells. Invest Ophthalmol Vis Sci 1996;37:2165-76. 93. Takagi H, King G, Ferrara N, Aiello L. Hypoxia regulates vascular endothelial growth factor receptor KDR/Flk gene e xpression through adenosine A2 receptors in retinal capillary endothelial cells. Invest Ophthalmol Vis Sci 1996;37:13111321. 94. Ash JD, Overbeek PA. Lens-specific VEGF-A expression induces angioblast migration and proliferation and stimulates angiogenic remodeling. Dev Biol 2000;223:383-98. 95. Mino RP, Spoerri PE, Caballero S, Player D, Belardinelli L, Biagionni I, Grant MB. Adenosine receptor an tagonists and retinal neova scularization in vivo. Invest Ophthalmol Vis Sci 2001;43:3320-4. 96. Grant MB TR, Caballero S, Ozeck MJ, Davis MI, Spoerri PE, Feoktistov I, Biaggioni I, Shryock JC, Belardinelli L. Adenosine receptor activation induces vascular endothelial growth factor in human retinal endothelial cells. Circ Res 1999;85:699-706. 97. Smith LE, Kopchick JJ, Chen W, Knapp J, Kinose F, Daley D, Foley E, Smith RG, Schaeffer JM. Essential role of grow th hormone in ischemia-induced retinal neovascularization. Science 1997;276:1706-1709. 98. Bussolino F, Mantovani A, Persico G. Molecular mechanisms of blood vessel formation. Trends Biochem Sci 1997;22:251-6. 99. Daniel TO, Abrahamson D. Endothelial signal integration in vascular assembly. Annu Rev Physiol 2000;62:649-71. 100. Feoktistov I, Biaggioni I. Adenosine A2B receptors. Pharmacol Rev 1997;49:381-402. 101. Linden J. Molecular approach to ad enosine receptors: receptor-mediated mechanisms of tissue protection. Annu Rev Pharmacol Toxicol 2001;41:775-87.

PAGE 173

155 102. Lutty GA, Merges C, McLeod DS. 5' nucleotidase and adenosine during retinal vasculogenesis and oxygen induced retinopathy. Invest Ophthalmol Vis Sci 2000;41:218-29. 103. Braun N, Lenz C, Gillardon F, Zimmermann M, Zimmermann H. Focal cerebral ischemia enhances glial expression of ecto-5'-nucleotidase. Brain Res 1997;766:213-26. 104. van Calker D, Muller M, Hamprecht B. Adenosine regulates via two different types of receptors, the accumulation of cyclic AMP in cultured brain cells. J Neurochem 1979;33:999-1005. 105. Londos C, Cooper DM, Wolff J. Subclasses of external adenosine receptors. Proc Natl Acad Sci U S A 1980;77:2551-4. 106. Daly JW, Butts-Lamb P, Padgett W. Subclasses of adenosine receptors in the central nervous system: interaction with caffeine and related methylxanthines. Cell Mol Neurobiol 1983;3:69-80. 107. Bruns RF, Lu GH, Pugsley TA. Character ization of the A2 adenosine receptor labeled by [3H]NECA in ra t striatal membranes. Mol Pharmacol 1986;29:33146. 108. Gao Z, Chen T, Weber MJ, Linden J. A2B adenosine and P2Y2 receptors stimulate mitogen-activated protein kina se in human embryonic kidney-293 cells. cross-talk between cyclic AMP and protein kinase c pathways. J Biol Chem 1999;274:5972-80. 109. Rivkees S, Reppert, S. M:RFL9 en codes and A2B adenosine receptor. Mol Endocrinol 1992;6:1598-1604. 110. Pierce KD, Furlong TJ, Selbie LA, Shine J. Molecular cloni ng and expression of an adenosine A2b receptor from human brain. Biochem Biophys Res Commun 1992;187:86-93. 111. Palmer TM, Stiles GL. Adenosine receptors. Neuropharmacology 1995;34:68394. 112. Montesinos MC, Gadangi P, Longaker M, S ung J, Levine J, Nilsen D, Reibman J, Li M, Jiang CK, Hirschhorn R, Recht PA, Ostad E, Levin RI, Cronstein BN. Wound healing is accelerated by agonists of adenosine A2 (G alpha s-linked) receptors. J Exp Med 1997;186:1615-20.

PAGE 174

156 113. Ongini E, Fredholm BB. Pharmaco logy of adenosine A2A receptors. Trends Pharmacol Sci 1996;17:364-72. 114. Marquardt DL, Walker LL, Heinemann S. Cloning of two adenosine receptor subtypes from mouse bone marrow-derived mast cells. J Immunol 1994;152:4508-15. 115. Linden J, Thai T, Figler H, Jin X, Robeva AS. Char acterization of human A(2B) adenosine receptors: radio ligand binding, western blot ting, and coupling to G(q) in human embryonic kidney 293 cells and HMC-1 mast cells. Mol Pharmacol 1999;56:705-13. 116. Olah ME. Identification of A2a adenosine receptor domains involved in selective coupling to Gs. Analysis of chim eric A1/A2a adenosine receptors. J Biol Chem 1997;272:337-44. 117. Palmer TM, Stiles GL. Structure-function analysis of inhibitory adenosine receptor regulation. Neuropharmacology 1997;36:1141-1147. 118. Bruns RF. Adenosine antagonism by purines pteridines and benzopteridines in human fibroblasts. Biochem Pharmacol 1981;30:325-33. 119. Brackett LE, Daly JW. Functional charac terization of the A 2b adenosine receptor in NIH 3T3 fibroblasts. Biochem Pharmacol 1994;47:801-14. 120. Feoktistov I, Biaggioni I. Characteriz ation of adenosine receptors in human erythroleukemia cells and platelets: further evidence for heterogeneity of adenosine A2 receptor subtypes. Mol Pharmacol 1993;43:909-14. 121. Webb R, Sillis, MA., Chovan, JP., Blawie rczak, JL., Francis, JE. CGS 21680: a potent selective adenosine A2 receptor agonist. Cardiovasc Drug Rev 1992;10:26-53. 122. Hide I, Padgett WL, Jacobson KA, Daly JW. A2A adenosine receptors from rat striatum and rat pheochromocytoma PC12 ce lls: characterization with radioligand binding and by activation of adenylate cyclase. Mol Pharmacol 1992;41:352-9. 123. Chern Y, Lai HL, Fong JC, Liang Y. Multiple mechanisms for desensitization of A2a adenosine receptor-mediated cAMP elevation in rat pheochromocytoma PC12 cells. Mol Pharmacol 1993;44:950-8.

PAGE 175

157 124. van der Ploeg I, Ahlberg S, Parkinso n FE, Olsson RA, Fredholm BB. Functional characterization of adenosine A2 receptors in Jurkat cells and PC12 cells using adenosine receptor agonists. Naunyn Schmiedebergs Arch Pharmacol 1996;353:250-60. 125. Feoktistov I, Biaggioni I. Adenosine A 2b receptors evoke interleukin-8 secretion in human mast cells. An enprofylline-sens itive mechanism with implications for asthma. J Clin Invest 1995;96:1979-86. 126. Jarvis MF, Schulz R, Hutchison AJ, Do UH, Sills MA, W illiams M. [3H]CGS 21680, a selective A2 adenosine receptor agon ist directly labels A2 receptors in rat brain. J Pharmacol Exp Ther 1989;251:888-93. 127. Nakane T, Chiba S. Adenosine constr icts the isolated and perfused monkey coronary artery. Heart Vessels 1990;5:71-5. 128. Alexander SP, Cooper J, Shine J, Hill SJ. Characterization of the human brain putative A2B adenosine receptor expressed in Chinese hamster ovary (CHO.A2B4) cells. Br J Pharmacol 1996;119:1286-90. 129. Kenakin TP, Bond RA, Bonner TI. Defi nition of pharmacological receptors. Pharmacol Rev 1992;44:351-62. 130. Stehle JH, Rivkees SA, Lee JJ, Weaver DR, Deeds JD, Reppert SM. Molecular cloning and expression of the cDNA for a novel A2-adenosine receptor subtype. Mol Endocrinol 1992;6:384-93. 131. Dixon K, Gubitz, AK., Sirinathsingh ji, DJS., Richardson, PJ., Freeman, TC. Tissue distributions of adenosine recptors mRNAs in the rat. Br J Pharmacol 1996;118:1461-1468. 132. Iwamoto T, Umemura S, Toya Y, Uchi bori T, Kogi K, Takagi N, Ishii M. Identification of adenosine A2 receptor-cAM P system in human aortic endothelial cells. Biochem Biophys Res Commun 1994;199:905-10. 133. Peakman MC, Hill SJ. Adenosine A2B-receptor-mediated cyclic AMP accumulation in primary rat astrocytes. Br J Pharmacol 1994;111:191-8. 134. Peakman MC, Hill SJ. Adenosine A1 recep tor-mediated inhibition of cyclic AMP accumulation in type-2 but not type-1 rat astrocytes. Eur J Pharmacol 1996;306:281-9.

PAGE 176

158 135. Mogul DJ, Adams ME, Fox AP. Differen tial activation of ad enosine receptors decreases N-type but pot entiates P-type Ca2+ cu rrent in hippocampal CA3 neurons. Neuron 1993;10:327-34. 136. Liang BT, Morley JF. A new cyclic AM P-independent, Gs-mediated stimulatory mechanism via the adenosine A2a recep tor in the intact cardiac cell. J Biol Chem 1996;271:18678-85. 137. Strickler J, Jacobson KA, Liang BT. Direct preconditioning of cultured chick ventricular myocytes. N ovel functions of cardiac adenosine A2a and A3 receptors. J Clin Invest 1996;98:1773-9. 138. Murthy KS, McHenry L, Grider JR, Makhlouf GM. Adenosine A1 and A2b receptors coupled to distinct interactive signaling pathways in intestinal muscle cells. J Pharmacol Exp Ther 1995;274:300-6. 139. Elfman L, Lindgren E, Walum E, Fr edholm BB. Adenosine analogues stimulate cyclic AMP-accumulation in cultured neuroblastoma and glioma cells. Acta Pharmacol Toxicol (Copenh) 1984;55:297-302. 140. Altiok N, Balmforth AJ, Fredholm BB. Adenosine receptor-induced cAMP changes in D384 astrocytoma cells a nd the effect of bradykinin thereon. Acta Physiol Scand 1992;144:55-63. 141. Fiebich BL, Biber K, Gyufko K, Berger M, Bauer J, van Calker D. Adenosine A2b receptors mediate an increase in in terleukin (IL)-6 mRNA and IL-6 protein synthesis in human astroglioma cells. J Neurochem 1996;66:1426-31. 142. Blazynski C. Discrete distributions of adenosine receptors in mammalian retina. J Neurochem 1990;54:648-55. 143. Schulte G, Fredholm BB. Human adenos ine A(1), A(2A), A(2B), and A(3) receptors expressed in Chinese ha mster ovary cells all mediate the phosphorylation of extracellu lar-regulated kinase 1/2. Mol Pharmacol 2000;58:477-82. 144. Fredholm BB, Lindgren E, Lindstrom K, Nordstedt C. Alpha-adrenoceptor stimulation, but not muscarinic stimul ation, increases cyclic AMP accumulation in brain slices due to protein kinase C mediated enhancement of adenosine receptor effects. Acta Physiol Scand 1987;131:543-51.

PAGE 177

159 145. Zamecnik PC, Stephenson ML. Inhibition of Rous sarcoma virus replication and cell transformation by a sp ecific oligodeoxynucleotide. Proc Natl Acad Sci U S A 1978;75:280-4. 146. Blake KR, Murakami A, Miller PS. Inhi bition of rabbit globin mRNA translation by sequence-specific oligodeoxyribonucleotides. Biochemistry 1985;24:6132-8. 147. Ohkawa J, Koguma T, Kohda T, Taira K. Ribozymes: from mechanistic studies to applications in vivo. J Biochem (Tokyo) 1995;118:251-8. 148. Heuer TS, Chandry PS, Belfort M, Ce lander DW, Cech TR. Folding of group I introns from bacteriophage T4 involves internalization of the catalytic core. Proc Natl Acad Sci U S A 1991;88:11105-9. 149. Inoue T, Cech TR. Secondary structure of the circular form of the Tetrahymena rRNA intervening sequence: a technique for RNA structure analysis using chemical probes and reverse transcriptase. Proc Natl Acad Sci U S A 1985;82:648-52. 150. Zaug AJ, Been MD, Cech TR. The Tetrahymena ribozyme acts like an RNA restriction endonuclease. Nature 1986;324:429-33. 151. Herschlag D, Cech TR. Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 2. Kinetic descripti on of the reaction of an RNA substrate that forms a mismatch at the active site. Biochemistry 1990;29:10172-80. 152. Tsui LC. The spectrum of cystic fibrosis mutations. Trends Genet 1992;8:392-8. 153. Michel F, Hanna M, Green R, Bartel DP, Szostak JW. The guanosine binding site of the Tetrahymena ribozyme. Nature 1989;342:391-5. 154. Michel F, Westhof E. Modelling of th e three-dimensional architecture of group I catalytic introns based on comparative sequence analysis. J Mol Biol 1990;216:585-610. 155. Kole R, Altman S. Properties of purif ied ribonuclease P from Escherichia coli. Biochemistry 1981;20:1902-6. 156. Bothwell AL, Stark BC, Altman S. Ribonuc lease P substrate sp ecificity: cleavage of a bacteriophage phi80-induced RNA. Proc Natl Acad Sci U S A 1976;73:19126.

PAGE 178

160 157. Plehn-Dujowich D, Altman S. Effectiv e inhibition of influenza virus production in cultured cells by external gui de sequences and ribonuclease P. Proc Natl Acad Sci U S A 1998;95:7327-32. 158. Yuan Y, Hwang ES, Altman S. Target ed cleavage of mRNA by human RNase P. Proc Natl Acad Sci U S A 1992;89:8006-10. 159. Cedergren R. RNA--the catalyst. Biochem Cell Biol 1990;68:903-6. 160. Perrotta AT, Been MD. A pseudoknot-like st ructure required for efficient selfcleavage of hepatitis delta virus RNA. Nature 1991;350:434-6. 161. Perrotta AT, Been MD. Cleavage of o ligoribonucleotides by a ribozyme derived from the hepatitis delta virus RNA sequence. Biochemistry 1992;31:16-21. 162. Hampel A, Tritz R, Hicks M, Cruz P. 'Hairpin' catalytic RNA model: evidence for helices and sequence requirement for substrate RNA. Nucleic Acids Res 1990;18:299-304. 163. Joseph S, Burke JM. Optimization of an anti-HIV hairpin ribozyme by in vitro selection. J Biol Chem 1993;268:24515-8. 164. Esteban JA, Walter NG, Kotzorek G, Heck man JE, Burke JM. Structural basis for heterogeneous kinetics: reengi neering the hairpin ribozyme. Proc Natl Acad Sci U S A 1998;95:6091-6. 165. Haseloff J, Gerlach WL. Simple RNA enzymes with new and highly specific endoribonuclease activities. 1988. Biotechnology 1992;24:264-9. 166. Pley HW, Flaherty KM, McKay DB. Three-dimensional structure of a hammerhead ribozyme. Nature 1994;372:68-74. 167. Chowrira BM, Berzal-Herranz A, Bu rke JM. Ionic requirements for RNA binding, cleavage, and ligati on by the hairpin ribozyme. Biochemistry 1993;32:1088-95. 168. Hampel A, Tritz R. RNA catalytic properties of the minimum (-)sTRSV sequence. Biochemistry 1989;28:4929-33. 169. Dembinska O, Rojas LM, Chemtob S, Lachapelle P. Evidence for a brief period of enhanced oxygen susceptibility in the rat model of oxygen-induced retinopathy. Invest Ophthalmol Vis Sci 2002;43:2481-90.

PAGE 179

161 170. Chowers I, Banin E, Hemo Y, Porat R, Fa lk H, Keshet E, Pe'er J, Panet A. Gene transfer by viral vectors into blood vesse ls in a rat model of retinopathy of prematurity. Br J Ophthalmol 2001;85:991-5. 171. Smith LE, Wesolowski E, McLellan A, Kostyk SK, D'Amato R, Sullivan R, D'Amore PA. Oxygen-induced retinopathy in the mouse. Invest Ophthalmol Vis Sci 1994;35:101-111. 172. Zucker M, Mathews, C.C. ,Turner D.H. Algorithms and Thermodynamics for RNA Secondary Structure Prediction: A Practical Guide : Kluwer Academic Publishers; 1999. 173. Kashani-Sabet M, Funato T, Florenes VA, Fodstad O, Scanlon KJ. Suppression of the neoplastic phenotype in vivo by an anti-ras ribozyme. Cancer Res 1994;54:900-2. 174. Li X, Gervaix A, Kang D, Law P, Sp ector SA, Ho AD, Wong-Staal F. Gene therapy targeting cord blood-derived CD 34+ cells from HIV-exposed infants: preclinical studies. Gene Ther 1998;5:233-9. 175. Choi KS, Lee TH, Jung MH. Ribozyme-medi ated cleavage of the human survivin mRNA and inhibition of antiapoptotic f unction of survivin in MCF-7 cells. Cancer Gene Ther 2003;10:87-95. 176. Luzi E, Papucci L, Schiavone N, Donnini M, Lapucci A, Tempestini A, Witort E, Nicolin A, Capaccioli S. Downregulation of bcl-2 expression in lymphoma cells by bcl-2 ARE-targeted modi fied, synthetic ribozyme. Cancer Gene Ther 2003;10:201-8. 177. Beck J, Nassal M. Efficient hammerh ead ribozyme-mediated cleavage of the structured hepatitis B virus encapsidation si gnal in vitro and in cell extracts, but not in intact cells. Nucleic Acids Res 1995;23:4954-62. 178. Quigley GJ, Rich A. Structural dom ains of transfer RNA molecules. Science 1976;194:796-806. 179. Robinson GS, Pierce EA, Rook SL, Foley E, Webb R, Smith LE. Oligodeoxynucleotides inhib it retinal neovascularizati on in a murine model of proliferative retinopathy. Proc Natl Acad Sci U S A 1996;93:4851-6. 180. Leeds JM, Henry SP, Bistner S, Scherrill S, Williams K, Levin AA. Pharmacokinetics of an antisense oli gonucleotide injected intravitreally in monkeys. Drug Metab Dispos 1998;26:670-5.

PAGE 180

162 181. Bennett MR, Schwartz SM. Antisense ther apy for angioplasty restenosis. Some critical considerations. Circulation 1995;92:1981-93. 182. Ferre-D'Amare AR, Zhou K, Doudna JA. Cr ystal structure of a hepatitis delta virus ribozyme. Nature 1998;395:567-74. 183. Walter NG, Burke JM. The hairpin ribozyme: structure, assembly and catalysis. Curr Opin Chem Biol 1998;2:24-30. 184. Dahm SC, Derrick WB, Uhlenbeck OC. Ev idence for the role of solvated metal hydroxide in the hammerhead cleavage mechanism. Biochemistry 1993;32:13040-5. 185. Narlikar GJ, Herschlag D. Mechanistic aspects of enzymatic catalysis: lessons from comparison of RNA and protein enzymes. Annu Rev Biochem 1997;66:1959. 186. Snyder RO, Flotte TR. Production of clini cal-grade recombinant adeno-associated virus vectors. Curr Opin Biotechnol 2002;13:418-23. 187. Liu XL, Clark KR, Johnson PR. Production of recombinant adeno-associated virus vectors using a packaging cell line and a hybrid recombinant adenovirus. Gene Ther 1999;6:293-9. 188. Lai CM, Lai YK, Rakoczy PE. Adenovirus and adeno-associated virus vectors. DNA Cell Biol 2002;21:895-913. 189. Lu QL, Bou-Gharios G, Partridge TA. Nonviral gene delivery in skeletal muscle: a protein factory. Gene Ther 2003;10:131-42. 190. Walther W, Stein U, Fichtner I, Malc herek L, Lemm M, Schlag PM. Nonviral in vivo gene delivery into tumors using a novel low volume jet-injection technology. Gene Ther 2001;8:173-80. 191. Harashima H, Shinohara Y, Kiwada H. In tracellular control of gene trafficking using liposomes as drug carriers. Eur J Pharm Sci 2001;13:85-9. 192. Liu F, Huang L. Development of non-viral vectors for systemic gene delivery. J Control Release 2002;78:259-66. 193. Templeton NS. Cationic liposome-mediated gene delivery in vivo. Biosci Rep 2002;22:283-95.

PAGE 181

163 194. Sorensen TU, Gram GJ, Nielsen SD, Hans en JE. Safe sorting of GFP-transduced live cells for subsequent culture using a modified FACS vantage. Cytometry 1999;37:284-90. 195. Stoeber K, Tlsty TD, Happerfield L, Thomas GA, Romanov S, Bobrow L, Williams ED, Williams GH. DNA replication licensing and human cell proliferation. J Cell Sci 2001;114:2027-41. 196. Ohta S, Koide M, Tokuyama T, Yokota N, Nishizawa S, Namba H. Cdc6 expression as a marker of prolif erative activity in brain tumors. Oncol Rep 2001;8:1063-6. 197. Sarman B, Toth M, Somogyi A. Role of endothelin-1 in diabetes mellitus. Diabetes Metab Rev 1998;14:171-5. 198. Fadel BM, Boutet SC, Quertermous T. E ndothelial cell-sp ecific regulation of the murine endothelin-1 gene. J Cardiovasc Pharmacol 2000;35:S7-11. 199. Bu X, Quertermous T. Identification of an endothelial cell-specific regulatory region in the murine endothelin-1 gene. J Biol Chem 1997;272:32613-22. 200. Feoktistov I, Biaggioni I. Adenosine A2B receptors. Pharmacol Rev 1997;49:381-402. 201. Yakel JL, Warren RA, Reppert SM, North RA. Functional expression of adenosine A2b receptor in Xenopus oocytes. Mol Pharmacol 1993;43:277-80. 202. Minet E, Michel G, Mottet D, Raes M, Michiels C. Transduction pathways involved in Hypoxia-Inducible Fact or-1 phosphorylati on and activation. Free Radic Biol Med 2001;31:847-55. 203. Maxwell PH, Ratcliffe PJ. O xygen sensors and angiogenesis. Semin Cell Dev Biol 2002;13:29-37. 204. D'Angio CT, Finkelstein JN. Oxygen regul ation of gene expr ession: a study in opposites. Mol Genet Metab 2000;71:371-80. 205. Michiels C, Arnould T, Remacle J. Endothelial cell responses to hypoxia: initiation of a cascade of cellular interactions. Biochim Biophys Acta 2000;1497:1-10.

PAGE 182

164 206. Dor Y, Porat R, Keshet E. Vascular endothelial growth f actor and vascular adjustments to perturbations in oxygen homeostasis. Am J Physiol Cell Physiol 2001;280:C1367-74. 207. Eldar-Finkelman H. Glycogen synthase ki nase 3: an emerging therapeutic target. Trends Mol Med 2002;8:126-32.

PAGE 183

165 BIOGRAPHICAL SKETCH Aqeela Afzal was born in Lahore, Pakistan in 1972. She was raised in England (UK), California (USA), and Kuwait. She received her Bachel ors degree in 1995 in Medical Technology from Kuwait University. Sh e then went to the State University of New York at Buffalo and received a Masters in Clinical Laboratory Sciences in 1997. In 1999, she came to Gainesville, Florida and joined the Ph.D. program in Veterinary Medical Sciences. List of Publications. Dandona, P., Mohanty, P., Ghanim, H., A ljada, A., Browne, R., Hammouda, W., Prabhala, A, Afzal, A, Garg, R. The Suppr essive effect of di etary restriction and weight loss in the obese on the generati on of reactive oxygen species by leucocytes, lipid peroxidation and protein carbonylation. J. Clin. Metab 2001. Jan 86(1):35562. Lewis, P., Afzal, A. Human Ocular Histology. Advance 2001; 53-56. Afzal, M., Afzal, A., Jones, A., Armstrong, D. A rapid method for the quantification of GSH and GSSG in biological samples. Methods. Mol. Biol. 2002; 186:117-22 Afzal, A., Afzal, M., Jones, A., Armstrong, D. Rapid determination of glutamate using HPLC technology. Methods. Mol. Biol 2002; 186:111-5. Shaw, LC., Afzal, A., Lewin, AS., Timmers, A ., Spoerri, PE., Grant, MB. Reduction of the expression of the IGF-1 receptor by ri bozyme cleavage results in reduction of pre-retinal angiogenesis. Invest. Ophthal. Vis. Res Accepted. Afzal, A., Shaw, LC., Caballer o, S., Spoerri, P., Lewin, AS., Zeng, D., Bellardinelli, L., Grant, MB. Reduction in pre-retinal neova scularization by ribozymes that cleave the A2B adenosine receptor mRNA. Circ Res Submitted.

PAGE 184

166 Abstracts Afzal, A., Iwabuchi, S., Ellis, EA., Tama i, K., Samuelson, D., Armstrong, D. Localization of lipid peroxides at sites of oxidative stress in ocular tissues by the tetramethylbenzidine reaction. Invest. Ophthalmol. Vis. Sci. 41:S904, 2000 Armstrong, D., Aljada, A., Higa, H., Ghanim H., Afzal, A., Iwai, S., Browne, R., Dandona, P. Activation of signal transducti on in the retina by lip id hydroperoxide. Invest. Ophthalmol. Vis. Sci 42:S243, 2001. Iwai, S., Cahallers, S., Higa, A., Afzal, A., Ue da, T., Fukuda, S., Iwabuchi, S., Grant, M., Armstrong, D. Increased MMP activity in rabbit virtreous following exposure to lipid hydroperoxide (LHP). Invest. Ophthalmol. Vis. Sci 42:S573, 2001. Harding, R.J., Kallberg, M., Lewis, PA., Ellis, EA., Afzal, A., Samuelson, D. Immunocytochemistry of endothelin-1 recepto rs A and B in iridocorneal angles of the dog and monkey. Invest. Ophthalmol. Vis. Sci 42:S328, 2001. Afzal, A., Shaw, LC., Caballero, S., Ellis EA., Grant, MB. The development of hammerhead ribozymes that specifically cleave the A2B receptor mRNA. Invest. Ophthal. Vis. Sci 2002. 43:E-Abstract 3711 Samuleson, D., Kallberg, M., Lewis, P., Ellis, A., Afzal, A. Locali zation of endothelin-a receptor in iridocorneal angl es of glaucomatous dogs. Invest. Ophthal. Vis.Res. 2002. 43. 43 E-abstract 1053. Afzal, A., Shaw, LC., Caballero, S., Ellis, A., Zeng, D., Bellard inelli, L., Grant, MB. The development of hammerhead ribozym es that specifically cleave the A2B receptor mRNA. American Diabetes Association, San Francisco, CA, Jun 2002. Spoerri, PE., Shaw, LC., Bead le, C., Afzal, A., Pan, H., Grant, MB. IGF-1R and VEGFR-1 hammerhead ribozymes affect gl ucose induced tight junction protein modifications in cultured huma n retinal endothelial cells. Invest. Ophthal. Vis. S ci. 2003. 44; E-Abstract 3911.


