Environmental Neurotoxins and Parkinson's Disease - the Alpha Synuclein Connection

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Title:
Environmental Neurotoxins and Parkinson's Disease - the Alpha Synuclein Connection
Physical Description:
1 online resource (77 p.)
Language:
english
Creator:
Murlidharan,Giridhar
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University of Florida
Place of Publication:
Gainesville, Fla.
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Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Biomedical Engineering
Committee Chair:
Ogle, William
Committee Co-Chair:
Fernandez-Funez, Pedro
Committee Members:
Okun, Michael S
Ormerod, Brandi K.

Subjects

Subjects / Keywords:
alpha -- environmental -- neurotoxins -- parkinson -- synuclein
Biomedical Engineering -- Dissertations, Academic -- UF
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Biomedical Engineering thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
Parkinson's disease (PD) is the most common movement disorder and is characterized by the loss of dopaminergic neurons in the substantia nigra and the presence of proteinacious aggregates enriched in Alpha-Synuclein called Lewy bodies. Most PD has a sporadic origin, however, recent studies have shown that environmental toxins, including pesticides, induce Alpha-Syn aggregation and may cause PD symptoms. For instance, paraquat and rotenone induce Alpha-Syn up-regulation and aggregation, and elicit loss of dopaminergic neurons in mice and rats respectively. The fact that PD has high incidence in rural areas made us wonder whether other pesticides commonly used in farming also had the ability to induce Alpha-Syn aggregation. For my Master's thesis I developed a cellular assay to identify pesticides that induce Alpha-Syn aggregation. I treated differentiated human neuroblastoma SY5Y cell culture expressing a Alpha-Syn-GFP fusion with the 21 most commonly used pesticides in Florida?s orange industry for 24 hrs. Then, I visualized the Alpha-Syn aggregation in fixed cells under the microscope. My findings are that three herbicides (2, 4 D-isopropylamine salt, norflurazon and diuron), three insecticides (diflubenzuron, pyridaben and carbaryl) and four fungicides (mefonoxam, azoxystrobin, thiabendazole and imazalil) induce Alpha-Syn aggregation. I then combined the pesticides that do not induce Alpha-Syn aggregation with a low dose of diuron, a potent inducer of Alpha-Syn aggregation, and found that two more insecticides (abamectin and sulfur) and 2 herbicides (simazine and glycophosate) induce Alpha-Syn aggregation. Also, the herbicide isopropylamine shows a distinctive change in the aggregation pattern. These findings demonstrate that 14 out of 21 pesticides tested have the ability to induce Alpha-Syn aggregation, thus suggesting a strong link to PD. These pesticides should be further tested in rodents for a better assessment of their ability to induce PD symptoms. Understanding the risks associated with the current farming practices should contribute to reducing the incidence of sporadic PD in rural and farming environments.
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
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Description based on online resource; title from PDF title page.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Giridhar Murlidharan.
Thesis:
Thesis (M.S.)--University of Florida, 2011.
Local:
Adviser: Ogle, William.
Local:
Co-adviser: Fernandez-Funez, Pedro.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-02-29

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UFE0043396:00001


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1 THE ALPHA SYN U CLEIN CONNECTION By GIRIDHAR MURLIDHARAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS F OR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011

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2 2011 GIRIDHAR MURLIDHARAN

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3 my parents

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4 ACKNOWLEDGMENTS When I landed in the University of Florida in f all of 2009 for my MS in Biomedical Engineering my sights were trained on doing a thesis on a facet of genetics and molecular biology as a prelude to the consummation of my ultimate goal -doctorate. It was my good fortune that Dr. William Ogle, Assistant Professor, BME, UF recommended me to an interview for gradu ate research assistantship at the Fernandez Funez and Rincon Limas laboratory at the Department of Neurology, UF Words fail me in adequately expressing my sense of gratitude to Dr Ogle for playing a catalytic role at a crucial stage of my career. Biggest thanks to Dr Fernandez Funez who put me through the paces and gave me ample opportunities to do it myself even while keenly but unobtrusively guiding me all the way in completion of my thesis. Verily, both Dr. Fernandez Funez and Dr. Rincon Limas have been my friends, philosophers and guides, giving me a solid grounding in the intricacies and applications of molecular biology in neuroscience, a subject in which both of them are an acknowledged authority. I would remember them as my gurus wherever I am. Addi tional thanks go to Dr. Brandi Ormerod Assistant Professor, BME, UF, for accepting to be a member of my thesis committee and helping me out with her expertise in cell culture techniques. No less inspiring was the day to day help and guidance received from the post doc researchers Jonathan Sanchez, Kurt Jensen and Yan Zhang especially during the initial days of my joining the lab when I had to work first time with the techniques and equipments most of which I had only read about during my undergrad days in India. It was fun working with Masters student Jose Herrera and undergraduate students Barbara, Stephanie, Daniela, Serena and Nabiha. With people from Spain, India, Cuba, Columbia, Mexico China and Pakistan, our lab was in a way truly cross cultural, an d made for a lot of fun and backslapping camaraderie so essential in a research lab if only to relieve one of taut nerves.

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5 A special word of thanks is due to Dr. Irene Malaty MD, Movement disorders center, UF, who allowed me the privilege of shadowing he r in the course of my Clinical preceptorship class Seeing first hand the treatment regime indeed gave me a fair bit of idea on diagnosis and Dr. Michael S. Okun, Co director, Movement disor der centre, UF kept the project going with his constant encouragement with funds and more importantly with words and gestures of encouragement that kept my spirits high. Suguna Jairam, my confidant and alter ego, has been a source of great strength as inde ed have been my parents back home in India. That I have taken to the path of research during the development phase of my career has been a sort of dream come true for them. Keen academics themselves, my parents created in me an insatiable but healthy thirs t for scientific knowledge during my salad days. As I stand at the cusp of completion of my thesis, I realize more than ever before the stellar role played by them in shaping my career. A constraint of space prevents me from acknowledging individually the roles of other colleagues in the collaborating lab s and friends here in Gainesville who encouraged me at every point throughout this project. Special thanks to friends and folks back in India, I will be failing in ir contr ibutions, direct and indirect, compendiously even while remaining guilty of not acknowledging them individually.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ........................... 8 LIST OF FIGURES ................................ ................................ ................................ ......................... 9 LIST OF ABBREVIATIONS ................................ ................................ ................................ ........ 12 ABST RACT ................................ ................................ ................................ ................................ ... 13 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .................. 15 ................................ ................................ ................................ ................ 15 Symptom ................................ ................................ .......................... 15 ................................ ................................ ......................... 16 ................................ ................................ ............................. 16 Sporadic and Genetic Causes ................................ ................................ .......................... 16 Synuclein ................................ ................................ ......................... 17 Synuc lein and PD ................................ ................................ .. 18 2 MATERIALS AND METHODS ................................ ................................ ........................... 21 Cell Culture ................................ ................................ ................................ ............................. 21 Cell D ifferentiation ................................ ................................ ................................ ................. 21 RNA Extraction ................................ ................................ ................................ ...................... 21 RT PCR ................................ ................................ ................................ ................................ ... 22 Quantitative PCR ................................ ................................ ................................ .................... 23 Synuclein GFP DNA Amplification ................................ ................................ ................... 24 Transformation Using SURE2 Cells ................................ ................................ ............... 24 Midiprep ................................ ................................ ................................ .......................... 25 Synuclein GFP DNA ................................ ............................... 25 Cell Viability Assay ................................ ................................ ................................ ................ 26 Syn Aggregat ion ................................ ................................ .......... 27 Pesticide Treatment ................................ ................................ ................................ ......... 27 Triple Immunostaining of SY5Y Cells ................................ ................................ ............ 28 Pesticide Combination Treatment ................................ ................................ .......................... 29 Determination of Aggregation Negative Diuron Concentration ................................ ..... 29 Combination Treatment ................................ ................................ ................................ ... 29 Statistical Analysis ................................ ................................ ................................ .................. 29 Cell Viability Data Analysis ................................ ................................ ............................ 29 Syn Agg regation Experiments Data Analysis ................................ .............................. 30

