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Gene Expression Analysis during West Nile Virus Disease, Infection, and Recovery

Permanent Link: http://ufdc.ufl.edu/UFE0041523/00001

Material Information

Title: Gene Expression Analysis during West Nile Virus Disease, Infection, and Recovery
Physical Description: 1 online resource (247 p.)
Language: english
Creator: Bourgeois, Melissa
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: brain, central, encephalitis, gene, microarray, transcriptome, west
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Gene Expression Analysis During West Nile Virus Disease, Infection, and Recovery It was the goal of this project to profile gene expression during West Nile virus (WNV) infection in the central nervous system (CNS) of horses. It was hypothesized that there are gene pathways whose expression changes in a significant and consistent manner due to WNV as a result of exposure status, survival/immune status, and CNS location. To test this hypothesis, the equine CNS transcriptome was sequenced, these sequences were used to create a custom microarray, and this array was used to analyze gene expression in the thalamus and cerebrum of three different groups of horses (nai umlautve/WNV exposed, vaccinated/WNV exposed, and normal). Statistical and pathway analysis was performed on this data to identify genes and gene pathways of interest. In total, 41,040 genes and contigs were sequenced and annotated from the transcriptome- 1,280 of which were novel to the equine genome project. Significant differences (p < 0.05) in gene expression were seen due to exposure in 9,020 genes, survival in 7,395 genes, and location in 7,649 genes. Pathways analysis revealed that many genes mapped to neurological and immunological categories, which were found to be downregulated in WNV infection. Detailed analysis of the immunological pathways revealed that both innate and adaptive components of the immune response were involved in the response to WNV infection, and that higher levels of expression of these pathways were correlated with survival. PTX3 (pattern recognition response to viruses), interleukin-15 production, and the JAK/STAT pathway were found be be upregulated by WNV infection. Infection with WNV also led to an increase in the expression of SOCS3, a negative feedback inhibitor of the JAK/STAT pathway, possibly reflecting evasion of innate immunity by the virus. Apoptosis was found to be upregulated due to WNV infection, providing expression level evidence of neuropathology due to viral infection. Transcriptional genes also demonstrated changes in expression levels due to WNV infection. Detailed analysis of neurological pathways revealed that transcripts in both the glutamate and dopamine signaling pathways were decreased in expression in the WNV infected brain, providing evidence of glutamate excitotoxicity and pathology associated with a lack of dopamine. In addition, many of the transcripts mapped to non-infectious neurological disease functions, including mental disorders and degenerative neuropathies, suggesting a correlate between the neuropathology induced by viral infection of the CNS and the neuropathology seen in non-infectious neurological disease. This project provided novel insights into global gene expression during WNV infection, and led to a better understanding of how the CNS responds to viral infection. Confirmation studies looking at individual transcripts of interest will be performed. This data will be used to contribute to potential therapeutics and diagnostic options for WNV and other viral encephalitides.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: 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 Melissa Bourgeois.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Long, Maureen T.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041523:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041523/00001

Material Information

Title: Gene Expression Analysis during West Nile Virus Disease, Infection, and Recovery
Physical Description: 1 online resource (247 p.)
Language: english
Creator: Bourgeois, Melissa
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: brain, central, encephalitis, gene, microarray, transcriptome, west
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Gene Expression Analysis During West Nile Virus Disease, Infection, and Recovery It was the goal of this project to profile gene expression during West Nile virus (WNV) infection in the central nervous system (CNS) of horses. It was hypothesized that there are gene pathways whose expression changes in a significant and consistent manner due to WNV as a result of exposure status, survival/immune status, and CNS location. To test this hypothesis, the equine CNS transcriptome was sequenced, these sequences were used to create a custom microarray, and this array was used to analyze gene expression in the thalamus and cerebrum of three different groups of horses (nai umlautve/WNV exposed, vaccinated/WNV exposed, and normal). Statistical and pathway analysis was performed on this data to identify genes and gene pathways of interest. In total, 41,040 genes and contigs were sequenced and annotated from the transcriptome- 1,280 of which were novel to the equine genome project. Significant differences (p < 0.05) in gene expression were seen due to exposure in 9,020 genes, survival in 7,395 genes, and location in 7,649 genes. Pathways analysis revealed that many genes mapped to neurological and immunological categories, which were found to be downregulated in WNV infection. Detailed analysis of the immunological pathways revealed that both innate and adaptive components of the immune response were involved in the response to WNV infection, and that higher levels of expression of these pathways were correlated with survival. PTX3 (pattern recognition response to viruses), interleukin-15 production, and the JAK/STAT pathway were found be be upregulated by WNV infection. Infection with WNV also led to an increase in the expression of SOCS3, a negative feedback inhibitor of the JAK/STAT pathway, possibly reflecting evasion of innate immunity by the virus. Apoptosis was found to be upregulated due to WNV infection, providing expression level evidence of neuropathology due to viral infection. Transcriptional genes also demonstrated changes in expression levels due to WNV infection. Detailed analysis of neurological pathways revealed that transcripts in both the glutamate and dopamine signaling pathways were decreased in expression in the WNV infected brain, providing evidence of glutamate excitotoxicity and pathology associated with a lack of dopamine. In addition, many of the transcripts mapped to non-infectious neurological disease functions, including mental disorders and degenerative neuropathies, suggesting a correlate between the neuropathology induced by viral infection of the CNS and the neuropathology seen in non-infectious neurological disease. This project provided novel insights into global gene expression during WNV infection, and led to a better understanding of how the CNS responds to viral infection. Confirmation studies looking at individual transcripts of interest will be performed. This data will be used to contribute to potential therapeutics and diagnostic options for WNV and other viral encephalitides.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: 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 Melissa Bourgeois.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Long, Maureen T.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041523:00001


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1 GENE EXPRESSION ANALYSIS DURING WEST NILE VIRUS DISEASE, INFECTION, AND RECOVERY By MELISSA ANN BOURGEOIS DVM 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 2010

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2 2010 Melissa Ann Bourgeois

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3 To my parents and my sister, for their unfailing support and unconditio nal love throughout the years

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4 ACKNOWLEDGMENTS I thank the members of the Long lab at the University of Florida, including Maureen Long, Sally Beachboard, Katie Maldonado, Kathy Seino, and Deanne Fanta for their invaluable assistance over the entire course of this project. I also thank the members of my commi ttee, Maureen Long, Nancy Denslow, Paul Gibbs, David Allred, David Bloom, and James Maruniak for their wonderful insight and contributions to the evolution and progression of this project. I thank the members of the UF Interdisciplinary Centers for Biotec hnology and Research Gigi Ostrow, Li Liu, and Savita Shanker for their help in accomplishing different portions of this project And finally, I thank my wonderful family and friends for their unfailing support thro ughout the entire course of my graduate degree This project was funded with a grant from the USF Centers for Biological Defense.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 11 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 14 2 LITERATURE REVIEW ................................ ................................ .......................... 18 West Nile virus ................................ ................................ ................................ ........ 18 Epidemiology ................................ ................................ ................................ .... 18 Ecology and Host Range ................................ ................................ .................. 19 Molecular Epidemiology ................................ ................................ ................... 22 Viral Structure and Life Cycle ................................ ................................ ........... 23 Experimental Models of Infection ................................ ................................ ..... 25 Clinical Signs in Horses ................................ ................................ .................... 30 Clinical Signs in Humans ................................ ................................ .................. 31 Immune Response ................................ ................................ ........................... 32 Neurological Response ................................ ................................ .................... 39 Pathology ................................ ................................ ................................ ......... 40 Diagnostics ................................ ................................ ................................ ....... 40 Treatment ................................ ................................ ................................ ......... 43 Vaccination ................................ ................................ ................................ ....... 44 High Throughput Pyrosequencing ................................ ................................ .......... 47 Microarray Analysis ................................ ................................ ................................ 48 3 DEEP SEQUENCING, ANNOTATION, AND ANALYSIS OF THE EQUINE CENTRAL NERVOUS SYSTEM TRANSCRIPTOME ................................ ............. 51 Methodology ................................ ................................ ................................ ........... 51 Sample Collection ................................ ................................ ............................ 51 RNA Extraction and Quality Analysis ................................ ............................... 51 First Strand cDNA Synthesis and Amplification cDNA Library Construction ... 53 Normalization of the cDNA Library ................................ ................................ ... 54 High throughput Pyrosequencing of the Normalized cDNA Library .................. 56 Library Annotation and Analysis ................................ ................................ ....... 57 Results ................................ ................................ ................................ .................... 59 Nucleic Acid Quality Assessment and Normalized cDNA Library Titration ....... 59 cDNA Library Quality Assessment ................................ ................................ ... 59 Normalized cDNA Library Sequencing Results ................................ ................ 60

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6 BLAST and GO Analysis ................................ ................................ .................. 61 Comparison of Species NR/NT NCBI Database ................................ ............. 63 Novel Genes Analysis ................................ ................................ ...................... 63 Biomarker Discovery ................................ ................................ ........................ 64 Analysis Against the Human EST Database ................................ .................... 65 Discussion ................................ ................................ ................................ .............. 65 Future Work and Issues to be Addressed ................................ ........................ 66 Conclusions ................................ ................................ ................................ ............ 68 4 GENE EXPRESSION ANALYSIS OF THE CENTRAL NERVOUS SYSTEM OFHORSES DURING WNV INFECTION WITH REGARDS TO EXPOSURE, SURVIVAL, AND LOCATION ................................ ................................ ................. 83 Methodology ................................ ................................ ................................ ........... 83 Microarray Probe Design ................................ ................................ .................. 83 Sample Collection ................................ ................................ ............................ 83 RNA Extraction ................................ ................................ ................................ 85 cDNA Creation and Dye Labeling ................................ ................................ ..... 86 Hybridization and Scanning of Arrays ................................ .............................. 87 Normalization and Statistical Analysis ................................ .............................. 88 Gene Ontology Enrichment ................................ ................................ .............. 89 Pathway Modeling ................................ ................................ ............................ 89 Microarray Valid ation ................................ ................................ ........................ 90 Results ................................ ................................ ................................ .................... 92 Study Design ................................ ................................ ................................ .... 92 Array Normalization ................................ ................................ .......................... 93 Statistical Analysis ................................ ................................ ............................ 93 Gene Ontology and Pathways Analysis Overview ................................ ............ 95 Exposure Status ................................ ................................ ............................... 95 Immune/Survivor Status ................................ ................................ ................. 100 CNS Loca tion ................................ ................................ ................................ 104 Analysis of Overlap Between Exposure, Survival/Immunity, and Location ..... 107 Array Validation ................................ ................................ .............................. 108 Conclusions ................................ ................................ ................................ .......... 121 5 CONCLUSIONS ................................ ................................ ................................ ... 170 APPENDIX A RNA QUALITY DATA ANALYSIS ................................ ................................ ......... 175 B LIST OF HIGHLY UPREGULATED TRANSCRIPTS RECOGNIZED BY IPA ....... 178 C LIST OF HIGHLY dOWNREGULATED TRANSCRIPTS RECOGNIZED BY IPA 198 LIST OF REFERENCES ................................ ................................ ............................. 199 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 247

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7 LIST OF TABLES Table page 3 1 Experimental tissues used to create the normalized cDNA library .................... 70 3 2 Dilutions of duplex speci fic endonuclease used to normalize the cDNA library .. 70 3 3 Average RNA quality data for samples ................................ ............................... 71 3 4 Data from 454 sequencing titration run ................................ ............................... 71 3 5 Data from 454 sequencing runs Newbler Assembler ................................ .......... 72 3 6 Data from 454 sequencing runs Paracel Transcript Assembler .......................... 73 3 7 Summary of BLAST results for five separate databases ................................ .... 74 3 8 Average scores for equine databases ................................ ................................ 74 3 9 Recognized biomarkers for disease ................................ ................................ ... 75 3 10 Biomarkers identified from novel genes ................................ .............................. 76 3 11 Summary of BLAST analysis of sequenced equine transcriptome to the human expressed tag sequence database ................................ ......................... 77 4 1 Probe groups for inclusion on the microarray ................................ ................... 125 4 2 Samples used to obtain RNA for dye labeling and hybridization to the array ... 125 4 3 Samples and analyses for the array ................................ ................................ 126 4 4 Number of significant genes for each analysis ................................ ................. 126 4 5 Canonical pathways for all analyses ................................ ................................ 127 4 6 Functions for all analyses ................................ ................................ ................. 129 4 7 Transcriptional regulators with increased expression ................................ ....... 132 4 8 Transcriptional regulators with increased expression ................................ ....... 136 4 9 Transcripts for all analyses mapped to neurological CPs ................................ 141 4 10 Transcripts in glutamate signaling pathway for all analyses ............................. 143 4 11 Transcripts in dopamine signaling pathways for all analyses ........................... 145 4 12 Transcripts for all analyses mapped to immunological CPs ............................. 146 4 13 Transcripts in IL 15 production and signaling for all analyses .......................... 148

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8 4 14 Transcripts in IL 9, IL 22, and JAK/STAT signaling for all analyses ................. 149 4 15 Functions for genes common to all groups ................................ ....................... 150 4 16 Validation of the array ................................ ................................ ....................... 151 A 1 RNA quality data for all samples used to create the cDNA library .................... 177 B 1 Transcripts increased in expression recognized by IPA ................................ ... 178 C 1 Transcripts decreased in expression recognized by IPA ................................ .. 198

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9 LIST OF FIGURES Figure page 2 1 Life cycle of West Nile virus. ................................ ................................ ............... 50 2 2 Structure of West Nile virus genome. ................................ ................................ 50 3 1 Representative e lectropherogram for RNA samples ................................ .......... 78 3 2 Percent positive identity of sequences matching to the equine da tabases. ........ 79 3 3 Gene ontology classification of cell death.. ................................ ......................... 80 3 4 Gene ontology classification of physiological processes.. ................................ .. 80 3 5 Sequence count by species group for the NCBI NR/NT databases. ................... 81 3 6 Average length of novel sequ ences. ................................ ................................ ... 82 3 7 Novel gene categories based on gene ontology function. ................................ .. 82 4 1 Box plots for Loess normalization. ................................ ................................ .... 152 4 2 Heat map and dendrogram of all arrays demonstrating similarity in gene expression. ................................ ................................ ................................ ....... 153 4 3 Fold change analysis of signficant genes (p<0.05). ................................ .......... 154 4 4 Number of genes that mapped to GO categories for all analyses. ................... 155 4 5 Distribution of genes among GO categories. ................................ .................... 156 4 6 Top 25 canonical pathways for all analys es.. ................................ ................... 157 4 7 Neurological canonical pathways for all analyses. ................................ ............ 158 4 8 Glutamate receptor signaling pathway. ................................ ............................ 159 4 9 Dopamine receptor signaling pathway.. ................................ ............................ 160 4 10 I mmunological canonical pathways for all analyses.. ................................ ....... 161 4 11 IL 15 production pathway. ................................ ................................ ................ 162 4 12 IL 22 and JAK/STAT pathways for exposure. ................................ ................... 163 4 13 Neurological functions by category for all analyses.. ................................ ........ 164 4 14 Neurological functions all analyses. ................................ ................................ .. 165

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10 4 15 Immunological and cell death/apoptos is functions for all analyses. ................. 166 4 16 Canonical pathways for significant genes common to all analyses. ................ 167 4 17 Neurological functions for significant genes common to all analyses. ............. 168 4 18 Immunological functions for significant genes common to all analyses ............ 169

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11 LIST OF ABBREVIATION S BLAST Basic Local Alignment of Sequences CNS Central nervous system DNA Deoxyribonucleic acid EST Expressed sequence tag GO Gene ontology IPA Ingenuity Pathways Analysis NCBI National Center for Biotechnology Information PFU Plaque forming unit RNA Ribonucleic acid WNV West Nile virus

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12 A bstract of Dissertation Presented to t he Graduate School of t he University o f Florida in Partial Fulfillment o f t he Requirements f or t he Degree o f Doctor o f Philosophy GENE EXPRESSION ANALYSIS DURING WEST NILE VIRUS DISEASE, INFECTION, AND RECOVERY By Melissa Bourgeois May 2010 Chair: Maureen T. Long Major: Veterinary Medical Sciences It was the goal of this project to profile gene expression during West Nile virus ( WNV ) infection in the central nervous system ( CNS ) of horses. It was hypothesized that there are gene pathways whose expression cha nges in a significant and consistent manner due to WNV as a result of exposure status survival /immune status and CNS location T o test this hypothesis, t he equine CNS transcriptome was sequenced these sequences were used to create a custom microarray and this array was used to analyze gene expression in the thalamus and cerebrum of three di fferent groups of horses (nave/WNV exposed, vaccinated /WNV exposed, and normal ). Statistical and pathway analysis was performed on this data to identify genes and gene pathways of interest. In total, 41,040 genes and contigs were sequenced and annotated from the transcriptome 1,280 of which were novel to the equine genome project. Significant differences (p<0.05) in gene expression were seen due to exposure in 9,020 genes, survival in 7,395 genes, and location in 7,649 genes. Pathways analysis revealed that many genes mapped to neurological and immunological categories which were found

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13 to be downregulated in WNV infection Detailed analysis of the immunological pathways revealed that both innate and adaptive components of the immune response were involved in the response to WNV infection, and that higher levels of expression of these pathways were correlated with survival. PTX3 ( p attern rec ognition response to viruses), i nterleukin 15 production and the JAK/STAT pathway w ere found be be upregulated by WNV infection. I nfection with WNV also led to an increase in th e expression of SOCS3, a negative feedback inhibitor of the JAK/STAT pathway possibly reflecting evasion of innate immunity by the virus. Apoptosis was found to be upregulated due to WNV infection, providing expression level evidence of neuropathology due to viral infection. T ranscriptional genes also demonstrated changes in expression levels due to WNV infection. Detailed analysis of neurological pathways revealed that transcripts in both the glutamate and dopamine signaling pathways were decreased in expression in the WNV infected brain, providing e vidence of glutamate excitotoxicity and pathology associated with a lack of dopamine. In addition, many of the transcripts mapped to non infectious neurological disease functions, including mental disorders and degenerative neuropathies suggesting a corr elate between the neuropathology induced by viral infection of the CNS and the neuropathology seen in non infectious neurological disease. This project provided novel insights into global gene expression during WNV infection, and led to a better understan ding of how the CNS responds to viral infection. C onfirmation studies looking at individual transcripts of interest will be performed. T his data will be used to contribute to potential therapeutics and diagnostic options for WNV and other viral encephali tides.

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14 CHAPTER 1 INTRODUCTION West Nile virus (WNV) is one of the leading causes of arboviral encephalitis in the United States in both horses and humans. Like other encephalitic flaviviruses, WNV can be devastating in its ability to cause long term neurological deficits and even deat h. Since the introduction of WNV to New York in 1999, the virus has spread rapidly throughout both North and South America In the United States alone, 25,748 clinical cases of disease have been confirmed in horses [1] and 29,624 cases of clinical disease have been reported in humans with 1,161 human deaths [2] A number of vaccines are currently available for horses and have been shown to be efficacious in preventing clinical disease. The availability of these vaccines ha s been one of the major reasons for the decline in equine cases. Vaccines are in clinical trials for humans, but are not yet available for use. However, despite the presence of these vaccines, further research into WNV is necessary as future outbreaks of this disease may still occur in nave environments and/or due to mutation. W est N ile virus can be used as a model to understand the ecology, epidemiology, and path o physiology of many arboviral and encephalitic diseases, and this knowledge can be used to improve our response to arbovirus epidemics and epizootics In addition, there is a need for ante mortem tissue specific diagnostics that can rapidly differentiate previous exposure from current infection in animals and humans There is also a lack of kn owledge with regard to pathophysiology and immunopathology, especially in volving host pathogen interactions during WNV infection Specifically, there is a large gap of knowledge regarding how the central nervous system (CNS) responds to viral infection an d in understa nding which gene pathways are dy sregulated due to viral encephalitis. This is

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15 especially evident regarding n euroin vasion, neurovirulence, and the neurological immune response. Finally, and likely most important in the nave host, there are few, if any, effective therapeutic interventions for any viral encephalitis leading to lifelong disease disability and even death in animals and humans G ene expression analysis on a global scale can provide detailed information on host pathogen inter actions and can lay a scientific foundation for future diagnostic tests and therapeutic options that are necessary to address deficiencies in knowledge of infectious disease Deep sequencing for the construction of microarrays is one such methodology and tool through which new knowledge can be rapidly generated Tissue specific, validation m icroarrays can then be used to investigate the levels of gene expression within and between hosts The s e data can provide detailed information on the host response to infection and the host response by global analysis of gene expression. Analysis of gene dysregulation can then be targeted toward the analysis of pathways of genes changed due to viral encephalitis The enhanced u nderstanding this will provide can be used to increase our understanding of and ability to combat viral encephalitis. The long term goal of this research was to develop methods and generate host expression data at the level of brain and spinal cord to develop more rapid diagnostic tests and interventional strategies for viral encephalitis. The short term goal of this project was to sequence the equine brain transcriptom e and use tissues from a model of WNV infection and encephalitis in horses to create a custom equine high density microarray for profiling of gene expression during WNV infection. It was hypothesized that there are genes that change in a consistent manner during WNV disease, i nfection,

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16 and recovery. Specifically, there should be a difference in gene expression between exposure to WNV, survival from WNV infection, and location in the brain. This hypothesis was explored in four specific aims. In the first aim, the transcripto me from the central nervous system of the horse was sequenced, annotated, and analyzed. In the second aim, samples from horses experimentally infected with WNV (vaccinated and non vaccinated) and horses not exposed to WNV (negative control s) were hybridiz ed to 4x44,000 spotted microarray s based upon the afore mentioned sequences. These data w ere then subjected to statistical analysis for the third specific aim to determine whether there were differences in gene expression between: 1. the cerebrum and thal amus of horses infected with WNV (not vaccinated), 2. between vaccinated and non vaccinated horses infected with WNV, and 3. between non vaccinated horses infected with WNV and untreated horses not exposed to WNV. The gene expression levels of the array w ere validated with relative quantitation reverse transcription real time PCR on six genes that were shown to be significantly differentially regulated between nave and non nave horses exposed to WNV. The probes on the array were validated by BLASTing th e array probe sequences against the EqCab2 genome. Finally, in the fourth specific aim, these data were used in pathway analysis and gene ontology enrichment to determine the common pathways of genes that were changed between and amongst analyse s due to v iral infection. This exploration of gene expression during WNV infection in the horse is the first step in the development of new therapies, diagnostics, and new preventative strategies This information will eventually be combined with other compon ents of a systems biology approach combining interdisciplinary scientific fields to gain a better

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17 understanding of host pathogen interactions (such as proteomics and cell biology) to verify the global analysis conducted herein. This information could eve ntually be used to combat not only outbreaks of WNV, but also as a model to understand and reduce the impact of viral encephalitis in general.

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18 CHAPTER 2 LITERATURE REVIEW West Nile virus Epidemiology West Nile virus (WNV) was first discovered in 1937 in the West Nile province of Uganda. Before 1999, the virus was considered enzootic in Africa, the Middle East, the Mediterranean, and West/central Asia with periodic incursions into Europe. Few cases of WNV actually resulte d in neurological disease or death. Exceptions included an outbreak in the 1960s in France, a 1996 1997 outbreak in Romania, a 1996 outbreak in southern Russia, and a 1998 outbreak in Israel, all of which resulted in neurological disease and death in eith er horses, humans, or birds. [3, 4] In 1999, a single poi nt introduction of WNV occurred in North America in New York City. [5, 6] Because thi s occurred into a nave environment, it provided a unique opportunity to monitor how an arbovirus interacts with and adapts to a new environment. By 2001, the virus had been documented in 21 states, the Cayman Islands, and Canada. By 2002, WNV had reache d California on the western coast of the US, 5 Canadian provinces, Hispaniola, Guadeloupe, and Mexico. In 2003, the virus was isolated in 22 Mexican states, Central America (Belize, Guatemala, and El Salvador), Cuba, Puerto Rico, and the Bahamas. By 2004 the virus had spread to South America with documentation in Trinidad and Columbia. And in 2006, WNV had reached as far south as Argentina. WNV is now the most widely distributed Flavivirus, present on all continents except Antarctica. [7 9]

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19 Ecology and Host Range West Nile virus is a seasonal dis ease, with case occurrence corresponding with peak mosquito vector activity. In northern, temperate climates, peak activity occurs in the summer months. In tropical and subtropical climates, disease activity is high year round, although it may fluctuate slightly dependent upon rainfall. [10] One of the major questions in the epidemiology of WNV is how the virus overwinters, especially in Northern latitudes. Previous studies have isolated WNV from overwintering female Culex pipiens mosquitoes. [3, 11] In addition, as noted above, the virus undergoes year round transmission in warmer climates, and may be transported back to temperate locations by migratory birds. And finally, either chronic infections in birds or non traditional reservoir hosts may in cubate the virus during warmer months. It should be noted that for viral transmission to occur, a minimal threshold temperature of 14.7 o C is required with higher temperatures increasing amplification of virus linearily. [12] The Flaviviridae are traditionally thought to be maintained in nature in a bird mosquito bird cycle. In North America, over 60 species of mosquito have been found to be capable of transmitting WNV Based on host feeding and vector competency studies, it appears that Culex species are the main vector species responsible for the transmission. The primary Culex species varies according to geographic region with Cx. pipens the Northeastern vector, the C x. tarsalis the western vector and, putatively, C x. quinquefasciatus and nigripalpu s, the Southern vector s [10, 13 18] Early feeding studies and blood meal analysis on Culex species demonstrate that these mosquitoes preferentially feed on birds and only occasionally feed on mammals, except wh ere many mammalian hosts are available (Rios L, unpublished data). So while Culex species may play a role as a bridge vector for WNV, other mosquito species are likely to

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20 be involved in the life cycle involving mammalian disease. [19] The species that appear to be capable of functioning as bridge vectors include Aedes spp, Anopheles spp, and Coquillettidia spp [10, 13, 19, 20] Once the virus enters the mosquito, it replicates in the fat and endothelial cells of the posterior midgut. At around the fourth day post infection (PI), the virus enters the hemocoele and sp reads to other parts of the body including the head and salivary glands. Thus the virus is incubated by the mosquito for approximately seven days PI before it can be transmitted. [4] During this time, the virus undergoes a variety of interactions with the mosquito and the environment dependent on temperature, seasonality, and climate conditions. The virus has also been shown to be capable of trans ovarial transmission, albeit at a low rate. [21] Birds serve as the main reservoir host for WNV and develop high levels of viremia at >10 7 PFU/mL post infection. Between 74 and 100% of mosquitoes become infected when feeding on hosts with viral titers >10 7 while on ly 0 36% of mosquitoes become infected when feeding on hosts with viral titers >10 5 [22] It was originally thought that Corvidae (i.e. cr ows, blue jays, ravens) served as the major reservoir for the virus. However, while these birds develop high viremias, they quickly succumb to clinical disease. L aboratory infection of Corvidae with the NY99 strain of WNV resulted in the clinical signs o f lethargy reluctan ce to fly, depress ion anorexi a and sporadic neurological signs (i.e. ataxia ) four days post infection (pi) By day five PI all birds either spontaneously died or were euthanized Viral titers in all birds measured >10 7 PFU/mL. [23] In another study in which birds wer e infected via mosquito bite, oral ingestion, or contact, the Corvidae were highly susceptible to disease. The birds demonstrated clinical signs of lethargy, ruffled feathers, and unusual posture with a high

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21 viremia and death 24 hours after the onset of c linical disease. [24] Therefore while Corvidae function as excellent sentinels for moni toring the introduction of WNV into an area, they do not survive long enough to function as effective reservoirs. A recent study on bl ood meal analysis of trapped mosquito species demonstrated that Culex mosquito species do not preferentially feed on the Corvidae. Instead, blood meal analysis on Culex mosquito species revealed that these mosquitoes preferentially feed on passerines including the American robin, the Northern cardinal, the common grackle, a nd the house finch. [19, 25] Yet minimal mo rtality due to WNV has been noted in these birds during these surveillance studies. In another study, robins, house finches, and sparrows were infected with WNV orally, via mosquito bite, and by direct contact with other infected birds. These birds devel oped a high viremia (10 5.4 10 8.9 PFU/mL) with few clinical signs and minimal mortality. [24 ] Thus, these particular passerine birds have been designated the major potential reservoir for WNV, rather than corvids. These findings are consistent with the sister North American flavivirus, St. Louis encephalitis (SLE),. [26, 27] Other arboviruses have been shown to be maintained in non avian reservoirs (i.e. Venezuelan equine encephalitis). Experimental peripheral infections with WNV i n investigate the possibility of unknown reservoir and incidental hosts. Infections of bats, pigs, and dogs have resulted in low levels of virus (10 1 10 3 PFU/mL) and the absence of clinical signs and it is unlikely that these hosts function in nature as effective reservoirs [28 30] Alternatively, experime ntal infection of cats resulted in mild, non neurological signs of disease with moderate viremias of 10 3 10 4 PFU/mL, demonstrating that cats

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22 may be capable of viral amplification and transmission. [28] The possibility of rodents as a rese rvoir is of particular concern due to the role of wild rodents in VEE. Experimental peripheral inoculation of fox squirrels with WNV resulted in clinical signs in only 1 of 11 subjects with levels of viremia ranging from 10 1.7 10 6.1 PFU/mL [31] Similar results were obtained i n chipmunks, with peripheral inoculation resulting in no clinical signs with virus titers of 10 3.9 10 6.7 [32] Reptiles may also serve as a reservoir for WNV infection. In laboratory infection of 24 juvenile alligators, all became infected and were able to sustain this viremia for up to 8 days with 88% developing WNV titers >10 5 PFU/mL. In addition, there was evidence of oral and cloacal shedding of WNV with contact transmission. [33] In a naturally occurring outbreak in Florida, neurological disease in farmed alligators was noted, with serum viral titers ranging from 10 3.6 10 6.5 PFU/mL. [34] Therefore there is evidence that wild rodents and reptiles ma y serve as WNV reservoirs in nature, altering the traditional view of a bird mosquito bird maintenance cycle (see Figure 2 1). Molecular Epidemiology West Nile virus is classified as a Flavivirus within the genus Flaviviridae. The genera Flaviviridae encompasses a wide range of viruses, most of which are spread through mosquitoes and ticks (arthropod borne diseases). Other, related viruses of veterinary import within this genera include Pestiviruses ( bovine viral diarrhea and classical swine fever ) as well other Flaviviruses including Japaneses encephalitis virus (JEV) and Kunjin virus (KJ). Closely related flavivirus primarily of human importance include SLE and tick borne encephalitis (TBE) There are two distinct phylogenetic lineages of West Nile virus (lineage 1 and lineage 2). The virus that was introduced into North America was of lineage 1

PAGE 23

23 genotype and is generally considered to be more pathogenic than lineage 2 viruses (primarily rest ricted to Africa) Although both lineage 1 and 2 viruses can result in neuroinvasive disease lineage 2 viruses are responsible for outbreaks of flu like disease [8] Genetic mutations within the viruses are responsible for the differences in virulence and phylogenetic classification, and include alterations in the envelope (E) protein and nonstructural (NS) proteins. [7, 8, 35, 36] This can be seen in North America, where limited evolut ion of WNV has occurred during the spread of the virus to the west. [37 40] Viral Structure and Life Cycle WNV is an enveloped, single stranded positive sense RNA virus, approximately 50 nm in size. The genome of WNV is approximately 11,000 base pairs in length and codes for 10 viral proteins, in cluding 3 structural proteins and 7 non structural proteins Capsid pre Membrane Envelope NS1 NS2A NS2B NS3 NS4A NS4B NS5 (see Figure 2 2) [41] The NS proteins are largely involved in viral replication, while the structural proteins mainly function in maintenance of the virion and are responsible for the majority of host immunogenicity. The first step in viral infection occurs when domai n III of the E protein binds to as yet uncharacterized host cell receptors although some host proteins have been demonstrate d to assist movement of the virus in host tissues [42] Receptor mediated endocytosis occurs, and the virus enters the host cell within a low pH vesicle. The virus is then released from the vesicle into the cellular cytoplasm as a single str and of positive sense RNA. Translation occurs first, since viral proteins are required for subsequent RNA replication steps. Host cell elongation initiation factors (eIF) bind to n the traditional initiation,