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 E20110109_AAAAXA INGEST_TIME 2011-01-09T15:13:49Z PACKAGE UFE0000624_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 24721 DFID F20110109_AABNPB ORIGIN DEPOSITOR PATH afzala_a_Page_033.QC.jpg GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
69bfc41ca542fc6a070c11b734ffa991
SHA-1
84f1c4c89497a111c280a2162bc5923915718ed7
5526 F20110109_AABNON afzala_a_Page_025thm.jpg
58a8c7a19b9f77750db97d09688fbe3f
1caabc37a549ad78a4f6961a719ae14d5d99f150
9406 F20110109_AABNNY afzala_a_Page_018.QC.jpg
7e822cb2bdaf5f6e49d7d1ac06b86320
6cc46f386b0c2b1678a4a19ce456aa0d881db5b8
871890 F20110109_AABMKW afzala_a_Page_007.jp2
1641c6b2507323055f1556a20aa7af29
aa4b82a83f628ba9147d777c7e9ceaf917ae2c12
101597 F20110109_AABMLL afzala_a_Page_022.jp2
b87a4df585195bc7615cf0a85f6adc8e
c080e0cb3ab0ee6105f182e28ef2ffc45eb27ec6
5953 F20110109_AABNPC afzala_a_Page_033thm.jpg
85f1a5ae56a2ff0f5ea9cc582f6dbe04
3f502887d9f230fced814931c14cf4f3f60a1c68
26486 F20110109_AABNOO afzala_a_Page_026.QC.jpg
f7c0097e8355965a047a5f40b790595f
0b664c17f9b873da53c8ffb440093e7d8425e5d0
2274 F20110109_AABNNZ afzala_a_Page_018thm.jpg
6448a22d0aec69ec652bab308e87bf94
c7b105fa22becebb8861d8fbf130d58a776f38e6
1051966 F20110109_AABMKX afzala_a_Page_008.jp2
31bea61cd19d1efbf60284b1f25e5866
93fa6f50c3ae2575fa2d9f6e4a190410e5e01ac5
106465 F20110109_AABMMA afzala_a_Page_037.jp2
9a5c745840574dc519be0cb706f9ef6a
5bcaea30e67590f780d31a7ab9cc21bc49635dc8
107385 F20110109_AABMLM afzala_a_Page_023.jp2
d64a942c4b73384ca5e426ff9f2a816c
98e37bb7d2d083182b3750c1d8b870f795534279
12736 F20110109_AABNPD afzala_a_Page_034.QC.jpg
f263ab381f0856b1d62a1156d28479d0
17f57e9fc01485975ab557df13d233a6b6470d5d
6351 F20110109_AABNOP afzala_a_Page_026thm.jpg
8a23211f24e0a26291a06cb37c0f8489
2a3e59a1450a7f577cd4a6008ef19d5e7cbfcb00
1051973 F20110109_AABMKY afzala_a_Page_009.jp2
91f97902869d7e917eb7e7ec1c1596e3
cb4d3da160306bcf9679d9515ef91f3525d629f7
1051981 F20110109_AABMMB afzala_a_Page_038.jp2
067136e6ed0a72b5e7e636cd769b9502
e546522f5080f01d2f21d64a256fdc921f734906
1051982 F20110109_AABMLN afzala_a_Page_024.jp2
c4aecb798682b574e3564e676b599030
26fb201f284765b06c958fdd4cd828aef18c84ed
4292 F20110109_AABNPE afzala_a_Page_034thm.jpg
0b4417bc33bb15d05bdd6e0e57f61603
815bb56499a67e470e279a79e8377bbb0f9e7b5a
25123 F20110109_AABNOQ afzala_a_Page_027.QC.jpg
b2fc0b9172ae21c0c69dc95aeaf9a6ed
30aa6227444ffbb55675450b63f656003a9ba9db
1051970 F20110109_AABMKZ afzala_a_Page_010.jp2
ed5c17aaba1542c901fe4fa755b39418
b927224ed051f75cc1ecb306417b7d9e29809a27
621416 F20110109_AABMMC afzala_a_Page_039.jp2
ea24fdb72b5642dd932490d3de79a2e7
98897eb95d27f124e6c0ffb76539b41347f4b812
95246 F20110109_AABMLO afzala_a_Page_025.jp2
e0c91c11dbbd2517ae2af09d7b8e176e
a60d3d1ad709eda03b1a4e16d286f26c10183015
25974 F20110109_AABNPF afzala_a_Page_035.QC.jpg
94bd218aba83f825c48fd0b08c2112f7
61aab6c8594ea5c00d6a26f2dd145959f449e786
105192 F20110109_AABMMD afzala_a_Page_040.jp2
8033b1c74431bab3964355f549dffc17
41b3fb22e62271115ee202c5b69b27b33dcac36d
6162 F20110109_AABNPG afzala_a_Page_035thm.jpg
13b85f42354de9ee15e946d8b18afde1
0d3533ceaf1b23dd223eebc0c4bb3b4fd6841ed5
6240 F20110109_AABNOR afzala_a_Page_027thm.jpg
baeb6a5f324215a3842561588051f4bc
17bbc6f6be9bb217af5c72b692db9fe4028ea160
654054 F20110109_AABMME afzala_a_Page_041.jp2
4dd2525ab4582292033fb0291f026335
11f01d459d498e20b4a4e7657481b9131917e4ab
106902 F20110109_AABMLP afzala_a_Page_026.jp2
39ca9400a1723e8254532a2219810b92
62cb940e2886753a8106e9916aeac725dcd333ba
9405 F20110109_AABNPH afzala_a_Page_036.QC.jpg
af79f7f1bac9cddbc6b53c5c63c9daa0
322e328e589f7dfb4f09abc66a9a1fea60a2e2b7
24109 F20110109_AABNOS afzala_a_Page_028.QC.jpg
972da510745ce270406e8284538e7e5d
bf4e80e4730502e21c35ca4e2d4dfee9908e3123
86566 F20110109_AABMMF afzala_a_Page_042.jp2
a72e390c9e4a5b34205aadd5b62b15fb
0c0c8d49919723d5822195feae5e98d85e692256
103537 F20110109_AABMLQ afzala_a_Page_027.jp2
bec545fcaf9da00d29a1a1dcbf6d04a8
5e2f895ced32ebfa03b25d3c147b0c218e7b46e8
3136 F20110109_AABNPI afzala_a_Page_036thm.jpg
596b2c1f058be8b5b16d841be448f066
9f6e37eb59ac52de3efd6dcdb8ba26b888e83098
5755 F20110109_AABNOT afzala_a_Page_028thm.jpg
1dade16ac231b458330a692c1aa195b8
2e2982adbfd05181dcd11170a25fd6029bdf89e7
1051983 F20110109_AABMMG afzala_a_Page_043.jp2
6db438865237230ebc88df278bbf9527
15747dcfe10e91d25d836c1b2fc2cba02e8b6fbd
97242 F20110109_AABMLR afzala_a_Page_028.jp2
2a4e881d6a5c5e3bf8f0994def69cfc3
08e2177840316709bb5cfe4afbbcec28e6d82caf
25429 F20110109_AABNPJ afzala_a_Page_037.QC.jpg
91827c5f582698e46311e6ed47a58c62
766dedadd580f3bbdb617c4337da7e72cfd13c30
23436 F20110109_AABNOU afzala_a_Page_029.QC.jpg
296c1d3c59eb6c3e37820a08eee7a2aa
9f36e10c4beedeab035e8d90f449813c98cdbd26
104207 F20110109_AABMMH afzala_a_Page_044.jp2
7726c458107ac8936fa0222e61514f76
bc61001825051ad8a1121b833c58a66c8d1769cb
97993 F20110109_AABMLS afzala_a_Page_029.jp2
3e753929a971aadb3c79628b5bd5fa00
2aa40385d6795822666669c55d2b4352ff91a977
6108 F20110109_AABNPK afzala_a_Page_037thm.jpg
961b4e882db5640cee03de716a9dac83
c27c0e6fc7ca04f79d92dbf42e32c8cbc67de875
5821 F20110109_AABNOV afzala_a_Page_029thm.jpg
aebf232b8e289cd44fc71b02f3cd35c0
29cb7983c4c6b7f3601ba1a479c55f553e72fabf
864655 F20110109_AABMMI afzala_a_Page_045.jp2
a1691af7f45eac9f358169b63896e8cb
dfa123c45cd83e81acbbdbb02b7d8777ce2138de
838058 F20110109_AABMLT afzala_a_Page_030.jp2
c4f664bfb7ad61a656bd0ae661916bad
7ab5745ba6eb78bb413924b24e971233d28bb058
17905 F20110109_AABNPL afzala_a_Page_038.QC.jpg
e53f58be2317da253ebdc00c7a85b6a8
d33febaf591d92271b238ebf9883444ad13d2f36
12330 F20110109_AABNOW afzala_a_Page_030.QC.jpg
0cbac623aa82a1ce9c272faff5f7875e
247c3235943767be8ca2af0c947eaa6856dda1e2
338322 F20110109_AABMMJ afzala_a_Page_046.jp2
09db05ac7a6ffc4fc36d8b0a5dce3ccd
d016634af0958c6c502e528cc8aa3b146b949443
109086 F20110109_AABMLU afzala_a_Page_031.jp2
d9d3dfd98700838f49a3f86960d37a00
79ec77b457963a2bf575a9b8bf65db797630a068
4960 F20110109_AABNQA afzala_a_Page_045thm.jpg
e7e18f189c5197c411e55849ffb8aa90
b74dede304024f719322469c4345bd248403ed1d
5601 F20110109_AABNPM afzala_a_Page_038thm.jpg
fe3fc3e57995d7e37a22692ace0a3453
f8ba2f0ae6a32232bce5d0e4d6af8eaac4fb4bc3
25998 F20110109_AABNOX afzala_a_Page_031.QC.jpg
0f52cf681e1a2ffe64a11d8f45fea3d1
ae4bceb49ccfa523baeabc00446f187f159437da
107956 F20110109_AABMMK afzala_a_Page_047.jp2
c32c544839e0ef46805df66129c6c4c6
8faf45ef398d8d244e028b30faa3c3125a98cf5d
586298 F20110109_AABMLV afzala_a_Page_032.jp2
c49da7144fc7b9d9154feb739f5643f8
f445fb6649b56afc9c02b7c952a6fb509924e6fa
8693 F20110109_AABNQB afzala_a_Page_046.QC.jpg
a189865886777238ee9ffc7c2381d947
b5bf253fcbbc618a875cfd9d5f1791538e5e3488
12002 F20110109_AABNPN afzala_a_Page_039.QC.jpg
b53ff95f469368dc59338676daffaa2f
c273bada0cc00df8aaeea25593854a3608f2445d
6278 F20110109_AABNOY afzala_a_Page_031thm.jpg
f3f225c4d78f710569a455ceb2bcd141
545883d97b0afd20e64a067f610790440614472b
964525 F20110109_AABMML afzala_a_Page_048.jp2
c4ea51376747b68eb242d5887a873e33
e26efb02041273c28ea8b52e09d0da5057bbb900
101643 F20110109_AABMLW afzala_a_Page_033.jp2
ad63cab1d9d066e9f6081dab751d0302
b2c733368f9268c6920f0df7233a33e4823011ad
2492 F20110109_AABNQC afzala_a_Page_046thm.jpg
d93150bdd888244d3c03e6cc293c6e47
83d9fa3ddf28095f22469e145409facce134cd6f
3987 F20110109_AABNPO afzala_a_Page_039thm.jpg
bedc2aaf941762842f88792cf0cb2415
aa5a6644526cfd192ad592b2dcb5a8e61d37b9c3
12411 F20110109_AABNOZ afzala_a_Page_032.QC.jpg
8a5fae807795bd72e0d41cb883dcb190
5aefa752acd08db6df7a51226404f27e25e1dae7
100751 F20110109_AABMNA afzala_a_Page_063.jp2
a4098564da4a148c0d3f70239253c63e
7a4624c82199590ead1667ce86987218c6849f36
109369 F20110109_AABMMM afzala_a_Page_049.jp2
5c54af74f89b831ca3605b158431620d
cbb2111cc2bad2eb558fb909e70612f448ff8b84
738157 F20110109_AABMLX afzala_a_Page_034.jp2
17868549d71ec3f3731637e87392efc4
3a42082f886538cc4ebb6ad1c093eac0823e8df1
26115 F20110109_AABNQD afzala_a_Page_047.QC.jpg
2dd5f8b6e75658901f6a40c07fa14f31
13284e3b7ae876a7cc61e621732639c66dbd7808
25134 F20110109_AABNPP afzala_a_Page_040.QC.jpg
79155c236376ee1368f4ec1bcdcbea2b
d132fe8262f32432e7f3b8428eebed53709ccb2c
1037913 F20110109_AABMMN afzala_a_Page_050.jp2
067615b86885fa8f3804d582261ad2ef
1d43d742b2cdf7e14b0fd2acec1e08b638254116
108998 F20110109_AABMLY afzala_a_Page_035.jp2
5c991d47a1de9b83dd3b316bb1f9d9a2
0260a72041cd3380d7d291d3e5ddc18bdce9c0ad
648444 F20110109_AABMNB afzala_a_Page_064.jp2
a81ba5ca32237749ee437b7d5aa3f3c6
959d7d10db396e5d236f2e9045e8ec22ab0deb0e
6362 F20110109_AABNQE afzala_a_Page_047thm.jpg
7866d575b6e9a95a1ea638ff6aa5cdb4
887dcbae6da05591ac8476a34ec46257c45f30ec
6135 F20110109_AABNPQ afzala_a_Page_040thm.jpg
dc4421d7fc31bbcfe81076688f367326
172f6b15a618c5972110e586ad005b197564c371
961469 F20110109_AABMMO afzala_a_Page_051.jp2
f5dbf8134aef120857773a2d7e5a0be5
b1b627f26f7e40794a7f2ebc78e0793c17712d98
396817 F20110109_AABMLZ afzala_a_Page_036.jp2
67968bfefdfa27933496e640bb829921
5bd557c844a5f836186dc9d999eacd537a426d14
107384 F20110109_AABMNC afzala_a_Page_065.jp2
e36ddbfcb6467ce768e360beed47c39f
0006c0f62ba14151ea2763420b43fe5349f8e6c8
21728 F20110109_AABNQF afzala_a_Page_048.QC.jpg
a0e85fa84456ed36901416e9c100b169
870d2e3b06653ad07f4a200ebe1ca6ee57272425
12664 F20110109_AABNPR afzala_a_Page_041.QC.jpg
f485514cfb31d277057a61ad37e5611f
8f3d91ca9c8dfd50d5e91eada619e5a40c952df5
648206 F20110109_AABMMP afzala_a_Page_052.jp2
9033540bc196d9560bbc6e5effffdd02
f8c2ca5fa3192d9855ce253e7ee2a1a6c3124481
109813 F20110109_AABMND afzala_a_Page_066.jp2
6138f8e2f25cb8b38b8901437bf8ed1f
243c91e6107f60b909af504dc5b651063af32ca3
5776 F20110109_AABNQG afzala_a_Page_048thm.jpg
295ab4f00278439cc756d6fd623375aa
ba93a2906970b3f3621103effd4bca7c057255ac
99818 F20110109_AABMNE afzala_a_Page_067.jp2
266b8cd8e9dc4dbf2dc337408a9b473a
eb11fc262472b9095b40b37381e3104f4cb084e2
26677 F20110109_AABNQH afzala_a_Page_049.QC.jpg
a4534701861a5cc5699e1288d0a80d62
8e20aa9dd23432c087189764246217ab043ea52f
3847 F20110109_AABNPS afzala_a_Page_041thm.jpg
36855b1ba24d8638ee98fde52610c9ce
63f082f50d64cdd6fd0d90539865aaf901dfb43e
1051943 F20110109_AABMMQ afzala_a_Page_053.jp2
64c07a5db7cfd51d43ac630a666f9767
9a70c7686ce7cb3dce48205f7973c61f8c1e6fbf
633703 F20110109_AABMNF afzala_a_Page_068.jp2
2b913a575e4d9ce14ec040f90d68f935
d342edf7c5a70806bebacb1ac0bc87f23559dee6
6193 F20110109_AABNQI afzala_a_Page_049thm.jpg
7b688ac67d2aaf79442d0563a7afdbc2
d72302696a9d956a77e10228b2c2471ee6a18639
20325 F20110109_AABNPT afzala_a_Page_042.QC.jpg
2c6cff6950e1d09657e38da7396a39fa
3877eecb32ba617c02f4ca430dafaf7bfc046744
F20110109_AABMMR afzala_a_Page_054.jp2
7f2284f7e66e3a124f99b9cb430fe2bb
f07d8a628e8c8a12087580889a2ee89d8df9b516
106033 F20110109_AABMNG afzala_a_Page_069.jp2
71c043a6efdf711c1720b86c12d56860
c2f2059f72500b12bf0ffe932e2e3fe46439480b
24550 F20110109_AABNQJ afzala_a_Page_050.QC.jpg
9f1e3d4b917bf8da719b731bfae60a5e
e8701dd0b3896afca6e4075235a2256759798ac7
4937 F20110109_AABNPU afzala_a_Page_042thm.jpg
6db953a84ecbaadb1608f3aba46583ac
548650781745198763dd4992299f51b8a37dea20
664569 F20110109_AABMMS afzala_a_Page_055.jp2
3810dd6d3108c8d09f57540f53d121d1
c028c817e83acf2c0d415f168010fb84f9a2e269
92852 F20110109_AABMNH afzala_a_Page_070.jp2
8d7203e4fbcb6d3cd3b6d532a87f434d
6e893ffbbefeffa7b2938398d2354ed72d2936a6
5734 F20110109_AABNQK afzala_a_Page_050thm.jpg
b08858f1ff7ce2334dfeeec7a83c33b1
556d129086957fc73492e642ace2b468a4c6d7e6
18916 F20110109_AABNPV afzala_a_Page_043.QC.jpg
479f06486246c798e833a57f53a63458
6af20a87122dd65897cc9df16c6b5b68cced08ce
1051946 F20110109_AABMMT afzala_a_Page_056.jp2
b2e8ae348ab115d00478494b8a867a19
d19dd3d11d83dfa5bd8236fc274ce5149c371da0
957045 F20110109_AABMNI afzala_a_Page_071.jp2
6cb75338fdaa8fbf9c7a913e75aeb7fc
6d44950591c196699b0ead1af64acc2c6a778b00
22571 F20110109_AABNQL afzala_a_Page_051.QC.jpg
76f30859814c33a41be7e5e1f76fa248
96f84f08824f28dfe2f229e34d7dbb897e18fd39
5241 F20110109_AABNPW afzala_a_Page_043thm.jpg
7bfa26ed85b6f573f8e1d684b74279b0
522d47ca015b1f171600b13ba8e95d9c8d4c5a7f
96261 F20110109_AABMMU afzala_a_Page_057.jp2
174b298ebfb0de27031a298260dd236a
d61153e5935c5dd6520e564c07f98594bc9c89cf
1051985 F20110109_AABMNJ afzala_a_Page_072.jp2
6e3d74791fbbb12e0c5d18d24d511e11
e9cd80c7a81334875b9f130d6e28820737ea7afa
2530 F20110109_AABNRA afzala_a_Page_058thm.jpg
ca1fc577b029495e2733f772a6a543e6
342b21a4cdcfb1b3a7249e8463615cc7111b24c5
5674 F20110109_AABNQM afzala_a_Page_051thm.jpg
73c8834bb141c209aa9aaae3d841b16c
091b46b72fc33091a18dd3b0f81687a5ac418766
24963 F20110109_AABNPX afzala_a_Page_044.QC.jpg
8be63ff7dc9a212d272ecdd6d9927ec6
d1636da1281ba23c1700ad91d328a84b9a8c7a88
461206 F20110109_AABMMV afzala_a_Page_058.jp2
2b1ab2771c06df1241c695460d9fa936
24a3c1bd8a44cc6334c61c08726a5bdbd7fbac43
1002657 F20110109_AABMNK afzala_a_Page_073.jp2
4634219ac0ca2ff67a9ee2f2d36b29dc
77821deb7609d1e912f07d0842f9a66d3c0166c3
11723 F20110109_AABNRB afzala_a_Page_059.QC.jpg
8b6a2578edec20fce0067b8fa4cd96da
ed75732f7cb2ddc88400f3332d4051492105fc5c
14319 F20110109_AABNQN afzala_a_Page_052.QC.jpg
d3d8945f863be41bcb9a08e1faee83d3
fdd156c6fc9a75dccf4c0a8f2f6666c51cdad43a
5887 F20110109_AABNPY afzala_a_Page_044thm.jpg
07040d2bd6858a6e11535b6832cf562c
2cf15c9d56becb0955d6c86ad0ebcdecf6e96ec7
603806 F20110109_AABMMW afzala_a_Page_059.jp2
ede456e1a62b6fb255cefb04bd1ab852
8f56e98cf25e01c1a0377c33b9980acce1492966
104098 F20110109_AABMNL afzala_a_Page_074.jp2
919558cd3820e120f26a623c923847c7
96890d337e2d8ee8cebafd30134657946be66de3
4020 F20110109_AABNRC afzala_a_Page_059thm.jpg
d3a445b819a76c51481becfab76251b6
bb3b93fe40fbdcc832d93976e0fa4dd56e76b530
4810 F20110109_AABNQO afzala_a_Page_052thm.jpg
943c4adab07786c3dd0e7a03084b296f
287ec00d1263977ab8d2574843b62d8fafac0aa1
20773 F20110109_AABNPZ afzala_a_Page_045.QC.jpg
559407982d2b31c130da66822a4df9ee
8a9036d038cde5fe9f610cac3ac3986355be9a88
1051967 F20110109_AABMMX afzala_a_Page_060.jp2
2ded5cbe4d2df83bc9df4fd96e1f78ad
69a8e676cf91aa34b43dad5f306b8ba3781c0745
190765 F20110109_AABMOA afzala_a_Page_090.jp2
3dbc62af5d2099ae7ad6bbd42bcaf7fd
9090bd00fb4146184766e1105a6e36348c81629b
758396 F20110109_AABMNM afzala_a_Page_075.jp2
a09f94770bc496ddc155919d1cb95942
59c3ecd82fa27e6a76c8b0b587fb46b3393ffb50
26827 F20110109_AABNRD afzala_a_Page_060.QC.jpg
73c7243505f349e54b323c503a6e36ea
b8ca0ea2b948f5be25a1ed57b093433a57a98e13
27551 F20110109_AABNQP afzala_a_Page_053.QC.jpg
a0d2dfdc4decfdd255b88bbff4063749
46b83a9240a9c04e9289d1bd816dece12a336577
873706 F20110109_AABMMY afzala_a_Page_061.jp2
56fdf3545bc74ce2853defa9171fd3b8
886620153c0b48f134d562aed033d6cdb25ed9ac
283268 F20110109_AABMOB afzala_a_Page_091.jp2
b8f81483a2776d23016f992054d3d8b4
393a021571edb9b318a48bf0b48455c116159893
862075 F20110109_AABMNN afzala_a_Page_076.jp2
a30160ed511f9b64702f710e6da22a94
56a42c269063b2fd3f5e3bda6f4d0b00225616fe
6543 F20110109_AABNRE afzala_a_Page_060thm.jpg
afcfedfe56ff3eb8941cd833b912aca9
1aaecbe149d1bf7fd57e62b502749621a7d4f8ea
6436 F20110109_AABNQQ afzala_a_Page_053thm.jpg
12aed0f2e656a17e8e9f895ecd0b2dc6
42a6e4834e3399de6d6f9d603a98bfe2772ea88f
106511 F20110109_AABMMZ afzala_a_Page_062.jp2
f917b22878a6b6d568d19967979f7db9
b92727e6c33b6144dceb1284866a56e849b915b4
108379 F20110109_AABMOC afzala_a_Page_092.jp2
77ca3e26c82b41eaa3ffc2a95730ae49
6680484de592195b3e2ef66f7e5437cc27f987e2
97543 F20110109_AABMNO afzala_a_Page_077.jp2
a0ae08c6fee2f9186690cd04a3a38226
c43cd7678d64f57dca6d80d18bd4f5c0c300cc1e
14045 F20110109_AABNRF afzala_a_Page_061.QC.jpg
c8a06f6d1b47b890fd482bec334fdc4b
8e39c1e566bf5a3af15d8d5ecd5ffda63d7de08f
25633 F20110109_AABNQR afzala_a_Page_054.QC.jpg
3f4397df1d3598f34be995f387111c12
8b736678755691922956979e42ef4794b35945f4
328047 F20110109_AABMOD afzala_a_Page_093.jp2
5a8b024b6dbc7d1d340253d7ec28004c
93b938cfe7ba626d17ada7a75917586f2dc75e7c
105825 F20110109_AABMNP afzala_a_Page_078.jp2
cbec2587618063bb58bdcac23fe1cc62
9a7c3eb5ca438dd41e021f0c2d8c0f891aa88d07
4359 F20110109_AABNRG afzala_a_Page_061thm.jpg
96942b8973195b7f7c9f62f1d30acb46
70e2315bd718b6559a766c7bbdc85581da374f14
6228 F20110109_AABNQS afzala_a_Page_054thm.jpg
cc594912cb5753470f2a799a3b149b74
48c9d9fbfa999ebabac8f2237d01df896d4e0442
724355 F20110109_AABMOE afzala_a_Page_094.jp2
92dde3127feab0ffd22c59ef63948fad
7079f82463eb4c6a18e72667c5fbbe5811e071e2
113856 F20110109_AABMNQ afzala_a_Page_079.jp2
dfceefe7400aa6eeb8c5f44b0d52877c
4ecfab5f1fa737fba5230fafd22273d336cafbd1
25171 F20110109_AABNRH afzala_a_Page_062.QC.jpg
d6db4c554ef01ce24f9c049705fb51e6
539455b1b07d5aa01d2d1e2327ba93afdb7a4793
110041 F20110109_AABMOF afzala_a_Page_095.jp2
07f926b497fd8b6ae5fcfb9c1fafe68f
8d9cbfc30518104d9bf46933754781f82b1fddec
5926 F20110109_AABNRI afzala_a_Page_062thm.jpg
ca42c6c879fdfdb0b3555c7b38fb7b04
993b90594b66db0ea8f3edd406a14d2979adc146
12006 F20110109_AABNQT afzala_a_Page_055.QC.jpg
1ca7cefc2361d0df917930966c919b6a
42c60c0762277734df1595937436648136c0f9d8
105573 F20110109_AABMOG afzala_a_Page_096.jp2
d57c13bdb026feb8a129897b96aa289e
c0f301bbdeb441db313bb63ce135d20467e7c14f
105429 F20110109_AABMNR afzala_a_Page_080.jp2
f77fa993a0d1b9079dcfb2d6dfada196
a55e3fb260d60dae4a32ea66fae1a2b9c3aa47db
23684 F20110109_AABNRJ afzala_a_Page_063.QC.jpg
601227c1097465198902239567904532
dffcd65c9d9924299647688d242324af22ba7f71
4098 F20110109_AABNQU afzala_a_Page_055thm.jpg
ab22a26dd497653392aa25976ae1238c
dbcd2d209f75ec4bb74af3e06a6e51a351a4ddbd
613277 F20110109_AABMOH afzala_a_Page_097.jp2
037010c120d988f1c22d306690b97210
0019fe868b294fe39eedce894fccc6f02db7b6c7
420589 F20110109_AABMNS afzala_a_Page_081.jp2
78ee830802b56b0cb8bb48c2509d5787
44636014dedbc8bd83710b9f9af7dccacd2100dc
5653 F20110109_AABNRK afzala_a_Page_063thm.jpg
49a5752d8d75434bf156f9eea71f0223
e8b530ecefaad8715822a8a4159fc065dc744537
27853 F20110109_AABNQV afzala_a_Page_056.QC.jpg
4997dba1ec8c8bc729045f80bb50c34e
5bb4fc970e62982e22bf188d859ea3882e2756db
285300 F20110109_AABMOI afzala_a_Page_098.jp2
3772da9a27eed6a131d6e596a1db06d2
2f10cb3e2ed5ab04f493c8424fca2ff23fbaf47b
77417 F20110109_AABMNT afzala_a_Page_082.jp2
c0c41098d708dae5c661609af724c7c0
d9003cbe998456dcf21be4c2c7f4afed1d94bed2
13147 F20110109_AABNRL afzala_a_Page_064.QC.jpg
1f1d7441d6aaee361ad721bc39783719
ff721cfeb696b7105a1ce0618638bd1b056dc4a9
6368 F20110109_AABNQW afzala_a_Page_056thm.jpg
f2409c21e6893a5464066f774f54e615
5e0f22aa40e37fd8957af322c1bb4d9096fbfe0e
419593 F20110109_AABMOJ afzala_a_Page_099.jp2
3764bb5e325e1816edf77ff11d386e9f
a1e6f7c44572b0303c62aeca345425ce78961d03
281652 F20110109_AABMNU afzala_a_Page_083.jp2
289604c3b7d07892086a4cad4f12e18d
00c116cd7bc290e9c397bc6f54f931c24c1bf2fe
2936 F20110109_AABNSA afzala_a_Page_071thm.jpg
eb50bfa0d640e09ae30a99d53e231591
8fd19a3ecfc19de07cec5d9a91bae71d757aba25
4460 F20110109_AABNRM afzala_a_Page_064thm.jpg
1a41852c2071d76cee9334320db33473
e5503c53344bea668b9dc035b2b37685979e508b
23331 F20110109_AABNQX afzala_a_Page_057.QC.jpg
057cc2c5b8c779d509e11b4b28a2b317
b0a2107f79e7c4966e5536cc6d065b2aa5d1dc91
472894 F20110109_AABMOK afzala_a_Page_100.jp2
b1e1b8ff1abe7fd756f36bc4d5be8168
1ba45aa65e7f789d734d829c7c76d12b6ce4b42a
332249 F20110109_AABMNV afzala_a_Page_084.jp2
e03ca3aab5ef6af90c628952f382a11f
cdc9213e4fbe5052067cde8a57e84fe65d373c7f
13489 F20110109_AABNSB afzala_a_Page_072.QC.jpg
3f697293f72f3e21070becddfe987076
092f89e8d509ed75d1d61c8ac00269ed6a7ba83c
25491 F20110109_AABNRN afzala_a_Page_065.QC.jpg
691ef823e409ba703017c99ec6dc6040
557b89e7d291174ed9d8d5f4ab13a662cdeaa97d
5743 F20110109_AABNQY afzala_a_Page_057thm.jpg
8d0d48dd4bcc8729ba18f953f6ff9588
1d451de3fd2df90b0b9a0eca237e7915a1b725d2
109430 F20110109_AABMOL afzala_a_Page_101.jp2
9fb7bdcd0bd3286bb21767184f822846
db7eed7b766d6b1f8ee143748e2d361e38c0e054
88567 F20110109_AABMNW afzala_a_Page_085.jp2
3f9c4c5423c49b49cd1284b10a8f14e7
ecb6808043615ed68ab0ca4b07bee90e3243bd9c
3729 F20110109_AABNSC afzala_a_Page_072thm.jpg
1f89d78c8390c84a9301a037a7bad369
acfbf3b0247b91b2ad2df3babaa04a28da7c6a5f
6157 F20110109_AABNRO afzala_a_Page_065thm.jpg
79dd82a084cfec4fdc96d977ca0e2f14
97b98a90af575b0f19d118002fef468da2163976
7889 F20110109_AABNQZ afzala_a_Page_058.QC.jpg
0f5ae0daa8a49d5c56861fc9fdc2e492
718c869818d55a7c58a667274a30ec52e47df956
109004 F20110109_AABMPA afzala_a_Page_116.jp2
16ba82fe00ac05c23fe8e6ae0055d890
38a9107b240ab7166e6613190a4a9a7afa24da73
538264 F20110109_AABMOM afzala_a_Page_102.jp2
893246f5bca3f2e70bd4610193e4d4cb
adee69f3c04f653738f631c1a358de6f4e42e1c9
260767 F20110109_AABMNX afzala_a_Page_086.jp2
c3c913307ec62e596edb4e1af9b11bb0
cb7266de0fec4466f1e067013ddb6f27ff803931
11661 F20110109_AABNSD afzala_a_Page_073.QC.jpg
087caf1554e77075df8425ded4505687
04625acbea786ccdf58268550607fc314c9eb347
26299 F20110109_AABNRP afzala_a_Page_066.QC.jpg
dc5f18175441633141dff08d3e4cfdcf
6093d31298c3f3225107fec0bf9ed56c41a2db56
29713 F20110109_AABMPB afzala_a_Page_117.jp2
01d5ce2b76c814b2e08e7f5439322af7
657c06963bafa258eec25fd7a523b88035147550
92637 F20110109_AABMON afzala_a_Page_103.jp2
b815559d0ff59e9748e76c7076a7b158
b103f1b82c0b948e8c60cae28f64fb37f3bc96ce
114247 F20110109_AABMNY afzala_a_Page_087.jp2
5f28321e8008d9e3a71bbe4002f2938a
49798fc82227caed3a1526cd7d7c26217e1a2878
3343 F20110109_AABNSE afzala_a_Page_073thm.jpg
b76d8d41475c7eb4c943dee033bd0653
8a73a6862732f96e2089f628c1e90c6e061b19b1
6460 F20110109_AABNRQ afzala_a_Page_066thm.jpg
1063683b22e46e60d30421aead042aaa
18a09d32a03927cc0a82155899bb44fe2fa24f6d
79528 F20110109_AABMPC afzala_a_Page_118.jp2
a8e1ff9ed1d50ea83126d9b057d1f91e
1b0e8ed758b0ef1049ff80783e0d99e029b3eb74
109263 F20110109_AABMOO afzala_a_Page_104.jp2
1f07d2d0222c1b579b34c8d7fab00d0b
eae37927c08f59660de20f87adf9bc40ea7ba558
659721 F20110109_AABMNZ afzala_a_Page_088.jp2
2789175c13ccd3c09c99e4dfa807f754
fafbd0d3305660ad7275243a9fe5daa5163bcffe
25096 F20110109_AABNSF afzala_a_Page_074.QC.jpg
f83b7d31e2ba8d306394d049756241ad
0344174fd92819d5fd1e30f65319118456710ed2
23643 F20110109_AABNRR afzala_a_Page_067.QC.jpg
edf9696dfdf69b3ca17a6ad72bc015ce
2e8c9dab7ecba3869c8cde0302866c07fa210db1
291923 F20110109_AABMPD afzala_a_Page_119.jp2
04414fd9cde9b372c03bee64751043a3
fac341eba21e2040de684023d447a99aa36d2c1b
106363 F20110109_AABMOP afzala_a_Page_105.jp2
a9a7543998415cb1eb1ce869b01059cb
ee9a214bb9399e2ebdb06c691e3aec4216de8cdf
6201 F20110109_AABNSG afzala_a_Page_074thm.jpg
1e2d651066bd67b0886f30b3a56613d4
2b572c3b061a856865ba8d0624f817d00bc90de1
5835 F20110109_AABNRS afzala_a_Page_067thm.jpg
7591554d29f6addf9d99b000881d07d0
d688da5ee5083b97f5b61621a23ec777c9f4e5c0
302493 F20110109_AABMPE afzala_a_Page_120.jp2
4294b48763abaa98f1a44f5826788d76
101325f97c3ea3e33654139105abd6ae9de82b02
98240 F20110109_AABMOQ afzala_a_Page_106.jp2
933bc945989a7ac59e1974f785720675
e62072d89eb4c146425e3afbeea2f7fb0c67321d
13019 F20110109_AABNSH afzala_a_Page_075.QC.jpg
e355019803fc10d7ce5f3b5a9c9c5b77
6e3c8949f5926436ac3dec83567528515bc55f32
11083 F20110109_AABNRT afzala_a_Page_068.QC.jpg
cd5007e5a354748243a55125c42fa73f
2dc996069cafc2e6f97c5af1f1813513ac42d7ca
103645 F20110109_AABMPF afzala_a_Page_121.jp2
ea65c8a52dd036b5a05420c3d8db5baa
10c9a2675ddded73e83478aec16351dad494cfa3
110942 F20110109_AABMOR afzala_a_Page_107.jp2
1b7e2cc4af7dedfaccf97645fb7c7c6d
b76ab65d07a409858d03e2c04d219a6a4db05a57
3679 F20110109_AABNSI afzala_a_Page_075thm.jpg
fac4791aef00dad9df3b30352fe4644e
530018c0ba991e92c68c7ae9e7b3f1f01e1a1ab9
518416 F20110109_AABMPG afzala_a_Page_122.jp2
89d2a9fdafa2d2b170873773675fbbf5
20fd87e81931aa33fed8e7e2f16dc2892ecf1804
13912 F20110109_AABNSJ afzala_a_Page_076.QC.jpg
467b14acb3ccf0f13b837cfe90d592bd
95a23824e4a6f5fe770ae2ea0e31839f3adeed47
3670 F20110109_AABNRU afzala_a_Page_068thm.jpg
253784d546288661db3fbafc26e83066
8a8693042d02dc3a197357dabb5f9df0f0f45c1c
370793 F20110109_AABMPH afzala_a_Page_123.jp2
49f468694b5f8db57241bfcaae6fdd4d
664f13816bdb28de5bcc1cb97f2d3f99b7de4ece
101094 F20110109_AABMOS afzala_a_Page_108.jp2
e2493b7ffcb124e11c0f983c5bfd6513
40800d410d7b9fc8a66a095ccdf277559315ca09
4028 F20110109_AABNSK afzala_a_Page_076thm.jpg
08ddf815e97a1329afaafd885271d825
f8dcce9244f1f25a205af312a523c0b8ad9dbf41
25455 F20110109_AABNRV afzala_a_Page_069.QC.jpg
f44464f42f49a02c3096113d79d3610a
20bf2b4625ce0aec789a5a3d923f6b4e2c27db5d
93169 F20110109_AABMPI afzala_a_Page_124.jp2
2058cb99fc762ff2be49850815c6c6f4
8ea911bb9285d9ecb8bfa10d858cb1b584eae50b
100045 F20110109_AABMOT afzala_a_Page_109.jp2
6dce79e0df0aca21b5cb70747b2d8ab6
c4bc4ea4cae1889606869af17fa2419110076e1c
23613 F20110109_AABNSL afzala_a_Page_077.QC.jpg
a20e887e48ce745ccd551e6d549dc71b
2b3101c706b4690f55fd7ee1507e7ec903e82bb1
6011 F20110109_AABNRW afzala_a_Page_069thm.jpg
6ce3995e4139921d739f52434b12804e
2e548cfd324601970b121efb5472ff7e2f75441a
380211 F20110109_AABMPJ afzala_a_Page_125.jp2
94557ca102fdb29de9f340d907a22426
fb361bbe34fe6607b251f0d529a4a34089624864
105056 F20110109_AABMOU afzala_a_Page_110.jp2
81097c7ec928868ed671dde1c2b4a4c4
0313ee5e75bd31fed7af2a05104b968b34fca903
5162 F20110109_AABNTA afzala_a_Page_085thm.jpg
286c9abbecef9cf8a0e17f5ba284ca88
a18b792eccb6c935d226371ed677bb3d1d68d942
5732 F20110109_AABNSM afzala_a_Page_077thm.jpg
ea45ede635eb5ee662a230c6225d563e
b3899748ce41c54591f9e31dc8b25978ef4299c5
22250 F20110109_AABNRX afzala_a_Page_070.QC.jpg
ef37ccbdb757ecf914a943e8fa49f72e
17a8f06e130a53df0adc68192d5896cdbd402e25
324137 F20110109_AABMPK afzala_a_Page_126.jp2
ac2582af37b292bedb6042a76dcba8bc
58ff826e5d0d591e9c29294ae255095ec2dc8b85
101026 F20110109_AABMOV afzala_a_Page_111.jp2
f993b1f22b1d8b2f34139bc31238d5cc
b6de2a22d3c448b12db3aced58ff6f41946645b9
7780 F20110109_AABNTB afzala_a_Page_086.QC.jpg
4d1c182a2aa4a6fe47e777fc47158c08
25ac8bdac90d34e43c775653066e3e0852767a25
25532 F20110109_AABNSN afzala_a_Page_078.QC.jpg
ce693f50a126d4833b4f0d9899b3467f
e91dc84f6476a55e62af54f3b37b1c41ba0359fe
5474 F20110109_AABNRY afzala_a_Page_070thm.jpg
b04b0c89d3a53fa295c23e6ab93f846f
bb8e0466e2c384d16dba6465280ec921db5d245a
99763 F20110109_AABMPL afzala_a_Page_127.jp2
55c73e769c68acd7b9ee53375023406c
651b2880843f0d7bcd3572b33610f3362c668a18
103376 F20110109_AABMOW afzala_a_Page_112.jp2
4080007cd269a2eed478890456154061
2421b44db73328739ce41fd777f9fb0ca321e851
2440 F20110109_AABNTC afzala_a_Page_086thm.jpg
99bf65103db3acff1e91b07c141f9f46
4811359a2d9e5003134b39284e5a9a5b1933a04a
6088 F20110109_AABNSO afzala_a_Page_078thm.jpg
66ebedc7f18a5f48039544ecd8fdd7e2
b3cd09740a3aaf7b697885a2c842000ee7b21440
10970 F20110109_AABNRZ afzala_a_Page_071.QC.jpg
af6574ad4de18e390c62ed142d243044
2b39d543bc33133808f83c05350b4f8ec6208c95
330378 F20110109_AABMPM afzala_a_Page_128.jp2
c18c81fad44071a2536c3f642f48c50a
e9c771bfd0181d7c4f4cd77c5386dd0eee2bdc5f
100800 F20110109_AABMOX afzala_a_Page_113.jp2
d44ac1b0f91be1805a4520c49a3b3824
61b544122727de9d2f11268c251ba87bc9595b1c
164044 F20110109_AABMQA afzala_a_Page_142.jp2
0336096483239136cb31e7264f27b6c0
0c2bc40dcea47fa95547eb77c55413d1ad03bde9
4830 F20110109_AABNTD afzala_a_Page_087.QC.jpg
4e1233edf837c9d718a06a0834338760
841b0ddbd5a2984007913ba6fb161abb266c6138
26709 F20110109_AABNSP afzala_a_Page_079.QC.jpg
13d5a015fa437d8e1ec44e8a506ddcc8
9bf6b9e44547021fa37abc0ff98b5062aedd5118
97646 F20110109_AABMPN afzala_a_Page_129.jp2
40a9de8169f69c93bbd5d8cfd664c7d9
dbfe356bcd7cb8ddf432ac505937361e818b9e2a
106944 F20110109_AABMOY afzala_a_Page_114.jp2
bdfee54589875f1763740d0cc6661488
51891af87ca3df4c58673c7f42eeee3635fa8edf
88409 F20110109_AABMQB afzala_a_Page_143.jp2
df207860bc4eee0f4856726f3df8f941
793537b0e5d0a35d743cf143a5d50aec750f1a30
1955 F20110109_AABNTE afzala_a_Page_087thm.jpg
4f19e77b73557655f3b2d73e287176ad
59d097ec2fb53c320f5928c0bfff02297070e294
24773 F20110109_AABNSQ afzala_a_Page_080.QC.jpg
52e39d06f3297f1201e08bb7706ab42a
85b13068bdf742b5ae78db758d743035d2064430
919201 F20110109_AABMPO afzala_a_Page_130.jp2
835c008066c9df1ee13cbc4d1aad87f1
e4e68139535e8e71d804df6f5136f22897c1787c
95317 F20110109_AABMOZ afzala_a_Page_115.jp2
3a3cde36265e14313a5f0ecec2ca8302
bde7486db93ca29f51e93d51e9162c70590d3aec
322262 F20110109_AABMQC afzala_a_Page_144.jp2
d986d7fd91519c0b0a3bee092214e70e
159a8664a0ed67c5f9c6a0eca340912b0ad66618
12852 F20110109_AABNTF afzala_a_Page_088.QC.jpg
de70e02dc6a8f8ace2095ec05fa5b4e6
713dbe3793e6dc38ae9c2d53007ac10a8d43ac04
9992 F20110109_AABNSR afzala_a_Page_081.QC.jpg
3e15b92d9c01fc54271aba8e43e34759
298b49eb0d9e5028cdc1c4c96e99115144ccdd2b
974075 F20110109_AABMPP afzala_a_Page_131.jp2
35f2eca7c53705ea0640f531896a87dc
4eed6b1c71ce22d69856c316cd39699593a86aec
1051788 F20110109_AABMQD afzala_a_Page_145.jp2
4bed1c896a3b9161b39027e06e4c5234
2ad88c2da24769a4690ba0c7fc6f4fc284316bc5
4302 F20110109_AABNTG afzala_a_Page_088thm.jpg
01d212ec1f9eea505338e926bbfc0ce7
05cf1d8fdee660f1731fa1b4f43feffa43ce5c33
3320 F20110109_AABNSS afzala_a_Page_081thm.jpg
ac34a8ccabf6b823581a9b88a88ff437
526738050304ce220b38bf86cd2d68a3ffc848a7
89758 F20110109_AABMPQ afzala_a_Page_132.jp2
6790a969070357d96cb0b2d86907216a
bd6d18cae1fcfac6a71067f613cd334bba6aa187
1051978 F20110109_AABMQE afzala_a_Page_146.jp2
a6b784cb3cdabfea7cf8f39cf9fb679d
9b62782dcc7f4c5458836a5b31dfa8e8b110a88d
22819 F20110109_AABNTH afzala_a_Page_089.QC.jpg
5786fcb2fee101d68551d07b44aae695
cdf0a8f1e53cfe118357104aa0337764b931d4d0
18030 F20110109_AABNST afzala_a_Page_082.QC.jpg
8e56857cdc0a3a789d34ed0e78329770
7dd410079624364fed45ab558d3e56f595cde68d
258534 F20110109_AABMPR afzala_a_Page_133.jp2
fe1344c4bb35d9a363fa47bc911d9e37
421d622fcb337538defb6ce05676e8b504f16cd4
1051826 F20110109_AABMQF afzala_a_Page_147.jp2
ab3e80afbe42e080282407e030fe4b04
74a40a6576e4bfc22d9361801cf709468e151e3f
5481 F20110109_AABNTI afzala_a_Page_089thm.jpg
fe507676c1044a0193644a61ebc7b498
38d7d246269295b84b35b19139179052b55c9efd
4554 F20110109_AABNSU afzala_a_Page_082thm.jpg
25882853ee90221b1b0272df6641161c
6062978242dce3ad640c5adec5e8d4ede6fc73c4
417765 F20110109_AABMPS afzala_a_Page_134.jp2
9e5b4729efc25fb8c9aba435439fe6df
46995007356c56c0315f9bff5f880ddda0afd335
331181 F20110109_AABMQG afzala_a_Page_148.jp2
b123092f2c3a55aa6d0f526355ef24e5
f6c89428ade6d66f7822a694f6cc4130855d7d67
6530 F20110109_AABNTJ afzala_a_Page_090.QC.jpg
9085373872f1f1e865eb9e2c6c181d89
12213949d2eac9cc8e5ae4dedfa32d3d5bbcf5d9
98374 F20110109_AABMQH afzala_a_Page_149.jp2
993780db46b02d39df0aa64d39a7d9d3
d107de4c2908ce52db9835d6811fe2b0f4f602a9
2369 F20110109_AABNTK afzala_a_Page_090thm.jpg
2bf2a396c90b9b84651ab5dde159dea5
7784baebe7f4fdd087f4ace707cc0ba949a4b358
7222 F20110109_AABNSV afzala_a_Page_083.QC.jpg
194a23863300c5be0d338ded1a97b772
00ff799de05617ab68b55911c968cabaec5d0bd2
426547 F20110109_AABMPT afzala_a_Page_135.jp2
1ee57710b5e3c629215062cde42d5120
b3643951798e07cd03c8e34e69ee012fdce04dfd
108552 F20110109_AABMQI afzala_a_Page_150.jp2
2eff3daae0e60eb9177bb755c92fcdab
a91bae4416f5beb27b3d93f2517591ec92eda581
8842 F20110109_AABNTL afzala_a_Page_091.QC.jpg
5a737f61d234c821be108beeb37d417e
c0e04eb51150d580343ff288ec08216f249f7046
1930 F20110109_AABNSW afzala_a_Page_083thm.jpg
91b598fa04241c2259e11f21726cb8e7
4164e7ccb9bf9770d6cf32e9c86f186719f5c40c
100560 F20110109_AABMPU afzala_a_Page_136.jp2
989fabf52a74e26e1b335608f68f7bfa
ba2ec35ed4a52282a39c59cf531168a456d6d9b2
1051950 F20110109_AABMQJ afzala_a_Page_151.jp2
275911980e1a9b587618455b26eed566
cf324a2fbd9b36ebaa9b8c6119dd220a1df5ff0f
2201 F20110109_AABNUA afzala_a_Page_098thm.jpg
12aa8635a10d1108b0ad535b3efd9d4d
d92e365b811e0f21c8dce6ed032027856f923eaf
2570 F20110109_AABNTM afzala_a_Page_091thm.jpg
92c9ba94271ff7a04e14d9a0ff2ecea1
89b9048cc3be3bfd835a0ca4ce897d017e698339
9681 F20110109_AABNSX afzala_a_Page_084.QC.jpg
fe1b85895ece3d33bce336757757199e
792183c8863a10bf86923a9ae61784e41c91ec01
990844 F20110109_AABMPV afzala_a_Page_137.jp2
33025f5067c09911d5e195a005db68a6
f5e8d815c01f0dc90a0e0a664714ae879a123fcd
1051986 F20110109_AABMQK afzala_a_Page_152.jp2
124d329e48b92996c58f92fb9450bf49
866fee93b14f2eb728974118edca50775966c4cd
9968 F20110109_AABNUB afzala_a_Page_099.QC.jpg
a9e559b53c8d4046cf19d2e3e07c072e
79aa59a50b5c1c8624beaa90f8cc51b38314849f
25684 F20110109_AABNTN afzala_a_Page_092.QC.jpg
7b9f056add37949f40df8c3c3340bc60
4d8c0a79a6aa92c216341fe4cba3bf4dd4158587
2835 F20110109_AABNSY afzala_a_Page_084thm.jpg
43981e0fa15952239bb0e2a6db89d1c3
cae6277e4e5c0ab63733ddf66e8948f92674d856
402540 F20110109_AABMPW afzala_a_Page_138.jp2
2f89063e907c961185bf566b80155fed
e6b4b6ac47f864090e0e9e71cf34e47dfae967b4
102319 F20110109_AABMQL afzala_a_Page_153.jp2
d10277fc73afec6b71de8dcd6b3a88fc
7d7587085e4766d6cdd0ba63b6756c21f1cb68a6
2655 F20110109_AABNUC afzala_a_Page_099thm.jpg
4adabd61942bb15c4b4da84418573c33
f13a44182383c0d09d74ce27ebd321f40f1d746c
6267 F20110109_AABNTO afzala_a_Page_092thm.jpg
a119dda97a9cc327bf3e89c696b9930a
494d3298f4ec3759daa8c2553402da58d1c9ee2b
21684 F20110109_AABNSZ afzala_a_Page_085.QC.jpg
b3342112e8c691c79a852028ca5e1072
e018deb3c2490e1c8531ffb71cbd7346a2c8b5cd
93550 F20110109_AABMPX afzala_a_Page_139.jp2
5dcfeae8120111fb5b9b5bf97d7f307f
96c441e7b7c80bc1838f70146d2221d259d08b72
97901 F20110109_AABMRA afzala_a_Page_168.jp2
2e93a87cde028631695fdcf3ba2c5d14
73c408cb2eaf55b8a00029619fe7af0835717ad0
101442 F20110109_AABMQM afzala_a_Page_154.jp2
3b5b1227d7fd3ad641e9813a31fb699f
59355bd9c12101bbb46c15f117bd6e769e300604
10469 F20110109_AABNUD afzala_a_Page_100.QC.jpg
b7d7ab0752dbdc5323931cbfbcd477be
1ae679cb5842db5e40e2da23d5ce28b2fb57171f
9641 F20110109_AABNTP afzala_a_Page_093.QC.jpg
67e4cbcef7c978c6d45c77e5947b5203
2826d2786d44e2ad47dbb875aa4b837fe16b8e85
381998 F20110109_AABMPY afzala_a_Page_140.jp2
231f7b2def62eac62c444695eabe1b35
2e38b12bd75943a8eb0c2f666d5ebcf6fda5f528
106005 F20110109_AABMRB afzala_a_Page_169.jp2
37d10212fed512a99881952797526d78
be08e675b3e9f62a3517a5b8fdbaa1c804cd89c4
689807 F20110109_AABMQN afzala_a_Page_155.jp2
c127e03d141d4158d8382b4243af7ca3
a30b7e8a50c5c6f1586f87e0a61682b3ffdb7667
3483 F20110109_AABNUE afzala_a_Page_100thm.jpg
135bd4a495af7d0c7b7f67e4ef97d0cc
7bfce99771838714137f49263e99ed76fc43fcea
2854 F20110109_AABNTQ afzala_a_Page_093thm.jpg
63415959678c394ce151a13cd1dc4003
534db677a1e94f940594b7f4eef095227a91a257
108979 F20110109_AABMRC afzala_a_Page_170.jp2
ffe07ccda66d46f27e90140a704a3d81
25d96d47a549ce4e1aa4e884b638766a6b4d44c2
110924 F20110109_AABMQO afzala_a_Page_156.jp2
dae31d41ffc9997b53bffc0d270991df
9e8a4e74ca99bd051aba138ba2a57a0204857019
479736 F20110109_AABMPZ afzala_a_Page_141.jp2
ee44b2a503490d33b3f0e7a9d950a346
c54ca88897bdbd5e5ee18d075880b7a131676147
24719 F20110109_AABOAA afzala_a_Page_178.QC.jpg
95c87f8a8034dbe73dfcc7d888e2b251
821bd04ac364394e990936bde5d8e684de647159
25940 F20110109_AABNUF afzala_a_Page_101.QC.jpg
8a2b7694a9d8652071131c055dccb0b2
e3490afaecc953272de6f22765d6f9100a3f3e68
16345 F20110109_AABNTR afzala_a_Page_094.QC.jpg
4e1b83637fdbf05fceee5a063a51e11d
5572212612385be8063c061f9d68c03e00adffe8
112608 F20110109_AABMRD afzala_a_Page_171.jp2
ef7d9ab6b010619fe84db2f6836bd546
1bd4a8a8270c57513a5ae775a3dc1882a129d128
112858 F20110109_AABMQP afzala_a_Page_157.jp2
a225e4b7ffb56a58f54d79718ed902a0
d05d8d71259f2650b04a12167cea773fad7693d9
6022 F20110109_AABOAB afzala_a_Page_178thm.jpg
6edae2b98e2cab71e4042f10d1bca8c6
877375d513ff021b8e044d626011be0ac8cd0def
6314 F20110109_AABNUG afzala_a_Page_101thm.jpg
00a719c35f6629bfe1b623190d99053c
0c66af23a429266c81a95483f31b19bb46b92f1d
4800 F20110109_AABNTS afzala_a_Page_094thm.jpg
b9175886857cc5786a2f9eba3e0ae11f
55b876f3ba87a6d8313ab72fc64dfa300c8225e0
117328 F20110109_AABMRE afzala_a_Page_172.jp2
4935aafe07b18771de7a234205f0e7e8
3c98cb3862729b881ef86e876022faa554956667
103127 F20110109_AABMQQ afzala_a_Page_158.jp2
3aeb20f85ccab71ccbc3e1726cd02db6
c61e828633915de504b5b33e0a69340d18d48c70
12229 F20110109_AABNUH afzala_a_Page_102.QC.jpg
685cfd8a372cb96c45e661360bd5ad75
1ae369222a198d1b3b66ce30ce31856014263272
26474 F20110109_AABNTT afzala_a_Page_095.QC.jpg
b029e766b9ca4b42ea9ef06b07a26730
3330a6fe98637d3e03c10846b250c5b56f47125e
111206 F20110109_AABMRF afzala_a_Page_173.jp2
de2ad2b1681962c72ccc3fc1e4b5ce0f
ec766e04eb8c570c364b1686b96f7d53037e9986
417792 F20110109_AABMQR afzala_a_Page_159.jp2
198f830798ad60062b9dc823536b5824
d9cf916a4933b4d7ad97a841e0fc0c0721cde064
24474 F20110109_AABOAC afzala_a_Page_179.QC.jpg
ce6594c2ccef54e92bf4221b5baad4f1
f4a563802feedd81a23556be9ac05d3a6d533bcb
3566 F20110109_AABNUI afzala_a_Page_102thm.jpg
a5cfe038325cf94290f6367dfbc1c39d
5edebffd777cf4020da20fa432889d9e7779e6b0
6326 F20110109_AABNTU afzala_a_Page_095thm.jpg
8784b5370dfbb7e2bdceb14dce1199e2
2d4499f27a3a0074316a3c22dc8a32c513d9ccb4
113186 F20110109_AABMRG afzala_a_Page_174.jp2
b35c073d4ad2ed960942f13469944b44
8f36f8a936e12b2e1e60ea666bdff7fabb3449ed
99168 F20110109_AABMQS afzala_a_Page_160.jp2
94c0bce1e7cc04e4a5f8324a8a2ee993
132c01d6e146f59101cf348132bfa850f80cebc0
6397 F20110109_AABOAD afzala_a_Page_179thm.jpg
43dd033dd3f51726bdb305b8b4615b7d
9df965223d7d21ae05bb3939bfc2bc62f6553890
22037 F20110109_AABNUJ afzala_a_Page_103.QC.jpg
b9834a1e480c527666564a7577628f56
d55e212e9cdd74ca4d75db4440cf5bc97793e6a0
25315 F20110109_AABNTV afzala_a_Page_096.QC.jpg
2e50fedb2c2831246d13fb8b84571f3a
ff3f1667edf204a045a54137a3804f2174321489
114848 F20110109_AABMRH afzala_a_Page_175.jp2
9ca8e8e3f23f0cfa17f2a645844379b3
7506118be030db1cba9b19e1be1fe47e82383568
107800 F20110109_AABMQT afzala_a_Page_161.jp2
33b4c062b3c6b47b59ae24a3e6994310
db98fd4dcf376f3ed7c3470666b7af8f595ededd
24518 F20110109_AABOAE afzala_a_Page_180.QC.jpg
5d06f75e32789b9532e61d5f4b6bd82a
17f7802fef0e3550f15dc71ed1c2c61777d71526
5644 F20110109_AABNUK afzala_a_Page_103thm.jpg
8d3a98cf6cf3968a36c433de80668727
5760e848905506b72e987f3ee1889ce81cc7562c
115344 F20110109_AABMRI afzala_a_Page_176.jp2
9073ddc5a766bd0a21e154d4fd6aa96b
fc32d58de6ce0502c844cc8b8fa8b0a1e236436e
5728 F20110109_AABOAF afzala_a_Page_180thm.jpg
0c1e7a7539b8ab9e8d23939131421fbf
2fd2037ca3728af7cb3bc15b58df7fba3f3918f3
26540 F20110109_AABNUL afzala_a_Page_104.QC.jpg
029450a31102ccfbdbcd59417e982034
543ce09260388ebe73106ed61e60bc3f7dc07b6c
6126 F20110109_AABNTW afzala_a_Page_096thm.jpg
17c6a706d4df7bcf0216ae01dfaa29ac
11129db8ac85a443334540d4f0f096136338ed37
113195 F20110109_AABMRJ afzala_a_Page_177.jp2
30e06a96842d9f7571d6d33821060788
e716d38cd07182f811c4dfe8c00330614360985e
1051969 F20110109_AABMQU afzala_a_Page_162.jp2
455b8f7087b2fa850c27dc056cd33ec3
8898fba20c8ac47aaca489f9848e935b45735688
22197 F20110109_AABOAG afzala_a_Page_181.QC.jpg
038f44a159d86857ed4dbf7bac53391a
721d274725b2ac2ea360156b372922baef6ad718
6107 F20110109_AABNVA afzala_a_Page_111thm.jpg
e424f444fd1d5eab80bbe279f7ee7cf1
00c4da7282b69423b03027dc284d7dc6d5a8ea81
6553 F20110109_AABNUM afzala_a_Page_104thm.jpg
c5f3c53f4c0bef7aa1d85a86349cda7d