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7 3 RESULTS ................................ ................................ ................................ ............................... 32 Differentiation of SY5Y Cells ................................ ................................ ................................ 32 Morphological Changes ................................ ................................ ................................ ... 32 Gene Expression Q PCR ................................ ................................ ................................ 32 ................................ ......................... 33 Cell Viability Data ................................ ................................ ................................ .................. 33 Positive Controls ................................ ................................ ................................ ............. 34 Orange Industry Fungici des Induced Cell Toxicity ................................ ........................ 35 Orange Industry Insecticides Induced Cell Toxicity ................................ ....................... 35 Orange Industry Herbicides Induced Cell Toxic ity ................................ ......................... 36 Syn Aggregation ................................ .......................... 36 Negative Control (No Pesticide Treatment): ................................ ................................ ... 38 Positive Control Environmental Neurotoxins: ................................ ................................ 38 Syn Aggregation ................................ ................................ ............ 38 Syn Aggregation ................................ ................................ .......... 39 Syn Aggregation ................................ ................................ ............ 40 Combination Syn Aggregati on ................................ ............................. 40 Aggregation Negative Diuron Concentration ................................ ................................ 40 Combination Treatment of Pesticides ................................ ................................ ............. 41 Syn Aggregation ................................ ............................. 42 4 DISCUSSION ................................ ................................ ................................ ......................... 63 APPENDIX A S YN A GGREGATION I NDUCING P ESTICIDES ................................ ........................... 67 B S YN A GGREGATION N ON I NDUCING P ESTICIDES ................................ ................. 71 LIST OF REFERENCES ................................ ................................ ................................ ............... 72 B IOGRAPHICAL SKETCH ................................ ................................ ................................ ......... 77

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8 LIST OF TABLES Table page 3 1 Syn aggregation ..... 48 3 2 Syn aggregation ..... 49 3 3 Syn aggregation ... 49 A 1 Syn Aggregation Inducing Pesticides ................................ ................................ ............. 67 B 1 Syn Aggregation Non Inducing P esticides ................................ ................................ .... 71

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9 LIST OF FIGURES Figure page 2 1 Q PCR setup for Tyrosine Hydroxylase gene expression in differentiating SY5Y cells (Reference/Housekee ping gene GAPDH) ................................ ................................ 31 2 2 tau (Reference/Housekeeping gene GAPDH) ................................ ................................ ......... 31 3 1 Morphological changes during differentiation of SY5Y cells ................................ ........... 43 3 2 Q PCR data: Tyrosine Hydroxylase gene expression analysis. Statistical significance tween two adjacent days. p>0.05, ** 0.0050.05, ** 0.0050.05, ** 0.0050.05, ** 0.0050.05, ** 0.0050.05, ** 0.0050.05, ** 0.0 050.05, ** 0.0050.05, ** 0.005
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10 3 11 Syn aggregation analysis; 24 hr Negative control NO Pesticides treatment ................. 50 3 12 treatment ................................ ................................ ................................ ............................ 50 3 13 Syn aggregation analys ................................ .................. 51 3 14 ................................ ................. 52 3 15 Syn aggregation anal ................................ ................... 53 3 16 Syn aggregation non inducing herbicides; 24 hr treatment ................................ ............ 53 3 17 Insecticide Diuron Syn aggregation analysis; 24 hr treatment ................................ ................................ ................................ ............................ 54 3 18 Herbicide Diuron Syn aggregation analysis; 24 hr treatment ................................ ................................ ................................ ............................ 54 3 19 Negative control No pesticide treatment on cells for 24 hrs; Cell 1 ............................... 55 3 20 Negative control No pesticide treatment on cells for 24 hrs ; Cell 2 ............................... 55 3 21 Rotenone ind Syn aggregation ................................ ................................ ................ 55 3 22 Syn aggregation ................................ ................................ ................. 56 3 23 Syn aggregation ................................ ................................ .................. 56 3 24 Syn aggregation ................................ ................................ ..................... 56 3 25 Syn aggregation ................................ ........................... 57 3 26 Syn aggregation ................................ .......................... 57 3 27 Syn aggregation ................................ ....................... 57 3 28 Syn aggregation ................................ ................................ 58 3 29 Syn aggregation ................................ ...................... 58 3 30 Insecticid Syn aggregation ................................ ............................. 58 3 31 Syn aggregation ................................ ............................... 59 3 32 Syn aggregation ................................ ................................ ..... 59 3 33 Syn aggregation ................................ ........................... 59

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11 3 34 Syn aggregation ................................ ................................ ... 60 3 35 Syn aggregation ................................ 60 3 36 Syn aggregation ........................... 60 3 37 Syn aggregation .................... 61 3 38 Insecticide Abamectin + Diuro Syn aggregation ....................... 61 3 39 Syn aggregation .............................. 61 3 40 Herbic Syn aggregation .......................... 62 3 41 Syn aggregation ................................ ........................ 62

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12 LIST OF ABBREVIATION S 6 OHDA 6 hyd roxydopamine BBB Blood brain barrier cDNA Complementary DNA DBS Deep brain stimulation DMEM EDTA Ethylenediaminetetraacetic acid FBS Fetal bovine serum GAPDH Glyceraldehyde 3 phosphate dehydrogenase GFP Green fluorescent pr otein LB Lewy bodies LDOPA L 3, 4 dihydroxy L phenylalanine LiCl Lithium Chloride MPP+ 1 methyl 4 phenyl pyridinium PD PBS Phosphate buffered saline Q PCR Quantitative polymerase chain reaction RT PCR Reverse transcriptase polymerase ch ain reaction SN Subthalamic nucleus SNPc Substantia nigra pars compacta TH Tyrosine Hydroxylase Syn synuclein

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13 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements f or the Degree of Master of Science THE ALPHA SYNUCLEIN CONNECTION By GIRIDHAR MURLIDHARAN August 2011 Chair: William Ogle Cochair: Pedro Fernandez Funez Major: Biomedical Engineering PD) is the most common movement disorder and is characterized by the loss of dopaminergic neurons in the substantia nigra and the presence of proteinacious Syn) called Lewy bodies. Most PD has a sporadic origin, howeve Syn up regulation and aggregation, and elicit loss of dopaminergic neurons in mic e and rats respectively. The fact that PD has high incidence in rural areas made us wonder whether other Syn aggregation. For my Syn aggregation. Syn GFP fusion with the 2 1 Syn aggregatio n in fixed cells under the microscope. My findings are that three herbicides (2, 4 D isopropylamine salt, norflurazon and diuron), three insecticides (diflubenzuron, pyridaben and carbaryl) and four fungicides (mefonoxam, azoxystrobin, thiabendazole and im Syn aggregation. I then combined the pesticides that do not

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14 Syn aggregation, and found that two more insecticides (abamectin and sulfur) and 2 herbicides (simazine a nd Syn aggregation. Also, the herbicide isopropylamine shows a distinctive change in the aggregation pattern. These findings demonstrate that 14 out of 2 1 pesticides tested Syn aggregation, thus suggestin g a strong link to PD. These pesticides should be further tested in rodents for a better assessment of their ability to induce PD symptoms. Understanding the risks associated with the current farming practices should contribute to reducing the incidence of sporadic PD in rural and farming environments.

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15 CHAPTER 1 INTRODUCTION isease PD ) was first characterized by James Parkinson in his in 1 817 (1) PD is the second most prevalent neurodegenerative disorder next only to and the most common movement d isorder in the world A ffect ing about 1 million people in the US and about 5 mill ion worldwide PD has a worldwide life time risk of 2% and it has been predicted with the current demographic conditions that the cases w ill double by 2050 (2) PD is characterized by (a) l oss of dopamine synthesizing / dopaminergic neurons in the Substantia Nigra of the mid brain, and (b) the appearance of proteinacious aggregates ( L ewy bodies) in the remaining dopaminergic neurons (3) Dopamine is a neurotransmitter that has been associated with a variety of functions such as cogniti on, motivation, voluntary movements, mood, attention, reward and punishment. The loss of dopamine production in the brain exhibits itself primarily in the form of loss of motor control in the body of PD patients The p rimary symptoms of PD, those exhibited by all PD patients include : postural instability, bradykinesia (slowness of motion), tremors, rigidity and gait disturbance (4) In addition, some patients present with less common secondary symptoms that cover the complex spectrum of parkinsonism : speech trouble, loss of facial expression, fatigue, difficulty in swallowing, microgr aphia (small and illegible handwriting), sexual dysfunction, pain, dementia, sleep disturbance, fear, hallucinations, compulsive behavior, loss of smell and bladder disturbances (5,6) S ome of these symptoms have be en associated with the side effects of the medications given to the PD patients. The non dopaminergic neuronal loss in the brain is

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16 considered responsible for the non motor symptoms of P D, like cognitive dysfunction, loss of memory etc (2,6) Treatments of Disease Today, t he most effective treatments for PD available work either towards delivering the dopamine precursor Levodopa to the brain or suppres s Levodopa metabolism during its journey to the dopam inergic neurons (2) Surgery in PD has also evolved over the years. Patients with higher degree of constant motor dysfunction are good candidates considered for deep brain stimulation (DBS) This procedure involves mild electrical stimulation of the subthalamic nucleus (STN) and the glo bus pallidus interna of the brain using electrodes Although not a cure, DBS provide s symptomatic treatment of PD improving the quality of life of PD patients. D isease Sporadic and Genetic C auses The f amilial nature of PD was br ought to light by many epidemiological studies A bout 10 30% of the PD patients have positive family histories while first degree relatives of PD patients are at a 2 7 fold increased relative risk of PD (7,8) Out of the 16 PD loci (Park 1 16), the role of mutations in five have been confirmed with various models whereas the eleven others are being investigated further for their contribution in PD pathogenesis (9) The confirmed PD genes are Syn uclein gene ( SNCA ) parkin PTEN induced putative kinase 1( PINK1 ) DJ 1 and leucine rich repeat kinase 2 ( LRRK2 ). The genetic cases of PD that show mutations in the dominantly inherited genes i.e. SNCA and LRRK2 always exhibit L ewy body patholo gy in the dopaminergic neurons whereas the recessively inherited genes parkin PINK1 and DJ 1 do not always exhibit Lewy bodies (10) (11) Disorder s of this kind have been a hurdle in explaining many sporadic occurrences of the disease with genetic influen ce