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24 elongation, and t ermination steps of translation occur form ing a viral polyprotein coding Capsid preMembrane Envelope NS1 NS2A NS2B NS3 NS4A NS4B NS5 ion to occur. In subsequent translational events, the viral NS2B/NS3 protease carries out the cleavage of the polyprotein. [43, 44] RNA dependent RNA polymerase (RdRp) binds to conserved stem loop structures of incompletely characterized even t. [45, 46] Other viral proteins involved in this replication complex include NS1 and NS4A. The viral NS3 helicase acts to un wind/stabilize the RNA genome. [47] Replication of a negative sense strands serve as the template for the creation of positive sense viral RNA genomes. Replication of positive sense RNA str ands occurs when the same viral and viral genome). [48 50] The re is evidence that only a few negative strands are created, and that multiple replication events occur simultaneously on one of these strands to produce a large amount of positive sense RNA viral genomes. These positive sense RNA strands have two functio ns which include serving as a translational template for the creation of more polyproteins and as the viral genome that is released from the cell. protein. [51] Viral packaging occurs once enough viral proteins and genomes have been created in the golgi apparatus. The capsid (C) protein is arranged in an icosahedral

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25 symmetry around the positive sense RNA with the assistance of NS2A. [52] The preMembrane ( prM ) and E proteins are then arranged around the viral capsid. The prM conformation of the M protein protects the low pH mediated membrane fusion domain of the E protein during transit in a vesicle from the cell. Once released by exocytosis, the prM protein is cleaved to the M (mem brane) form. [41, 53] The virus is then ready to bind to and infect new host cells. Experimental Mod els of Infection Controlled stud ies of WNV infection are difficult due to a lack of well defined models and difficulty in reproducing infection in the mammalian host. Horses and other incidental hosts, including humans, have a high rate of subclinical di sease (or exposure) with only a small proportion developing clinical signs of infection. [24, 28 32, 54 56] Conversely some small mammal models demonstrate extreme susc eptibility to disease with high mortality rates and short survival times. [23, 24, 57] Both of these scenarios make studying the pathogenesis of WNV difficult as a low rate of clinical signs requires a large number of subjects and the small mammal models may have very different immunopathological mechanisms of disease. Thus many experimen tal protocols have limited proven translational applicability and questionable extrapolation to disease in naturally affected hosts. Mosquito or peripheral experimental challenge of the horse is one example of a model in which there is limited reproduct ion of clinical disease with a symptomatic:asymptomatic ratio of 1:12. [54] Yet experimental inoculation of the horse should be an ideal model for studying WNV, since equines develop clinical disease and pathology during WNV infection simila r to humans. In response to this problem, an intrathecal model for WNV laboratory infection in horses has been developed. This

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26 method uses a sterile technique to directly inject the virus (~10 5 PFU) into the subarachnoid space of the CNS at the atlanto o ccipital junction of the horse while under anesthesia resulting in clinical disease in 100% of nave subjects. [58, 59] Peripheral inoculation with vaccines developed against WNV has been successful in protecting against clinical disease induced by direct inoculation of the CNS and serves as the basis for studying the pathogenesis of WNV in this current research. Current ly accepted methods of viral infection include intradermal, intramuscular, intraperitoneal, intravenous, and intrathecal needle injection, as well as mosquito infection. Each of these methods has problems associated with their use. Needle injection is meant to mimic mosquito bite infection. However, needles cannot simulate the biological factors associated with mosquito bite infection that may influence disease, includin g differing bite locations, mosquito associated factors (vector capacity, salivary proteins and enzymes, incubation temperature), and locale specific host response. Work aimed at understanding mosquito/virus interactions traced the path of WNV through the mosquito. As previously described, the virus undergoes initial viral replication, spread ing to the hemocoele, head, and salivary glands in a 7 day cycle. [4] During this time, the virus may undergo as yet uncharacterized interactions with the vector that cannot be replicated in a mechanical inf ection model and may affect how the virus affects the laboratory model. Yet the mosquito infection model is not ideal either. The amount of virus inoculated into the host will vary depending on the number of mosquitoes that bite the host and the stage of viral replication within the vector, thus confounding the results of experiments which require known infectious doses. This is supported by different

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27 arbovirus experiments involving LaCrosse virus in deer and in another involving WNV in chickens where unexpectedly higher viremias were induced with mosquito infection compared to needle inoculation. [57, 60] There are also a variety of environmental factors that influence viral replicative ability in the mosquito host that it may be difficult to control in the laboratory, including ambient temperature, seasonality, and climate conditions.. [12] Other studies have demonstrated a lack of correlation in the pathological lesions of WNV between mosquito, needle, and oral routes of infe ction. [55] Therefore, while a lack of biological factors is problematic with mechanical methods of WNV infection, the lack Birds are an example of a laboratory model that demonstrates both low rates of infection/subclinical disease and high rates of infection/mortality. This is dependent upon the species of bird affected. The family of birds C orvidae (crows, blue jays, ravens, etc.) appear to be the most severely affected clinically with high viral titers, a short incubation period, and a high mortality rate. Clinical signs of lethargy reluctan ce to fly, depress ion anorexi a and sporadic neurological signs (i.e. ataxia ) by four days post infect ion (PI) during l aboratory infection intravenously with 10 3 PFU/mL of the NY99 strain of WNV was seen in crows and blue jays By day five PI all birds either spontaneously died or were euthanized Viral titers in the blood of all birds measured >10 7 PFU /mL consistent with field infection levels. [23] In another study in which birds were infected via mosquito bite, oral ingestion, or contact, the Corvidae were highly susceptible to disease. The birds demonstrated clinical signs of lethargy, ruffled feathers, and unusual posture with a high viremia and death 24 hours post onset of

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28 clinical disease. [24] Thus laboratory infect ion of Corvidae appears to simulate natural infection based on clinical signs and viral titers, but these birds make a poor laboratory model due to their extreme susceptibility to disease. Young chickens appear to be susceptible to WNV, and are easier to obtain, thus they make a better substitute model for understanding mechanisms of pathogenesis in the avian host. Adult chickens are exceptionally resistant allowing for understanding successful innate responses to WNV. [57, 61, 62] Laboratory investigations into the use of raptors as laboratory models have not been consistent. In the wild, raptors appear to be highly susceptible to WNV. In a surveill ance study, 61 out of 149 (66%) total raptors submitted to a clinic tested positive for WNV through real time PCR. Clinical signs in these birds depended on the species of raptor. G reat h orned o wls presented with neurological signs of head tremors, head inc oordination, and ataxia, while red tailed h awks presented with the more general signs of emaciation, dehydration, depression, and variable mild neurological abnormalities [63] This is in contrast to one study which compared mosquito, needle, and oral infection of kestrels, hawks, and owls. No clinical signs were noted in any species yet high viral titers (>10 5 PFU/mL) were obtained with pathological lesions that varied according to species and route of infection. [55] Passerines appear to maintain a subclinical disease with high viral titers for an extended period of time in the wild and in the laboratory setting. B lood meal analysis of mosquito species has demonstrated that Culex mosquito species (the main species responsible for WNV transmission among birds [13, 15] ) preferentially feed on pa sserines including the American robin, the Northern cardinal, the common grackle, and the house

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29 finch. [19, 25] Yet minimal mortality has been noted in th ese birds due to WNV in surveillance studies and laboratory studies support this finding. In one study, robins, house finches, and sparrows were infected with WNV orally, through mosquito bite, and through contact. These birds developed a high viremia (1 0 5.4 10 8.9 PFU/mL) with few clinical signs and minimal mortality. [24] Experimental p eripheral infection of both nontraditional and traditional laboratory mammal species has been performed to investigate their effectiveness as laboratory models of WNV. Infections of bats, pigs, and dogs have resulted in low levels of virus (10 1 10 3 PFU/m L) and the absence of clinical signs. [28 30] Therefore, it is unlikely that these hosts will serve as effective laboratory models. This is in contrast to laboratory mice and cats which develop both a moderate to high viremia and clinical signs. Needle inoculation of cats results in mild, non neurological signs of disease with viremias of 10 3 10 4 PFU/mL. [28] Peripheral inoculation of WNV in the laboratory murine host (C57BL and BALBc) resul ts in a high viremia and clinical signs of encephalitis, anorexia, depression, and death. [64] The susceptibility of laboratory mice to WNV is in distinct contrast to wild mice that are genetically resistant. Laboratory mice contain a point a denylate synthetase gene in the flv locus on chromosome 5 whic h makes them susceptible to WNV infection. [64 66] Therefore the mouse, due to its small size and the ease with which genetic manipulations can be performed, remains one of the main models for studying WNV infection. However, the applicability of the mouse model to other species is controv ersial due to inbreeding to obtain genetic mutants which may have deleterious effects on such factors as the immune response. In addition, the infection itself results

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30 in CNS infection that is distinct from that of humans and horses in terms of pathology and virus localization. Other rodent models have been explored as an effective laboratory model. To date, all have demonstrated subclinical signs with high levels of viremia, providing evidence of their possible role as effective reservoir hosts but ques tionable role as an accurate laboratory representation of WN encephalitis in the outbred host. Experimental peripheral inoculation of fox squirrels with WNV resulted in clinical signs in only 1 of 11 subjects with levels of viremia ranging from 10 1.7 10 6. 1 PFU/mL [31] Similar results were obtained in chipmunks, with peripheral inoculation resulting in no clinical signs and virus titers of 10 3.9 10 6.7 [32] F inally, e xperimental infection of the golden hamster induces high viral titers up to 10 4.7 and fatality but no clear indication of infection through clinical disease. Also this model is pathologically different in that hamsters develop a high degree of tissue necrosis in the CNS, which is minimal i n natural infection of horses and humans. [56] Clinical Signs in Horses In the horse, the majority of W NV infections are subclinical. It is estimated, based on experimental mosquito challenge and epidemiological analysis, that about 10% of horses naturally exposed to WNV actually develop clinical disease. [67] Clinical sig ns usually begin between nine and eleven days of infection. [58, 67, 68] Initially, these include the general systemic signs of fever (38.3 o C 39.4 o C), anorexia, and depression. The onset of neurological disease is usually abrupt and there are changes in behavior or mentation with an insidious onset of motor deficits. [69, 70] Horses exhibit signs consistent with an encephalomyelitis (diffuse inflammation of the brain and spinal cord disease) exhibited by a combination of mentation and spinal cord abnormalities and defects in cranial nerves. Spinal cord abnormalities inclu de a stiff stilted gait (which can

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31 be mistaken for lameness but is likely bradykinesia ), ataxia (involving two or more limbs, symmetric or asymmetric), flaccid paralysis (lower motor neuron disease), paresis, and recumbency. Several hours or days of m uscl e fasciculations (most notable around the muzzle but can involve the entire body) are often noted. Cranial nerve abnormalities include weakness of the tongue (CNXII hypoglossal), muzzle deviation (CNVII facial), head tilt and/or difficulty balancing (CN VIII vestibulocochlear), and difficulty swallowing (CNIX glossopharyngeal). Changes in mentation include a change in sensorium with intens e reactions to environmental sounds and motion [71] In addition, changes in behavior including severe aggression, somnolence, and coma may be seen. Approximately 30% of horses with clinical signs of disease die spontaneously or are humanely euthanized (this number incre ases to 100% if the horse is recumbent). The remaining 70% of horses with clinical disease recover between three and seven days after the onset of clinical signs However, approximately 30% of the horses that recover will recrudesce within two weeks and can go through a short or prolonged bout of the the previously described clinical signs Of the horses that completely recover, 10% will retain long term complications, including weakness, ataxia, and fatigue. [10, 54, 58] In these studies, many owners indicated that horses also seemed to have evidence of personality chan ge. All the former may have le d to loss of use of the animal for the owner. Clinical Signs in Humans Natural infection in humans has been documented from mosquito bites, transfusions, breast feeding, intrauterine transmission, and organ transplants. [72] Most

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32 infections with WNV in humans are subclinical, with clinical signs developing in about 20 40% of people infected and an average incubation period of two to 14 days. The most common clinical presentation consists of flu like complaints of illness, oftenaccompanied by skin rash, increased body temperature, headache, myalgia, vomitin g, diarrhea, and fatigue. Approximately 1% of people progress to neurological signs which can be divided into brain like symptoms of encephalitis/meningitis (including disorientation, seizures, tremors, ataxia, photophobia, stiff neck, Parkinson like synd rome, etc.) and/or acute flaccid paralysis (loss of use of breathing, limbs, paresis, and complete paralysis). Movement disorders are consistent with bradykinesia with the lesion of paralysis consistent with anterior horn syndrome. Recovery among these p atients is variable, ranging from complete recovery to chronic long term deficits. The case fatality rate is approximately 8% in neurologically affected patients. Other complications, though rare, include chorioretinitis, rhabdomyolysis, myositis, autono mic involvement, hepatitis, pancreatitis, myocarditis, orchitis, uveitis, and vitritis. [73] Immune Response After the bite o f an infected mosquito, WNV is inoculated peripherally into the skin and muscle. Dendritic cells and other antigen presenting cells (APCs) take up the virus through recognition of the viral envelope proteins with conserved pattern recognition receptors an d induce an innate immune response. Toll like receptor (TLR) 3 has been shown to play an important role in this regard, although the question of whether the receptor is protective or increases neuroinvasion of the virus has yet to be resolved. [74, 75]

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33 Type I interferons (IFN and IFN ) that are produced by the APCs in response to activation of the pat tern recognition receptors (PRR s ) oligo a denylate synthetase (OAS) and p rotein kinase R ( PKR ) Eukaryotic translation initiation factor 2 alpha kinase 2 (human gene)]. OAS enzyme transforms the RNaseL protein to an active form that degrades viral RNA and prevents viral replication. Protein kinase R is also activated by the type I interferons. This protein acts to phosphorylate eukaryotic initiation factor 2 (eIF 2) and prevent translation initiation. The APCs also produce IFN which functions to upregulate MHC I expression and enhance natural killer ( NK ) cell activity The virus is transported by the APCs t o the regional lymph nodes to initiate an adaptive immune res ponse This is largely driven by the cytokine expression pattern of the macrophages and dendritic cells, as well as by NK T lymphocytes. Expression of IL 12 in association with IFN secretion by macrophages and dendritic cells induces the differentiat ion and proliferation of of T helper type 1 (Th1) lymphocytes. These cells secrete interleukin (IL) 2 and IFN which, along with NK T cell production of IFN activates and enhances the activity of CD8+ T lymphocytes. Once activated, CD8+ T lymphocytes undergo positive feedback through the self secretion and stimulation of IFN and IL 2. Activated CD8+ T lymphocytes kill virally infected cells by inducing apoptosis in target cells expressing the proper MHC I/ PAMP combination. Apoptosis can be induce d by T lymphocytes through two pathways: the perforin/granzyme pathway and the Fas/FasL pathway (TNF and TGF secreted by microglia can also induce the Fas/FasL pathway). The perforin/granzyme pathway is a direct pathway wherein the perforin acts to cr eate membrane pores which allow the

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34 granzymes to enter the target and start cellular degradation. This pathway has been shown to be essential in the WNV immune response. [76] The Fas/FasL pathway induces apoptosis by initiating an intracellular caspase cascade (cytoplasmic or mitochondrial) which activates proteins that degrade cellular nucleic acids. Both mechanisms of apoptosis result in the death of an infected cell. Lab oratory studies have demonstrated increased mortality and viral loads in CD8+ T lymphocyte deficient mice. In addition, CD8+ T lymphocyte deficient mice that did survive had long term, low levels of persistent viremia. [77] E x pression of IL 4/5/10 by macrophages and dendritic cells drives the differentiation and proliferation of T lymphocyt es cells into a Th2 phenotype These cells function to stimulate the B lymphocyte response (including B lymphocyte phagocytic activity, ant ibody isotype switching and somatic hypermutation) and also may inhibit the Th1 cell response depending on species. In addition, B lymphocytes initially produce IgM antib odies to WNV and then undergo a class switch to produce IgG. [78 81] Antibodies are important in fighting viral infection through multiple mechanisms, the most important of which includ e vir us neutralization ( binding to the virus to prevent binding to cell receptors and uptake of v irus ), antibody dependent cell mediated cytotoxicity (binding to the virus to enhance Fc receptor recognition from NK cells and phagocytosis of the virus), and complement activation. Numerous studies have shown that antibody production against WNV is essential in protecting against disease. Mice deficient in IgM production (C57BL/J6 IgM / ) were unable to combat WNV infection and succumbed to disease even at low viral titers. Subsequent passive transfer of WNV specific antibodies was able to protect against disease in susceptible

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35 mice. [82] In another study, polyclonal antibody was passively transferred to wild type, B lymphocyte deficient ( MT), and B lymphocyte and T lymphocyte deficient (RAG) nave mice. Wild type mice were protected compared to c ontrols and were able to clear viral infection. However, while administration of the antibodies to MT and RAG1 mice did reduce morbidity and mortality, these mice were unable to completely clear viral infection. [83] Thus, vaccination leads to a specific antibody response that is essential to protect against viral infection. Th e onset of this host immune re sponse corresponds with the viremia that occurs between two and four days post infection. If the host is unable to neutralize the virus, the virus may gain access to the CNS by breach ing the blood brain barrier S everal ways in which this might occur hav e been postulated The first includes disruption of the barrier when endothelial cells are exposed to pro inflammatory cytokines released by macrophages and dendritic cells of the innate immune system. These cytokines, including tumor necrosis factor ( TN F ) IL 6, and IL 12, induce blood vessel dilation and the endothelial cell expression of cellular receptors such as E Selection and ICAM 1 for leukocyte migration. [80] The second hypothesis involves the infection of circulating peripheral mononuclear cells that migrate acros s the blood brain barrier. [80, 81] This is supported by research demonstrating that mice deficient in TLR 3 cell receptors found on immune cells have decreased neurological disease compared to wild type mice [80, 81] A third hypothesis involves the use of peripheral nerves such as the olfactory nerve to travel to the CNS. [80] Once the virus gains access to the CNS, a combination of the neuronal cell response, innate and adaptive immunity, and the virus life cycle likely produce the neuropathological change responsible for clinical disease in the host. Most of the

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36 mechanisms have be en solely derived from murine models of WNV that heavily rely on genetically modified strains. The virus initially binds to host T lymphocyte receptors on microglia including TLR 3, RIG 1, MDA 5, and integrins which begins the immunological cascade. It h as been demonstrated that T lymphocytes lacking in TLR 3, RIG 1, and MDA 5 receptors have diminished cytokine and antiviral responses compared to cells that have these receptors. [84 86] Binding to the cell receptors activates a signaling cascade invol ving transcription factors such as interferon regulatory factor (IRF) 3, NF B, and MAPK, which lead to the production of type I interferons (IFN and IFN ). Lack of the IRF 3 signaling pathway in vitro leads to a more virulent WNV phenotype. [85] These IFNs then bind to cell receptors and induce an antiviral response through secondary signaling, including the JAK/STAT pathway. Mice that completely lack both IFN and IFN exhibit increased mortality (100%) with a decreased time to de ath compared to WT mice (mortality 62%). In addition, IFN / / mice had higher viral loads in a greater number of tissues compared to WT mice. [87] The type 1 IFNs induce Oligo a denylate synthetase which leads to RNAseL production and the degradation of viral RNA, as well as the transcription of PKR kinase which leads to the production of eIF 2 and inhibition of viral transcription. Mice that are PKR / and RL / demonstrate increased mortality (90%) compared to WT mice (30%) as well as increased viral loads in multiple tissues. [80] In vitro RNaseL / cells produced 5 10 times higher viral loads than RNaseL +/+ cells. [88] The majority of the time, the antiviral response by microglial cells is beneficial to the host. However, microglial cells also produce a variety of inflammatory mediators that lead to host cell damage. These include nitric oxide synthetase (NOS), reactiv e

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37 oxygen species (ROS), IL 6, IL 12, TNF TGF phospholipase A, and matrix metalloproteins. These inflammatory mediators produce damage both directly and indirectly through signaling cascades. Nitric oxide synthetase induces the production of peroxyn itrite which damages cell lipids and proteins, and potentiates glutamate excitotoxicity. The ROS molecules, hydrogen peroxide and hydroxyl radicals, and matrix metalloproteinases (MMPs) also lead to generalized cellular damage. Interleukin 6, IL 12, and TNF induce inflammation and the migration of CD8+ T lymphocytes which can lead to cell damage. Tumor necrosis factor and transforming growth factor (TGF) can lead to apoptosis. P hospholipase A increases the production of arachadonic acid, eventuall y leading to prostaglandin, thromboxane, prostacyclin, and leukotriene production with subsequent inflammation. The production of these detrimental mediators by microglial cells has been studied in JEV infection of mice. [89] Thus microglial cells, while essential for the innate immune response, can also lead to neuropathology in the host. Neuronal infection by WNV also leads to host cell damage, both directly through the actions of the virus, and indirectly through the host immune response. During infection, neuron s express the chemokines, CXCL10 and CCR5 which drive the recruitment of CD8+ T lymphocytes [90] Lack of CXCL10 has been shown to decrease CD8+ T lymphocytes recruitment, increase viral titers in the brain, and increase morbidity and mortality in experiments involving mice. [90] This, combined with IFN secreted by NK T lymphocytes and IFN and IL 2 secreted by Th1 cells, functions to recruit CD8+ T lymphocytes to sites of viral replication and upregulate MHC I expression. CD8+ T lymphocytes once activated, underg o positive feedback through

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38 the self secretion and stimulation of IFN and IL 2. As previously mentioned, CD8+ T lymphocytes kill virally infected cells [ 76, 77] However, CD8+ T lymphocyte activity in the CNS may be detrimental to the host. In one study, mice infected with a high viral load of WNV (10 8 PFU/mL) suffered 100% mortality with a six day mean survival time. This was in comparison to mice that were infected with a low viral load (10 3 PFU/mL) which suffered 27% mortality with a mean survival time of 11 days. Histopathology of the brains reve aled neuronal degeneration and inflammation consisting predominantly of CD8+ T lymphocytes. When 2 microglobulin mice ( 2 M ) mice lacking MHCI and incapable of recruiting CD8+ T lymphocytes were infected in the same study with high titers (10 8 PFU/mL) of virus, mean survival time was increased. But when these same mice were infected with low titers of virus (10 3 PFU/mL), increased mortality but prolonged mean survival time was noted. [91] Thus CD8+ T lymphocytes appear to function in both the recovery from and pathology of WNV infection. Since WNV preferentially infects the neuronal cell bodies of the thalamus, midbrain, hindbrain, and spinal cord, the gray matter of th ese regions is most affected leading to virus and immune induced neuropathology. Another mechanism of neuronal cell death, partially contributed to by T lymphocy t e mediated apoptosis, may be neuronal excitotoxicity. [92 96] This mechanism is suspected t o be involved in WNV pathology, since there can be extensive apoptosis and necrosis with limited virus load. This phenomenon has been extensively studied in neuronal HIV infection [92 95] and Sinbis virus infection. [96] Briefly, glutamate is the primary excitatory neurotransmitter in the nervous system. Upon release (exocytosis) from the presynaptic cleft, glutamate binds to ionotropic (NMDA, AMP A) and

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39 metabotropic (G protein coupled) cell receptors on the post synaptic cleft. This binding results in the opening of calcium channels (ionotropic) as well as the activation of the PIP 2 /IP 3 /DAG pathway (metabotropic) both of which result in a net inc rease in intracellular calcium. This increase in calcium leads to an action potential and the propagation of neuronal signals. When too much glutamate is present, excessive calcium accumulates in the neuron and leads to the production of toxic substances such as phospholipase A and NO S Excessive levels of glutamate in the synaptic cleft can be due to apoptosis of neighboring neurons (i.e. due to CD8+ T cell mediated killing), lack of energy and/or oxygen (leading to failure of glutamate re uptake with e nergy dependent glutamate transporters), and loss of GLUT 1 receptors ( function in glutamate reuptake). [97] This has been investigated in a study looking at the expression of the principle excitatory amino acid transporter (EAAT) in the spinal cord during WNV infection. This study found decreased EAAT expression with increased astrocyte expression during WNV infection, providing indirect evidence for glutamate excitotoxicity. [98] Neurological Response In contrast to the immune response to viral infection, few st udies have been performed to increase our understanding of the response of the CNS to WNV infection. The majority of work in this area has involved an examination of the clinical signs and pathological distribution of lesions during WNV infection and the subsequent histopathology associated with these lesions. [99 101] Limited work has been performed looking at the neurological response of the host at the molecular/genetic level. This has mainly consisted of the afore mentioned study examining the expression of glutamate transporters in the spinal cord during WNV infection. [98] Another study focused on the

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40 detection of biomarkers in CSF fluid. [102] However, little or no work has been performed to look at the response of the nervous system on the level of gene expression and the downstream effects of these changes. This includes changes in neurotransmitters, receptors, and structural components of the nervous system. Work in this area could lead to a greater understanding of how the CNS responds to infection with WNV and other viral encephalitides, and measures that may be taken to combat these affects. Pathology Clinicopathologically, WNV causes a polioencephalomyelitis mainly involving the midbrain, hindbrain, and spinal cord. This is characterized grossly by an increasing number of lesions progressing from the d iencephalon through the hindbrain and down to the spinal cord. Spinal cord lesions become progressively worse caudally. Congestion of the meninges and hemorrhagic foci may be seen. Histopathologically, inflammatory lesions characterized by layer of mono cellular perivascular cuffing are present. These layers of monocellular cells may also be present in the gray matter (gliosis). These lesions are the most severe in the basal ganglia, thalamus, pons, and medulla (midbrain and hindbrain) Gliosis and mon ocellular perivascular cuffing are also present in the spinal cord and become worse caudally. Few, if any, lesions are seen in the cerebrum and cerebellum of the outbred hosts, horses and humans further emphasizing the predilection of this virus for cell body enriched tissues of the midbrain, hindbrain, and spinal cord. [10] Diagnostics Since no clinical signs are pathognomonic for WNV infection, all horses that are suspected of being infected with WNV should undergo ancillary diagnostic testing to rule out other diseases. This should include a complete blood count (CBC) and serum

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41 biochemistry as well as a cerebrospinal fluid analysis (CSF). Blood analysis is usually normal, although there may be a lymphopenia, elevated muscle enzymes secondary to traum a, and hyponatremia. Cell and protein counts in the CSF may be elevated, usually consisting of an elevated mononuclear cell population, protein concentration of N<70mg/dL, and mild xanthochromia. [103 106] Differentials for neurological disease in the horse suspected of WNV infection should include hepatoencephalopathy, rabies, alphavirus infection (EEE, WEE), equine protozoal myeloencephalitis (EPM), leukoencephalomalacia, tremorigenic toxicities, equine herpesvirus 1 (EHV 1), botulism, hypocalcemia, and verminous meningoencephalmyelitis. [10] These diseases and metabolic conditions may be ruled out with the pertinent testing but the similarity of these conditions can make accurate antemortem diagnosis difficult The preferred test developed by the National Veterinary Services Laboratory for the detection of WNV in the horse is the IgM capture enzyme li nked immunosorbent assay (MAC ELISA). This test detects whether the horse has had recent exposure to WNV by testing for the presence of IgM antibodies. High levels of IgM antibodies are usually present post exposure to WNV at the time of clinical diseas e in the horse and last for approximately 6 weeks. After this time, circulating levels of IgM WNV antibodies decline and are replaced by IgG antibodies. The sensitivity and specificity of this test is 81% and 100%. Briefly, the test works by coating the wells of a charged plate with some type of anti horse IgM antibody (i.e. goat). The sample of interest is placed on the plate, and the antibodies present in the sample bind to the anti horse antibodies. WNV antigen is then added, which binds only to the IgM WNV antibodies present that are

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42 themselves bound to the anti horse antibody. Finally, an anti WNV monoclonal antibody linked to some type of identifier is added to the well, binds the WNV antigen, and a color change is induced. Those animals that co ntain WNV IgM antibodies thus have a color change, while animals negative for WNV IgM antibodies do not. The test takes approximately 24 48 hours to run and relies upon the presence of clinical disease. The quantity of this response has no correlation to severity of disease and does not predict survival [10, 107] In th e unvaccinated horse, the plaque reduction neutralization test (PRNT) can be utilized This test can be used to diagnose WNV infection if there is a four fold rise in paired neutralizing antibody titers. Briefly, cells are grown to confluent monolayers on plates. Samples of interest containing dilutions of the serum sample to be tested are mixed with known concentrations of virus. The mixed samples are cultured on the plates under agar to prevent spread of viral plaques. The wells are stained and the nu mber of plaques per well, per dilution are counted. Horses with high levels of neutralizing antibody will have no plaques even at high dilutions of the serum (i.e. 1:64 or lower) due to the antibody binding to and preventing the virus from infecting the c ells. Horses with little or no neutralizing antibodies will have wells demonstrating a cytopathic effect (plaques) even at low dilutions (i.e. 1:2). In this manner, the titer of the antibody can be determined (the lowest dilution at which there are no vi ral plaques). If this test is run sequentially (i.e. at 1 and 4 weeks) and there is a four fold rise in the antibody titers, then the horse has been recently exposed to WNV. T his test has fallen out of favor for diagnosis of WNV infection due to the fact that vaccination produces antibodies which confound the results and the length of time that it takes to complete (1 2 weeks) This

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43 test is now mainly used to determine the titer of antibodies to WNV in the subject of interest. [10, 107, 108] Other accepted methods of testing for the virus post mortem include real time PCR to detect viral antigen, viral culture to isolate the virus, and immunohistochemistry to detect viral antigens. All of these tests are used post mortem, since it is very difficult to detect the virus in the live animal this is only possible during the viremic phase at approximately 2 4 days after the onset of clinical signs. If these forms of testing are desired, whole brains (especially the midbrain and hindbrain) and/or spinal cord should be submitted to a testing laboratory chilled and in proper containers for bio containment. [10, 109] Treatment Currently, there is no effective anti viral treatment for WNV and t reatment can only be focu s ed on providing supportive care. Horses that present with clinical signs of WNV should be placed on flunixin meglumine ( 1.1mg/kg every 12 hours intravenously ) This appears to reduce the muscle tremors and fasciculations associated with WNV. There is c ontroversy in the use of corticosteroids in horses affected with WNV due to the possibility of enhancing the viral load both peripherally and in the CNS. R ecumbent horses generally require more aggressive therapy, due to the high mortality associated with recumbency. For the short term relief of anxiety, acepromazine can be used (0.02 mg/kg IV or 0.05 mg/kg IM). For long term tranquilization, detomidine hydrochloride (0.02 0.04 mg/kg IV or IM) can be used. Therapies that have yet to be tested and proven efficacious include the use of IFN and WNV specific IV immunoglobulin. [10]

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44 Vaccination Inactivated Whole Virion/Subunit Vaccines. Killed and subunit vaccines are often used in veterinary medicine for their safety record ( will not revert to live virus) and for their potential in over the counter marketing. The first licensed vaccine (Innovator, Fort Dodge Animal Health, Overland Park, KS), available since 2001, is a killed West Nile vaccine consisting of a formalin inactivated whole virion. The adjuvant present in this vaccine is MetaStim a proprietary oil, non aluminum, dual phase adjuvant. [110, 111] This vaccine is curren tly labeled for the control of viremia of WNV infection in the horse. Efficacy and duration of immunity studies using this vaccine demonstrated that 18 out of 19 vaccinated horses did not develop a detectable viremia. It should be noted that the duration of protection against WNV clinical disease has yet to be tested in the clinical challenge model. [ 112] Initial field studies indicate that there is a limited antibody response in 30% of horses with a rapid decrease in the level of antibodies by 5 to 7 months after vaccination. [113] No subunit vaccines are currently licensed for use in the horse. Inactivated whole viri on vaccines do not actively replicate once administered in the host. Thus multiple vaccinations are required for the nave equine. However, the earliest vaccine series within foals should not begin before 3 months of age due to possible interference wit h maternal antibodies. Foals born to vaccinated mares should receive a 3 dose series of this vaccine. The first dose should be administered at 4 6 months of age with the second dose following 4 6 weeks after the first. The third dose should be given a t 10 12 months of age before the major vector season. In foals residing in areas with high vector activity (i.e. the Southeast), the initial dose should be administered at 3 months of age. In foals