752b78994163044520293ced116e78890ee6ea2a
12151 F20110109_AABNTX afzala_a_Page_097.QC.jpg
37d96b174e311a7c4f83c1a7dd9984a5
09dc0c20bc233cae41e8a5830713d9ec86c6a0a0
112397 F20110109_AABMRK afzala_a_Page_178.jp2
366c1fb70812fb7d67df32bc717636ea
940726bf74f00e3299a461a8e395343e00ff1020
487293 F20110109_AABMQV afzala_a_Page_163.jp2
06a51205fab8e46f774991981d6c9554
5d5d50faf796ae75d024dac0946d2997f8c18bde
5581 F20110109_AABOAH afzala_a_Page_181thm.jpg
0b5828ece2a9ccf18121e2a1e77efa75
2e2c333f2fffe563d41383ea68baacfff2edbf04
25156 F20110109_AABNVB afzala_a_Page_112.QC.jpg
601c3b49f3557c02e5f7d5e8b88f99c1
95af9b0b3ad217f571e502994f7f8a1dc7833501
24720 F20110109_AABNUN afzala_a_Page_105.QC.jpg
88d6f6104a28d4d4b08b07828052cf39
dad3126503414f829938e5935e82efc8bccfc719
4121 F20110109_AABNTY afzala_a_Page_097thm.jpg
1c358ce69f66d77885af96b8d6b61276
76b72200d401157144774587881739aea5efbe96
118315 F20110109_AABMRL afzala_a_Page_179.jp2
ae8735230812779be41fb624a8d931a0
1397b2977de899d39475474a91ccdea9ba21fca5
871417 F20110109_AABMQW afzala_a_Page_164.jp2
a33f666314460e245994af733b4a7e23
24d9985685d4edb0401ad6a2ee5c1fefbe780a22
5314 F20110109_AABOAI afzala_a_Page_182.QC.jpg
d4f5609270ae1cfaded92150864c8e0b
d06c4f48116b301247b0d5d3b07b9f7d0f97b9a7
6363 F20110109_AABNVC afzala_a_Page_112thm.jpg
b7a0299045c446b0df0f2c44f4138a1b
d431182c430b81679ab0b297e79bdf6bc804193b
6529 F20110109_AABNUO afzala_a_Page_105thm.jpg
9c43c39aa041efb329cf2ffd6c5ed6f1
6ae2572d73e869329156ab93d318306146c1ccfc
8328 F20110109_AABNTZ afzala_a_Page_098.QC.jpg
33645e2ecc71726042c5c0394ae1d25e
67ed9a3e4d4ca0fa25fb11b88ddaebcee7b693d9
107640 F20110109_AABMRM afzala_a_Page_180.jp2
2e035223f031328bc52d3d7f87472ea3
3512a33e07926e68cb91076fd78217ef603b887e
83151 F20110109_AABMQX afzala_a_Page_165.jp2
2640f6054580309c2d2f368041346798
2719971dbc3b8e0fba0bd215a7363f120de94794
25271604 F20110109_AABMSA afzala_a_Page_011.tif
6787f84ae5e863377e3f5edb2527acd7
f94b875f7731b2381f3d8d72ab554bc582660055
1236 F20110109_AABOAJ afzala_a_Page_182thm.jpg
b7e5be1bbde466ce2c3b8871caa4ba8d
888808e0473a5f7fdd5053fd7bd3cdeb6c498943
24607 F20110109_AABNVD afzala_a_Page_113.QC.jpg
73ab616ac00161617a0a9a9be20d83ae
f7c0f647441a831da320f757601fb044a79f5e46
23583 F20110109_AABNUP afzala_a_Page_106.QC.jpg
959d4422d570ba84c6a769149f0299f4
21f13ac5868e613f0f25088887bdcb285438816d
103245 F20110109_AABMRN afzala_a_Page_181.jp2
ba2f1d8610828f2fb392f314213d5586
d1b1ef164851f57df42c09fe386509ae5d46e06f
105511 F20110109_AABMQY afzala_a_Page_166.jp2
75b4283c6d4ef93cab000e8f4f568210
c9ae44faa9e86e611294195a3c71e23064b6675f
1053954 F20110109_AABMSB afzala_a_Page_012.tif
82c752a0c8748cd32bf63b4724463b04
792bdbd874049e7cfb686afdd75da15e8e236ebd
20854 F20110109_AABOAK afzala_a_Page_183.QC.jpg
b8a266528c92abd678ea66dfd2d7ef6c
fcdacf3168fb9ae4ae19d0f5cf4372abc507d5d0
6249 F20110109_AABNVE afzala_a_Page_113thm.jpg
8b02368eedc113fa5e7dec47980e0366
7aa22a05e8a9f7c18150849bba846ea38a929877
5853 F20110109_AABNUQ afzala_a_Page_106thm.jpg
76c0e4928d522fa106cceba7b7f1a8ff
8ce7d65f2c82fc5f5ef0be50597fa3757e32d18f
21403 F20110109_AABMRO afzala_a_Page_182.jp2
a5d1342ddb2153132176d71813dc34fd
4844b92a5b326f274d5bbf4954f5d29f6fd42355
109742 F20110109_AABMQZ afzala_a_Page_167.jp2
da40e636ef70442cf8675af3db9f5dd8
18fbbc4f2254784773edd7fc852b9cc0477c8b0b
F20110109_AABMSC afzala_a_Page_013.tif
4053a699e83ba09677940f11f9d3297d
de63aff38ded675a938f22d0868f37ff72b5154f
5125 F20110109_AABOAL afzala_a_Page_183thm.jpg
e82823cc657a59d93679b12a6c826038
9f4a172c8febbbfc4c9b9e3a8847a64da678e86e
26591 F20110109_AABNVF afzala_a_Page_114.QC.jpg
0cecb3f454c4549354aa8bd1ffb0e4fd
3fdab03f977c0864af7c92445e4133b9a257f509
26672 F20110109_AABNUR afzala_a_Page_107.QC.jpg
3e24b5bf60a0939dd44c521db838e59d
aacafc1a5d40640a4e36d42665465cec8e4de929
92994 F20110109_AABMRP afzala_a_Page_183.jp2
fb3926e9122cefb52c41f33f782e9a75
be74e964ce91d2013f94cd74be66ad16005bac90
F20110109_AABMSD afzala_a_Page_014.tif
bbe59da8ab8b92b2d006966bcc54d665
a791d8f94b401d3517f53aa1583613bc079310ae
21326 F20110109_AABOAM afzala_a_Page_184.QC.jpg
536dea0ad99ef6bffa47a877bb2eb22e
6ec51b60b44eeeee819bf7929b16b5f8c44e2276
6180 F20110109_AABNVG afzala_a_Page_114thm.jpg
1f11951884c571405e4fdd628c1da625
e66d9476779a92cf9e740eb152f808e52bd7a538
F20110109_AABNUS afzala_a_Page_107thm.jpg
57262652be110bbb2e3d57f6314afdc4
b0e1f0ae96cfe77ada4e10295a09afc2182fe1fb
100048 F20110109_AABMRQ afzala_a_Page_184.jp2
91d3ae40c05b695f13484725a54a3ac2
b8f5212b1e4e92f5d66c0b5e67e914715edcd111
F20110109_AABMSE afzala_a_Page_015.tif
f67d74fe2fcd115dcf0c1cfc290cc492
301da52dd7d09eb507e8facaaa8c2f450f9c3870
4903 F20110109_AABOAN afzala_a_Page_184thm.jpg
794c695314842323726c4121b036a72b
9661c84b692ca84b40dde7507a4a9f15d0739e26
23672 F20110109_AABNVH afzala_a_Page_115.QC.jpg
336a03c11863002b26d57d8ef5401f1e
c5671eac1133dc71da93fbb6efe66c77dc5ea53c
25348 F20110109_AABNUT afzala_a_Page_108.QC.jpg
707e53adc5456821f345a50ae9ff4068
0fa51c1f2aca98e4fecfa480e0b1549768ddfbfa
F20110109_AABMRR afzala_a_Page_001.tif
a2472b36ca6e6928ea98ba5f612c785e
fbef75c6a1663288e7bdf6a036728b5192efed73
F20110109_AABMSF afzala_a_Page_016.tif
8e6e3dd47dbdb2cb8d982a9214bb9196
73942aa7f4930d0226a96bac6d084a5b46202bad
211537 F20110109_AABOAO UFE0000624_00001.mets FULL
e0b7b65ad4a38f51abe7f7111f286e5f
2ed427e33316f405b830917a4084bf2649aeeb59
5692 F20110109_AABNVI afzala_a_Page_115thm.jpg
990fe3414d55f1fa5e10c65cf3af6089
d74daf9f14c3457f9351c03609a6c9d4b15d931c
6002 F20110109_AABNUU afzala_a_Page_108thm.jpg
e1efa58be8088a2ca8a50ffd73979824
13548b52fe85d5332d13efa059b57dbd84673781
F20110109_AABMRS afzala_a_Page_003.tif
0197f6a03d3028a9b5903f115c3cc813
0b74e7574be83d5fc644f1dfe0f2431c9c6fbf93
F20110109_AABMSG afzala_a_Page_017.tif
2f1b7d5d5f26b0402df729cce0d4e626
98dde498eaa2aad4d761d7cc99748e6654907906
26179 F20110109_AABNVJ afzala_a_Page_116.QC.jpg
74e4082dc34efc80a1e772dfb5a040ea
735933b4d23693ee36fafbde6a1194f2294c19f5
24643 F20110109_AABNUV afzala_a_Page_109.QC.jpg
d87a19866c8b500652dc178fa6e2add4
e609d5d30cb1a99dabd677c7b91aa309474f67f0
F20110109_AABMRT afzala_a_Page_004.tif
a8574a80356eab322f9e622e61be934b
3e8523362b03a2769cf35e8741d57addc8e4d51f
F20110109_AABMSH afzala_a_Page_018.tif
452c6124946d3fa6f5e8ecbbee6cf052
2f9b9fd62a0434beb284fca75a1c76584d008295
6275 F20110109_AABNVK afzala_a_Page_116thm.jpg
d47403e87bb0b742d99cee8e9855fd29
5b4766e6d734c6d93eff713dc22cc1cab9daa325
5889 F20110109_AABNUW afzala_a_Page_109thm.jpg
9eea27c5a5c49c735c0b529045fdc4a2
702e5fda1f64dd58525a953efab5818c1e669e13
F20110109_AABMRU afzala_a_Page_005.tif
b66ddeacf0c5e61d859aa2060911b529
490a5cacc65bca0e27b97c11e4f7f6a5edf62c1e
F20110109_AABMSI afzala_a_Page_019.tif
06de0b2c7693f88ee8dbee1bcda2f0a1
bb506bbbda5bd6f3d2a61abd5dc67343adeff719
7561 F20110109_AABNVL afzala_a_Page_117.QC.jpg
d1ce028f40035db1828f6bf6d9d9f1cd
f8b948884d47800565b2f7b95ad9526bdc517597
F20110109_AABMSJ afzala_a_Page_020.tif
c4399dec4cf69b62b683111f6803caba
7bba0a6ee470ffa383a27afae213187cbcafdb1c
5483 F20110109_AABNWA afzala_a_Page_124thm.jpg
8c0779b7a5c96c972a8ca2012d3586be
a6eb246f9f4fcdebb9b40094f3dfd597dbff091a
1984 F20110109_AABNVM afzala_a_Page_117thm.jpg
0d5bb80e657a92c36ec905f108ed0698
d2c88a31c76bdb6f7b26692a1ab9bb2dfb4d39ff
25957 F20110109_AABNUX afzala_a_Page_110.QC.jpg
221cfc263980b26c79e34ab30e09d05a
4cccfcdda04ec18dee87315f701ca1364e7884b6
F20110109_AABMRV afzala_a_Page_006.tif
58c75076d3820d0b2fe5b5d33ec5b65d
c822f510a74da9b6b9f2c87997ee6e91c182ea46
F20110109_AABMSK afzala_a_Page_021.tif
062bb333b0c29a5090bddc3f6c0ac499
c12605059a32e90ccd37a6c2e9a6d62b86f7a361
10939 F20110109_AABNWB afzala_a_Page_125.QC.jpg
7db5047271893381aa8436357a10fa1c
f18e02251b8c239f49f94b81cc4b179402b900c3
19233 F20110109_AABNVN afzala_a_Page_118.QC.jpg
74c2cd0bc548d06560ebd83b12452a72
3e3f02bca2e74c47ad10d9c5dff4086d64c62b00
6202 F20110109_AABNUY afzala_a_Page_110thm.jpg
e08a0fbb7700f3c41567ca56510fd7d9
6ec3b851c6087c3e0eb6674a4e256a3545d865a2
F20110109_AABMRW afzala_a_Page_007.tif
4557b01eb2e67423422f52cd392e4f38
b2cf93ed0282a35609e1c6fac8de6c6db3a5d038
F20110109_AABMSL afzala_a_Page_022.tif
8875d307bcb278d86942481e71406a7f
0ca2cd246eb3e9039e081f6cd31adfbd7661577d
3403 F20110109_AABNWC afzala_a_Page_125thm.jpg
b3a27b8affe03904350bc7159e31240e
9011451b0b4a4ddcb90ef6e465b298c85e75bb94
4940 F20110109_AABNVO afzala_a_Page_118thm.jpg
9ba086a2877e465493523dd23a34818f
49e746c71720c34710f658e51a8f7ffbfe6fa3ad
24970 F20110109_AABNUZ afzala_a_Page_111.QC.jpg
abff3fa9c039acfbd34d8be392b26a20
075aa49dcfee4b8a4a9347d0a55fd8072d9bd01e
F20110109_AABMRX afzala_a_Page_008.tif
70940d937be6662295df3ac073401cde
c8f3571245ea13718f0e56eb1efa8b6fc68a3a8f
F20110109_AABMTA afzala_a_Page_037.tif
702e356d81e60316d4d1a5fbd7bc168b
178e2c7ecdcf5cab0d5903f92f34aa90b9b259c6
F20110109_AABMSM afzala_a_Page_023.tif
c18f246678c8bd0a06b8eec90199a2a4
69782257264247e31e3c6d00794805d89a5d1a11
10021 F20110109_AABNWD afzala_a_Page_126.QC.jpg
d3b5813537fb8e8e62483746eac2ee58
5385c038d41ccd151720230ade9c0b6939641777
8219 F20110109_AABNVP afzala_a_Page_119.QC.jpg
ddfc2abcba0c7e9d2d98d71c8b28ff12
138a6bd96b5ec4fdb58e051b1f3d502cec01862c
F20110109_AABMRY afzala_a_Page_009.tif
0fc47c318f53671320519a9bc439554c
1ad7758e387553c7747d92d759721c9b68268ae0
F20110109_AABMTB afzala_a_Page_038.tif
26b664ce19fa830063459fd76c4b8b8d
3815e10dbce2c744a25d10be4780e151dfb18b58
F20110109_AABMSN afzala_a_Page_024.tif
7cd4ddbc6be03c4c490d03e5e7198c66
d0c947dd4f8e213311585da9eaaf50b722d96835
3331 F20110109_AABNWE afzala_a_Page_126thm.jpg
6af3c1a30112c82bece518f7f0771895
d3af892a9100f60ab7208c7e06284bc053e294c2
2557 F20110109_AABNVQ afzala_a_Page_119thm.jpg
fbaf524708d206a0a15389ce30b50161
dcceb6f79f9197513604fbf99530eb84ee1912ad
F20110109_AABMRZ afzala_a_Page_010.tif
33ccb8dd8310b599cf70dc8dce1137a6
1c357834c2cac7e82c1a4e81c462860054cce7c3
F20110109_AABMTC afzala_a_Page_039.tif
0fffa7ccfd92a24eeecbed34f3daedbd
bb25b915022ab53d1432646d61ccc4c36043899d
F20110109_AABMSO afzala_a_Page_025.tif
f2cfec95da87d14cb91b6e78f336d169
4ec2d7d25d72df473e2ae39c37333e97af4e7e34
25181 F20110109_AABNWF afzala_a_Page_127.QC.jpg
e9b063763d0accc49312949cea4c200f
b280d605d24c2ffbaa822235ea9e8acf2086f0d4
8436 F20110109_AABNVR afzala_a_Page_120.QC.jpg
2636e72f61f4c633eee5e267e154e2a2
66f5e308f9ac3ea59ef86055c3386842313e83fe
F20110109_AABMTD afzala_a_Page_040.tif
49a9cd01602b64a36ed38b39f8ed33c9
9116a2f2687fa403fc91dbddf18fb69ac288e073
F20110109_AABMSP afzala_a_Page_026.tif
d1b80ab47e9f9855851fcdf6562b20fe
8de485327a3db09530ddf391a918c2f4a4d7e2e9
5965 F20110109_AABNWG afzala_a_Page_127thm.jpg
4e08359a5ad89efd17ff1c77e9ac9eba
e1ac06e1107eba4c567203559a2df4159468b5cc
2474 F20110109_AABNVS afzala_a_Page_120thm.jpg
6c0e4f3503d18c2817e659fe14ee550b
2dd2b434c4b8f2d94b7a7cb8c70437b328ca5e00
F20110109_AABMTE afzala_a_Page_041.tif
9bffb34c113efd3799e2bd8ea1596ee6
4249a99971ab892c1df94bf934b49dbeb9ded8b0
F20110109_AABMSQ afzala_a_Page_027.tif
6059e702ed43f18528f102d87cf059bb
07f6f09be75b5a77790addb5d2ae5fda7e2db7f2
10548 F20110109_AABNWH afzala_a_Page_128.QC.jpg
58fde4686c776a00e14f3ac79d33f2c0
3e01ba3390c2b62280940b3d005bbfa06ccefaf6
25153 F20110109_AABNVT afzala_a_Page_121.QC.jpg
5003a65caa6519e577ca38ff9fc7489c
464215979b5035e7ddd9bd68cd47ed525392108a
F20110109_AABMTF afzala_a_Page_042.tif
e41a0de90473c0a7e15a4d2a1e8dc77f
c78e0c5f48382e6bfc2c568b0e8f9296f1262c93
F20110109_AABMSR afzala_a_Page_028.tif
282c57ac5bb9d6c588182a7acd84648b
9940b3c0c09ff2082aa56a2cd462aed199f0bd7e
3227 F20110109_AABNWI afzala_a_Page_128thm.jpg
9c7c4a8a0ad18fac504dc1cd3eee1944
3b3fe233a542d34327633f1bb454ae7fa9cd9a3e
6392 F20110109_AABNVU afzala_a_Page_121thm.jpg
bdb8f0e98e4336b95e4013656b83a99c
d5882cbad9b0e15df106b66034975c91dcbd7605
F20110109_AABMTG afzala_a_Page_043.tif
2f453315dd64c930cef40a50b457afd9
2f3976dbd200c6e11775a8a5cb16cbb0a262a30c
F20110109_AABMSS afzala_a_Page_029.tif
2706d234a3298b586917dfcc97759940
ebac2a075b50f76f686c44274eff647f8abe3aa9
23372 F20110109_AABNWJ afzala_a_Page_129.QC.jpg
990f2f71fd0e733d75f7ade7ecc31b43
0eb1aed53d9a7cc4b7d8bb5e9ecbfe13eff9ef6e
7959 F20110109_AABNVV afzala_a_Page_122.QC.jpg
4e0542a9c844705e8b9d6a76cc208f41
2b6f5c59684654ca81c6112bed5b2a4229d11edc
F20110109_AABMTH afzala_a_Page_044.tif
2e3534ffb1a30327283701e73b8e9868
c572b587a08aa7f6b655efb3c1c591c8fc859434
F20110109_AABMST afzala_a_Page_030.tif
df8a31b2e88ed74157b2ebff2b3b73ad
753f2311d517f5a3a91272f1440606d2b45c3d6e
5652 F20110109_AABNWK afzala_a_Page_129thm.jpg
a2fd26ee5a93d001e910257b691121d8
a876e5ada2ec159c0250eacb3fd6783d8851febb
2235 F20110109_AABNVW afzala_a_Page_122thm.jpg
339b0ae1312a159a39607d696036024d
58e0701ad8625a341778077a8e1c8ecee167b934
F20110109_AABMTI afzala_a_Page_045.tif
3f40002dc47e7a26c7ee2433801e8dd8
aa507f7e4537255ebdedd139962dd8db52a6baca
F20110109_AABMSU afzala_a_Page_031.tif
4c6228cc09cefc4b8ee0d1a49698710d
13bab1bf71d68f81b34d9cb4709dc4d450e7ecb8
13838 F20110109_AABNWL afzala_a_Page_130.QC.jpg
f85cc17ce882373795263ee789979020
e8d759593dde424cdc42bdd9c65296cdb7ac496e
11580 F20110109_AABNVX afzala_a_Page_123.QC.jpg
f0dbe92d45effcfb13981513dc19d1b2
a6fc08d3d5ce100d4b25f240b32f8403fd6d48ad
F20110109_AABMTJ afzala_a_Page_046.tif
e84dd935bfc731028c89b7df1de2e93b
9e8c6f1f24122fc809d5ba26e88804977344eb6b
F20110109_AABMSV afzala_a_Page_032.tif
760383fa9766877ed24cb5560748b9ce
020e5eea26fea2038a25c8e6514f3d8209578420
5033 F20110109_AABNXA afzala_a_Page_137thm.jpg
26a3ad30b698f4fa4438ecc69a1c8e01
e5134473ecd86d788e4efdae862ac0b3b8510486
4128 F20110109_AABNWM afzala_a_Page_130thm.jpg
3729ec715d89dc5f827a6cdcf091f0a2
fdc134827be21e638da3865c968e8697d2f0a833
F20110109_AABMTK afzala_a_Page_047.tif
3f26ba5bfbbf93bb86756b3e0b57c899
73f64978fdcbca668ca0c560ea51ba1bf30657ca
11800 F20110109_AABNXB afzala_a_Page_138.QC.jpg
2d56dd37d1b8dda22912de4ecdf2665c
f1a627b09089cf918a6533dd42bb2e170659104d
12695 F20110109_AABNWN afzala_a_Page_131.QC.jpg
7f66f7471cea606a20d4a4d6fc849ba9
da643a3d95346fad9a0087269a3fff729d2f09b9
3502 F20110109_AABNVY afzala_a_Page_123thm.jpg
8d88a0542da837139f00e5d3224a5109
b09809087369ccd7366c3b01056f0106e680f277
F20110109_AABMTL afzala_a_Page_048.tif
94922e4702817588710e3704af5c4e1f
d09d91361b8d7ab46e866bb98a52ea17d70c5270
F20110109_AABMSW afzala_a_Page_033.tif
759f882a6f433043f1398c238e357454
0b079125e68d40f534517048f70fd269513afc00
3512 F20110109_AABNXC afzala_a_Page_138thm.jpg
3d946e57cfaa4c6bf08b18451746f82c
c67da25bf9194b32557576164cc3a659d9b4801f
3937 F20110109_AABNWO afzala_a_Page_131thm.jpg
3adb310a5f27b531aaedbce3a730548b
cf0895d7b82bb6bae2a621973564bdb764515f3a
22774 F20110109_AABNVZ afzala_a_Page_124.QC.jpg
9c492fa0ff1870073f76418b8a0bfb4f
dfd29b65c0f606b2a7d50c1fde56806800f6125c
F20110109_AABMUA afzala_a_Page_063.tif
34b5c805eac2bc6ad5d36b2ed0623c4d
799063f5d24fb3f65606debc04332e7384532ac1
F20110109_AABMTM afzala_a_Page_049.tif
1ba37f88cf44b03281f6460ba5094bcd
03a34e8594f151ce276ae1c6c6f95463b0fcdd15
F20110109_AABMSX afzala_a_Page_034.tif
4d1ba73ed6611cec8a5c68e16d1118ef
f7f3b20c8a0233c51b957739eec9fb6013b6783e
22856 F20110109_AABNXD afzala_a_Page_139.QC.jpg
cabb98c4568416618969041efb749f83
19d8045ff20025e6e7200192d1c28b47382a5d5d
21369 F20110109_AABNWP afzala_a_Page_132.QC.jpg
53b2a16cd251ccac97c044e2f6dfeaad
2cb7f465c1f865d8daa97620a9a7b1550d3b3072
F20110109_AABMUB afzala_a_Page_064.tif
fa812dac3e5a4a88971593c48dda9507
32ff98232f832d9781f8160ac80d466512937dbb
F20110109_AABMTN afzala_a_Page_050.tif
dfcc53c4fb01445e74e7130eec86bce2
7cd92c3f748b716c13bd1c7a8005b3390f858216
F20110109_AABMSY afzala_a_Page_035.tif
d6627d82a3859f88fb46442f8919c731
70d575f5d58326543884ab890604150ed8724a81
5389 F20110109_AABNXE afzala_a_Page_139thm.jpg
bd3b3ede24cd7a7cab2a708263067e80
f22f742849136ceb04a836f9623b86a41e9ad574
5409 F20110109_AABNWQ afzala_a_Page_132thm.jpg
0228bffd8a1311f53f3627ba5d20f06d
629c1ee7aca589c2615850d859163aa7a757b9dd
F20110109_AABMUC afzala_a_Page_065.tif
2e0e363ce24eea70b64dfd7f268725e9
d6a672639d697ad1cbca8e7b451042e511f2aeb0
F20110109_AABMTO afzala_a_Page_051.tif
b9a6599b12dc1fa7564cd186d1e3d504
a275931b10bfa261bbadb0baad3c2a14a84f990d
F20110109_AABMSZ afzala_a_Page_036.tif
1a49739bc75cc7eb4a580900b824f987
2066e3cf1df2fdbad042251e6e9d2f38699f648d
10037 F20110109_AABNXF afzala_a_Page_140.QC.jpg
af51d701732b4e4620033449204cfd16
11add842c74c4212df4d0da3f16c27c180ff644d
5748 F20110109_AABNWR afzala_a_Page_133.QC.jpg
eeeae34f2fef19e76f306f6a19efd735
77c6ce97b3963abc4401fbdaae176a33b1ce96d5
F20110109_AABMUD afzala_a_Page_066.tif
780f5dd9bc13c32a8e47f872d494bedb
f923b0e078e0496a86ab361eaf9c387790bf6d98
F20110109_AABMTP afzala_a_Page_052.tif
e027c998cbd5086c65b511e237756589
534ce9cb81c7dc599f92d5ae9d8fba34ca3a0dc2
3520 F20110109_AABNXG afzala_a_Page_140thm.jpg
8b87ec121e9f1cf8e6ddd9eea343fe58
ad93a127d42d27bd73da28f5393b61a1f0a0f54e
2359 F20110109_AABNWS afzala_a_Page_133thm.jpg
cda4cad7921abae97281fff7e04ab3a4
1abfa401112d833ca09c373ae71258c83e515aa7
F20110109_AABMUE afzala_a_Page_067.tif
8fa9893af3fa803dee477ac11af9a14e
033df1769ead5d91956d31a3b24cf541f9a70f70
F20110109_AABMTQ afzala_a_Page_053.tif
601c413a6b7577c117d0f555f9823063
32fb0a34a230d0f6726ce1bf1db5a965f0bd2c25
11263 F20110109_AABNXH afzala_a_Page_141.QC.jpg
ffec001fdb4cd5ba1b7cbd5e3b2ae60e
3c3759621339f9944a03e1fb541b3c65f431be11
9473 F20110109_AABNWT afzala_a_Page_134.QC.jpg
c6a59b4616fdb5382d9a9364ddc3c473
819e02c5a9a66e660ad297bb7185ce0f3558b543
F20110109_AABMUF afzala_a_Page_068.tif
29e4df8c89d10ea19881f9071c3610b2
b7c30712e22908ad58958c48cee22b74a91fc11e
F20110109_AABMTR afzala_a_Page_054.tif
e6c5ce85f8eccfd0987356c157419d15
6279bbfbcd69df4ce75c7cec2fe53f7dc7b9543d
5700 F20110109_AABNAA afzala_a_Page_036.pro
48f93012eb9f53f29939fb33348b93ac
79f619fb84673e246a1533238572dcd796620d9a
3791 F20110109_AABNXI afzala_a_Page_141thm.jpg
f48bf9e6977a93bb6e4f2d0dafae7609
195d78a772c26893e8dc9e96964274ab82d8a219
3425 F20110109_AABNWU afzala_a_Page_134thm.jpg
9e74d797b254d3344e041b60b6cd8ac3
c094ac57931488e2b2ab85994e5c3de8d9158d39
F20110109_AABMUG afzala_a_Page_069.tif
2a7d437eefc97bb8d676b27f24a47d87
63d381e1f60a48fede2c9b8f5d84f7cc66978f5c
F20110109_AABMTS afzala_a_Page_055.tif
a7eaf2a14c0eb0e639d92c8296a6c738
b060f1e4468d4c26c5615c890ad0c14b52be754e
49835 F20110109_AABNAB afzala_a_Page_037.pro
d17b351487ad7a2b68ea4621682a688e
a93c311b54d2c1c817195958e18fe59c1195e979
6809 F20110109_AABNXJ afzala_a_Page_142.QC.jpg
7a65ce823b290eaf5d0dc955edff6333
c86c9a45d5ea36c2516b697701ba1138e165ecf2
12073 F20110109_AABNWV afzala_a_Page_135.QC.jpg
c69ed44efd39bbe6476ab2b81bfb1cb6
a2856a65fa533465440cdb815db278545acd4649
F20110109_AABMUH afzala_a_Page_070.tif
6d18cc6c0b6f925889f3b37cdc6fa511
9ba5b56b3c76bb6ad89ad53805d04f9013144688
F20110109_AABMTT afzala_a_Page_056.tif
0abf461d8059eb442465b11025218d5a
9770a1721bbfcdac5c58ca90b438acbf8113f47a
14664 F20110109_AABNAC afzala_a_Page_038.pro
8d8812dd7385b8ef95063f3145bbd4c6
75bea12aa1095ab58829390b3b95bf8527e81a48
2350 F20110109_AABNXK afzala_a_Page_142thm.jpg
da5a2febc5f449d7f1e1ad186d7baa20
bbc8e765049b716fd0d6f3d85667f6a52e23987b
4354 F20110109_AABNWW afzala_a_Page_135thm.jpg
44be94134d4aa95546fc752d240223a1
05bfe1b5265ea7d8f6e06a0cebe11e2599a77bf6
F20110109_AABMUI afzala_a_Page_071.tif
70490dc5d0d6fc2da54076411b12043a
635fdfb38556f18086faed7ca8ab682d52344ca7
F20110109_AABMTU afzala_a_Page_057.tif
e0cf6675e40ff5b64791b1a539107e22
01ade35dd7aa0994fac481b9ac959818de14dd03
21842 F20110109_AABNXL afzala_a_Page_143.QC.jpg
66464b204701f4726a8a8efeac3578d9
08effc3cd1757a9bb20ce673e38328d9b8175a1d
24124 F20110109_AABNWX afzala_a_Page_136.QC.jpg
616baeb998c5c48b0c7aceef68c2235e
625f2d05c917bcaf9b9c1326fa2a24b78bd4a33d
F20110109_AABMUJ afzala_a_Page_072.tif
d6ab36babb36b47f83badd5c426b0d41
0836b53307746fcb3d0ffba95b7dac64669069d7
F20110109_AABMTV afzala_a_Page_058.tif
af3365e4d6021b5c56b2ad16e99940d1
b308d28c90b7ef2a3b71875d7dbbfd3b105e5174
6499 F20110109_AABNAD afzala_a_Page_039.pro
b95a54c5bca0836853f75a818a782d87
7e3379ff1c09ab9a9e4890b40386aede86f76b8b
26135 F20110109_AABNYA afzala_a_Page_151.QC.jpg
6ecdfbf12bf31d815f1293381a52d21a
55d33544b4b008af80cf804a95d0d2b3f95519a2
5153 F20110109_AABNXM afzala_a_Page_143thm.jpg
cc83f1438bee11351df2db72abe59ac9
e916f4911dc83b6f920af5555c49305b87820fed
5799 F20110109_AABNWY afzala_a_Page_136thm.jpg
d1d519cfa41cc5d325fca196c617ef08
a56144ce2c9c90f11ce90f24d8b12b63a3965a81
F20110109_AABMUK afzala_a_Page_073.tif
2571c21c88ff7483414f5c58f48ab65e
e55b7e956b735d9af2e7b8f88dfc43ebc2a69e22
F20110109_AABMTW afzala_a_Page_059.tif
74aa9099df6dcb57497fdc395a718e36
8b005b90c777e7e4f0faff8bbead00af8c57c641
49839 F20110109_AABNAE afzala_a_Page_040.pro
2ecaf5363708f711987ad96e12dee775
8c4106e92ff3b76a42502316cf995219bbbfd115
6355 F20110109_AABNYB afzala_a_Page_151thm.jpg
56fdd56bc367a2fcf930394759c78a0e
cd9e49a0d1424ee3d95cc6fe891f2d65691b9777
4272 F20110109_AABNXN afzala_a_Page_144thm.jpg
c18f4c6ab38b8d60ab88f73afc9ab170
a4d0bc024070793454c7495ad69ac092683af1db
F20110109_AABMUL afzala_a_Page_074.tif
01611cff3da4ef085eec4061ce680364
15bb11ec10b1d8b9eb006723e83a6ed5c0b2ebfc
9129 F20110109_AABNAF afzala_a_Page_041.pro
d40dc83dccaacb32dd781d92e55efe0e
747c0a9b7397ffde586358b3d4f37b7eb7c45469
26919 F20110109_AABNYC afzala_a_Page_152.QC.jpg
6cf4668545decea27527fa67f3569062
d2f768f90f4a88961d2f93d644b6dd5d53c18d1c
19159 F20110109_AABNXO afzala_a_Page_145.QC.jpg
05e322fbf9bbb5ddbb72323662076298
075bebb2b2f404d95a6a7d98ae3838e88ff77500
16229 F20110109_AABNWZ afzala_a_Page_137.QC.jpg
64034a8f57e37e266dd87aaaf687f4c9
a239c764a829ad371c5aa46bb4378636b4a0fb04
F20110109_AABMUM afzala_a_Page_075.tif
41db997c98e68e4315711371d8f91d88
dd2a7859377a8f44fe2aba19ecb4d8e702e415df
F20110109_AABMTX afzala_a_Page_060.tif
e485347514c936316e2786aa4af5e4f1
e297e29a2cfa0eb88aa076378a6dcbdf4e470e69
38978 F20110109_AABNAG afzala_a_Page_042.pro
7c862106c5aae6c8f691c58b414e2a42
384077ed6df812bff896c9638fbb621c0d1ef7c6
F20110109_AABMVA afzala_a_Page_089.tif
1fe386357de1ab8f3f41fee8f44b328a
e79d0a7f138db21f560d4d367e4be8eee44570ef
6480 F20110109_AABNYD afzala_a_Page_152thm.jpg
78d015738d33353238f832a05440437c
465a2ebd368032f498bab465330104e31cbfb278
5476 F20110109_AABNXP afzala_a_Page_145thm.jpg
8f2fa6cc0bbcf657b919105edcf77e26
01a11f2b87b5021d711a351b968056f2b407a6ff
F20110109_AABMUN afzala_a_Page_076.tif
492f932f8c069377ea07b1b785850c4b
06ed91a6ffd59eaa49c2def7b8920718b29d485d
F20110109_AABMTY afzala_a_Page_061.tif
a2477e7bdde611b76b4d4c7d3dcbd2de
97f86d7e943fbcf5288d741446562e0acfab2b3c
18074 F20110109_AABNAH afzala_a_Page_043.pro
0edf8ccfc9eec88983215a989f527ad0
3b0bd8e67ac8617a70c00f5c90f3d9ea586b42f2
F20110109_AABMVB afzala_a_Page_090.tif
98c8a9c83fb9adcfc5dc1229e2896b83
f5de1b8c0d4095d9d6ccd3bf02fba6a8eb488829
24261 F20110109_AABNYE afzala_a_Page_153.QC.jpg
4401f3135fa6e03376e4b84bbaa8a5da
6fa4a6251e6eb0bb7a4e4ac8f3e92b94f0746434
16898 F20110109_AABNXQ afzala_a_Page_146.QC.jpg
df03767f3a9bd0829a391f73f0d00b32
afb01528893228a4badf810315a81b9e5c6eafee
F20110109_AABMUO afzala_a_Page_077.tif
23980b7ef50ce2d022d8489a2ba2eec7
a5b510da656e4153423a0d3e4d436d87184a5235
F20110109_AABMTZ afzala_a_Page_062.tif
567dbcd2b73f50710800d82633a05e2f
222c80877853b20331b50af3bfc0b792fd065af2
46831 F20110109_AABNAI afzala_a_Page_044.pro
959e7f70bef5c0ea86d14f25fb3284b9
0b6d838e6de42596d02dd77fb91d85fde7470945
F20110109_AABMVC afzala_a_Page_091.tif
e5ea6b07db14718870dc0063f877a353
cd8de283bce16b8152a517b6a5cca74304462ff5
5814 F20110109_AABNYF afzala_a_Page_153thm.jpg
ca11cef7094d7f32d71293c4091e835b
840d455ef16c8df3c2fcd10842167b75f5503d2e
5034 F20110109_AABNXR afzala_a_Page_146thm.jpg
774bbe6f7f49352c7e2d2611c2039cf9
51b49ce922e903e9d5c1263880d7624d676afa2b
F20110109_AABMUP afzala_a_Page_078.tif
981d295964bb4245ddfb0772073ea666
996a914115482b139582d3fe46ca2ef083183f5b
37067 F20110109_AABNAJ afzala_a_Page_045.pro
d1f83410312a6b2828e54145c81e8515
cd356c1d38cb81c47a7c0d1c12e147c9a6766457
F20110109_AABMVD afzala_a_Page_092.tif
5369080a53cf32c4f7c5896b4d273832
021eb643fd4afc6283938ce42fe9c673a4998f53
24809 F20110109_AABNYG afzala_a_Page_154.QC.jpg
3b458ffee63090902caa9257c0ec4063
7120cea5723477e5ed2d1c896e289b01eccb79a4
15066 F20110109_AABNXS afzala_a_Page_147.QC.jpg
f6fa1c221687f749cd6a0cdca46aeef0
14ab8d58efdc422edbe3d08f6cc1951ac6a8510f
F20110109_AABMUQ afzala_a_Page_079.tif
cc8adec91243e7d1f1ea98a0dc7cf9cb
0b18a4171f0e0f621cf79c1cc0ffaa403ca286fb
14204 F20110109_AABNAK afzala_a_Page_046.pro
87ea585670a9e8cd8ad5564a3f9f2fe1
f7762f7d8162de457142f9e602742288b634eabf
F20110109_AABMVE afzala_a_Page_093.tif
c61eb075908db9c05c383c615e24de56
3d781474347f1bcf55038beda046c659b90b8a91
5769 F20110109_AABNYH afzala_a_Page_154thm.jpg
4fe552d5d2b6351135b07ff35357d86b
1153d39490af7752b2de14504ccc33d0a3b730af
4406 F20110109_AABNXT afzala_a_Page_147thm.jpg
aed40325a5bb0ed816c14284e1cf40b7
9a55196233914f09abc1ce660666743d3b5c52c5
F20110109_AABMUR afzala_a_Page_080.tif
5a5e619bc08085f38541d65bd6aabe48
5f2dc4c22b7a19bf2ef0f06ab100a118e8c8da58
46054 F20110109_AABNBA afzala_a_Page_063.pro
03ca873ab4fd997c03ed1b718672407d
fcdb967676398901538ea3d06659ffb0a6b9a6a7
49028 F20110109_AABNAL afzala_a_Page_047.pro
13e4e62fe848f8a84f3d91072d663ec6
397e5e8067434c8b4ac1b24f39ca4a77a6d828c3
F20110109_AABMVF afzala_a_Page_094.tif
0a77542551e130ce0379d867aeaadc85
4e18f0eeed545d60f3716ba69b86949102c9fe91
12127 F20110109_AABNYI afzala_a_Page_155.QC.jpg
69fbba4cb36b44e66f8283a88db715ad
8c689863821440af1305376dfdbd391663d20718
9816 F20110109_AABNXU afzala_a_Page_148.QC.jpg
3e94b9ab439158225460b538d1b4d31c
e90d146125f54df31982331ff3e79f585dcbf549
F20110109_AABMUS afzala_a_Page_081.tif
82488cf339a1f4f0e9ba3769ca7f3966
a6333df365bb6988ca6a278b5943f64df2cc39e4
12070 F20110109_AABNBB afzala_a_Page_064.pro
61160a26bf14690e45ba74e39137a518
406b7cbb7d2dcce44d41eea087f5624fcb83848a
43487 F20110109_AABNAM afzala_a_Page_048.pro
1beb03dfe95e4147cf4fc387b8d3901b
c57c5c3f04f325bbdc37922f25f8aa04b2db9dad
F20110109_AABMVG afzala_a_Page_095.tif
305b9fbd3976b5707ee51c1bfbd0bcbf
6c1e35b2e24d086ba0ef496a90dce0576ecd7787
3736 F20110109_AABNYJ afzala_a_Page_155thm.jpg
e2fef0f8241ae0050d9d7ddbb43996a1
d585f12d3eea5779a64af8d08fa8f35d6cd20b4e
3741 F20110109_AABNXV afzala_a_Page_148thm.jpg
3ca777c69f97d62fe4b93047068be170
7000d77c4681fb40f39551c8dcf6931ab3f5ae93
F20110109_AABMUT afzala_a_Page_082.tif
5baacb115c3f16e253c7d1e7006b2a86
bd6fdad3fd1f60cc6ab3e761b4cafb9b906e848d
49346 F20110109_AABNBC afzala_a_Page_065.pro
39603ecc28429eabb3a4510dc7606b6f
4babc7ebcd122223792cce81ad1355103aa211f3
50423 F20110109_AABNAN afzala_a_Page_049.pro
9295fff89938df7c76ef45cd1c22517b
d2af7d1296d95d7bc8151f30d19785ab776c5274
F20110109_AABMVH afzala_a_Page_096.tif
2e4c55d47cf607a0f7fa05815bcce5b1
38d203edeed62209205422cfaa5413c311ce57c6
27181 F20110109_AABNYK afzala_a_Page_156.QC.jpg
6de8c3b2904be02a9f4d942489a631f5
9639ca712390c20cfec4aef8e93a631cadabf027
23640 F20110109_AABNXW afzala_a_Page_149.QC.jpg
824b2f2f0c21afe9b17118cb468b0df8
cc7ec610c6df3040ed2dabfdae2b0f3dce9f6086
F20110109_AABMUU afzala_a_Page_083.tif
2e062a3f7ccfee4616784e6cc8e37689
8e015d179bd3d80ab925b3cb3e1cc53c53f2cea7
51395 F20110109_AABNBD afzala_a_Page_066.pro
af229368126c33cd3de092d0baa34feb
5f6958f066c5740bff672d67b2962d47248e9b95
46610 F20110109_AABNAO afzala_a_Page_050.pro
b37cf743cfa0fc51ac4892ebd7e0f737
3f9552dc6df1096e7251583fe4b2a28e4a7516e4
F20110109_AABMVI afzala_a_Page_097.tif
55f206b42474d74b3cdf9f4d4ea808ad
9933b4572ab22bc2883fa381d20c87089c55ab8a
6462 F20110109_AABNYL afzala_a_Page_156thm.jpg
ed02a0ae5a13938c0cf4bba5afe1b8a2
8725170bdcb926ff0f607678a2a4dc3916cc0d36
5582 F20110109_AABNXX afzala_a_Page_149thm.jpg
52ef459c3157b72c0596784d7e18bf66
5b25be9f9f1cdf2bf0d664dd4fd930a7dc01c1bd
F20110109_AABMUV afzala_a_Page_084.tif
8475f3c028204781edd72f98e59ce2d4
2fe6df0318dc3ddd43df359b262d3231ccafa559
42297 F20110109_AABNAP afzala_a_Page_051.pro
5324dae93c915f8b882d7358ec8290ff
0829773c9e5a3b6203eeae39958878cc117b11bb
F20110109_AABMVJ afzala_a_Page_098.tif
227c1005b1404721fbab3e84fa191f22
c327b72445ac92c60593245fe29b1e35a187a6da
5349 F20110109_AABNZA afzala_a_Page_164thm.jpg
cbeaa5eda1e446d61d60effe14d3a14d
6453f4dbf281f63b8de78ca5a0f979c8c76e1b6c
27791 F20110109_AABNYM afzala_a_Page_157.QC.jpg
6d04a8cbf41b474516d30cbc4b49daad
54056c63f1fabf29fc4bf54d99569411284843f5
26581 F20110109_AABNXY afzala_a_Page_150.QC.jpg
058e0485ab0c3eade764612c3cb60a62
93274007654c53f3325976567dec3aeb234aee68
F20110109_AABMUW afzala_a_Page_085.tif
53d3d06f9b1e9a08f6578eddf4109e43
f18a43f439516790ea00fe776d80a08f3f96a315
45858 F20110109_AABNBE afzala_a_Page_067.pro
ebdffc0930a291cea51a5a1fef5ce29d
50dada9e9bbb89ae92a2431fa095ea04efc8e15f
11127 F20110109_AABNAQ afzala_a_Page_052.pro
2b1f2ae2d5bdb70f867b3fd9048a2947
8ecaaef3eed35ffd8616c7698eb419f2563d91a4
F20110109_AABMVK afzala_a_Page_099.tif
e0d41f1c917c6b36530b78a8ca4b76c5
0b09c2541250c0a673b0f193d3705ff34d769a01
17939 F20110109_AABNZB afzala_a_Page_165.QC.jpg
8ec28e0e919ed947d3b25aee6b6c1539
412f7c9348b13a2d20c49b4e586c12bc31a7a979
6297 F20110109_AABNYN afzala_a_Page_157thm.jpg
3731c6586562fdc3f1b731049567d8c8
61ef59e2be33426b464e2e18e4620c96da11ea3a
6241 F20110109_AABNXZ afzala_a_Page_150thm.jpg
2cdc7bd945279883f94d73b94f035d2e
d498bfed5bce5a7057e9fff93274f039060dde30
6119 F20110109_AABNBF afzala_a_Page_068.pro
901a14cdf5ed1b15f5a37e25c9713626
56f5813dc870dd7b940083a5fa896e7618d46649
50540 F20110109_AABNAR afzala_a_Page_053.pro
2683970c76ba0380d84a5bd8316d1ee8
b3825967294e4d5cdee3aae29852840fbc4f9b04
F20110109_AABMVL afzala_a_Page_100.tif
fd3355c69f24b8e180979a5cc10a2a2f
8781e27d6ca1afab4b23deb8aa6969965ff855b3
F20110109_AABMUX afzala_a_Page_086.tif
f89da17a1f56308301ac24c5d7134e26
57a7a12cd029fa791e6d7d04c8071a670459e9d7
5141 F20110109_AABNZC afzala_a_Page_165thm.jpg
d5eaec4588b248d2495db7a673d56855
17a7a366c3097aa599018a9b2ae963065830d08b
5954 F20110109_AABNYO afzala_a_Page_158thm.jpg
c35987a4675d969b8aabe73a87fc200e
9524fc100f2c67bfc27c3d61db7e6a80167c5dff
48923 F20110109_AABNBG afzala_a_Page_069.pro
a64243ef95380b0d6643615447debfc8
8435e6f0339ff7010246732f059d64b813009a92
F20110109_AABMWA afzala_a_Page_115.tif
92aff56bc48bbb17cb63c65c7e7a1bc3
bb5aa467a373c83a9d2a9a3cc848b5f6d6e2b80d
47823 F20110109_AABNAS afzala_a_Page_054.pro
0ba7301ae9b88520dd4f5b57340bbd41
41a9dede37ecc2a33dc84eabe6bfb596ddfca601
F20110109_AABMVM afzala_a_Page_101.tif
743b4b996f50b33840e10cc1f7190c66
c41720267c28fe4f4ec813338e09d637991e7393
23088 F20110109_AABNZD afzala_a_Page_166.QC.jpg
58945faf0530ec117f77a958dbb4bee3
e5ff59c00b8326974e992a84869f9b2f2f514d14
10258 F20110109_AABNYP afzala_a_Page_159.QC.jpg
c9d876685a58e9135f0c16325f62a2b8
245cc0af659893632332814069f491b8acc54f87
41493 F20110109_AABNBH afzala_a_Page_070.pro
94460658701b81bddaa1bd3bdbad0cd9
6dca65d1f938c48e04c5e4f8478015aa605968f4
F20110109_AABMWB afzala_a_Page_116.tif
952977c9bb5dfe4e6893b06151d18f70
6eb9054eae51b85cff3121c3e70658d24886d0db
11664 F20110109_AABNAT afzala_a_Page_055.pro
b1f5ff0674c3404459fc97874bf43c8a
024b5eddeba7e413af4ac82596f05172b3cffb7a
F20110109_AABMVN afzala_a_Page_102.tif
fcb3a97ae284d93ef3fa459229868e2a
bfd1254aba4beb4a67659ac5368246ad36c62bb1
F20110109_AABMUY afzala_a_Page_087.tif
07266c27e6e86cea25329453b0f31f1f
740b9707f16017fd55febcac66b1462b1b94680c
5923 F20110109_AABNZE afzala_a_Page_166thm.jpg
503661e2104043689350c8ac7901b836
dcf35827322e8772ca395a8e5b82b891df2f461c
3350 F20110109_AABNYQ afzala_a_Page_159thm.jpg
5df0e1867ba3d0cdf266466577e0afee
cf0f110e5e440507d5d1e45106ef56c124544913
6982 F20110109_AABNBI afzala_a_Page_071.pro
490eb5558911b62f0674707f16d0a87d
1d9b9a39f28c355605114c717e79a86ec6c1fe10
F20110109_AABMWC afzala_a_Page_117.tif
32b06de05739eb1ddcf0f20b01820535
c4c41db1671653cef95c2511de699b8b634de47a
51906 F20110109_AABNAU afzala_a_Page_056.pro
6df7baf52c8c1b92a04b33d512f64be8
12c408040b63172ff311a7717e5d49afb3f5992f
F20110109_AABMVO afzala_a_Page_103.tif
8ab4d5c4e4a2855f1cdbbf380a321153
00a450717c8362605234f02a7a1b370809f09fc8
F20110109_AABMUZ afzala_a_Page_088.tif
11658f5663c1d36c0a96a1063276d77a
eefcbf241c9f1bb412625b2046502cc5cc491d4e
24117 F20110109_AABNZF afzala_a_Page_167.QC.jpg
35479edc907146e6c85848b84bd0ab83
8aae76717eca42c069d556718f8a22d0bcdccd25
23566 F20110109_AABNYR afzala_a_Page_160.QC.jpg
ced70c355684a3a4223d86a1b1e49512
a6a60cb3c16330f3f515ef98916a0cdbde087925
7134 F20110109_AABNBJ afzala_a_Page_072.pro
c1298f4ec7476296c636209eb1e67b69
6ef43da2056d67f03f19b22375ad973102ba92d8
F20110109_AABMWD afzala_a_Page_118.tif
73629be306deb07a46d590d97eb5b4e9
d8cec34dc595776050754260ccbb89dee996a541
43041 F20110109_AABNAV afzala_a_Page_057.pro
1c5050bf1128147b78f402e9a915a1a1
080a7f5cf7555d7fc05374adb58cf8809550bf68
F20110109_AABMVP afzala_a_Page_104.tif
a56de93852582a51d4d65fb781302dea
5e5a78182fc5d97df5f037b94a2ae2ed97ed9a89
5900 F20110109_AABNZG afzala_a_Page_167thm.jpg
212fadac1468e0908f75edefe33f4a77
911c760a9b179e003185d0eaeeb29e454e5943a4
5771 F20110109_AABNYS afzala_a_Page_160thm.jpg
68abe4ddea5404c8ef0ee51af75df118
181153f908c7703c342c72a83fac259ae7342a50
7342 F20110109_AABNBK afzala_a_Page_073.pro
ded28a484f85808bf1612c498bec9cfa
8f1d9819efbb2911fdf25c76eadb65243435e385
F20110109_AABMWE afzala_a_Page_119.tif
b43c73f88ec925210ece1f5d95da9bc3
7c0e21b60b008449475f04346ea3b47fd9e4950d
10524 F20110109_AABNAW afzala_a_Page_058.pro
290019c4b0f97f5f850e8502932f4745
7bb644e13abbfea3cad7a2429a1248835103df45
F20110109_AABMVQ afzala_a_Page_105.tif
4298d177889d4069e449e99bf0d44597
96081526966ca9745cd6667a89747439605987b1
20511 F20110109_AABNZH afzala_a_Page_168.QC.jpg
0eb8e64fb20f023196a9b3c7ef4b81a4
14059a11fc68386a8d63991afb3a40c517fa40b3
26022 F20110109_AABNYT afzala_a_Page_161.QC.jpg
2b48f9f7f23c0632f4115f992364b35d
6a194498a341642e5c2c88f6f88ed12200edf074
48646 F20110109_AABNBL afzala_a_Page_074.pro
bbce66604c8228ec3caf4be8e10fe35f
98920d044c78e66a9ada39d932917737c5a5e97f
F20110109_AABMWF afzala_a_Page_120.tif
b102ae2e899630a706524c6238189e6b
ca5464bb1dca0554b8d3d372c632de3de8d4bd59
10519 F20110109_AABNAX afzala_a_Page_059.pro
9bc6527dd979d20bf772fca3f0596782
326aa826823e061153322b2397c408df44890cb9
F20110109_AABMVR afzala_a_Page_106.tif
c8ff473a525c1a7bc551efd281d27cb8
b69ac060cb505f35b9b2bfc8081d1510e896e37e
6769 F20110109_AABNCA afzala_a_Page_090.pro
1a68bdd74240f74a61a6234c43910363
ee5d416d6eea33dd28a890dd3d5835942745f9e0
5551 F20110109_AABNZI afzala_a_Page_168thm.jpg
b67e8db1cc2a1383afd5b79e962c0413
0cb82cf7b97cc2f20f41b4fbd116ff6197002888
6211 F20110109_AABNYU afzala_a_Page_161thm.jpg
4ccc3bb8647aaf048f641f7e6887284f
1eb4eb49327d4f99a2aa8741ee853fb82903dbb6
11421 F20110109_AABNBM afzala_a_Page_075.pro
adbc3e9188719003841a401c0e96e50a
7c7b1bac6dea276b0ffcd3c946c94c9d6d8e55ea
F20110109_AABMWG afzala_a_Page_121.tif
d17bbd8e39faceb1860e7d12898febaa
3af188df3bebd6dd3cf324879ad21feb2a61ea1f
14052 F20110109_AABNAY afzala_a_Page_061.pro
abb0c28a646c17b9afb880fd88b5f28e
43e7ce23114a49b2b5e07d4a6eff64b1e36ff124
F20110109_AABMVS afzala_a_Page_107.tif
f306ea8ca30daa9ac82d924b008119c3
2b1c3d3c4999bf0540183c5157c3bcacddb6ba2b
10530 F20110109_AABNCB afzala_a_Page_091.pro
eda5188cb51b4e5e407279479ce5732d
b50e9f6e3cf9fe660a5321aaf4ff8df8f6ff5fa3
24079 F20110109_AABNZJ afzala_a_Page_169.QC.jpg
fa25b98f218ea08fd1aa5e8debc3b6f8
f54c5fcfd84dfaf2737d2d8586fb54082a365785
26995 F20110109_AABNYV afzala_a_Page_162.QC.jpg
9c515ee9c5165cb748cc592e4afb72df
2f764904929624980899f2e0afa4a6aa4f15b3dd
44849 F20110109_AABNBN afzala_a_Page_077.pro
c1a482abaee860439bd0553a151f220e
6eaec92f16a59444aee3640bf46508453aa6fda3
F20110109_AABMWH afzala_a_Page_122.tif
a90a91ccc346107d5a39dc1d9f7405ae
f5fcdc94e0d167f2bf40e3fe51d04de041d7b457
48466 F20110109_AABNAZ afzala_a_Page_062.pro
58e0bf229d880f85fe05336466c40438
4c29d83ca7709e33e957cad46e367cb8b6f77564
F20110109_AABMVT afzala_a_Page_108.tif
0861a18e8476842bfb1582c731fa60d9
041a60279fc524a9dadc599beaf0bb06f0cfba25
49605 F20110109_AABNCC afzala_a_Page_092.pro
27b817c791ca2d00319bb7215bd322db
bbd1c4aba68e68691d5c533fd7f685938e617c23
24789 F20110109_AABNZK afzala_a_Page_170.QC.jpg
4ae2257719c0a8b907af99dae6ae4e88
1420ccf9e8fb764b8c83730f496e013b311d865c
6487 F20110109_AABNYW afzala_a_Page_162thm.jpg
0921e73942de7bf53af19d4fe06d5d85
b941fa71143047b969280d359f891a181810af41
49099 F20110109_AABNBO afzala_a_Page_078.pro
5a79352b91000d4f63fca3093d8df93b
b9b48f5a598ffc7746a9c429193fcb2894531819
F20110109_AABMWI afzala_a_Page_123.tif
cfcba970dee4cb21c346bbf09c1221db
84d283e844f96abc07ea7e3db47aa204a8d78be1
F20110109_AABMVU afzala_a_Page_109.tif
88a3766dfc790f1476d3f778e275d605
c7b8b330c68b96f5bb39e90b16b6950983c80e24
9760 F20110109_AABNCD afzala_a_Page_093.pro
6fbd9a815fa1589b79ab3f05fd65e8e7
207ed9074b52b83ac94f1d75c4d36efd1eb914d8
5895 F20110109_AABNZL afzala_a_Page_170thm.jpg
5fba03fe8dc1b983d3c6c9b812aeef13
f3472a0c6f52ea7e5855f2df105b878b9338bad0
12550 F20110109_AABNYX afzala_a_Page_163.QC.jpg
f749bb1f1a7c8ac55f2dfa88e5d7b7ba
affe8cad52bb3b00ab308b41336b626b4ce1aa9f
53301 F20110109_AABNBP afzala_a_Page_079.pro
7a60b9dd322bde0b71b44016f06dfd33
b9b4cf5b00fa02af2dbb603413bb3b544774b4e8
F20110109_AABMWJ afzala_a_Page_124.tif
ac7484ac729e3c2f7524eecf1bc74cf6
850f5e6a90fa4bd495114e412ba989ca21b3e4dc
F20110109_AABMVV afzala_a_Page_110.tif
d221e950340ea2e638cfff8de4e29185
14eaab28e886bdbf122980810c44cfa31898193d
21296 F20110109_AABNCE afzala_a_Page_094.pro
c1eb66a07a153246b46357b109e0078c
8933e41e14cea5841e501b08cb1c3c9f69cfa1cf
24221 F20110109_AABNZM afzala_a_Page_171.QC.jpg
638fc343cc5be27505e7c08170aac457
4e7fd9e156eb45a8620953acaf412d26a377ac59
3257 F20110109_AABNYY afzala_a_Page_163thm.jpg
0265b04c204529ab2fc03300e9a07b89
39a5b13baae22fb562826193d071f68ffe941d3c
48374 F20110109_AABNBQ afzala_a_Page_080.pro
e86e5ebe1835e0351c1c55cb3cf19f88
08748dd219aba295b5df96fe7195e586ea2f5aa7
F20110109_AABMWK afzala_a_Page_125.tif
83cf6c6e9eb60e2cd51544159e9f8955
40c174bb90facb06bae9da1ac4c812a54b39754e
F20110109_AABMVW afzala_a_Page_111.tif
c38aee1cb9e5b9cb29e16399930b174a
cd76bac78d1f21845da1a7767b0ddbaab25d6db5
6024 F20110109_AABNZN afzala_a_Page_171thm.jpg
63e6f774cd2f59833616d653568db3fc
7a21e013fa6b46cc59a2b265f516fbb9669c9ede
16298 F20110109_AABNYZ afzala_a_Page_164.QC.jpg
a805e32b26bd8a50f154a5fc97d6d473
b0bb7501e87606167c11c6d8559138559b5251f9
6300 F20110109_AABNBR afzala_a_Page_081.pro
d9332878b16b727f4e1441cb1bc846ea
0e466f0ad935239a37b4379e28568ca1b2e59805
F20110109_AABMWL afzala_a_Page_126.tif
e1ade22e7151cf84585052698c6476ad
3a87e23a732d0bf72a2ea7ebe8848437d95289c0
F20110109_AABMVX afzala_a_Page_112.tif
b43f90d53d75f150a94270be1ef68c7b
0078927f219bea372f9be57f1c4c5d926a37f581
51056 F20110109_AABNCF afzala_a_Page_095.pro