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17 T he overall risk due to these rare genetic factors accounts for a small fraction of PD cases whereas the majority of the cases still remain idiopathic/sporadic (10,12,13) It is therefore important to probe in to other factors that have been reported or are expected to play a role in PD pathogenesis. Lewy Bodies and Synuclein An important pathological feature of PD is the appearance of L ewy bodies which are neuronal cytoplasmic proteinacious inclusions associated with structures called lewy neurites. The fact that L ewy bodies contain poly ubiquitinated proteins lik Syn is a direct evidence for the possible role of dysfunction of protein degradation pathways like the Ubiquitin proteasome system (UPS) in P D pathogenesis. Syn is a 140 amino acid long protein that is commonly found in presynaptic neurons of the ma mmalian brain. It is a protein with unknown function and was first found in synaptic vesicles of neurons (14) Syn exists in fibrils of which have been reported in the brains of patients with L ewy body disease (15 22) PD models have been used to observe that the disease is characteristically acc ompanied by changes in the expression levels and distribution of Syn in the affected dopaminergic neurons (23) The identification of the mis sense mutations A53T and A30P in Syn in two kindreds with autosomal dominant inherited P D provided a strong link between Syn and PD (24 26) Syn gene multiplication (duplications and triplications) were also observed in early onset familial PD cases. This supports the role of the higher levels of wild type Syn in PD pathogenesis. Transgenic mice models ex pressing mutant (A53T and A30P) and wild type human Syn ubiquitously, showed motor impairment but very few cases of L ewy body pathology or nigral cell loss were observed (10) On the other hand, Yamada et al in 2004 (27) reported that Syn in the Substantia nigra of ra ts induced about 50% loss of dopaminergic

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18 neurons along with Lewy body pathology These models support the idea that the abundance of Syn protein in the L ewy bod y aggregates of the PD a ffected dopaminergic neurons is a diagnostic hallmark of PD (28 31) Many factors seem to facilitate the aggregation of Syn (32) S ome of the mechanisms /agents that ha ve b een suggested to be responsible for the aggregation of Syn in the L ewy bodies are phosphorylation at amino acid serine 129; C terminal truncations, oxidative stress, interactions with metals or certain proteins or lipids; and pesticides (10,33 35) The genetic PD cases have also shown the aggregate s of Syn in proteinacious inclusions (25,26,36) Syn aggregation is not only confined to PD but is also seen in L ewy body de mentia, and other neurodegenerative disorders (32) It can therefore be predicted that aggregation of Syn might be the missing link in the puzzle surrounding all these disorders. Environmental Factors Synuclein and PD The twin studies involving mono and heterozygotic twins showed that environmental factors are more likely to be responsible for PD than the genetic factors (37 39) Many epidemiological studies have proved t hat factors like rural living, well water drinking and pesticide exposure can increase the risk of PD (40) A set of 19 studies showed that pesticide exposure doubles the risk of PD (41) An imp ortant example is 6 hydroxydopamine (6 OHDA) a compound whose structural similarity to d opamine cause d competitive inhibit ion at the substrate binding site of dopamine transporters in a n on h uman primate model (42) Additionally, e nvironmental neurotoxins have been reported to cause parkinsonian symptoms in animal models (4,43) The insecticide r otenone is a n aturally occurring complex ketone that can easily cross the blood brain barrier. It is also a strong inhibitor of the complex I of mitochondria. 50% of r ats exposed to rotenone develop ed features typical of PD like intracellular inclusions positive for Syn and Ubiquitin and behavioral features similar to human PD (44,45) In the

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19 a herbicide that is also created as a byproduct of the narcotic drug heroin, was reported to induce classic parkinsonian sympto ms in a group of young addicts when they injected the mselves with 1 methyl 4 phenyl 1,2,3,6 tetrahydropyridine (MPTP) contaminated heroin (46,47) T his is one of the key reports link ing environmental factors to PD pathogenesis. Non human primate and rodent models of MPTP exposure ha ve show n its ability to cause PD symptoms ( 48) e.g. mitigation of symptoms in response to dopaminergic precursors like L D OPA (L 3,4 dihydroxy L phenylalanine) and dopamine receptor agonists (49,50) Mice and rat models have shown lesser sensitivity to MPTP as compared to the primate models (51,52) however they show extra nigral dopaminergic neuronal loss and enteric tyrosine hydroxylase positive neuronal loss (53) Paraquat i s a nother commonly used herbicide ( 1, 1 dimethyl 4,4 bipyridinium ) that is structurally similar to MPTP (54) Paraquat induce s dopaminergic loss in the substantia nigra, reduced ambulatory activity (neurobehavioral PD characteristic) and also cross es the blood brain barrier (BBB) in mice models (55 ) Interestingly, p araquat injected in the SN of mice results in transcriptional activation of SNCA an Syn in a dose dependent fashion (56) Finally, the last pesticide that has been associated with parkinsonian symptoms i s the organochloride dieldrin. Dieldrin induced neurotoxic and dopamine depleting effects were first shown in duck, dove and rat models (57 60) Th at dieldrin was capab le of causing Parkinsonian symptoms was clear f rom the reports of increased residual dieldrin concentration in PD affected brains and selective dopaminergic toxicity in primary mesencephalic cultures It was also found that dieldrin treatment promotes the Syn fibril formation (16,61 64) These findings proved that the up regulation and aggregation of Syn as a consequence of treatments with the se environmental toxins i.e. pesticides we re p o tential mechanisms of

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20 pathogenesis of PD due to the gene environment interactions (56) In this work, we have develop ed a cell culture based assay for visualizing and quantifying pesticide induced Syn aggregation and their potential role in PD pathogenesis

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21 CHAPTER 2 MATERIALS AND METHODS Cell Culture SH SY5Y cells were grown in Corning 25 cm 2 c anted neck flasks. The media used was G ibco D 10% h eat i nactivated fetal bovine serum (FBS) + 1% p enicillin s treptomycin. The cells were maintained inside a 5% CO 2 37 C incubator until they grew to about 90% confluence. At this point, the cells were trypsinized ( t rypsin EDTA 1x, Mediatech), incubated for 5 mins inside the 37 C 5% CO2 3 canted neck flasks. The media was changed every other day in order to keep the cells healthy for experiments. Cell Differentiation D ifferentiation in the SH SY5Y human neuro blastoma cells was induced by a combined action of retinoic acid and mitotic inhibition by reduction of f etal b ovine s erum (FBS). The SH SY5Y differentiation media was DMEM I nvitrogen) + 1%FBS + 1% penicillin streptomycin + 10M all trans retino ic acid ( S igma). The reduction in the serum and addition of retinoic acid to induce neuronal differentiation of SY5Y cells was taken from Lopes et al (65) During differentiation the media was changed every day. RN A Extraction The collection of the cells was carried out by trypsinizing the cells with 2 mL of t rypsin EDTA per canted neck flask and incubation for 5 mins in the 37 C 5% CO2 incubator. 6 mL of media for the cell differentiation was then added to collect the cells. The solution with cells was then centrifuged at 4 000 rpm for 10 mins. The supernatant was discarded and the pellet was p hosphate b uffered s aline (PBS) from Mediatech. The RNA extraction from the day 0, day 4, day 5, da y 6 and day 7 old cells was carried out with

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22 RNAqueous Kit (Ambion). 350 L of lysis buffer was added to the p ellet of cells (thawed from 80 C freezer) and resuspended. 350 L of 64% ethanol was added, tubes were vortexed for 2 mins and centrifuged at 13 000 rpm for 30 sec. The supernatan t was transferred to the filter and c entrifuged at 13 000 rpm for 30 sec. 700 L of wash solution # 1 was added and centrifuged at 13 000 rpm for 30 s ec. 500 L of wash solution#2/3 was added and centrifuged at 13 000 rpm f or 30 sec. The solution in the collection tube was now discarded and the column alone was centrifuged at 13 000 rpm for 30 sec to remove any leftover wash solutions. A new collection tube was now used to elute 40 L of elution solution through the filter, centrifugation was done at 13 000 rpm for 30 sec. The volume of elution was measured and half that volume of LiCl was added for precipitation and th e solution was incubated at 20 C for 1 hour. The tubes were now centrifuged at 13 000 rpm for 15 mins. The supernatant was now removed and discarded. The pellet was washed twice with cold 70% ethanol and re centrifuged to aspirate away leftover supernatant. The pellet was now air dried for 10 mins. Finally, 30 L of Cellgro water was now added and the resuspens ion was done after 15 mins. RT PCR The reverse transcriptase polymerase chain reactions were carried out with superscript III first strand synthesis system from invitrogen to obtain the cDNA from the RNA. 400 ng of RNA was initially used to obtain the cDNA The primers used for the RT PCR were ; a) T yrosine hydroxylase Forward b) Tau c) GAPDH Fo GAG TCA ACG GAT TTG GTC GT All the following steps (ex c e p t where mentioned, were carried out on ice).