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45 born to unvaccinated mares, the 3 dose series of vaccine s should start at 3 4 months of age (extra first dose at 3 months of age in foals living in areas with high vector activity) with a second dose 4 weeks after the initial dose. The third dose should be given at 10 12 months of age; again, before the onset of the peak vector season. For adults with an unknown vaccination history and/or no history of vaccination, a 2 dose series of vaccines should be given with the second dose given 4 6 weeks after the first. These horses should be given a booster before th e onset of the vector season. In adult horses with a known history of vaccination, one vaccination is required prior to the onset on the vector season (usually in the spring) each year. However, if the horse lives in an area with vector activity year rou nd (i.e. the Southeast), two doses of vaccine are recommended each year one before the onset of peak vector activity in the spring and a booster in the late summer/fall. [114] Modified Live Vaccines Modified live virus vaccines are considered more desirable due to their ability to mimic natural infection without causing clinical disease, thus inducing long term immunity to viral pathogens. Two recombinant live vector preparations are licensed for commercial use in horses. The canarypoxvirus vector vCP2017 (CP WN; Recomb itek Merial, Duluth, GA) was the first licensed in 2004. This vaccine expresses the WNV membrane (prM) and envelope (E) genes under control of and packaged with a carbopol adjuvant which is a proprietary cross linking polymer adjuvant that has a depot e ffect to slow release of antigen. This adjuvant is considered superior to the aluminum based adjuvants as it is proposed to induce both humoral and T cell responses. After a single dose of CP WN, 90% of horses demonstrated protection against viremia. [115]

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46 In foals born to both vaccinated and unvaccinated mares, a 3 dose series of vaccines should start with the initial dose at 5 6 months of age (in areas with high v ector activity, an extra dose at 3 months of age for foals born to unvaccinated mares, and the first dose at 3 months of age in foals born to vaccinated mares should be given). The second dose should be given 4 weeks after the first and the third dose at 10 12 months prior to the onset of peak vector activity. In adult horses with an unknown vaccination history, a 2 dose series of vaccines should be given 4 6 weeks apart with a booster prior to the onset of peak vector activity. In horses with a history of vaccination, annual boosters should be given before peak vector activity. If living in areas with high mosquito activity year round (endemic for WNV), two vaccines should be given one in the spring and one in the late summer/early fall. [114] A nonadjuvanted, single d ose attenuated WNV, live flavivirus chimera (WN FV) vaccine (PreveNile Intervet, DeSoto, KS) became available in 2006. [116] This vaccine expresses the envelope (E) and membrane (prM) of WNV in the yellow fever vaccine 17D (YF17D). The safety, efficacy and duration of immunity of the veterinary chimera (YF WN) were investigated. [58, 117] This vaccine, given at 20X and 100X immunogenicity dosages did not revert to virulence or have a detectable viremia despite the development of neutralizing antibody after vaccination. In foals born to both vaccinated and unvaccinated mares, a 2 dose series of vaccines starting at 5 6 months of age with a second dose administered at 10 12 months of age before the onset of peak vector activity should be given. In areas of high vector activity, an extra dose should be given to foals born to unvaccinated mares at 3 months of age and the primary vaccination given at 3 months of age for foals born to

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47 vaccinated mares. For adult ho rses with an unknown vaccination history, one dose should be given initially and then a booster dose administered every year before the onset of peak vector activity. In horses with a known vaccination history, vaccination should occur yearly before the o nset of peak vector activity. [114] High Throughput Pyrosequencing Pyrosequencing is a high throughput sequencing methodology that can generate reads (sequenced transcripts) of 250 base pairs with greater than 100 million base pairs per run. Briefly, the nucleic acids contained in a library of interest are randomly fragmented by nebulization. Linkers (A and B) are attached to each end of the fragments. Avidin biotin purification is used to select only the fragments containing both A and B linkers. The ssDNA containin g the linkers are attached to DNA capture beads and immersed in water in oil microreactors. Clonal amplification is performed in each microreactor such that each DNA capture bead contains multiple copies of a specific ssDNA fragment. The DNA beads contai ning the libraries are placed into individual 44 mm wells. The sequencing reactions then occur, in which each base is cycled individually. A chemiluminescent signal is emitted with the correct basepair matching. Specifically, ATP is generated when added sulfurylase reacts with the pyrophosphate released from the reaction extension. The ATP then reacts with added luciferase and luciferin to generate light plus oxyluciferin, which is read by the computer as correct incorporation. The use of high throughpu t sequencing technologies, including pyrosequencing, is becomingly increasingly common as a method to sequence eukaryotic and prokaryotic genomes. [118, 119] Recently pyrosequencing has also gained popularity as an analysis tool in other genetic methodologies, involving both disease and non diseased

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48 states. These include the identification and ana lysis of epigenetic changes in cancer, [120 124] the discovery of disease causing mutations, [125 127] and the identification and analysis of small interfering and micro RNAs. [128 131] Applications have also demonstrated the usefulness of pyrosequencing as a means of analyzing the transcriptome for expression profiling and new gene discovery in both plants and animals. [132 135] This is especially useful in newly assembled genomes where annotation of the information, as well as the prediction of ne w genes, may be incomplete. Thus the information gathered from pyrosequencing experiments can be used in a variety of applications. One such example of where high throughput pyrosequencing would be useful in a newly assembled genome is the horse genome project. Assembly of the horse genome was completed in 2007 with 6.8X coverage of the approximately 2.7 billion base pair genome. [136] However, while the sequencing of the genome is complete, work on the annotation and identification of the sequences is still in progress S equencing the genome does not provide information on which genes are expressed and at what frequency. A nalysis of the transcriptome of newly sequenced organisms can provide invaluable information that will help to dev elop a comprehensive picture of Microarray Analysis Microarrays are useful tools to analyze the nucleic acid components of organisms. The number of applications for arrays has increased in recent years, and has included such diverse functions as pathogen identification [137, 138] microRNA/siRNA discovery [139, 140] and exon analysis [141 143] One of the more common applications of microarrays is in the analysis of gene expression for a systems biology appoach, especially in regards to diseased states. [144, 145] This is particularly useful when

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49 analyzing gene expression on a global scale, as interactions between pathways of interest can be targeted to understand how pathogens affect the host and defin ed as an integrated model. Microarrays have facilitated the study of the Flaviviridae in multiple applications including detection of variants of Dengue virus (DV) in human samples [146] differentiation between different flaviviral and other viral infections [147, 148] and mutations in the structu ral regions of the WNV genome [149] Microarrays have also been applied to analyze gene expression at both the cell cult ure and organism level for DV, JEV, and yellow fever virus (YFV) infection. [150 155] However, the analysis of gene expression during WNV infection has been limited. The only study involving whole organism gene expression analysis was performed in mice, an anima l that is not naturally susceptible to WNV infection. The study consisted of analyzing the differences in gene expression between mice infected with strains of WNV differing in neurovirulence, and thus did not incorporate gene ontologies into an integrate d model. As expected, genes involved in immunological, neurological, and apoptotic functions were differentially regulated. Forty seven genes were shown to be upregulated in highly neuroinvasive strains. [156] Thus there is still a lack of knowledge in how infection with WNV causes global changes in gene expression in naturally affect hosts. This information could be used to gain a bette r understanding of host pathogen interactions.

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50 Figure 2 1 Life cycle of West Nile virus. Blue arrows represent how the virus is known to be maintained in nature, in a bird mosquito bird cycle, with periodic infections of dead end hosts (horses and humans). Orange arrows represent other, recently discovered methods of transmission through which th e virus may be maintained in nature. Figure 2 2 Structure of West Nile virus genome. The genome codes f or 10 proteins. These include 3 structural proteins (capsid, prem embrane, and envelope) and 7 non structural proteins (NS1, NS2A/B, NS3, NS4A/B and NS5). The protein coding region is flanked by two untranslated regions

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51 CHAPTER 3 DEEP SEQUENCING, ANN OTATION, AND ANALYSI S OF THE EQUINE CENT RAL NERVOUS SYSTEM TRANS CRIPTOME Methodology Sample Collection Ti ssues to create the cDNA library were obtained from the archived tissues samples of three groups of two horses each (total of 6 individuals) and consisted of 1) nave horses infected intrathecally with 1 X 10 5 WNV, 2) non nave horses vaccinated utilizing a modified live attenuated Yellow Fever (YF) chimera vaccine for protection against WNV (Prevenile, Intervet Schering Plough) and infected intrathecally with 1 X 10 5 WNV and 3) horses that were not infected or vaccinated ( see Table 3 1). Experimental infection and vaccination of horses occurred according to previously published data. [58] Horses from groups 1 and 2 were euthanized ( University of Florida IACUC protocol s #F077, #F093, #D163) if demonstrating clinical sign s or at the end of the study (day 21 ) if not demonstrating clinical signs. Horses from group 3 were normal healthy horses, not infected with WNV and were euthanized due to other causes. All horses were necropsied immediately upon euthanasia. Tissues were snap frozen in dry ice and ethanol and stored at 80 o C until RNA extract ion was performed. Eight tissues were collected from each horse and included cerebrum, cerebellum, thalamus, midbrain (rostral and caudal colliculus, tectum, and tegmentum), hindbrain (pons and medulla oblongata), cervical spinal cord, lumbar spinal cord, and spleen. RNA Extraction and Quality Analysis Total RNA was extracted from the tissues listed in Table 3 1 (48 total samples). A 30 mg piece of tissue was weighed out for each sample on dry ice. The tissues were homogenized using manual disruption a nd placed in 1 mL of g uanidium thiocyanate

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52 (Trizol Invitrogen, Carlsbad, CA). The samples were vortexed and allowed to remain at room temperature for 5 minutes to allow complete dissociation of the nucleoprotein complexes. Two hundred L of molecular grade chloroform (Fisher Scientific ) was added to each sample. The samples were placed at room temperature for 2 minutes, then centrifuged at 12,000 x g at 4 o C for 15 minutes. The chloroform and centrifugation step were repeated to ensur e complete removal of the lipids. A 0.5 mL aliquot of isopropanol alcohol was added to each sample and incubated at room temperature for 5 minutes. The samples were centrifuged at 12,000 x g at 4 o C for 10 minutes to precipitate the RNA. One mL of 75% et hanol wa s added to each pellet, mixed, and centrifuged at 7,500 x g for 5 minutes at 4 o C The ethanol was poured off and the pellets air dried for 5 minutes. RNAsecure (Ambion, Austin, TX) diluted to a 1X concentration was heated on a heat block at 6 0 o C for 5 minutes and 75 L added to each pellet to inactivate any residual RNases. The pellets were incubated at 60 o C for 10 minutes in RNAsecure and cooled to room temperature. For DNase treatment, 7.5 L of 10X DNase buffer and 1 L of rDNase (Ambion Austin, TX) was added to each sample. Samples were incubated at 37 o C for 1 hour. After incubation, 7.5 L of DNase inactivating reagent (Ambion, Austin, TX) were added to each sample and the samples were incubated at room temperature for 2 minutes. T he samples were centrifuged at 10,000 rpm for 2 minutes, removed from the inactivating reagent, and placed at 80 o C until quality assessment. RNA quality assessment was performed using a microfluidics platform. One L of each RNA sample was placed on a n ano drop machine (ND 1000, Nanodrop Technologies, Wilmington, DE). The concentration and 260:280 ratio of each sample

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53 was assessed. One L of each RNA sample was then run on the Agilent 2100 Bio analyzer (Santa Clara, CA) to assess the degree of RNA degr adation. Briefly, the sample was incubated with fluorescent dyes, run on a gel, and excited with a laser to generate an electropherogram. From this electropherogram, the ratio of the 28S and 18S ribosomal peaks was obtained and software extraction perfor med. A RNA integrity number (RIN) was generated with high values (6 10) corresponding with tall 28S and 18S rRNA peaks and a low baseline, indicating minimal degradation of the RNA and a high quality sample. Low values (1 5) corresponding with a shift in the he ight of the 28S and 18S rRNA peaks and an increasing baseline, indicating a large amount of RNA degradation and low quality samples. Only samples with a RIN >6 were used for the study. An analysis of all techniques to determine the best technique for the isolation of high quality RNA can be seen in Appendix A. First Strand cDNA Synthesis and Amplification cDNA Library Construction The RNA isolated from the tissue samples from each horse were po oled to create 5 samples total The RNA was converted to ful l length, double stranded cDNA using the SMART PCR cDNA synthesis kit, the Advantage 2 PCR kit, and the PowerScript Reverse Transcriptase (Clontech, Mountainview, CA). To create the first strand of cDNA, 1 g of RNA sample was mixed with 1 Primer II A, 1 L SMART II A Oligonucleotide mix and DI Water for a total volume of 5 L. The contents of the tube were mixed, centrifuged briefly, and incubated in a thermocycler at 70 o C for 2 minutes. Two Ls 5X first strand buffer, 1 L DTT, 1 L dNTP mi x, and 1 L MMLV Reverse Transcriptase were added to each tube. The tubes were mixed, centrifuged briefly and incubated at 42 o C for 1 hr in an air incubator. T ris E DTA buffer

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54 (10mM Tris, 1mM EDTA) at a volume of 40 L was added to each tube and the tubes were heated at 72 o C for 7 minutes. One L of cDNA was added to 9 L of deionized water t o amplify the cDNA Seventy four L DI water, 10 L 10X Advantage 2 PCR Buffer, 2 L 50X dNTP, 2 L 50X Advantage 2 Polymerase Mix was added f or a total volume of 90 L. The tubes were vortexe d and centrifuged The tubes were held at 95C for 1 min ute then cycled 17 times with the following parameters: 95C for 15 sec onds 65C for 30 sec onds and 68C for 6 min utes The amplified cDNA was then analyzed using a micro fluidics platform to determine concentration and purity. The cDNA was purified using a proprietary PCR purification kit ( QIAquick PCR Purification Kit Qiagen, Valencia, CA) Proprietary binding buffer (P B1) at a volume of 450 L was added to the 90 L cDNA sample from SMART PCR cDNA Synthesis kit and placed in a QIAquick spin column. The sample s were centrifuged at 13,000 rpm for 30 60 s. The flow through was discarded, and 0.75 mL of proprietary clean sing buffer ( PE ) was added to the column s and centrifuged at 13,000 rpm for 60 s. The flow through was discarded and the column centrifuged 13,000 rpm for an additional 1 minute. The DNA was then eluted by adding 50 L water (pH 7.0 8.5) to the center of the membrane and centrifuging the column at 13,000 rpm for 1 min. A micro fluidics platform was used to check the concentration and purity of the sample. Normalization of the cDNA Library The purified cDNA sample was normalized to ensure equal expression of all transcripts ( cDNA Normalization Trimmer Kit Evrogen Moscow, Russia ) All tubes containing the cDNA samples (5 total) were combined and 1000 ng of purified cDNA

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55 was placed into a 2 mL Sarstedt tube. Three M olar sodium acetate (0.1 volumes) pH 4.8 was added, then 2.5 volumes of 98% ethanol was added and the tube vortexed. The sample was centrifuged for 15 min at 13,000 rpm and the supernatant removed. One hundred L of 80% ethanol was laid over the pellet. The tubes were centrifuged at 1 3,000 rpm for 5 minutes. The supernatant was removed and the ethanol wash repeated. The pellet was air dried for 15 min at room temperature, then dissolved in sterile water to the final cDNA concentration of 100 150 ng/ L Eight L s of ds cDNA were combined with 4 L 4X h ybridization buffer and 4 L s terile water to begin hybridization. The contents were mixed and 4 L aliquoted into each of four tubes. The tubes were centrifuged at 14,000 rpm for 2 min, incubated in a thermal cycler at 98 o C for 2 min, and incubated at 68 o C for 5 hours. Proprietary d uplex specific endonuclease (DSN) treatment was performed. Two tubes were created one with a 1:1 ratio of DSN storage buffer to 1 L of DSN solution, the other with a 3:1 ratio of 3 L of DSN storage buffer to 1 L of DSN solution. The DSN master buffer was preheated at 68 o C and 5 L was added to each tube containing hybridized cDNA. The tubes were centrifuged and incubated at 68 o C for 10 min. The DSN enzyme was added to each tube as specified in Table 3 2 DSN functions to degrade double stranded cDNA which should only be present after denaturing and reannealing with the abundant transcripts. The tubes were incubated in the thermal cycler at 68 o C for 25 ed to each tube, and the tubes were mixed and centrifuged The tubes were incubated in the thermal cycler at 68 o C for 5 min, then placed on ice. Twenty Ls of sterile water was added to each tube.

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56 The amplification steps of the normalized cDNA were pe rformed. A master mix was prepared ( 40.5 L Sterile water, 5 L 10X PCR Buffer, 1 L 50X dNTP mix, 1.5 L Evrogen PCR primer M1, and 1 L 50X Polymerase Mix ) and 49 L added to 1 L of each diluted cDNA sample (from DSN treatment, see above). The tubes were mixed and centrifuged briefly Amplification was performed by cycling 18 times at 95 o C for 7s, 66 o C for 30 s, and 72 o C for 6 min. The samples were run on a gel and the well s co ntaining normalized samples were combined. A second amplification step was then L of sterile water. The tube was mixed and centrifuged briefly For a control, 2 L of control cDNA was aliquote d into another tube and combined with 20 L of sterile water. The tube was mixed and centrifuged briefly Two L of each of these diluted samples (control and normalized) were then mixed with a master mix consisting of 80 L Sterile water, 10 L 10X PCR Buffer, 2 L 50X dNTP mix, 4 L Evrogen PCR primer M2, and 2 L 50X Polymerase Mix. PCR was performed on both tubes cycling 12 times at 95 o C for 7s, 64 o C for 10s, and 72 o C for 6 min The concentration and purity of the normalized equine W N V libr ary was determined using a micro fluidics platform and run on a gel to assess concentration and purity. High throughput Pyrosequencing of the Normalized cDNA Library The library was sequenced using the high throughput sequencing system Gene Sequence 20 (454 Life Sciences, Branford, CT) located in the UF I nterdisciplinary C enters for B iotechnology R esearch High Throughput DNA sequencing core lab. An initial titration run was performed to ensure transcript normalization followed by two full sequencing ru ns. Briefly, the cDNA library was randomly enzymatically digested,

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57 adaptors added, and the pieces immobilized on streptavidin coated Sepharose beads. The beads were immersed in water in oil microreactors and subjected to theromocycling parameters for cl onal amplification. Th e beads were added to microwells Reaction beads containing sulfurylase and luciferase and primers corresponding to the adaptor sequences were added to each micro well The primer sequences used for the sequencing runs were as follo ws : A 5' CCA TCT CAT CCC TGC GTG TCC CAT CTG TTC CCT CC C TGT CTC AG 3' B 5' CCT ATC CCC TGT GTG CCT TGC CTA TCC CCT GTT GCG TGT CTC AG 3' Each dNTP was added in a flow through sequence to the micro well s and luminescence reactions captured by camera and the Peak Height Determination Software (Pyrosequencing AB). Library Annotation and Analysis The sequence data was initially assembled using sequence assembly software ( Newbler 454 Life Sciences, Roche Applied Science, Indianapolis, IN ) and the EqCab2 and E nsembl databases Singlets and contiguous, non redundant sequences (contigs) were identified. A second sequence assembly software program was used for final assembly of data ( Paracel Transcript Assembler Paracell Inc, Pasadena, CA) PTA was used to per form additional sequence cleaning, sequence clustering (including seed clustering matching to known mRNA sequences clustering matching to similar sequences and pairwise comparisons ) and assembly for the final development of a set of nonredundant sequences (contigs) and the identification of genes Annotation of these sequences was performed with the Basic Local Alignment Search Tool ( BLAST ) quest algorithm for storage, management, and analysis of expressed sequence tag ( EST ) sequences. This

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58 consisted of a homology search using BLASTX (protein) and BLASTN (nucleotide) against the NCBI databases (NT Nucleotide Database, NR Protein Database), and equine databases (Horse draft genome d atabase, predicted proteins in the horse genomes, and EST collection for the horse). Only genes that were positively identified by BLAST with Expect Values (E value ) below 10 4 were used to compile the results. BLAST results were further cleaned and stored in BlastQuest, a database developed by the ICBR (UF, Gainesville, FL) that facilitates management of BLAST results and the Rare Ontology Consortium (GO) term browsing. A query for the top 100 BLAST hits for each contig against the NCBI Gene databas e was performed ( Assembly Filter UF ICBR, Gainesville, FL ). This provided annotation information, gene function, and metabolic pathway associations based on GenMAPP and KEGG pathway database maps. The GO terms and pathway information associated with the lowest e value and consistent with the NCBI databases were assigned to the query assembly process. Contigs with the highest agreement were maintained and the least similar sequences were eliminated. Sequence orientations were determined by software inst ruments, AssemblyFilter and ESTscan (EMBnet, Switzerland). Sequences were analyzed and grouped according to GO function. Sequences were f urther analy zed for species composition The complete sequenced transcriptome was run against the human expressed se quence tag database ( Fisher cluster UF, Gainesville, FL) to determine sequence homology between the human and horse An E value of < 10 4 was set and only one match per sequence generated.

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59 Results Nucleic Acid Quality Assessment and Normalized cDNA Lib rary Titration The tissues from which RNA was isolated to create the cDNA library were obtained from three groups of two horses each (total of 6 individuals ) ( Table 3 1 ). The quality and purity of the RNA sample was determined using the RNA Integrity Number and 260:280 ratio, respectively. Only samples with a RIN > 6 and 260:280 ratio > 1.75 were used to create the cDNA library. The average RIN for all RNA samples was 7.37, and the a verage 260:280 ratio was 1.97 ( Table 3 3, Figure 3 1 ). cDNA Librar y Quality Assessment The concentration s and the purit ies of the individual cDNA libraries were also measured. For the nave/non exposed cDNA sample, the concentration was measured at 512.24 ng/uL with a 260:280 ratio of 1.82. The non nave/exposed cDNA samples had concentrations of 474.31 ng/uL and 644.69 ng/uL with 260:280 ratios of 1.84. The nave/exposed sample had concentrations of 452.86 ng/uL and 640.41 ng/uL with 260:280 ratios of 1.85 and 1.83, respectively. Successful normalization was confi rmed via a titration sequencing run in which five to ten percent of the library was sequenced and 6,197 reads were assembled from 226,271 total bases analyzed. Vector filtration resulted in a total of 1,474 reads (23.8% contamination) for a total of 386,7 40 (31.5%) bases. From this clean up, 4,723 reads were assembled comprised of 839,531 bases. After grouping according to occurrence, there were 273 completely assembled reads, 678 partially assembled reads, 3,113 singletons, 460 repeats, and 199 outlier s. All of the sequences were then analyzed for overlap to establish the presence of contigs. 308 contigs were identified with an

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60 average length of 208.1 base pairs. Only 4 large contigs were identified (average length 634.8 base pairs), confirming that normalization was effective ( Table 3 4 ). Normalized cDNA Library Sequencing Results Newbler assembly software analysis resulted in the assembly of 514,462 sequences from a total of 49,857,586 bases after linker contamination was removed. In total, 70,8 28 sequences were classified as fully assembled reads while 64,823 sequences were classified as partially assembled reads There were 276,760 sequences defined as singletons with 93,504 sequences defined as repeats and 8,497 sequences clas data, 16,895 contigs (sets of overlapping DNA sequences) compri sed of 4,720,747 bases were assembled. These contigs ranged in size from 93 to 2,827 base pairs with an average length of 279.4 base pairs. Large contigs (1,902) were also assembled with an average length of 818 base pairs ( Table 3 5 ). Sequences from three input sequence sets (16,895 contigs obtained from the Newbler assembly, 443,584 sequence sets left from the 454 sequencing run partially assembled reads, singletons, ou tliers, and repeats and 22,748 sequences from the Equus Caballus 2 genome database) were fed into the Paracel Transcript Assembler (PTA) for a tot al of 483,227 sequences ( Table 3 6 ). A total of 188,885 final sequences were clustered after cleaning. Clu sters were determined by the amount of overlap between similar sequences (100 bp minimum) and 61,499 sequences matching with based on annotation), pairwise comparison (comparing every sequence with every

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61 stering (determining similarity based on sequence overlap). Pairwise comparisons resulted in 75,413 of these being seed clusters and 11,634 clusters were formed. Fr om these clusters, 11,621 cluster contigs and 8,058 seed cluster contigs were identified. In addition, 2,098 cluster singlets and 37,654 seed cluster singlets were identified. Using PTA, 19,670 contigs and 75,413 singlets were identified. Combining the results from Newbler and PTA analyses and utilizing known sequences from the equine genome databases, 19,987 contigs and 21,053 genes were assembled. Unassembled sequences were not considered for further analysis. Thus in total, 41,040 sequences were us ed in the BLAST analysis [19,679 PTA contigs (=11,621 non seeded contigs + 8,058 seeded contigs), 308 newbler contigs and 21,053 unassembled EquCabv2 genes]. These sequences have been submitted to GenBank [157] for public access (study # SRP000619). BLAST and GO Analysis A BLAST search was run against five separate databases including the NR database, NT database, EquCab2 chromosomes database, EquCab2 predicted genes database, and EquCab2 ab initio predicted genes by GenScan. The e value was set at 10 4 for the 41,040 sequences searched and 31,357 good sequence hits were obtained with this criterion ( Table 3 7 ). Approximately 73.7% of the sequences identified in this project were matched in the equine chromosome database and at least one of the equine p redicted genes databases, while 23.1% of the sequences recognized in this project were only identified in the equine chromosome database. Completely novel

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62 genes that have as yet to be identified by the equine genome project were represented by 3.1% (1,278 ) of the sequences identified. The average HspScores indicated a high degree of alignment of the sequences with those in the equine databases, at 492.3956, 1326. 042, and 643.1346 ( Table 3 8 ). The average hit length for the databases was 501.524, 1,328.6 9, and 647.97 base pairs respectively, indicating that the sequences identified were of a significant length. The majority of sequences for all databases demonstrated positive identity greater than 95%. For the EqCab2 Chromosomes database, 40,145 of the 40,973 sequences had a positive identity of >95% ( Figure 3 2 a,b ). In the EqCab2 Predicted Genes database, 40,264 of the 40,999 sequences had a >95% identity. And finally, for the EqCab2 ab initio Predicted Genes database, 39,650 of the 40,977 sequences h ad a percent positive identity >95%. The genes were then grouped into Gene Ontology categories with particular interest in the genes involved in the immune system, the CNS, and programmed cell death (apoptosis). For the three major GO categories, 27,355 genes grouped in molecular function, 25,582 genes grouped in cellular component, and 24,351 genes grouped in biological process. In the GO category of cell death under biological process, 1,119 genes were identified, with 1,046 (93.5%) of these involved in apoptosis ( Figure 3 3 ). In the GO category of organismal physiological processes, 5,278 genes were classified, including 1,920 (36.4%) in neurophysiological processes and 1,272 (24.1%) in the immune response ( Figure 3 4 ). In addition, 569 sequences we re classified as having neurotransmitter receptor activity (including dopamine, neuropeptides, benzodiazepines, and acetylcholine) while 571 sequences were

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63 classified as having neurotransmitter binding activity. These results demonstrate that this library successfully identified genes in the horse that are involved in the immune response and genes that are neurologically specific. Comparison of Species NR/NT NCBI Database Top species counts for the NCBI databases were compared against all sequence hits With an e value < 10 4 for all databases, there were 39,257/41,040 accurate hits for both the NR and NT NCBI databases. For the NR database alone, there were 30,011 value= 0) hits, while for the NT database, there horses comprised the greatest number of hits (18,927 NR and 25,887 NT) with the species groups humans, primates, canines, and bovines containing the next greate st number of hits (see Figure s 3 5 a,b ) Some discrepancy was noted between the different databases, and is likely explained by the database program which selects only the top hits in the results list. Novel Genes Analysis Further analysis was performed on the 1,280 (3.1%) sequences not recognized by any of the equine databases. Of these novel sequences, 709 (55.4%) were classified into gene ontology databases. With overlap, 579 could be classified into biological processe s, 592 into cellular component, and 619 into molecular function. The average length of all these sequences was 595 base pairs with a range of 50 8,802 b ase pairs ( Figure 3 6 ). In order to eliminate redundancy of genes, the genes were grouped into 16 cate gories based on their GO function wherein multiple overlapping GO functions were grouped within each category ( Figure 3 7 ). Many of the novel genes were associated

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64 with the CNS and included transport (97), signal transduction (91), neurological genes with structural and physiological functions (86), protein modification (82), and transcription (77). Biomarker Discovery Using Ingenuity Pathways Analysis software, biomarker discovery analysis was performed on the 41,040 sequences. This analysis searc hes for transcripts that may correlate with molecules present in clinical samples that can be used to detect certain diseased or physiological states. For all biomarker categories, 3,227 potential biomarkers were discovered ( Table 3 9 ). For specific disease processes, most biomarkers were in the categories of neurological disease (496), although categories such as inflammatory disease, organismal injury and abnormalities, hematological disease, immunological disease, and inflammatory disease also had large numbers of possible biomarkers. This analysis provided a prediction of clinical samples which would likely contain biomarkers with 1,319 detectable in blood or serum samples. Of the 1,280 novel genes, 11 (0.94%) were found to be potentially useful biomarkers ( Table 3 10 ). The majority of these biomarkers are found in nervous tissue (9/11) with 4, Tet oncogene 1 (TET1), structural maintenance of chromosomes 1A (SMCA1), embryonic ectoderm development (EED), and adenine phosphoribosyltransferase (APRT ), predicted in blood and urine. Two of the genes are known to be associated with neurological disease ( glutamate receptor, ionotrophic, AMPA 4 GRIA4 and neurofilament, medium polypeptide NEFM) and one of the genes with inflammatory disease/organismal d amage ( adenine phosphoribosyltransferase APRT).

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65 Analysis Against the Human EST Database Sequences were run against the human expressed se quence tag database. With an E value cut off < 10 4 31,473 equine transcriptomic sequences matched against the hu man EST sequence database (8,050 contigs, 6,296 seed contigs, and 17,127 singlets). This represented a percentage match of the equine transcriptome to the human transcriptome at 69.27% (contigs), 78.13% (seed contigs), and 80.17% (singlets), indicating a high degree of sequence homology between the human and equine transcriptome. Equine sequences demonstrated good match to the human EST database with an average percent identity of 90.17%, an average bit score of 512.91, and an average alignment length of 424.85 (see Table 3 11 ). Discussion This is the first project to sequence the neurological transcriptome of the equine. These data will be invaluable for future studies involving a variety of applications for both pathological and nonpathological studies in the equine and other host s For this project, 41,040 sequences were identified by BLAST analysis in 5 sequence databases There was overall consensus amongst the NCBI databases as to the hits on species, with the vast majority of sequences matching to the horse. Dogs, primates, humans, and cattle also had a large number of hits. Further analysis of the sequenced transcriptome revealed that 9,504 of the identified sequences were missed by equine predicted databases, and 1,280 of the identified sequenc es have yet to be discovered in the equine genome project. This is most likely due to the incomplete annotation of the annotation and analysis, transcriptomic sequences that sh ould be recognized by the equine databases may be recognized in other organisms Another possibility for the

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66 discrepancy could be differences between the equine genome and transcriptome (i.e. splice variants). These issues are likely to improve with time and increasing annotation of the equine genome project. Over 27,000 of the identified sequences grouped under Gene Ontology classifications. Identification is dependent upon the GO database classifications which at this point are incomplete. Continue d study of genes will lead to improvements within the GO database and more comprehensive identi fi cations. Not surprisingly, for this project, GO classifications were enriched for neurological sequences. B iomarker analysis performance will be important in the future to target comprehensive systems biology approaches. For this project, 3 ,227 potential biomarkers were identified with 496 of these involved in neurological disease. These biomarkers will be useful in the future once confirmed, as many of the biomarkers for the genes that were identified are accessible in submitted clinical samples (urine, blood, plasma, CSF). Future work will need to be conducted to ensure the presence of these biomarkers. This portion of the project also demonstrated high s equence homology between the equine and human EST database, showing that the horse may be useful in the study of the human organism. Future Work and Issues to be Addressed Some of the major limitations of this portion of the project involved the proces s of annotation Because the horse genome seque ncing project is relatively new, correctly identifying transcripts can be a challenge. This project highlighted this complication, as novel sequences were identified in the transcripts sequenced that should have been present in the equine genome project. Another major issue is the identification of transcript variants. Because of the software programs used to annotate the data, and

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67 because the databases containing the reference sequences are limited in their scope, it is not possible to iden tify every po ssible SNP or alternatively spliced transcript In addition, it is difficult to map transcripts that originate in repetitive regions due to the short read length of the sequences and the non unique regions of DNA to which they are being mapped. Finally, b ecause of the short reads generated by pyrosequencing (250 base pairs or less), aligning the sequences correctly and not discarding transcripts with similar sequences can be an issue. These problems demonstrate that pyrosequencing is a powerful tool for t ranscriptomic analysis, but it is only as good as the databases against which it can be referenced. The major problems that were encountered during this project were in preserving the RNA and cDNA library quality. RNA quality was preserved by immediately freezing the tissues at 80 o C upon harvesting, storing the samples at 80 o C until use, working quickly with the samples in an RNase free environment, storing the samples in RNAsecure, and analyzing the quality of the samples with the Agilent 2100 bioanaly zer. For the cDNA library quality, there was a problem with linker contamination. This appears to be a problem with the kits used to create the library, and was solved with software removal of the linker sequences. Future work for this portion of the pr oject will involve continued analysis of the annotated library. This will include data mining on the sequences to identify transcripts of interest. Of particular interest are microRNA sequences and transcript variants. PCR and Sanger sequencing will als o be performed on the identified biomarkers to validate the results. It would also be of interest to attempt to identify the biomarkers in stored

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68 serum samples. The annotation and analysis of the equine brain transcriptome provided a wealth of data that will continue to provide valuable data. Conclusion s This portion of the project used pyrosequencing technology to sequence the transcriptome of the central nervous system of horses (nave and non nave) challenged with West Nile virus and untreated, normal controls. In total, 41,040 sequences were identified by BLAST analysis in 5 sequence databases ( NR database, NT database, EquCab2 chromosomes database, EquCab2 predicted genes database, and EquCab2 ab initio predicted genes by GenScan ). There was overal l consensus amongst the NCBI datab ases as to the hits on species, with the vast majority of sequences match ing to the horse Dogs, primates, humans, and cattle also had a large number of hits. Over 27,000 of these sequences grouped under Gene Ontology cl assifications. These sequences were enriched for those genes involved with the nervous system. Of these sequences, 9,504 sequences were identified that were missed by equine predicted databases, and 1,280 genes were identified that have yet to be discove red in the equine genome project. B iomarker analysis was performed on all of the sequences and 3,227 recognized potential biomarkers were identified with 496 of these involved in neurological disease. These will be important targets for system biology st rategies utilizing genomic and proteomic techniques. Biomarker analysis performed on the novel sequences identified 11 potential biomarkers. Many of the biomarkers for all genes and novel genes that were identified are accessible in submitted clinical sa mples (urine, blood, plasma, CSF) Thus this project identified many genes that are specific to the neurological system, are completely novel to the horse, and have potential applications as biomarkers. Finally, the comparison of the equine transcriptome sequenced in this

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69 project to the human EST project demonstrates high sequence homology between the ESTs of the two species, and provides evidence that data generated from equine studies can be directly applicable to human studies. This project demonstrat es the importance of gene expression studies to supplement the limitations of current sequence databases. This is the first report of the use of pyrosequencing to analyze the transcriptome of the equine with contribution of genes novel to the equine genom e project. Thus i t appears that next generation sequencing, including pyrosequencing, is an effective tool to annotate the transcriptome of organisms. In addition, pyrosequencing is a useful tool to identify novel sequences and possible biomarkers for di sease in the analysis of the transcriptome of organisms, even those with annotated genomes.