6794742ff5bdb30f525d480299f4db5f
615592dc30790906f435b0a23f31bb6aaa637e8c
23795 F20110109_AABNZO afzala_a_Page_172.QC.jpg
ff4fab3385a89750017bd20fe88355e9
8bf3ca4651fb29ff6317cec2af6f21df36a8cdbf
34418 F20110109_AABNBS afzala_a_Page_082.pro
de7e02cd89b86da003ae4d998ce11de5
880239c47d0e145eda68f2d4aa0dc4620af248bc
F20110109_AABMWM afzala_a_Page_127.tif
0fe3a49530d285b893868a151f52d2a0
43bbd5944f5f6a9341b14f4c3bf6fcab30d924c7
F20110109_AABMVY afzala_a_Page_113.tif
e7f6a060f53116838ef55c701bb6614e
da1fea2cdaf4aacec7d320168dd4f2f9d34cee63
48352 F20110109_AABNCG afzala_a_Page_096.pro
885b80b60b6a86cafa67e2362039614a
b857233c1fdcfbe5320d37cb784a4fd3cfce7f1b
F20110109_AABMXA afzala_a_Page_141.tif
5e7e679dd7459559ab651e22a29cea7c
6b38aee6870b2dc459e85ce86d4e80be0c55f458
5706 F20110109_AABNZP afzala_a_Page_172thm.jpg
0e39625703f2f9aeb7ca6bc74e24c674
8bacd3d0736a1f049867989ea43f8cedf60c87e6
46656 F20110109_AABNBT afzala_a_Page_083.pro
0a12f0c93fdeef2730a432d130990ab1
a3f1ec1e0fff98ab0663cf53b49b20f708594a61
F20110109_AABMWN afzala_a_Page_128.tif
7b88406260d06ebde6b8d753f5046762
62b9c5a1b0964ea682ca52646d5d8b528e1b87f7
6115 F20110109_AABNCH afzala_a_Page_097.pro
475bd8c84899022ef5a1ef685539f3d7
83f96a4e71cda0a0b50b4208b076882ace14bbc2
F20110109_AABMXB afzala_a_Page_142.tif
99d1d71eef52d8f76920f6a2c51f7755
1abe218ee5bc92ae31e8e3927482ab5662a1746e
22960 F20110109_AABNZQ afzala_a_Page_173.QC.jpg
3678dcc0e2a83fe203f85aa52fe8b566
216b59a661ede9171c2387c21c2b8ba0e07ce133
F20110109_AABMWO afzala_a_Page_129.tif
04a89ef7c415680c186ee7a201c5dce7
5307e612ff489d03a4fe1bb5c2662051c1e714ec
F20110109_AABMVZ afzala_a_Page_114.tif
38fc80e6ee214cb160977c4655b1f138
b66422bd470bb7beb734bd0854ef097b00fe53e1
7891 F20110109_AABNCI afzala_a_Page_098.pro
71f98719e8f46bdd5458391c7c555fcf
5ac1b930971c0a0755e6751ef73808a048cf3831
F20110109_AABMXC afzala_a_Page_143.tif
8006a04bbf964146e656a1e9ce02cf18
87f8fc345ba28602ddcfea59dded86cb252b98a4
12701 F20110109_AABNBU afzala_a_Page_084.pro
ce2d0e80d4ec1a91f5c412e511c99352
5deadb406c930ce5f00acf80dec06301f4c607d6
5770 F20110109_AABNZR afzala_a_Page_173thm.jpg
22b16fefc1777c00679067a0580b1a95
0b9bafb9de497ae2e130691c95b721a3e7f9f3eb
F20110109_AABMWP afzala_a_Page_130.tif
6ed7791ec8bc81bcce3181c3c2dbdb29
9cc6631bc32ccfc7f1c0098c451ad2367b581d0a
16051 F20110109_AABNCJ afzala_a_Page_099.pro
0663f6f9472b343bafb8ab5fd8343ba5
2f23642989d72f0e8ae1af4f263d5ec9860f88e6
F20110109_AABMXD afzala_a_Page_144.tif
bdfddc0cc5b28f30d3fcbce56ef00127
4dab9dc65f0abddf5ed9925ea52284efc8eeae42
40364 F20110109_AABNBV afzala_a_Page_085.pro
edc86873cb016d921f32696a4246162b
fefefd2a09e94c100a6b80bcd12e6c8e0f6b9945
23536 F20110109_AABNZS afzala_a_Page_174.QC.jpg
11d6156544813f82c32e153913685687
4827ae1caa4ab9641d056d7015d525dd358783e3
F20110109_AABMWQ afzala_a_Page_131.tif
21f58c8c2ed0a76a44769a695948894c
0fd543afa72e66a4177b5735e8ba9d22f4dc760c
14540 F20110109_AABNCK afzala_a_Page_100.pro
ad9e0d309be47a7fb23798cace7a8891
b2bf2562b94db9d802bff8a460b2ad2451a38ed5
F20110109_AABMXE afzala_a_Page_145.tif
438e693e7a482d0f97c50a01299521b6
c2d4248f5de7f91054665aa37aa1c6d9d828eae4
1919 F20110109_AABNBW afzala_a_Page_086.pro
4c1181d43eb3c1c1221328c471208a19
c15802b94e2499173574bf3e030868b197db0405
6076 F20110109_AABNZT afzala_a_Page_174thm.jpg
81a4c4d27e886b5c594f833e4f30cd7e
bf7759915eccc20c71a52f62822a41c499a372e2
F20110109_AABMWR afzala_a_Page_132.tif
ecfbe2330755f46001e108bdce265e33
e4f68d36713b0672c57b786fe1e7fbc8c4ba07b4
49778 F20110109_AABNDA afzala_a_Page_116.pro
00ae9f8a830ef0311b8b844dfeeb27dc
9368c2b591fbc51903955ed12dc67dffd13653c4
51320 F20110109_AABNCL afzala_a_Page_101.pro
2699534c66b223c0a8d67a3e43952427
5fc5c4c1287a1b3376d6fd24956aab88e85d2d81
F20110109_AABMXF afzala_a_Page_146.tif
32b5f57c1e6153bf2bdb70b91cb1e6cb
b6d96aacf6f2c1e7e37ce3f47fff6e2d06bea7d0
1901 F20110109_AABNBX afzala_a_Page_087.pro
ef00e278c5eb9c5fa839bf111f242d4a
bed9fefb83b1379cc2fa750dc177041d0606280d
23575 F20110109_AABNZU afzala_a_Page_175.QC.jpg
a5f8f608f7e894b37b5b6e1da4be6bf4
dffa10765a536bf05e48af3bedc966f540b74a78
F20110109_AABMWS afzala_a_Page_133.tif
06d1d9400fe9d1a18b12d8c643d4b8d9
2439e94f6c58b3b00f4f3ad0599dd4afdee03964
12027 F20110109_AABNDB afzala_a_Page_117.pro
b160c385e9bfaa1945c31e3a160fd09e
eee1e1193545f43ee87b83687124515bda6417af
11512 F20110109_AABNCM afzala_a_Page_102.pro
e6b2c99d80e1c74dcbd28897e114ca2a
7c2c4527987e4d37f4577b4ae67ef63a05d4002c
F20110109_AABMXG afzala_a_Page_147.tif
6f693adc25ead2e25e55d9ce67d1f21f
f139e4b4c1f33770f827698895e54194b45ae8c0
3540 F20110109_AABNBY afzala_a_Page_088.pro
c2054504e98e3d1f9dac997548a62359
bb29f1d4ab1e3733edf3af5bf4210edadd6f6162
6205 F20110109_AABNZV afzala_a_Page_175thm.jpg
3bf44e204633d65adf7a260f85aaff71
b9e38e6b12fc2fd891c48e78575bac2072c387b9
F20110109_AABMWT afzala_a_Page_134.tif
ffc43f81bd7c4a24924a695ebb6cfa6a
fcbe00a5a36a65c39bc26cbecc45ccdce2d8496e
35278 F20110109_AABNDC afzala_a_Page_118.pro
d35fda437944957553274b812ed23d0b
d0072ddd83d3551670f7ef846908edd2d2afe01d
41603 F20110109_AABNCN afzala_a_Page_103.pro
c34f195ffe6a5b41855164d860dc6fa8
2a5798bbb0ee197622407bb6ff61972f794a3a60
F20110109_AABMXH afzala_a_Page_148.tif
8459829829b0b9251427b7fde092e219
ab5f28389c6dce4d4edd8d98ce0733927ed2890f
42286 F20110109_AABNBZ afzala_a_Page_089.pro
1c1751906b07652d18ebbfc85fd74a59
9e807b80aef0f50b2c558becf29fd444b25290ad
24089 F20110109_AABNZW afzala_a_Page_176.QC.jpg
6602470a3085501211648133789bcab3
33fa523eda2e175e47cacb01ab50d321d0ebf095
F20110109_AABMWU afzala_a_Page_135.tif
9a83a1f3584051c9b891c735a84b2ec1
65ae221181ff74e970a7c919ac55ca68952d6b75
9533 F20110109_AABNDD afzala_a_Page_119.pro
42812a32d450945b447f8aa0439b4ce1
60889c1f02f154bc71e18020f669ffaeb185526d
49461 F20110109_AABNCO afzala_a_Page_104.pro
96dd476a37664cc8c483da6b489148b8
0cfb35918d2d31c73dedabb02f130b6e822c1f33
F20110109_AABMXI afzala_a_Page_149.tif
9619a0c252075b6a18470ec2e68a2eaa
a3836e504e97d4ecba6fdb70bae111f5c6e7a578
5913 F20110109_AABNZX afzala_a_Page_176thm.jpg
0827691609a0cbc381dcd436c82b47d3
651cd42a436bb1f6a710e9b5d0d14087ac9929cf
F20110109_AABMWV afzala_a_Page_136.tif
62563f945c1128a8424c0bd72396438d
9fca85d062bb9dbfe77e191bc29d7449374e30e0
9299 F20110109_AABNDE afzala_a_Page_120.pro
c93c06435915a3b7bdea72cbf8b29a4c
c17ba977f4131a3c400773f731cd9ec3579a4e38
48665 F20110109_AABNCP afzala_a_Page_105.pro
d7a5f05328c3ac7086befe410bda58db
6d0f9ba880e350af01b25157994632eee27091fe
F20110109_AABMXJ afzala_a_Page_150.tif
157e07d6952776537463b2cb28d0cf77
9bc2d43b1b8290afd316720ae95d441b8c85b642
25356 F20110109_AABNZY afzala_a_Page_177.QC.jpg
1360877a35fdf7e8224197d17b53bf74
866c76e5a32ac79d8a8d7af0f458098c2ad1e296
F20110109_AABMWW afzala_a_Page_137.tif
1a32c570594215b89d049203125ba508
ee0f061ab29b81d191929358db41b7073ba8b7b1
47558 F20110109_AABNDF afzala_a_Page_121.pro
100bf9ecc9445313d2700b7ff897fc19
c1816853ba81170e61b6510810ebf0b2cefcb420
43591 F20110109_AABNCQ afzala_a_Page_106.pro
652914358c6dac1ba7460bc4f5320d9a
f7ca6038989afddbd511d51d5d6d04f774b4ddc9
F20110109_AABMXK afzala_a_Page_151.tif
14bac320a702d9aeabd80439df506d00
2f029fd19c90634382335042e6b7967a8b9f7aad
6071 F20110109_AABNZZ afzala_a_Page_177thm.jpg
e2897239aaa370903a556db98806798e
f73ced89cee650a2df1cf916ea9a4673a47401ca
F20110109_AABMWX afzala_a_Page_138.tif
a77426ddf2b19c316a80cec377dd8a53
73234de389d6ddbe40dc9091516999a740505947
49812 F20110109_AABNCR afzala_a_Page_107.pro
7aa22afbc1f01d6c1a0eea64cd40ef93
d6f9050854b777b3af0aafbec4a2714ebba46060
F20110109_AABMXL afzala_a_Page_152.tif
a96a30a0b0ea3567e3845b193acc2bcd
5e49f9acb1bfcc15ef068801e27a398b1e192da0
F20110109_AABMWY afzala_a_Page_139.tif
f56b1279dada2060128cd8cd80b86237
1a45e99a83a5b254ffcf305bdb888d4f7a53e408
10338 F20110109_AABNDG afzala_a_Page_122.pro
67597dcc618e0230d5c1ab6e6c97172a
f4c5fd5f8b6c2ff379f3a35ac844000e9ccde216
F20110109_AABMYA afzala_a_Page_168.tif
647bffb1b4da8d64413bc04251ca031f
d2e1c487eb6591880da0d3439e7141a9f40a4d8e
45248 F20110109_AABNCS afzala_a_Page_108.pro
f9355b96a41c2cb21bc9ab3d98d42572
b38efe7dd830c7608d01939ab9802971cd80a784
F20110109_AABMXM afzala_a_Page_153.tif
75c001316d8d9d3f8941407af2c576f7
fd58407540d2775dddd40afa172483fdbc194922
F20110109_AABMWZ afzala_a_Page_140.tif
0a03448cf60279b152e386a1ddbd9571
f6a7bec89c9169362040a7224755ae223eedb53a
8874 F20110109_AABNDH afzala_a_Page_123.pro
a2fbbae41af27b46e177e50f68a5c0d6
9700f8e0172c448f1c602aff1295bf781a24c488
F20110109_AABMYB afzala_a_Page_169.tif
4194d20aaa4e0434323a4c08029b1210
ebd58bf6eeadca90e227f30cf39fc94916942ab1
45265 F20110109_AABNCT afzala_a_Page_109.pro
eeafe190a90c396bb44ca45903f2bace
9092a26063dcaf067c8569895657f01a3abb8a8d
F20110109_AABMXN afzala_a_Page_154.tif
8dbfdb3f43017f0937ee2d5167ec3564
419afaec32f241506c4763d220f965e9af9bf735
42676 F20110109_AABNDI afzala_a_Page_124.pro
09609d342292aad3a16162a17fe0586f
4a632704b4229cf469dd53089375c9aeb99360c6
F20110109_AABMYC afzala_a_Page_170.tif
d12ca868667d808778e8be14978bb1af
2457a912b82ddb142e3aeac54c7d3ca660d76a34
47775 F20110109_AABNCU afzala_a_Page_110.pro
39af850f65ca7ba144debe2e086e01d8
1c0b79587a93a613f96b5d13897e8d9b5ef883be
F20110109_AABMXO afzala_a_Page_156.tif
e1ccdebc3c11a1a3e4f3a3aa5a1ee590
e6f2f2e260336a8be672e0c933c4d544270102a7
6350 F20110109_AABNDJ afzala_a_Page_125.pro
786cbd0516928d178e6f4c7e3c5e8621
bcecc0f624b8dd90796264ce96d82307f7d2e8bd
F20110109_AABMYD afzala_a_Page_171.tif
0319ce63d1799313713dbabebdf6f0e9
edaa4f2f1a78a1f4f3d3ad930e000b1304a30407
44716 F20110109_AABNCV afzala_a_Page_111.pro
a832f34232f53b251c6cfe01f4de313c
0c3e88d509c73a6cbb7146e443971e815a4de3e8
F20110109_AABMXP afzala_a_Page_157.tif
f2c0601d24786433955437cecf749650
6c5de8dbd9ff243f385a9988bc30036f0239217e
6252 F20110109_AABNDK afzala_a_Page_126.pro
36c095dbf67041e6fd396899c79ba656
9b5e5f76961a2d5295c6657eb394486c714559dc
F20110109_AABMYE afzala_a_Page_172.tif
eec5df8eff05dc7e960c1b9a8ccc048e
cce22ac7fe3b0a860e248b29d4239c33d331a8df
47103 F20110109_AABNCW afzala_a_Page_112.pro
9c6d7d6afd22c7d0938864c976701ece
c8b7375e493faa71545cc13994e647b7e2709d71
F20110109_AABMXQ afzala_a_Page_158.tif
6677e9b18cc2c848788f94c974c5c4fa
8a051b602ddb65c34e867aaabe843ca1166b2ca0
46306 F20110109_AABNDL afzala_a_Page_127.pro
5899bfc2cf4cd469a774b9ddd69ff710
b09fc3bddca8db1a5f8cb90120e9be253f4e705e
F20110109_AABMYF afzala_a_Page_173.tif
076994c31f27277fa423fcf6bfff2ae8
03bf4ef126cd7414ec735225741c10acad2b2cb7
46059 F20110109_AABNCX afzala_a_Page_113.pro
03b871aac2faa94678ce43b7d76f0bc0
423579f3a092c61eb513c7af5beac1f7440dfb8b
F20110109_AABMXR afzala_a_Page_159.tif
1abdc440aa0058dbbdd74c587c10241d
abce9703f47c55edcd17960939b9b26b816fdca7
38830 F20110109_AABNEA afzala_a_Page_143.pro
f6f4a9f0d22226096b503044bdc08c1c
9f7636a488fc8b8c1afbc6b9f727abad79abc304
11485 F20110109_AABNDM afzala_a_Page_128.pro
b14cbf0a577f87d4c91d90d1163fb47d
a9d99f3fd29c23a732cd18a4c80b1cdc55cea1e4
F20110109_AABMYG afzala_a_Page_174.tif
dc40ee3ab607f095470aa80c7102a91c
e8491b7e796c79c852ba63ca34d070a105ee142d
48174 F20110109_AABNCY afzala_a_Page_114.pro
98ca996a60bdf5aec31cc81c53615398
acec14c8ed5a759f4ac23497379cf71721eee02e
F20110109_AABMXS afzala_a_Page_160.tif
c62f03d5f2a954d6ad367fdce9fe6e6b
0d42c8ce9596ee8f2f54d4ab60018397a32c7dfc
8367 F20110109_AABNEB afzala_a_Page_144.pro
1337a9843c3b94a384e4eb9d2b38cee8
b5624ba4114ffd45bbdb8f6c948ae8c58cae8e85
44929 F20110109_AABNDN afzala_a_Page_129.pro
5631b4f0f9a077d1e2c2ff7a99079053
692f81c8e1ecaeb529850b3a13a44ebf392c057f
F20110109_AABMYH afzala_a_Page_175.tif
f98b51eb8b27eca5e89a4470475029ae
99fb1e2656f27aad8d178c1c304dbfeadf572fd8
42155 F20110109_AABNCZ afzala_a_Page_115.pro
3d33e3673b31b7c291c7e9600fd60e55
f20c07f238707b795551973255532b61326c23e9
F20110109_AABMXT afzala_a_Page_161.tif
88d34a08419b164542177c330c4b4b6b
b56c1ae83b6d9ffa839d6ae1fa1d879b7d6bb7d5
9903 F20110109_AABNEC afzala_a_Page_145.pro
77fc3fd60ad064d6ec6d56b8cc58cb0f
d0b46fe02b26a932b95fbc833e263b16ba30be6b
2381 F20110109_AABNDO afzala_a_Page_131.pro
ad1a0f130d33c27401dabb72aa43ca68
f1af17aeb68428f4239491d4abf2da4076320378
F20110109_AABMYI afzala_a_Page_176.tif
fc1a15e0c1431adaa4defab1e3dc50bf
8789263d70f392f2a9866cd7545023e20953cfac
F20110109_AABMXU afzala_a_Page_162.tif
813d185a275dab2922ba0795d5f2f278
83492cbbac3bafd8dbd8336c3591b8d3b91df831
7336 F20110109_AABNED afzala_a_Page_146.pro
862c8f8739173305440490f955fd3992
2644d5b958c00cfe4f1e583941f2ab4d48ea102a
40103 F20110109_AABNDP afzala_a_Page_132.pro
8712490c1295843c2e73951658dbcd52
b14c382455e0306269738fb5dbc834209503a50e
F20110109_AABMYJ afzala_a_Page_177.tif
bcfd4e2a8ece7a22aa8ef5b4f26e3d06
6dbfdc706e716bcb0f475cd479d47b7c250338b0
F20110109_AABMXV afzala_a_Page_163.tif
0e9e0b130376d004ea13622896a67990
cf8b4314e4cef02cbb02ed25fabac0b59dcb70d8
F20110109_AABNEE afzala_a_Page_147.pro
0418ea5db87481f528454c36f501f0c0
23310cd746bd16af4d675c48794d0f6401f17446
1113 F20110109_AABNDQ afzala_a_Page_133.pro
94fab1d31784e69f056fd32f7c370412
790dca108a10e7eef00627edd588d3789e7b258c
F20110109_AABMYK afzala_a_Page_178.tif
ce0c1ac792b80ee3d2331864b4070a3d
0dc7f4f87fcc30b94cc8ef829637e944ca8a9568
F20110109_AABMXW afzala_a_Page_164.tif
500cd0dbac0e4b617dc766ecb4178ae3
8c1b8c4c22ddc23ac0df2482c41202252485734d
F20110109_AABNEF afzala_a_Page_148.pro
c2e6dc4f77bd496d8a06a301741c4730
c490105f78c2a258210edfa6ae29e97186cbf957
8197 F20110109_AABNDR afzala_a_Page_134.pro
af3109c13083826332ac1f703f3b3375
9795f8f7557d48773f42b3e3a1dfc293a8c00bfa
F20110109_AABMYL afzala_a_Page_179.tif
9cd87bd794f6a8202f64d8d59814dd3a
7cd54ff5fcadd0b91ca3a02073c49f1cbbe1859f
F20110109_AABMXX afzala_a_Page_165.tif
297e8a080336307bbf6f543581cf48d3
cd4cc522b0afa7c57ef33c1cf7abcebf45d032d6
44678 F20110109_AABNEG afzala_a_Page_149.pro
ee13e33f97aa560a811e43786b269ece
0a04e820f83487f96ddeb80859e86022f3d3c993
75543 F20110109_AABMZA afzala_a_Page_010.pro
3cde3fa00c6451b46e77aa78cc7afd7b
3a74cd1f7734430e6b73138af0a129a47cf8ae09
5780 F20110109_AABNDS afzala_a_Page_135.pro
515e7cec9a6bd2df1759d6c95796ca5b
2bacf668814710ab3a9f02c67bf0c1ae3861ad18
F20110109_AABMYM afzala_a_Page_180.tif
95e2f8c8f7f489ef758aed3643f0bf26
1d7c1597a7b84960a1376dc92bf4807021d42348
F20110109_AABMXY afzala_a_Page_166.tif
3694347b3c6b826ec08ddd963cc4db09
22f7b7573d8151ca001675e02d87654f8446cc31
9820 F20110109_AABMZB afzala_a_Page_011.pro
de8f2c0a9110150256a6391342b3c016
0702d749ad74d4878d1792e797592583741208ed
45387 F20110109_AABNDT afzala_a_Page_136.pro
7c2c1a7739350457c94bef5a2744f264
a99e4ef90126296b6799a6b8ba95da4a8a844915
F20110109_AABMYN afzala_a_Page_181.tif
6ba4a5b94ccf3f76fa61f43c49a6e638
a465c17476c03290455cf4a122ccb27aa8bae162
F20110109_AABMXZ afzala_a_Page_167.tif
2c4c14bc832332cce3ab2187bf9aebad
10b9a165bb9406fbaf04fd80eee2402682fd1452
49938 F20110109_AABNEH afzala_a_Page_150.pro
2ccbf74345d1a9ead88a322faabd072c
008fb295db27d8bc166d2b9b68a7a2538e866c5f
21043 F20110109_AABMZC afzala_a_Page_012.pro
4ce0b83808afc0e755f2be5d092a19da
e2c1e6c6bb0c24d994c7973a9c9653d87707d9b9
15429 F20110109_AABNDU afzala_a_Page_137.pro
6f98c1557a8c5ddb662d5b9803801bb4
b7035af2353db59ef2eff275c843eda5205bb0f5
F20110109_AABMYO afzala_a_Page_182.tif
94c4bf1173e7ef2af34cc6724842f8f3
8c4d74a5c8874b568c676d7cf903095dddb7495e
49458 F20110109_AABNEI afzala_a_Page_151.pro
de3d40ad3d8aa609405a077a9bec2cff
a8fd3d2eb54736810865dfe593cf18dc87c02ac9
21551 F20110109_AABMZD afzala_a_Page_013.pro
7b17af6c885fbad6f6ed0e872815fc25
9547cf90669988e1445e582cea294397ccfef99a
9104 F20110109_AABNDV afzala_a_Page_138.pro
f74b40085d2ace45aabee9e49381e223
30935d72ea8a1be5383814cf7e38190265d569bb
F20110109_AABMYP afzala_a_Page_183.tif
5895d1479be601748d791fd7ddb55c9b
9f7b6bc051f1b90a9e96c1c06c60e32708e52031
52751 F20110109_AABNEJ afzala_a_Page_152.pro
582c9e50973a0f34405d6e304fec0c93
38bde2fd89d64595f04e6b4c9fae7448cc31387a
22329 F20110109_AABMZE afzala_a_Page_014.pro
b1b0ef3ada82018fbcc8619a2a14d3ef
a7d9c9ed4b6977191b726d4fdcab80e1840bfb55
42057 F20110109_AABNDW afzala_a_Page_139.pro
468ba93d351d0ec2c057546c7c71d276
83651da131d5a9e3dd6b0e68648bd299d7600f43
F20110109_AABMYQ afzala_a_Page_184.tif
75a7efbe8daece08358d8c6b7e1ce9d0
bdc3c7412fc8ecf132c1b0c38cffd3b6d78d8939
46996 F20110109_AABNEK afzala_a_Page_153.pro
caad1aaaaf9f38473bb152b474f16062
e4b2ea534e4ce78f6afbf442bd2066af451c2176
21312 F20110109_AABMZF afzala_a_Page_015.pro
9be22cd93fd72beb5461cd4f8fe0b413
59aae828076b908c1eda825c6bc8afe9762e58c3
4534 F20110109_AABNDX afzala_a_Page_140.pro
aebd33bcf41922ec6fd849c3509296c2
ae05096b6353c77f9518bd7407ba34bb481d6e8f
8508 F20110109_AABMYR afzala_a_Page_001.pro
568a7900f789fa4ce0d2f2e7c0d84e41
b03845e0edc26af7db4175f339d5fcd4900db010
49569 F20110109_AABNFA afzala_a_Page_170.pro
7a7252d70bca0406f548b05b976a1089
cf441db386fc4139321d7b43451518f30afa9250
47030 F20110109_AABNEL afzala_a_Page_154.pro
c3d43681d5d0d8119654e18a29935074
d05fef1dea347b3cf583005ad63604b8277e0911
18715 F20110109_AABMZG afzala_a_Page_016.pro
0f3afa3495d7873eab27701b106fd8dd
c307166ef4b12f8fb6108fa810f621b57b852130
5356 F20110109_AABNDY afzala_a_Page_141.pro
1b6beb6fc3b17814251014769f3c51e7
aa82b96b10673cec76b039bc1fc45ba2b25685a4
879 F20110109_AABMYS afzala_a_Page_002.pro
58b5a3a50b595a4b34d1c3cf7e895abf
5b7237add0c583b93d8081cb20ecd24f756c5127
51621 F20110109_AABNFB afzala_a_Page_171.pro
42fa022d4a8c3a698481df17e41ecbb1
3295e716ce5bade673a96f8974676d2d8a0446ec
13497 F20110109_AABNEM afzala_a_Page_155.pro
535b97699f57132e51ade5ca0efb9441
262f84a5a895bdd486902cb6669d53f3a3849cf7
39597 F20110109_AABMZH afzala_a_Page_017.pro
76dbcaaf72306c2878850a0f9a0643c6
c32626faf1922d02c45a0ba4227fcba3687d3dc9
F20110109_AABNDZ afzala_a_Page_142.pro
37edce5732a7fabb4d6c4709a6a500d7
b23bc14ca40a495b96c7741dbc5c7364fba5bd71
40446 F20110109_AABMYT afzala_a_Page_003.pro
002878d5f7a6c0f22c4479d5d6800798
ec6030d4b325c6391fd3e98505deabffb4560f7d
53774 F20110109_AABNFC afzala_a_Page_172.pro
2bc7cd989b23102230f320df19a209c0
8dfa43e7f2534bfe8bb9af97f3dde8fb6c7a3280
51857 F20110109_AABNEN afzala_a_Page_156.pro
66ee50bf63f612ae7494b7397758b265
1b87abc39babe7e519a4da57478ebf1de42a31f5
15433 F20110109_AABMZI afzala_a_Page_018.pro
f86019d75f1e5e1676b1426b68c6c532
e7b9e87182fef3e5ac5d30d519d3fb0d7440347d
38045 F20110109_AABMYU afzala_a_Page_004.pro
1927448c1ccb8f1ab12d8a9a38cc0bee
e048646290d4528dd39af7d6bb13d726082bb1dd
49972 F20110109_AABNFD afzala_a_Page_173.pro
93a9df98f71e940ce094afda2c9d48db
3736399ca65b7cf2688f942aba254ce7251487f0
52655 F20110109_AABNEO afzala_a_Page_157.pro
5079eb8e30794b4a48bbefeccf745c8f
2d425e9ba244292dfafa18e6240a5a3b92728bf0
37422 F20110109_AABMZJ afzala_a_Page_019.pro
b85c229fcb4319cf908bb26e92d4e733
31d1ed2377dd8b3e6a7a4413aee6461942f4a31a
84406 F20110109_AABMYV afzala_a_Page_005.pro
ea2df780c1ab8567234b8d0146bcaecb
9096c7d84f751c2a47210ca53b639a73a7835d3a
51494 F20110109_AABNFE afzala_a_Page_174.pro
99c4e3ec3dc18215105dea88f885d3d9
a85243d52b6f2abbed1fdb6361b5767a9bf03c8f
47469 F20110109_AABNEP afzala_a_Page_158.pro
743f9d48847b909241fb799ea5cdd2f2
e72450d2018dbe70729c645481da2b399e0b006a
6190 F20110109_AABMZK afzala_a_Page_020.pro
14dffc1920d85707a3a6e3252d461318
101d995ae9658ebdf44b0b693173c75fe3cbf018
112094 F20110109_AABMYW afzala_a_Page_006.pro
9ec73986f7b38533408bfe827015c700
9534244b97b29753a8ba503734bd2f31ddb1d71a
52525 F20110109_AABNFF afzala_a_Page_175.pro
5f5e9796810d0bf3de4fb662018b359f
bf2936a3be2ab72b9f9b2435b08dc1261450ebca
11847 F20110109_AABNEQ afzala_a_Page_159.pro
fde5d2b031993f046305100a6f1a16b6
4489be515a2c849a99a1257ef1a11a9b4933f21a
49066 F20110109_AABMZL afzala_a_Page_021.pro
1bfb9d17bb369dff584f23eec78b13b7
fcc6577f3016250d3f54116affd581a6a409f654
32885 F20110109_AABMYX afzala_a_Page_007.pro
7cf800f85c5a168cac829c6daac7b26e
0e17befeaa1643faaa2942e3296859b3387c8698
52913 F20110109_AABNFG afzala_a_Page_176.pro
e2f0a42cce55e831ea20df32018b498e
9261974d1ae97aa92c3eec743b9b9d6e01bd31a0
45201 F20110109_AABNER afzala_a_Page_160.pro
2a87558d8a8302853c2b7c0c6ebb2405
945f343fadd29e8cfaa54c2550a912874f301a92
48841 F20110109_AABMZM afzala_a_Page_022.pro
a35b4f44752254d99a82a6e18f9ce68f
529f9fb9990322532fe748f32521968c03122ad4
58499 F20110109_AABMYY afzala_a_Page_008.pro
6e537b5cfeb2869906965984fd3c893a
ad4fa24b440315ddf2f99d601ef6a098c6a1e2de
51458 F20110109_AABNFH afzala_a_Page_177.pro
c13f058b894e04bfcbe4cc7205e3b414
d94d733f0a1cfd66bfb2f0e78802b1aa8a0e9fc0
49813 F20110109_AABNES afzala_a_Page_161.pro
49a5840001805a027dd8efcae36b13e4
aa25dd6e54df9b9e50a75706cf7bd9134eb85aec
51144 F20110109_AABMZN afzala_a_Page_023.pro
3ec4613419762f0a1dc7b772aa108cdc
d83c181d64353dc49e6feea577a9947da87f87a7
69952 F20110109_AABMYZ afzala_a_Page_009.pro
4cbfd8fa56a976d9d3e574a3bc2747c6
ab4c69d1800934c24b17c3e45291c79ff764ae20
21044 F20110109_AABNET afzala_a_Page_163.pro
89f1f1d10a5b0703616ddeb25a00ca69
5a2b4be96a5a30f1851313314045c1cb1d15c4ed
15804 F20110109_AABMZO afzala_a_Page_024.pro
01b061aecee906b8b18c552a664983ee
6f0440676ece16dd4393521f256e957d67d24b89
50708 F20110109_AABNFI afzala_a_Page_178.pro
9b46c896e9c8d373df9aa655b0249ce3
9c2866895c2877946d7d6cf4fd337a93f9fa72fa
11008 F20110109_AABNEU afzala_a_Page_164.pro
0d7d9cb22c332454bccdf06ad2ad9e9e
7bbf7179b7e36a7a5f9a9bcbee882cb304f086d0
44474 F20110109_AABMZP afzala_a_Page_025.pro
a7de4ae852c93fbd2869821152eff371
fedca790ec7211bf201b57a90c5682725ba47efb
55115 F20110109_AABNFJ afzala_a_Page_179.pro
e9566e53b14eba0645a7e0cb3a9df304
c2052f915477577c2288ccc509e1bd775934aa74
36308 F20110109_AABNEV afzala_a_Page_165.pro
561c7376332737174b7f8973a43d8b7b
c7dc8b7174b85f20d26dd4d96a6783d02f46ec5c
51675 F20110109_AABMZQ afzala_a_Page_026.pro
e065f455a52a4a2337c52d0917f6e0a5
8be9d27b3a2aa203bfcd48417895a52e96c42667
46500 F20110109_AABNFK afzala_a_Page_181.pro
fc04eecdfaa7d4b03e2f928c86fc071d
db2df9cc678aa468859eec5859d847f61b144f13
47616 F20110109_AABNEW afzala_a_Page_166.pro
367880679f97044c924bbadd863bccd8
890715f528fd5465e5effa8c0e198729f37bc631
50469 F20110109_AABMZR afzala_a_Page_027.pro
2b10029f152d5115f979dd8dc46b7029
c7ed3549952a2aff37be2f00d4f2b3b0805c0467
931 F20110109_AABNGA afzala_a_Page_013.txt
d6ed1330ae4b23daeed265e0449fd042
a9945c7de5b3f0eb6e1b2eb9a1beedb3c7150ad3
7991 F20110109_AABNFL afzala_a_Page_182.pro
bcf8fd2f924b234455778db8199d3616
cc786c86890fc1761539a79f2a9d2b05e7551fd3
50176 F20110109_AABNEX afzala_a_Page_167.pro
0190384a451afadafaac9c5080708d7c
cd4d141ebf9b6a73c36f8ad66147d78eebecce89
43907 F20110109_AABMZS afzala_a_Page_028.pro
1cc423d58b160102d5bf9e910bfaa50c
37ee7f29d8a67798197717c33b7229f72b38bd6d
963 F20110109_AABNGB afzala_a_Page_014.txt
6a83b8e10461335508c8d27b89330fb3
204ea6bdc096fc019bd9cf1055943aeceee25b01
43313 F20110109_AABNFM afzala_a_Page_183.pro
3a817c001ab73f739c8139f5e9886a28
453110f592ffba2971dfc672df4563cea2f9d2bb
44213 F20110109_AABNEY afzala_a_Page_168.pro
677e97c53f5c1ef661399e76275b04a9
efd20e6b88dadb1e7dc6eb2e03cfa6af7068ec56
44893 F20110109_AABMZT afzala_a_Page_029.pro
db20be60430bff20060405ae21dfbb5b
12278f11e5a612eab24ff74a5e485d9e1144f47f
51585 F20110109_AABMDA afzala_a_Page_162.pro
b28b5fa8d054646b0e88a5c91e72a4c0
1fc4c6a8ea9e07428e71681be7ea87bf41f09fff
972 F20110109_AABNGC afzala_a_Page_015.txt
4da5520c9b3e3146da854f4c2f99265a
b28aa57d866c68c2a4c39cb11dcbf6d8cfd82354
48133 F20110109_AABNFN afzala_a_Page_184.pro
bd3a303f31003ed3e1ee83bc3742659a
9361d955a9eeb02bd6224fe19098ee2f7dad477f
48256 F20110109_AABNEZ afzala_a_Page_169.pro
6effeb9a6d5e72e9a8c4e87678cc05d6
a4b7fb4038f612604a44aee9b524e9cdb3a41b5a
48300 F20110109_AABMDB afzala_a_Page_180.pro
a824f57b70e679a636d3ee2d044b12d9
cae1a500184b4b1f34cd4af9f3a75e59496dc411
815 F20110109_AABNGD afzala_a_Page_016.txt
877e6d93c4c6dfff4e4d4856f41aaf65
8a52896e0b39e3699d093abb0c404f2b34de4f90
470 F20110109_AABNFO afzala_a_Page_001.txt
4931116563c3071cbd46b569624de77e
4f62dca852f1879edb14d8730a13361cb4a98411
3660 F20110109_AABMZU afzala_a_Page_030.pro
e271d792840b3a9799b6ef0ceb644959
77baa0ae0df2fc04475da7678d2d337cb98379a4
11620 F20110109_AABMDC afzala_a_Page_144.QC.jpg
883529c4d55cb443f9c0db0aabea7f67
d0b03da4a262dfd496f78ef758a1ed7035484925
1733 F20110109_AABNGE afzala_a_Page_017.txt
94b5ee2d5ec941cba98504f1c89e0b5a
fdc23b62537e0c1bca96d50249ace7340412b886
90 F20110109_AABNFP afzala_a_Page_002.txt
1ca404922addc79e0bec94d080dce1af
40dd6fcbef69761d991daae355e3832a8104af7a
51375 F20110109_AABMZV afzala_a_Page_031.pro
f00cf28fd618a5bf9b265d66d43588c7
5b259b919ae0f4f0493e85a95a5157bbefe43293
28501 F20110109_AABMDD afzala_a_Page_018.jpg
e9fd3e38a34abff361de4e2514d38b8b
abf4a866973469f86ef5aea5d0abefbdb5fc7c98
616 F20110109_AABNGF afzala_a_Page_018.txt
066118ee15f30fd676703e5ffaaa04ac
75991b32a70e5b7f11b3e6577d8865b501465d46
1641 F20110109_AABNFQ afzala_a_Page_003.txt
1163081670925ef7294be11ae7aae541
82aabaa529e512343f5b8ae8d7487919209ac616
4823 F20110109_AABMZW afzala_a_Page_032.pro
5902efd669579787cdb57f368108cc34
0ff5e9cfb3aa28fa6a862f18d5b036deffd321f2
51801 F20110109_AABMDE afzala_a_Page_060.pro
ace75441af86f93f791a06e57e051fd6
d3b40ac9fbb6ba38617c829fc9663b94995d3c2e
1597 F20110109_AABNGG afzala_a_Page_019.txt
4a99c8c443d82ae6f28613ad0b1a329d
e1de2046d913eb6b4726139cc785e9eb9df9ea96
1531 F20110109_AABNFR afzala_a_Page_004.txt
0d5fb823a341cb6fb9947dc0aa342705
7519e4034cf47932b34ab9631ab069c8be94caa4
47266 F20110109_AABMZX afzala_a_Page_033.pro
b3e2f667e5387111370736416548f1c9
cfcda478dc5915749296454e67c37361d6787a3b
24830 F20110109_AABMDF afzala_a_Page_158.QC.jpg
b5ec74c5ef22de572c88003da06f7f1a
8a3d770566db3be13b9f572b5501e5597c83892d
336 F20110109_AABNGH afzala_a_Page_020.txt
a11c751f0e25034b89c98a7d9e7c4cf9
59c049bd4d4127806ba3b97a8afc1a78884a6a5b
3559 F20110109_AABNFS afzala_a_Page_005.txt
abcf5cf539803e7618ce2f7b597a5528
3ef196ba1ddac292fb3b19ca06f82b73266a0d9f
4304 F20110109_AABMZY afzala_a_Page_034.pro
673c953766c4a050604c0d15af59f42a
1fc104954bf75cded9b279df373ec201dfdaff54
29058 F20110109_AABMDG afzala_a_Page_128.jpg
9eb76a141e46e95e29ee2bf254b59e36
1a8350e206241979e4892194a95c8f340c7a7202
1939 F20110109_AABNGI afzala_a_Page_021.txt
ada49b7073fef238541d0063f4e3e441
a41dbf736d3c52aaa2cea44bf2311aec1280844c
4516 F20110109_AABNFT afzala_a_Page_006.txt
ba63e7432e6ebdf256649699f80f2c00
2d24257910afb4c32788d4072b18ded7097acd02
50821 F20110109_AABMZZ afzala_a_Page_035.pro
7bb6679583aafff2cdad21d6c32801cd
b679289be3b6320916ea383a016291c9c186c8de
1326 F20110109_AABNFU afzala_a_Page_007.txt
888ee574942bad2078e7d43e433f9843
0d821bdbee5c58bb27e58daaeb917ac42770d366
1928 F20110109_AABNGJ afzala_a_Page_022.txt
ea1bc53e0fa3988f3d5baf8344661966
61ece5ef651723cf5ef36fc4eef84d6b3e4f4fc1
2307 F20110109_AABNFV afzala_a_Page_008.txt
c1c3aaace10cd4697e51c180130b16c8
239ee87d119fffbcc93477f67dfe46850df10587
68934 F20110109_AABMDH afzala_a_Page_085.jpg
4ac396f6954014db4fe8ba77ad6aa139
1d834e09a1f13a016b4ac6d9932e488ee35d88e6
2013 F20110109_AABNGK afzala_a_Page_023.txt
ca52d9ba71ad3ef035813161154fb800
00b40b8e764efe2aaff2598fbcc1b65fda829630
2740 F20110109_AABNFW afzala_a_Page_009.txt
92947c78c350c68a5328d1ec95769b9b
f353772d7b407f1e4f58a622610494f038827c4f
6376 F20110109_AABMDI afzala_a_Page_079thm.jpg
27c97626484eb98e3776967165cbea72
8dc777e25da25eb8c33b0a6ed385b58d3c6c646b
803 F20110109_AABNGL afzala_a_Page_024.txt
34481f94b4a6629528adcc007a8b6018
5b5800432ce1fdb37fd7f3e29170a08eade78182
2999 F20110109_AABNFX afzala_a_Page_010.txt
f37f462a41f508a743df92726181cb2c
08f17501f8313ce337d86dc038721d448c4700c2
327 F20110109_AABNHA afzala_a_Page_039.txt
cd6693ad378546372ffd244992c81b01
8512bdbd9923bf1be334b6199a0d89f55a8e4f59
9306 F20110109_AABMDJ afzala_a_Page_076.pro
8a7f4963121e3d8b60d4c2c01bbd56aa
78cfd33d39a2f6870d4820fa1d2f423113905752
1780 F20110109_AABNGM afzala_a_Page_025.txt
0130688577664f6548b21a7132f313c0
77707103643ddcc5e08d2edfcc5d0c18ef97bc0e
19598 F20110109_AABMCW afzala_a_Page_130.pro
6d06d7381e764a82158f466a52e33fcf
dc161fc094cbf8ad55f7e3b53534018df1389970
400 F20110109_AABNFY afzala_a_Page_011.txt
203b75b0830a562e3aabd34ad39b3ec2
74a7d9641c04ae32817e2baa7584212ec302cb52
1975 F20110109_AABNHB afzala_a_Page_040.txt
310a27590cf81de5ebf69481c034bbb0
80ce5655c41ddcd7ba8429321c5557ebba43b52c
F20110109_AABMDK afzala_a_Page_002.tif
a36262809c8cebd88327a82545776c4e
7d6d8abbced146879a9ae3f2b67af78a0d49a921
447 F20110109_AABNHC afzala_a_Page_041.txt
461b66c1ffcc15588fc1473cb3480b4a
5da6a72979aec251423c403ed4b4f680dfa676d2
2040 F20110109_AABNGN afzala_a_Page_026.txt
ff6ef00c3d2f836dae17622ab7385dd2
0ed466a42cc2f1d953858ba96b3f953d154d5277
93681 F20110109_AABMCX afzala_a_Page_089.jp2
ed7f9cbc593e559c6d54cc3398b7471d
bef83b983cefc50eafc5668e62769ca93e106ac6
993 F20110109_AABNFZ afzala_a_Page_012.txt
8db614d3688c335626910baaea2a4972
a3db7f578a9d8634d860482920f151ac6267e547
92074 F20110109_AABMEA afzala_a_Page_009.jpg
5eb4363af62c151035554fcc26f739f6
15059e849b8df3de6318331356ecf0c79e5ca124
1865 F20110109_AABMDL afzala_a_Page_154.txt
d15bea44eaf15bb51837126ac461a9d6
8dd19ecb17dc9f7a957fa581911bdea45f89df33
1595 F20110109_AABNHD afzala_a_Page_042.txt
15c54823ec9a639222d6c951981ddd2e
18c2961486c0096f2b941556e22875fd35501f39
1992 F20110109_AABNGO afzala_a_Page_027.txt
7ad53fd10ff3be04c734dce1b34a3728
cb3ba584e524aa576c83f6ddd75c546ba6e3510b
F20110109_AABMCY afzala_a_Page_155.tif
a3bbf4a04fea63ad03daeaaf60255963
fc9570a78c32c7d743e28d5c7f9f2e0a66c2684e
105919 F20110109_AABMEB afzala_a_Page_010.jpg
51a857a95e8bbb0a95b6105251e5baad
26fd18f1c687fe3180ee9261ddba85a850b0211d
5973 F20110109_AABMDM afzala_a_Page_169thm.jpg
b59b9b5995078826585335470a8dd07e
9d88a187ceca2fa4e9f6e6851b928066b7fed69c
F20110109_AABNHE afzala_a_Page_043.txt
c952dd0775b31ad6f68f95303d605653
a8f281d92d078950e38ab8740375ad3ea0a1e654
1792 F20110109_AABNGP afzala_a_Page_028.txt
1bf4bcf1365c96d10550d66da688016e
b59465eb40a4e4bb4f485316da49ed8a2334f6e0
32338 F20110109_AABMCZ afzala_a_Page_097.jpg
ced9fb356c4874c1802d4bc24424de21
2725e6e9f9cbf1eb16750981908ee7171d13959b
16311 F20110109_AABMEC afzala_a_Page_011.jpg
f70f5229aae3790a5b6f3fa72e63b9dc
d072be9fdfa753c04a29f4f4ac1b9e94885a2aad
F20110109_AABMDN afzala_a_Page_080thm.jpg
bf6f3b3538481411c6bb329d4fe422f6
510be6b6cbb305cfb777e97030ef292aaefc25c5
1866 F20110109_AABNHF afzala_a_Page_044.txt
af01d579074fad7f5b633454d3f6f721
554344495e16e2974f0cb572d7eafe55ee5a4f53
1812 F20110109_AABNGQ afzala_a_Page_029.txt
36b695e6e7d7eebfc6560ccb66d3c2aa
a4de7a86a89250bf05f490f8e584938fb0155e4b
36452 F20110109_AABMED afzala_a_Page_012.jpg
f13ca735556a4548199c52c10c0d2027
2440af978d1abb901bb1dbf601a7cde30fdd32b1
4230 F20110109_AABMDO afzala_a_Page_030thm.jpg
dd598e16b3a35fe08269c8f566a388a4
fb4ec9707da2ebf6bd5b9b6d4465026fb156d9a7
1478 F20110109_AABNHG afzala_a_Page_045.txt
653a351a0587bfe0028ef8bce793f5b0
9890c71e9044901e84df32a634e57e7e5d6c7d58
41652 F20110109_AABMEE afzala_a_Page_013.jpg
34f73a1540fae2b9af5817bb03722fb4
15ff32cfb5949de5eb3efdba6fbc3f5168268ca8
274210 F20110109_AABMDP UFE0000624_00001.xml
774e8b0e1114e06e97076601acd6fa21
eab936c3fdab9db3bbb496ce937925597dda838c
234 F20110109_AABNGR afzala_a_Page_030.txt
2f582b62de2b72d608d3f7cbaeda232f
4483405016df17a401803b80f08db332fbed04f1
687 F20110109_AABNHH afzala_a_Page_046.txt
0e09b6f62fb5edaf68ddba13319173a6
bf13e0b652080460a570aa6f5e45ad4b726d3794
41692 F20110109_AABMEF afzala_a_Page_014.jpg
78df87fdaa5323b2222aff4e33ee909f
55665c0b95ae17886f0dd8c027d0c77579384ea4
2022 F20110109_AABNGS afzala_a_Page_031.txt
d1d78967589b5c6b330dfffa627334cb
da8d9cde4102bfff02ef570bfd140effcf79e4ff
F20110109_AABNHI afzala_a_Page_047.txt
4e0957432f7aec3bbf6a7367a12c1869
29921893de6be26af555fe52e430f31263edf84f
36384 F20110109_AABMEG afzala_a_Page_015.jpg
7d00b4301885a64015ddf4360362b3e7
1aa3709ba23d627cb78db67aacf5d3e297202fe7
290 F20110109_AABNGT afzala_a_Page_032.txt
bc36ac4525bdb37d18e5f81e248266ae
8f42a6d31a16f1ffbfd94aa710835b8b122d3712
1732 F20110109_AABNHJ afzala_a_Page_048.txt
6de96eaf9a463100ae6d7bc453ded3d8
b3d73fc558aad7b083b220f6bae92d2b05989b05
35281 F20110109_AABMEH afzala_a_Page_016.jpg
0872c8944c615fb1bd0907f042f9038f
9c2e968824d2272b9fe18e55e9aed334b9ce7487
22121 F20110109_AABMDS afzala_a_Page_001.jpg
9cbf7d90424d6eacb108bfad564df388
4af49b18f35f2646bacd110ffc35c796c4d4bef4
1867 F20110109_AABNGU afzala_a_Page_033.txt
65d09402e7d61d5261377563541abcd6
86ffb972d428c72200a7fcdfa2d968b5a697df48
F20110109_AABMDT afzala_a_Page_002.jpg
d43a23843f4038a5e4533cbe7c42750b
e4ef91a650e9eefb155e6849aaa7bb0635038e10
255 F20110109_AABNGV afzala_a_Page_034.txt
219e535e8feb1b50ac8eb998e1aee833
2e848f5b979d85483be9c9687c323cd3c38f6d77
1991 F20110109_AABNHK afzala_a_Page_049.txt
77b00576395ee3e82b24b6db80e26f41
ef799fa0d7315e23d59017961b0233e281eeded6
70852 F20110109_AABMEI afzala_a_Page_017.jpg
a156ee13b0b72acbaf16f73df07899e2
4867d3521cc74620269052d291b88518f08b1198
69003 F20110109_AABMDU afzala_a_Page_003.jpg
29faebc9fb34d39f072987f51d31efbc
a6aa168ade2310a2b03bc0b26115e10e0a92ceb0
2036 F20110109_AABNGW afzala_a_Page_035.txt
464109523b67a35664b7c97a18976160
4c310e5cdd54c9ac7fd066c99eb54e05e628617e
1948 F20110109_AABNIA afzala_a_Page_065.txt
961d32d324b7d82cb43baf5517e3f74d
11e22663e6519b8c7d922bb16206d8d7688b2576
1844 F20110109_AABNHL afzala_a_Page_050.txt
90c2af1d1caabe387aff2530e821b83e
489032216e3fea0c30af0f4a8827208add5d50dc
63710 F20110109_AABMEJ afzala_a_Page_019.jpg
d0645742154d074f81ab3952fc256957
db102c22b63955c9e990489cb7926c8869af4883
65849 F20110109_AABMDV afzala_a_Page_004.jpg
c38716180a92a7be1e98c17a164b02ee
853831757ed9ce2f578ebf14b4285566ea3c4477
289 F20110109_AABNGX afzala_a_Page_036.txt
0326aa001809880aae1d91e4cc29026d
53fb971364a5343a845031b5817a05fdf74c4184
2016 F20110109_AABNIB afzala_a_Page_066.txt
e661adc867328c18042cdd8e1c301322
f7d290c846105a9679c7a366b41dffaea2d99d32
1725 F20110109_AABNHM afzala_a_Page_051.txt
5ac5a0998cbccf71c88059ffafb7d5b7
d3a34cd6c31a54410bb1b62b82b41e86fcf01ec2
43348 F20110109_AABMEK afzala_a_Page_020.jpg
3a33b0d99962b538655206cf2e371d6f
17c7fbdadc0bf6d92d2342a5faa60b1b041a247a
81195 F20110109_AABMDW afzala_a_Page_005.jpg
0d601369eb81d14fd8b9e6bdf702a586
9673bd5915ccf291921ea2e1cf5d8eb51c48c62d
1963 F20110109_AABNGY afzala_a_Page_037.txt
5cd3d58f721f93ef90b8d582a20e836f
b499f55961b94d03140b5f6f716ad34fdb5d6a44
1816 F20110109_AABNIC afzala_a_Page_067.txt
d284c53164e1e4646e87b857f7e77314
cc58c5b2698cb8739c1829d0838d13a7acd702b4
515 F20110109_AABNHN afzala_a_Page_052.txt
656c8564e16ba4f640a3cdef3c0902fe
e1b14730c6a9db50c4f1696c80268831eb100879
27693 F20110109_AABMFA afzala_a_Page_036.jpg
bfba407003c4c18a8a4690564e96b5a8
fdb34fa490253809a3cfa91f564c16e5e174c0e7
78793 F20110109_AABMEL afzala_a_Page_021.jpg
8f7559bb937a7d9a997a3ed31d80d46b
62f9e4c2cd7b6e1fc11122b9541b34fa56a05a71
119131 F20110109_AABMDX afzala_a_Page_006.jpg
94f12597c924f50c93d47dc82c21905c
71f4f5212b3142a295c582f33e379e0843b3ec8e
613 F20110109_AABNGZ afzala_a_Page_038.txt
1b4eaf292ba568aefb1be839ec243e68
3922bd870382b790c623522a828896c0487d0956
452 F20110109_AABNID afzala_a_Page_068.txt
6ed150d83b4f3d5926fff20d08141c13
f2324cf7807b9a931775c51863b851fcd9f01aa0
1987 F20110109_AABNHO afzala_a_Page_053.txt
c8e5aef42569ca88e26ac25d75b29ec4
43f58e55b440eaa1e6d4f5fea17e8014c8b2eaa9
81574 F20110109_AABMFB afzala_a_Page_037.jpg
41cfddd6a7836c0cf92fbb5a2dbfae82
f151b2a46d79a2aa7fd24ff7ee4b64b575b4cf7b
78156 F20110109_AABMEM afzala_a_Page_022.jpg
a5b072baeeaf15d3e18e61468f9a0c99
d8ea9682b2a54a3ddabbf6a8cb877db4682ea5e0
38838 F20110109_AABMDY afzala_a_Page_007.jpg
6c9b4fa07c00c31a6e0e6ca9e466ff36
4e27d0b52907a8a00ec4547006c04c8f4bb22017
1941 F20110109_AABNIE afzala_a_Page_069.txt
6b15a3fd6a811d75d49c4ba08086e28d
ff79030f047572588808df6734e51b4d0952a2b6
1883 F20110109_AABNHP afzala_a_Page_054.txt
ce9062012c53aa7c10cb50ff5fbad3b5
7851de423e5559790ea93a9cc1d6168bc9e2b0e3
55989 F20110109_AABMFC afzala_a_Page_038.jpg
cdd07fa9e721611eafd7803f5df833d8
38476730179ca4640a022edb1d3846228ed4da33
83733 F20110109_AABMEN afzala_a_Page_023.jpg
ecd3bda382d5cfd803f03d979af21300
d61e24092bfe2d651ec1a65235a156f0521c72cb
71565 F20110109_AABMDZ afzala_a_Page_008.jpg
c551cedb1e13e54eeeb4f4918594b012
5e9cab8f25439158688821ba296b921ff59f01c6
1658 F20110109_AABNIF afzala_a_Page_070.txt
a6a9196d42060bd8a9e206cb70877dc5
26d9686156dbc3f905389eb27cd3d6a79717079c
612 F20110109_AABNHQ afzala_a_Page_055.txt
5329acb4613dfdb9e80064c0b2832b3b
87e6158ba9820f448328039053c3f6eac17ac089
36688 F20110109_AABMFD afzala_a_Page_039.jpg
8393581300cf0ebc03fb7ed555ff9b2e
7645355f749726270a8aedace664e3626fe4f16a
63036 F20110109_AABMEO afzala_a_Page_024.jpg
9380510de8bb7e23a0498b5de0679db1
1bd7e22395a51bbaa17325792ea4d9f432cc7f1f
352 F20110109_AABNIG afzala_a_Page_071.txt
ee199f67e07aadd5fd7535e8a4258cb4
0e1d95a3ef4d07771e94d226c026acf761ce502b
2033 F20110109_AABNHR afzala_a_Page_056.txt
0f7372dcac71b994e70c48add55f8713
4fd99379d76fdef977c4a99fb57f22bc0673f8e8
80935 F20110109_AABMFE afzala_a_Page_040.jpg
f0910ede36ba1f516957e78cb28e6975
69232318878a51ab2c9cd2ea420e5e91d5a935a9
74271 F20110109_AABMEP afzala_a_Page_025.jpg
2663834a45427ab2abc080416745be8d
56a592e5011cb2681cd1c7435e275b0984a306c0
370 F20110109_AABNIH afzala_a_Page_072.txt
9be154158255a5948cf1d62ca4d727c1
9aab486df4484b84b24c55191498aeef04da854b
1712 F20110109_AABNHS afzala_a_Page_057.txt
f1a7b5479c72d77e7056f5270ec6f62d
a3e7d1d9d69fa6a96b03e0a61b7db42f7224d0ff
40510 F20110109_AABMFF afzala_a_Page_041.jpg
62aba17ca19cd85a5cd6dc015b3de68f
d6e6e3385c695dc27bf98fc737137fc1015a5505
84376 F20110109_AABMEQ afzala_a_Page_026.jpg
adae74d6a78326b530bc60d288f1d3e3
07216c34787dfe8336da0666e33b75e4e9ce483d
364 F20110109_AABNII afzala_a_Page_073.txt
ce132da74e1a7beb47a79b78b056e439
7117f2c33eea354703c1f80464d73a00c4950d94
638 F20110109_AABNHT afzala_a_Page_058.txt
d9ca7da613562ee373705e1b0cfbbd7f
fa6490422b96672734ee712755bd9e8a109dc904
65810 F20110109_AABMFG afzala_a_Page_042.jpg
e53ab5ec531fae6cf2d1b45b098171cf
8c5d57c331d2bbe3759f73c38baa79d24917a664
80497 F20110109_AABMER afzala_a_Page_027.jpg
29147e3b517bea274c412aa8490de20b
6af019c54e78a60e6519f9c4ef9ae8b2a2ac284d
1937 F20110109_AABNIJ afzala_a_Page_074.txt
bf2a7128948c0b10439b2cdf259ded85
57b794dc360513de099807268383159ffc535254
527 F20110109_AABNHU afzala_a_Page_059.txt
7b7d9599b68129a18c44a6a68418ff52
dfcb3c81f9ea77cc6e3a03615373d218e850e7ea
59897 F20110109_AABMFH afzala_a_Page_043.jpg
100b01d957583cdd4e4e5cfcebf63ac3
b1317fcae69c6545c46eb0f60430abe3dadd8467
74787 F20110109_AABMES afzala_a_Page_028.jpg
56c9338ed582fbd2d44012a84967779f
7236cd721d54b557537ccce9c713eb70b8ee0df4
790 F20110109_AABNIK afzala_a_Page_075.txt
96ef22905a80ac217b8946bcfa1026f8
ea51959f31e76a2be1cb5ef4968e831982dc5f0a
2037 F20110109_AABNHV afzala_a_Page_060.txt
23252a9cccaffcb7ef74139eb2940af4
fb13b2e103edca7100fad7e9aa4627eb1167478f