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23 The RNA+Primer mix constituted of the RNA isolated f rom SY5Y Cells, 50 M oligo(dT) 10 mM deoxy ri bonucleotide triphosphate (dNTP) mix and diethylpyrocarbonate ( DEPC ) treated water in concentrations provided with the kit. The RNA+Primer mix was then transferred to 65C for 5mins (using the thermomixer) and then on ice for 2 min. The complimentary DNA ( cDNA ) Synthesis mix was now prepared with 10x RT buffer, 25 mM MgCl 2 0.1 M DTT, RNase OUT (40 M/ L ) and Superscript III RT (200 M/ L ). The concentrations used were as per the protocol provided with the kit. 10 L of the cDNA synthesis mix was now adde d to the 10 L of the RNA+Primer mix. The fi nal mix was now incubated at 50C for 50 mins and then at 85C for 5 min. The cDNA was stored at 20C until further use. The PCR reaction was now carried out using cDNA obtained from the RNA isolated from the ce lls at Days 0, 4, 5, 6 and 7 post induct L make a total reaction The PCR reaction was setup according to the followi ng steps. a) Denaturation at 94C for 2 mins. (b) Denaturation at 92 C for 15 secs, Annealing at 55C f or 30 secs and Elongation at 72C for 1 min. Step (b) was set for 35 cycle s. (c) Fi nal elongation at 72C for 7 mins. (d) The final PCR product was kept at 4C until used. The PCR products were then run in a 1% agarose electrophoresis gel. Quantitative PCR The quantitative PCR was carried out using SYBR Green PCR master mix from Applied B iosystems. The PCR mix contained: 12.5 specific the RT PCR of 400 ng of initial RNA from cells at the various time points in differentiation and

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24 ive PCR was run at 7500 fast 96 well setup. Comparative CT and standard 2 hr Q PCR settings were activated. The reference sample was Day 0 QPCR reaction and the control gene was GAPDH. The layouts of the Q PCR plates ( Figures 2 1 and 2 2) show that columns 1 to 6 contain the PCR amplification reactions of TH and tau genes and 6 12 for GAPDH gene respectively, e.g. row 1 consists of 6 copies of the negative control sample (without the cDNA) f or the TH gene amplification and 6 of negative control of GAPDH primer mediated PCR amplifications respectively. The rest of the rows contain 6 copies each of day 0, 4, 5, 6 and 7 cDNA PCR amplifications. Syn uclein GFP DNA Amplification Transformation Using SURE2 Cells Synuclein GFP DNA construct was provided by Dr. Eli e zer Masliah, UCSD (66) A 25 L aliquot of SURE 2 cells w as thawed on ice for 10 mins. 2 L ME was added to the aliquot and the mixture was allowed to thaw further for 20 mins. About 30 Synuclein GFP DNA were added to the aliquot in a 1 L volume. The aliquot w as now incubated on ice for 30 mins. The aliquot w as then heat pu lsed for a period of 30 secs in a 42 C water bath. The aliquot w as then kept on ice for 2 mins and 0.9 mL of SOC medium (at room temperature) was then added to it The aliquot w as finally incubated at 37C for 1 hour in a horizontal/ slanted position or ma ximum growth of the competent cells with shaking at 225 250 rpm. 200 L of culture f rom the aliquot w as plated on ( Isopropyl D thio galactoside) IPTG XGal ( F isher B io reagents) containing agar plate for transforming the S URE 2 cells with the experimental DNA The white colonies (containing the Syn GFP DNA construct) were picked from these plates after 14 hrs of incub ation of the plate at 37 C The colonies were grown in 35 mL of LB b roth + a mpicilin medium in 50 mL Corning screw cap tubes and were shaken in the

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25 orbital shaker for 14 16 hrs in the 37C incubator. 500 L of the culture was first removed and was mixed wit h 500 L of autoclaved g lycerol for making the glycero l stock which was frozen at 80C for the DNA amplification in future The rest of the culture was used for the Midiprep. M idiprep The Midiprep was carried out with the Qiagen Midiprep kit. The culture was first centrifuged at 5 000 rpm for 15 mins in a 50 mL Corning screw cap tube All the supernatant was removed. Pellet was resuspended in 8 mL of buffer P1. 8 mL of buffer P2 was then added and the tube w as immediately and gently inverted 5 6 times to m ix the solution. The tube was then kept at room temperature for 5 mins. 8 mL of buffer P3 was then added and the solution was mixed (by inversion) until it was clear with white particle s floating on the top. The tube was then centrifuged for 15 m ins at 8 0 00 rpm. Tip 100 column was first equilibrated by running 5 mL of the buffer QBT over it. The e ntire supernatant was loaded into the equilibrated Tip 100 filter column (Note: It is important that none of the white particles enter the column as they can bloc k it ). The column was then washed twice with buffer QC. The DNA was then eluted into a 15 mL Corning culture tube with 8 mL QF buffer. 5.6 mL isopropanol was then added and mixed to the eluted solution. The mixture was then microcentrifuged (in 2 mL Eppend orf tubes, in multiple batches, by removing the supernatant and adding 2 mL of the mixture), 10 min at 13 000 rpm. The pellet was then washed with 70% e thanol. The pellet was finally air dried for 10 mins in the 37 C incubator and then resuspended in 100 L of DNAse treated CellGro water. Syn uclein GFP DNA Once the amplification of the Syn GFP DNA was completed, the DNA was checked for the correct length using digestion analysis. The mixture of the 75 100 ng of the DNA, restriction enzyme E co R I (1 L ), restriction buffer (1 L ) and water to make the final volume of 10 L was prepared. The digestion was carried out at 37 C for 5 hrs, and then the reaction as stopped by

PAGE 26

26 incubation of the mixture at 75C for 20 mins. The DNA was then run on a 0.8% agarose electrophore sis gel to check for the expected band sizes post digestion with E co RI restriction enzyme. Cell V iability A ssay All cell viability assays were carried out in Nunc # 237105 black bottom 96 well micro plates. For this assay, the total number of cells initia lly plated on the wells was 15 000. The cells were counted with the h emacytometer from F isher scientific. CellTiter Glo luminescent cell viability assay from P romega was used to determine the cell viability of differentiated cells after 24 hours of pestic ide treatment s The following steps were followed for this assay. The o paque walled multi plates were prepared for the reaction and were used to differentiate the cells 7 days prior to the experiment and 70% of the differentiation media was changed every d ay. Control wells without the cells were prepared to obtain background/reference luminescence values. The CellTiter Glo buffer and the lyophilized CellTiter Glo s ubstrate w ere thawed and equilibrated at room temperature for 30 mins prior to the beginning of the assay. 5 mL of the given buffer was then added to the lyophilized CellTiter Glo substrate. The solution was then mixed well by vortexing for 5 7 mins on maximum speed. This form ed the CellTiter Glo reagent (Stored at 20 C in the amber bottle pro vided with the kit when not being used). Once the differentiation of the cells was complete, they were treated with pesticides and other environmental neurotoxins whose effect on viability was being checked with the CellTiter Glo assay. Now an equal volum e of the Cell Titer Glo reagent (100 L for 96 well plates) was added to the media in each well. The contents of the plate were then mixed in an orbital shaker for 2 mins to induce cell lysis. The plates were incubate d at room temperature for 10 mins to s tabilize the luminescent signal. The luminescence was finally recorded using a

PAGE 27

27 Luminometer from Bio Tek The integration time used was .25 1 second. Every sample had 4 replicates and the average luminescence value from these four wells was considered. Pest icide T reatment and Syn A ggregation Imaging of the cells was carried out in g lass bottom 96 well micro plates MGB096 1 2 LG L from M atrical The n umber of cells plated initially for these experiments w as 35 000. The cells were allowed to grow on the glass bottom plates for a period of 14 16 hrs. The cells were then transfected with the Syn GFP DNA construct using the Lipofactamine 2000 kit from I nvitrogen. Pools of plasmid DNA (400 ng per well) + Opti DMEM (25 L per well) mix and Lipid (1 L per well) + Opti DMEM (25 L p er well) were prepared according to the number of wells transfected. The pools were then kept at room temperature for 5 mins. The two pools were then mixed in one 2 mL E ppendorf tube or a 10 mL C orning cell culture tube by gentle swirling and kept at room temperature for 20 mins. 52 L of the pools was then added to each well containing cells to be transfected. The plates were then incubated for 24 hrs at the 37C 5% CO2 incubator. After 4 hrs of the transfection step, t he media in the wells was removed an d fresh media was added The cells were now differentiated before further treatment with pesticides Pesticide Treatment All the pesticides commonly used in the orange industry in Florida were bought from Sigma except for 2, 4, D isopropylamine a herbicid e that was obtained from Agri Star. Stock solutions of the pesticides at suitable concentrations and in suitable solvents ( Table 3 1 3 2 and 3 3 ) we re first prepared at 100 x or 1000 x the required concentrations and we re added in right proportions to the w ells containing differentiated and transfected SY5Y cells. The pesticide treatments were carried out and the cells were incubated in the in 37 C 5% CO2 incubator for 24 hrs for all the individual and combination treatments.