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70 Table 3 1 Experimental tissues u sed to c reate the normalized cDNA l ibrar y Sample c ategory (n) Sample t ype (#) Experiment s pecifics Vaccinates/Immune + e xposed (n = 2) Cerebrum (2), cerebellum (2), thalamus (2), midbrain (1), hindbrain (2), cervical spinal cord (2), lumbar spinal c ord (2) Day 0 non control horses vaccinated A Day 365 challenged with WNV B intrathecally Day 365 386 monitored for clinica l signs Day 386 (21 days PI) euthanasia, necropsy, tissue collection Unvaccinated/Non immune + e xposed (n = 2) Cerebrum (2), c erebellum (2), t halamus (2), midbrain (2), hindbrain (2), c ervical spinal c ord (2), lumbar s pinal c ord (2) Day 0 mock vaccination Day 365 challenged with WNV B intrathecally Day 365 374 monitored for clinical signs Day 372 374 (7 9 days PI) euthanasia, necropsy, tissue collection Unvaccinated + n on exposed (n = 2) Cerebrum (1), cerebellum (1), midbrain (1), hindbrain (1), cervical spinal cord (2), lumbar spinal c ord (1) Normal h orse A Live chimera WNV vaccine containing the prM and E proteins of WNV expressed in a YF17D vector B WNV NY99 strain 10 5 pfu/mL Note: This table describes the experiments conducted on the horses whose tissues were used to create the normalized cDNA library There were three groups of two horses each (column 1). Neurological tissue and spleen were collected from each horse and used for RNA isolation (column 2). Column 3 describes the experiment details, including dates of vaccination, infection, and euthanasia/necropsy where applicable. Table 3 2 Dilutions of d uplex s pecific e ndonuclease u sed to normalize the cDNA l ibrary C omponent Tube 1 S1 DSN1 Tube 2 S1 DSN1/2 Tube 3 S1 DSN1/4 Tube 4 S1 Control DSN Enzyme in Storage Buffer 1 uL DSN Dilution 1 uL DSN Dilution 1 uL DSN Storage Buffer 1 uL

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71 Table 3 3 Average RNA q uality d ata for s amples Note: The average RNA quality data for the sample groups is illustrated. The RNA Integrity Number (RIN) wa s used to assess the degree of RNA degradation. The average 260:280 ratio wa s used to determine the purity of the sample. Table 3 4 Data from 454 s equencing t itration r un Reads Number Total reads 6,197 Total bases 1,226,271 Average length 197.8 Min. length 45 Max. length 379 Vector filtered reads 1,474 (23.8%) Vector filtered bases 386,740 (31.5%) Reads to be assembled 4,723 Bases to be assembled 839,531 Completely assembled reads 273 Partial assembled reads 678 Singletons 3,113 Repeats 460 Outliers 199 Total contigs 308 Total bases 67,174 Average length 218.1 Min. length 101 Max. length 901 Total large contigs 4 Total bases 2,539 Average length 634.8 Min. length 529 Max. length 901 Note: Successful normalization of the cDNA library was confirmed with a titration run in which five to ten percent of the library was sequenced. Software was used to filter the vectors (linkers added by the cDNA library construction kits) to clean up the seque nces for BLAST analysis. From this clean up, 4,723 reads were assembled comprised of 839,531 bases. T he 4,723 reads were grouped according to occurrence as completely assembled reads, partially assembled reads, singletons, repeats, and outliers. All of the sequences were analyzed for overlap to establish the presence of contigs. 308 contigs were identified with o nly 4 large contigs identified confirming that normalization was effective. Sample Number of Tissues Concentration (ng/uL) RIN 260:280 Ratio Nave + Non exposed 8 411.63 (152 652.53) 7.1 (6 7.8) 1.96 (1.8 2.08) Non nave + Exposed 16 829.55 (122 1551) 7.4 (6.7 8.4) 1.99 (1.87 2.06) Nave + Exposed 16 439.02 (182 968) 7.6 (6.7 8.4) 1.95 (1.75 2.04) Total Average 7.37 1.97

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72 Table 3 5 Data from 454 s equencing runs N ewbler Assembler Note: Newbler assembly software was first used to assemble the sequences. 514,462 sequences were assembled from a total of 49,857,586 bases after linker contamination was removed. Sequences that could be linked from beginning to end were classified as areas of ind and standalone sequences coding for areas of individual genes at a frequency of less overlapping D NA sequences) composed of 4,720,747 bases were assembled. Reads Number Total # of reads 826,176 Total # of clean reads 514,412 (62.3%) Total # of bases 95,486,897 Total # of clean bases 49,857,586 (52.2%) # of fully assembled reads 70,828 # of partially assembled reads 64,823 # of singletons 276,760 # of repeat reads 93,504 # of outlier reads 8,497 # of all contigs 16,895 # of bases covered 4,720,747 Avg. contig size 279.4 Min. contig size 93 Max contig size 2,827 # of large contigs 1,902 # of bases covered 1,557,286 Avg. large contig size 818 Min. large contig size 500 Max. large contig size 2,827

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73 Table 3 6 Data from 454 s equencing r uns Paracel Transcript Assembler Reads Number N o. of current input sequences 483,227 N o. of sequences removed during cleanup 294,342 (609 were EquCabv2 genes) N o. of sequences kept after cleanup 188,885 (22,139 were EquCabv2 genes) N o. of sequences in seed clusters 61,499 N o. of sequences pairwise compared 127,386 N o. of singlets after pairwise compared 75,413 N o. of problem sequences 54 N o. sequences in clusters 51,919 N o. of seed clusters 21,421 N o. of clusters 11,634 Largest cluster cl.007 (2,998) 2 nd largest cluster cl.015 (2,721) Largest seed cluster sd.17584 (112) 2 nd largest seed cluster sd.3613 (105) No. final assemblies 134,844 No. of cluster.contigs 11,621 No. of cluster singlets 2,098 (4 genes + 19 newbler contig + 2,075 reads) No. of seed cluster.contigs 8,058 No. of seed cluster singlets 37,654 ( 21,021 genes + 29 newbler contig + 2,498 reads) No. of PTA contigs 19,679 (11,621 + 8,058) No. of singlet 75,413 (28 genes + 260 newbler contig + 75,125 reads) No. of contigs 19,987 (=11,621 + 8,058 + 19 + 29 + 260) No. of genes 21,053 (=4 + 21,021 + 28) Note: Data from the sequencing runs using Paracel Transcript Assembly software. After clean up, 188,885 sequences were used for assembly. Sequences were placed into seed clusters where applicable (61,499). Sequences that were not identified as pairwise comparison, and clustering. In total, there were 134,844 final assemblies. These were identified as 19,987 contigs and 21,053 genes for a total of 41,040 sequences submitted for BLAST analysis

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74 Table 3 7 S ummary of BLAST results for five separate d atabases Count % NR/NT EquCab 2 Chr. EquCab2 Predicte d Genes EquCab2 GeneSca n Newbler c ontigs Cluster c ontigs Seeded cluster c o ntigs EquCab2 g enes 28,789 70.1% 58 412 7,760 20,559 817 2.0% 37 360 219 201 652 1.6% 11 641 0 0 5,391 13.1% e value* 65 5,326 0 0 2,462 6.0% e value** 61 2,401 0 0 1,651 4.0% 50 1,601 0 0 677 1.6% e value* 13 299 79 286 287 0.7% e value** 9 273 0 5 314 0.8% 4 308 0 2 41,040 308 11621 8058 21053 = e value < 1e 20 ** = e value < 1e 4 Note: The BLAST search was run against five separate databases. The e value (likelihood that the hit would happen due to chance) was set at 10 4 for the 41,040 sequences searched. 73.7% (30,258) of the sequences identified in this project had bee n previously identified by the equine chromosome database and one or both of the equine predicted genes databases; 23.1% (9,504) of the sequences recognized in this project were missed by both of the EqCab2 predicted genes databases but were identified in the equine chromosome database; and 3.1% (1,278) of the sequences identified in this project were missed by all equine databases. Therefore, 26.2% of the sequences annotated in this project are novel to at least one equine database, while 3.1% of the sequ ences identified in this project are completely novel to the horse. Table 3 8 Average s cores for equine d atabases EqCab2 Chromosomes EqCab2 Predicted Genes EqCab2 ab initio Predicted Genes by GeneScan Average HspScore 492.3956 (17 11,585) 1326.042 (14 23,780) 643.1346 (15 11,553) Average BitScore 976.5968 (34.193 22,966.1) 2629.181 (508 47,141) 1275.415 (30.2282 22,902.7) Average Hit Length 501.524 1328.69 647.97 Note: The average scores for the equine databases indicate a high degree of sequence alignment with the sequences that matched. [ HspScore (high scoring segment pair) measures degree of local alignments with no gaps. Higher scores indicate better alignment. BitScore statistical accounting of the raw alignment score which is th e sum of the substitution and gap scores. Higher scores indicate better alignment. Average hit length the length of the sequences that align. ]

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75 Table 3 9 Recognized biomarkers for d isease Biomarker Category Number of Biomarkers Percent of Genes All biomarkers 3,227 7.9% Neurological disease 496 1.2% Infectious disease 71 0.17% Inflammatory disease 159 0.39% Organismal injury and abnormalities 155 0.38% Metabolic disease 87 0.21% Infection mechanism 70 0.17% Immunological disease 163 0.40% Antimicrobial response 17 0.04% Antigen presentation 133 0.32% Inflammatory response 151 0.37% Hematological disease 216 0.53% Uncategorized disease 1817 4.43% Note: Using Ingenuity Pathways Analysis software, biomarker discovery analysis was performed on the 41,040 sequences. For all biomarker categories, 3,227 (7.9%) potential biomarkers were discovered. This table demonstrates the disease categories or biological responses that the biomarkers are associated with.

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76 Table 3 10 Biomarkers i dentified from novel g enes ID Symbol Entrez Gene Name Location Type Samp le Disease EDL92759 APRT Adenine phosphoribosyltransfera se Cytoplas m Enzyme Nervous tissue, u rine Inflammator y response, o rganismal injury and abnormality NP_062542 C21ORF62 Chromosome 21 open reading frame 62 Unknown Other Nervous tissue BAF84809 EED Embryonic ectoderm development Nucleus Transcriptio n regulator Nervous tissue, b lood NP_00109930 0 EME1 Essential meiotic endonuclease 1 homolog 1 (S. pombe) Nucleus Other Nervous tissue EDL85231 GRIA4 Glutamate receptor, ionotrophic, AMPA 4 Plasma Membran e Ion channel Nervous tissue Neurologica l disease, o rganismal injury and abnormality XP_00107772 9 LOC687257 Hypothetical protein LOC687257 Unknown Other EDL00021 MOBKL3 MOB1, Mps One Binder kinase activator like 3 (yeast) Cytoplas m Other Nervous tissue NP_005373 NEFM Neurofilament, medium polypeptide Cytoplas m Other Nervous tissue Neurologica l disease BAF84610 SMC1A Structural maintenance of chromosomes 1A Nucleus Transporter Nervous tissue, blood, plasma/s eru m EAW54299 TET1 Tet oncogene 1 Nucleus Other Blood, p lasma/ s eru m XP_00147281 8 ZFP422 RS1 Zinc finger protein 422, related sequence 1 Unknown Other Nervous tissue Note: Genes novel to the equine genome were also submitted to IPA for biomarker discovery analysis. Of the 1,280 genes, 1 1 (0.94%) were found to be potentially useful biomarkers. The majority of the biomarkers are found in neurological tissue (9 /1 1 ) but 4 can be fou nd in blood and urine samples. In addition, 2 of the genes are known to be associated with neurological disease and 1 of the genes with inflammatory disease/organismal damage.

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77 Table 3 11. Summary of BLAST a nalysis of sequen ced equine transcriptome to t he human e xpressed sequence tag d atabas e Note: Contigs, seed contigs, and singlets from this project were BLASTed against the human EST database. In total, 31,473 sequences matched to the human EST database (row 1) with an e value < 10 4 Contigs Seed Contigs Singlets Number of Matches E < 10 4 8050/11621 6296/8058 17127/21361 Percent Homology Match 69.27% 78.13% 80.17% Average E value 1.44636E 05 2.38671E 06 8.09623E 06 Average Bit Score 189.0196894 907.9956004 519.9152099 % Identity 89% 91% 90% Alignment Length 187.9218634 698.33831 435.6858761

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78 F igure 3 1 Representative e le ctropherogram for RNA s amples RNA samples were assessed for the degree of degradation using the Agilent 2100 Bioanalyzer. Degradation was determined by the ratio of the height of the 28S:18S ribosomal peaks and the height of the baseline by software analysis of the electrophe rogram. The sample was then assigned an RNA Integrity Number (RIN) with high degrees of degradation corresponding to a RIN<6. Only samples with RIN>6 were used for this study.

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79 Figure 3 2 Percent p ositive i dentity of sequences m atching to the equine d atabases. A ) A ll the percent positive identity scores up through 100%. B) A ll the percent positive identity scores excluding 100%. The majority of sequences for all databases demonstrated positive identity greater than 95%. EqCab 2 Chromosomes database 40,145 / 40,973 sequences (97.9%) had a positive identity of >95%. EqCab2 Predicted Genes database 40,264 / 40,999 sequences (98.2%) had a >95% identity. EqCab2 ab initio Predicted Genes database 39,650 / 40,977 sequences (96.7%) had a percent positive identity >95%. 0 5000 10000 15000 20000 25000 30000 35000 40000 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 Number of Sequences Percent Positive Identity A EqCab2 Chromosomes EqCab2 Predicted Genes EqCab2 GenScan ab initio Predicted Genes 0 1000 2000 3000 4000 5000 6000 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 Number of Sequences Percent Positive Identity B EqCab2 Chromosomes EqCab2 Predicted Genes EqCab2 GenScan ab initio Predicted Genes

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80 Figure 3 3 Gene o ntology c lassification of cell d eath These were categories included under biological processes. The majority of genes involved in cell death were involved with apoptosis (programmed cell death 1,046 genes). Figure 3 4 Gene o ntology classification of p hysiological p rocesses These categories were included un der biological process The majority of genes were involved with neurophysiological processes (1,920) and the immune response (1,272). 18 22 33 1046 Cell Aging Cell Killing Cytolysis Programmed Cell Death 206 261 351 489 779 1272 1920 Muscle Contraction Cell Activation Regulation of Physiological Processes Organismal Movement Other Immune Response Neurophysiological Processes

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81 Figure 3 5 Sequence count by s pecies g roup for the NCBI NR/NT d atabases. A.) Sequence count for the NR database. The majority of sequences mapped to the horse, with other prominent groups including the human, primate, canine, and bovin e. B.) Sequence count for the NT database. The majority of sequences in this database also mapped to the horse, with other prominent groups including the human, primate, canine, and bovine. 0 4000 8000 12000 16000 20000 Other/Unclassified Synthetic construct Plant Lower Insect Fish Amphibian Avian Rodent/Lagomorph Other Land Mammal Aquatic Mammal Swine Bovine Equine Feline Canine Primate Human 191 161 5 113 17 36 40 42 1042 178 146 299 1544 18927 14 2796 2158 2072 A 0 10000 20000 30000 Synthetic construct Plant Lower Insect Fish Amphibian Avian Rodent/Lagomorph Other Mammal Aquatic Mammal Swine Bovine Equine Feline Canine Primate Human 30 2 118 1 40 13 3 153 76 4 802 2116 25887 57 2449 3564 3811 B

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82 Figure 3 6 Average length of novel s equences. The majority of sequences annotated were less than 1000 base pairs, with an average length of 595 base pairs. Figure 3 7 Novel g ene categories based on gene o ntology f unction. G enes were grouped into general categories based on GO classifications. The categories of transport, signal transduction, neurological, protein modification, and transcription were represented the most. 0 100 200 300 400 500 600 700 800 721 220 81 49 38 35 21 12 12 9 9 9 2 8 5 5 0 1 4 1 9 2 0 0 2 0 0 4 1 4 0 0 0 0 1 1 Number of Sequences Length of Sequences 0 10 20 30 40 50 60 70 80 90 100 41 67 35 86 82 91 52 19 77 97 57 27 61 15 60 56 Number of Genes Gene Categories by GO Classification

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83 CHAPTER 4 GENE EXPRESSION ANAL YSIS OF THE CENTRAL NERVOUS SYSTEM OF HORSES DURING WNV IN FECTION WITH REGARDS TO EXPOSURE, SURVIV AL AND LOCATION Methodology Microarray Probe Design Probes consisted of oligonucleotides (60 mer) fabricated by a patented algorithm (Agilent Technologies, Santa C la ra CA ) based on the annotated equine brain library and a 44,000 gene array (Agilent Tech nologies Santa Clara, CA ) was constructed Preference was given to probes with the greatest length, greatest abundance, and lowest e value (10 4 ) within a cluster (set of similar sequences) All designed probes were included with one replicate each in 1 ) annotated, 2) annotated minus orientation, 3) unannotated, and 4) recovered genome categories ( see Table 4 1) Several p robes consisting of neurological, immunological, and cell death gene ontolog y categories were considered to be of particular import ance and replicate s were include d on the array. Uniquely designed probes (250) designed by the manufacturer ( Agilent ) were also included as technological controls on the arrays. Sample Collection Tissues for analysis were derived from horses used in an experimental intrathecal challenge model wherein nave horses develop ed grave West Nile (WN) encephalitis (100% nonsurvivorship) and all vaccinate d horses d id not develop clinical disease (100% survivorship) Specifically, brain tissues used to create the cDNA for dye labeling were obtained from the se archived samples of three groups of six horses each (total of 18 individuals) and consisted of 1) nave horses infected intrathecally with 1 X 10 5 WNV 2) non nave horses vaccinated utilizing a modified live attenuated Yellow

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84 Fever (YF) chimera vaccine for protection against WNV (Prevenile, Intervet Schering Plough) and infected intrathecally with 1 X 10 5 WNV and 3) horses that were not infected or v accinated ( Table 4 2 ). Experimental infection and vaccination of horses occurred according to previously published data. [58] Horses from groups 1 and 2 were euthanized ( University of Florida IACUC protocol s #F077, #F093, #D163) if demonstrating clinical signs or at the e nd of the study (day 21) if not demonstrating clinical signs. Horses from group 3 were normal healthy horses, not infected with WNV and were euthanized due to other causes (lameness, age, etc.) All horses were necropsied immediately upon euthanasia. Tissues were snap frozen in dry ice and ethanol and stored at 80 o C until RNA extraction was performed. Tissues used in the array included cerebrum and thalamus (one section from each horse for a total of 36 samples) Three analyse s were established to test the hypothesis that the re are gene pathways whose expression cha nges in a significant and consistent manner due to WNV as a result of exposure status, survival/immune status, and CNS location. The analysis and the breakdown of the samples can be seen in T able 4 3. With respect to the experimental analyse s, th ree e subhypotheses were generated to analyze if there was a difference in gene expression between the nonvaccinated/exposed and untreated horses (exposure) the nonvaccinated/exposed and vaccinated/expose d horses (survival) and the nonvaccinated cerebrum and nonvaccinated thalamus (location) In gene expression status of those animals that recover from grave WN encephalitis through induction of vaccine mediated i mmunity

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85 gene expression status of naive animals undergoing grave encephalitis. RNA Extraction Total RNA was extracted from the tissues listed in Table 4 2 (36 total samples). A 30 mg piece of tissue was weighed out for each sample on dry ice. The tissues were homogenized using manual disruption and placed in 1 mL of g uanidium thiocyanate (Trizo l Invitrogen, Carlsbad, CA). The samples were vortexed and allowed to remain at room temperature (RT) for 5 minutes to al low complete dissociation of the nucleoprotein complexes. Two hundred L of molecular grade chloroform ( Thermo Fisher Scientific Waltham, MA ) was added to each sample. The samples were placed at room temperature for 2 minutes, then centrifuged at 12,000 x g at 4 o C for 15 minutes. The chloroform and centrifugation step s were repeated to ensure complete removal of the lipids. A 0.5 mL aliquot of isopropanol alcohol was added to each sample and incubated at room temperature for 5 minutes. The samples were centrifuged at 12,000 x g at 4 o C for 10 minutes to precipitate the RNA. One mL of 75% ethanol wa s added to each pellet, mixed, and repellet ed using centrifugation (7,500 x g 4 o C, 5 minutes). The ethanol was poured off and the pellets air dried for 5 minutes. RNAsecure (Ambion Austin, TX) diluted to a 1X concentration was heated on a heat block at 60 o C for 5 minutes and 75 L was added t o each pellet to inactivate any residual RNases. The pellets were incubated at 60 o C for 10 minutes in RNAsecure and cooled to room temperature. For DNase treatment, 7.5 L of 10X DNase buffer and 1 L of rDNase (Ambion, Austin, TX) was added to each sam ple. Samples were incubated at 37 o C for 1 hour. After incubation, 7.5 L of DNase inactivating reagent

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86 (Ambion, Austin, TX) were added to each sample and the samples were incubated at room temperature for 2 minutes. The samples were centrifuged at 10,0 00 rpm for 2 minutes, removed from the inactivating reagent, and placed at 80 o C until quality assessment. One L of each RNA sample was placed on a nano drop machine (ND 1000, Nanodrop Technologies, Wilmington, DE). The concentration and 260:280 ratio o f each sample was assessed. cDNA Creation and Dye Labeling Dye labeled cDNA was created using Cy3 dye (One Color Microarray Based Gene Expression Analysis kit, Agilent Technologies). The first strand cDNA was created using 3000 ng of RNA in 9 L or les s was aliquoted into individual tubes. 2.5 L of T7 promoter primer was added to each tube and the tubes were incubated at 65 o C for 10 minutes, then placed on ice for 5 minutes. The proprietary master mix (Agilent Technologies) was added t o each tube ( 8.5 L ) consisting of 4 L of 5X first strand buffer (pre warmed at 80 o C), 2 L of 0.1M DTT, 1 L of 10mM dNTP mix, 1 L MMLV, and 0.5 L RNAse inhibitor The tubes were incubated at 40 o C for 2 hours, heated to 65 o C for 15 minutes, and incubated on ice fo r 5 minutes. The amplification mixture for dye incorporation consist ing of 30 L of master mix (15.3mL of nuclease free water, 20 L 4X transcription buffer, 6 L 0.1M DTT, 8 L NTP, 6.4 L 50% PEG pre warmed at 40 o C for 1 minute, 0.5 L RNAse OUT, 0.6 L inorganic pyrophosphate, 0.8 L T7 RNA polymerase, and 2.4 L cyanine 3 CTP dye) was added and each tube was incubated at 40 o C for 2 hours. The dye labeled cDNA was then extracted using a propriety kit ( RNeasy Qiagen, Valencia, CA). The samples were br ought to a total volume of 100

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87 L by adding 20 L of nuclease free water and 350 L of the kit buffer ( RLT) was added to each sample and thoroughly mixed with a pipette. Ethanol (100%, 250 L) was added to each sample and mixed thoroughly with a pipette. Seven hundred L s of each sample were transferred to the kit colum n, and the columns were centrifuged at 13,000 rpm for 30 seconds at 4 o C. The columns were transferred to a new collection tu be and 500 L of buffer ( RPE ) was added to each column. The columns were centrifuged for 60 seconds at 13,000 rpm at 4 o C, and the eluant discarded. The buffer RPE and centrifugation steps were repeated. The columns were transferred to a new collection t ube and air dried for 2 minutes. To each column 30 L of RNase free water was added The columns were incubated for 1 minute at room temperature and were then centrifuged for 30 seconds at 13,000 rpm at 40 o C and this step was repeated with the same sampl e. The specific activity and yield of the samples were determined using a microfluidics platform ( Nano drop Technologies, Thermo Scientific) Both the concentration and the incorporation of the dye were measured. The formula Specific Activity = [(Conc entration of Cy3)/(Concentration of cRNA)] 1000 = pmol Cy3 per g cRNA was used to determine whether the sample would be used for hybridization to the array. Only samples with a specific activity > 8 were used. Hybridization and Scanning of Arrays Hy protocol. Individual, non pooled cDNA samples were hybridized to the arrays. B riefly, t he prioprietary blocking agent was prepared to a 10X concentration and incubated at 37 o C for 5 minutes. Individual tubes were prepared combining 1.65 mg of dye labeled

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88 cDNA, 11 mL of 10X blocking agent, nuclease free water, and 2.2 mL of the proprietary 25X fragmentation buffer for a final volume of 55 mL. The tubes were incubated at 60 o C for 30 minutes and then placed on ice for 1 minute. Fifty mL of the proprietary 2X hybridization buffer was added to each tube. The samples were mixed, centrifuged at 13,000 rpm for 1 minute at 25 o C, and placed on ice. The gaskets were placed into the c hambers and 100 mL of sample added to each chamber The array slides were placed on top of each gasket. The chambers were closed in the hybridization oven and rotated at 10 rpm for 17 hours at 65 o C. After the hybridization, the arrays were disassemble d in wash buffer 1. The slides were washed for 1 minute in proprietary wash buffer s at 37 o C. The excess liquid was dried off and the slides washed for 30 seconds in the proprietary stabilization and drying solution. The slides were scanned and data coll ected using the proprietary software ( Feature Extraction Agilent) Normalization and Statistical Analysis JMP Genomics version 4.0 ( S.A.S. Institute Cary, NC) was used to analyze the data. All files were transformed (log 2 ) and normalized using Loess n ormalization techniques. Normalization was checked using distribution analysis consisting of box plots, correlation heat maps, and overlayed kernel density elements ; and principal component analysis consisting of 2D, 3D, and scree plots. A two way analys is of variance ( ANOVA ) was performed (location and treatment were independent variables) and possible interactions between location (cerebrum and thalamus) and treatment (vaccinated, not vaccinated, normal) were tested in the ana l ysis addressing location, exposure, and survival (p<0.05). Only thalamus was compared between the two analyse s addressing exposure and survival due to differences in gene expression

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8 9 between the cerebrum and thalamus. Variability was estimated in the software via linear regression and Pearson correlation coeffiecient and the R square and residual variance tables were generated for each array. A significant genes lists was generated and a hierarchical clustering was performed. Gene Ontology En richment Probes for the analyse s of location, exposure, and survival that matched to the gene ontology categories of biological process, cellular component, and molecular function were identified. Gene ontology categories (as derived from the original an notation of the cDNA expression library, Fisher Cluster, University of Florida) that involved the neurological system, immunological system, apoptosis/cell death, and transcription/translation were targeted. The three analyse s were analyzed based on the n umber of significantly different genes that group ed into the se GO categories. Pathway Modeling Significant genes (p<0.05) for all three analyse s were fed into Ingenuity Pathways Analysis software (Ingenuity Systems, Redwood, CA). Only genes that were con tained in the database were mapped, and fold changes >1 and < 1 considered. Network modeling to determine interactions between significant genes, canonical pathways analysis to determine genes involved in known pathways, and disease/physiological function /location annotation was performed on significant genes. transcript s and p values considered in ranking of pathways, networks, and functions. This process was performed on all significant genes as well as on the gene ontology enriched datasets.