79122 F20110109_AABMFI afzala_a_Page_044.jpg
6bea791773dd8f01efb44480974e4b48
85c8100a7bcd10e9ba10859d01380658b84effa1
75667 F20110109_AABMET afzala_a_Page_029.jpg
f4265cb198ac6cb08c6c058ad3396c7f
7676c14dd9066d58053efd73b8e37a04ba8ab7de
802 F20110109_AABNHW afzala_a_Page_061.txt
e556f4b5a8efda883471b4eda87256a5
74fbce6991905be20c5c8517b72faa2974df0964
35450 F20110109_AABMEU afzala_a_Page_030.jpg
0d96f2992841773b18e0f851f6260fe4
72c1a46794d7362bb71c2fc02189afc12c852a2d
573 F20110109_AABNJA afzala_a_Page_091.txt
57da4bb51dda0758e754b0662cc8939e
5805d4a5fc797f8f8ff489e4955079eb139847f5
797 F20110109_AABNIL afzala_a_Page_076.txt
babc5cca621b119d88d05fae18f19993
4ce6fba56f68b71afae797e9d2eb8908cc8f1ea0
1960 F20110109_AABNHX afzala_a_Page_062.txt
ebcbfb041579bd618a37f76caa0d8123
af086bdf8415ebf397b6a53aff9b03e76afbc261
65675 F20110109_AABMFJ afzala_a_Page_045.jpg
7fe9d78f3b19010ee793cbee0f88f112
0f8f9f15aa5a13ee496c90bc4cd554a9504e2837
83582 F20110109_AABMEV afzala_a_Page_031.jpg
d97abcedd74c174518d33d1b6fc1da6d
3da35cdf388950f2d82809414193a65d13e534d0
1954 F20110109_AABNJB afzala_a_Page_092.txt
f0fc58b8c96dfe461ddeaa080250dd27
6d0344260e150aa5e05cef0b9329c0e7f83a78ff
1776 F20110109_AABNIM afzala_a_Page_077.txt
917b3943422033cdc4ee035c0daa506c
97ed9f5dcb0ca11f6db0f7ad97b2542c327aa7bc
1823 F20110109_AABNHY afzala_a_Page_063.txt
6ba88eea85976ee69d9ff0bcce1e67a1
813c64778673b85cdbf83603bf60b5971b1898c0
28982 F20110109_AABMFK afzala_a_Page_046.jpg
61d3a539d0cd9da17e04bd5f536c217c
20c23b24bc28c579093a99d826b8deeedd356d50
35980 F20110109_AABMEW afzala_a_Page_032.jpg
f2dbad91d94f089dd3d8f6a4d50ad1e1
c20197cb7822be1148dcaf495aec2ddcb7b443cd
450 F20110109_AABNJC afzala_a_Page_093.txt
762bafd15a5171b425f139c5d69af601
126e237c0ac5adada71f2a2b2b68f0b95e9e86dd
F20110109_AABNIN afzala_a_Page_078.txt
ebc821e64f2ead8d7aca3c5ab87e9729
61826cdbd2260ebe2be4b51c53331c998736607f
552 F20110109_AABNHZ afzala_a_Page_064.txt
3b30b55ec97198040e8929fa74d50045
9670bb5528894657ff372d927f4fbe0fd1f86542
84298 F20110109_AABMFL afzala_a_Page_047.jpg
fb6756c35c4332e03d8d7718b2494fac
654265bcf874bf3b56620fc8349087c7f4d3ffe5
77727 F20110109_AABMEX afzala_a_Page_033.jpg
4e0d513217045ad42d878aecc399cd9b
b962e5e07c5d9802d2b25fab3a5c0f33c387bd2b
80942 F20110109_AABMGA afzala_a_Page_062.jpg
915ba3dcea8e063fb24d67bedd0fb3c7
5b589d288ccd4ec632aa7b7283e0ca14e7e28b9c
1015 F20110109_AABNJD afzala_a_Page_094.txt
db91f194c66dde3391ef3ea1dc73d640
c6aecb0d489789b69d0ee7631be7ad0555bb3502
2088 F20110109_AABNIO afzala_a_Page_079.txt
21aa539a9418d0f0cdc5d88f45e8e35b
7e2f119ae9304465d86fc59f2197abde90bd3b01
72550 F20110109_AABMFM afzala_a_Page_048.jpg
d610123328cfe27ee97f38eddb5b0072
b1dfb2bad4d53eb38ec75716ca844a4b8d424cff
38218 F20110109_AABMEY afzala_a_Page_034.jpg
f676638feccae24396cfa89f22d12708
f4eae6c6891db908a498d2d62c6061c1c6d44d2e
77950 F20110109_AABMGB afzala_a_Page_063.jpg
7c67e608f1f8a3f819e6a837fc5984eb
cc3a6ee05846695ecba4b065fe56fbdf0d1d11e4
2039 F20110109_AABNJE afzala_a_Page_095.txt
33f12f6bc7aea0796dc02ba2a81114a9
25eacf400474992286552b0f845fb356d5f8b53e
1912 F20110109_AABNIP afzala_a_Page_080.txt
24e6dcc78c6f0ed86675f0f41feddf1b
2eb5d5625499dd0c4f179bbd621196bf515f803c
83348 F20110109_AABMFN afzala_a_Page_049.jpg
09594bffc2db452d2da83b554a0b82e9
29b95b37492812c62effcd3a95ca9adb954b11c1
83564 F20110109_AABMEZ afzala_a_Page_035.jpg
c9d494e98a7cfc1184017b938462ab6f
6e12f792002edb7b30855f75061a7061f8d920d6
39721 F20110109_AABMGC afzala_a_Page_064.jpg
92ce7ee47f9ce399a67163ea1ff30257
6381352580d0e83121713e510ad7fd2e46d91e1a
1911 F20110109_AABNJF afzala_a_Page_096.txt
0a162b0ac8a3057e529e579072b8a596
9bce8e842786adaf7877231f68b68a8eb8915583
365 F20110109_AABNIQ afzala_a_Page_081.txt
a830501ca499258a6bf01fcf991db426
4a8c556ffa86208301e240b70165dfb8e32f462d
78749 F20110109_AABMFO afzala_a_Page_050.jpg
aeeea129bc63862b3e69f374c76bd786
24aa8041d2827d8140ffc18a3507c0d22613bf91
83265 F20110109_AABMGD afzala_a_Page_065.jpg
f19e65da9f164666afb90383794c12ce
644709cb0344153c23fc25a6dbe19c9f3e4c2ae7
330 F20110109_AABNJG afzala_a_Page_097.txt
e8e0eb6470a36418a357e8a132e1b765
419b868af92ce3f9ea365b8a551055cf9f931ef7
1462 F20110109_AABNIR afzala_a_Page_082.txt
032347d16780349bbac063863b34e758
3bbca3c9ad0f3b113f1c1d327ab6159be933f963
75352 F20110109_AABMFP afzala_a_Page_051.jpg
e437e896cfa144c1dc50de115b379596
40154d6a225ee57e6a0f5c53fff3511aeb9db7ff
84045 F20110109_AABMGE afzala_a_Page_066.jpg
1df503eb28496a3c58afbb40cdb0dd85
e30c631c79b3e2d2eba2d32269047574753f0e43
332 F20110109_AABNJH afzala_a_Page_098.txt
3365306c627a27da55051b0582c3cca2
2a886df17ee488e46dbe86ca3461eaa4517a0b3a
2171 F20110109_AABNIS afzala_a_Page_083.txt
397551e00ef34949b315d3b9ef4988c7
3cc565710725e2942860e5af4b80d9d759992561
41566 F20110109_AABMFQ afzala_a_Page_052.jpg
cd76709750b36c8e77f2402939efc120
a0803575090ef797e8f1bb6b5839e60432f461ca
75742 F20110109_AABMGF afzala_a_Page_067.jpg
67476dc13d721c5158916e3605e7691b
ded3023d9a677e5f1304b22f8b708fbe25e88418
929 F20110109_AABNJI afzala_a_Page_099.txt
458fed4ef6b4e84267b1a0debf5d7e89
c49bfc516f9639ff69e50c930f3c402e67996a21
675 F20110109_AABNIT afzala_a_Page_084.txt
ac9d072a6fa5a80809da72235d93ba34
ee51b79402a5127abb0365fcc925d2532281054c
88293 F20110109_AABMFR afzala_a_Page_053.jpg
76bd894d00c4195ebb0dbe2ab81f277d
b6b7560726e31db0c6c9633f5e44744a9662364c
33124 F20110109_AABMGG afzala_a_Page_068.jpg
7f98a257474ffc1aee2ef6fd45a62df6
e4d0e19b48e998c8085691215b2a68c12ee073da
1150 F20110109_AABNJJ afzala_a_Page_100.txt
630abcc3cb50ace23ee287696a09ff9f
86d1ce33cad3a13f76b95358598f82d0e995d708
1614 F20110109_AABNIU afzala_a_Page_085.txt
a1b0322244f63fb7b5862bdb4288e0ad
129207d209a2bb36e346a2cae8226e57b99e1473
83419 F20110109_AABMFS afzala_a_Page_054.jpg
c0498224872f0a15c541dc3745f95fa5
afc5f9cf6cdf80605734891c40515e9f9a0f071c
81146 F20110109_AABMGH afzala_a_Page_069.jpg
7adfc386686071dc069af11e86a2f279
74e717f1a2897c9f0a80a03215392c57f3475792
2017 F20110109_AABNJK afzala_a_Page_101.txt
fbf54bd773c741725c3252d9c8e4cd9e
cae7ccff45863e6da6124f046cbf524d77194405
106 F20110109_AABNIV afzala_a_Page_086.txt
b8b488d6e17ea35347aec791706c9dfd
c49d1584e8d76b22465b655c7e965095779dcb2a
37795 F20110109_AABMFT afzala_a_Page_055.jpg
ff100098f8b704345c71d7b75dc79c6b
c2c5f82618b9a5644c57aa9317c5eb57ad644d13
70948 F20110109_AABMGI afzala_a_Page_070.jpg
54befb352219b91de023b108e96cb08e
5de79f9ce5d0fae655156796faa45d146aa8db3c
579 F20110109_AABNJL afzala_a_Page_102.txt
ee689820b120abba2f3293340c84542c
15da7d9cdd177b3101e6ef6ce50cb270f76f1dc8
155 F20110109_AABNIW afzala_a_Page_087.txt
b4d2f648af7117e4f5d4955c28c9e2fa
65f8019026ad77502789054a41301ed951fc1454
88012 F20110109_AABMFU afzala_a_Page_056.jpg
9ccceedacba9698353d368152b8f3a04
55ed19404aef2cff267f429b4aec6d647174365e
40632 F20110109_AABMGJ afzala_a_Page_071.jpg
84dbc381a09b10c3db2ccaf2b6a85876
d5c61d0f02f4f0f53bd5e5a07c1c156eb25f5006
562 F20110109_AABNKA afzala_a_Page_117.txt
211d750f699ff2dd257926609a2af5a8
19eea18ffd130a2ad89584eea0e258bd739b28f7
150 F20110109_AABNIX afzala_a_Page_088.txt
1b7024712411d680fc7cddf7f6551b00
f9ffec2fe8e93aa5956e93499cfa1a35e615f473
73425 F20110109_AABMFV afzala_a_Page_057.jpg
1846b2de7e6da98fa8a16a07185c7f6f
9c80712278f29c3de68c5fe58be6d28ecac358ae
1516 F20110109_AABNKB afzala_a_Page_118.txt
b34c6cc56b73c49d3264374682187d01
6f43b1239d2d63dd2b348f98b2a6749ab9b44c76
1742 F20110109_AABNJM afzala_a_Page_103.txt
5bfc0ba87a7bd23fba341e26a2c66a21
fb285cfe26151b312c2324aa443140f94a65a92a
1693 F20110109_AABNIY afzala_a_Page_089.txt
49b31b61aac6c8f4c56a73aa5355184e
41f3c3928f8e57c71dfde1bc884d8bc1ffc06579
26825 F20110109_AABMFW afzala_a_Page_058.jpg
5b30c81ea3c6cf45eb3d7c6d2b1fb3f0
b4bc8381565a263811fd727010126bd7442f16c9
49425 F20110109_AABMGK afzala_a_Page_072.jpg
8b97d69ae8598311b71871302723ab50
551fcdca76928e9810e11807beaeee1b0d14bcfc
678 F20110109_AABNKC afzala_a_Page_119.txt
a7fc2243e1e83ca189f7389a2c75bff8
eb5617b2124b4f449604440d228bb33338bd5005
2005 F20110109_AABNJN afzala_a_Page_104.txt
a200502e3700d22010c9c4292a6860fa
0f9b8b3532cce8cfad6581f25fa5df4a49f2e61c
374 F20110109_AABNIZ afzala_a_Page_090.txt
e75c2d29a50b015b2c627dc352a98ac2
df37c1beca9588fa7eaef4f357856218f481171e
34165 F20110109_AABMFX afzala_a_Page_059.jpg
95b75beaf47d3c5ed21c219ef654a7bb
740b8ee97165b5f5bb5f36841434ed00c598e5b0
72638 F20110109_AABMHA afzala_a_Page_089.jpg
503c60867af8addd2cd93f5edb8f8aa3
2badaa9e60dba22cc5381f64e2cafca6416d4c31
44609 F20110109_AABMGL afzala_a_Page_073.jpg
8cb8859fc3e5fefb52d9af25116793fc
0f6d033927a8987357e2c518839c3a2946488972
430 F20110109_AABNKD afzala_a_Page_120.txt
a4b7aa973ed52eb39bb017d57309b54d
692c23d40eb88491e1180b5fbc602c07523d7ee3
1950 F20110109_AABNJO afzala_a_Page_105.txt
c2f0d724c016848b5f4547347028c4cf
37675bc983b1f92a6114d24142f0355c43d7360b
86545 F20110109_AABMFY afzala_a_Page_060.jpg
b920f0ae1a92fc3125bd0266e288998c
d8798c1c518cd817c6ad5f13dad1a4b8b40ffc17
19018 F20110109_AABMHB afzala_a_Page_090.jpg
f6aed018f6cc420574d727b845ca554c
6dbb671bdfe6b60e774d7c36e3fce4c896a19c2f
80002 F20110109_AABMGM afzala_a_Page_074.jpg
94dc18b43c62212367b3cc1c5202de21
ce5835537cc8ceb9ceedac22c61e1fbb769309aa
1907 F20110109_AABNKE afzala_a_Page_121.txt
407f1a366a8bae312ed4be6a35a2ea95
9201fead57d56478988a498e43d69fe0586131b8
1770 F20110109_AABNJP afzala_a_Page_106.txt
fee0ef5376822330d27fcae3916c9c72
7ce1e46d793708828a5c49cf8082306ae6384451
43187 F20110109_AABMFZ afzala_a_Page_061.jpg
47f6392d46ca1e2e118ecf7897e010d5
2c6f9cebf6bb0bc6fd37fac1eae4b6bee98c4c61
26935 F20110109_AABMHC afzala_a_Page_091.jpg
bab0ae699e8da1555ddd147893c59abd
113b5335bfe3fd495d13e3674b48d6240a2be45e
40385 F20110109_AABMGN afzala_a_Page_075.jpg
c6e355355b687ec5a3a55e4647722a60
197d4628116ab8ec727ab07a96a08df50361e409
463 F20110109_AABNKF afzala_a_Page_122.txt
d9ef419a7cc61b04663b3cf7602861f2
f75c924c54ec90e1c44e4aee2242f470ca76394c
1973 F20110109_AABNJQ afzala_a_Page_107.txt
8964173f199f945fce8632785fade655
ac100ebd7bfc79b11216e355e7fccecbc2a5c722
83083 F20110109_AABMHD afzala_a_Page_092.jpg
68b0bbf486981fe35b4e040b67a547e7
20cd5f52a795e0a5878c1ff48998b856d3b9dcba
43506 F20110109_AABMGO afzala_a_Page_076.jpg
e9216526c04dac2273e95c2f4c821659
6f626ca80ae6f9fc6a65e9bd8803aad3d238568c
437 F20110109_AABNKG afzala_a_Page_123.txt
5c80749c1099d4c061b086b70891fed6
a9da16c9dc1fafac01fb4ff267d10ddcf7271788
1815 F20110109_AABNJR afzala_a_Page_108.txt
9f39c5034ef3b71314dae1e14503b6f4
bf004fa13843ba3101ff0b6f5472c059508e549a
30106 F20110109_AABMHE afzala_a_Page_093.jpg
e52a17d5fefe1ba9d2bbcab90868431e
ace6331bc2023dbe7753a32aacf87a144ef49e92
76280 F20110109_AABMGP afzala_a_Page_077.jpg
1ed3e84042461155542dcd7404d27001
d2221edd7543cca7a4cf7b32f2950af741da9f0d
1729 F20110109_AABNKH afzala_a_Page_124.txt
f0d66533fb6e2b19ff3b83b7354102af
975ba21f67288d3f9e963b2629b99bd1a51edeff
1811 F20110109_AABNJS afzala_a_Page_109.txt
7d8381a971b3ab58f94bb64f3c875298
1740b57afcfc70a18caa9454b4d82e8dbb1cc738
55808 F20110109_AABMHF afzala_a_Page_094.jpg
4bc54b67c4ac7ed959f3366212e46a16
565dd456deba7b7e38f0fa4379460fd5343f5d5a
80241 F20110109_AABMGQ afzala_a_Page_078.jpg
d7b4c0a1f24edcb930ad308f19513067
e2be87c1ed6c09e2c29248bd47dd455926baea50
288 F20110109_AABNKI afzala_a_Page_125.txt
e30b12dc75be79be4de7857afb94571c
e3ab1d7ba271e3f9288e8018d0b4885e0861366d
1932 F20110109_AABNJT afzala_a_Page_110.txt
736bca56a97c93f5d88a0e97b8e88a7a
e3e0a285129f3184fb309935aa5de87c81b0c6e9
85369 F20110109_AABMHG afzala_a_Page_095.jpg
2a2d37813e04c16fadd7e9ca3f6fafb8
d373ce0c6bf81092f06534b93f7652d54e615d5d
86107 F20110109_AABMGR afzala_a_Page_079.jpg
655318ac386e617972e63dff9cbbb7b9
0c3ac823fd3414eb0ada3d5e3af5d019aae11fc5
F20110109_AABNKJ afzala_a_Page_126.txt
a3df25c4d54939db2950ad3a53513295
c26bf847de7f62c3d694cb92c0f4a3c3f056898e
1820 F20110109_AABNJU afzala_a_Page_111.txt
6b82b8eae6eae210408fbb4082afdbd1
bee225cdc48c0ce344f68797bad4b4187f9c26b0
81724 F20110109_AABMHH afzala_a_Page_096.jpg
5a74b9c73ccab90556140ade5050e9b3
557d0bf44445b8471d9602a3494cf2d4330ecdee
79538 F20110109_AABMGS afzala_a_Page_080.jpg
361d70b318bce2049b48fb8f18fc9e35
42d4fae23d1d68f957f1ea321cefa45ab3196c17
1843 F20110109_AABNKK afzala_a_Page_127.txt
2554b476f5eb7263a3c3dc23d994498b
25d456bda386f8e9ab3596999e753f4de2b2907a
1884 F20110109_AABNJV afzala_a_Page_112.txt
243e82e8edbd51e770a9fd93aa594e04
ab14c87d5ab71bd5db32af2ae42e429ce14510b5
25954 F20110109_AABMHI afzala_a_Page_098.jpg
2caf885ae6ee41612402c7ce5969db65
4d6d1106d6fe70bdcfc64df302e39cef1b0c36cb
29336 F20110109_AABMGT afzala_a_Page_081.jpg
f4b0eed258823d26e72d5abddd5550c9
9db7b52f844acf258ab7449878c5e4935e8be8ff
563 F20110109_AABNKL afzala_a_Page_128.txt
5e55dc5f38900e1ffd177367e3825917
b631ed9aa54c355ef1ef20db989eb87e12be6528
F20110109_AABNJW afzala_a_Page_113.txt
ff97848561a7d81667b94845cfd85347
4adeb770b1619f5a7d5206029ccf08d86adb344f
30507 F20110109_AABMHJ afzala_a_Page_099.jpg
c6cf0272617de2f3f1ece14b7fb416c2
3e3e947f05577b6dfa1820ef75023c4508842c00
59460 F20110109_AABMGU afzala_a_Page_082.jpg
1b0b00f8aa767b579418703a5ab5066b
b9012933bd15638cf7d90437101bf833eeeab468
1559 F20110109_AABNLA afzala_a_Page_143.txt
c5b4f299f9da490f682087ca39139c28
423c3e55e89bbd706c2fc458a5a0d32561e605ab
1851 F20110109_AABNKM afzala_a_Page_129.txt
7dd592461dfb2af1f2db204b28607530
b35ca93fb2c43a8060b6c9ffc3e2d24839d2345a
1902 F20110109_AABNJX afzala_a_Page_114.txt
6a112d1c536201ebb2ac029b6dab314b
d7f26c703203acabbf8fd338d7da280de61ff53a
33389 F20110109_AABMHK afzala_a_Page_100.jpg
1edec6e8b605f7f8f98aa8bc14fe6186
862d1b576efd26cb9c786e566382ee9dd5c11d6a
22407 F20110109_AABMGV afzala_a_Page_083.jpg
7c4d2a23052ecf7d7e35b6aac338e2ea
c5723f53fbf211638a1a8d18f4a6d0326a1ea3f0
369 F20110109_AABNLB afzala_a_Page_144.txt
994a4a8255b304552ef99fcb89f9ff34
8261188d4fdc11f4ba337e29b82a2758e68eba1b
1731 F20110109_AABNJY afzala_a_Page_115.txt
8c04d3efff4d2f8a0fed6232627aaa6e
7e0504b2352d1bb53542a351b0f24eb3bcfbb716
30543 F20110109_AABMGW afzala_a_Page_084.jpg
1f18f1bec3c58fd6e7e1cb3e977b446d
80d1e40c4c8f107e4ee8f0b02841c846072df642
570 F20110109_AABNLC afzala_a_Page_145.txt
c1937250c9a8d7817539e74ae5fa5c8a
c524ac3a77d4f42625144ea4b5e734a972eb8a45
888 F20110109_AABNKN afzala_a_Page_130.txt
86519301d2af95833f323a81529d2fb9
d61855a5c4efc5ff15738d837feea63c5e1d5123
2000 F20110109_AABNJZ afzala_a_Page_116.txt
86c07ee1c9968c1c2f7e3569e16aba73
8c009716b8988518a1e676598d8c8c1ed7bde5ad
84776 F20110109_AABMIA afzala_a_Page_116.jpg
9666a718546ca5e49260e27e4d944892
b978ef6b271518ef27b815714df3d1e0a039ae3a
F20110109_AABMHL afzala_a_Page_101.jpg
995adf01330cc21d97d0e439924a7e3d
28233e2cae6efb42fcfb6b46667b914ebee3ae04
23273 F20110109_AABMGX afzala_a_Page_086.jpg
a22188d8657e178972b7b321ee052275
93526cf830568af66a17481fc368a5e817f81344
388 F20110109_AABNLD afzala_a_Page_146.txt
78993d15f002f158882d66049507f506
6a697b7016f712607e4d8dc551fe412ede4373b7
134 F20110109_AABNKO afzala_a_Page_131.txt
38494074f5d0895c7cde2026d0ccb085
ad48e66ce5c1e5f89f953722cdf10dc7269e7dcd
22226 F20110109_AABMIB afzala_a_Page_117.jpg
6fb2073851ca33712d2c158a9a97c015
21304ea064bfb9e934aa574deca3239ca10c4fce
36878 F20110109_AABMHM afzala_a_Page_102.jpg
bd15eb34f8a636a9535a54847be910a2
09d8da4e904725d12432dbd137d81d07fd34bb8b
13108 F20110109_AABMGY afzala_a_Page_087.jpg
4fe867984692ec04a53a5b6f5c6c37a0
849668ce9358cfbdb27cc0c06899d215a4ec2527
278 F20110109_AABNLE afzala_a_Page_147.txt
a800a2d5527478a456689e10f7999f26
6edd18488fecedd1a94314a956d05dd6a158369c
1632 F20110109_AABNKP afzala_a_Page_132.txt
6580b0fdaae26b686d0a37c21acffc2c
c2fa501d497b7320017666a363b8c60851fd8d82
61006 F20110109_AABMIC afzala_a_Page_118.jpg
25c023ec7a09ca0eae2a3ab3c8903b61
b33982947e8bb9d596a70cdbb874c4097c6ee079
72175 F20110109_AABMHN afzala_a_Page_103.jpg
4fb2a03110c4b89b013bff7a017c143b
8759adc7ade9ef70a465bdb6500c5ec4ef3b4a1b
34147 F20110109_AABMGZ afzala_a_Page_088.jpg
11b8f960939925cb1b43b3346150c2a1
fd02901b5c1871672d6fdf2808e9e3594c61626e
306 F20110109_AABNLF afzala_a_Page_148.txt
f62f3c8d46a89ca212516d1abe86c7eb
4889984f3dfa42e5396a9472af032f01bfafd365
105 F20110109_AABNKQ afzala_a_Page_133.txt
28f4e02d77dda675aa9285610c2ae00a
215bccff03ca81516ace8100f816e392c1b4e4e4
22594 F20110109_AABMID afzala_a_Page_119.jpg
a748b9192db9688b4e7df8d99f23d587
23e0539b32157f43c3ebcc96f967bd2a9e631856
84623 F20110109_AABMHO afzala_a_Page_104.jpg
e427954a3cd96ff436b06e178a371643
7fca7b3e662366ff9e23bafe049883902f627063
F20110109_AABNLG afzala_a_Page_149.txt
e32149771e272f7b1dcffa89cb13b99c
79ab573a00faff58ada708bd41d036034fab7c87
435 F20110109_AABNKR afzala_a_Page_134.txt
19e87c1cdee2ee2892a9e97b5cf854c9
49982a556208d7653c7b3ed251971f87c6d086bc
82401 F20110109_AABMHP afzala_a_Page_105.jpg
2494d2ff68ad9d1c985c8b024101b75c
1f3c498c570b795ab164aaf78de47df74844fc3e
23626 F20110109_AABMIE afzala_a_Page_120.jpg
2cbb3f4a44d97a392f9a43c6d9c822bd
41395c5f0be919a286687e87f2bd9327a778af71
1983 F20110109_AABNLH afzala_a_Page_150.txt
8aedf32ef3d4a78679d929e7ddd2e726
a1a13402536742be130cae93154a494bc33e7a5a
334 F20110109_AABNKS afzala_a_Page_135.txt
9afeab32015bd35f6501ad8ad66bcf88
37d8b01d2d3622d00a432b5ba62713d08686b1ff
75545 F20110109_AABMHQ afzala_a_Page_106.jpg
9d47cc17e5e06c27d9b4053a773845a0
e2d3e195ed3ce410ac2f5f294bb9d73921906280
79293 F20110109_AABMIF afzala_a_Page_121.jpg
8a990bcb45bf01ff08fcb94bbb690527
d7c9a9b9c45afd203927495b4577de9cbfa0c51a
1952 F20110109_AABNLI afzala_a_Page_151.txt
f10a3c83ae4c8fd15766913eb24a4fbb
8d60f6fbe724ee0bb85178e7bfd9a89883ddcdba
1826 F20110109_AABNKT afzala_a_Page_136.txt
930e50f2bdbb983815d1c79bfeaa61ed
439e7fa0ab56ec1888d2cc87ca8fd94d106338d3
85963 F20110109_AABMHR afzala_a_Page_107.jpg
870333dc0b9b4141813a39575d3830eb
7187851218fd7448a87b995b509d69a746cdf3b2
27257 F20110109_AABMIG afzala_a_Page_122.jpg
d02c6af68ce12259c51936aa6ed268b6
0d8ae560ab4b1940b51159ef3b7a329bcd70fe82
2069 F20110109_AABNLJ afzala_a_Page_152.txt
ea87bcfa36e0ce1e74bf0ec493d0dc90
c77851f5e95c62a11c42c1a2039bf70484e7ea3c
740 F20110109_AABNKU afzala_a_Page_137.txt
8842ec5085c659f2534571807be14555
e19b0973a490431f4600fb9be83431aab94bf9ab
78563 F20110109_AABMHS afzala_a_Page_108.jpg
8f62aa25811c0ccb2e8000dd1408b593
5057eff627e565bafd2602d15816b6318b237f93
31999 F20110109_AABMIH afzala_a_Page_123.jpg
0e9b0b0e94796bb384333e3d806dce04
aa4a687b841702b3f433f0a24b519837d24140c1
1880 F20110109_AABNLK afzala_a_Page_153.txt
bc34804e30f6357172db0fb0d365d73d
0d823733224e61ab6e49c77cb6bb5d403d1c2b93
410 F20110109_AABNKV afzala_a_Page_138.txt
3e2affc2460cf7f70c3ff11b1a6c213a
76780c55f616befc44e1cc5f6b2441b8cf0f0404
78347 F20110109_AABMHT afzala_a_Page_109.jpg
cf8959914e2579aa0f38fcbde5d86565
59cbf7b7355142aa24f047d0d58e7cc470ac4d3d
71501 F20110109_AABMII afzala_a_Page_124.jpg
9b7450bcee18a162fc941d241e6cb192
9a929467b622623e2d32d42be2081ee6455085aa
921 F20110109_AABNLL afzala_a_Page_155.txt
6e2fa7586ec8a2108e35ea0814f7a5d9
e3c099c1391c206ec80ae5f0d67f411122762010
1699 F20110109_AABNKW afzala_a_Page_139.txt
2d1f0e66a6e2ae7e2726d9add09ce991
aeeaa10f3253b3c222a79e8c9ef4a85596b0e274
83014 F20110109_AABMHU afzala_a_Page_110.jpg
3cbe81b7dac2c5ff0945963302a6d8cd
89b389c885b04826b5a927f911da268ca4f0cd30
30418 F20110109_AABMIJ afzala_a_Page_125.jpg
58d9ab0feb7bcedb98e0a7594256ac6c
9aff810f8af34f1ec31b5dfeecc9853babc08fe4
2074 F20110109_AABNMA afzala_a_Page_170.txt
1f44cb63817ee7400a550320267cc95e
8d6f41199ac6c2d25fde5f7851bdf37d1a8d2ce4
F20110109_AABNLM afzala_a_Page_156.txt
b57670636a3196fe19274a5c9eedba0d
5a2664546e0fa5642a2d47461cb9901bea4fec1a
252 F20110109_AABNKX afzala_a_Page_140.txt
42a87e9d22ac208a1b407914747623ad
9306156a19ed016009bf261b4975277f33eef31b
79053 F20110109_AABMHV afzala_a_Page_111.jpg
895195407139bdb1357f43cc659f91e7
e18e2a4e045a955f19aea7a454b9e2addadf7211
27747 F20110109_AABMIK afzala_a_Page_126.jpg
550374d730a0bf96b8adbbf900a561b5
db6f6716dc3d534e77a3e991b1b2a13485eacadf
2153 F20110109_AABNMB afzala_a_Page_171.txt
1a645593740d5852dee3e50155d4922e
52353471f866438bde66f8ae2da5340cfd69e564
2091 F20110109_AABNLN afzala_a_Page_157.txt
ec9a4a7b96d575b019e8acd2cdc27b4a
ea2876b630cc9a1323624433513d82bd1a8aaf1d
291 F20110109_AABNKY afzala_a_Page_141.txt
a247527a6b5badd4febc98c55310353a
8e27b4dd93341f3c4df2c26cb5bd04697a1a999d
80060 F20110109_AABMHW afzala_a_Page_112.jpg
6ccacdcaa3176cad9b5e6918b9680f83
2580ad81938c6edc77c89af247104ead1a429a6e
78330 F20110109_AABMIL afzala_a_Page_127.jpg
e98c580a0a9ca7b405e3cd9ddb6ea10e
60df81c47bb045334f7a646b7c57028678f5d700
2243 F20110109_AABNMC afzala_a_Page_172.txt
31a45f7ea14fe423b143449df4eab838
665a8808cf6649d24225eb61aef47328453e9b39
508 F20110109_AABNKZ afzala_a_Page_142.txt
0f6b87b59a8806fd035c04fe2e14a57d
8026635e34bda4f8bc5837d594250cd81f6db348
78850 F20110109_AABMHX afzala_a_Page_113.jpg
5709f808b455c6332eb8a2f94ff05d55
638589e6deb36c2898d7e5f4b2cb99fd68b4a74a
69251 F20110109_AABMJA afzala_a_Page_143.jpg
d2e2e15e9c8828e20f4943049e406c4f
10072234f6404605d23950f400f962ebf4806717
2063 F20110109_AABNMD afzala_a_Page_173.txt
d2e4519fa2d6bbe1babfad219f29b8b6
ca3b16edcb55b68aac10f376282fe11f9fa32ced
1874 F20110109_AABNLO afzala_a_Page_158.txt
0224147ffa65c16dc688e37e9cc994e1
8df08d73277d872b42f0be290aec0724fa8ab823
81873 F20110109_AABMHY afzala_a_Page_114.jpg
e3d7aa5edd7a264c82524a23b6ea8eb7
3bd016b45a5cd8b7e208af93d822984ef07bb8db
32415 F20110109_AABMJB afzala_a_Page_144.jpg
3b78e0332ec017896d620ea906098ed5
378f5d065c20e719f7650b1c7cb70a60cce45214
76835 F20110109_AABMIM afzala_a_Page_129.jpg
6643c145c89f1f0960efe687d53bbfb2
ecbbee7af05c970275dce624e181dde44f5466c6
2122 F20110109_AABNME afzala_a_Page_174.txt
0c45cf73188c10a1f94df7f3fbe2553f
58dd8c99da05ea10c9e5594a2b889a21ee40e127
631 F20110109_AABNLP afzala_a_Page_159.txt
81f4cd905c868ed9d4779641a1d03967
0474f1c9adddda0bf69610378374e0b55012944d
73764 F20110109_AABMHZ afzala_a_Page_115.jpg
fde73f8fc5ab3c1989cf584730b00a7b
cdefcb95527053099f6bad56d7bf9eba80b0f172
63342 F20110109_AABMJC afzala_a_Page_145.jpg
5dcb5eb6361118f3d1482812a3284043
4ab94f99fb8fb6eab33d3d5f60f696e2596d9520
47741 F20110109_AABMIN afzala_a_Page_130.jpg
d2cdd9e7b44687ee664a134d8c6b2fe1
3d987ed8840047be287c79cd58fb994dca2f1247
2158 F20110109_AABNMF afzala_a_Page_175.txt
828f4ef128abc73d2f940e6e85910c50
35a3ccc09c19feb52c67b35d697431a38134dfe3
1825 F20110109_AABNLQ afzala_a_Page_160.txt
c3f32611c406c7034c31a1aa62806a5c
9b207128dc99b666a161effe0aaee8a001ba1353
55705 F20110109_AABMJD afzala_a_Page_146.jpg
b34494a080798c1518428fbccada2252
1ada85fde5fd4c076381c300edc7a028ddaf9064
39926 F20110109_AABMIO afzala_a_Page_131.jpg
81f3c14403e59796d14f8690d79cec72
d2b8ab7a7d6fcfb30035c9b9b9a00383ba794680
2190 F20110109_AABNMG afzala_a_Page_176.txt
9ef8adb04b5e752cc012ab685295f85f
4ec3426b9d722e2e4fb32dd05567d0e074946738
1970 F20110109_AABNLR afzala_a_Page_161.txt
f3546afe34093c63f1ba679e6b2bcdde
441f5b6f54e93b2c20387236469de3404e536bfe
46802 F20110109_AABMJE afzala_a_Page_147.jpg
8039c5cad447f5a7a684c28260c23d19
0f278736057630208cfb7c8da306684e6abe7883
69087 F20110109_AABMIP afzala_a_Page_132.jpg
a76a1660d4b836635acc66e387ee2344
4c26db0fa0716b50a52b3251680b7e9e05a2aee6
2107 F20110109_AABNMH afzala_a_Page_177.txt
5e5c0b852e0c59caff4d538b0eea2598
8b01793d1f7b5187f774547a6068bec18065e80a
2023 F20110109_AABNLS afzala_a_Page_162.txt
0c03a09fbc832bfaf653a5766f290f08
ac67a0e57e2acf8534cc8664fc301f59bdf7bbba
27031 F20110109_AABMJF afzala_a_Page_148.jpg
adadc8fb1c30f0c414aef2ce5578ab8a
2c7b9cfa2600a458e8474e0a7befcbc0d332e058
15902 F20110109_AABMIQ afzala_a_Page_133.jpg
1c0609bdf51a9db86143a170133b99bc
819be5a8dfc79d58a1ee4bfffff7c552b045c768
F20110109_AABNMI afzala_a_Page_178.txt
226435b60c5c0852721d826ee26a99e1
ec17c9e1339e42b81eaac9185d45dcbcf9bb1bf3
846 F20110109_AABNLT afzala_a_Page_163.txt
90caee90cda51fb53821ddc1c123a51a
b8601780a66a667a7e85ead5e96bea064a178d08
76147 F20110109_AABMJG afzala_a_Page_149.jpg
53f53cc158bbe0dd19a0780c5f366622
484402a16b9c025878e7a876d86c9f91d8bd9555
27106 F20110109_AABMIR afzala_a_Page_134.jpg
5ab4bc4f3185156cb6c27c970084c70c
80b2cbb5db761c6479bba434f7ab5ff064cfdcb7
2265 F20110109_AABNMJ afzala_a_Page_179.txt
ef779759c775469dc4e3d132fb7f2305
060126c6fcb21f8818786c435a1f9fe208bbde74
624 F20110109_AABNLU afzala_a_Page_164.txt
8d305102d5801330812da30d1087a4bb
7d1a0bf45e09a38dd7758346efae5366a67c34b1
82585 F20110109_AABMJH afzala_a_Page_150.jpg
a0e8984125c60b6ee47e268b734fb142
df5ece80b0bff6d0641622d67da29e0527bb9507
31953 F20110109_AABMIS afzala_a_Page_135.jpg
77dbaf095531bd92f14f4587d925ae13
42928bb31077afb39b1432934b878be27bacddb1
1988 F20110109_AABNMK afzala_a_Page_180.txt
b21a877b678096be9d13074e6a3a4f38
dd0f50f6f4385f22fa998d1222a1bf8162c016a8
1553 F20110109_AABNLV afzala_a_Page_165.txt
0e0b92a889f37dae20c004f32cf7e3c6
a2cb899bbe7307923c2d6fcd04fa0f303c16ff88
83616 F20110109_AABMJI afzala_a_Page_151.jpg
555a858488399f6acff33c72399b996e
a7e3baf352abb4075e14b7187641316a6892d618
77142 F20110109_AABMIT afzala_a_Page_136.jpg
636848532d668a57a1d9c1e80347ff2d
ab9708f3819bcc9ff6da628f8a2a9340c45b3c0c
1935 F20110109_AABNML afzala_a_Page_181.txt
a2d921f893421d0f7a0ffdbf0261d999
93802cd4c0c3834094a79010cf6c44cb4a7f5ac8
1999 F20110109_AABNLW afzala_a_Page_166.txt
22fcab4b32b6914d397722bacb98ee98
87d350486b04c03c971c921576e191439008243f
87280 F20110109_AABMJJ afzala_a_Page_152.jpg
d559e3e47f5132893d7158bde4f73b64
4e0aba8d2cf9c265df6e64a03f2afd5dd98afae9
49565 F20110109_AABMIU afzala_a_Page_137.jpg
b11a0be8ef73cade45bec97404264f0b
fba9e87e8ba53bed6b65eaf79dd5d7c40515c06e
25560 F20110109_AABNNA afzala_a_Page_006.QC.jpg
83ac1cca8a8054f09c94694d944e5217
d83be2596cc47e0902b191308df9022eac57b901
375 F20110109_AABNMM afzala_a_Page_182.txt
78320ce9f671ae5f871019933c7e7f53
8ba1053e0255aabf7d2157b0b48583967233d97b
2092 F20110109_AABNLX afzala_a_Page_167.txt
9421274da08658423cd66d7172b1a885
53dbc7d6113fa392cf6f28065c413166adb674c3
78086 F20110109_AABMJK afzala_a_Page_153.jpg
ac90260b64708216fd97909a7c5750e7
38e3eb2a4e0c5bd60bed4e47b73270a6b6d24ada
35027 F20110109_AABMIV afzala_a_Page_138.jpg
81d0cf6113ea9bf3b66f035faf1c1bc9
2e32171312b127e94e196e57a20fe6b4dae9834f
5626 F20110109_AABNNB afzala_a_Page_006thm.jpg
a13cb4f62aa17eff50849d7848dddab4
ede3853109e141f72abba38a686b351c70de7835
1759 F20110109_AABNMN afzala_a_Page_183.txt
e8fa81deba79a60ab6f8516b5a82d901
693f775e24abea75276f123faec69afd68727bb3
1856 F20110109_AABNLY afzala_a_Page_168.txt
0e7ecbdf7fbb66b8099d76d91c5f74a6
a53eb81202fcb110eacfc0434e0fbebf218e359f
79175 F20110109_AABMJL afzala_a_Page_154.jpg
63251e0568dbde4a56c9040c7450056b
92afe92c058d64653873ef4e999e8fd42a141452
71729 F20110109_AABMIW afzala_a_Page_139.jpg
f5e7d0ea34b6aaeb2cdbf117476dc939
c4c6c4b81af72e742e1fbeae9e0cffb770b372c5
9250 F20110109_AABNNC afzala_a_Page_007.QC.jpg
4e73939793499745b0c1ebc3ec9062e4
74349389a7efe85953deb924a674d2d580dea8d8
1969 F20110109_AABNMO afzala_a_Page_184.txt
291af0fbec6396845e0e7169ca1ea9f9
b706a06a4ae1fe2eb097529bfa400343e8170a05
2011 F20110109_AABNLZ afzala_a_Page_169.txt
131c9562f6e9c6ffdc4937289e557432
104dd89cb66b476b2be822ab121109de02ba38d8
81657 F20110109_AABMKA afzala_a_Page_169.jpg
701a39557b31139fd9e0aafd7fb6a002
104e60a5d3abc9a6cc2bb2f57de47aa82cff1e98
39579 F20110109_AABMJM afzala_a_Page_155.jpg
5c3c083d1f8d3dc25170ba8d1e62c2fc
563f56d0ae34560deb3b557274af2f0007f4d1b0
25566 F20110109_AABMIX afzala_a_Page_140.jpg
fef46b4573bc28c13acf04b66acffa2b
79bc41dbdb9e51db01a28d8e26bd69dc4c30a7d7
F20110109_AABNND afzala_a_Page_007thm.jpg
c1f720b69ab4aa95c90199f822cd9b99
0b64934b8858921f48d9a415d03efbcf9937af68
83231 F20110109_AABMKB afzala_a_Page_170.jpg
e72cb38ca6efb8c5a92c1e203891c288
3c8720d084a60832a7d2ca0047014a183336e152
30491 F20110109_AABMIY afzala_a_Page_141.jpg
282e07b183746fb7df3f4501a334d6f9
1b43fcc82cafcbac19ead8d77b47a530b1c3c617
19790 F20110109_AABNNE afzala_a_Page_008.QC.jpg
948a198ba48722c82383336bcc665784
401b6fa16df3b3b85001f5c8ff6f80627880130f
4187456 F20110109_AABNMP afzala_a.pdf
bc6d064c424899abe95cb8339545b1fb
29acbccd9987e3f53a653447b6c3e85900f3b50d
85027 F20110109_AABMKC afzala_a_Page_171.jpg
2d97edc59d33fa4c1fc833f53311ff16
51e2283bfda520350c5c1a2246d9a89a49d2ecc5
87108 F20110109_AABMJN afzala_a_Page_156.jpg
c7e4d4b3b8dd21ee063d658620db79c5
5c8c6245218607a9bd174675527dccd0fd4d97ea
18100 F20110109_AABMIZ afzala_a_Page_142.jpg
6d31af4607be2b692a8bbc27753c7526
250a4de8087ce338e2f89af31d0180dd34395787
4862 F20110109_AABNNF afzala_a_Page_008thm.jpg
9a1930e99de58e4e86595ed0ca27378d
de6e88efc3e983c4df247725a213fb15e1a6b65b
6784 F20110109_AABNMQ afzala_a_Page_001.QC.jpg
469fb7f08929b6818d93c6bd3807cb68
7f77a042a446c76a8421b89339f832e487785a70
89176 F20110109_AABMKD afzala_a_Page_172.jpg
4936392033c702d02e34b7d2e9db50e1
84f8ef3b1b00b7e81ab87557f9c43ad198be1a86
86218 F20110109_AABMJO afzala_a_Page_157.jpg
0b4a2041d4c06b9c151619f20e81ace1
ae4dac26b131378f0091a6eda777f6cec6ca2628
24767 F20110109_AABNNG afzala_a_Page_009.QC.jpg
dc94d56b6de4066955e219ec3efb90dc
2e78a58fb3072cbd35bf1dca67527be589b794b0
1818 F20110109_AABNMR afzala_a_Page_001thm.jpg
dab52ef7808e2cc5b0c273e35aa597f1
8097e10e13accc929df9cb20c2590b3abc8f810f
84975 F20110109_AABMKE afzala_a_Page_173.jpg
10abc062961e2717befa30bf2a7da97e
25e4c479446b6af8acc7500002ad61e5e8e080b0
79115 F20110109_AABMJP afzala_a_Page_158.jpg
0a15a7722782cc2cf45e14b9871dab99
f01f317c5d840beee0a19e5b5c74304c7f2a0613
6053 F20110109_AABNNH afzala_a_Page_009thm.jpg
2309a97d895cdf696c27c2f7ecaca5ad
0c0aa03154e9521908c7bca5bc11a4834b8eddd6
985 F20110109_AABNMS afzala_a_Page_002.QC.jpg
84103e7dc63e4a5ffc6d9f66d78a67d3
89e32c49cb9f982f5a8f0be9b6b0928efdaf51bf
86350 F20110109_AABMKF afzala_a_Page_174.jpg
df36051f4631e49978c19c571c5897ac
2b16cc1b9402c0b250e97110eaef192b6a23a5f5
31780 F20110109_AABMJQ afzala_a_Page_159.jpg
8a124eb9f9b2eec8d9706783d2bc6309
b12b45f16ac8bc57d2b632550f54c2b2bedb0c2a
28144 F20110109_AABNNI afzala_a_Page_010.QC.jpg
f8f9858668de0fe4d070a1a2977a38db
e9defb1228ad3ebbda956a14510522b8efac65b0
F20110109_AABNMT afzala_a_Page_002thm.jpg
c281862c7e7d1cb32f5125e72e987287
f9e8ae5d0b1112b992b59bcfaa48f69e880910b3
90139 F20110109_AABMKG afzala_a_Page_175.jpg
e149290d7bf6ba51d9089ecbfd05a1ff
ae343b394bac6364ba2840363840478c3315f55a
76154 F20110109_AABMJR afzala_a_Page_160.jpg
fe58f1726592001c840ab849c60847f5
8af9773694740155dda2955a69d1b23cea4d889f
6652 F20110109_AABNNJ afzala_a_Page_010thm.jpg
bf1156d90ffe5ea75a6ed11b1763fcca
ef318759b861794d794310d6ea435038537ffba4
22026 F20110109_AABNMU afzala_a_Page_003.QC.jpg
040013cf39bd54fe6a7778cf88f77773
cdcb777271e0007d3f329f14d005a6e7bfdec1e4
89571 F20110109_AABMKH afzala_a_Page_176.jpg
22fcc94e4a78bd263901643ac72fad50
bc61b27811785611215a34bd615ebafaf2781368
82318 F20110109_AABMJS afzala_a_Page_161.jpg
cb8b9a02fe21c69882692fa59736ae52
ab0553d76841c51f1f584cb6baac7222e684bec0
4722 F20110109_AABNNK afzala_a_Page_011.QC.jpg
d5f4d8ce3570959a92199b66367ac783
0d1db9080b3f3523cdafb2f8b382ddd60dccad79
5148 F20110109_AABNMV afzala_a_Page_003thm.jpg
bd20b9548be1e6c403c7126c0fc8d03c
ebc8f1e7cd354bca6bdc7de440c8a341abeff737
86704 F20110109_AABMKI afzala_a_Page_177.jpg
df504ad3b9ada22c944b810cde41ece1
418725c0e05819ebcd93ca70d35f172eb1c087eb
87172 F20110109_AABMJT afzala_a_Page_162.jpg
d3ad4c9242e13f236abc47f3c144f3ff
04ce85add9ff9563f5066a9d8837496e4164c5f8
1438 F20110109_AABNNL afzala_a_Page_011thm.jpg
18a136da6c073233973578ee4d901c8e
395a8e77906c7fc26a01560524b571e66c59e525
20742 F20110109_AABNMW afzala_a_Page_004.QC.jpg
966e615443616c4dde0a1b8d3121c8cc
66551579cec677a5819f0232dd1c49b9920b1c59
86254 F20110109_AABMKJ afzala_a_Page_178.jpg
d429c0a326bfe4513e6b40ee7f2efbf6
ca493832672d652183f9eec156a46f1b9acc994b
39911 F20110109_AABMJU afzala_a_Page_163.jpg
0a1adecf53f4a45d3ef5da3fd7b91e61
a02f98c1f753a4cfea880b2f806e13daeea8035c
19536 F20110109_AABNOA afzala_a_Page_019.QC.jpg
554a98e40c2ada981757497e6453e094
7ba54605ac41622646ebcaff7afc90a0ec8fef38
11857 F20110109_AABNNM afzala_a_Page_012.QC.jpg
6d513df74a55a42e537d52da86053cfa
87d8f260073c4d762eb4283e54b4e5de793db98f
5053 F20110109_AABNMX afzala_a_Page_004thm.jpg
252e18e0dc4802850d9050595f932a71
1397a1e673adb903700670683b7231f9f5ab2175
91765 F20110109_AABMKK afzala_a_Page_179.jpg
792024cefeccb09c96d0cb9126992c50
08e17de59b114fdd1f7ed43c018dbb87f55ac647
44931 F20110109_AABMJV afzala_a_Page_164.jpg
7566077f6551d2dfaea9121ced2ee882
6fcac7fefcc2f037b40d027f376b1f46fda58487
4886 F20110109_AABNOB afzala_a_Page_019thm.jpg
0ed24bd9f8b4c50f0ae40e361604b41b
5f0efae73cbb94666d4e1015fc75536780f93d49
3371 F20110109_AABNNN afzala_a_Page_012thm.jpg
14e89eaeee3b30cbd16fa5ae422e93aa
ed54fea0904bd537206f1ead3202008c0f94bfbd
17866 F20110109_AABNMY afzala_a_Page_005.QC.jpg
f7679c402716c06a2b30205f8fc67617
f8486ddd4f8a5123beb1ffc03e2176d403d25612
83695 F20110109_AABMKL afzala_a_Page_180.jpg
513f4ff792d5da84e708609b0f28c2ad
43b20c99881be1d2ee54b0a2d2577de705732ac3
63566 F20110109_AABMJW afzala_a_Page_165.jpg
3e3ce7161e623f1bab3004bfdf2e7104
192d819f7c4bf12919d4873bc61cfedb4fa13b32
13873 F20110109_AABNOC afzala_a_Page_020.QC.jpg
614dbd10d8d98ce86b57f0d90f635605
4a36d6e5d98fed5e0cb6eacdf9ff295abf1957b8
13202 F20110109_AABNNO afzala_a_Page_013.QC.jpg
cbc5c691fbb6350cb253356a50ca2f2d
8b6a870b6f9acb9ad05a8b2ef1d1b398070ae89a
4161 F20110109_AABNMZ afzala_a_Page_005thm.jpg
775f8ec55214f429df5ade0afafa623f
9b63cdc91690d917c810fc0605d39a1214fbfd6b
80814 F20110109_AABMKM afzala_a_Page_181.jpg
1b280862c7fb2069edce5b465ae45270
6ad817064996a4b00250a33dd5c5ceec5fc67a40
80713 F20110109_AABMJX afzala_a_Page_166.jpg
01b72661105b542de222fa808361a2e2
42d036f797a0acc2b2a25ffdc8fd7d3703f0b8ac
346946 F20110109_AABMLA afzala_a_Page_011.jp2
00c16fe7d6726fd0606cc7910ca457b4
8d5631996b6a4f27dce428f0ff6e7c22473a13e1
4138 F20110109_AABNOD afzala_a_Page_020thm.jpg
9caaf2d696c87920f7ff63f8e9f6c304
d9fb8525b3433413fcef88e6bebbe939fce8f0e9
3627 F20110109_AABNNP afzala_a_Page_013thm.jpg
7396568b8624ebfa8de3cbda82e63d50
65abca350a20fa3a8943a93d7c7ce42f44763ef4
17373 F20110109_AABMKN afzala_a_Page_182.jpg
049beb3da0d0d73f6d84ca2468d222e4
6c784eb0f070379501b627ea157aa366a95e6a52
82513 F20110109_AABMJY afzala_a_Page_167.jpg
2d3c24b68a10a4a64219f63c0843932c
d50e6048c674eea874c68954ed56c71ed4aeacb9
48711 F20110109_AABMLB afzala_a_Page_012.jp2
22afc8c395a95c09b028e957d9e2674c
1c347f45c92b7cdfc93512747c44ace85a392bb3
24613 F20110109_AABNOE afzala_a_Page_021.QC.jpg
4f5be2edb2a3cf9f52806373ba197a2d
988f8570acd270beec92a0456b7879152fdb0f56
75146 F20110109_AABMJZ afzala_a_Page_168.jpg
a1480b8c6eeca0ef472d979baa795690
fce6efd8b263fb2bc4f849d989aa3330c568e25b
54454 F20110109_AABMLC afzala_a_Page_013.jp2
e099d07457f93c473d039255f152f987
7e2483643f5f261f835a56ab60fbb421aa090641
6256 F20110109_AABNOF afzala_a_Page_021thm.jpg
071debbd38021b009448b9da2a6f35a8
a21543174c2a910e84478a6cefd60e88f569f1a3
13471 F20110109_AABNNQ afzala_a_Page_014.QC.jpg
3bed946225831fbe08a0be1adf0421f2
61ead99b66dd2445dc587b175a3da3062f7ac384
74605 F20110109_AABMKO afzala_a_Page_183.jpg
8f87f5dfa08a6904746a9d0dd209d143
d562995abc5df3f3894e1e7e5c4c362b5a0a457a
53811 F20110109_AABMLD afzala_a_Page_014.jp2
4fc1ebff829553ef28d7c6b1ddba885b
922b4e8081b6f41f8b2fbb629d01054d9ee7f0fd
24112 F20110109_AABNOG afzala_a_Page_022.QC.jpg
bf1bff5ef7687eab9beab0f7d7e8b3fb
f9da916c2d78bb06117d94f38c2af39de938e695
3696 F20110109_AABNNR afzala_a_Page_014thm.jpg
2e090b23de586af517de873ab71be264
85972b76ec1f5802b1c8ee09d2aa5b3d04bd771e
78999 F20110109_AABMKP afzala_a_Page_184.jpg
94008add9437abb1eea1d9ce6c9c7b21
53ce40d43dec50f81532c0cb3342de26ab9b8030
46545 F20110109_AABMLE afzala_a_Page_015.jp2
b173ce5bb55975e3dbea2dd3c4eacf3e
65d00d13f4899cccd5b07c28066ad5a745b84971
6096 F20110109_AABNOH afzala_a_Page_022thm.jpg
351f3dcdb9c1c8c1c8afced0450ff216
4317a2498cdb48eb2f8e6f48b22eed366d0cbf43
11679 F20110109_AABNNS afzala_a_Page_015.QC.jpg
84b69a3600a4d797f089f58e53793434
1ccf4153e265c45e2e8f3ffff99f24f48fe733fa
24690 F20110109_AABMKQ afzala_a_Page_001.jp2
7ba876d04fc4b50a55bb70dc4c8fb177
c16806c20f84fea2693541a55fad19b5431d143e
455494 F20110109_AABMLF afzala_a_Page_016.jp2
e844172a66175e80cb7373665e8029d7
829d4e197c6047d720ec2c47a36765b494a0974b
26252 F20110109_AABNOI afzala_a_Page_023.QC.jpg
494d4afdeb586f32c1138ad1ca973866
a008072e13fa1d1d2d9ed7885c0be67cf27fc088
3410 F20110109_AABNNT afzala_a_Page_015thm.jpg
51aa871e125501f865d42ce6095c1bcf
e2047aa198aa405bed182de664ce68715f25d3d6
4789 F20110109_AABMKR afzala_a_Page_002.jp2
787385f6f908af04b5798e61ace70be6
1d3664811471296d236ac736a4c10e643ba06317
88664 F20110109_AABMLG afzala_a_Page_017.jp2
899d57e08bde3a75f068efe8a92847ad
61a75ec838022bbff49d2063f430f0bce0db337e
6437 F20110109_AABNOJ afzala_a_Page_023thm.jpg
47e2105484703ac54b04bd77e3469584
fd2b75cce71bf59aa21ae3fa9fa9c0e3c3101e0c
11686 F20110109_AABNNU afzala_a_Page_016.QC.jpg
391722edb5b98333afaf6dabc0d2f781
0fccb7f69603ba11e6c38310bb4ba56f270570fa
87424 F20110109_AABMKS afzala_a_Page_003.jp2
c72a99191e38fdb3c19cdc2d39097551
c6778c50ee327cced3f56f09ff0c5c5cb472fa9c
36277 F20110109_AABMLH afzala_a_Page_018.jp2
4980f190716f8030d4a0748fe807357a
2e0d4a4138edb12887d8d9084fc9f03820f0b230
17388 F20110109_AABNOK afzala_a_Page_024.QC.jpg
a4f14503e5cf9d2c74b5a19145ce7789
e0b6db82b137f4d6912531005fabf16720ddab62
3261 F20110109_AABNNV afzala_a_Page_016thm.jpg
f37b531e75a340edcd4887832e664c38
8ea0aef608e5140e301b7ce774d8c003dceee395
84965 F20110109_AABMKT afzala_a_Page_004.jp2
31177435a7eb939afd200bd68ec305d1
35c1cd3c1a68f80c4babda980d543db37489d935
83005 F20110109_AABMLI afzala_a_Page_019.jp2
9fa10ea8515d73bc8eba636517ab2716
5d0d673e95bb4e4c2200bb2a0e6e7d34ddfd8165
4639 F20110109_AABNOL afzala_a_Page_024thm.jpg
d6846c52c24748431b26aefa4c079ae0
c8b9f01fff11ec9f95f008090600a909873fa36c
20793 F20110109_AABNNW afzala_a_Page_017.QC.jpg
fb005fb49d50a36b0e89fa3b93e9ce91
e625883ad8877cc310c6ca6f41be4a9867f8f2d7
1051964 F20110109_AABMKU afzala_a_Page_005.jp2
c4702425211efbefa840b0cb32bb404e
b1a37dd047dc9310f32034c10b1581a5dbf9e643
1051977 F20110109_AABMLJ afzala_a_Page_020.jp2
2a2c84c1d351e003432dbf316b936268
0e0102b68932096bbbcd6ff0988327517a5d9709
4282 F20110109_AABNPA afzala_a_Page_032thm.jpg
f055f1642b5aae6aa1dcd39e2bad2c79
73fb07117eaa65dc578971eb13b8cac63fda27c1
22661 F20110109_AABNOM afzala_a_Page_025.QC.jpg
c4eb52133b77e4c2b10caab5f22ddccc
1d35572b1c137ba455b1f4ee15c6628648fce455
5007 F20110109_AABNNX afzala_a_Page_017thm.jpg
ba79cc2adc83fcc6859b9b132d1f4f16
04cc1f1bec3e98536e4f073df74b731ded0b4fdf
1051962 F20110109_AABMKV afzala_a_Page_006.jp2
2d4e62960f2ba8a123ee7123f86ad3d9
8e23b6701791238664266beda8af040c944aa732
103350 F20110109_AABMLK afzala_a_Page_021.jp2
998ad72600b38ea8fbf8e3606f803e58
4f05a1d013c0b620db9a645d61258a38ecc4c20f