PAGE 28

28 Triple Immunostaining of SY5Y Ce lls Stock solution o f MitoT racker red CMXRos from P romega was prepared with DMSO to a final concentration of 1 mM. The solution was kept at 20C freezer and was covered in aluminum foil ( as the dye is sensitive to light ) and was thawed at room temperatur e 30 min prior to use. The working concentration of the MitoTracker ( 100 nm ) was prepared in the SY 5Y cell culture media. The Mito Tracker was added to the wells and was incubated in the 37C 5% CO2 incubator for 45 min s The plates were then exposed to a s less ambient light as possible. The cells were then washed with PBS twice over 5 7 min s The cells were then fixed in 200 L fixation solution per well (40% forma ldehyde in 1x PBS) and left at room temperature for 20 mins. The cells were then washed with PBT (0.3% TritonX 11 in 1x PBS), thrice over 5 10 mins. 200 L of bl ocking solution per well (0.5% bovine serum albumin (BSA) in PBT) was then added and the plates were left at room temperature for 30 mins. The primary antibody (r at monoclonal a nti GFP, S igma ) was added at 1:200 dilution to the wells and was kept at room temperature for 2 hrs. The cells were then washed with PBT solution thrice over 5 10 mins. The secondary antibody (Goat Anti rat fluorescein isothiocyanate ( FITC ) secondary antibody) was then added at 1:600 dilution and the cells were kept at room temperature for 30 mins. The cells were finally washed twice with PBT over 5 10 mins and the nucleus staining DAPI was then added at 1:1000 dilution in PBT. The cells were kept at room temperatu re for 10 mins before the PBT was replace d with 200 L PBS in each well. The cells were then ready to be imaged with triple staining of MitoTracker Syn GFP and DAPI.

PAGE 29

29 Pesticide Combination Treatment Determination of Aggregation Negative Diuron Concentration The pesticides that were found to be capable of inducing Syn aggregation in the differentiated SY5Y cells were called aggregation positive and the rest were called aggregation negative pesticides. In this experiment, the a ggregation positive herbicide diuron was used as a sensor for combinat orial pesticide treatment. To find the concentration of diuron to be used as the sensor in the combinat oria l pesticide treatment we added 1/ 10 0, 1/ 5 0, 1/ 20, 1/10 and 1/5 times of the treatment concentration of diuron (1M) i.e. 10 nM, 20 nM, 5 0 nM 100 nM and 200 nM respectively to the transfected and differentiated cells From this we found the concentration of diuron that was incapable of causing any aggregation of Syn Combination Treatment We added the 10 nM of the herbicide d iuron ( determined from the previous experiment to be aggregation negative ) with the treatment concentrations of all the insecticide s, herbicides and fungicides from T able 3 1 3 2 and 3 3 Following the same steps of the individual pesticide s induced aggregation experiments these combinations of the 21 pesticides and diuron in the concentration at which it is incapable of inducing ag gregation of Syn were added to the differentiated SY5Y cells expressing the Syn GFP DNA construct. The imaging and analysis of the cells post pesticide combination treatments were done in the exact same way as for the in dividual pesticides. Statistical A nalysis Cel l Viability Data Analysis T he CellTiter Glo system produces luminescence corresponding to the number of cells in each well of the cell culture plate. The lumin escence data was collected as four replicates of e ach individual treatment. The final value of the cell viability was calculated using the mean of

PAGE 30

30 the four luminescence value s with the stand deviations shown as error bars The mean luminescence value of every treatment was normalized to the mean luminescence value of the NO pesticide treatment wells This way we could analyze the difference in the number of cells in the wells induced due to the various treatments. Syn Aggregation Experiments Data Analysis The cells were treated with the toxins (both individually and in combinations) at particular c oncentrations i n triplicate s. After the cells in the plates were treated with the toxins for 24 hrs, fixed and immunostained, we took approximately 10 15 images of each well. Approximately 5 Syn GFP aggregates were counted according to the three size categories. We first obtained the aggregation data for the NO pesticide treatment wells ( n egative control treatment). T he mean percentage of the 3 categories of aggregates induced due to the nega tive con trol treatments was obtained along with the mean percentage of expression of the protein in the diffuse form throughout the cells along with the standard deviation values We then calculated the mean and standard deviation values of the number of Syn aggregates of each size category induced due the treatment s of the cells with the various pesticide s and plotted the comparison graphs between the experimental and the negative control treatment of the differentiated SY5Y cells.

PAGE 31

31 Figure 2 1 Q PCR s etup for Tyrosine Hydroxylase gene expression in differentiating SY5Y cells (Reference/Housekeeping gene GAPDH) Figure 2 2 Q PCR setup for tau gene expression in differentiating SY5Y cells (Reference/Housekeeping gene GAPDH)

PAGE 32

32 CHAPTER 3 RESULTS Differen tiation of SY5Y Cells PD specifically affects the dopaminergic neurons of the brain. In order to make the SH SY5Y human neuroblastoma cells a relevant model for this PD study, we needed the cells to exhibit characteristic features of dopaminergic neurons. Differentiated SY5Y cells ha d been shown before to express properties typical of dopaminergic neurons (65) This process of differentiation of the SH SY5Y cells to a dopaminergic neuron like cell culture was induced by reducing th e concentration of FBS and introduction of all trans retinoic acid in the cell culture media. The f ollowing changes were observed to determine the number of days taken for the acquisition of dopaminergic characteristics post introduction of the differentiation conditions. Morphological Changes The ex pected morphological change during t he dopaminergic neuronal differentiation of SY5Y c ells is extensive neurite growth. T hese changes were visible starting from the d ay 2 post differentiation w ith no further difference s in the morphology through days 4, 5, 6 and 7 post differentiation (Figure 3 1 ). Gene Expression Q PCR The number of days taken for the SY5Y cells to undergo dopaminergic neuronal differentiation could not be determined precisely by the morphological changes of the cells. We then used gene expression analysis of two genes whose expression is expected to increase during differentiation. Tyrosine hydroxylase (TH) is the rate limiting enzyme of dopamine synthesis and tau is the microt ubule stabilizing protein necessary for neurite formation during differentiation. We amplified the two genes to determine the degree of dopaminergic differentiation. GAPDH

PAGE 33

33 (Glyceraldehyde phosphate dehydrogenase) was used as the housekeeping gene, i.e. it is expected to show no change in the expression level during differentiation. The quantitative RT PCR was carried out to quantif y the relative change of expressions of TH and tau genes with respect to GAPDH at time points day 0, 4, 5, 6 and 7 post differen tiation. The expression of the TH and tau w as normalized to d ay 0. The Q PCR data confirm s that the levels of TH and tau are the highest seven days post differentiation (Figures 3 2 and 3 3 ) The gene expression of TH on d ay 7 is approximately three times the expression on d ay 6. With t hese experiments our assay conditions were setup and SY5Y cells were allowed to differentiate for 7 days before the pesticide treatments. Common Pesticides Used in Orange Industry The aim of this project was to dev elop a cell based assay to analyze and quantify the ability of pesticides used in the orange industry of Florida to induce aggregation of Syn in differentiated SY5Y cells We obtained r eports with the list of pesticides used by the orange industry farmers of Florida over the last 8 10 years from Dr. Davis Daiker and Dr. Dennis Howard, Florida D epartment of A griculture and C onsumer S ervices We could narrow down a list of 7 herbicides, 8 insecticides and 7 fungicides from these reports that were being used most common ly b y the farmers of the state of Florida each year. Tables 3 1, 3 2 and 3 3 show the list of orange industry fungic ides, herbicides and insecticides that we tested using our assay. Cell Viability Data Before we could start the aggregation experiment, i t was important for us to find the concentrations of these pesticides that were cytotoxic to the day 7 differentiated S Y5Y cells in our as say so that we could use a safe non toxic optimal concentration for evaluating aggregation