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90 Microarray Validation For the purposes of the initial validation of the utility of this microarray, several highly significant genes (six) were selected to 1) verify the accuracy of the probe hybridization, and 2) verify the accuracy of the relative expression values detected by the probe. To verify the relative expression values, only transcripts that were significantly upregulated or downregulated (p<0.05, fold change >2, < 2) in the exposure analysis were picked for analysis. A total of six transcripts were targeted to be used as primer sets in the validation experiment and included a denylate synthetase (2,5 OAS) complement component 1 (CC1) TNF receptor ligand (TNFR) interleukin 6 (IL 6) DEAD Box 60 (DB60) and defensin 4 ( DB4) with actin (ACT) as the endogenous control. Two sets of primers were designed using primer design software (ABI Primer Express version 3.0, Applied Biosystems). The primers are available upon request. The first s et of primers was designed to amplify a larger segment of the gene. Conventional PCR was performed using a proprietary master mix (Readymix Taq PCR Mastermix with MgCl 2 Sigma Aldrich, St.Louis, MO). For each primer reaction, 25 L of the reagent mix, 1 L each of forward and reverse primer (10mM), 5 L of sample, and 18 L of water were added to each respective PCR tube. The samples were held at 94 o C for 2 minutes, then cycled 25 times at 94 o C for 1 minute, 50 o C for 2 minutes, and 72 o C for 3 minutes. Th e samples were then held at 72 o C for 5 minutes and cooled at 4 o C. The reactions were run in triplicate for each set of primers. The three tubes from each reaction were combined and purified using a PCR purification kit (QIAquick PCR purification kit, Qiagen, Valencia, CA). Briefly, 5

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91 volumes of the kit binding buffer (PB1) was added. The samples were mixed and placed on the kit column. The tubes were centrifuged at 13,000 rpm (>10,000g) for 60 seconds. The eluant was discarded and 750 L of wash buffer (PE) was added. The columns were centrifuged at 13,000 rpm for 60 seconds, the eluant discarded, and the columns centrifuged a gain at 13,000 rpm for 60 seconds. Thirty Ls of water was added to each column membrane and the columns centrifuged again at 13,000 rpm for 60 seconds. The concentration and purity of the samples were determined using a microfluidics platform. The reac tions were resolved utilizing a 0.9% agarose gel and imaged under standard UV conditions. If a band(s) was visualized, t h e samples were submitted to the UF Interdisciplinary Centers for Biotechnology Research for Sanger sequencing. Sequencing results wer e checked against expected gene sequences. Once the correct sequence was validated, amplified samples were run under the thermocycling conditions listed above using a second set of nested primers. The presence of a band of the correct length was verified on a 0.9% agarose gel. For each target, a standard curve was generated using 5 two fold dilutions and triplicate wells The slope of the reaction and the R square was calculated via the proprietary software (ABI 7900, Applied Biosystems). The primer ef ficiency was checked using the equation efficiency = 10^( 1/slope). These primers were then used in real time, relative quantitation PCR in a SYBR green assay (Fast SYBR Green Master Mix, Applied Biosystems) to validate the findings of the level of expres sion demonstrated via array. Using proprietary conditions, 10 L of Fast SYBR Green Master Mix, a variable amount of each primer dependent on reaction efficiency 3000 ng of cDNA, and water up to a volume of 20 L were added to

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92 each well with replicates o f three wells performed on each sample. The plate was centrifuged and the real time PCR reactions (7500 Fast Real Time PCR System, Applied Biosystems) were performed using the reaction parameters consisting of a hold at 95 o C for 20 seconds, followed by 40 cycles consisting of a 40 cycle reaction at 95 o C for 3 seconds and a 40 cycle reaction at 60 o C for 30 seconds. Relative quantitation analysis was performed using the proprietary software for calculation of the comparative Ct method ( Applied Biosystems software for the 7500 Fast machine ) wherein 2 is used for the comparison of relative quantitation between the thalamus of vaccinated/exposed horses and nonvaccinated/exposed horses To verify the accuracy of the probe hybridizations, the probe seque nces were BLASTed against the equine genome (Fisher Cluster, UF ICBR, Gainesville, FL). Only sequences with e values <10 4 were generated. Sequences were checked for percent identity and sequence alignment. Results Study Design Three microarray experiments were completed to answer the question of whether there were differences in gene expression in WN encephalitis according to exposure status, vaccination status, and CNS region. The analyses and the breakdown of the samples can be seen in T able 4 3. With respect to the experimental groups, three subhypotheses were generated to analyze if there was a difference in gene expression between the nonvaccinated/exposed and untreated horses (exposure), the nonvaccinated/exposed and vaccinated/exposed ho rses (nonsurvival), and the nonvaccinated cerebrum and nonvaccinated thalamus (location). In particular, gene expression values from the thalamus (6) of the nonvaccinated/exposed horses was

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93 compared to expression values from the thalamus (6) of the untrea ted group to determine if there was a difference in gene expression due to exposure to WNV. Gene expression values from the thalamus (6) from the nonvaccinated/exposed horses was compared to gene expression values from the thalamus (6) of the vaccinated/e xposed horses to determine if there was a difference in gene expression between nave horses which succumb to WNV and those that are immune, do not develop significant disease and survive from WNV infection. Since the thalamus undergoes a higher viral loa d as determined by our previous studies [47 ,48 ] gene expression values from the thalamus (6) of nonvaccinated/exposed horses was compared to gene expression values from the cerebrum (6) of the same group to determine if there was a difference in gene expre ssion during exposure to WNV in these two different regions of the brain. Array Normalization Loess normalization was performed on all arrays and confirmed by distribution analysis. F igure 4 1 illustrates the normalization of each individual array. For analysis of the distribution and variability of the data itself, correlation and principal components analysis for all groups demonstrated that the majority of variance was accounted for with the first three principal components (x, y, and z) with Eigenva lues ( percents of variability ) of the each component, 11.09 (30.81%), 4.94 (13.71%, and 3.21 (8.94%), respectively. In addition, the mean of the R 2 was 0.939392 (range 0.8781 0.9871) for all arrays. A heat map and dendrogram was generated between all arrays (see Figure 4 2). Statistical Analysis Analysis of mean relative difference in gene expression using an ANOVA with interactions between treatment and location revealed significant differences in gene expression (p < 0.05) for all analyses (exposure, nonsurvival, and location). To

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94 determine which tissues should be compared in the exposure and nonsurvival groups (i.e. thalamus only or pooled thalamus and cerebrum), differences in gene expression were analyzed between the thalamus and cerebrum of the n ormal, nonexposed horses. The degree of fold change (relative fluorescent intensity) was analyzed for all differentially regulated genes. In total, 7,321 genes were significantly different between the two locations (6,911 after duplicate removal). There fore, for the exposure and nonsurvival groups, only thalamus was compared. This data is summarized in Table 4 4. The same 3,421 were significantly altered in all three analyses and overall, 4,000 (44%), 3,472 (46%), and 3,811 (49%) genes were expressed a t levels > 1.0 and < +1.0 for the exposure, nonsurvival, and location groups. Exposure Status. For exposure status, significant differences in gene expression in the thalamus were seen between nonvaccinated/exposed and normal, nonexposed horses for 9,0 20 genes (12,029 without duplicate probe removal). When analyzed solely by fold change, 2,936 genes decreased by < 1.0 (395 < 2.0) and 2,084 increased by > +1.0 (749 > +2.0) in the exposed nonvaccinated horses compared to the nonexposed normal horses. Immune/Survival Status. For immune/survival status, significant differences in gene expression in the thalamus were seen between nonvaccinated/exposed horses (nonsurvivors) and vaccinated/exposed horses (survivors) in 7,395 genes (9,978 without duplicate probe removal). In the nonvaccinated, nonsurvivors, 2,123 genes were decreased by < 1.0 (225 < 2.0) while 1,800 were increased by > +1.0 (666 > +2.0) compared to the vaccinated, survivors.

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95 CNS Location. Where analyzed by location in the brain, signi ficant differences in gene expression were seen between the cerebrum and thalamus of nonvaccinated horses exposed to WNV (location) for 7,649 individual genes (10,555 without duplicate probe removal). For the location analysis, 2,053 genes were decreased b y < 1.0 (609 < 2.0) while 1,827 were increased by > +1.0 (406 > +2.0) ( F igure 4 3) when the thalamus was compared to the cerebrum in nonvaccinated, exposed horses Gene Ontology and Pathways Analysis Overview Gene ontologies were mined for all signific ant genes based on those categorized in public accessed databases provided by NCB I Because of the sheer diversity of GO in this analysis, neurological, immunological, and apoptosis were GO categories specifically chosen for deeper data analysis by pathwa ys analysis Ingenuities Pathways Analysis Software (IPA) was used to identify putative physiological interactions between genes that were significantly changed Canonical pathways, functions, and networks were determined using the Fisher exact test wit h a p<0.05 and fold change < 1.0, >+1.0. Fewer than 25% of significant genes mapped to the IPA database for all groups. Of the genes that did map, identification was based on the NCBI nucleotide database gene IDs. Canonical pathways were identified to d emonstrate interactions between significantly changed genes. Functions (disease and physiological) were identified based on the transcript s and pathways identified as significantly changed. Transcript s of significance were also targeted for all analyses to identify those that may be of import in future studies. Exposure Status Gene o ntology. The first subhypothesis asked whether there was a difference in gene expression due to exposure to WNV between nonvaccinated horses exposed to

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96 WNV and normal horses not exposed to WNV. For this exposure analysis, genes that were found to have significant differences in expression were classified into gene ontology (GO) categories. With overlap, 6,009 genes were classified under biological process, 6,454 genes were classified under cellular component, and 6,646 genes were classified under molecular function (F igure 4 4 ). The genes that mapped to GO categories were then group ed according to the functions of transc ription/RNA processing, neurological genes, immunological genes, and cell death/apoptosis. T he most genes mapped to GO processes of transcription/RNA processing ( 2,022 ) with the second most genes mapping to neurological c ategories (1,081) G enes also map ped to i mmunological categories (983 ) and cell death/apoptosis (430 ) (Figure 4 5) Canonical pathways. Canonical pathways were first examined for interactions between multiple significant transcript s. For the canonical pathways assessment of the expos ure analysis, the majority of pathways were involved with some aspect of cell signaling for a variety of locations/functions ( T able 4 5, F igure 4 6 ). Seven of the top 25 pathways (based on the p value) were classified as neurological pathways (81 transcr ipt s) with 2 of the top 25 pathways classified as immunological pathways (25 transcript s). The neurological canonical pathways were analyzed for exposure and 1 7 pathways were identified (Figure 4 7 ). Specific neurological pathways that demonstrated dysr egulation for the exposure analysis included neurotransmitter pathways and signaling pathways These included glutamate receptor signaling (Figure 4 8 ), dopamine receptor signaling (Figure 4 9 ), axonal guidance signaling, CREB signaling in neurons, synapt ic long term depression, amytrophic lateral sclerosis,

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97 synaptic long term potentiation, GABA receptor signaling, reelin signaling in neurons, neuropathic pain signaling in dorsal horn neurons, semaphorin signaling agrin interactions at neuromuscular junctions, neurotrophin/TRK signaling, CNTF signaling, serotonin receptor signaling, and circadian rhythm signaling. The immunological canonical pathways were analyzed for the exposure analysis and 4 7 pathways were identified (Figure 4 10 ) When examining all of the CPs identified, pathways involved in the innate and adaptive response were present. The immune pathways that were upregulated in the exposure analysis (i.e. due to WNV) included the IL 15 signaling pathway (Figure 4 1 1 ), the IL 22 signaling pathway, the IL 9 signaling pathway, and the Interferon Signaling Pathway (Figure 4 1 2 ). Multiple pathways involved in apoptosis were also dysregulated in the exposure analysis. These included the retinoic acid mediated apopto sis signaling, calcium induced T lymphocyte induced apoptosis, cytotoxic T lymphocyte mediated apoptosis of target cells, induction of apoptosis by HIV1, and April mediated signaling. Functions Functions were assessed for the exposure analysis which l inks the top transcript s in each pathway to their related disease states and normal function. The functions were distributed amongst many analyse s, but of particular note are the number of functions involved with neurological and immunological pathways as well as cell death ( T able 4 6). For the exposure analysis, 4 categories were identified involving neurological functions (2,326 transcript s), 10 categories were identified involving immunological functions (1,830 transcript s for exposure), and 1 category was identified as involving cell death (1,153 transcript s exposure).

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98 The functions involving neurological categories were further analyzed. Most genes group ed under neurological disease when compared to nervous system development and function, behavior and psychological disease (Figure 4 1 3 ). When further analyzed by specific disease, genes mapped to mental disorders ( including and schizophr enia ), as well as degenerative neuropathies ( including progressive moto r neuropathy, ) (Figure 4 1 4 a,b) The functions involving immunological pathways were grouped with cell death/apoptosis for analysis. For the exposure analysis, the most genes were categorized under inflammation (992 transcript s). Both innate (inflammatory response, antigen presentation, immune cell trafficking) and adaptive (humoral immune resp onse, cell mediated immune response, cytotoxicity, immune cell trafficking) aspects of immunity were identified. Cell death and apoptosis categories were also seen for the exposure analysis, with 1,299 total genes involved with cell death, and 1,006 total genes involved with apoptosis (Figure 4 1 5 ) Transcripts Significantly upregulated and downregulated transcripts for the exposure analysis were identified. Transcripts that were increased in expression by 1 fold or more or decreased in expression b y 1 fold or less and mapped to the IPA database were analyzed for the exposure analysis ( A ppendix B). In total, for the upregulated transcripts 37 out of 543 transcripts (6.8%) were transcriptional regulators (Table 4 7 Appendix B). For the downregula ted transcripts 84 out of 1,031 transcripts (3.9%) were transcriptional regulators (Table 4 8 Appendix B). Specific transcriptional

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99 transcripts upregulated of note included signal transducer and activator of transcription (STAT1 ) interferon regulatory f actor 2 (IRF2 ), and interferon regulatory factor 3 (IRF3), ets variant 7 (ETV7), basic leucine zipper transcription factor, ATF like (BATF ), eomesodermin homolog (EOMES ), zinc finger, NFX1 type containing 1 (ZNFX1 ), activating transcription factor 3 (ATF3 ), and W W domain containing transcription regulator 1 (WWTR1). Transcriptional transcripts of particular note that were downregulated included SUB1 homolog (SUB1), nuclear factor I/A (NFIA), ankyrin repeat and SOCS box containing 1 (ASB1) (Table 4 7 4 8 ) Specific neurological transcripts were also significantly changed in expression (Table 4 9). For the exposure analysis, a total of 176 transcripts were downregulated and 43 transcripts were upregulated. Transcripts involved with n eurotransmitter pathways including glutamate receptor signaling (Figure 4 8 Table 4 10) and dopamine receptor signaling (Figure 4 9 Table 4 11) were of particular note. This included a decrease in the expression of NMDA glutamate receptors (GRIN), metabotropic glutamat e receptors (GRM8, HOMER 3 ), kainate glutamate receptors (GRIK 1 ), ionotropic glutamate receptors (GRIA 1,4 ), and glutamate clearance receptors (SLC1A) (glutamate receptor signaling) a decrease in the expression levels of the dopamine receptor D5 (DRD5), ade nylate cyclase, protein kinase, protein phosphatase, and tyrosine hydroxylase, and an increase in the expression levels of monoamine oxidase (MAO). C atenin (cadherin associated protein), delta 2 (neural plakophilin related arm repeat protein) (CTNND2 ) was also highly upregulated This molecule is specific to the brain, and functions to connect cell junctions and cytoskeletal architecture with signaling pathways (Appendix B ).

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100 Transcripts involved in the immune response for the exposure analysis were also significantly dysregulated. In total, 176 immune transcripts were downregulated, while 130 transcripts were upregulated (Table 4 1 2 ). The most notable was pentraxin 3 (PTX3), upregulated over 9 fold, which functions in the pathway of pattern recognition receptors in recognition of viruses and bacteria. Other upregulated immunological transcripts included DEAD (Asp Glu Ala Asp) box polypeptide 58 (DDX58 ), zeta chain (TCR) associated protein kinase 70kDa (ZAP70 ), Fc fr agment of IgG, low affinity IIIa, receptor (CD16a) (FCGR3A ), complement component 1, r subcomponent (C1R ), CD8a molecule (CD8A ), interleukin 4 induced 1 (IL4I1 ), interleukin 7 (IL7 ), CD5 molecule like (CD5L ), CD4, CD3, and interleukin 15 (IL15 ). Transcrip ts that mapped to specific immunological pathways of interest that were significantly expressed included those that mapped to the IL 15 pathway ( upregulation of IL 15, IRF3, STAT1, and TYK2; downregulation of phosphoinositide 3 kinase, regulatory subunit 1 (alpha) (PIK3R)), and those that mapped to the IL9, IL22, and JAK/STAT pathways (upregulation of SOC S 3, STAT1, TYK2; downregulation of PIK3R) ( Figures 4 1 1 4 1 2 ; Tables 4 13, 4 14). Apoptotic transcripts were also upregulated in the exposure analysis, i ncluding poly (ADP ribose) polymerase family member 14 (PARP), caspase 4 (CASP4) retinoid receptor (RXR) and retinoic acid nuclear receptor (RAR ) (Appendix B) Immune/Survivor Status Gene o ntology. The second subhypothesis asked if there was a differenc e in gene expression in the nonsurvivors which were not vaccinated and exposed to WNV compared to the survivors (100%) that were vaccinated and exposed to WNV. [58, 59, 117] The genes that were found to be significantly different in expression were classified into gene ontology (GO) categories. A total of 5,120 genes were classified under biological

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101 process, 5,462 g enes were classified under cellular component, and 7,696 genes were classified under molecular function (F igure 4 4 ) with overlap Analysis was then performed to analyse the GO categories according to the functions of transcription/RNA processing, neurolo gical genes, immunological genes, and cell death/apoptosis. T he most genes mapped to GO processes of transcription/RNA processing ( 1,864 ) with the second most genes mapping to immunological c ategories (850) G enes also mapped to neurological categories ( 840 ) and cell death/apoptosis (338 ) (Figure 4 5) Canonical pathways. Similar to the analysis to exposure status, the majority of canonical pathways engaged cell signaling for a variety of cell types, functions and transcripts ( T able 4 5, F igure 4 6 ). Ten of the top 25 pathways (based on the p value) were classified as neurological pathways (156 transcripts ). None of the top 25 pathways were identified as immunological pathways The neurological canonical pathways were analyzed for nonsurvivorship and 19 path ways were identified (Figure 4 7 ). Specific neurological pathways that demonstrated dysregulation for the exposure analysis included neurotransmitter pathways and signaling pathways. Like the analysis for exposure status, these included glutam at e receptor signaling (Figure 4 8 ), dopamine rec eptor signaling (Figure 4 9 ), CREB signaling in neurons, synaptic long term depression, amytrophic lateral sclerosis, synaptic long term potentiation, GABA receptor signaling, neuropathic pain signaling in d orsal horn neurons, semaphorin signaling, neurotrophin/TRK signaling, CNTF signaling, serotonin receptor signaling, glutamate metabolism, and circadian rhythm signaling. Other pathways identified in this analysis included axonal guidance signaling

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102 neureg and agrin interactions at neuromuscular junctions. Forty nine pathways involved in both the innate and adaptive immunity were identified as associated with immune/survivorship sta tus (Figure 4 10) The immune pathways that were upregulated (as in the analysis of exposure status) included the IL 15 (Figure 4 11), IL 22, IL 9, and IFN signaling pathways (Figure 4 12). Multiple pathways involved in apoptosis were also dysregulated a nd the previous analysis included the retinoic acid mediated apoptosis signaling, calcium induced T lymphocyte induced apoptosis, and death receptor signaling. Functions In the assessment of f unctions associated with nonsurvivorship, multiple transcrip ts were identified in f unctions that were distributed amongst many analyse s ( T able 4 6). Five categories were identified involving neurological functions (2,246 transcripts ), nine categories were identified involving immunological functions (1,542 transcri pts ), and one category was identified as involving cell death (1,082 transcripts exposure). In the analysis of specific neurological categories, more genes grouped under neurological disease when compared to nervous system development and function, beha vior, and psychological disease (Figure 4 13 ). When further analyzed by specific disease, genes mapped to the similar mental disorders and degenerative identified by the previous analysis (Figure 4 14 a,b) For deeper analysis of immunological and apopt osis functions similar functions were identified as those involved in the exposure analysis. Mo st genes were categorized under inflammation (832 transcripts ) with both innate and adaptive immune

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103 functions identified. Cell death and apoptosis categories were also seen for the nonsurvival analysis, with 476 total genes involved with cell death, and 340 genes involved with apoptosis (Figure 4 15). Transcripts Individual, significantly upregulated and downregulated transcripts analyzed for their assoc iation with nonsurvivorship. Transcripts that were increased in expression by 1 fold or more or decreased in expression by 1 fold or less and modeled in the IPA database (Appendix B). For transcriptional regulators, 35 of 543 transcripts (6.4%) were upre gulated (Table 4 7, Appendix B) while 46 of 1,031 transcripts (4.4%) were downregulated (Table 4 8, Appendix B). Specific transcriptional genes of interest that were upregulated include STAT1, IRF2 IRF3, ETV7, BATF, BATF EOMES ZNFX1 ATF3 and WWTR1. Transcriptional genes of interest that were downregulated included SUB1, NFIA, and ASB5 (Table 4 7, 4 8). In the subanalysis of neurological transcripts (Table 4 9), a total of 209 downregulated transcripts and 42 upregulated transcripts were i dentified, and similar to the analysis of exposure status, were primarily composed of transcripts involved with neurotransmitter pathways including glutamate receptor signaling (Figure 4 8, Table 4 10) and dopamine receptor signaling (Figure 4 9, Table 4 1 1). This included a decrease in the expression GRIN, HOMER3, GRIK1/ 2, GRIA1 / 2 / 3, SLC1A, AC, PK, and PP, and an increase in the expression levels of MAO and CTNND2 Further analysis of the transcripts involved in the immune response in the nonsurvivors i dentified 215 immune transcripts which were downregulated, while 116 transcripts were upregulated (Table 4 12). Upregulated transcripts included PTX3 (7.7 fold increase over vaccinates), DDX58 ZAP70 r eceptor CD16a FCGR3A C1R CD8A

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104 IL4I1 IL 7 CD5L CD4, CD3, and IL15. Transcripts that mapped to separate immunological pathways that were significantly changed in express ion included those that mapped to the IL 15 pathway ( upr egulation of IL 15, IRF3, and STAT1 ; downregulation of PIK3R, PTK2B, and mitogen act ivated protein kinase 1 (MAPK1) ) The transcripts that mapped to I L 9, IL 22, and JAK/STAT pathways included upregulation of SOC S3, STAT1 with downregulation of PIK3R, MAPK1, and protein inhibitor of activate d STAT, 2 (PIAS2)) (Figures 4 11, 4 12 ; Tables 4 13, 4 14). Apoptotic transcripts were also upregulated for the nonsurvivors and included (PARP) and caspase 4. (Appendix B) CNS Location Gene o ntology. The third subhypothesis asked whether there was a difference in gene expression due to location in the brain during WNV infection between thalamus and cerebrum of the nonvaccinated exposed horses. With overlap, 5,200 genes were classified under biological process, 5,675 genes were classified under cellular component and 5,715 genes were cla ssified under molecular function (F igure 4 4 ). The genes that mapped to GO categories were then grouped according to the functions of transcription/RNA processing, neurological genes, immunological genes, and cell death/apoptosis. M ost genes mapped to GO processes of transcription/RNA processing ( 1,664) with the second most genes mapping to immunological c ategories (798) G enes also mapped to neurological categories (447 ) and cell death/apoptosis (349 ) (Figure 4 5) Canonical pathways. For the canoni cal pathways assessment of the analysis of significantly different genes depending on CNS location analysis, the majority of pathways were involved with some aspect of cell signaling also ( T able 4 5, F igure 4 6 ).

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105 Seven of the top 25 pathways were identified as neurological (125 transcripts ) while two of the top 25 pathways were identified as immunological pathways (40 transcripts ). The deeper analysis of the neurological canonical pathways significant for locatio n sixteen pathways were identified (Figure 4 7). Specific neurological pathways that demonstrated dysregulation also included neurotransmitter pathways and signaling pathways. These included glutamate receptor signaling (Figure 4 8), dopamine receptor si gnaling (Figure 4 9), axonal guidance signaling, CREB signaling in neurons, synaptic long term depression, amytrophic lateral sclerosis, synaptic long term potentiation, GABA receptor signaling, reelin signaling in neurons, neuropathic pain signaling in do rsal horn neurons, semaphorin signaling agrin interactions at neuromuscular junctions, neurotrophin/TRK signaling, CNTF signaling, serotonin receptor signaling, and circadian rhythm signaling. Forty ei ght i mmunological canonical pathways (Figure 4 10) involving both the innate and adaptive response were present. The immune pathways that were upregulated in the thalamus compared to the cerebrum (Figures 4 11, 4 12) included the same previously identifie d signaling pathways (IL 15, IL 22, IL 9 and IFN). Multiple pathways involved in apoptosis were also dysregulated in the location analysis. These included the retinoic acid mediated apoptosis signaling, calcium induced T lymphocyte induced apoptosis, cyto toxic t lymphocyte mediated apoptosis of target cells, induction of apoptosis by HIV1, and apoptosis signaling, and myc mediated apoptosis signaling. Functions Functions were assessed for the location in the CNS which links the top transcripts in each pathway to their related disease states and normal function ( T able 4 6). For the location analysis, five categories were identified involving

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106 neurological functions (3,242 transcripts ), ten categories were identified involving immunological functions (1,5 58 transcripts ), and one category was identified as involving cell death (719 transcripts ). The further analyses of specific neurological function were similar to that mapped for both the exposure and immune status analyses (Figure 4 13 ) mental disorders and degenerative neuropathies (Figure 4 14 a and b) The functions involving immunological pathways were grouped with cell death/apoptosis for analysis. Like the previous analyses, most genes were categorized under inflammation (834 transcripts ), with involvement of both innate and adaptive immunity. Cell death and apoptosis categories were also seen for the location analysis, with 210 total genes involved with cell death, and 184 total genes involved with apoptosis (Figure 4 15). Transcrip ts Significantly upregulated and downregulated transcripts dependent upon location in the CNS were identified and modeled (also see Appendix B). In total, for the upregulated transcripts 38 of 543 transcripts (6.9%) were transcriptional regulators (Tab le 4 7, Appendix B). For the downregulated transcripts 40 of 1,031 transcripts (3.8%) were transcriptional regulators (Table 4 8, Appendix B). Specific transcriptional transcripts changed of note were similar to that of both the exposure analysis and the immune/survivor analysis (Table 4 7, 4 8). These included STAT1 IRF3, ETV7, BATF ZNFX1 ATF3 and WWTR1 that were upregulated, and SUB1 and ASB1 that were downregulated. Specific neurological transcripts were also significantly changed in expression (Table 4 9). For the location analysis, a total of 176 transcripts were downregulated and 43 transcripts were upregulated. Transcripts of note were similar to the previous

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107 analyses (Figure 4 8 and 4 9, Table 4 10 and 4 11). This included a decrease in the glutamate signaling expression of GRIN1/2A/2B/3A, HOMER1, GRIK1, GRIA1/2/3/4, and SLC1A. Similar decreases were seen in the dopamine signaling pathway transcripts AC and PP, with an increase in MAO. The p rotein CTNND2 was also highly upregulated Transcripts involved in the immune response for the location analysis were also significantly dysregulated. In total, 266 immune transcripts were downregulated, while 210 transcripts were upregulated (Table 4 12). In this case PTX3 was upregulated over 4 fold The other upregulated and downregulated immunological transcripts that were identified were similar to the analyses involving exposure and immune status These included upregulation of DDX58 ZAP70 FC GR3A C1R CD8A IL4I1 and IL7 Transcripts that mapped to specific immunological pathways of interest that were significantly expressed included those that mapped to the IL 15 pathway (upregulation of IL 15, IRF3, JAK3, and STAT1; downregulation of PIK3 R, PTK2B, MAPK1, and MAPK1), and those that mapped to the IL9, IL22, and JAK/STAT pathways (upregulation of JAK1, PIK3R3, and STAT1; downregulation of PIK3R 1,2, MAPK1, MAP2K1 ) ( Figures 4 11, 4 12; Tables 4 13, 4 14). Apoptotic transcripts were also upregu lated in the location analysis and were similar to that of immune/survivorship status, including poly (ADP ribose) polymerase family member 14 (PARP), and caspase 4 (CASP4) (Appendix B) Analysis of Overlap Between Exposure, Survival/Immunity, and Locati on Genes common to all pathways (3,423 genes) were analyzed by Fisher exact test (IPA). For canonical pathways analysis, four pathways (23 transcripts ) involving the neurological system were identified and nine pathways (61 transcripts ) involving the

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108 immunological system were identified (Figure 4 16). Significant genes were then analyzed for related functions (T able 4 15 ). The majority of these transcripts modeled for cell death (646), with genetic disorder containing the second highest number of tra nscripts (629) and neurological disease the third most modeled (479). The neurological functions were analyzed separately (Figure 4 17 ). Neurological functions that were common to genes in all analyses substantiated the separate findings of all of the se parate factor analyses. These included mental disorders (schizophrenia, bipolar affective disorder) and degenerative neuropathies (progressive motor neuropathy, sclerosi s). For immunological and cell death functions, many genes were also categorized under inflammatory disorders, cell death, apoptosis and immunologica disease (F igure 4 18 ). Other functions of significance included cytotoxicity, infectious disease, humor al immune response, cell mediated immune response, inflammatory response, immune cell trafficking, and antigen presentation. Array Validation The correct sequences (checked against the sequences from the transcriptome) were identified for the primer pai rs 6, DEADBox60, Def 4, and TNF r. The housekeeping gene and the other genes were significantly up regulated. Primer efficiencies were established for all primer pairs using s tandard curves analysis and efficiency calculation, with efficiencies ranging between 85 and 97%. Real time relative quantitation PCR was then run in triplicate on the thalamus from 6 of the vaccinates and 6 of the non vaccinates, with actin as the endo genous control. The results of the relative

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109 quantitation can be seen in Table 4 16. As expected, there was a relative increase in expression for all primer pairs when comparing the vaccinates to the nonvaccinates. This correlated with the microarray dat a, which showed an increase in fold change for all of the transcripts chosen. Probe sequences were analyzed by comparison to the most recent version of the EqCab2 genome using the basic l ocal a lignment s earch t ool (BLAST, Fisher Cluster, University of F lorida, Gainesville, FL) to determine the accuracy of each sequence to detect single genes as opposed to gene families. In total, 42,843 oligonucleotide probes were analyzed and 40,113 probes matched to one sequence with 100% identity (93.6%). Of these, 3,700 (9.2%) matched more than once to a genomic sequence implying possible binding to a gene family. The majority of these which matched to multiple sequences were identified as belonging to one chromosome. In addition, 2,687 probes matched at <100% ide ntity (average 97.5% identity). Forty three probes did not match, and were most likely present as controls since the sequences could not be detected in the sequenced library (Table 4 17). Discussion This experiment was the first study to analyze glob al gene expression during WNV disease, infection, and recovery in the CNS of natural, outbred equine hosts. The data generated from this project provides invaluable insight into WN encephalitis in both equine and human hosts, and is a useful platform for future studies. The majority of previous work that has been conducted to understand WN encephalitis has been conducted in murine models. Rodents are not natural hosts for WNV and demonstrate clinical disease and pathology that differs greatly from natural host infection. In

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110 contrast, horses (a natural host) demonstrate similar clinical disease and pathological distribution of lesions that closely mimic natural WNV disease in humans. Thus the equine model is recognized as a highly useful tool to study WN encephalitis for the purpose of gaining insight into both horse and human WN disease. The main hypothesis, that there are families of genes that are changed in a consistent manner in horses undergoing WN encephalitis was investigated. In the actually a nalysis of the microarray data, three subhypotheses were investigated to explore whether there was a difference in gene expression based on the state of exposure, immunity/survival, and location in the CNS. All three analyses demonstrated highly similar c hanges in the canonical pathways, functions, and transcripts Because there was high amount of overlap in our findings from these analysis, and there were interactions between factors, either these findings support a generalized model of WNV encephalitis based on exposure status, recovery, and CNS pathology or the state of WN infection without regard to immunity and recovery has been primarily modeled. This is entirely feasible because this model is one of grave overwhelming encephalitis. Thus subtle di fferences between noninfection and recovered WNV horses may not be appreciated based on the experiment design and analysis. Alternatively, It is possible that immunity from WN encephalitis after exposure is similar to a completely nave, nonexposed state. Additionally, it is likely that the time of sample collection (21 days for vaccinated/exposed horses, 7 9 days for nonvaccinated/exposed horses) influenced gene expression in immune horses. However, overall, it appears that horses that are exposed to WNV demonstrate similar chan ges in gene expression, which are highlighted by the changes in the

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111 thalamus. The s e data indicate that measurement of changes in gene expression in the thalamus correspond to localization of the virus since the thalamus was a primary focus for viral localization in WN infection in these horses at the time of disease. [58, 59, 117] Th is allows for development of neuronal c ell specific models for further investigations of the transcriptome and proteome of WNV infection. A total of 17 neurological canonical pathways were identified across the three analyses, the majority of which were involved with signaling within the nerv ous system. The functions identified from these pathways mapped to mental disorders ( bipolar enia, and depression ) and degenerative neuropathies ( progressive motor neuropathy, di sease, neurodegeneration, amytrophic lateral sclerosis, and multiple sclerosis ). These diseases are also highly associated abnormalities of transmitter and synaptic transmission in the thalamus and hypothalamus. Neurotransmitter pathways were one of the t op dysregulated pathways for all groups, including glutamate pathways. Glutamate is the primary excitatory neurotransmitter in the neurological system. Previous work has demonstrated that an excess of glutamate at the synaptic cleft can lead to apoptosis of neurons through glutamate excitotoxicity. [92 98] This can be caused by release of excessive levels of glutamate from the pre synaptic neuron, downregulation of glutamate receptors on the post synaptic neuron, and downregulation of glutamate uptake receptors. In this study, the nonvaccinated group of h orses exposed to WNV demonstrated gene expression changes consistent with glutamate excitotoxicity. These included a decrease in the expression levels of NMDA glutamate receptors, metabotropic glutamate receptors,

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112 kainate glutamate receptors, ionotropic g lutamate receptors, and glutamate clearance receptors. Thus it appears that infection with WNV leads to a downregulation of glutamate receptors on the post synaptic neuron as well as glutamate uptake receptors on glial cells. This may lead to an increase in glutamate levels in the synaptic cleft, apoptosis through glutamate excitotoxicity, and contribute to the neuropathology associated with WNV infection. Further study involving the detection and quantification of these transcripts from neuronal cells i nfected with WNV is necessary before any firm conclusions can be drawn. Dopamine was another neurotransmitter pathway that was significantly changed in all three groups. Dopamine is a stimulatory neurotransmitter that functions, among other things, in th e control of voluntary movement (lack of dopamine leads to the inability to control voluntary movement like syndrome with bradykinesia and incoordination). [158] In the nonvaccinated group of horses exposed to WNV, a decrease was seen in the expression leve ls of the DRD5 as well as the downstream affector transcripts AC, PC, and PP. In addition, tyrosine hydroxylase, which catalyzes the conversion of tyrosine to dopamine, was downregulated. The expression of monoamine oxidase (MAO), which functions to brea kdown dopamine, was increased in the nonvaccinated exposed group. Thus exposure to WNV may lead to a decrease in dopaminergic receptors and subsequent downstream signaling, a decrease in enzymes to create dopamine, as well as an increase in MAO. This res ults in a total decrease in available dopamine, which may explain many of the clinical signs seen in WNV infection studies targeting the actual transcripts are necessary.