Permanent Link: http://ufdc.ufl.edu/UFE0000624/00001

Material Information

Title: Reduction in pre-tetinal neovascularization by ribozymes that cleave the A2b receptor mRNA
Physical Description: Mixed Material
Creator: Afzal, Aqeela ( Author, Primary )
Publication Date: 2003
Copyright Date: 2003

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0000624:00001

Permanent Link: http://ufdc.ufl.edu/UFE0000624/00001

Material Information

Title: Reduction in pre-tetinal neovascularization by ribozymes that cleave the A2b receptor mRNA
Physical Description: Mixed Material
Creator: Afzal, Aqeela ( Author, Primary )
Publication Date: 2003
Copyright Date: 2003

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0000624:00001


This item has the following downloads:


Full Text












REDUCTION IN PRE-RETINAL NEOVASCULARIZATION BY RIBOZYMES
THAT CLEAVE THE A2B RECEPTOR mRNA


















By

AQEELA AFZAL


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


2003


































To my son, Faris Wasim.















ACKNOWLEDGMENTS

It is a pleasure to thank the many people who have made this dissertation possible.

Many people have been a part of my graduate education as friends, teachers and

colleagues. My mentor, Maria Grant, has been all of those. It is difficult to overstate my

gratitude for her. She has instilled in me the qualities of being a good scientist. Her

infectious enthusiasm for clinical research has been a major driving force during my

career at the University of Florida. This dissertation is a small tribute to an exceptional

woman from a student who is still anxious to learn from her.

My sincerest thanks are also due to Lynn Shaw. He patiently taught me all the

techniques I needed to complete my work. He also spent countless hours editing and

doing the graphics for this dissertation. His insightful comments were crucial for editing

the many drafts into the final dissertation. My thanks are also due to Polyxenie E.

Spoerri who taught me all the tissue culture techniques I needed to complete this

dissertation. Thanks also to Sergio Caballero, Rehae Miller and past and present

members of the Grant lab: Tom Ruzich, Nilanjana Sengupta, Christopher Beadle, Hao

Pan

I would like to thank my committee members: Dr. Don. A. Samuelson (Professor

of Veterinary Medicine); Dr. Dennis. E. Brooks (Professor of Veterinary Medicine); Dr.

John. B. Dame (Professor of Veterinary Medicine); Dr. Donald. A. Armstrong, Dr.

Elizabeth C. Uhl (Clinical Assistant Professor of Veterinary Medicine) and Dr. Harm J.









Knot (Assistant Professor of Pharmacology and Therapeutics) for their guidance over the

years.

My son, Faris Wasim, has been a great source of inspiration. Being tired of not

being able to fulfill his requests when I wanted to and missing him has been the best

motivation for completing this dissertation. My husband, Wasim Asghar, has also shared

this exciting journey with me. He has provided constant support and encouragement

throughout my graduate career.

A very special thanks to the two people to whom I owe everything I am today, my

parents, Mohammed Afzal and Mussarat Afzal. Their unwavering faith and confidence

in my ability and in me is what has shaped me to be the person I am today. Thank-you

for everything. My thanks are also due to my sister, Aneela Afzal, and brother Yaseen

Afzal, for their support and countless hours of babysitting. My family opened their hearts

to me and my little one and made it possible for me to come to work knowing that he was

in good hands.

In addition, I would also like to thank the Department of Pharmacology and

Therapeutics at the College of Medicine at the University of Florida, and the College of

Veterinary Medicine at the University of Florida for their financial support during my

graduate career.

Thanks also to the men and women who donated their eyes to our research. Their

gift has made it possible for us to understand several eye diseases and prevent them in the

future.
















TABLE OF CONTENTS
page

A C K N O W L E D G M E N T S ................................................................................................. iii

LIST OF FIGURES ................................................. ........ ........... viii

LIST OF ABBREVIATION S ............................................................ ......... ...... xii

ABSTRACT ........ .............. ............ .. ...... .......... .......... xvii

CHAPTER

1 BACKGROUND AND SIGNIFICANCE...................... ....... ...... ...............

A n ato m y o f th e E y e ............ .................. ................................................... ...............
T he R etina ........................................................ 4
B lood Supply to the R etina......................................................... ............... 8
T he B lood R etinal B arrier .......................................................................... ....
R etinopathies ................................................... ............. ........... 10
Age Related M acular Degeneration ........................................ ............... 10
D iabetic R etinopathy .................. ................. ...... .. .................... ................ .. 11
Non-Proliferative Diabetic Retinopathy (NPDR) ...............................................11
Proliferative Diabetic Retinopathy (PDR)................. ............................. 15
R etinopathy of P rem aturity .............................................................. ..................... 17
Treatm ent of Retinopathies. ........................................ .......................... 19
Angiogenesis ................................................. ................ ............... 24
Extracellular m atrix (ECM ). ............................................. ............... 26
E CM degradation ..................................... ............... ..... ..... 26
Bound Factors ...................................................................... ......... .................... 30
In te g rin s .................................................................................................. 3 0
E pH receptors. ........................................................................................ 3 1
V ascular endothelial (VE) cadherins.............................. ............... 32
G row th Factors. ................................................................33
A ngiopoeitins ........................................33
Vascular Endothelial Growth Factor .........................................35
Fibroblast Growth Factor ................................................38
Platelet D erived G row th Factor ................................................................... 39
Transform ing G row th Factor- ....................................................... 39
A d en o sin e ...............................................................4 4
A denosine and the R etina ........................................................................ ........... 47



v









Adenosine Receptors ................ .... ...... ...... .......... .. .. ...... ................. 49
Pharmacology of the A2B receptors............... .............................................59
Distribution of the Adenosine Receptors ....................................... ...............60
Intracellular Pathways Regulated by A2B Receptors ......................................62
R ib ozy m es ............................................................................ 64
Self Splicing Introns ........................... ..................... ...............64
G group I Introns .....................................................................64
G group II Introns .................................................................................. 67
R N ase P R N A .........................................................67
Sm all Self C leaving R ibozym es ................................... .................................... 71
H hepatitis D elta V irus ............................................ .. .. .. ...... ........... 7 1
H airpin Ribozym es .................. ............................ .... .. .. .. ........ .... 71
H am m erhead R ibozym es.......................................................... ............... 71
E xperim mental A im ....................... .... .................. .. .. ......... ......... 77

2 M ETHODS AND M ATERIALS ........................................ .......................... 85

Defining Location of the Target Sequence............................................................85
Preparation of the Target Oligo-Nucleotide .................................................... .....86
Time Course of Cleavage Reactions for Mouse and Human Targets (Hammerhead
R ib ozym es) .........................................................................86
M multiple Turnover K inetics ............................................................................... 87
Cloning of the Hammerhead Ribozymes into the rAAV Expression Vector............. 88
Sequencing of the Clones ........... .................... .......... .... ............... 88
Human Retinal Endothelial Cell (HREC) Tissue Culture .......................................89
LDL U ptake of the HREC .................................................................................... 90
Transfection of HREC using DEAE-Dextran...............................................91
Transfection Efficiency using DEAE Dextran for HREC's ............. ...............92
Cell M migration A ssay ......... ....... ....................... ......... ...... .. ........ .... 92
M orphology of H EK C ells................................................... ............................. 93
Transfection using Lipofectamine on HEK 293 cells..............................................93
Transfection Efficiency for HEK Cells using Lipofectamine Reagent ....................94
cAM P Assay on Transfected HEK 293 Cells.................................. ............... 94
Total Retinal RNA Extraction for PCR....... ............ ................. ............. 97
R eal T im e P C R ...................... .. .. ......... .. .. ..... .... ................................97
A n im a ls .................................. ........... ..... ................... .. ....... ................... 9 8
Intraocular Inj section into the Mouse Model of Oxygen Induced Retinopathy ..........98
Statistical A nalysis................................................... 99

3 RESULTS ........ .......... ........................... ..... 100

D eterm ining Accessibility of the Target Site ................................... ... ..................100
Time Course of Ribozyme cleavage.............................. ...............103
M multiple Turnover K inetics ..................... ........................... ....................... ... 106
Cloning of the Hammerhead Ribozyme into an rAAV Expression Vector.............. 109
Sequencing of the C lones ............... ................. ................................................
Cell Cultures ..................... ........ .. ....................... ............... 111









Transfection of HREC ................. ..................................... ............. 114
Transfection Using Lipofectmaine on HEK Cells.......... ................................118
CAM P Assay on Transfected HEK Cells...................................... ............... 121
R eal T im e P C R ...................... ........ .................. .... ....................... ........12 5
Effect of A2B Ribozymes on Neovascularization in the ROP Mouse Model ...........125

4 D ISC U S SIO N ........... ................................... .............. ................ 13 1

Ribozymes As Tools To Study Gene Expression ....................................................132
Delivery Of The Ribozyme In vivo.............................................. ... ............... 135
Prom other C considerations ................................................ .............................. 139
F future Stu dies ...................... .. .. ......... .. .. ..... ..... ................................ 142

5 LIST OF REFEREN CE S................................................. ............................. 147

BIOGRAPHICAL SKETCH ........................... ............................................... 165
















LIST OF FIGURES


Figure pge

1-1 Cross sectional view of the components of the eye ................................................2

1-2 The ten layers of the retina ....................................................................... 6

1-3 A funds shot of ARM D ............................................... .............................. 12

1-4 N on-proliferative retinopathy ...................................................................... ....... 14

1-5 New blood vessel growth around optic nerve in PDR .......... .............. ..........16

1-6 ICROP definition of retinopathy. ................................................... ...................18

1-7 T he five stages of R O P ............................................................................. ............20

1-8 L aser treatm ent of the eye. .......................................................................... ...... 21

1-9 Cartoon showing cryotherapy application to the anterior avascular retina .............23

1-10 The process of angiogenesis......................................................................... ...... 25

1-11 PAs hydrolyze plasminogen to plasmin. ........................................ ............... 28

1-12 Angiopoeitins are ligand for the Tie 1 and Tie 2 receptors............... ................ 34

1-13 The vascular endothelial cell growth factor (VEGF R2) signaling pathway. .........37

1-14 The FGF receptor and signaling pathway. .................................... .................40

1-15 The PDGF receptor and signaling pathway. ................................. .................41

1-16 The TGF- receptor signaling pathway. ...................................... ............... 43

1-17 Intracellular and extracellular production of adenosine.................... ...............46

1-18 Role of the high and low affinity adenosine receptors........................ ...............50

1-19 Homology of the A1 receptor for human and mouse ............................................53

1-20 Homology of the A2A receptor between human and mouse.................. ................54









1-21 Homology of the A2B receptor between the human and the mouse. ........................55

1-22 T h e A 2B receptor.............. .......................... .. .................. ................ ............ 57

1-23. T he A 2A receptor .............. ............................ .............. ... ...... ......... 58

1-24 The A 2B signaling pathw ay. ........................................ ......................................63

1-25 The secondary structure group I introns .................................................65

1-26 Splicing mechanism of the group I introns ....................................................66

1-27 Secondary structure of Group II introns................. ...........................................68

1-28 The splicing mechanism of the Group II introns..........................................69

1-29 Cleavage of the tRNA 5' leader sequence by Rnase P. ..........................................70

1-30 Self-cleaving ribozymes resolve concatemers formed by rolling-circle replication
into individual genom ic m olecules ............................................... ............... 72

1-31 Structure of the hairpin ribozym e......................................................... ... ........... 73

1-32 Structure of the hammerhead ribozyme. ............................................................75

1-33 The hammerhead ribozyme cleaves its substrate by a transesterification
reaction ..............................................................................76

1-34 Cleavage of the A2B receptor by a ribozyme prevents translation of the protein.....79

1-35 Target sequences of the human and mouse A2B ribozymes 1 and 2.......................80

1-36 Hammerhead ribozymes for the A2B Rzl and Rz2.........................................81

1-37 The p21Newhp Vector with the CMV enhancer and beta actin promoter...............82

1-38 Tim e course for the R OP m odel........................................ ........................... 84

3-1 Theoretical tertiary structures of the active A2B Rzl generated by the mfold
program ........................................................................... 10 1

3-2 Theoretical tertiary structures of the active A2B Rz2 generated by the mfold
program ........................................................................... 102

3-3 Time course autoradiograph of a 10% polyacrylamide 8M urea gel showing
products of cleavage of the A2B Rz2 on the mouse target.................. ........ 104

3-4 Tim e course analysis data. ............................................ ............................. 105









3-5 Time course of the ribozyme with increasing target concentrations...................107

3-6 Time course cleavage reaction with varying temperatures (37 C/25C) and
magnesium concentrations of 20mM/lmM. ............. ......... .............108

3-7 Kinetic analysis of the ribozymes. ................................................110

3-8 Sequence of the active and inactive versions of the A2B ribozymes at the site of
insertion within the p21N ewHp vector ........... ........................... ....... ........... 112

3-9 Pebble stone morphology of the HREC .................................... ....................... 113

3-10. LD L uptake of H REC .......................... ..................................... ............... 115

3-11 The GFP plasmid. This plasmid was driven by a CMV enhancer and a chicken
beta actin promoter ........... ............................ ......... ..... .......... 116

3-12 Transfection efficiency of the HREC ...................................... ......... ............... 117

3-13 Theory of m migration assay ..................... ......... ............................ ............... 119

3-14 Migration data for the cells transfected with the active and inactive versions of
the A2B receptor and the vector control. 10% FBS/DMEM is the positive control
and D M EM alone is the negative control ..............................................................120

3-15 Transfection Efficiency of HEK cells. ............. ........................... ..... ............ 122

3-16 HEK cells transfection efficiency following passage 1. ........................................ 123

3-17 cAMP accumulation in HEK cells transfected with the control, active A2B Rz2 and
inactive A2B Rz 2 ...... ....... ..................................... .. ............. 124

3-18 Real time RT-PCR results showing relative levels of the adenosine A2A and A2B
receptor mRNAs isolated from HEK cells transfected with plasmid DNA ..........126

3-19 The mice eyes were embedded in paraffin and three hundred serial sections were
d on e. ........................... ............................................... ........ 12 7

3-20 Injection with the control plasmid prior to exposure to high oxygen shows a high
number of endothelial cell nuclei surrounding blood vessel lumen.....................128

3-21 Injection with the active A2B ribozyme prior to high oxygen exposure significantly
reduced the pre-retinal neovascularization.................................. ...... ............ ...129

3-22 Injection of the active and inactive versions of the A2B Rz2 and the vector control
in the R O P m house m odel........... ......... ...... ......... ........................ ............... 130

4-1 Entry of the AAV and transferring into the cell................. ...............137









4-2 Diagram of the expression cassettes fusion protein and alkaline phosphatase
(A lk Phos). ................ .. ...... .......................................... .................. 141

4-3 A2B signaling pathway with theoretical downstream effects, which have yet to be
confirm ed .......................................................................... 14 6















LIST OF ABBREVIATIONS

a-LDL Acetylated 1,1'-Dioctdycl-3,3,3',3' tetra methyl indocarbocyanin
perchlorate
ABAM Antibiotic antimycotic mix

A1 Adenosine receptor type 1

A2A Adenosine receptor type 2A

A2B Adenosine receptor type 2B

A2B Rzl Adenosine receptor type 2 ribozyme 1

A2B Rz2 Adenosine receptor type 2 ribozyme 1

A2R Adenosine receptor type 2

A3 Adenosine receptor type 3

ADA Adenosise deaminase

AK Adenosine kinase

AMP Adenosine monophosphate

ANG-1 Angiopoeitin 1

ANG-2 Angiopoeitin 2

ARMD Age Related Macular Degeneration

ARNT Aryl hydrocarbon receptor nuclear translocator

ARVO Association for Research in Vision and Ophthalmology

ATP Adenosine triphosphate

avP3 Alpha v beta 3 integrin









avP5

bFGF

BSA

cAMP

CAT

CGS21680

CHA

CHO

CMV

DMEM

DMSO

DNA

DPSPX

DTT

ECM

EDTA

EGS

Eph receptor

FAK

FAT

FBS

Flt

GAGs


Integrin

Basic fibroblast growth factor

Bovine serum albumin

3c, 5c-cyclic monophosphate

Chloramphenicol acetyltransferase

A2A agonist. 2- {4[(2-carboxylethyl)-phenyl]ethylamine}-5'-N-
ethylcarboxamidoadenosine
Cyclohexyladenosine

Chinese hamster ovary cells

Cytomegalovirus

Dubellco's modifeid eagle medium

Dimethyl sulfoxide

Deoxyribonucleic acid

Non-seletive adenosine receptor antagonist. 1,3-dipropyl-8(p-
sulfophenyl)xanthine
Dithiothreitol

Extracellular matrix

Ethylenediamine tetraacetic acid

External guide sequence

Ephrin receptor

Focal adhesion kinase

Focal adhesion targeting sequence

Fetal bovine serum

VEGF fms like tyrosine kinase

Glycosaminoglycans









GC

GCL

GFP

GPI

HBSS

HDV

HEK 293

HIF

HIV

HRE

HRECs

HSPGs

IACUC

IB-MECA

ICROP

ILM

INL

IP3

IPL

KDR

LAP

MMP

NAD


Guanosine cytosine content

Ganglion cell layer

Green fluorescent protein

Glycosylphosphatidylinositol

Hanks balanced salt solution

Hepatitis delta virus

Human embryonic kidney cells

Hypoxia inducible factor

Human immunodeficiency virus

Hypoxia response element

Human retinal endothelial cells

Heparan sulfate proteoglycans

Institution Animal Care and Use Committee.