PAGE 34

34 of Syn protein in the cells. The optimal treatment concentrations were determined by a cell viability as say The CellTiter Glo system from P rom ega works by causing cell lysis and production of a luminescent signal proportional to the ATP content in the cell culture thus providing an indirect measure of the number of cells We first determined the luminescence values of the blank wells (without c ells) and positive control wells (with cells and No pesticide treatment) so that we could obtain the baseline For the orange industry pesticides, we chose treatment concentrations of 0.1, 1, 5, 10 and 100 data. Considering the concentration of pesticide at which we observed more than 30 40% loss of cells as toxic, w e found the treatment concentration of each pesticide safe to be used in the aggregation experiments In the following sections, we show the toxic concentrations and the treatment concentrations of the pesticides that we found from our experiments Positive Co ntrol s As positive control s we used four pesticides that ha d been previously reported to induce Syn protein aggregation. These pesticides were rotenone, paraquat, dieldrin and MPP+. Rotenone: The number of cells reduced to approximately 60% of the initial value after the 24 hr treatment with 0.1 but the toxicity does not significantly i ncrease due to increase in concentrations to 2.5 Based on its toxicity t he concentration used in the (Figure 3 4 A ) Due to p araquat treatment t he number of cells reduced to approximately 60% of the initial after the 2 Based on its toxicity t he concentration used in (Figure 3 4 B) There was a sudden drop in the number of cells to close to 0 M treatment of dieldrin. Based on its toxicity t h e concentration used in

PAGE 35

35 (Figure 3 4 C ) The toxicity of MPP+ was only significantly visible at the 5 treatment. Based on its toxicity t he concentration used in the aggregation experiments was 100 (Figure 3 4, D) Orange Industry Fungicides Induced Cell Toxicity M efonoxam, azoxystrobin and sodium phenylphenate caused complete cell death within our test concentrations at 5, 100 and 100 Based on the toxicity of these fungi cides, the ir concentration used for the three aggregation experiments were 0.1 (Figures 3 5 ) Fungicides copper sulfate and copper hydroxide were not as toxic as the first set of fungicides but caused 50 70% reduction in the number of cells within our t est concentrations of 0.1 to 100 Based on the toxicity of these fungicides, t he ir concentration used in both the aggregation experi (Figures 3 6 ) Fungicides thiabendazole and imazalil were the least toxic as they reduced the number of ce lls by only 20 30% within our test concentrations Based on the toxicity of these fungicides, their concentrations used in the aggregation experiments were 1 and 0.1 (Figure 3 6 ) Orange Industry Insecticides Induced Cell T oxicity The next class of orange industry pesticides of Florida that were tested for cell toxicity using the cell viability assay was insecticides. A bamectin, pyridaben and fenbutatin oxide were highly toxic and killed 80 95% of the cells within our test concentrations of 0.1 to 100 Based on the toxicity of these insecticides, t he ir concentrations used in the aggregation experiments were 0. (Figure 3 7 ) D iflubenzuron, carbar yl, chloripyrifos and sulfur were not as toxic as the first set but reduced th e total number of cells by 30 60%. Based on the toxicity of these insecticides, t he ir concentrations used in the aggregation experiments were 1, 0. 1, 1 and (Figures 3 7 and 3 8 ) A ldicarb was the least

PAGE 36

36 toxic in the insecticides as it was able reduce the number of cell s only by approximately 20%. The concentration of this insecticide used in t he aggregation experiment was 1 (Figure 3 8 ) Orange Industry Herbicides Induced Cell Toxicity The next class of orange industry pesticides of Flor ida that were tested for cell toxicity using the cell viability assay was herbicides. The herbicides norflurazon a nd simazine were the most toxic, as they could reduce the number of cells by approximately 60% of the initial value. Based on the toxicity of these herbicides, t he ir concentrations used in the aggregation experiments were 0. 1 and 1 M respectively (Figure 3 9 ) The herbicides Bromacil, Diuron and Glycophosphate were not as toxic as the first set of herbicides, although they reduced the number of cells by 40 50% of the initial value. Based on the toxicity of these insecticides, t he ir concentration used in the three aggregation experiments was 1 M (Figures 3 9 and 3 1 0 ) The herbicide 2, 4, D isopropylamine was the least toxic of the 7 herbicides tested (including Paraquat, data shown in the positive controls section) as it could reduce the number of cells by only approximately 25%. The concentr ation of this herbicide used in the aggregation experiments was 10 M (Figure 3 9 ) O range Industry Pesticides Ind uce Syn Aggregation The next part of the project was to screen the set of 2 1 pe sticides with our assay and to Syn aggregation. The pesticide induced aggregation assay was divided into the following phases : a) transfection of the SY5Y cells with Syn GFP DNA, b) addition of differentiatio n media to the SY5Y cell culture and differentiating the cells over 7 days, c) treatment of the differentiated cells with pesticides for 24 hrs and d) immunostaining and imaging the cells using fluorescence microscopy. The Syn GFP construct was kindly pr ovided to us by Dr. Eliezer Masliah ( UCSD ) (66) and was amplified by transformation purified by Midiprep and analyzed by digestion analysis.

PAGE 37

37 The transfection efficiency and continuous expression of the Syn GFP gen e over the differentiation was tested using No pesticide treated control cells. Once the transfection of the DNA into our SY5Y cells was carried out and the day 7 differentiation of the cells was complete, we treat ed the cells with the optimal concentratio ns of each pesticide. All the treatments were carried out in triplicates to avoid experimental variability and to obtain quantifiable data After the pesticide treatments for 24 hr the cells were fixed and immunostained with triple stain ing : MitoTracker : Specifically stains the mitochondria. This staining was done in order to visualize the mitochondrial damage GFP antibody staining: Syn GFP construct was visible in the live cells, the signal was very weak an d quenched in a few seconds Using the antibody staining against GFP, we could obtain a more robust signal that enabled us to obtain clear images of the cells. DAPI: 4',6 diamidino 2 phenylindole is a fluore scent marker that stains the AT rich regions of t he DNA and is used as a nucle ar dy e. DAPI was used for observing changes in the nucleus of the cells by the 24 hours of treatments as the nucleolus disintegrates in highly stressed/dead cells. The following section shows the quantified data of Syn aggregation s due the treatments with various pesticides. The quantification of all the aggregates induced by various pesticide treatments were based on size. The aggregates in the differentiated SY5Y cells were counted according to their diameters an d were split into three categories 0.5 diameter.

PAGE 38

38 Negative Control (No Pesticide Treatment) : In the negative controls we observed that approximately 90% of the cells accumulated Syn protein in its diffuse form (Figure 3 1 1 ) App roximately 5% of the cells exhibited small aggregates size Figure s 3 19 and 3 2 0 show two representative examples of the cells. Positive Control Environmental Neurotoxins: These are the compounds that are Syn aggregation. Rotenone and d ieldrin showed that approximately 70% and 65% of the aggregates were 0.5 respectively (Figures. 3 1 2 A and C ) We conclude that these pesticides predominantly induce small and medium sized aggregates (Figures 3 2 1 and 23) (Figure 3 1 2 B ) We conclude that paraquat produc es larger aggregates along with less er small and medium sized aggregates (Figures 3 2 2 ) and change in mitochondrial morphology to globular structures representing damage MPP+ showed that approximately 30% of the aggregates were 0.5 (Figure 3 1 2 D ) We conclude that MPP+ predominantly produces Syn protein (Figures 3 2 4 ) Both paraquat and MPP+ produced a change in the mitochondrial morphology from a healthy fragmented hair like pattern seen in negative control no p esticide treatments, to an aggregated and round appearance representing damage Fungicide Induced Syn Aggregation Syn aggregation, we followed the same procedure as described in the posit ive controls. Four out of the seven

PAGE 39

39 fungicides tested showed ability to induce Syn aggregation in our experiments. The rest of the fun gicides were unable to cause aggregation. Mefonoxam and thiabendazole showed similar patterns. Approximately 50% and 45% of the aggregates were 0.5 the fungicides respectively (Figure 3 1 3 A and C) We conclude that they are capable of producing larger aggregates along with small and medium sized aggregates o n statistically significant level (Figures 3 25 and 3 27 ) Azoxystrobin and imazalil showed that approximately 50% and 70% of the aggregates were 0.5 respectively (Figure 3 1 3 B and D) We conclude that these fungicides produce predominantly small and medium sized aggregates (Figures 3 26 and 3 28 ) Imazalil treatment also changed the mitochondrial morphology from a healthy appearance seen in negative control no pesticide treatmen ts, to a round and aggregated appearance. Insecticide Induced Syn Aggregation Three out of the eight insecticides tested showed the ability to induce Syn aggregation in our experiments. Diflubenzuron and pyridab en showed similar patterns. Approximately 80% and 98% of the aggregates were 0.5 1 (Figure 3 1 4 A and B ) We conclude that these insecticides predominantly produce small aggregates of Syn protein (Figures 3 29 3 3 0 ) Carbaryl showed a different aggregation pattern as compared to the other inse cticides. Approximately 45% of the aggregates induced were 0.5 ( Figure 3 1 4 ( C) We conclude that these insecticides predominantly produce small