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113 Clini cal neurological disease in horses caused by WNV is characterized by a combination of spinal cord, midbrain/hindbrain, and mentation abnormalities. Specifically, a stiff stilted gate (perhaps similar to bradykinesia), ataxia, flaccid paralysis, paresis, r ecumbency, muscle fasciculations, cranial nerve abnormalities, changes in personality, and hyperesthesia are often noted. Long term in horses that recover, muscle wasting is often seen along with residual neurological deficits. [10] These clinical signs mim ic many of the clinical signs seen in some human neurological disorders, progressive motor neuropathy, neurodegeneration, amytrophic lateral sclerosis, and multiple sclerosis For this study, it was fou nd that many of the pathways and transcripts previously shown to be dysregulated due to these diseases are also abnormally expressed during WNV infection. Thus neurological infection with WNV in horses appears to mimic many of the seemingly non infectious neurological disorders seen in man. Why this occurs is not known. Perhaps the non infectious neurological disorders actually have an origin in viral infection. Or perhaps the brain can only behave and react in a certain manner no matter the stimulus or insult. Regardless of the speculation as to why, this study was the first to demonstrate that infection with WNV leads to dysregulation in known neurological gene pathways, including those involved with neurotransmission and downstream signaling. This c orresponds with clinical signs of disease in affected hosts, and also suggests a correlate between the neuropathology induced by viral infection of the CNS and the neuropathology seen in non infectious neurological disease. The similarities between the three analyses can also be seen when examining the immunological pathways and functions. Previous work in elucidating the

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114 immunological pathways involved with WNV infection have focused on individual pathways, mainly involving the adaptive response. Howe ver, this is limiting in that a comprehensive picture of how the host responds to infection with different branches of the immunological system has not been formed. This study demonstrated that both an innate (inflammatory response, antigen presentation, immune cell trafficking) and adaptive (humoral immune response, cell mediated immune response, cytotoxicity, immune cell trafficking) immune response are present in all analyses. In general, the majority of immune transcripts and pathways were downregulat ed in the nonvaccinated horses exposed to WNV. Thus there is evidence that a balanced immune response is downregulated during WNV infection at the peak of clinical disease. In contrast, certain immune pathways appeared to be upregulated during WNV infe ction in nonvaccinated horses exposed to WNV. The Interleukin 15 signaling pathway is one of these pathways. IL 15 has been shown to stimulate CD8+ T cell and natural killer cell activation and proliferation; activate memory T cells; prevent apoptosis; a nd phosphorylate the JAK kinases and STAT3, STAT5, and STAT6. [159 162] IL 15 has been shown to be particularly important in providing a protective immune response to viral infection. This study was the first to provide evidence for the upregulation of the IL 15 pathway during WNV infection. For all three analyses, IL 15 was upregulated over 2 fold, as well as STAT1 (transcription factor) which was upregulated over 2 3 fold. Interestingly, the downstream elemen ts of IL 15 were downregulated in the unvaccinated horses exposed to WNV. There could be many explanations for this. The virus could be blocking the downstream effector elements of the IL 15 pathway to prevent the host immune response to the virus. Ther e could also be other elements in

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115 the IL 15 pathway that are not yet elucidated. It is also possible that this finding is only a reflection of the timing when the nave horses exposed to WNV were euthanized (at the onset of clinical signs) and a beneficia l response from IL 15 to viral infection could not be realized in these horses. Thus it appears that IL 15 is upregulated in response to WNV infection, and while it may play a key role in recovery from viral infection, its dysregulation may be a key compo nent of the immunopathology of this disease. Continued work targeting the quantification of IL 15 levels during viral infection at different time points is necessary for further clarification of this data. Other pathways that were upregulated in non vacc inated horses exposed to WNV were the IL 22, the IL 9, and the interferon signaling pathways with IL 22 and IL 9 activating similar transcripts Both of these pathways activate JAK and TYR transcripts which in turn phosphorylate and activate STAT (Signal Transducers and Activators of Transcription) specifically STAT1, STAT3, and STAT5. These STAT transcripts induce the expression of ISGs (interferon stimulated genes) through a variety of mechanisms, and lead to the induction of an innate antiviral respo nse. [163] As expected, expression of these JAK/STAT transcripts is upregulated during WNV infection in the unvaccinated horses exposed to WNV. Of interest as well is the finding that the SOCS3 (suppressor of c ytokine signaling 3) is also upregulated in the exposure and survival analyses. SOCS3 functions as a negative feedback inhibitor on the JAK/STAT pathway, thereby inhibiting the innate immune response. [164] This has not been documented previously in WNV, but has been shown in other studies to be upregulated by viral infection [165, 166] Upregulation of SOCS3 allows the virus to escape the innate immune response and has also been shown to lead to chronic infection and in flammation. Thus it is possible that

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116 while the JAK/STAT pathway is upregulated in response to WNV infection for the activation of innate immunity, WNV may induce the expression of the SOCS3 molecule to suppress this pathway and evade the innate immune res ponse. Many transcripts identified as having various functions were also significantly dysregula ted. One group included transcriptional regulators. Transcriptional regulators with increased expression included STAT1, IRF2, IRF3 ETV7, BATF EOMES ZNFX1 ATF3, and WWTR1 Understanding these transcripts is important for understanding how the host responds at the cellular level to WNV infection. The general transcriptional regulator, ETV7 may be involved in the cellular response to WNV, the immune respon se to WNV, or may be involved with WNV replication. The Th 17 response is regulated by BATF [167] and leads to inflammation and tissue injury, consistent with the clinicopathological findings due to WNV. The EOMES transcription factor has been shown to be st imulated by IL 2 and involved in the differentiation of CD8+ T cells. [168] This is consisten t with upregulation of the cellular immune response to WNV. Again, both ZNFX1 and WWTR1 are general transcription factors, inducing components of t he immune system. The molecule ATF3 has been shown to be an early response gene that copes with cell stressors and can induce apoptosis, [169] possibly coinciding with the pathology of WNV in these analyses. And finally, STAT1, IRF2, and IRF3 are transcript ional regulators that are involved in the innate immune response to viral infection. [163] Besides the value of understanding the host response to infection, identifying the transcriptional genes that are upregu lated during viral infection is important to understand how the virus itself may undergo transcription. The exact

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117 mechanism of this process has yet to be identified, but recognizing the host transcriptional regulators that are upregulated is an important first step. Downregulation of transcriptional regulators was also noted for all three analyses. These included ASB1 ASB5 SUB1, and NFIA The ASBs function to suppress the SOCS (suppressor of cytokine signaling) transcripts This coincides with the evidence of the upregulation of the SOCS3 transcript mentioned in the previous section to combat the innate immune response of the JAK/STAT pathway by the virus. The general transcriptional factor SUB1 (SUB1 homolog) is implicat disease. Downregulation of the nuclear factor, NFIA, (nuclear factor I/A) is notable in that it contradicts previous findings for upregulation during adenovirus transcription. Other, nontranscriptional genes were also highly upregula ted and downregulated. The most highly upregulated transcript for all analyses (9 fold for exposure, 7.7 fold for survival, and 4.2 fold for location) was PTX3 (pentraxin 3). This molecule has many functions, including an integral role in the pathway of pattern recognition receptors in recognition of viruses and bacteria. [170, 171] This gene is induced by IL 1b, and functions in the phagocytosis and opsonization of antigens, as well as in the inflam matory response. Thus infection with WNV and recovery from disease may be associated with an increase in this molecule that plays an integral role in innate immunity. Another transcript that was highly upregulated in all analyse s was CTNND2 a brain specific molecule which functions to connect cell junctions and cytoskeletal architecture with signaling pathways. [172] This could provide evidence that dysregulation of neurological tissue, such as that induced during WNV infection, leads to re

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118 arrangem ent of neuronal architecture and the induction of signaling. This may also be important in viral entry into the cell. A variety of single transcripts involved in the immune response were also upregulated in all analyses. These included DDX58 ZAP70, F CGR3A, C1R CD8A IL4I1, IL7, CD5L, CD4, CD3, and IL15 to name a few. Therefore the immune response appears to play a role in both infection with and recovery from WNV. Apoptotic transcripts were also upregulated in all analyse s PARP and CASP4, while som e apoptotic transcripts were upregulated in only the exposure analysis and these include RXR and its receptor, RAR. Understanding which transcripts are upregulated or downregulated during viral infection is important. This provides a glimpse into the a ffect of the virus on individual transcripts and, with further studies, could lead to the elucidation of many unanswered questions. Specifically, it may identify transcripts that are used by the virus to bind to and enter the cell. It may also identify t he cellular components that the virus uses for transcription and translation. Because this study involved samples from the peak of infection and samples during the recovery phase from infection, transcripts that are universally upregulated are of particul ar import in identifying candidate biomarkers and important genes. Future Work and Issues to be Addressed. The main limitation of this project was in the samples used for the study. This is particularly evident for the immune status/ survival analysis. For humane reasons, all horses demonstrating clinical signs of disease had to be euthanized immediately. Therefore samples were collected from non vaccinated exposed horses at the height of clinical disease approximately 4 7 days

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119 post exposure. The samp les collected from vaccinated horses exposed to WNV were taken 14 days post exposure. Not only was the collection of the samples from each of these analyses at different points in the disease process, there was no ability to collect samples over time. Th erefore, the interpretation of data is somewhat limited. Nonetheless in this model none of the protected horses exhibited clinical disease, virus was not isolated from any of these protected horses, nor was there evidence of significant pathology in these horses. For the exposure analysis, the major limitation was the untreated horses. In the non vaccinated, exposed analysis, all horses were age matched (all 1.5 years of age at euthanasia) and breed matched. In addition, the environment was controlled (confined to a research laboratory for the majority of their life). The untreated horses consisted of a more diverse population of individuals not matched for age (ranging from 1 year to geriatric) or for breed. The circumstances under which the individ ual animals lived were not controlled. Finally, the individuals were all being euthanized for different reasons (limb deformity, age, etc.) and one had a known enlargement of the pituitary gland. Thus there may have been an inherent variability in the ho rses and this may have introduced enough variability so limited detection of differences between normal horses and recovered horses occurred. Another major limitation of the study involves the inherent problems with microarrays. There were problems wit h dye incorporation (likely a result of the chemistry of the manufacturer) such that multiple dye labeling experiments needed to be performed at times for certain samples. The probes on the arrays are only 60 oligonucleotides in length, yet transcripts wi ll bind to the probes if there is a 25 base pair

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120 match. Therefore there may be transcript interference when binding to some of the probes. The last major limitation to be discussed is in the pathway analysis of the data. The IPA database is an excelle nt resource for building pathways and networks between transcripts of interest and identifying diseased states. However, the program is only as good as the databases that it references. Fewer than 25% of the significant genes in this study were actually identified by the IPA database. The only way in which to solve this problem would be to use a program with a larger database from which to design pathways and networks a resource that is not yet available. In addition, the program is biased toward the d iseases and pathways that are recognized in the software. For some of the transcripts that occur in the brain are recognized as similar or the same as those in the li ver pathway, they will be mapped accordingly. Future work with this project will involve continued analysis of the array expression data. This will include continuing to analyze the pathways, functions, and networks identified by the IPA database, as wel l as feeding this data into other pathway modeling tools. The array will also need to be validated with parallel experiments. Ideally, this would consist of performing a study with WNV in a different host (i.e. a mouse model of infection) and analyzing t he gene expression of those tissues on the array. Other studies that would help to validate the array could include immunohistochemistry on molecules that are shown to be significantly upregulated or downregulated with fixed tissue specimens from the same horses. Finally, there are serum samples at different time points over the course of disease from the same horses

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121 analyzed on the arrays. These samples could be used to identify significant biomarkers and to better understand peripheral gene expression over the time course of the disease. Conclusions For this portion of the project, the sequenced and annotated transcriptome of the equine CNS was used to create a 4 x 44,000 custom spotted oligonucleotide microarray. This array was used to analyze gene e xpression in the CNS to answer whether there was a difference in gene expression due to 1) exposure status to WNV (infected vs noninfected), 2) immune/survival from WNV infection, and 3. location in the brain during WNV infection. Statistical analysis was performed on the data to identify genes that were significantly up or downregulated. Significant genes were then analyzed statistically utilizing a systems biology approach to detect interactions between genes to generate biological models of WNV infect ion and disease. A large number of genes were identified as significant when looking at the three different analyses (9,020 for exposure, 7,395 for survival, and 7,649 for location). Gene ontological analysis was performed on the data from all three an alyses. Most genes mapped to transcription/RNA processing (5,550) with the second most genes mapping to neurological categories (3,065) for all analyse s. A large number of genes also mapped to immunological categories (2,631) and cell death/apoptosis (1, 117) The GO data was supported by the pathways analysis, which found the most genes modeled within signaling pathways, many of which were involved with transcription. After cell signaling (which was not specified to location) the majority of significan t genes were modeled within neurological pathways and disease functions. This analysis indicates that components of both the glutamate and dopamine pathways

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122 were down regulated in the immunologically susceptible horses undergoing challenge to WNV. This f indings provides preliminary evidence that glutamate excitotoxicity and dysregulation of the neurotransmitter, dopamine, likely contribute to the clinicopathological features of WNV. Many of the transcripts for all the analyse s consisted of several components of previously characterized mental disorders and degenerative neuropathies This may be evidence of simple overlap of functional processes in the brain or suggest a more complex relationship between viral infection of the CNS and the clinicopat hological states of neuropsychiatric and neurodegenerative disorders. The other major set of pathways that were shown to be dysregulated in this investigation were those involved in the immunological response. Components of both the innate and adaptive immune response were found in all comparative analyses. The majority of immunological pathways were downregulated in the non vaccinated exposed horses compared to the others (as well as in the thalamus) providing evidence that rather than upregulation of normal protective immune responses in the nave host, the virus likely interacts to block induction of several responses. Apoptosis was also upregulated in WNV susceptible host undergoing grave challenge, a finding consisting observed in vitro and in the rodent models, but with limited validation in the natural host undergoing infection. Under grave WNV challenge, specific immunological and transcriptional pathways of note included upregulation of the IL 15 production and signaling pathways, the JAK/STAT pathway, and the SOCS3 transcripts From this data, it may be hypothesized that induction of IL 15 may be part of the immunopathogenesis of grave

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123 WNV infection. Further, it could be hypothesized that WNV blocks induction of immunity by induction of the suppressive SOCS pathway to downregulate JAK/STAT Specific transcripts that were significantly upregulated and downregulated were also identified that may indicate dysregulation as part of the pathogene sis of disease. Transcription genes increased in expression involved in the immune response included STAT1, IRF2, IRF3, BATF, and EOMES. Transcription genes decreased in expression included NFIA, SUB1, ASB1, and ASB5. Individual transcripts not involve d in transcription were also upregulated by a significant amount. One was PTX3 ( a C reactive protein ) which has been shown to be involved in the pattern recognition response to viruses and bacteria. Whether or not this is part of antigen processing and p resentation by macrophages or induced by other components of the immune response such as cytokines or cellular debris is an open question. The important gene, CTNND2 catenin is derived is important in cell adhesion and dendritic growth. Whe ther or not induction of this gene is cause by the virus to aid infection or represents a physiological repair response in the virally injured CNS is another issue worth y of investigating. Understanding which of these transcripts that are upregulated or d ownregulated during viral infection and, which, of the corresponding proteins can be detected in plasma or serum may lead identification of candidate biomarkers. This study also provides initial validation of the array which can be continued by investigat ion of these pathways through generation of new hypotheses and study of the array under other diseases conditions.

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124 In summary, the microarray proved to be a useful tool to understand changes in gene expression patterns during WNV infection. Significant changes were identified in transcriptional, neurological, immunological, and apoptotic pathways with associations made between viral encephalitis and non infectious neurological disease based on a systems biology approach. Future work will involve furthe r data mining and validation of the array, as well as the identification of transcripts and pathways that can be targeted for therapeutics and diagnostics.

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125 Table 4 1 Probe groups for i nclusion on the m icroarray Probe g roup # of probes Replicates Important 3,883 1 Annotated 28,600 1 Annotated_minus 1,567 1 Unannotated 3,906 1 Recovered_genome 5,444 1 Control 250 1 Note: (Annotated). Probes were individually selected to be included twice on the array (Important analyse ) and included sequences involved in neurological, immunological, and transcription al processes, as well as cell death. Probes that were determined to be Unannotated probes and probes recovered from the EqCab2 genome sequencing project were also inclu ded. 250 Agilent controls were incorporated. Table 4 2 Samples used to obtain RNA for dye labeling and hybridization to the a rray Sample c ategory (n) Sample t ype (#) Experiment s pecifics Vaccinated/Immune + Exposed (n = 6 ) Thalamus (6) Cerebrum (6) Day 0 non control horses vaccinated A Day 365 challenged with WNV B intrathecally Day 365 386 monitored for clinical signs Day 386 (21 days PI) euthanasia, necropsy, tissue collection Unvaccin ated/Non immune + Exposed (n = 6 ) Thalamus (6) Cerebrum (6) Day 0 mock vaccination Day 365 challenged with WNV B intrathecally Day 365 374 monitored for clinical signs Day 372 374 (7 9 days PI) euthanasia, necropsy, tissue collection Untreated (n = 6 ) Thalamus (6) Cerebrum (6) Normal Horse A Live chimera WNV vaccine containing the prM and E proteins of WNV expressed in a YF17D vector B WNV NY99 strain 10 5 pfu/mL Note: Horses from three analyse s (vaccinated/exposed to WNV, nonvaccinated/exposed to WNV, and untreated) were used in the study. RNA was extracted from the cerebrum and thalamus from each of the horses (total of 36 samples) and used to create Cy3 dye labeled samples that were hybridized to the arrays.

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126 Table 4 3 Samples and a nalyses for the a rray Analys is Samples Tissue type Exposed to WNV? Exposure Status Not vaccinated 6 horses Thalamus X Untreated 6 horses Thalamus Survival/Immune Status Not vaccinated 6 horses Thalamus X Vaccinated 6 horses Thalamus X CNS Location Not vaccinated 6 horses Thalamus X Not vaccinated 6 horses Cerebrum X Note: A total of 12 tissues were compared for each of the analyse s/questions asked. The questions included determining if there was a difference in gene expression due to exposure to WNV, recovery from WNV infection, and location in the brain. Table 4 4 Number of significant genes for e ach a nalysis Analyse s Samples Before duplicate removal After duplicate removal Exposure Status Nonvaccinate vs Control 12,029 9,020 Survival/Immune Status Nonvaccinate vs Vaccinate 9,978 7,395 CNS Location Nonvaccinate Cerebrum vs Thalamus 10,555 7,649 Note: The number of significant genes for each analyse (with and without duplicate removal) was determined. An ANOVA with interactions (p<0.05) was used to determine significance.

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127 Table 4 5 Canonical p athway s for a ll a nalyses Exposure s tatus S urvival /Immune s tatus CNS l ocation Canonical Pathways log(p value) Transcripts log(p value) Transcripts log(p value) Transcripts Adrenergic Signaling 1.89E+00 11 2.21E+00 11 4.35E+00 14 Amyotrophic Lateral Sclerosis Signaling* 1.75E+00 12 1.64E+00 14 4.75E+00 15 Antiproliferative Role of Somatostatin Receptor 2 3.49E+00 11 Axonal Guidance Signaling* 1.84E+00 29 alanine Metabolism 1.93E+00 8 Breast Cancer Regulation by Stathmin1 4.95E+00 23 Butanoate Metabolism 1.55E+00 8 Calcium Signaling 2.68E+00 21 Calcium Signaling 3.45E+00 19 cAMP mediated Signaling 1.71E+00 18 2.75E+00 18 4.04E+00 19 adrenergic Signaling 1.99E+00 16 2.52E+00 16 5.05E+00 18 Caveolar mediated Endocytosis Signaling 1.75E+00 8 CDK5 Signaling* 1.89E+00 11 1.76E+00 9 Corticotropin Releasing Hormone Signaling 1.55E+00 13 2.36E+00 13 CREB Signaling in Neurons* 2.41E+00 19 2.84E+00 21 6.23E+00 24 CXCR4 Signaling^ 3.71E+00 18 Dopamine Receptor Signaling* 2.41E+00 11 1.72E+00 8 EGF Signaling 3.92E+00 9 Endothelin 1 Signaling 2.07E+00 19 4.08E+00 20 G Beta Gamma Signaling 1.78E+00 11 2.11E+00 11 4.18E+00 14 Glutamate Receptor Signaling* 1.70E+00 9 5.02E+00 15 5.77E+00 13 GNRH Signaling 1.90E+00 15 G Protein Coupled Receptor Signaling 3.40E+00 24 7.28E+00 29 IL 10 Signaling^ 1.62E+00 8 Leptin Signaling in Obesity 2.77E+00 11

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128 Table 4 5. Continued Exposure status Survival/Immune status CNS location Canonical Pathways log(p value) Transcripts log(p value) Transcripts log(p value) Transcripts Leukocyte Extravasation Signaling^ 1.73E+00 17 4.38E+00 22 Melatonin Signaling 2.40E+00 11 Molecular Mechanisms of Cancer 2.88E+00 31 3.82E+00 31 Neuregulin Signaling* 5.60E+00 16 Neuropathic Pain Signaling In Dorsal Horn Neurons* 3.44E+00 17 7.30E+00 19 Phenylalanine Metabolism 1.75E+00 6 Phospholipase C Signaling 1.57E+00 22 1.73E+00 17 Protein Kinase A Signaling 1.65E+00 26 Rac Signaling 3.60E+00 14 Relaxin Signaling 2.40E+00 15 5.39E+00 19 Renin Angiotensin Signaling 1.75E+00 12 4.16E+00 15 Role of NFAT in Cardiac Hypertrophy 4.09E+00 21 Role of NFAT in Regulation of the Immune Response^ 1.71E+00 17 SAPK/JNK Signaling 1.78E+00 11 Semaphorin Signaling in Neurons* 2.05E+00 7 Sphingosine 1 phosphate Signaling 1.78E+00 11 Synaptic Long Term Depression* 1.82E+00 18 2.84E+00 20 5.21E+00 20 Synaptic Long Term Potentiation* 1.75E+00 12 2.60E+00 16 6.03E+00 18 Thrombin Signaling 2.14E+00 22 2.28E+00 20 4.28E+00 22 Valine, Leucine and Isoleucine Degradation 1.71E+00 9 Note: All significant canonical pathways for all analyse s are listed. The denotes pathways involved with the nervous system (11) while the ^ denotes pathways involved with the immunological response (4)

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129 Table 4 6 Functions for a ll a nalyses Function Category Transcripts Exposure Status Transcripts S urvival /Immune Status Transcript s CNS Location Amino Acid Metabolism 61 75 178 ^ Antigen Presentation 2 8 Auditory and Vestibular System Development and Function 7 Auditory Disease 12 Behavior 237 195 301 Cancer 648 918 893 Carbohydrate Metabolism 61 48 4 Cardiovascular Disease 744 505 618 Cardiovascular System Development and Function 61 37 44 Cell Cycle 174 239 390 ^ Cell Death 1153 1082 719 Cell Morphology 214 230 326 Cell Signaling 107 60 164 ^ Cell mediated Immune Response 42 42 25 Cell To Cell Signaling and Interaction 361 453 455 Cellular Assembly and Organization 364 319 420 Cellular Compromise 32 11 8 Cellular Development 375 270 501 Cellular Function and Maintenance 140 63 104 Cellular Growth and Proliferation 806 721 424 Cellular Movement 546 671 699 Connective Tissue Development and Function 71 31 16 Connective Tissue Disorders 562 435 506 Dermatological Diseases and Conditions 20 11 18 Developmental Disorder 22 12 14 DNA Replication, Recombination, and Repair 46 16 35 Drug Metabolism 4 2 Embryonic Development 28 22 16 Endocrine System Development and Function 5 7 Endocrine System Disorders 518 502 566 Gastrointestinal Disease 404 345 328 Gene Expression 130 404 18 Genetic Disorder 1544 1269 1498 Hair and Skin Development and Function 2 2 4 Hematological Disease 12 22 13 Hematological System Development and Function 169 318 217 Hematopoiesis 40 81 50 Hepatic System Development and Function 3 3

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130 Table 4 6. Continued Function Category Transcripts Exposure Status Transcripts Survival/Immune Status Transcript s CNS Location Hepatic System Disease 13 13 ^Humoral Immune Response 20 18 29 ^Hypersensitivity Response 7 ^Immune Cell Trafficking 16 6 24 ^Immunological Disease 575 569 577 ^Infection Mechanism 12 21 3 ^Infectious Disease 164 54 58 ^Inflammatory Disease 965 798 824 ^Inflammatory Response 27 34 10 Lipid Metabolism 52 27 4 Lymphoid Tissue Structure and Development 7 2 Metabolic Disease 547 524 573 Molecular Transport 176 132 239 *Nervous System Development and Function 578 670 981 *Neurological Disease 1316 1210 1626 Nucleic Acid Metabolism 59 6 16 Ophthalmic Disease 2 2 18 Organ Development 36 14 64 Organ Morphology 24 9 20 Organismal Development 2 8 2 Organismal Functions 13 15 25 Organismal Injury and Abnormalities 11 16 61 Organismal Survival 155 140 126 Post Translational Modification 2 200 238 *Psychological Disorders 195 164 322 Renal and Urological Disease 2 19 Renal and Urological System Development and Function 7 2 Reproductive System Development and Function 6 2 2 Reproductive System Disease 2 3 63 Respiratory Disease 2 53 6 Respiratory System Development and Function 3 RNA Post Transcriptional Modification 2 4 Skeletal and Muscular Disorders 886 728 793 Skeletal and Muscular System Development and Function 92 39 14 Small Molecule Biochemistry 181 129 206 Tissue Development 102 245 234 Tissue Morphology 45 58 11 Tumor Morphology 5 18

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131 Table 4 6. Continued Function Category Transcripts Exposure Status Transcripts Survival/Immune Status Transcript s CNS Location Vitamin and Mineral Metabolism 24 14 64 Note: The number of transcripts for significant functions for all analyse s are listed. The denotes functions involved with the nervous system (6) while the ^ denotes functions involved with the immunological system (11).

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132 Table 4 7 Transcriptional regulators with increased e xpress ion Symbol Entrez gene n ame GenBank Location Fold change e xposure s tatus Fold c hange s urvival /immune s tatus Fold c hange CNS l ocation ALX3 ALX homeobox 3 NM_006492 Nucleus 2.05 ANKFY1 A nkyrin repeat and FYVE domain containing 1 XM_511280 Nucleus 1.329 1.508 1.017 ATF3 A ctivating transcription factor 3 NM_001046193 Nucleus 3.255 4.896 2.049 ATF6 A ctivating transcription factor 6 XM_513949 Cytoplasm 1.225 BATF Basic leucine zipper transcription factor, ATF like BC032294 Nucleus 5.086 4.364 2.596 BHLHE41 Basic helix loop helix family, member e41 NM_001002973 Nucleus 1.421 BLZF1 Basic leucine zipper nucl ear factor 1 XM_001136772 Cytoplasm 1.103 CSDA Cold shock domain protein A NM_003651 Nucleus 2.243 3.216 1.808 CTNNB1 Catenin (cadherin associated protein), beta 1, 88kda XM_845101 Nucleus 1.126 DDX54 DEAD (Asp Glu Ala Asp) box polypeptide 54 XM_001152033 Nucleus 1.207 ELF1 E74 like factor 1 (ets domain transcription factor) XM_852043 Nucleus 1.163 1.872 ELK1 ELK1, member of ETS oncogene family XM_548979 Nucleus 1.242 ELK3 ELK3, ETS domain protein (SRF accessory protein 2) XM_001146216 Nucleus 1.871 1.151 1.465 EOMES Eomesodermin homolog (Xenopus laevis) XM_001165845 Nucleus 3.849 3.344 ETV6 Ets variant 6 NM_001987 Nucleus 1.07 ETV7 Ets variant 7 XM_001172937 Nucleus 5.863 6.179 2.475 FOXP2 Forkhe ad box P2 NM_014491 Nucleus 1.598

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133 Table 4 7. Continued Symbol Entrez gene n ame GenBank Location Fold change e xposure status Fold c hange s urvival /immune status Fold c hange CNS l ocation GBX2 Gastrulation brain homeobox 2 XM_543300 Nucleus 1.721 3.717 GTF2E1 General transcription factor IIE, polypeptide 1, alpha 56kda NM_001103294 Nucleus 1.51 1.872 ID4 Inhibitor of DNA binding 4, dominant negative helix loop helix protein XM_001170946 Nucleus 1.363 IKZF2 IKAROS family zinc finger 2 (Helios) NM_016260 Nucleus 1.414 IRF2 Interferon regulatory factor 2 XM_532847 Nucleus 2.61 1.797 IRF3 Interferon regulatory factor 3 AK292027 Nucleus 1.31 1.935 1.086 IRX1 Iroquois homeobox 1 XM_001251876 Nucleus 5.101 IRX3 Iroquois homeobox 3 NM_001104996 Nucleus 3.398 IRX4 Iroquois homeobox 4 NM_001098466 Nucleus 1.02 ISL1 ISL LIM homeobox 1 XM_001150633 Nucleus 2.327 JUNB Jun B proto oncogene NM_001075656 Nucleus 2.201 2.017 KHDRBS1 KH domain containing, RNA binding, signal transduction associated 1 CU210913 Nucleus 1.116 KLF6 Kruppel like factor 6 AK151769 Nucleus 1.746 LASS2 LAG1 homolog, ceramide synthase 2 NM_001034667 Nucleus 1.029 LBH Limb bud and heart development homolog (mouse) NM_001099152 Nucleus 2.376 2.11 1.208 LRRFIP1 Leucine rich repeat (in FLII) interacting protein 1 BC083492 Nucleus 1.42 1.516 MAX MYC associated factor X XM_847808 Nucleus 1.087 1.382 MED21 Mediator complex subunit 21 XM_534858 Nucleus 1.056 1.001

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134 Table 4 7. Continued Symbol Entrez gene n ame GenBank Location Fold change e xposure status Fold c hange s urvival /immune status Fold c hange CNS l ocation MYB (includes EG:4602) V myb myeloblastosis viral oncogene homolog (avian) D26147 Nucleus 1.557 1.081 NCOA3 Nuclear receptor coactivator 3 XM_543039 Nucleus 1.112 NFE2L2 Nuclear factor (erythroid derived 2) like 2 XM_857112 Nucleus 1.037 1.337 NFIC Nuclear factor I/C (CCAAT binding transcription factor) XM_542179 Nucleus 1.626 NFIL3 Nuclear factor, interleukin 3 regulated NM_001075240 Nucleus 1.802 2.263 1.836 NFKBIE Nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, epsilon XM_583214 Nucleus 1.482 PAX3 Paired box 3 AC118213 Nucleus 2.534 PAX4 Paired box 4 NM_006193 Nucleus 2.674 PRRX1 Paired related homeobox 1 NM_006902 Nucleus 1.319 1.421 PURA Purine rich element binding protein A XM_001251355 Nucleus 1.225 REL V rel reticuloendotheliosis viral oncogene homolog (avian) XM_531836 Nucleus 1.124 1.88 2.204 SAP30 Sin3A associated protein, 30kda XM_843990 Nucleus 1.382 1.973 1.588 SFRS2 Splicing factor, arginine/serine rich 2 XM_852679 Nucleus 1.075 SHOX2 Short stature homeobox 2 AK145063 Nucleus 3.957 SIM2 Single minded homolog 2 (Drosophila) XM_001169429 Nucleus 1.408 SOX10 SRY (sex determining region Y) box 10 DQ896471 Nucleus 1.049 SOX2 SRY (sex determining region Y) box 2 XM_516895 Nucleus 1.016