Selective A3 adenosine receptor agonist. N6 (3-iodobenzyl)Ado-5'N-
methyl Uronamide
International classification of Retinopathy of Prematurity

Inner limiting membrane

Inner nuclear layer

Inositol triphosphate

Inner plexiform layer

VEGF kinase insert domain

Latency associated peptide

Metalloproteinases

Nicotinamide adenine dinucleotide









NBTI Nitrobenzylthioinosine

NECA N-ethylcarboxyamidoadenosine

NFL Nerve fibre layer

NPDR Non proliferative diabetic retinopathy

5'NT 5' Nucleotodase

OLM Outer limiting membrane

ONL Outer nuclear layer

OPL Outer plexiform layer.

PAs Plasminogen activators

PAl-1 Plasminogen activator inhibitor-1

PAI-2 Plasminogen activator inhibitor-2

PBS Phosphate buffered saline

PDGF Platelet derived growth factor

PKC Protein kinase C

PLC Phospholipase C

PDR Proliferative diabetic retinopathy

rAAV Recombinant adeno associated virus

RBCs Red blood cells

ROP Retinopathy of Prematurity

RNA Ribonucleic acid

rRNA Ribosomal RNA

RNasin Ribonuclease inhibitor

RPE Retinal pigment epithelium









R-PIA

SAH

TBS

TGF

Tie 1 and 2

TIMPS

TNF-ac

tPA

tRNA

TR

uPA

VE cadherin

VEGF

VEGF-R1

VEGF-R2

WBCs

XAC

XDH

XO


Selective Al receptor agonist. R-phenylisopropyl-adenosine

S-Adenosylhomocysteine

Tris buffered saline

Transforming growth factor

Angiopoeitin receptors 1 and 2

Tissue inhibitors of matrix metalloproteinases

Tumor necrosis factor alpha

Tissue type plasminogen activator

Transfer RNA

Inverted terminal repeats

Urokinase type plasminogen inhibitor

Vascular endothelial cadherins

Vascular endothelial growth factor

Vascular endothelial growth factor-receptor 1

Vascular endothelial growth-receptor 2

White blood cells

Xanthine amine cogener

Xanthine dehydrogenase

Xanthine oxidase















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

REDUCTION IN PRE-RETINAL NEOVASCULARIZATION BY RIBOZYMES
THAT CLEAVE THE A2B RECEPTOR MRNA

By

Aqeela Afzal

May 2003

Chair: Dr. M.B. Grant
Cochair: Dr. D. Samuelson
Major Department: Veterinary Medical Sciences

Tissue hypoxia and ischemia initiate events that lead to pre-retinal angiogenesis.

Adenosine modulates a variety of cellular functions by interacting with specific cell

surface G-protein coupled receptors (Ai, A2A, A2B, A3) and is a potential mediator of

angiogenesis. The A2B receptor has been implicated in the mediation of angiogenesis. The

lack of a potent, selective A2B receptor inhibitor has hampered its characterization. Our

goal was to design and characterize a hammerhead ribozyme that would specifically

cleave the A2B receptor mRNA and examine its effect on retinal angiogenesis. Active

and inactive ribozymes specific for the mouse and human A2B receptor mRNAs were

designed and cloned in expression plasmids. HEK 293 cells were transfected with these

plasmids, and A2B mRNA levels were determined by quantitative RT-PCR. Human

retinal endothelial cells (HREC) were also transfected, and cell migration was examined.

The effects of these ribozymes on the levels of pre-retinal neovascularization were

determined using a mouse model of oxygen-induced retinopathy. We produced a









ribozyme with a Vmax of 10.8 pmole min1 and a kcat of 36.1 min1. Transfection of HEK

293 cells with the plasmid expressing ribozyme resulted in a reduction of A2B mRNA

levels by 45%. Transfection of HREC reduced NECA stimulated migration of the cells

by 47%. Intraocular injection of the constructs into the mouse model reduced pre-retinal

neovascularization by 54%. Our results suggest that the A2B receptor ribozyme will

provide a tool for the selective inhibition of this receptor, and provide further support for

the role of the A2B receptor in retinal angiogenesis.


xviii














CHAPTER 1
BACKGROUND AND SIGNIFICANCE

The formation of blood vessels is a fundamental process that can be broken down

into two basic pathways. The first is vasculogenesis, which is the formation of new

blood vessels such as seen in embryogenesis. The second is angiogenesis, which is the

formation of blood vessels from pre-existing blood vessels. Angiogenesis is common in

both normal physiological processes (pregnancy, menstruation, wound healing) and

disease states (cancer, retinopathies, psoriasis). The focus of this study is the process of

angiogenesis in retinopathies, including diabetic retinopathy, the leading cause of

blindness in adults, and retinopathy of prematurity (ROP).

Anatomy of the Eye

The two eyes in humans are oriented to facilitate binocular single vision, which

results from the forward position of the eyes and the chiasmal crossing from axons of

ganglion cells. Axons from the right visual field carry impulses to the left optic tract and

vice versa. The eye contains the elements that take in light and converts them to neural

signals. For protection, the eye is located within the bone and connective tissue

framework of the orbit. The eyelids cover and protect the anterior surface of the eye and

contain glands, which produce a lubricating film (tears).1

The globe has three spaces within it: the anterior chamber, posterior chamber and

the vitreous chamber. 1 (Figure 1-1)










conjunctiva


ciliary body
\,
iris
aqueous
humor


vitreous humor
/ retina
optic nerve





macula

choroid


cry stalline
lens


extraocular
muscle


Figure 1-1. Cross sectional view of the components of the eye









The anterior and posterior chambers contain aqueous humour, which is produced by the

ciliary body and provides nourishment for the surrounding structures. The vitreous

chamber is the largest space in the eye and lies adjacent to the inner retinal layer and

contains the gel-like vitreous humor.1

The eye is made up of three layers: an outer fibrous layer, a middle vascular layer

and an inner neural layer (retina).1 The outer fiber layer is a dense connective tissue that

provides protection for structures within, maintains the shape of the eye, and provides

resistance to the pressure of the fluids inside the eye. The sclera is the opaque white of

the eye, and the cornea is transparent and allows light to enter the eye where the lens

refracts it to bring light rays into focus on the retina.1

The middle layer of the eye is made up of three structures. The iris acts as a

diaphragm to regulate the amount of light entering the pupil. The ciliary body produces

components of the aqueous humor and has muscles that control the shape of the lens

during accommodation. The choroid is an anastomosing network of blood vessels with a

dense capillary network.1

The principle functions of the choroid are to nourish the outer retina and to provide

a pathway for the vessels that supply the anterior eye. The choroid is an egress for

catabolites from the retina, which diffuse through Bruch's membrane into the

choriocapillaris. The suprachoroidal space provides a pathway for the posterior vessels

and nerves that supply the anterior segment. 12 The choroid also plays a role in the

maintenance of intraocular pressure due to the high blood flow in its vessels. The

choroid has the largest sized and the greatest number of vascular channels in the eye, and

the amount of blood flowing through these channels at any time has an effect on the









intraocular pressure. The choroid also provides a regular smooth internal surface for the

support of the retina. The smoothness of Bruch's membrane is important in maintaining

the exact relationship between the retinal pigment epithelium (RPE) and the outer

segments of the adjacent photoreceptor rods and cones. 1,3

The Retina

The retina is located between the choroid and the vitreous, and extends from the

circular edge of the optic disc, where the nerve fibers exit the eye, to the ora serrata and is

continuous with the epithelial layers of the ciliary body.1,4 The retina is a thin, delicate

and transparent tissue that lines the inner eye. The neural retina is attached loosely to the

choroid through the pigment epithelium. Externally, the RPE contacts the collagen and

elastic tissue of Bruch's membrane of the choroid. Bruchs membrane is an elastic layer

that stabilizes the RPE and the photoreceptors. Internally, the retina lies next to the

vitreous. Anteriorly, the RPE gives rise to the ciliary body, and posteriorly, all the retinal

layers terminate at the optic disc except the nerve fiber layer. The retina is thickest at the

equator and thins at the ora serrata.1

The retina can be divided into the central retina and the peripheral retina. The

central retina is thick and includes the macula, fovea and foveola. The macula has a

yellow appearance due to xanthophyl (a carotenoid), which is found in the ganglion cells.

The peripheral retina includes the remainder of the retina from the macula to the temporal

or nasal side. The ora serrata is the extreme periphery of the retina. It is the junction

where the retina ends and gives rise to the teeth like processes that form the ciliary body.

The peripheral retina ends at the ora serrata and forms the teeth like processes that form

the base of the ciliary body.1









Under the light microscope, ten layers of the retina can be differentiated (Figure 1-

2): RPE, rod and cone layer, outer limiting membrane (OLM), outer nuclear layer

(ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer

(IPL), ganglion cell layer (GCL), nerve fibre layer (NFL) and the inner limiting

membrane (ILM). The visual pathway consists of three interconnecting neurons and

some receptor cells. The neurons include the bipolar cells, located within the retina; the

ganglion cells, located in the inner retina, the axons of which go through the optic nerve

to the chiasm and end in the lateral geniculate nucleus; and the third neuron is from the

geniculate body to the occipital cortex.1

The rods and cones are the sensory receptors. The outer segments have

photopigments, which are excited by light, resulting in a visual response. The cell bodies

of the rods and cones lie in the ONL and the axons synapse with dendrites of bipolar cells

in the OPL. The dendrites of the bipolar cells extend to the OPL and synapse with axons

of rods and cones; their axons extend to the IPL and synapse with dendrites of the

ganglion cells from the NFL. The INL also has horizontal and amacrine cells. The

horizontal and amacrine cells in this layer provide horizontal integration.1 The RPE is a

single layer of uniform cells. It is located in the outer circumference of the retina and

extends from the edge of the optic disc to the ora serrata. The cells of this layer are

hexagonal shaped and carry a brown pigment. These cells may be multinucleated,

especially in the ora serrata. The RPE provides metabolites to the receptors and removes

the outermost ends of external segments of the photoreceptors. If the RPE cells are

damaged or diseased, these cells are not replaced; instead, adjacent cells slide laterally to

fill the space of the necrotic cells. The RPE cells possess microvilli on










choroid

pithel ium
outer s5egments

inner segments


outer nuclear
layer (ONL)

outer plexiform
layer (OPL)

inner nucle-ar
layer (I NL)

inner plexiform
layer PL)

ganglion cell
layer (CCL)
optic fiber layer
(OFL)


So-------------------o




photoreceptor


Horizontal cell


Figure 1-2. The ten layers of the retina include: retinal pigment epithelium, rod and cone
layer, outer limiting membrane, outer nuclear layer, outer plexiform layer,
inner nuclear layer, inner plexiform layer, ganglion cell layer, nerve fibre
layer and the inner limiting membrane


I II I


_f,%









the apical surface that interdigitate with the photoreceptors. The RPE is critical to

vitamin A metabolism and photoreceptor maintenance.

The rod and cone layer lies external to the OLM. It has a thick inner segment and

thin outer segments joined by a slight constriction. The cell membrane is continuous

between the constrictions. The outer segments have parallel processes, which are short in

cones and long and thin in rods. 1

The OLM under a light microscope has a thin fenestrated membrane-like

appearance. However, the OLM is not a basement membrane. Electron microscopy

revealed it to be a zonula adherens between the photoreceptors and the Miller cells. The

zonula adherens probably serve to keep the highly elongated photoreceptors in place.1

The ONL has the cell bodies of the rods and the cones. The axons of the rods and

cones synapse in the OPL with bipolar and horizontal cells.1

The OPL is a reticular structure, which is a transition zone between the receptors

(neuroepithelial). The OPL is a layer of synaptic contacts between photoreceptors,

bipolar cells and horizontal cells. The axons of the rods end here in spherules (oval

shaped) and those of the cones end in pedicles (broad conical swellings). The spherules

are invaginated and synapse with bipolar dendrites or horizontal cells, and can make 2-4

contacts. The pedicles, on the other hand, contact many dendrites of horizontal cells and

bipolar cells.1

The INL is a band of nuclei belonging to horizontal cells, bipolar cells, and

amacrine cells. The Muller cells provide support and nutrition to the retina. They

surround capillary walls and extend from the ILM to the extracellular membrane.l









The IPL is a junction between the first order neuron (bipolar cells) and the ganglion

cell layer. This layer contains the nuclei of displaced ganglion or amacrine cells and

processes of the Muller cells.1

The GCL contains the cell bodies of ganglion cells, which are thin in the nasal area

and thicker near the macula. The axons of the ganglion cells run internally and then

become parallel to the inner surface of the retina to give rise to the NFL and the optic

nerve fibres.1 The NFL has the axons of the ganglion cells and is thickest around the

optic nerve.1 Branching processes of the Miller cells and a basal lamina like structure

secreted by them forms the ILM. The macula is the center of the retina (area centralis)

and is divided into the fovea (cone dominated), parafovea (ganglion cell dominated) and

the perifoveal retina (single layer of ganglion cells).1

Blood Supply to the Retina

The retina has the highest rate of metabolism of any tissue in the body and thus has

a dual blood supply from the retinal and choroidal capillaries. If either of these sources is

interrupted, ischemia develops and leads to loss of function. The outer retina is supplied

by the choriocapillaris and the central retinal artery supplies the remainder. The retinal

artery is different from other arteries and does not have an internal elastic lamina but does

have a prominently developed muscularis.1'5

The outer retinal layers receive their nutrition from the choroidal capillary bed;

metabolites diffuse through Bruch's membrane and the RPE into the neural retina. The

central retinal artery provides nutrients to the inner retinal layers. The artery enters the

retina through the optic disc, usually slightly nasal of center, and branches into a superior

and inferior retinal artery, each of which further divides into nasal and temporal branches,

and these vessels continue to bifurcate. The nasal branches run a relatively straight









course toward the ora serrata, but the temporal vessels arch around the macular area en

route to the periphery. Two capillary networks exist within the retina. The deepest one

lies in the inner nuclear layer near the outer plexiform layer, and the superficial one is in

the nerve fiber or ganglion cell layer. The OPL is avascular and thought to receive its

nutrients from both retinal and choroidal vessels.1'5

Retinal arterial circulation is terminal; therefore there is no direct communication

between the retina and other vessel systems. The junctions of endothelial cells in retinal

vessels are tight or occluded. Thus, to enter or leave the retina, most substances require

active transport across the endothelial cells. The outer retinal layers receive their nutrition

from the choroidal capillary bed; the central retinal artery provides nutrients to the inner

retinal layers. These vessels are distributed to the four quadrants of the retina.6 The retinal

artery and vein to a particular quadrant supply most of the quadrant. If arterial supply to

a retinal quadrant is interrupted, infarction of that section occurs. The retinal capillaries

supply the inner two thirds of the retina; the choroidal circulation supplies the remaining

outer retina via regulated transport across the pigment epithelium.1'5

The Blood Retinal Barrier

The epithelial portion of the blood-retinal barrier is the retinal pigment epithelium.

This barrier separates the choroidal tissue fluid, which is similar to plasma, from the

retinal tissue fluid. Tight junctions that exist between the endothelial cells of the retinal

vessels and similar tight junctions in the RPE maintain the blood retinal barrier. Thus,

the retinal vessels are impermeable to the passage of molecules greater than 20-30 kDa,

and small molecules such as glucose and ascorbate are transported by facilitated diffusion

through the RPE.1,5,7









Vascular beds are situated to provide nourishment. To avoid problems with the

presence of blood vessels in the outer retina, the outer layers of the retina receive their

nourishment from the choriocapillaris.1

Retinopathies

The retina of the eye is uniquely situated to provide optimal vision. Blood supply

to the retina is also strategically placed to avoid any hindrance of the visual pathway.

Retinopathies (diseases affecting the retina) disrupt this balance and lead to loss of vision.

Retinopathies affecting humans include: age related macular degeneration (ARMD),

which primarily affects the aging population; diabetic retinopathy (DR), which primarily

affects the working population; and retinopathy of prematurity (ROP) which primarily

affects the newborns.

Age Related Macular Degeneration

ARMD is a disease which affects the RPE and leads to blindness in the aged

populations.8 There are two forms of ARMD: dry and wet.9-11

Dry ARMD is characterized by the presence of soft drusen and pigmentary

abnormalities. Drusen is an amorphous acellular debris present within the basement

membrane of the RPE. It is seen as 'yellow' spots within the macula. Low amounts of

drusen are a consequence of age; however, a larger amount present within the retina is

indicative of ARMD.10 Drusen leads to mild vision loss and increases the risk of

progression of the disease to the wet form of ARMD.8'9

The wet form of ARMD (also known as the exudative or the neovascular phase) is

characterized by choroidal neovascularization, RPE detachment and disciform scarring.

The wet form of ARMD leads to rapid vision loss. The choroidal neovascularization









(CNV) leads to the formation of immature blood vessels which result in leakage of serum

and blood and loss of central vision.9'12 (Figure 1-3)

Diabetic Retinopathy

Diabetes Mellitus affects millions of people worldwide and is the leading cause of

blindness in working age adults.13,14,15 There are two forms of diabetes mellitus: Type I,

which typically affects juveniles and is known as insulin dependent diabetes mellitus, and

type II, which is the adult onset form of diabetes and is known as non-insulin dependent

diabetes mellitus.15 Diabetes also leads to systemic complications such as kidney failure,

hypertension and cardiovascular disease.16,13 DR is the most frequent diabetic

complication. Eye problems due to diabetes can be asymptomatic and if left untreated

can lead to serious visual loss. The longer a patient has diabetes, the more likely they are

to develop diabetic retinopathy.13'16'17'15 Diabetic eye disease can be divided into two

phases: background diabetic retinopathy (non-proliferative phase) and proliferative

diabetic retinopathy (PDR).

Non-Proliferative Diabetic Retinopathy (NPDR)

In NPDR small retinal blood vessels are damaged. NPDR is the result of two

major processes which affect retinal blood vessels, vessel closure and abnormal vessel

permeability.13 The vessels leak fluid (edema) and later blood (hemorrhage) into the

retina. Macular edema is the most common cause of reduced vision in patients with non-

proliferative diabetic retinopathy and is seen as milkiness of the retina surrounded with

exudates (yellow clumps). 16,15 These exudates are the result of fat or protein leaking out

of the vessels. Water is quickly reabsorbed into the vessels




















































Figure 1-3. A. A funds shot showing drusen (yellow). B. Wet form of ARMD
showing blood leakage. (National Eye Institute)









or tissue under the retina. However, the fatty material is absorbed very slowly and thus

left behind surrounding the leakage site.16,18 (Figure 1-4)

Vessel closure may be due to blood cell clumping, damaged endothelium, swelling

of an abnormally permeable vessel wall or compression of the capillary by surrounding

retinal swelling. Diabetic patients have closure/non-perfusion of capillaries, which leads

to a decreased oxygen supply. In areas surrounding the area of non-perfusion capillaries

dilate to compensate for the decreased oxygen supply. Small focal dilations

(microaneurysms) of retinal capillaries also develop due to weakened capillary walls,

thus allowing for bulging. 13 When multiple areas of the retina have lost their blood

supply, angiogenic factors are released which stimulate proliferation of new blood

vessels. These new blood vessels are small and fragile, therefore, cause bleeding and the

formation of scar tissue within the retina. Small arterial closures follow capillary closure,

and deprive larger regions of the retina of blood supply. This is seen as 'cotton wool

spots' on the retina in fluorescein angiography.16

Blood vessels in the body are usually fenestrated allowing fluid to pass through

vessel walls. These openings are small enough to allow water and ions to pass through,

while preventing the passage of blood cells and larger proteins. In contrast, retinal blood

vessels have tight junctions between the endothelial cells of blood vessel. Therefore, all

fluids and molecules exiting the vessels have to pass through the cell. This lack of

fenestration helps to keep the retina thin and dehydrated for proper function. These tight

junctions form the blood retinal barrier, which partitions the neural retina from the

circulation and protects the retina from circulating inflammatory cells.16 The tight

junctions are formed by a number of proteins such as: occludin and claudin. These






















































Figure 1-4. A. Non-proliferative retinopathy. Hemorrhage (arrowhead) short arrow
microaneurysm, larger arrow exudates. B. Macular edema. (National Eye
Institute)









proteins limit the flow of fluid between endothelial cells. Diabetic patients have a lower

amount of occludin at the tight junctions in the retinal endothelial cells and can leak

fluid.16

Proliferative Diabetic Retinopathy (PDR).

Proliferative diabetic retinopathy is the stage of diabetes characterized by

angiogenesis on the surface of the retina. 13 Patients can have NPDR for years before

progressing to PDR. PDR is diagnosed by the presence of proliferating blood vessels

within the retina or optic disc. These vessels grow on the retinal surface or into the

vitreous cavity and take on a frond-like configuration as they grow.19 (Figure 1-5) The

new blood vessels form due to the closure of retinal capillaries, which leads to ischemia.

As patches of the retina are deprived of oxygen and nutrients, vasoproliferative factors

are released which diffuse into the vitreous cavity. These factors stimulate growth of

new vessels throughout the retina.15

The new blood vessels are not located in the same location as the ischemically

damaged retina and are very fragile and bleed into the vitreous. A small amount of blood

may be removed in a few weeks and larger blood hemorrhages may take a few months.

If dense blood from multiple recurrent hemorrhages occurs, then vision may not be re-

stored since the residual inflammatory debris and dead cells cannot be removed. Another

complication of PDR is traction retinal detachment. New vessels grow and regress and

lay down fibrous scar tissue, which contracts and shrinks as it matures. If the

neovascularization is on the surface of the retina then contraction of the fibrous scar

distorts the retina. However, if the vessels grow into the vitreous and contract, retinal

detachment occurs which leads to blindness. 13,15



















































Figure 1-5. New blood vessel growth around optic nerve in PDR (Top). Hemorrhage
from new blood vessel growth (Bottom). (National Eye Institute)









Retinopathy of Prematurity

Retinopathy of prematurity (ROP), also known as retrolental fibroplasia, is a

potentially blinding condition affecting the retina of newborns. In the 1950s, it was

associated with the use of high oxygen levels in neonatal units.20 Modem neonatal care

has curbed the incidence of ROP, but because the survival rate of low-birth-weight

infants is increasing, the exposure of surviving babies to high oxygen levels is also

increasing and ROP is still a relevant clinical problem.21'22

ROP causes more blindness among children in the world than all other causes

combined. It begins after removal from high oxygen conditions and may progress rapidly

to blindness over a period of weeks.23 Active growth of the fetal eye occurs between the

last 12 weeks of full term delivery (28-40 weeks of gestation). At 16 weeks of gestation,

blood vessels gradually grow over the surface of the retina. Vessels reach the anterior

edge of the retina and stop progressing at about 40 weeks of gestation.20'21'24'25

The international classification of ROP (ICROP) defines retinopathy by several

distinct criteria: location, extent, stage, and plus disease.26 Location refers to the location

of the damage to the retina relative to the optic nerve. Normally retinal vessels begin

growth at the optic nerve and gradually move toward the edge of the retina. Vessels

further from the optic nerve are more mature. To standardize the location of ROP, the

retina is divided into three zones: Zone I is centered on the optic disc and extends from

the optic disc to twice the distance between the disc and macula; Zone II is a concentric

ring around zone I and extends to the nasal ora serrata (the edge of the retina on the side

toward the nose); and zone III is the remaining crescent of retina on the temporal side

(side towards the temple) (Figure 1-6). The extent of ROP is described by the clock

hours






18


12


Temporal
ora serrata






9-




Macular
center


Nasal ora serrata







tc

Optic nerve


Figure 1-6. ICROP definition ofretinopathy. The retina is divided into three zones:
Zone I, Zone II and Zone III.









of the retina involved in the ROP. For example, if the ROP extends from 1:00 to 5:00,

the extent of ROP is 4 clock hours.24'27

ROP is a progressive disease that begins with some mild changes in vessels and

may progress on to more severe changes. The five stages of ROP describe the

progression of the disease (Figure 1-7). Stage 1 is characterized by a demarcation line

between the normal retina (near the optic nerve) and vascularized retina. In stage 2, a

ridge of scar tissue rises up from the retina due to growth of abnormal vessels. This ridge

forms in place of the demarcation line. In stage 3, the vascular ridge grows due to spread

of abnormal vessels and extends into the vitreous. Stages 4 and 5 refer to retinal

detachment; stage 4 refers to a partial retinal detachment caused by contraction of the

ridge, thus pulling the retina away from the wall of the eye; and stage 5 refers to complete

retinal detachment. Plus disease is a very severe form of ROP which is characterized by

the abnormal growth of blood vessels near the optic nerve.24'28

Treatment of Retinopathies.

Spot laser photocoagulation is used for the treatment of ROP.29 This uses an

argon/diode laser to burn spots on the peripheral and middle portions of the retina. When

laser light hits blood or pigment, it is absorbed as heat energy and produces a small burn.

The laser treatment leads to a decrease in the level of vasoproliferative factors produced

by the ischemic retina. The avascular retina is treated using a small laser spot (Figure 1-

8). The laser spot directly treats the retina and the underlying tissue, thus reducing

inflammation and results in less damage to other ocular structures. Destruction of small

patches of the ischemic retina reduces the oxygen demand and decreases the

vasoproliferative factor production. Laser treatment also thins the pigmented tissue under

























Stage 1 ROP is characterized by a demarcation Stage 2 ROP; the white line is replaced by a
line. The orange vascular retina is on the left ridge of scar tissue R. The arrow shows a tuft
and the gray peripheral retina is on the right of new vessels.
separated by the white line.


Stage 3 ROP. The size of the ridge has Plus disease shows dilation and tortuosity of
increased (between arrows) and the growth of blood vessels near the optic nerve.
the ridge extends into the vitreous.


Figure 1-7. The five stages of ROP (National Eye Institute)

































Figure 1-8 Laser treatment of the eye. The laser spot directly treats the retina and the
underlying tissue. Laser treatment thins the pigmented tissue under the retina
and allows more oxygen to diffuse in from the vessels under the retina









the retina thus allowing better oxygen diffusion in the retina. Laser treatment increases

oxygen supply, lowers the demand for oxygen and lowers the incidents for new vessels

growth.30,28 Laser photocoagulation also causes less pain than other therapies. Currently

laser treatment is the best option for the treatment of retinopathies.14,31,29,32

Cryotherapy is also one of the treatments available for the treatment of

retinopathies.33'34 This technique involves placing a cold probe on the sclera until an ice

ball forms on the retinal surface. Multiple applications are done to cover the entire

vascular area (Figure 1-9). This thins the tissue under the retina (by destroying it) and

allows easier oxygen diffusion through the retina.31 Due to the pain involved in

cryotherapy, anesthesia has to be administered which is a risk factor for premature

infants. If no anesthesia is administered, cardiac arrest follows. Another complication is

hemorrhage due to excessive bleeding.31'33'35'36

If laser photocoagulation or cryotherapy is unsuccessful, a scleral buckle may be

used.37 This involves surgery and is used if there is shallow retinal detachment due to the

contraction of the ridge. A silicone band is tightly placed around the equator of the

eye thus producing a slight indentation on the inside of the eye.38 This indentation

relieves traction of the vitreous gel and allows the retina to flatten back onto the wall of

the eye. The silicone band is then removed a few months later to allow the eye to

grow.14,31,39 If the scleral buckle is not sufficient, vitrectomy may be performed. Small

incisions are made into the eye, the vitreous removed and replaced by saline. This

technique also has had limited success. Current available therapies for the different types

of retinopathies have had limited success. The underlying cause of retinopathies is

angiogenesis































Figure 1-9. Cartoon showing cryotherapy application to the anterior avascular retina. A
cold probe is placed on the sclera till an ice ball forms on the retinal surface.
Multiple applications are done to cover the entire vascular area. This
treatment thins the tissue under the retina and allows easier oxygen diffusion
through the retina









(abnormal blood vessel formation). Development of other effective therapies for the

diseases requires an understanding of the process of angiogenesis.28,33,40-42

Angiogenesis

Vasulogenesis is the formation of new blood vessels. Precursor cells (angioblasts)

differentiate into endothelial cells which later link to form blood vessels. Angiogenesis

on the other hand is the sprouting of blood vessels from pre-existing blood vessels.43 The

vasculature of the retina undergoes both vasculogenesis and angiogenesis. The

superficial retinal vessels, which originate at the optic disc, are formed by the process of

vasculogenesis and the process of angiogenesis later forms the capillary beds.

The process of angiogenesis involves endothelial branching, sprouting, migration,

proliferation and anastomosing with endothelial cells in existing vessels.43-45 (Figure 1-

10) Vascular endothelial cells form a monolayer throughout the entire vasculature. They

are polarized cells with an apical surface and a basal surface, which is surrounded by a

basal lamina.46-49 Mural cells wrap around this structure and are contractile cells, which

regulate vessel diameter and consequently blood flow.47'50 On large vessels they are multi

layered and referred to as smooth muscle cells. On capillaries mural cells are sparse and

usually referred to as pericytes.48'50 The extracellular matrix, bound factors and the

soluble growth factors all play an important role in the process of angiogenesis.















Endothelial cells




VEGF
Capillary formation
EC proliferation
EC migration
EC protease production







Pericvles-

C


PDGF-B
PDGF-B Recruitment of

-"-* pericytes


VEGF

rGF-pl Differentiation

'Ang1 Stabilization


?- -- -- -- -- -- --U


-T I


ri


* +Angl
Stabilization


+Ang2
+VEGF

Sprouting


+Ang2 -
VEGF

Regression .,:

', '
.m
I'r


-AngI
% Destabilization




St.

4


Figure 1-10. The process of angiogenesis. The process of angiogenesis involves
endothelial branching, sprouting, migration and proliferation. Vascular
endothelial cells form a monolayer throughout the entire vasculature.
Pericytes wrap around these cells.









Extracellular matrix (ECM).

The ECM surrounds and provides mechanical support for blood vessels. The

activated endothelial cells cerate gaps in the basement membrane, which allows them to

sprout into the ECM. The ECM is composed of two compartments: the interstitial

matrix and the vascular basement membrane.51'52

The interstitial matrix consists of fibrillar collagen and glycoproteins (e.g.

fibronectin, laminin). Fibronectin attaches cells to a variety of ECM components, and

laminin anchors cell surfaces to the basal lamina. Collagen provides structural support, is

synthesized by fibroblasts and is the most abundant protein comprising the ECM. There

are 12 types of collagen and types I, II and III are the most abundant types of collagen in

the ECM.51'52 The vascular basement membrane lies between the endothelial cells and

pericytes. It is composed of type IV collagen, which forms the basal lamina upon which

the endothelium rests, and heparan sulfate proteoglycans.51

Proteoglycans are glycosaminoglycans (GAGs) linked to proteins. Cell surface

heparan sulfate proteoglycans (HSPGs) function as endothelial cell receptors that

recognize the ECM. They are present in the basement membranes and cell surfaces.

These proteoglycans modulate the response of endothelial cells to basic fibroblast growth

factor (bFGF), vascular endothelial growth factor (VEGF) and other heparan binding

angiogenic factors by sequestering these molecules in the ECM. Heparatinases trigger

the release of these growth factors from the ECM and make them available for

angiogenic stimuli.51,53

ECM degradation

Endothelial cells degrade the surrounding ECM by the release of plasminogen

activators (PAs) and matrix metalloproteases (MMPs).54 The PAs hydrolyze plasminogen









to plasmin, which is a general protease that can digest most proteins. (Figure 1-11) It

also converts latent collagenase into active collagensase which can then degrade collagen

type I, II and III. 54 There are two types of PAs: tissue type PA (tPA) and urokinase-type

PA (uPA).55 Both PAs utilize the same substrate, plasminogen, and both have two

specific inhibitors, plasminogen activator inhibitor-1 (PAl-1) and plasminogen activator

inhibitor-2 (PAI-2). PAI-1 is produced by endothelial cells to inhibit PA activity to

ensure a balanced degradation of the ECM. uPA and PAI-1 are also upregulated by

angiogenic factors such as basic fibroblast growth factor (bFGF) and vascular endothelial

growth factor (VEGF)54-56

Matrix metalloproteases (MMPs) are zinc dependent endopeptidases which are

secreted as zymogens and proteolytically activated by other MMPs or plasmin.51 MMP

expression may also be regulated by growth factors such as VEGF, bFGF and TGF-1.51'56

MMPs degrade components of the ECM and are subdivided into: collagenases,

stromelysins (cleave laminin and fibronectin), matrilysins, gelatinases (cleave collagen

type IV), membrane type (MT) MMP and other MMPs. Endothelial cells, smooth muscle

cells and fibroblasts produce collagenase 1 (MMP-1), stromelysin (MMP3), gelatinase A

(MMP-2), gelatinase B (MMP-9), matrilysin (MMP-7), and MT1-MMP (MMP-14,

which has fibrinolytic activity).54'55












(PAI) -


tPA
uPA



--\


Plasminogen Plasim P
PA




ro-Plasminogen


/


MMP



Angiostatin


Fibin
Degradation


Figure 1-11. PAs hydrolyze plasminogen to plasmin. Plasmin subsequently activates
matrix metalloproteases, which degrade the extra cellular matrix.
PA=plasminogen activator; uPA=urokinase type PA; tPA=tissue type PA;
PAI=plasminogen activator inhibitor; MMP=matrix metalloproteases;
TIMPs=tissue inhibitors of MMPs


pro-MMP


positive
feedback


pi


MMP


TIM
TIMPS


ECM
Degradation









Endothelial cells also produce tissue inhibitors of MMPs (TIMPs) that are specific

inhibitors of MMPs and modulate the degradation of the ECM. TIMPs are secreted

proteins, which inhibit MMPs in a 1:1 stoichiometry. They reversibly interact with the

catalytic domain of the MMPs to inhibit their activity. TIMPs differ in their ability to

interact with various MMPs. For example, TIMP2 inhibits MT-MMP and TIMP3

inhibits MMP9. TIMPs also bind to the heparan sulfate proteoglycans in the ECM and

concentrate them to the specific regions within the tissue. 56

Angiostatin and endostatin are naturally occurring anti-angiogenic molecules,

however, they are also produced by proteolytic cleavage by MMPs from the pro-forms of

plasminogen and collagen XVIII, respectively.5 The production of MMPs, however, is

cell and tissue specific. For example, bFGF and VEGF upregulate interstitial collagenase

(MMP-1) and also increase the formation of plasmin. Plasmin converts the inactive form

of MMP-1 to the active form. Gelatinase A (MMP-2) is upregulated by calcium influx.

It is responsible for the angiogenic switch and for the differentiation of the endothelial

cells into tubes. MMPs promote capillary tube formation, however, at high

concentrations, they have an opposite effect.

ECM degradation produces fragments, which have the opposite effect of the intact

molecule. For example, hyaluronan, a GAG found in the ECM, has anti-angiogenic

properties. However, when cleaved, it enhances the action of angiogenic factors.

Conversely, proteolytic degradation of fibronectin, plasminogen and collagen produces

fragments, which have both anti-angiogenic and angiogenic activity. MMP2 undergoes

proteolysis to produce PEX, which is the C-terminal non-catalytic domain of MMP2.

PEX is anti-angiogenic and inhibits the gelatinolytic activity of MMP2.









Bound Factors.

Activated endothelial cells are anchorage dependent for survival. In addition to

degradation of the ECM, the endothelial cells require bound factors to help in migration

towards the ischemic stimulus. These bound factors include integrins, Eph receptor

Eph/Ephrins complexes, and VE cadherins

Integrins.

Integrins provide the scaffolding for the cells to migrate upon and are used by the

endothelial cells to recognize the ECM.5 Integrins play a role in regulating cell growth,

differentiation and survival.59-63

Integrins are cellular receptors for ECM proteins and are expressed by all adhesive

cells.64 Integrins are composed of a and 3 chain heterocomplexes, which are integral

membrane glycoproteins. They have long extracellular domains, which are the ligand

binding regions. Eighteen different a subunits, and 8 3 subnits have been identified.

These subunits can associate in 24 known combinations. A short transmembrane region

follows the short intracellular domains of both the a and the 3 subunits and the

cytoplasmic tail of the beta subunit links the integrins to cytoskeletal actin of the

endothelial cell.62,63,65

Integrins are linked intracellulary to actin filaments by specific actin binding

proteins, such as Talin, alpha actinin, vinicluin and paxillin.66 Focal adhesion kinase

(FAK) is a protein with tyrosine kinase activity and is composed of a large kinase domain

flanked by an amino and carboxyl terminus. A region of the c-terminus, known as focal

adhesion targeting sequence (FAT) recruits FAK to paxillin. Integrin mediated cell

adhesion occurs when FAK is tyrosine phosphorylated.58









avP3 has a well-characterized role in angiogenesis. It mediates adhesion of cells to

vitronection, fibronectin, von Willebrand factor, osteopontin, tenascin and

thrombospondin. Although the v3Ps integrin is minimally expressed on normal resting

blood vessels, it is significantly upregulated in newly formed blood vessels within

tumors, in healing wounds and in response to certain growth factors. vPs3 expression is

upregulated in endothelial cells exposed to angiogenic factors and those exposed to

hypoxia. Integrins also help to target the activity of the MMPs, for example, avPs3

interacts with MMP-2 and also regulates signaling via the vascular endothelial growth

factor receptor -2 (VEGF-R2). Natural components of the ECM, such as, endostatin,

angiostatin, thrombospondin and tumastatin are all anti-angiogenic and exert their effect

by binding to the avP3 intergrin and disrupting the endothelial cell-ECM interaction. If

this integrin is disrupted using an antibody (LM609) or a peptide antagonist (cyclic

peptide 203, RGDfv), it results in the disruption of angiogenesis progression. VEGF and

bFGF are capable of inducing the expression of av3P integrin of endothelial cells.67'68

EpH receptors.

To discriminate cell partners from fibroblasts or inflammatory cells, the Eph

receptor is utilized. The Eph receptor is the largest receptor of the receptor tyrosine

kinase family (RTK) family.69'70 The receptors are divided based on ligand affinity into

class A and class B. The extracellular domain of the Eph receptor consists of the ligand

binding globular domain, cysteine rich region and 3 fibronectin type II repeats. The

cytoplasmic portion of the receptor consists of ajuxtamembrane domain, and a carboxyl

terminus. The ligands for these receptors are ephrins, which are also divided into

subclass A and subclass B. Ephrins subclass A are anchored to the plasma membrane by









glycosylphosphatidylinositol (GPI) anchor and ligands A1-A5 have been identified.

Ephrins subclass B have a short transmembrane domain and a short cytoplasmic tail.

Only three subclass B ephrins have been identified (B 1-B3). Both the receptor and the

ligands are membrane bound and therefore a signal is transduced in the receptor

expressing cells and the ligand expressing cells.70'71

Prior to cell-cell contact, the Eph receptor and ehprins ligand are loosely clustered

at the cell surface. Following cell-cell contact, the receptor and ligand heterodimerize

and tetramerize. These receptors are capable of bi-directional signaling (forward and

reverse signaling). Eph A receptor enhances the adhesion of cells and the number of

focal adhesion points and is known to be involved in forward signaling. Eph receptor B

is phosphorylated in the intracellular domain and is known to be capable of both forward

and reverse signaling.72

Vascular endothelial (VE) cadherins

Endothelial cells express at least three cadherins: N-, P and VE cadherin. N-

cadherin is diffusely spread across the cell, P cadherin is present in trace amounts and VE

cadherin is specifically localized to inter endothelial cell junctions. Beta catenine and

plakoglobulin are anchored to the cadherin through actin and a catinine. VE cadherin

mediates contact inhibition of endothelial cells by decreasing the amount of proliferation

and allows endothelial cell monolayers formation in the vessel wall. VEGF increases

endothelial cell permeability by phosphorylation of a tyrosine residue of VE cadherin.

This phosphorylation leads to dissociation of the VE cadherin and translocation of the

beta catenine/plakoglobulin complex to the nucleus to regulate gene transcritption.73-75









Growth Factors.

The process of angiogenesis requires co-ordination of several growth factors, which

play distinct roles in the process, examples include: angiopoeitins, vascular endothelial

growth factor (VEGF), fibroblast growth factor (FGF), platelet derived growth factor

(PDGF) and transforming growth factor (TGF).

Angiopoeitins

Angiopoeitins are secreted ligands for the two Tie receptors: Tie 1 and Tie2 (Tek).

Both receptors are endothelium specific,73 and have an extracellular domain composed of

two immunoglobulin like folds and three fibronectin repeats. The cytoplasmic region has

a tyrosine kinase domain interrupted by a short kinase insert.

Angiopoeitin-1 and angipoeitin-2 are ligands for the Tie receptors. Both ligands

can bind the receptors, however, only ANG1 can phosphorylate the receptor.76 ANG-2

inhibits Tie2, detaches pericytes and loosens the matrix surrounding the vessel. ANG-1

does not initiate endothelial network organization it stabilizes networks initiated by

VEGF by enhancing the interaction between endothelial cells and pericytes. Binding of

ANG1 to the Tie 2 receptor initiates cell survival through the PI3 kinase, Akt pathway.

Akt leads to the upregulation of survivin, which is an apoptosis inhibitor.

Phosphorylation of Tie2 leads to the phosphorylation of Dok. Dok then activates the Ras,

Nck, and Crk pathway, which are involved in cell migration, proliferation and

organization of the cytoskeleton. Molecules interacting with the Tie2 SH2 domains are

Grb2, SHP2 which modulate cell growth, differentiation, migration and survival.76

(Figure 1-12)









)


Organization Proliferation Endothelial Endothelial
of Cell Migration Cell Survival
Cytoskeleton

Figure 1-12. Angiopoeitins are ligand for the Tie 1 and Tie 2 receptors. Binding of
angiopoeitin 1 to the Tie 2 receptor leads to endothelial cells proliferation,
migration and cell survival. Angiopoeitin 2 inhibits Tie 2. ANG-
l=angiopoeitin 1; ANG-2=angiopoetin 2.









Vascular Endothelial Growth Factor

VEGF is a heparan binding potent endothelial cell mitogen. It promotes

endothelial cell survival via activation of the phosphatidylinositol 3-kinase (PI3K/Akt)

pathway and inhibits apoptosis.77 VEGF undergoes alternative splicing to produce 5

known isoforms: VEGF 121, VEGF 145, VEGF 165, VEGF 189 and VEGF 206.73 The

isoforms differ in storage in the ECM and their extracellular pathways.78 VEGF 121 and

VEGF 165 are secreted extracellularly, whereas VEGF 189, VEGF 206 and possibly

VEGF 165 are either cell or matrix associated due to their affinity for heparan sulfate.