PAGE 40

40 Syn protein but also produce a signific ant percentage of larger aggregates (Figures 3 31 ) Herbicide Induced Syn Aggregation Syn aggregation in our experiments. 2, 4 D isopropylamine salt showed that approximately 85% of the aggregates were 0.5 1 (Figure 3 1 8 A) We conclude that this herbicide induces predominantly small aggregates (Figures 3 32 ) Norflurazon showed that approximately 30% of the aggregates were 0.5 were 1 (Figure 3 1 5 B ) We conclude that this herbicide induces predominantly medium sized aggregates (Figures 3 33 ) Syn protein. Approximately 15% of the a ggregates were 0.5 (Figure 3 1 5 C) (Figures 3 34 ) Combination Pesticides Induced Syn Aggregation In order to observe any changes in the pattern or the ability Syn aggregation induc tion by the 21 orange industry pesticides and to mimic the conditions in the farms where the pesticides are sprayed in combinations we carried out two pesticide combination treatments. Here we tested all fungicides, insecticides and herbicides in combinations wi th a low concentration of the Syn aggregation that we found from our experiments, diuron. Aggregation Negative Diuron Concentration Aggregation negative concentration of diuron was that concentration at which it was Syn aggregation in ou r model. During the individual pesticide treatment

PAGE 41

41 the herbicide diuron as the treatment concentration and observed the induction of maximal Syn aggregation In order to find the aggregation negative concentration of diuron, w Syn protein at a 10 nM, 20 nM, 50 nM, 100 nM and 200 nM of diuron. W e found th is concentration to be 10 nM (Figure 3 4 0) We used this concentration of diuron in the combination treatments of pesticides Combination Treatment of Pesticides 10 nM of diuron was added to the treatment concentration of all the insecticides, fungi cides and herbicides ( T able s 3 1, 3 2 and 3 3) Syn GFP expressing differentia ted SY5Y cells were then prepared and treated with these combinations following all the steps similar to the individual pesticide treatments. We observed that two insecticides and two herbicides that Syn aggregatio n were now capable of the same. These were insecticides abamectin and sulfur, and herbicides simaz ine and glycophosate ( Figure s 3 35 to 3 3 9 ) These aggregations were quantified and analyzed. We observed that the insecticides abamectin and sulfur induced 6 0% and 70% of aggregates between 0.5 to 1 M and 40% and 30% of aggregates between 1 2 M in size respectively (Figure 3 1 7 ) Herbicides simazine and glycophosate induced 55% and 95% of aggregates between 0.5 and 1 M and 45% and 5% of aggregates between 1 2 M in size respectively (Figure 3 1 8 ) We also observed that the herbicide 2,4 D isopropylamine salt that predominantly induced aggregates of the size 0.5 during the individual treatment now induced approximately 60% 1 2 zed aggregates respectively during the combination treatment e xperiment as seen in Figure 3 35. Quantification of t he increase d seen in Figure 3 1 8 (A)

PAGE 42

42 Syn aggregations could also cause the change in the mitochondrial morpholog y from the normal appearence seen in negative control no pesticide treatments, to a round and aggregated appearance. The rest of the pesticides from T ables 3 1, 3 2 and 3 2 were unable to induce aggregation both during the individual treatments and the co mbination treatments of pesticides. Syn Aggregation Syn aggregation in Syn observed due to these treatments was predo minantly diffuse Syn aggregation non inducing herbicide, Bromacil (Figure 3 Syn expression due to these treatments and it showed similar patte rn in all the pesticides incapable of inducing Syn aggregation (Figure 3 16).

PAGE 43

43 Figure 3 1 Morphological changes during differentiation of SY5Y cells Figure 3 2 Q PCR data: Tyrosine Hydroxylase gene expression analysis Statistical sig nificance 0.005
PAGE 44

44 Figure 3 3 tau gene expressions between two adjacent days. p >0.05, ** 0.005
PAGE 45

45 Figure 3 4 Cell viability data from 24 hr treatments of differentiated SY5Y cells with positive control environmental neurotoxins. pesticide treatment ( 0 M concentrat ion ). p>0.05, ** 0.0050.0 5, ** 0.005
PAGE 46

46 Figure 3 6 Cell viability data from 24 hr treatments of differentiated SY5Y cells with fungicides used in orange industry treatment (No drugs). p>0.05, ** 0.005

0.05, ** 0.005
PAGE 47

47 Figure 3 8 Cell viability data from 24 hr treatments of differentiated SY5Y cells with i nsecticides used in orange industr y. pesticide treatment (No drugs). p>0.05, ** 0.0050.05, ** 0.005
PAGE 48

48 Figure 3 1 0 C ell viability data from 24 hr treatments of differentiated SY5Y cells with herbicides used in orange industry pesticide treatment (No drugs). p>0.05, ** 0.005
PAGE 49

49 Table 3 2 S yn aggregation P esticide F amily Solubility Concentration to be tested (M) Paraquat Bipyridylium family Water 0.5 2,4 D, isopropylamine salt Cholorphenoxy acid or ester Water 10 Bromacil Uracil family Water 1 Norflurazon Fluorinated Pyridazinone Family Acetone 0.1 Simazine s triazine herbicides family Acetone 1 Diuron Substituted urea family Acetone 1 Glyphosate Glycophosphate family Water 1 Table 3 3 Syn aggregation P esticide F amily Solubility Concentration to be tested ( M) Abamectin Avermectin Family Acetone 0.1 Diflubenzuron Substituted urea Family Water 1 Pyridaben Pyridazinone family Acetone 1 Fenbutatin oxide Organotin family Acetone 0.1 Sulfur Unknown Acetone 1 Carb aryl Carbamate family Water 0.1 Aldicarb Carbamate ester family Water 1 Chlorpyrifos Organophosphates family Water 1

PAGE 50

50 Figure 3 1 1 Syn aggregation analysis ; 24 hr Negative control NO Pesticides treatment Figure 3 1 2 Syn aggregation analys is ; 24 hr treatment A) Rotenone B) Paraquat C) Dieldrin D) MPP+

PAGE 51

51 Figure 3 1 3 Syn A) Mefonoxam B) Azoxystrobin C) Thiabendazole D) Imazalil

PAGE 52

52 Figure 3 1 4 Syn A) Diflubenzuron B) Pyridaben C) Carbaryl

PAGE 53

53 Figure 3 1 5 Syn A) 2,4 D B) Norflurazon C) Diuron Figure 3 16 Syn aggregation non in ducing herbicides; 24 hr treatment

PAGE 54

54 Figure 3 1 7 Combination Syn aggregation analysis; 24 hr treatment A) Insecticide abamectin + herbicide diuron B) Insecticide Sulfur + herbicide diuron Figure 3 1 8 Combination pes Syn aggregation analysis; 24 hr treatment A) Herbicide 2, 4 D + diuron B) Herbicide Simazine + diuron C) Herbicide Glycophosate + diuron

PAGE 55

55 Figure 3 19 Negative control No pesticide treatment on cells for 24 hrs; Cell 1 Figure 3 20 Negative control No pesticide treatment on cells for 24 hrs; Cell 2 Figure 3 2 1 Rotenone induced Syn aggregation Synuclein GFP MitoTracker DAPI Merged Synuclein GFP MitoTracker DAPI Merged Synuclein GFP MitoTracker DAPI Merged

PAGE 56

56 Figure 3 2 2 Paraquat induced Syn aggregation Figure 3 2 3 Dieldrin induce d Syn aggregation Figure 3 2 4 MPP+ induced Syn aggregation Synuclein GFP MitoTracker DAPI Merged Synuclein GFP MitoTracker DAPI Merged Synuclein GFP MitoTracker DAPI Merged

PAGE 57

57 Figure 3 2 5 Fungicide mefonoxam induced Syn aggregation Figure 3 2 6 Fungicide azoxystrobin induced Syn aggregation Fig ure 3 2 7 Fungicide Thiabendazole induced Syn a ggregation Synuclein GFP MitoTracker DAPI Merged Synuclein GFP MitoTracker DAPI Merged Synuclein GFP MitoTracker DAPI Merged

PAGE 58

58 Fig ure 3 2 8 Fungicide Imazalil induced Syn aggregation Fig ure 3 2 9 Insecticide Diflubenzuron induced Syn aggregation Fig ure 3 30 Insecticide Pyridaben induced Syn aggregation Synuclein GFP MitoTracker DAPI Merged Synuclein GFP MitoTracker DAPI Merged Synuclein GFP MitoTracker DAPI Me rged

PAGE 59

59 Fig ure 3 3 1 Insecticide Carbaryl induced Syn aggregation Fig ure 3 3 2 Herbicide 2, 4 D induced Syn aggregation Fig ure 3 3 3 Herbicide Norflurazon induced Syn aggregation Synuclein GFP MitoTracker DAPI Merged Synuclein GFP MitoTracker DAPI Merged Synuclein GFP MitoTracker DAPI Merged