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135 Table 4 7. Continued Symbol Entrez gene n ame GenBank Location Fold change e xposure status Fold c hange s urvival /immune status Fold c hange CNS l ocation SP1 Sp1 transcription factor XM_509098 Nucleus 1.155 1.625 1.364 ST18 Suppression of tumorigenicity 18 (breast carcinoma) (zinc finger protein) XM_001148965 Nucleus 1.307 STAT1 Signal transducer and activator of transcription 1, 91kda BC151378 Nucleus 3.021 3.763 2.384 TARBP1 (includes EG:6894) TAR (HIV 1) RNA binding protein 1 XM_514281 Nucleus 1.041 TCF12 Transcription factor 12 NM_001077885 Nucleus 1.157 TEAD1 TEA domain family member 1 (SV40 transcriptional enhancer factor) XM_001171565 Nucleus 1.786 1.319 TGIF2 TGFB induced factor homeobox 2 NM_021809 Nucleus 1.11 TP73 Tumor protein p73 XM_593064 Nucleus 1.171 TTF2 Transcription termination factor, RNA polymerase II XM_513683 Nucleus 1.17 1.74 VGLL2 Vestigial like 2 (Drosophila) BC118622 Nucleus 2.693 WWTR1 WW domain containing transcription regulator 1 XM_847454 Nucleus 3.107 2.939 2.33 ZFP57 Zinc finger protein 57 homolog (mouse) NM_001109809 Nucleus 1.025 ZIC1 Zic family member 1 (odd paired homolog, Drosophila) XM_516806 Nucleus 1.836 ZNFX1 Zinc finger, NFX1 type containing 1 XM_534452 Nucleus 3.531 3.311 1.255 Note: Transcriptional transcripts upregulated by 1 fold or greater for all analyses

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136 Table 4 8 Transcriptional regulators with increased e xpression Symbol Entrez g ene n ame GenBank Location Fold change e xposure s tatus Fold c hange s urvival / immune s tatus Fold c hange CNS l ocation AFF3 AF4/FMR2 family, member 3 X M_0011610 10 Nucleus 1.068 1.474 A NKIB1 Ankyrin repeat and IBR domain containing 1 XM_844926 Nucleus 1.194 ANKRD54 Ankyrin repeat domain 54 XM_538382 Nucleus 1.217 1.074 ARID4A AT rich interactive domain 4A (RBP1 like) XM_859819 Nucleus 1.76 2.145 2.296 ARNT2 Aryl hydrocarbon receptor nuclear translocator 2 AC101776 Nucleus 2.328 1.231 1.274 ARX Aristaless related homeobox XM_854885 Nucleus 1.721 ASB1 Ankyrin repeat and SOCS box containing 1 XM_516189 Nucleus 2.439 1.24 ASB5 Ankyrin repeat and SOCS box containing 5 NM_0010757 44 Nucleus 1.292 BCL11A B cell CLL/lymphoma 11A (zinc finger protein) NM_022893 Nucleus 1.539 BCLAF1 BCL2 associated transcription factor 1 XM_855478 Nucleus 1.121 CAND1 Cullin associated and neddylation dissociated 1 XM_531667 Cytoplasm 1.104 CASKIN1 CASK interacting protein 1 XM_848538 Nucleus 1.253 1.474 CBFA2T2 Core binding factor, runt domain, alpha subunit 2; translocated to, 2 XM_606138 Nucleus 1.097 1.196 CBFA2T3 Core binding factor, runt domain, alpha subunit 2; translocated to, 3 XM_546780 Nucleus 1.112 CLIP2 CAP GLY domain containing linker protein 2 XM_583422 Cytoplasm 1.084 1.056 1.116 CREBL2 Camp responsive element binding protein like 2 XM_0011533 86 Nucleus 1.359 CREG1 Cellular repressor of E1A stimulated genes 1 NM_0010759 42 Nucleus 1.883 CRTC1 CREB regulated transcription coactivator 1 XM_866768 Nucleus 1.268 1.098 CUX2 Cut like homeobox 2 BC151245 Nucleus 2.455 EBF1 Early B cell factor 1 CU012046 Nucleus 1.014 EGR4 Early growth re sponse 4 XM_540228 Nucleus 2.667 3.288 ETV5 Ets variant 5 NM_004454 Nucleus 1.36 1.263

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137 Table 4 8. Continued Symbol Entrez g ene n ame GenBank Location Fold change e xposure status Fold c hange s urvival / immune status Fold c hange CNS l ocation FBXW7 F box and WD repeat domain containing 7 NM_018315 Nucleus 2.149 FOXG1 Forkhead box G1 NM_005249 Nucleus 3.475 5.43 FOXO1 Forkhead box O1 NM_002015 Nucleus 1.012 FOXO4 Forkhead box O4 XM_529032 Nucleus 1.942 1.496 GBX2 Gastrulation brain homeobox 2 XM_543300 Nucleus 1.602 GRLF1 Glucocorticoid receptor DNA binding factor 1 NM_004491 Nucleus 1.153 HIVEP2 Human immunodeficiency virus type I enhancer binding protein 2 XM_518773 Nucleus 1.097 HTATSF1 HIV 1 Tat specific factor 1 EU176345 Nucleus 1.341 ID4 Inhibitor of DNA binding 4, dominant negative helix loop helix protein XM_0011709 46 Nucleus 1.647 JMY Junction mediating and regulatory protein, p53 cofactor NM_152405 Nucleus 1.662 KIDINS22 0 Kinase D interacting substrate, 220kda XM_532865 Nucleus 1.676 1.505 1.016 LCOR Ligand dependent nuclear receptor corepressor XM_584325 Nucleus 1.23 LHX5 LIM homeobox 5 NM_0011020 61 Nucleus 1.487 MED16 Mediator complex subunit 16 XM_849586 Nucleus 1.48 MEF2B Myocyte enhancer factor 2B NM_0011032 31 Nucleus 2.336 MEIS2 Meis homeobox 2 NM_170674 Nucleus 2.279 MEOX2 Mesenchyme homeobox 2 NM_0010980 45 Nucleus 1.714 2.945

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138 Table 4 8. Continued Symbol Entrez g ene n ame GenBank Location Fold change e xposure status Fold c hange s urvival / immune status Fold c hange CNS l ocation MLL2 Myeloid/lymphoid or mixed lineage leukemia 2 XM_543684 Nucleus 1.004 MYCN V myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian) XM_540091 Nucleus 1.166 1.441 1.682 MYOCD Myocardin AL669846 Nucleus 2.41 1.62 MYT1L Myelin transcription factor 1 like NM_0010937 76 Nucleus 2.568 NAB1 NGFI A binding protein 1 (EGR1 binding protein 1) AC006460 Nucleus 1.929 1.809 NFIA Nuclear factor I/A XM_536691 Nucleus 2.799 1.243 NFIB Nuclear factor I/B XM_531936 Nucleus 1.524 1.168 NFIX Nuclear factor I/X (CCAAT binding transcription factor) XM_862151 Nucleus 1.043 1.16 1.198 NKX2 8 NK2 homeobox 8 XM_584660 Nucleus 2.093 NPAS4 Neuronal PAS domain protein 4 XM_540832 Nucleus 1.881 2.893 PBX1 Pre B cell leukemia homeobox 1 XM_0011745 13 Nucleus 2.117 1.794 1.64 PIAS2 Protein inhibitor of activated STAT, 2 XM_612798 Nucleus 1.014 PROX1 Prospero homeobox 1 BX928753 Nucleus 1.367 RAI14 Retinoic acid induced 14 XM_0011512 40 Nucleus 1.081 RBM9 RNA binding motif protein 9 NM_0010825 79 Nucleus 1.303 RERE Arginine glutamic acid dipeptide (RE) repeats XM_536734 Nucleus 1.17 RFC1 Replication factor C (activator 1) 1, 145kda AY600371 Nucleus 2.124 RNF112 Ring finger protein 112 XM_546649 Nucleus 2.082 1.936

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139 Table 4 8. Continued Symbol Entrez g ene n ame GenBank Location Fold change e xposure status Fold c hange s urvival / immune status Fold c hange CNS l ocation RNF14 Ring finger protein 14 NM_0010815 40 Nucleus 1.112 SIN3B SIN3 homolog B, transcription regulator (yeast) XM_847635 Nucleus 1.559 SMAD4 SMAD family member 4 AC091551 Nucleus 1.72 1.399 SMAD7 SMAD family member 7 XM_512124 Nucleus 1.416 SMAD9 SMAD family member 9 XM_0011440 71 Nucleus 1.053 SOX10 SRY (sex determining region Y) box 10 DQ896471 Nucleus 1.251 SOX6 SRY (sex determining region Y) box 6 AC068405 Nucleus 1.351 SP4 Sp4 transcription factor XM_527679 Nucleus 1.065 SPEN Spen homolog, transcriptional regulator (Drosophila) XM_591419 Nucleus 1.019 SRF Serum response factor (c fos serum response element binding transcription factor) XM_847209 Nucleus 1.177 SRY Sex determining region Y AC146189 Nucleus 1.397 SS18L1 Synovial sarcoma translocation gene on chromosome 18 like 1 NM_0010780 95 Nucleus 1.89 1.064 1.138 SUB1 SUB1 homolog (S. Cerevisiae) NM_0011054 07 Nucleus 3.347 2.235 2.353 TCEA2 Transcription elongation factor A (SII), 2 XM_0011529 36 Nucleus 1.104 1.116 TCF4 Transcription factor 4 NM_003199 Nucleus 1.122 1.255 TCF7L2 (includes EG:6934) Transcription factor 7 like 2 (T cell specific, HMG box) AL158212 Nucleus 1.027 TLE2 Transducin like enhancer of split 2 (E(sp1) homolog, Drosophila) NM_003260 Nucleus 1.051 1.182

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140 Table 4 8. Continued Symbol Entrez g ene n ame GenBank Location Fold change e xposure status Fold c hange s urvival / immune status Fold c hange CNS l ocation TSHZ1 Teashirt zinc finger homeobox 1 XM_533368 Nucleus 1.13 TULP4 Tubby like protein 4 BC152476 Cytoplasm 1.67 YY1 YY1 transcription factor XM_510162 Nucleus 1.186 1.383 1.206 ZEB1 Zinc finger E box binding homeobox 1 XM_615192 Nucleus 1.093 ZEB2 Zinc finger E box binding homeobox 2 AY029472 Nucleus 2.033 1.285 ZFP57 Zinc finger protein 57 homolog (mouse) NM_0011098 09 Nucleus 2.323 ZIC1 Zic family member 1 (odd paired homolog, Drosophila) XM_516806 Nucleus 2.372 ZMYND8 Zinc finger, MYND type containing 8 XM_866938 Nucleus 1.082 ZNF219 Zinc finger protein 219 XM_867319 Nucleus 1.162 ZNF398 Zinc finger protein 398 AK290499 Nucleus 1.446 Note: Transcriptional genes downregulated by 1 fold or less for all analyses

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141 Table 4 9 Transcripts for a ll a nalyses mapped to n eurological CPs Exposure Status S urvival /Immune Status CNS Location Decreased Increased Decreased Increased Decreased Increased Agrin Interactions at Neuromuscular Junction 1 4 2 2 2 4 Amyloid Processing 4 1 3 1 3 3 Amyotrophic Lateral Sclerosis Signaling 11 1 14 0 13 2 Axonal Guidance Signaling 25 6 22 7 19 10 CDK5 Signaling 11 0 9 0 10 2 Cholecystokinin/G astrin mediated Signaling 5 2 7 2 6 1 Circadian Rhythm Signaling 2 1 4 1 5 1 CNTF Signaling 1 2 2 1 4 3 CREB Signaling in Neurons 15 4 19 2 20 4 D ocosahexaenoic Acid (DHA) Signaling 2 0 2 1 Dopamine Receptor Signaling 10 1 6 2 6 2 GABA Receptor Signaling 6 0 6 0 8 0 Glutamate Receptor Signaling 8 1 15 0 12 1 GNRH Signaling 12 3 11 1 13 2 Huntington's Disease Signaling 10 5 8 5 12 7 Melatonin Signaling 6 1 10 1 8 1 Neuregulin Signaling 6 3 10 6 Neuropathic Pain Signaling In Dorsal Horn Neurons 8 2 16 1 17 2 Neurotrophin/TRK Signaling 5 0 4 0 6 2 Reelin Signaling in Neurons 4 2 3 3 5 5

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142 Table 4 9. Continued Exposure Status S urvival /Immune Status CNS Location Decreased Increased Decreased Increased Decreased Increased Regulation of Actin based Motility by Rho 3 2 Semaphorin Signaling in Neurons 6 1 5 2 2 1 Serotonin Receptor Signaling 1 1 0 2 1 1 Synaptic Long Term Depression 14 4 17 3 17 3 Synaptic Long Term Potentiation 11 1 15 1 17 1 Total 176 43 209 42 218 65 Note: This table shows the number of transcripts that mapped to each pathway for all analyses The majority of the transcripts demonstrated a decrease in expression values. Transcripts were included if they demonstrated a fold change >1 or < 1.

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143 Table 4 10 Transcripts in glutamate signaling p athway for a ll a nalyses Symbol Entrez Gene Name GenBank Fold c hange e xposure s tatus Fold c hange s urvival / i mmune s tatus Fold c hange CNS l ocation CAMK4 Calcium /calmodulin dependent protein kinase IV XM_517873 1.976 GLS Glutaminase AC005540 1.009 GNB1 Guanine nucleotide binding protein (G protein), beta polypeptide 1 BC004186 1.638 GNG5 Guanine nucleotide binding protein (G protein), gamma 5 BC003563 1.359 GRIA1 Glutamate receptor, ionotropic, AMPA 1 XM_001169416 1.398 1.057 1.498 GRIA2 Glutamate receptor, ionotropic, AMPA 2 NM_000826 1.505 1.643 GRIA3 Glutamate receptor, ionotrophic, AMPA 3 NM_007325 2.33 2.719 GRIA4 Glutamate receptor, ionotrophic, AMPA 4 NM_000829 1.253 1.039 GRID2 Glutamate receptor, ionotropic, delta 2 AC022317 1.581 GRIK1 Glutamate receptor, ionotropic, kainate 1 NM_000830 1.098 1.933 2.626 GRIK2 Glutamate receptor, ionotropic, kainate 2 XM_866973 1.533 GRIN1 Glutamate receptor, ionotropic, N methyl D aspartate 1 AF015731 1.949 1.487 GRIN2A Glutamate receptor, ionotropic, N methyl D aspartate 2A XM_547132 2.836 1.631 2.369 GRIN2B Glu tamate receptor, ionotropic, N methyl D aspartate 2B AC007535 1.563 1.989 GRIN3A Glutamate receptor, ionotropic, N methyl D aspartate 3A XM_862276 1.032 GRIP1 Glutamate receptor interacting protein 1 XM_001162097 1.23 GRM8 Glutama te receptor, metabotropic 8 AC079957 1.856 HOMER1 Homer homolog 1 (Drosophila) XM_001139767 1.084 HOMER3 Homer homolog 3 (Drosophila) XM_541929 1.718 1.134

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144 Table 4 10. Continued Symbol Entrez Gene Name GenBank Fold c hange exposure status Fold c hange survival/ immune status Fold c hange CNS location SLC17A7 Solute carrier family 17 (sodium dependent inorganic phosphate cotransporter), member 7 NM_001098046 1.598 2.652 8.147 SLC1A2 Solute carrier family 1 (glial high affinity glutamate transporter), member 2 NM_004171 1.639 2.869 2.278 Note: The transcripts significantly changed in the glutamate signaling pathway are shown for all three analyses The transcripts were mainly glutamate receptors, although other components of the pathway, such as glutamate re uptake receptors, are also present. Alm ost all transcripts are downregulated, providing evidence that glutamate excitotoxicity may be present and damaging the neurons. Transcripts were included if they demonstrated a fold change >1 or < 1.

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145 Table 4 1 1 Transcripts in dopamine signaling p a thways for a ll a nalyses Symbol Entrez Gene Name GenBank Fold c hange exposure s tatus Fold c hange s urvival/ immune s tatus Fold c hange CNS l ocation ADCY1 Adenylate cyclase 1 (brain) NM_174229 1.789 1.898 ADCY2 Adenylate cyclase 2 (brain) XM_851103 1.683 1.223 2.027 ADCY5 Adenylate cyclase 5 NM_183357 1.258 1.379 ADCY8 Adenylate cyclase 8 (brain) XM_539166 1.021 1.954 ADCY9 Adenylate cyclase 9 BC151229 1.46 DRD5 Dopamine receptor D5 XM_604584 1.097 GCH1 GTP cyclohydrolase 1 XM_846790 1.296 IL4I1 Interleukin 4 induced 1 AY358933 3.176 3.265 1.405 PPP1R14A Protein phosphatase 1, regulatory (inhibitor) subunit 14A XM_867134 2.083 PPP1R3C Protein phosphatase 1, regulatory (inhibitor) subunit 3C BT030698 2.27 1.746 1.686 PPP2R2B Protein phosphatase 2 (formerly 2A), regulatory subunit B, beta isoform XM_001159292 2.348 1.14 PPP2R2C Protein phosphatase 2 (formerly 2A), regulatory subunit B, gamma isoform XM_001250700 1.243 1.378 1.446 PRKACB Protein kinase, camp dependent, catalytic, beta XM_862471 2.225 PRKAR2B Protein kinase, camp dependent, regulatory, type II, beta XM_001148361 1.627 TH Tyrosine hydroxylase BC149072 2.857 Note: The transcripts significantly changed in the dopamine signaling pathway are shown for all three analyses D opamine receptors(DRD5) and downstream signaling transcripts as well as enzymes that create dopamine (TH) were downregulated. Enzymes that degrade dopamine (MAO) were upregulated. Thus it appears that WNV infection decreases dopamine levels Transcripts were included if they demonstrated a fold change >1 or < 1.

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146 T able 4 1 2 Transcripts for a ll a nalyses mapped to i mmunological CPs Exposure Status S urvival /Immune Status CNS Location Down Up Down Up Down Up 4 1BB Signaling in T Lymphocytes 1 1 1 1 3 1 Activation of IRF by Cytosolic Pattern Recognition Receptors 1 3 0 5 Acute Myeloid Leukemia Signaling 4 1 Acute Phase Response Signaling 5 6 4 5 6 3 Amyloid Processing 3 1 B Cell Activating Factor Signaling 2 2 0 2 B Cell Development 0 3 B Cell Receptor Signaling 6 4 7 3 7 5 Calcium induced T Lymphocyte Apoptosis 4 3 2 3 2 4 Cardiac Hypertrophy Signaling 15 4 14 6 Caveolar mediated \ Endocytosis Signaling 6 1 6 2 2 3 CCR3 Signaling in Eosinophils 7 2 8 2 8 4 CCR5 Signaling in Macrophages 3 2 4 3 4 4 CD27 Signaling in Lymphocytes 2 1 CD28 Signaling in T Helper Cells 6 5 2 4 3 5 CD40 Signaling 3 1 4 1 Chemokine Signaling 8 1 7 1 Chronic Myeloid Leukemia Signaling 4 2 Clathrin mediated Endocytosis Signaling 13 1 7 1 9 3 CNTF Signaling 1 2 2 1 CNTF Signaling Complement System 0 3 0 2 CTLA4 Signaling in Cytotoxic T Lymphocytes 4 3 3 3 5 5 CXCR4 Signaling 13 3 10 4 13 5 Cytotoxic T Lymphocyte mediated Apoptosis of Target Cells 1 1 Dendritic Cell Maturation 3 6 4 5 Fc Epsilon RI Signaling 5 1 7 3 mediated Phagocytosis in Macrophages and Monocytes 4 2 6 4 1 1 2 2 FLT3 Signaling in Hematopoietic Progenitor Cells 4 2 fMLP Signaling in Neutrophils 7 4 6 4 7 5 GM CSF Signaling 3 2 4 1 6 2 iCOS iCOSL Signaling in T Helper Cells 6 4 4 4 4 4 IL 1 Signaling 7 3 IL 10 Signaling 3 5 1 3 IL 12 Signaling and Production in Macrophages 3 3 6 4 IL 15 Production 0 4 1 3 1 4 IL 15 Signaling 2 1 4 3 IL 17 Signaling 4 2

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147 Table 4 1 2. Continued Exposure Status Survival/Immune Status CNS Location Down Up Down Up Down Up IL 2 Signaling 3 0 5 2 IL 22 Signaling 0 3 1 2 1 2 IL 3 Signaling 4 2 4 2 6 4 IL 4 Signaling 2 3 IL 6 Signaling 4 2 3 2 IL 8 Signaling 11 3 9 7 9 8 IL 9 Signaling 1 2 1 2 2 3 Interferon Signaling 0 2 0 2 Leukocyte Extravasation Signaling 6 7 8 9 8 14 LPS/IL 1 Mediated Inhibition of RXR Function 7 7 LPS stimulated MAPK Signaling 4 1 5 2 7 2 Macropinocytosis Signaling 4 1 3 3 4 5 Mechanisms of Viral Exit from Host Cells 3 2 5 1 MIF Regulation of Innate Immunity 2 1 2 0 Natural Killer Cell Signaling 4 4 7 5 NF 4 3 5 6 NF 8 3 4 4 Oncostatin M Signaling 0 3 1 1 2 2 p38 MAPK Signaling 3 3 3 2 Primary Immunodeficiency Signaling 1 4 1 5 0 5 Production of Nitric Oxide and Reactive Oxygen Species in Macrophages 9 4 6 6 9 7 Regulation of IL 2 Expression in Activated and Anergic T Lymphocytes 2 4 Role of Macrophages, Fibroblasts and Endothelial Cells in Rheumatoid Arthritis 11 8 9 8 Role of NFAT in Regulation of the Immune Response 10 7 7 8 7 8 Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses 1 5 2 5 3 4 Role of PKR in Interferon Induction and Antiviral Response 2 1 1 2 Role of RIG1 like Receptors in Antiviral Innate Immunity 1 2 0 4 T Cell Receptor Signaling 4 6 4 4 4 6 T Helper Cell Differentiation 1 2 Toll like Receptor Signaling 3 2 TREM1 Signaling 2 1 Virus Entry via Endocytic Pathways 6 2 6 4 Total 176 130 215 166 266 210 Note: This table shows the number of transcripts that mapped to each pathway for all analyses The majority of the transcripts demonstrated a decrease in expression values. Transcripts were included if they demonstrated a fold change >1 or < 1.

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148 Table 4 1 3 Transcripts in IL 15 p roduction and signaling for a ll a nalyses Symbol Entrez g ene n ame Genbank Fold c hange exposure s tatus Fold c hange survival/ i mmune s tatus Fold c hange CNS l ocation IL15 Interleukin 15 AK290619 2.369 2.29 2.004 IRF3 Interferon regulatory factor 3 AK292027 1.31 1.935 1.086 JAK1 Janus kinase 1 X M_001161295 1.079 MAP2K1 Mitogen activated protein kinase kinase 1 XM_612526 1.4 MAPK1 Mitogen activated protein kinase 1 NM_002745 1.095 1.061 MAPK1 Mitogen activated protein kinas e 1 DQ508104 PIK3R1 Phosphoinositide 3 kinase, regulatory subunit 1 (alpha) NM_181504 1.062 1.339 PIK3R2 Phosphoinositide 3 kinase, regulatory subunit 2 (beta) XM_847313 2.44 2.341 PIK3R3 Phosphoinositide 3 kinase, regulatory subu nit 3 (gamma) XM_856294 1.537 PTK2B PTK2B protein tyrosine kinase 2 beta XM_543228 1.029 1.545 STAT1 Signal transducer and activator of transcription 1, 91kda BC151378 3.021 3.763 2.384 TYK2 Tyrosine kinase 2 XM_590006 1.504 Note: The transcripts significantly changed in the IL 15 production and signaling pathways are shown for all three analyses Transcripts involved in IL 15 production are upregulated for all analyse s (IL 15 and STAT1) while transcripts involved in signaling are downregulated. Transcripts were included if they demonstrated a fold change >1 or < 1.

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149 Table 4 1 4 Transcripts in IL 9, IL 22, and JAK/STAT signaling for a ll a nalyses Symbol Entrez g ene n ame Genbank Fold c hange exposure status Fold c hange survival/ immune status Fold c hange CNS location JAK1 Janus kinase 1 X M_001161295 1.079 MAP2K1 Mitogen activated protein kinase kinase 1 XM_612526 1.4 MAPK1 Mitogen activated protein kinase 1 NM_002745 1.095 1.061 PIAS2 Protein inhibitor of activated STAT, 2 XM_612798 1.014 PIK3R1 Phosphoinositide 3 kinase, regulatory subunit 1 (alpha) NM_181504 1.062 1.339 PIK3R2 Phosphoinositide 3 kinase, regulatory subunit 2 (beta) XM_847313 2.44 2.341 PIK3R3 Phosphoinositide 3 kinase, regulatory subunit 3 (gamma) XM_856294 1.537 SOCS3 Suppressor of cytokine signaling 3 NM_174466 1.535 1.809 STAT1 Signal transducer and activator of transcription 1, 91kda BC151378 3.021 3.763 2.384 TYK2 Tyrosine kinase 2 XM_590006 1.504 Note: The transcripts significantly changed in the IL 9, IL 22, and JAK/STAT signaling pathways are shown for all three analyses Transcripts involved in the upregulation of the innate immune response are increased (JAK1, TYK2, STAT1) while transcripts involved in inhibiting the innate immune response are also upregulated (SOCS3). Therefore there appears to be an induction of the innate immune response during viral infection that is counteracted in nave horses by an inhibition of the innate immune response. Transcripts were included if they demonstrated a fold change >1 or < 1.

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150 Table 4 15 Functions for genes common to all g roups Category # Transcripts p value Cell Death 646 0.007846 Genetic Disorder 629 0.006721 Neurological Disease 479 0.004563 Cancer 436 0.003866 Cellular Growth and Proliferation 409 0.015065 Cell Cycle 394 0.009029 Cellular Movement 388 0.00561 Inflammatory Disease 312 0.006328 Metabolic Disease 263 0.005236 Cell To Cell Signaling and Interaction 256 0.007265 Endocrine System Disorders 253 0.003244 Immunological Disease 238 0.005106 Hematological System Development and Function 217 0.012534 Skeletal and Muscular Disorders 203 0.003841 Cardiovascular Disease 172 0.01405 Cellular Assembly and Organization 165 0.007591 Gastrointestinal Disease 164 0.006634 Tissue Development 161 0.008721 Nervous System Development and Function 142 0.011708 Cell Morphology 109 0.010085 Cellular Development 94 0.017048 Tissue Morphology 92 0.012873 Cellular Function and Maintenance 74 0.01076 Psychological Disorders 68 0.015 Post Translational Modification 65 0.011553 Small Molecule Biochemistry 61 0.012521 Cardiovascular System Development and Function 58 0.015677 Carbohydrate Metabolism 52 0.007987 Hematological Disease 46 0.019348 Reproductive System Disease 44 0.017469 Cell Signaling 43 0.006106 Hematopoiesis 38 0.012244 Molecular Transport 38 0.010292 Infectious Disease 38 0.015653 Connective Tissue Development and Function 36 0.011979 DNA Replication, Recombination, and Repair 36 0.009334 Gene Expression 27 0.011831 Respiratory Disease 27 0.009232 Humoral Immune Response 24 0.010206 Skeletal and Muscular System Development and Function 24 0.02575 Embryonic Development 22 0.0234 Vitamin and Mineral Metabolism 21 0.008228 Cell mediated Immune Response 21 0.009502 Inflammatory Response 20 0.015859 Infection Mechanism 19 0.016993 Behavior 18 0.010367 Amino Acid Metabolism 18 0.006076 Immune Cell Trafficking 18 0.016015

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151 Table 4 15. Continued Category # Transcripts p value Cellular Compromise 16 0.020284 Organismal Development 16 0.023265 Lymphoid Tissue Structure and Development 14 0.021864 Developmental Disorder 13 0.010329 Lipid Metabolism 12 0.012967 Nucleic Acid Metabolism 11 0.020762 Antigen Presentation 9 0.020867 Connective Tissue Disorders 8 0.00695 Organ Morphology 8 0.016938 Organismal Injury and Abnormalities 8 0.016113 Renal and Urological Disease 8 0.026863 Organ Development 6 0.018 Tumor Morphology 5 0.01772 Reproductive System Development and Function 5 0.02464 Organismal Survival 4 0.00931 Endocrine System Development and Function 3 0.012347 Dermatological Diseases and Conditions 3 0.0166 Organismal Functions 2 0.0023 Hepatic System Development and Function 2 0.0196 Protein Synthesis 2 0.0196 Hair and Skin Development and Function 2 0.028 Digestive System Development and Function 1 0.028 Drug Metabolism 1 0.028 Hepatic System Disease 1 0.028 Renal and Urological System Development and Function 1 0.028 Visual System Development and Function 1 0.028 Note: The majority of functions were classified under cell death, genetic disorder, and neurological disease. However, the most significant classifications occurred in the neurological disease category which had the lowest p value. Table 4 1 6 Validation of the a rray Nonvaccinate average relative e xpression QPCR Vaccinate a verage r elative e xpression QPCR Average expression n onvaccinate: v accinate QPCR Nonvaccinate: vaccinate array e xpression 2'5'OAS + 1.689667 0.71533 + 2.4050 + 6.539663 Complement Component 1 r + 2.0895 0.2185 + 2.3080 + 1.886843 DEADBox60 + 1.7625 0.895 + 2.6575 + 5.651655 Defensin B4 + 0.365333 1.907 + 2.2723 + 6.99401 IL 6 + 1.342833 0.56933 + 1.9122 + 5.97945 TNF + 0.649 0.90583 + 1.5548 + 3.471118 Note: Comparison of the relative expression levels between the nonvaccinate and vaccinate thalamus and QPCR to array platforms.

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152 Figure 4 1 Box plots for Loess n ormalization. The green line indicates the mean of all arrays after normalization, while the red boxes indicate the range of response, the red lines in the boxes the median of each array, and the extended red lines standard deviations. All arrays normalized correctly.

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153 Fi gure 4 2 Heat map and d endrogram of a ll arrays demonstrating similarity in gene e xpression. Dark red indicates a high degree of similarity, while blue indicates a low degree of similarity.