VEGF is a mitogen for endothelial cells and each isoform has varying effects during

angiogenesis:73 VEGF 189 decreases lumen diameter, 121 and 165 increase lumen

diameter and increases vessel length. VEGF 165 binds to the ECM and releases bFGF

stored in the ECM, thus, bFGF and VEGF have a synergistic angiogenic effect. 78

There are three tyrosine kinase receptors for VEGF: VEGF R1 (Fltl), VEGF R2

(KDR/Flk-1) and VEGF R3 (Flt3).73 The receptors all have seven immunoglobulin like

extracellular domains, a transmembrane domain and an intracellular tyrosine kinase

domain, which is interrupted by a kinase insert.78 VEGFR1 and VEGR2 transduce

different signals to endothelial cells. VEGFR1 promotes cell migration and VEGFR2 is

mitogenic for the endothelial cells and also promotes migration. 73 Hypoxia upregulates

VEGFR1 and induces the expression of VEGF by endothelial cells. The increased

production of VEGF activates the VEGFR2 receptor phosphorylation and cell

proliferation. 78 Ligand binding to VEGFR1 leads to the activation of the small adaptor

proteins: Fyn, Yes and GAP. Ligand binding to VEGFR2, however, leads to

phosphorylation of the SHP-1 and SHP-2 adaptor proteins and PLC-gamma. PLC

gamma hydrolyzes phosphatidyl inositol 4,5-bisphosphate (PIP2) to form inositol









triphosphate (IP3) and diacylglycerol (DAG). The DAG remains associated with the

plasma membrane and activates protein kinase C (PKC). PKC is a soluble cytosolic

protein, which is activated by the increase in calcium concentration. Activation of PKC

leads to cell proliferation and permeability. VEGFR2 also leads to the activation of the

PI3 kinase/Akt pathway, which enhances cell survival. VEGFR2 plays a role in cell

migration by recruiting FAK. The MAPK pathway is also activated through Grb2, which

is an SH2 adaptor protein. It has two SH3 domains, which bind the guanine nucleotide

exchange factor SOS. SOS then leads to the activation of RAS. Activated RAS binds to

the N-terminal of RAF which phosphorylates MEK and phosphorylates MAP kinase.78

The activated MAPK pathway then leads to the activation of the intranuclear proteins

such as cyclin D which is important in the progression of the cell cycle from the G1

phase to the S phase.79 (Figure 1-13) VEGF also increases vascular permeability and

allows leakage of plasma proteins, formation of the ECM and upregulates the production

ofuPA and tPA and PAI-1 by endothelial cells. VEGF production is regulated by local

oxygen concentrations. Hypoxia upregulates production of VEGF by binding to the

hypoxia inducible factor (HIF).73

During retinal development astrocytes and neuronal precursors migrate away from

existing blood pre-existing blood vessels. As the distance between the astrocytes and the

pre-existing blood vessel increases, the astrocytes sense a state of hypoxia. Astrocytes

are more sensitive to hypoxia than neuronal cells and thus the astrocytes upregulate the

production of VEGF, which leads to angiogenesis. This upregulation of VEGF by the

astrocytes creates a concentration gradient of VEGF. This stimulates blood








VEGF


- DAG


PKC





Cell Proliferation
Vasopermeability


PLCy


I


0


E VEGFR



A) 1
P13K
SPaxillin

AKT
Pathway Cytoskeloton
Rearrangement
Cell Migration
Cell
Survival




S Angiogenesis


Ras

\
MAPK
Pathway



Gene Expression
Cell Proliferation


Figure 1-13. The vascular endothelial cell growth factor (VEGF R2) signaling pathway.
VEGFR2 activates several pathways all of which lead to angiogenesis.


I









vessel formation towards the astrocytes, which produce VEGF. In ROP babies are

placed in a high oxygen incubator because their lungs are not fully developed. The

hyperoxia inhibits the VEGF production by the astrocytes thus causing newly formed

blood vessels to regress. Once the babies are taken out of the incubator all the cells of the

retina sense hypoxia and upregulate the production of VEGF. This leads to abnormal

angiogenesis and unregulated blood vessel growth.78

Fibroblast Growth Factor

Fibroblast growth factor (FGF) is ubiquitously expressed as either basic FGF or

acidic FGF. FGF is either in the cytoplasm or bound to the ECM due to its intrinsic

affinity for heparan.73 FGF binds to four related receptors, which are expressed on many

cells. Ligand binding induces receptor dimerization. Endogenous heparan sulfate in cells

is required for the activation of FGF. The receptor for FGF has three immunoglobulin

like folds; two intracellular tyrosine kinase domains, a short transmembrane region and a

juxtamembrane domain, which is longer than any other receptor.80 The intracellular

domain has two phosphorylation sites.81 Ligand binding to the FGF receptor induces

tyrosine phosphorylation of an adaptor molecule, FRS2. The phosphorylated FRS2 then

allows binding of a small adaptor molecule GRB2. GRB2 is involved in the activation of

the GTP binding protein Ras. Since FRS2 does not have an SH2 domain, another adaptor

molecule, SHP-2 associates with FRS2 alpha in the active FGF receptor.81 The

importance of the association of this molecule with the FRS2 is not well defined. GRB2

exists with SOS, which catalyses the exchange of GDP for GTP on Ras for activation.

Therefore, SOS, facilitates the coupling of GRB2 to Ras.81 The activated MAPK then

leads to the activation of the intranuclear proteins such as cyclin D which progresses the

cell from the G1 phase to the S-phase.79 (Figure 1-14) This growth factor induces









processes in endothelial cells and stimulates proliferation and migration of endothelial

cells and pericytes, and production of PA by the endothelial cells. bFGF plays a role in

blood vessel remodeling by stimulating endothelial cells to form tube like structures.73'82

Platelet Derived Growth Factor

Platelet derived growth factor (PDGF) is a mitogen for smooth muscle cells73 and

potent chemoattractant factor for smooth muscle cells, monocytes and fibroblasts. PDGF

is a dimer consisting of two polypeptide chains: A and B. These chains combine to form

3 PDGF isoforms of PDGF AA or BB or heterodimers of PDGF AB.73'83 The PDGF

receptor consists of a single transmembrane domain which has intrinsic kinase activity.83

The receptor is also a dimeric mixture of the alpha and beta subunits.73 Ligand binding

induces receptor dimerization and transphosphorylates tyrosine residues in the

cytoplasmic domain of the receptor.83 Endothelial cells express the beta receptor and are

stimulated by PDGF-BB.73'83 PDGF-BB acts through the MAPK/ERK pathway to

stimulate c-jun/c-fos related genes in the nucleus to stimulate proliferation.83 PDGF also

acts through the PI3kinase pathway to activate PKB, which stimulates cell survival and

proliferation. PDGF also plays a role in angiogenic chords formation and stimulates

sprout formation. PDGF also mediates proliferation and migration of pericytes along

angiogenic sprouts.73 (Figure 1-15)

Transforming Growth Factor-P

Transforming growth factor beta (TGF-P) is produced by almost all cells and thus

its activation represents an important control mechanism.84 TGF-3 is hydrolyzed















FGFR


S FGF




Grb2 Sos

TK 1 Ras/Raf/MEK/MAPK pathway
TK1 \
Shp2

TK 2 Gene transcription in the nucleus



Figure 1-14. The FGF receptor and signaling pathway. Ligand binding to the FGF
receptor leads to tyrosine phosphorylation of adaptor molecules and activation
of the MAPK pathway. The MAPK pathway leads to endothelial cell
proliferation, and migration.





41


PDGF-R


I ,, ,I II i I u I u i i, i lUI
I, I h III i IIIII I i h, I I0-I lh






P13-K

/
SPKB

Survival


40 Grb2
SO


Sos







Ras-ERK


SProliferation


Figure 1-15. The PDGF receptor and signaling pathway. The PDGF receptor acts
through the MAPK pathway to stimulate proliferation and also stimulates
endothelial cell survival through the PI3-kinase/PKB pathway.









intracellularly by a furin peptidase to produce the carboxyl terminal peptide.73 This

peptide associates with the amino terminal to form the latency associated peptide (LAP).

The LAP dimerizes to form the mature TGF-3 which is then secreted in the inactive

form.73 Plasmin activates the latent complex. TGF-3 also produces Pal-1 which inhibits

plasminogen. Thus, showing that the action of TGF-P is self limiting.85

There are three different types of TGF-3 receptors designated, I, II and III. TGF-P

binds directly to the TGF-PII receptor. Binding of the II receptor is followed by the

recruitment of the TGF-3 I receptor. Both the receptors then form a stable complex and

receptor II then phosphorylates receptor I which induces the signal cascade of the

receptor.85 Once the TGF-3R2 is bound to the TGF-P31 receptor, the kinase activity of

receptor 1 is activated. This leads to the recruitment and the accumulation of the Smad

proteins, which are then phosphorylated by the receptor. The name SMAD is derived

from the genes encoding them. The genes were first identified in drosophila and C.

elegans. The drosophila gene was named MAD (mother against decapentapleigic) and

the gene from C. elegans was named SMA (small body size).86'87 (Figure 1-16)

TGF-P is a bifunctional regulator. At low levels, TGF-P stimulates angiogenesis,

and at high levels it inhibits angiogenesis.85 TGF-3 is found in the ECM, on endothelial

cells and on pericytes. It supports the anchorage independent growth of fibroblasts.73

TGF-P also controls cell adhesion by regulating the production of ECM and integrins.

Endothelial cell migration and formation of tube like structures are regulated by TGF-3.

TGF-P also upregulates the production of TIMPS, thus has anti-proteolytic activity. 73

TGF-P inhibits endothelial cell proliferation73 by blocking the effect of other mitogenic

growth factors and enhances pericyte differentiation. It helps to form the vessel

















TGF-P



TGF-P
Type I Type I Recept




\\ mad2

Smad2
Smad4' j I


Angiogenic Blood
Vessel Growth
4


Cell Growth
Cell Mobility
Angiogenesis


Smad2 Smad4
Smad2 Smad4


Smad4.Smad2


Figure 1-16. The TGF-3 receptor signaling pathway. Ligand binding to the TGF- 3
receptor II leads to the recruitment of TGF- P receptor I. The activated
receptor recruits the Smad proteins and stimulates angiogenesis at low levels
and inhibits angiogenesis at high levels of TGF-3 .


Arrested
Growth









wall by stimulating the production of the extracellular matrix, strengthens the vessel wall

and has matrix modulating effects and also stimulates tube assembly.73'87

Angiogenesis is a complex process, which involves the extracellular matrix, bound

factors and soluble factors. Of the soluble factors, VEGF plays an important role in the

early phases of angiogenesis. VEGF is an important mediator of compensatory

angiogenesis and is a potent mitogen induced by hypoxia and nucleosides such as

adenosine. 53,88,89 However, even though the angiogenesis process may solve the

nourishment aspect of the outer retinal layers if the choriocapillaris was impaired, it

would still cause vision impairment.90,90-94

Tissue hypoxia and ischemia initiate a series of events which lead to the

development of collateral blood vessels, followed by compensatory angiogenesis, which

is detrimental and results in aberrant blood vessels that are friable and prone to

bleeding.92'95 Mediators of compensatory angiogenesis include VEGF, which is a potent

mitogen induced by hypoxia and nucleosides such as adenosine.95-97 Depending on the

character of the ischemic stimulus, adenosine plays two roles: as an intracellular

signaling factor which promotes neovascularization following chronic hypoxia or

ischemia, and as an endogenous protective factor which is capable of protecting the retina

from acute ischemia. Adenosine also upregulates VEGF in retinal endothelial cells.

Therefore, adenosine may be a critical signal in the control of gene expression after

retinal ischemia.91'98'99

Adenosine

Adenosine is an endogenous nucleoside, which modulates many physiological

processes such as cardiac myocyte contractility, modulation of neurosecretion and

neurotransmission, cell growth and gene expression, regulation of intestinal tone and









control of vascular tone.100 Adenosine serves as a signal to increase energy supply and

demand by affecting cellular metabolic rates and tissue perfusion. Metabolites of

adenosine may also have significant physiological and pathological effects. The level of

adenosine available for these effects is determined by a number of factors including the

rate of production, transport and metabolism.100

Stimuli that mediate the local production of adenosine include hypoxia, ischemia

and inflammation. The endothelium is a barrier to adenosine, thus the adenosine formed

within the lumen of the blood vessels may be derived from nucleotides released from

platelets or endothelial cells. Ischemic parenchymal cells or nucleotides derived from

nerves or intestinal mast cells give rise to interstitial adenosine. This adenosine may

produce vasodilation via the A2A receptor on vascular smooth muscle cells, which are

especially accessible to the interstitial nucleoside. Adenosine may also be derived from

adenine nucleotides from many cell types by mechanisms which are not well

understood.100'101

Since AMP is derived from the breakdown of ATP, adenosine formation is closely

linked to the cellular energy state. Adenosine may be formed intra or extracellularly.

(Figure 1-17) The enzyme 5' nucleotidase (5'N) cataylzes the metabolism of ATP to

adenosine. S-adenosylhomocysteine hydrolase also catalyses the break down of S-

adenosylhomocysteine (SAH) into adenosine. SAH contributes significantly to

adenosine formation in the heart and ischemic conditions in the brain. Once formed,

intracellular adenosine is transported out of the cell to exert effects on specific cell

surface receptors. The transport of adenosine is bidirectional.










L-Homocystiene 5-N
A / Pi
SAH Hydroloase


Extracellular
,,,,,B,,.,


Tase iADP
[ AdAdenosine kinase
I' ATP


Adenosine
deaminase


Adenosine


T

U-
OLM^


02
02~ XDH or XO

02
Z 1 XDH orXO
02^^" T


Figure 1-17. Intracellular and extracellular production of adenosine. SAH hydrolase: S-
adenosyl homocysteine hydrolase. 5'NT:5'nucleotidase. XDH:xanthine
dehydrogenase. XO:xanthine oxidase


Intracellular









Ecto 5'N catalyses the breakdown of 5'AMP to adenosine, thus giving rise to

extracellular adenosine. The intracellular and the extracellular adenosine can be

differentiated from each other using specific inhibitors of ecto 5'N.100,101

Adenosine deaminase (ADA) and adenosine kinase (AK) catalyze the breakdown

of adenosine in the cytoplasm. Both ADA and AKA are found in the cytoplasm. ADA is

a 36 kDa protein which catalyzes the formation of inosine. ADA is heterogeneously

distributed in tissues and highest activity is during development. AK is a 38-56 kDa

monomeric protein. It is also widely distributed throughout the body. AK catalyses the

phosphorylation of adenosine to 5'AMP. If the intracellular adenosine is high, then AK

is inhibited.100'101

Five types of adenosine transporters have been classified according to sensitivity to

nitrobenzylthioinosone (NBTI), which is an adenosine transport inhibitor. Most of these

transporters are sodium dependent and are bidirectional. Following degradation of

adenosine, inosine leaves the intracellular environment and forms hypoxanthine.

Xanthine dehydrogenase catalyses the oxidation of hypoxanthine to xanthine and

subsequently to uric acid. Conversion of xanthine to uric acid also reduces NAD to

NADH. Xanthine oxidase genetrates superoxide and hydrogen peroxide, both of which

are damaging to cells. Endothelial cells stimulated by ischemia and reperfusion are key

sources of xanthine oxidase formation and activity.100

Adenosine and the Retina

Adenosine is heterogeneously distributed throughout the retina of various species,

such as rat, guinea pig, monkey, human and mouse.100 Adenosine immunoreactivity is

found in the ganglion cell layer, the inner plexiform layer and the inner nuclear layer.
91,102 Under resting conditions, endogenous purines in the retina are in the form of ATP









(70%) and adenosine (2%). During development, the retinal Muller cells provide

glycosaminoglycan to the extracellular spaces for angioblasts which provides a scaffold

for angioblast migration and organization. In developing and adult mammalian retina

Muller cells express 5' nucleotidase (5'N) ectoenzyme, a glycoprotein. This enzyme

catalyzes the hydrolysis of purine nucleotide monophosphates, to the corresponding

nucleoside. The 5'NT can metabolize all purine monophosphates, however, the major

product is adenosine. Adenosine is an intercellular communication molecule and is a

modulator of synaptic transmission in the brain and the retina, and is a local regulator of

blood flow in several organs. In the retina, adenosine is released in response to ischemia,

thereby modulating the blood flow in the adult and neonates. Adenosine is also

chemotactic and a mitogen for endothelial cells, and enhances endothelial cell migration

and tube formation.102 An increase in the 5'NT activity in cerebral ischemia was shown

by Braun et al.103 The pattern of 5'NT changes as the retina develops. In the early stages

of development, the greatest activity of 5'NT is found in the inner Miller cell processes.

When the inner retinal vasculature reaches completion (about 22 days of age), the inner

retina activity of the enzyme decreases and the activity in the outer retina increases (in

both plexiform layers).102

Lutty et al showed that at days 1-5, an increased adenosine immunorectivity is

found in the inner retina and the edge of the formed vasculature in the neonatal dog. An

increase in the adenosine product shifted toward the ora serrata as the vascular

development progressed radially. On day 8 the 5'NT is increased in the inner retina, and

on day 15 there is an increase in the adenosine immunorecativity in the nerve fibre layer

and the inner nuclear layer. When the radial progression of the inner retinal vasculature









is complete on day 22, the 5'NT and adenosine are decreased throughout the nerve fibre

layer and increased in the ganglion cell layer, the inner nuclear layer and the

photoreceptor inner segments.102 An increase in adenosine levels at most ages was found

to be proportional to an increase in the 5'NT activity. In summary, the 5'NT activity

shifts from the nerve fibre layer to the inner plexiform layer during development and the

adenosine location is also shifted. Thus the Miller cells provide a glycosaminoglycan

rich extracellular milieu for angioblast differentiation and also provide adenosine which

is a stimulus for blood vessel formation.100,102

Adenosine Receptors

Adenosine receptors have been implicated in mast cell activation, asthma,

regulation of cell growth, intestinal function, neurosecretion modulation and vasodilation.

Adenosine receptors modulate cAMP (adenosine 3c, 5c-cyclic monophosphate)

intracellulary. Based on their ability to inhibit or stimulate adenylyl cyclase, the

adenosine receptors were initially divided into A1 and A2 subtypes. 100,104,105 The A2

receptor was further divided into 2 subtypes based on the finding of a high affinity A2

receptor in the rat striatum and a low affinity A2R in the brain 106 Both of these receptors

activate adenylyl cyclase. The high affinity receptor was designated as A2A and the low

affinity receptor was designated A2B.100,107

Adenosine activates four different cell receptors: A1, A2A, A2B and A3. In most cell

types, adenosine activates the A1 receptor to lower oxygen demand, and activates the A2

receptors to increase the oxygen supply. Thus the A1 and A2 receptors act to rectify

imbalances between oxygen supply and demand.100,10s(Figure 1-18)








02 Supply/Demand



t
Hypoxia




%ar



Figure 1-18. Role of the high and low affinity adenosine receptors. The A2 receptors
increase oxygen supply. The A2A receptor leads to vasodilation and the A2B
receptors lead to angiogenesis









Ai, A2B and A3 adenosine receptors are N-linked glycoproteins, which have sites

for palmitoylation near the carboxyl terminus. Glycosylation has no effect on the affinity

of ligands for these receptors, thus these sites may be involved in targeting newly formed

receptors to the cell surface. All receptors can be readily deglycosylated upon incubation

with glycosidase.101 The molecular pharmacological and physiological relevance of the

Ai, A2A and A3 receptors is well known. However, the A2B receptor is not as well

characterized due to a lack of selective pharmacological probes and because this receptor

has a low affinity for adenosine.100

The A1 receptor was initially cloned from rat, human, bovine and rabbit. The A1

receptor has seven transmembrane domains and is 326 amino acids in length and is about

36-37 kDa. Mutations in the H 274 and H 251 region result in loss of agonist and

antagonist binding. Chimeric receptor constructs reveal transmembrane domains 5, 6 and

7 to be important for binding. In the brain, the A1 receptors couple to Gi and Gs and

inhibit the actions of adenosine.

The A1 receptor decreases membrane potential (by increasing K+ and Cf

conductivity), lowers neurotransmitter release (e.g. glutamate and dopamine) and

decreases calcium influx by stimulating calcium mobilization via the pertusis toxin

sensitive pathway through the activation of PLC beta with G protein 3/y subunit.101 All of

these effects of the A1 receptor lead to a decrease in neuronal excitability and

metabolism. Thus, the A1 receptor has a neuroprotective role in ischemic tissue.100

The A2A receptor was initially cloned from canine, rat and human and produced

responses which are anti-inflammatory.101 It has seven transmembrane domains

consisting of 410-412 amino acids and is about 45 kDa (comparable to Ai). Mutations in









H 274 and H 251 also lead to loss of agonist and antagonist binding. Adenosine relaxes

vascular smooth muscle via the A2 receptor mediated mechanism and thus increases

tissue perfusion.

In the retina, the vasodilatory effects of adenosine are mediated by A2 linked to

potassium ATP channels.100'101 Adenosine increases glyconeogenesis via the A2 receptor

and thus promotes an increase in the supply and demand ratio for metabolic substrates in

the retina. A2A decreases the superoxide release from activated neutrophils and inhibits

platelet aggregation. These are all anti-inflammatory actions, the importance of which in

retinal response to ischemia has not been established. Ideally, a drug that is an A2A

agonist and an A2B antagonist is needed to further understand the two receptors.100

The A2B receptor was initially cloned from rat hypothalamus109, human

hippocampus110 and mouse mast cells 100 The receptor was found in these tissues by PCR

with degenerate DNA oligonucleotides that recognized conserved regions of the G

protein coupled receptors. The human, rat and mouse A2B receptors share 86-87% amino

acid homology.109 The human A1 and the human A2A, and A2B receptors share 45%

amino acid homology.100 Closely related species such as rat and mouse share 96%

homology. The A1 receptors have 87% amino acid homology in various species (Figure

1-19) 111,112, the A2A receptors have 90% homology (Figurel-20). 113 while the A3

receptors differ significantly between species.111,112 Figure 1-21 shows the homology

between the human and the mouse A2B receptor.100
































Figure 1-19. Homology of the A1 receptor for human and mouse. The yellow sequences
indicate homology between the human and mouse Al receptor sequences.
The white sequences indicate non-homologous regions and the blue sequences
indicate conserved sequences.






































Figure 1-20. Homology of the A2A receptor between human and mouse. The yellow
sequences indicate homology between the human and mouse A2A receptor
sequences. The white sequences indicate non-homologous regions and the
blue sequences indicate conserved sequences.































Figure 1-21. Homology of the A2B receptor between the human and the mouse. The
yellow sequences indicate homology between the human and mouse A2B
receptor sequences. The white sequences indicate non-homologous regions
and the blue sequences indicate conserved sequences.









The membrane structure of the A2B receptors is that of a typical G protein coupled

receptor consisting of a 7 transmembrane domains connected via 3 extracellular and 3

intracellular loops. (Figure 1-22)100,110,114

Trans membrane domains have a high degree of amino acid homology in different

species. The human, mouse and rat A2B receptors have 2 potential N-glycosylation sites

in the second extracellular loop.109 The human N-linked glycosylation sites are Asp 153

and 163 which are in the second extracellular loop. Both of these sites are conserved in

all of the A2B sequences of all species that have been cloned.100,115

The A2A intracellular and the third intracellular loop are involved in coupling A2A

receptor to G proteins. 100,111 The third intracellular loop is a 15 peptide portion of the

A2A receptor which has 57% amino acid homology with the A2B receptor and also

determines the selective coupling with GS.100,116 Both A2A and A2B are coupled to Gs.

The A2A and A1 receptors have 27% amino acid homology and the A1 is not coupled to

Gs. Amino acids in the second intracellular loop may modulate the A2A receptor coupling

since lysine and glutamic acid are necessary for efficient A2A adenosine receptor Gs

coupling.100,116 Analogous lysine and glutamic acid residues are also present in the A2B

receptor. The major difference between the A2A and the A2B receptor is the long

intracellular C-terminal tail of the A2A. (Figure 1-23) This long tail is not involved in Gs

coupling to the receptor. Removal of the c-terminal tail of the A2A receptor does not

inhibit stimulation of adenylyl cyclase when truncated receptor is expressed in CHO

cell.100,111,116

Mutational studies of the A2A receptors have shown that the Thr 298 residue of the

C-terminal tail of the A2A receptor is located close to the seventh membrane













NH -) 9

3
3 3 3 0 )



nm e d i 3 8 lop
a3 P 3ai

13





ID39!3 )-COOH
3 3
.),33

Figure 1-22. The A2B receptor is a G protein coupled receptor consisting of a seven
transmembrane domain connected via 3 extracellular and 3 intracellular loops
flanked by an extracellular N- terminal and an intracellular C-terminal.






58





,33 3)

2 3, f
3 ,, ,3



V3
13.3 3


3 33)

333



S3 ) ))33 33






)-COOH

Figure 1-23. The A2A receptor structure consists of 7 transmembrane domains connected
via 3 extracellular and 3 intracellular loops flanked by an extracellular N-
terminal and a long intracellular C-terminal.









span and is essential for the development of rapid agonist mediated

desensitization.100,111,117 The threonine residue is also present in the human A2B (Thr 300),

however, its role in receptor desensitization has not been explored. A2B receptors can be

coupled to other intracellular signaling pathways in addition to Gs and adenylyl

cyclase.100

The A3 receptor was cloned from the human, rat and sheep. It is composed of 320

amino acids and has about 40-50% homology to the A1 and A2 receptors. It has low

affinity for alkylxanthine antagonists such as theophylline and caffeine (which is a classic

antagonist for A1 and A2). The non specific A3 antagonist IB-MECA inhibits adenyly 1

cylcase and increase PLC, calcium mobilization and decrease TNF-alpha. Higher

concentrations of adenosine are required to activate the A3 receptors than are required to

activate the A1 or the A2 receptors.100

Pharmacology of the A2B receptors

Highly selective and potent agonists designed for A1, A2A, and A3 receptors are

available and are important tools for the characterization of adenosine receptors. The

lack of a potent selective A2B antagonist hampers the characterization of its cellular

functions. 95,100 The most potent agonist for A2B is NECA.100,118-120 At a concentration of

2[LM, NECA produces half the maximal effect (EC50) for stimulation of adenylyl

cyclase.120 NECA is non-selective and thus activates other adenosine receptors with

greater affinity. The EC50 for the A1 and A2A receptors is in the low nanomolar range

and that of the A3 receptors is in the high nanomolar range. Therefore, the

characterization of the A2B receptor depends on the use of compounds, which are potent









selective agonists of other receptor subtypes. Therefore the A2B receptor is usually

characterized by exclusion.100

CGS 21680121 is an A2A selective agonist that can differentiate A2A and A2B

receptors.100,122-125 The A2A and the A2B receptors are both positively coupled to adenylyl

cyclase and are activated by the non selective agonist NECA. CGS 21680, on the other

hand, is ineffective on A2B receptors and as potent as NECA when activating A2A

receptors.100,120-122,126-128 R-PIA is an A1 selective agonist and the A2B receptor has low

affinity for it.100,119,120

The pharmacological characterization of the adenosine receptors is based on

apparent agonist potencies. This is not ideal as it depends on agonist binding to the

receptor and multiple processes of signal transduction. Therefore, for receptor subtype

identification, selective antagonists are preferable 100,129 Highly selective A2B antagonists

are not available. However, it is known that A2B has a low affinity for agonists, but a

high affinity for antagonists. Enprofylline (3-n-propylxanthine) is an anti-asthmatic drug,

and is the most selective, but not potent, A2B antagonist known. Other potent but non-

selective A2B receptor antagonists include 1,3-dipropyl-8 (p-sulfophenyl)xanthine

(DPSPX), 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), xanthine amine cogener (XAC)

and IPDX 95,119,120

Distribution of the Adenosine Receptors

Initially the A2B receptor mRNA was found in the rat. The highest levels of the

receptor was found in the cecum, bowel, bladder, followed by the spinal cord, lung,

epidydimus, vas deferens and the pituitary.100,130 Subsequently more sensitive RT-PCR

showed that the A2B receptors were present in all tissues of the rat, with the highest level

in the proximal colon and the lowest level in the liver.131 Primary tissue cultures have









different adenosine receptors present in the cells. This may be because there are different

populations of cells and each cell expresses a different type of adenosine receptor.100,132-
134 Studies on established cell lines also showed multiple adenosine receptor subtypes on

a single target.100,122,124,125 Also, studies on single cells show the presence of one or more

adenosine receptor subtype.135-137 Clonal cell lines also have co-expression of the A2A and

the A2B receptors.100,124 However, subsequent studies showed minute amounts of other

receptors too! Therefore it is possible that adenosine selective antagonists are needed to

better characterize the distribution of these receptors in cells. It is however, unclear why

there is simultaneous expression of multiple adenosine receptors in a single cell. Both A1

and A2A receptors have a high affinity for adenosine and need to be blocked before the

effects of the A2B receptor can be seen 100,135,136,138 However, this is not always the case

and may be a reason for discrepancies published in the literature. Elfman et al showed

that glial cells of rat astrocytes have A1 and A2B adenosine receptors which stimulate

cAMP.133,139-141 However, when the cells were stimulated by the non-selective agonist,

NECA, cAMP accumulation was seen even though there are A1 receptors present.

Therefore it may be that the importance of the A2B receptors is maximal where adenosine

receptor levels are high, such as, in tissues with a high metabolic demand or conditions

when oxygen is decreased. Both the A1 and the A2 receptors may modulate the response

to lower the concentrations of adenosine.100

The widespread unique localization of adenosine suggests that it is well positioned

to serve as mediator of important physiological and pathophysiological processes in the

retina. 91,100,142 In the retina, adenosine receptors are localized to the same retinal layers as

endogenous adenosine. In the mouse a tritiated A1 agonist, cyclohexyladenosine (CHA)









was used to localize the A1 to the inner retina (over the inner plexiform layer) and the A2

receptor was localized to the RPE (outer retina) and the outer and inner segments of

photoreceptors by using tritiated NECA.100,142 No A3 receptor has been found in the

retina. The location of adenosine receptor mRNA transcripts generally correlated with

the autoradiographic localization of the Al receptors, but not the A2 receptors.100

Intracellular Pathways Regulated by A2B Receptors

Adenosine receptors activate a diverse cascade of intracellular signaling. The A1

and A3 receptors inhibit adenylyl cyclase and stimulate PLCP by activation of pertussis

toxin sensitive G proteins Gi and Go.143 Adenosine binding to the A2A and the A2B

receptors couples them to Gs and adenylyl cyclase positively, however, the A2B receptor

is also active in other signaling pathways. The A2B receptor coupled to Gs can also

increase calcium transport into the cells by the cholera toxin sensitive pathway. This

pathway is cAMP independent even though it is coupled to Gs. 144,100 The A2B receptor is

also coupled to Gaq and leads to the activation of two distinct pathways. One of those

pathways lead to the activation of the MAPK pathway and the other pathway activates

the PI3 kinase/PkB pathway. (Figure 1-24)

Angiogenesis is a complex process and is the underlying cause of several

retinopathies. Currently available treatments for retinopathies are painful and have had

limited success. Since adenosine exerts its angiogenic effects upstream of VEGF, it is an

attractive target for inhibiting the process of angiogenesis. However a lack of selective

and potent A2B antagonists requires the use of molecular techniques to target the A2B

receptor. One such approach is the use of ribozymes to target receptors at the molecular

level.









Muller Cell

5'N



AMP
ADO -
C--->


Hypoxia


Figure 1-24. The A2B signaling pathway. The A 2B receptor couples to Gs and Gaq and
leads to an increase in calcium transport and also leads to the activation of the
MAPK pathway









Ribozymes

Ribozymes are catalytic RNA molecules that cleave other RNA molecules.

Ribozyme is short for ribonucleotide enzyme, which, catalyze the hydrolysis and

phosphopryl exchange at the phosphodiester linkages between RNA bases resulting in

cleavage of the substrate. 145,146 146,147

Ribozymes can be classified into 3 main groups based on function and size: self

splicing introns, RNase P, small self cleaving ribozymes.

Self Splicing Introns

Group I Introns

Self splicing introns can be divided into 2 classes: Group I intron and Group II

introns, based on the conserved secondary structure and splicing mechanisms (Figure 1-

25). Group I introns are found in a wide number of species, such as, eubacteria,

bacteriphages, fungal mitochondria, plant chloroplasts and rRNA of lower

eukaryotes.148'149'150 The splicing action consists of two consecutive

transphotoesterification reactions. In a transphotoesterification reaction, the number of

phosphodiester bonds remain constant, however, the position of the bonds changes.

(Figure 1-26)15'0151 Only the Tetrahymena large rRNA group I intron has been shown to

function without a protein in vivo. All other known group I introns require a single

protein co-factor to provide a scaffold that helps position the introns in a catalytic

conformation.152
















U A | A aLuaIwia 3'
A A r ian 1
AA U U
A uA A u A 709
A 5A A UAA
5"-a a U U AA
A-U a a A A A G
A-U u A A 9 A-U A-U
S U-A c A A u U.A U-A
A-U c A G c C-G UA
C-G u A U PIO0 C U- A-U
t. u U A a U-A UIA
UI u UAU AG-C 224 u A-U U-A
A U a 1 U-A 9 U-AGAG-C
C A .1 u-G P1 1 u A
A A u -U G U ....
AC-GAA a u-A U U G
U.A 305 acaEagacaG U A UA

G.GC ,, ,,5 ,,A P -: C-G
U j .A : U.A
350 6-G A. .U ..................... .
3$......... I ................
A-U. 440 U-A U
S A-U lJ :
BU U A.U A cc
CS AA A-U G A
AG-C U U C
AG A G A
UAA A
U-A A
U.A C-G U-A
A-U -A U
UA-A
U.A U-A G-C

UA C-G U-A
UA 28 U-A
A-U U-A
U.A U-A .........U-A
A-U U-G U-A
U.A 250 C-G, 6S0 A-U
U-A C-G P2 A.U
A.U G-U A-U
U-A C-G U-A
A-U UUU A-U
A UA U-A
U U.A U-A
A-U
A.U
U-A
A.U



U A
A A
U U


P9






P7

4P6
G
, 4 ACUGA
OUUUC GAUAG
AA






P3






P8


500


Figure 1-25. The secondary structure group I introns.










guanosine cofactor binds and
attacks phosphate at 5'-junction



i- cleavage at 5'-juction is followed by
ligation of guanosine cofactor to 5'-end



+ formation of helicies P1 and P10 aligns the
S3'-end of the intron with the guanosine
binding site


Nucleophillic attack by hydroxyl group at 3'-end of
r 1I uptream exon at eh 3'-phosphate of the intron
S liberates the intron and joins exons


Figure 1-26. Splicing mechanism of the group I introns









Group II Introns

Group II introns are self splicing introns found within nuclear pre-mRNA and in

the pre-mRNA of organelles from fungi and plants.153 (Figure 1-27) High concentrations

of magnesium and potassium ions are essential for their proper folding.153 Group II

introns also require a complex of proteins and small nuclear RNAs (SnRNA) for

cleavage. These components form the spliceocome. Group II splicing occurs via two

consecutive trans photoesterification reactions similar to group I introns. The main

difference in the splicing mechanism between the two introns is the nature of the

hydroxyl group, which initiates the initial phototransesterification reaction. In group I

introns, the reaction is initiated by the 3' hydroxyl group of the exogenous guanosine and

in the group II introns, the reaction is initiated by the 2' hydroxyl group of the internal

adenosine.154 (Figure 1-28)

RNase P RNA

RNase P is an endoribonuclease which removes the 5' leader sequence from

precursor tRNAs. RNase P has an RNA and a protein unit, both of which are essential to

its function. The RNA component is the catalytic component of the complex. The

protein subunit enhances the turnover rate of the reaction by acting as a scaffold for the

RNA that forces the RNA into a catalytic conformation.155,156 RNase P can recognize and

cleave 60 different tRNA substrates.157 RNase P recognizes the structure of the tRNA and

only a minimal tRNA structure is required for the creation of the RNase P cleavage site

(Figure 1-29). 158,157





















Domain


Figure 1-27. Secondary structure of Group II introns.
















































Figure 1-28. The splicing mechanism of the Group II introns.








RNase P


0,


immature tRNA mature tRNA
Figure 1-29. Cleavage of the tRNA 5' leader sequence by Rnase P.









Small Self Cleaving Ribozymes

Small Self cleaving ribozymes are nucleolytic RNA's and are found naturally.

They are associated with viruses and satellite RNA and can catalyze RNA cleavage

reactions in the absence of protein. 159 There are several types of small ribozymes, the

most extensively studied ones include: hepatitis delta virus (HDV), hammerhead and

hairpin ribozymes. The hammer head and hairpin ribozymes are derived from tobacco

ring spot virus satellite RNA.

Hepatitis Delta Virus

Hepatitis delta virus (HDV) is a short single stranded RNA found in patients

infected with human hepatitis B. It has a circular RNA genome, which encodes a

ribozyme in both orientations. HDV replicates through a rolling circle mechanism like

other self cleaving ribozymes (Figure 1-30), and the ribozyme is required for the cleavage

of the HDV genome into discrete units prior to packaging. 160,161

Hairpin Ribozymes

The hairpin ribozyme was originally found in the tobacco ring spot virus satellite

RNA. The hairpin ribozyme binds the substrate and forms a structure with 4 helices and

2 loops (Figure 1-31). The arms of the hairpin ribozyme hybridize to the substrate

molecule to from helix 1 (6 base pair) and helix 4 (4 base pair). Loop A has a BNGUC

target sequence required for cleavage, where B is G, C or U, and N is any nucleotide.162

There are no conserved nucleotides in any of the helices. 163,164

Hammerhead Ribozymes

The catalytic domain of the hammerhead ribozyme was discovered by comparing

self cleaving RNA sequences of a number of different viroid infectious RNA molecules.







00
repli n io i l g c Minus

5
+ 21
Plus _


00
Figure 1-30. Self-cleaving ribozymes resolve concatemers formed by rolling-circle
replication into individual genomic molecules










1 C
3'-NNNNNA
5'- N N N NNU
A


UG /
A 2
NNN
I I I
NNN
A
GA


N -5
N A-U-3'
C-G
C-G 3
A-U
AG-CC


A
A
B
A


A
U
A
A C-GGU
A-U
C-G
G-C 4
C-G
U-A
C-G
G A
UA


Figure 1-31. Structure of the hairpin ribozyme. The arrow indicates the site of cleavage.
The hairpin ribozyme binds the substrate and forms a structure with 4 helices
(1-4) and 2 loops (A and B).









Hammerhead ribozymes are small, approximately 34 base RNA molecules and

cleave RNA target in trans. The hammerhead ribozymes bind substrate to form a

structure, which consists of a stem and three loops and a catalytic core with a conserved

nine nucleotide sequence (Figure 1-32). A mutation in any of the conserved nucleotides

prevents RNA cleavage.165

The catalytic core of the hammerhead ribozyme has two functions: it destabilizes

the substrate strand by twisting it into a cleavable conformation and binds the metal

cofactor needed for catalysis.166 The hammerhead ribozyme cleaves the substrate by a

tranesterification reaction (Figure 1-33). The reaction requires the presence of

magnesium and water. The hydrated magnesium ion has two functions, both mediated by

water molecules. First, one molecule of water binds to one of the oxygen atoms of the

phosphate group, holding it in the proper orientation for the enzymatic mechanism.

Secondly, the environment of the active site lowers the pKa of another water molecule so

that it can donate a proton to the aqueous environment. In the transition state, five

oxygen atoms are arranged in a triangular bipyramid around the phosphorus atom. A

bond is formed between the 2' oxygen of cytosine 17 and the phosphorus atom.

Simultaneously a bond is broken between the phosphorous atom and the hydroxyl oxygen

of the next nucleotide, adenine 1.1. This leaves the cytosine with a 2'-3' cyclic

phosphate group. The 5'nucleotide recovers a proton from the aqueous environment,

completing a hydroxyl group. The reaction products diffuse away from the active site

leaving the ribozyme free to bind a second substrate molecule and complete another

reaction cycle. The hammerhead ribozyme recognizes substrate sequences on either side

of a NUX cleavage site, where N is any nucleotide and X is any nucleotide except G.















GAA


CGGC
GCCG
U A
II


I


GUAGU


NNNNN N-3'
NNNNNN-5'


Figure 1-32. Structure of the hammerhead ribozyme. The hammerhead ribozyme binds
substrate to form a structure, which consists of a stem and three loops and a
catalytic core with a conserved nine nucleotide sequence. Arrow indicates site
of cleavage.


3' 5'
N-N
N-N
N-N
N-N
N-N
A-U
X










A











To Adenine 1


;ine 17


H :OH

D"I"H O -Mg-
H I


To Adenine 1 1-CH2


'

H-OH;
To Adenine 1.1-CH2


a17

H
I

H


To Adenine 1.1-CH2


Figure 1-33. The hammerhead ribozyme cleaves its substrate by a transesterification
reaction. A. A molecule of water binds to an oxygen of the phosphate group.
B. Another water molecule donates a proton. A bond is formed between the
2' oxygen of cytosine 17 and the phosphorous atom. C. A bond is broken
between the phosphorous atom and the hydroxyl oxygen of adenine 1.1. D.
Cytosine remains with a 2'3' cyclic phosphate group. The 5' nucleotide
recovers a proton to complete a hydroxyl group. The reaction products then
diffuse away from the active site leaving the ribozyme free to bind a second
substrate molecule and complete another reaction.









The ribozyme anneals to the substrate mRNA by means of two flanking arms which

hybridize to form helices III and I. Cleavage occurs at the 3' end of the cleavage site.

Not all cleavage sites demonstrate the same efficiency. Generally, GUC is the most

efficient cleavage site, then CUC, UUC and AUC. The remainder of the cleavage sites

are cleaved at least 10 times less efficiently than the GUC site. The hammerhead and

hairpin ribozymes are being examined as gene therapies for a number of different

diseases because they are small and can be easily cloned and packaged into many of the

existing viral vectors for delivery to target cells. The advantage of the hammerhead

ribozyme is that it can recognize a greater number of cleavage sites than HDV or hairpin

ribozymes.167,168

Experimental Aim

Currently, the only available treatment for ROP is laser treatment of the retina,

which has limited success. The aim of this project is to design a hammerhead ribozyme

that will specifically target and cleave the A2B receptor mRNA resulting in a reduction in

expression of the A2B receptor protein and a reduction of cellular and physiological

functions affected by this receptor. We are using a hammerhead ribozyme primarily as a

tool to study the pathways that involve A2B. But this ribozyme can also be used as 'proof

of concept' for conventional drugs targeting the A2B receptor and, finally there is a

possibility that the hammerhead ribozyme itself could be used as a therapeutic agent.

The goal of this project was to examine the effectiveness of ribozymes in the

treatment of ROP. Previously we have shown that proliferative blood vessels have an

enhanced expression of the A2B receptor, therefore, there is justification to target this

protein in controlling the disease. Ribozymes were designed to specifically cleave the

mRNA of the A2B receptor to decrease the expression of the receptor protein. The









underlying hypothesis was that the cleavage of the mRNA of the A2B receptor at the

mRNA level would prevent translation of the protein and subsequently progression of

angiogenesis in ROP by preventing the growth of abnormal blood vessels. (Figure 1-34)

The selected target site was a short region of the mRNA for the A2B receptor. The first

step of the project was to design a ribozyme to cleave the sequence of the A2B receptor in

the mouse and the human. (Figure 1-35) Two hammerhead ribozymes were developed,

each of which had an inactive version with a single base mutation. (Figure 1-36)

The most efficient ribozyme was cloned into an rAAV construct (p21Newhp) for further

analysis. (Figure 1-37) The second step of the project was to develop in vitro assays to

examine the ability of the ribozyme to cleave the mRNA. These assays were used to

determine if the ribozymes would be effective for reducing pre-retinal neovascularization

in an oxygen-induced mouse model of retinopathy.

To test the ribozyme in vivo, the A2B Rz2 was intravitreally injected to the mouse

model. Several models for oxygen-induced retinopathy have been developed.

Dembinska169 and Chowers170 both used a rat model. The rats were placed in alternating

hypoxic and hyperoxic environments. The alternating environments lead to severe retinal

complications, which were not representative of retinopathy in human babies. Since the

timing and duration of the hypoxia was inadequate, it gave inconsistent results. In our

study we used a mouse model developed by Louis Smith.171 An advantage of using the

mouse retina is that in the newborn mouse the retinal vessel development stage is the

same as that of premature human babies. Also, normal retinal vascular development in

mice occurs within two weeks of birth, thus illustrating the evolution








A2B Receptor


Sr+ Ritbozyme
Transcription
41 g^-l

.........


Translation


Degradation

#kV -.0


Figure 1-34. Cleavage of the A2B receptor by a ribozyme prevents translation of the
protein.


'4 LOI ",








Species A2B Rzl Target A2B Rz2 Target
Mouse ACAUGUCUCUUUG CAUUGUCUAUGCC
Human AAGUGUCUCUUUG CAUUGUCUAUGCU

Figure 1-35. Target sequences of the human and mouse A2B ribozymes 1 and 2. Red
indicates a difference in sequence between the human and mouse species.










3'-U -A-5' 3'-U -C-5'
G-C G-A
U-A U-U
A-U A-U
C-G cleavage site C-G clca agc site

AAA-UC / A AA-UCA
G G UCUUUG-3' G G UAUGCC-3'
CcGGCG I I I 15 C CGGC I I I I
CI ii I AGAAAC i-5' I AUACGG-5'
U, GCCG C U GCCG C
U AAG U AUAGU
GUAG'Gc c


A2B Ribo2zme 1
2B


A2B Ribozyme 2


Figure 1-36. Hammerhead ribozymes for the A2B Rzl and Rz2. The target sequences
are indicated in red. For each ribozyme an inactive version of the ribozyme
was made with a C replaced by the G (arrow).











CMV enhancer
Beta-actin promoter
Exon



Intron
p21NewHp
6568 bp
HindillI (121)
Spelt (1931)
airpin Ribozyme
Nsii l2(0141
SV40 Poly(A)
PYF441 Enhancer
TR \HSV-tk

B SV40 PolylA) neoR


Hindlll Spel N
Hammerhead Hairpin
Ribozyme Ribozyme



Hairpin Cleavae
Site
Figure 1-37. A)The p21Newhp Vector with the CMV enhancer and beta actin promoter.
The hammer head ribozyme was cloned between the HindIII and Spel sites.
B) The hammerhead and the hairpin cleavage sites.