PAGE 60

60 Fig ure 3 3 4 Herbicide Diuron induced Syn aggregation Fig ure 3 3 5 Herbicide 2, 4 D + Diuron comb ination induced Syn aggregation Fig ure 3 36 Herbicide Simazine + Diuron combination induced Syn aggregation Synuclein GFP MitoTracker DAPI Merged Synuclein GFP MitoTracker DAPI Merge d Synuclein GFP MitoTracker DAPI Merged

PAGE 61

61 Fig ure 3 3 7 Herbicide Glycophosate + Diuron combination induced Syn aggregation Fig ure 3 3 8 Insecticide Abamectin + Diuron c ombination induced Syn aggregation Fig ure 3 3 9 Insecticide Sulfur + Diuron combination induced Syn aggregation Synuclein GFP MitoTracker DAPI Merged Synuclein GFP MitoTracker DAPI Merged Synuclein GFP MitoTracker DAPI Merged

PAGE 62

62 Figure 3 40 Herbicide Diuron 10 nM treatment unable to induce Syn aggregation Figure 3 41 Herbicide s incapable of inducing Syn aggregation Synuclein GFP MitoTracker DAPI Merged Synuclein GFP MitoTracker DAPI Merged

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63 CHAPTER 5 DISCUSSION PD is a complex neurodegenerative disorder with multiple etiologies. Environmental neurotoxins are the latest addition to the list of causative agents proposed for P D One of the most important connections between envir onmental neurotoxins and PD pathogenesis was found to be Syn aggregation C ytosolic aggregates of this synaptic protei n in the dopaminergic neurons are a characteristi c feature of PD pathogenesis (28 31) The rationale behind our work was to use this connection in developing a model to screen environmental neurotoxins to report their prospective role in Syn aggregation Epidemiological surveys have consistently linked rural living and the agricultural i ndustr y with increased risk of PD raising questions about the possible relation between specific environmental factors and PD (67) Pesticides from different chemical families like organ ochlorides (e.g. dieldrin), complex ketones (e.g. rotenone) and Syn aggregation and PD like symptoms in various animal models (4,68) This made us wonder if pesticides from other families Syn aggregation. Give n that t his work was partly funded by a family of orange farm ers with high prevalence of PD cases w e wanted to determine if the most common pe sticides used in the orange industry of Florida could Syn aggregation which would in turn explain the high incidence of PD among individuals exposed to these environmental toxins PD specifically target s dopaminergic neurons of the brain which s how characteristic Syn As our aim was to observe the changes in the distribution of Syn with a GFP tag for fluorescence imaging, we wanted to use a simple cell culture model that c ould be differentiated to exh ibit dopaminergic neuron like characteristics. The SK N SH derived SH SY5Y human neuroblastoma cells were best suited for this as it was

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64 evident from previous studies that this cell line showed neuronal characteristics upon diff erentiation with retinoic ac id (65) Syn GFP c DNA and differentiation for seven days using the combined effect of reduction of FBS and addition of retinoic acid, our Syn aggregation inducers The first evidence for this high sensitivity was that the positive control pesticides (rotenone, paraquat, dieldrin and MPP+) Syn aggregation according to diameters of the aggregates From the individual treatments of the most commonly used orange industry pesticides of Florida, we saw that 1 0 out of the 2 1 pesticides tested positive for Syn aggregation. T he fact more than 50% of the pesticides tested c aus ed one of the pathological h allmarks of PD was very interesting and somewhat surprisin g This finding demonstrated that pesticides from other biochemical families could also Syn aggregation. One of the main reasons which we considered responsible for this large number of positives was that we tested a collection of very effective p esticides Many pesticides inhibit regular cell function by mitochondrial and p roteosomal damage and it is not surprising that they are toxic to humans. The other possible factor was that our system consisted of a Syn protein which made it not very surprising that it behaved really sensitive to specific small changes in its environment to cause Syn aggregation. In order to mimic the process of pesticide usage in the farms and therefore the presence of these pesticides in th e se environment s we wanted to carry out combination treatments of the pesticides. W e treated the differentiated SY5Y cells with combinations of all 2 0 pesticides with a Syn aggregation inducer, the herbicide diuron The effectiveness of diuron as a herbicide has also ma de farmers from various agricultural industries

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65 to use it in many combinations with other pesticides. From th ese experiment s we reported four more pesticides (two insecticides and two herbicides) tha Syn aggregation and one herbicide that induced larger Syn aggregates. These experiments gave us a count of 14 orange industry pesticides out of 21 tested Syn aggregation. Although the mechanism of dopaminergic n euronal cell death in PD is still unclear oxidative stress and mitochondrial dysfunction are known to occur in PD affected brains (69 71) Evidences like enhanced mitochondrial pathology in the substantia nigra of Syn transgenic mice and occurrence of abnormally swollen and rounded m orphology of mitochondria in PD affected human brains have confirmed the mitochondrial damage during PD (72 74) To visualize morphological changes in mitochondria w e immunostain ed the differentiated SY5Y Syn GFP with a mito chondrial stain We observed that out of the 21 orange industry pesticides tested, all the herbicides and one fungicide Syn aggr egation coul d induce a swollen and rounded appearance of mitochondria. It would be very interesting to answer the important question of whether these pesticides have the capability of causing mitochondrial damage owing t o their biochemical structures free radical pro duction etc. by future studies Syn aggregation is very significant for better understanding of the link of PD to rural living and agricultural activity. However, the evidence collected in the cell culture model needs to be replicated in vivo It is important thus, that all the reported pesticides from this study be further tested with rodent models for their ability to induce classic PD symptoms. The findings from our work have been so promising that we believe that our assay should be u sed to evaluate different combinations of pesticides as well as other environmental toxins encounter ed in environment s such as agricultural and metallurgic

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66 industries and rural living We see our work as an important step in explaining the role of pesticid e exposure in rural sporadic occurrences of the most common movement disorder in the world, PD

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67 APPENDIX A SYN AGGREGATION I NDUCING P ESTICIDES Table A Syn Aggregation Inducing Pesticides P esticide Molecular formula Structure Paraquat C 12 H 14 N 2 2,4 D, isopropylamine salt C 11 H 15 Cl 2 NO 3 Norflurazon C 12 H 9 ClF 3 N 3 O Diuron C 9 H 10 Cl 2 N 2 O Simazine C 7 H 12 ClN 5 Glyphosate C 3 H 8 NO 5 P

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68 Table A 1. Continued Pesticide Molecular formula Structure Abamectin C 48 H 72 O 14 Diflubenzuron C 14 H 9 ClF 2 N 2 O 2 Pyridaben C 19 H 25 ClN 2 OS Sulfur S 8 Carbaryl C 12 H 11 NO 2

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69 Table A 1. Continued Pesticide Molecular formula Structure Mefonoxam C 15 H 21 NO 4 Azoxystrobin C 22 H 17 N 3 O 5 Thiabendazole C 1 0 H 7 N 3 S Imazalil C 14 H 14 Cl 2 N 2 O Rotenone C 23 H 24 O 6 Dieldrin C 12 H 8 Cl 6 O

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70 Table A 1. Continued Pesticide Molecular formula Structure MPP+ C 12 H 12 IN

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71 APPENDIX B SYN AGGREGATION N ON I NDUCING P ESTICIDES Table B Syn Aggregation Non Inducing Pesticides Pesticide Molecular formula Molecular Structure Bromacil C 9 H 13 BrN 2 O 2 Fenbutatin oxide C 60 H 78 OSn 2 Aldicarb C 7 H 14 N 2 O 2 S Chlorpyrifos C 9 H 11 Cl 3 NO 3 PS Copper Sulfate CuSO 4 Copper Hydroxide CuH 2 O 2 Sodium Phenylphenate C 12 H 9 NaO

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77 BIOGRAPHICAL SKETCH Born in Tamil Nadu, India, Giridhar Murlidharan did his schooling in New Delhi but SASTRA University beckoned him back to Tamil Nadu for his B.Tech (Engineering) in Biotechnology. He had the privilege of doing sho rt term projects at two of the prestigious institutes in India, All India Institute of Medical Sciences, New Delhi and Indian Institute of Technology, New Delhi as a part of his B.Tech curriculum. He wistfully recalls his two year MS in b iomedical engineer ing UF starting from fall 2009 as the best period in his career thus far. About to complete his MS thesis wo rk at the Fernandez Funez and Rincon Limas laboratory, Department of Neurology, UF, h e is embark ing on his PhD Biological and Biomedical Sciences i nterdisciplinary program starting fall 2011 at University of North Carolina, Chapel Hill. While genetics and molecular biology have been his areas of research interest, he is an avid sports fan and plays cricket, basketball and volleyball and besides being a playstation buff he is extremely fond of music.