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154 Figure 4 3 Fold c hange analysis of signficant g enes (p< 0.05). Transcripts that increased by greater than 1 fold and less than 1 fold were counted fo r all three analyses difference in gene expression between the nonvaccinated/exposed normal, nonvaccinated/exposed difference in gene expression between the thalamus and cerebrum of the nonvaccinated/exposed. Location Survival Exposure 0 500 1000 1500 2000 2500 3000 < 2 < 1 >+1 >+2 631 2101 1737 358 225 2123 1800 666 395 2936 2084 749 Location Survival Exposure

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155 Figure 4 4. Number of genes that mapped to GO categories for a ll a nalyse s. T he distribution of genes is relatively even, with slightly more genes overall in the exposure analysis compared to the non survival and location analysis For the represents the difference in gene expression between the nonvaccinated/exposed the difference in gene expression between the nonvaccinated/exposed expressio n between the thalamus and cerebrum of the nonvaccinated/exposed. 0 1000 2000 3000 4000 5000 6000 7000 8000 Exposure Survival Location 6009 5120 5200 6454 5462 5675 6646 7696 5715 Number of Genes Gene Ontology Groups Biological Processes Cellular Component Molecular Function

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156 Figure 4 5. Distribution of genes among GO c ategories. Most genes for all analyse s were classified under transcriptional categories, with neuro logical categories containing the second highest number of genes. For the purposes of this nonvaccinated/exposed expression between the nonvaccinated/exposed vac cinated/exposed, and and cerebrum of the nonvaccinated/exposed. 0 500 1000 1500 2000 2500 Neurological Immunological Transcriptional Cell Death/Apoptosis 1081 983 2022 430 840 850 1864 338 1144 798 1664 349 Number of Transcripts Exposure Survival Location

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157 Figure 4 6 Top 25 canonical pathways for a ll a nalyses The number of transcripts in the top 25 canonical pathways for exposure, survival, and location are graphed out. Neurological pathways are marked with an while immunological pathways are marked with a ^. T he majority of pathways are involved with signaling, then with neurologi cal functions. between the nonvaccinated/exposed the nonvaccinated/exposed vaccinated/exposed, between the thalamus and cerebrum of the nonvaccinated/exposed. 0.00E+00 1.00E+00 2.00E+00 3.00E+00 4.00E+00 5.00E+00 6.00E+00 7.00E+00 8.00E+00 0 5 10 15 20 25 30 35 Thrombin Signaling *CREB Signaling in Neurons *Synaptic Long Term Depression cAMP mediated Signaling Cardiac adrenergic Signaling *Synaptic Long Term Potentiation *Amyotrophic Lateral Sclerosis Signaling Adrenergic Signaling G Beta Gamma Signaling *Glutamate Receptor Signaling Corticotropin Releasing Hormone Signaling *Dopamine Receptor Signaling *CDK5 Signaling Endothelin 1 Signaling Renin Angiotensin Signaling Molecular Mechanisms of Cancer G Protein Coupled Receptor Signaling *Neuropathic Pain Signaling In Dorsal ^Leukocyte Extravasation Signaling Relaxin Signaling Phospholipase C Signaling PPAR RXR Activation ^Role of NFAT in Regulation of the GNRH Signaling SAPK/JNK Signaling Valine, Leucine and Isoleucine alanine Metabolism ^IL 10 Signaling Butanoate Metabolism Phenylalanine Metabolism *Axonal Guidance Signaling Protein Kinase A Signaling Calcium Signaling Leptin Signaling in Obesity Melatonin Signaling Sphingosine 1 phosphate Signaling Caveolar mediated Endocytosis Signaling *Semaphorin Signaling in Neurons Breast Cancer Regulation by Stathmin1 Role of NFAT in Cardiac Hypertrophy Calcium Signaling ^CXCR4 Signaling *Neuregulin Signaling Rac Signaling Antiproliferative Role of Somatostatin EGF Signaling log(p value) # Molecules Canonical Pathways Molecules Exposure Molecules Survival Molecules Location log(p value) Exposure log(p value) Survival

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158 F igure 4 7 Neurological canonical p athways for a ll a nalyses Canonical pathways identified as significant for each analyses w ere selected. T here was a high degree of ov erlap between all three analyses in neurological pathways. The location analysis contained transcripts that mapped to the most neurological pathways. The green line represents significance. For the purposes of expression between the nonvaccinated/exposed between the nonvaccinated/exposed ence in gene expression between the thalamus and cerebrum of the nonvaccinated/exposed. 0.00E+00 1.00E+00 2.00E+00 3.00E+00 4.00E+00 5.00E+00 6.00E+00 7.00E+00 8.00E+00 0 5 10 15 20 25 30 35 log(p value) # Molecules # of Molecules Exposure # of Molecules Survival # of Molecules Location log(p value) Exposure log(p value) Survival log(p value) Location

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159 Figure 4 8. Glutamate receptor signaling p athway. A. Expression pathway for the exposure analysis (nonvaccinated exposed normal), B. Expression pathway for the nonsurvival analyse (nonvaccinated exposed vaccinated exposed), C. Expression pathway for the location analysis (nonvaccinated exposed thalamu s nonvaccinated exposed cerebrum). The diagrams represents signaling in the synaptic cleft (square = pre synaptic neuron, oval = post synaptic neuron, circle = glial cell). Green represents downregulation of pertinent receptors, red represents upregula tion of pertinent receptors. WNV induces downregulation of glutamate receptors on the post synaptic cleft as well as glutamate uptake receptors on glial cells. This provides evidence that WNV induces glutamate excitotoxicity which likely contributes to n euronal pathology. C A B

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160 F igure 4 9 Dopamine receptor s ignaling pathway The graphic shown is for the exposure analysis but was similar in all analyses (see table 4 9). Green represents down regulation of transcripts red represents upregulation. Dopamine receptors (DRD5) and downstream signaling pathways as well as enzymes that create dopamine (TH) were downregulated, while MAO (enzyme that degrades dopamine) was upregulated. Thus it appears that WNV infection induces a decrease in the levels of dopamine.

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161 Figure 4 10 Immunological canonical p athways for a ll a nalyses The location analysis contained transcripts that mapped to the most immunological pathways. The green line indicates significance. For the purposes of thi s study, represents the difference in gene expression between the nonvaccinated/exposed vaccinated/exposed, and e in gene expression between the thalamus and cerebrum of the nonvaccinated/exposed. 0.00E+00 5.00E 01 1.00E+00 1.50E+00 2.00E+00 2.50E+00 3.00E+00 3.50E+00 4.00E+00 4.50E+00 5.00E+00 0 5 10 15 20 25 Role of NFAT in Regulation of the Immune Response Clathrin mediated Endocytosis Signaling IL 8 Signaling NRF2 mediated Oxidative Stress Response Leukocyte Extravasation Signaling Production of Nitric Oxide and Reactive Oxygen Acute Phase Response Signaling CD28 Signaling in T Helper Cells fMLP Signaling in Neutrophils B Cell Receptor Signaling iCOS iCOSL Signaling in T Helper Cells T Cell Receptor Signaling CCR3 Signaling in Eosinophils Calcium induced T Lymphocyte Apoptosis CTLA4 Signaling in Cytotoxic T Lymphocytes IL 3 Signaling Role of Pattern Recognition Receptors in Recognition CCR5 Signaling in Macrophages GM CSF Signaling Primary Immunodeficiency Signaling IL 15 Production IL 22 Signaling IL 9 Signaling 4 1BB Signaling in T Lymphocytes IL 10 Signaling IL 6 Signaling LPS stimulated MAPK Signaling Retinoic acid Mediated Apoptosis Signaling Activation of IRF by Cytosolic Pattern Recognition Role of PKR in Interferon Induction and Antiviral Role of RIG1 like Receptors in Antiviral Innate B Cell Activating Factor Signaling Interferon Signaling Chemokine Signaling Dendritic Cell Maturation Natural Killer Cell Signaling Virus Entry via Endocytic Pathways NF B Activation by Viruses Fc Receptor mediated Phagocytosis in IL 12 Signaling and Production in Macrophages Mechanisms of Viral Exit from Host Cells CD40 Signaling Complement System IL 15 Signaling IL 2 Signaling Fc RIIB Signaling in B Lymphocytes Induction of Apoptosis by HIV1 Toll like Receptor Signaling T Helper Cell Differentiation Cytotoxic T Lymphocyte mediated Apoptosis of Fc Epsilon RI Signaling Death Receptor Signaling MIF Regulation of Innate Immunity CXCR4 Signaling IL 1 Signaling Apoptosis Signaling IL 17 Signaling Regulation of IL 2 Expression in Activated and IL 4 Signaling B Cell Development CD27 Signaling in Lymphocytes Myc Mediated Apoptosis Signaling log(p value) # Molecules Canonical Pathways # of Molecules Exposure # of Molecules Survival # of Molecules Location log(p value) Exposure

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162 A B C Figure 4 1 1. IL 15 production p athway. A. Expression pathway for the exposure analysis (nonvaccinated exposed untreated), B. Expression pathway for the nonsurvival analysis (nonvaccinated exposed vaccinated exposed), C. Expression pathway for the location analyse (nonvaccinated exposed thal amus nonvaccinated exposed cerebrum). The diagrams represent the different methods of IL 15 production. Green represents downregulation of pertinent molecules, red represents upregulation of pertinent molecules. WNV induces upregulation of the producti on of IL 15. This likely contributes to the immune response against WNV.

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163 Figure 4 1 2 IL 22 and JAK/STAT pathways for e xposure. A. IL 22 signaling pathway and B. JAK/STAT signaling pathway comparing expression levels between non vaccinated /exposed horses and vaccinated/exposed horses (exposure analysis ). Red represents upregulation of gene expression, green represents down regulation of gene expression T he JAK/STAT pathway is upregulated during vi ral infection, demonstrating an innate immune response. However, the SOCS3 molecule is also upregulated, indicating that infection with the virus may lead to subsequent suppression of the JAK/STAT pathway and evasion of the innate immune response. The pa thways and levels of expression were similar for the non survival analysis The location analysis did not demonstrate upregulation of SOCS3. A B

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164 Figure 4 1 3 Neurological functions by category for a ll a nalyses As can be seen for all analyse s, the majority of transcripts were classified under neurological disease. in gene expression between the nonvaccinated/exposed repres ents the difference in gene expression between the nonvaccinated/exposed difference in gene expression between the thalamus and cerebrum of the nonvaccinated/exposed. Exposure Survival Location 0 200 400 600 800 1000 1200 1400 1600 Nervous System Development and Function Behavior Neurological Disease Psychological Disorder 332 146 1233 210 305 128 1091 196 575 206 1406 327 # Molecules

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165 Figure 4 1 4 Neurological f u nctions a ll a nalyses A. Neurological disease and p sychological disorders categories, and B. Nervous system development and function and behavior categories. The majority of transcripts mapped to neurological disease. The red bars represent the exposure analysi s, the purple bars the survival analysis and the orange bars the location analysis The diamonds represent the p value for exposure, the triangles the p value for non survival, a nd the squares the p value for location. For the purposes of the nonvaccinated/exposed gene expression between the nonvaccinated/ex posed vaccinated/exposed, thalamus and cerebrum of the nonvaccinated/exposed. 0 0.005 0.01 0.015 0.02 0.025 0 100 200 300 400 500 600 neurological disorder progressive motor neuropathy bipolar affective disorder neurodegenerative disorder Alzheimer's disease schizophrenia amyotrophic lateral sclerosis Huntington's disease Parkinson's disease multiple sclerosis epilepsy neuropathy major depression neurodegeneration hyperalgesia bipolar affective disorder schizophrenia seizures psychological disorder major depression anxiety stroke Neurological Disease Psychological Disorders p value # Molecules A 0 0.005 0.01 0.015 0.02 0.025 0.03 0 20 40 60 80 100 120 140 160 180 development neurological process neurogenesis synaptic transmission long term potentiation memory growth neurotransmission outgrowth quantity migration guidance differentiation morphogenesis plasticity behavior locomotion learning memory psychological process cognition Nervous System Development and Function Behavior p value # Molecules B

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166 Figure 4 1 5 Immunological and c ell d eath/ a poptosis functions for a ll a nalyses The majority of all transcripts mapped to the exposure analysis Both innate and adaptive immune categories are present, as well as both cell death and apoptosis. nonvac cinated/exposed nonvaccinated/exposed between the thalamus and cerebrum of the nonvaccinated/exposed 0.00E+00 5.00E 03 1.00E 02 1.50E 02 2.00E 02 2.50E 02 3.00E 02 3.50E 02 0 200 400 600 800 1000 1200 p value # Molecules Molecules Exposure Molecules Survival Molecules Location p value Exposure p value Survival p value Location

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167 Figure 4 1 6 Canonical pathways for significant genes common to a ll a nalyse s. The represents neurological pathways, while the ^ represents immunological pathways. A large number of genes mapped to both neurological and immunological categories. expression between the nonvaccinated/exposed between the nonvaccinated/exposed represents the difference in gene expression between the thalamus and cerebrum of the nonvaccinated/exposed. 0.00E+00 5.00E 01 1.00E+00 1.50E+00 2.00E+00 2.50E+00 3.00E+00 3.50E+00 0 2 4 6 8 10 12 log(p value) # Molecules # Molecules log(p value)

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168 Figure 4 1 7 Neurological functions for significant genes common to a ll a nalyse s. The functions identified include those involved with mental disorders and neurodegenerative disorders. represents the difference in gene expression between the nonvaccinated/exposed the difference in gene expression between the nonvacc inated/exposed represents the difference in gene expression between the thalamus and cerebrum of the nonvaccinated/exposed. 0 0.005 0.01 0.015 0.02 0.025 0.03 0 20 40 60 80 100 120 140 160 180 neurological disorder neuropathy progressive motor neuropathy bipolar affective disorder amyotrophic lateral sclerosis Huntington's disease Parkinson's disease schizophrenia multiple sclerosis seizures neurological process development long term potentiation synaptic transmission bipolar affective disorder schizophrenia Neurological Disease Nervous System Development and Function Psychological Disorders p value # Molecules # Molecules p value

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169 Figure 4 1 8 Immunological functions for significant genes common to a ll a nalyse s. A large number of immunological genes are common between all analyse s, as well as genes classified to cell death and apoptosis. For the nonvaccinated/exposed nonvaccinated/exposed represents the difference in gene expression between the thalamus and cerebrum of the nonvaccinated/exposed. 0 0.005 0.01 0.015 0.02 0.025 0 50 100 150 200 250 300 350 p value # Molecules # Molecules p value

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170 C HAPTER 5 CONCLUSIONS Many novel findings were made during the course of this project that will furth er our understanding of both equine genetics and viral encephalitis/neuropathology. In the first portion of this project, pyrosequencing technology was used to sequence the transcriptome of the central nervous system of horses. In total, 41,040 sequences were identified by BLAST analysis in 5 sequence databases Over 27,000 of these sequences group ed under Gene Ontology classifications. Analysis of th ese sequences revealed that they were enriched for those genes involved with the nervous system. Of the 41,040 sequences, 9,504 sequences were identified that were missed by equine predicted databases, and 1,280 genes were identified that we re completely novel to the equine genome project. B iomarker analysis was performed on all of the sequences and 3,227 recognized potential biomarkers were identified ( 496 of these involved in neurological disease 11 in the novel genes category) These will be important targets for system biology strategies utilizing genomic and proteomic techniques. Many of the biomark ers that were identified are accessible in easily accessible clinical samples (urine, blood, plasma, CSF). Thus this project identified many genes that are specific to the neurological system, are completely novel to the horse, and have potential applicat ions as biomarkers. Finally, the equine transcriptome sequenced in this project was compared to the human EST project and demonstrated high sequence homology (69 80%) between the ESTs of the two species This provides evidence that data generated from equ ine studies can be directly applicable to human studies. This portion of the project demonstrated that gene expression studies are necessary to supplement the limitations of current sequence databases. This is the first report of the use of

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171 pyrosequencin g to analyze the transcriptome of the equine with contribution of genes novel to the equine genome project. In the second portion of the project, the 41,040 sequences generated from the equine transcriptome were used to create a 4x44,000 custom oligonucl eotide microarray. This array was used to analyze gene expression in the brain of non vaccinated horses exposed to WNV, vaccinated horses exposed to WNV, and untreated horses. Specifically, three questions were asked was there was a difference in gene e xpression due to 1. Exposure to WNV (comparing the nonvaccinated/exposed horses to normal horses), 2. Survival from WNV infection (comparing the nonvaccinated/exposed horses to vaccinated/exposed horses), and 3. Location in the brai n during WNV infection (compar ing the thalamus of nonvaccinated/exposed horses to the cerebrum from the same horses). Statistical analysis was performed on the data using an ANOVA with interactions (p<0.05) to identify genes that were significantly up or down regulated. This d ata was then fed into IPA software to identify pathways, functions, and networks to map out the data. A large number of genes were identified as significant when looking at the three different analyses (9,020 for exposure, 7,395 for survival, and 7,649 fo r location). GO analysis was performed on the data from all three analyse s. Most genes mapped to transcription/RNA processing (5,550) with the second most genes mapping to neurological categories (3,065) for all analyse s. A large number of genes also m apped to immunological categories (2,631) and cell death/apoptosis (1,117) The GO data corresponded to the IPA analysis, which found that the most genes mapped to signaling pathways, many of which were involved with transcription. The

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172 second most numbe r of genes mapped to neurological pathways and disease functions. Detailed analysis revealed that components of both the glutamate and dopamine pathways were down regulated in the WNV infected brain, providing evidence of glutamate excitotoxicity and path ology associated with a lack of dopamine. In addition, many of the transcripts mapped to non infectious neurological disease functions, including mental disorders ( enia, and depression) and degenerative ne uropathies ( progressive motor neuropathy, sclerosis, and multiple sclerosis ). This study showed that WNV has an effect on the central nervous system of affected hosts by dysr egulating pathways involved with neurotransmission and downstream signaling. This corresponds with clinical signs of disease in affected hosts, and also suggests a correlate between the neuropathology induced by viral infection of the CNS and the neuropat hology seen in non infectious neurological disease. The other major set of pathways that were shown to be dysregulated by IPA analysis were those involved in the immunological response. Pathways involving both the innate and adaptive immune response wer e demonstrated in all analyse s. The majority of immunological pathways were downregulated in the non vaccinated exposed horses compared to the others (as well as in the thalamus) providing evidence that a balanced immune response is present in recovery fr om disease. Detailed analyses of the pathways revealed an increase in IL 15 production but a decrease in IL 15 downstream signaling in the WNV infected brain, providing evidence t hat IL 15 is part of a balanced immune response important in recovery from W NV infection. Another

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173 detailed analysis of the pathways revealed that WNV is likely able to evade innate immunity in nave horses by upregulating the production of the SOCS3 molecule which itself functions to block the JAK/STAT pathway and subsequent inna te immune stimulation. Apoptosis was found to be upregulated in the non vaccinated exposed analysis providing expression level evidence of neuropathology due to viral infection. Individual transcripts that were significantly upregulated and downregulated were also identified. For all analyse s, transcription regulators were both increased and decreased in expression. Transcription transcripts increased in expression involved in both the innate and ada ptive immune response included STAT1, IRF2, IRF3, BATF, and EOMES. Transcription genes decreased in expression included NFIA, SUB1, ASB1, and ASB5. ASB1 and ASB5 function to suppress the SOCS transcripts Thus the transcriptional immune response coincid es with the findings downstream mentioned above. Other transcriptional genes including those associated with the nervous system, were also affected. Individual transcripts not involved in transcription upregulated by a significant amount including PTX3, involved in the pattern recognition response to viruses and bacteria, and CTNND2 involved with the connection between neuron cytoskeleton and signaling. Understanding which transcripts are upregulated or downregulated during viral infection will be useful for the future identification of candidate biomarkers and important genes. Finally, the array was validated with real time reverse transcription PCR on six sets of primers. In summary, this was the first project to sequence the equine brai n transcriptome. This data was used to create a microarray platform that successfully analyzed gene expression during WNV disease, infection, and recovery. Novel

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174 pathways were revealed to be involved in the pathology of and defense against WNV infection. A link was made between infectious and non infectious neurological disease. A balanced immune response was shown to be important in recovery from WNV infection. This data will continue to be analyzed and will be used in the future to discover potential therapeutics and diagnostic options for viral encephalitis. The information gained from this project has furthered our knowledge of the neuropathology and neuroimmunology of viral encephalitis, and will continue to do so for years to come.

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175 APPENDIX A RNA QUALITY DATA ANA LYSIS The isolation of high quality RNA is difficult, due to the fast rate of degradation of RNA transcripts and the presence of RNases. Because of this, before any RNA is used during an experiment, the quality of the RNA must be as sessed. Previous work has demonstrated that RNA of low quality demonstrates up to a 7 fold difference in relative gene expression using Q PCR [173] and that RNA of low quality demonstrates different levels of gene expression and hierachical clustering when analyzed by microarray analysis [174] Traditionally, to assess the RNA quality before its use, the 28S:18S rRNA ratio is used. This technique relies on the fact that rRNA comprises over 80% of the cellular rRNA, and therefore the quality of the rRNA will reflect the quality of the cellular RNA. The sample is run on a gel and two bands (corresponding to a 28S band and a 18S band) are visualized using either traditional measurement techniques for gel bands or the Agilent 2100 bioanalyzer. The ratio of these two bands is then measured to determine the degree of RNA degradation. The ideal ratio is 2.7:1 (since 28S rRNA is approximately 5kb in siz e and 18S rRNA is approximately 2kb in size), but a ratio of 2:1 is considered the standard. However, there are problems with using this technique, especially when it is used to determine the quality of mRNA. rRNA quality does not necessarily reflect mRN A quality, as mRNA is smaller (less degradation) and has a faster turn over rate. Previous work has also shown there to be a large amount of variability in the assessment method [173, 175] and that the technique is affected by sample dilution.

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176 A new technique, called the RNA integrity number (RIN), was created to standardize the process of RNA integrity interpretation. This technique uses the A gilent 2100 bioanalyzer, a mi cro fluidics based platform for the analysis of RNA Small amounts of RNA are incubated with dye the s amples separated on microfabricated chips via gel electrophoresis (molecular weight separation) and a laser used to induce fluorescence. A computer captures the fluorescence images corresponding to the bands of RNA. If R NA degradation is present, a decrease in the 18S and 28S band ratio develops with a corresponding increase in the baseline signal between the two peaks A RIN is generated, which evaluates the en tire electrophoretic trace using a software algorithm This i ncludes the 28S:18S ratio, degradation of RNA (baseline) contamination of samples, etc This allows for the evaluation of the integrity of all of the RN A. Samples with a RIN of > 6 7 is the standard for high quality RNA, and high scoring samples demonstrate expected values. [176] In addition, the RIN has been shown to have less variability than other standardization methods, [173, 175] and ensures repeatability and reliability between experiments. This project only used RNA samples with a RIN >7. An example of an electropherogram trace for all samples can be seen in figure 3 1. Table A 1 shows the individual RI N numbers for the samples used to create the cDNA library.

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177 Table A 1 RNA q uality data for all samples used to c reate the cDNA l ibrary Sample Tissue Concentration (ng/uL) RNA Integrity Number 260/280 Ratio Uninfected Cerebrum 298 8 1.8 Cerebellum 596 7.8 1.9 Cervical SC 152 6.6 1.98 Lumbar SC 334 6 1.97 Cervical SC 524.5 7.2 1.97 Spleen 311.5 7.5 2.08 Midbrain 424.88 6.7 2.04 Hindbrain 652.13 7.2 1.94 Vaccinates Cerebrum 1551 8.2 1.95 Cerebellum 996.35 8.2 2.05 Cerv. SC 602.43 6.7 1.87 Lum. SC 369.45 7.7 1.96 Spleen 424 7 1.93 Hindbrain 456 6.9 2.02 Thalamus 848.48 6.7 2.03 Cerebrum 1042.72 8 2.05 Cerebellum 1404 7.8 1.99 Cerv. SC 323 7.4 1.95 Lum. SC 520.12 6.8 1.98 Thalamus 555.75 8.4 2.06 Spleen 122 7.8 1.92 Midbrain 1515.72 6.8 2.02 Hindbrain 1712.28 6.7 2.03 Non Vaccinates Cerebrum 700.59 7.7 1.99 Cerv. SC 256.12 6.9 1.99 Thalamus 316.38 7.7 1.99 Spleen 501.5 8.3 1.96 Midbrain 217.73 7.4 1.94 Hindbrain 236.08 7.4 1.97 Cerebellum 399 8 1.9 Lum. SC 236 6.7 1.9 Cerebrum 886 7.9 2.01 Cerebellum 968 8.4 1.96 Cerv. SC 152 7.1 2.01 Lum. SC 279 6.7 1.75 Thalamus 618 7.5 1.91 Spleen 468 8.4 1.95 Midbrain 425 7.6 1.97 Hindbrain 365 7.6 2.04 Samples used to create the cDNA library were assessed using the Agilent 2100 Bioanalyzer. Only samples with a RIN>6.5 were used in the library.

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1 78 APPENDIX B LIST OF HIGHLY UPREG ULATED TRANSCRIPTS RECOGNIZED BY IPA Table B 1. Transcripts increased in expression recognized by IPA Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation ABCA1 ATP binding cassette, sub family A (ABC1), member 1 NM_005502 Plasma Membrane Transporter 1.095 ABCC9 ATP binding cassette, sub family C (CFTR/MRP), member 9 NM_020297 Plasma Membrane Ion channel 1.395 ACN9 ACN9 homolog (S. Cerevisiae ) NM_001080352 Cytoplasm Other 1.964 ACSL5 Acyl coa synthetase long chain family member 5 XM_859627 Cytoplasm Enzyme 1.541 2.592 1.615 ACTN1 Actinin, alpha 1 DQ496098 Cytoplasm Other 1.733 ADAM17 ADAM metallopeptidase domain 17 XM_515293 Plasma Membrane Peptidase 1.237 ADAM9 ADAM metallopeptidase domain 9 (meltrin gamma) XM_614306 Plasma Membrane Peptidase 1.005 ADAMTS7 ADAM metallopeptidase with thrombospondin type 1 motif, 7 AY327122 Extracellular Space Peptidase 1.646 ADAMTSL1 ADAMTS like 1 NM_001040272 Extracellular Space Other 2.247 ADAMTSL3 ADAMTS like 3 CR926461 unknown Other 1.616 ADI1 Acireductone dioxygenase 1 XM_001153173 Nucleus Enzyme 1.021 AGBL1 ATP/GTP binding protein like 1 AC022817 unknown Other 1.863 AGFG1 (includes EG:3267) Arfgap with FG repeats 1 XM_516132 Nucleus Other 1.049 1.113 AGPS Alkylglycerone phosphate synthase XM_001154263 Cytoplasm Enzyme 1.328 AIM2 Absent in melanoma 2 XM_513914 Cytoplasm Other 2.316 2.376 1.469 AK3 A denylate kinase 3 D10376 Cytoplasm Kinase 1.321 AK7 Adenylate kinase 7 XM_537550 unknown Kinase 5.023 ALDH3A1 Aldehyde dehydrogenase 3 family, membera1 NM_001082420 Cytoplasm Enzyme 1.294 ALPK1 Alpha kinase 1 XM_545029 unknown Kinase 1.586 1.828 1.818 ALPK2 Alpha kinase 2 NM_052947 unknown Kinase 1.285 ALX3 ALX homeobox 3 NM_006492 Nucleus Transcription regulator 2.05 AMD1 Adenosylmethionine decarboxylase 1 BX640599 unknown Enzyme 1.439 ANKFY1 Ankyrin repeat and FYVE domain containing 1 XM_511280 Nucleus Transcription regulator 1.329 1.508 1.017 ANLN Anillin, actin binding protein XM_596461 Cytoplasm Other 1.249 AOX1 Aldehyde oxidase 1 BC105265 Cytoplasm Enzyme 1.034 1.653 1.22 APLP2 Amyloid beta (A4) precursor like protein 2 XM_001155586 Extracellular Space Other 1.521 ARHGAP15 Rho gtpase activating protein 15 AC092652 unknown Other 1.894 1.066 1.492

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179 Table B 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation ARHGEF12 Rho guanine nucleotide exchange factor (GEF) 12 XM_508820 Cytoplasm Other 1.235 1.008 ARHGEF5 Rho guanine nucleotide exchange factor (GEF) 5 NM_001110075 Cytoplasm Other 1.018 ARHGEF6 Rac/Cdc42 guanine nucleotide exchange factor (GEF) 6 XM_865157 Cytoplasm Other 1.011 1.167 1.303 ASAP3 Arfgap with SH3 domain, ankyrin repeat and PH domain 3 NM_001083446 unknown Other 1.774 ASXL2 Additional sex combs like 2 (Drosophila) XM_515337 unknown Other 1.211 1.186 1.18 ATF3 Activating transcription factor 3 NM_001046193 Nucleus Transcription regulator 3.255 4.896 2.049 ATF6 Activating transcription factor 6 XM_513949 Cytoplasm Transcription regulator 1.225 ATG16L2 ATG16 autophagy related 16 like 2 (S. Cerevisiae) BC146660 unknown Other 1.547 ATP11B Atpase, class VI, type 11B AC069431 Plasma Membrane Transporter 1.173 ATP6V0A2 Atpase, H+ transporting, lysosomal V0 subunit a2 AK289391 Cytoplasm Transporter 1.263 1.479 1.31 ATP6V0E1 Atpase, H+ transporting, lysosomal 9kda, V0 subunit e1 AF343440 Cytoplasm Transporter 1.096 ATP8B1 Atpase, class I, type 8B, member 1 XM_533394 Plasma Membrane Transporter 1.261 ATP8B3 Atpase, class I, type 8B, member 3 XM_849983 Cytoplasm Transporter 1.652 ATPAF1 ATP synthase mitochondrial F1 complex assembly factor 1 NM_001042546 unknown Other 1.059 ATXN1 Ataxin 1 BC011026 Nucleus Other 1.019 ATXN7L1 Ataxin 7 like 1 XM_001162005 unknown Other 1.497 1.225 AVEN Apoptosis, caspase activation inhibitor XM_510277 Cytoplasm Ion channel 1.118 B3GNT2 UDP glcnac:betagal beta 1,3 N acetylglucosaminyltransfer ase 2 NM_001102497 Cytoplasm Enzyme 1.16 B3GNT6 UDP glcnac:betagal beta 1,3 N acetylglucosaminyltransfer ase 6 (core 3 synthase) NM_001103307 Cytoplasm Enzyme 1.573 BAG3 BCL2 associated athanogene 3 XM_544046 Cytoplasm Other 1.184 2.262 1.611 BAT2 HLA B associated transcript 2 XM_581077 Cytoplasm Other 1.003 BATF Basic leucine zipper transcription factor, ATF like BC032294 Nucleus Transcription regulator 5.086 4.364 2.596 BCHE Butyrylcholinesterase AC009811 Plasma Membrane Enzyme 1.412 BHLHE41 Basic helix loop helix family, member e41 NM_001002973 Nucleus Transcription regulator 1.421 BIRC5 Baculoviral IAP repeat containing 5 NM_001001855 Cytoplasm Other 1.626 2.123 1.736 BLNK B cell linker XM_001159213 Cytoplasm Other 1.021 1.583 BLZF1 Basic leucine zipper nuclear factor 1 XM_001136772 Cytoplasm Transcription regulator 1.103 BTN3A2 Butyrophilin, subfamily 3, member A2 BC002832 unknown Other 2.562 4.179 2.671

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180 Table B 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation BTN3A3 Butyrophilin, subfamily 3, member A3 AK291722 unknown Other 2.897 2.54 1.684 C12ORF26 Chromosome 12 open reading frame 26 AC089998 unknown Other 1.042 C12ORF63 Chromosome 12 open reading frame 63 XM_849752 unknown Other 1.645 1.855 C13ORF31 Chromosome 13 open reading frame 31 XM_509657 unknown Other 1.271 C14ORF147 Chromosome 14 open reading frame 147 NM_138288 unknown Other 1.382 C14ORF159 Chromosome 14 open reading frame 159 NM_001102368 Cytoplasm Other 1.449 C14ORF80 Chromosome 14 open reading frame 80 XM_850440 unknown Other 1.449 C15ORF41 Chromosome 15 open reading frame 41 AC007429 unknown Other 1.33 C15ORF48 Chromosome 15 open reading frame 48 BC142379 Nucleus Other 2.988 3.454 2.32 C17ORF76 Chromosome 17 open reading frame 76 XM_001170539 unknown Other 1.32 C18ORF54 Chromosome 18 open reading frame 54 XM_001156992 Extracellular Space Other 1.153 1.785 C1ORF141 Chromosome 1 open reading frame 141 XM_001163680 unknown Other 1.292 1.535 1.105 C1ORF58 Chromosome 1 open reading frame 58 XM_600599 Cytoplasm Other 1.145 C1QA Complement component 1, q subcomponent, A chain XM_535367 Extracellular Space Other 1.602 1.985 C1R Complement component 1, r subcomponent XM_862090 Extracellular Space Peptidase 3.745 3.937 1.435 C4ORF22 Chromosome 4 open reading frame 22 XM_845542 unknown Other 1.899 C6ORF167 Chromosome 6 open reading frame 167 XM_612135 unknown Other 1.923 C6ORF72 Chromosome 6 open reading frame 72 XM_001173165 unknown Other 1.178 2.057 C7ORF57 Chromosome 7 open reading frame 57 NM_001079785 unknown Other 3.883 C8ORF37 Chromosome 8 open reading frame 37 XM_866332 unknown Other 1.162 CACNA1H Calcium channel, voltage dependent, T type, alpha 1H subunit XM_537016 Plasma Membrane Ion channel 1.305 CALB1 Calbindin 1, 28kda NM_001076195 Cytoplasm Other 2.684 CALCOCO2 Calcium binding and coiled coil domain 2 XM_537667 Nucleus Other 1.251 CAPN2 Calpain 2, (m/II) large subunit NM_001748 Cytoplasm Peptidase 1.115 CASP4 Caspase 4, apoptosis related cysteine peptidase EF636667 Cytoplasm Peptidase 3.771 4.336 2.227 CCDC50 Coiled coil domain containing 50 NM_001038147 unknown Other 1.115 1.962 1.385 CCDC60 Coiled coil domain containing 60 XM_865397 unknown Other 1.553 CCDC68 Coiled coil domain containing 68 XM_001135637 unknown Other 1.274 CCL1 Chemokine (C C motif) ligand 1 NM_001005252 Extracellular Space Cytokine 1.332 CCNL1 Cyclin L1 XM_875093 Nucleus Other 1.033 CD244 CD244 molecule, natural killer cell receptor 2B4 BC041607 Plasma Membrane Other 1.026 CD38 CD38 molecule XM_001160533 Plasma M Enzyme 2.073 3.989 2.73

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181 Table B 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation CD3E CD3e molecule, epsilon (CD3 TCR complex) NM_001003379 Plasma Membrane Transmembra ne receptor 1.68 2.895 1.533 CD44 CD44 molecule (Indian blood analyse ) XM_001151212 Plasma Membrane Other 1.166 2.443 1.773 CD46 CD46 molecule, complement regulatory protein NM_010778 Plasma Membrane Other 1.392 1.952 CD47 CD47 molecule AK050387 Plasma Membrane Other 1.045 CD5L CD5 molecule like XM_513905 Plasma Membrane Transmembra ne receptor 2.874 4.599 CD8A CD8a molecule BC151259 Plasma Membrane Other 3.548 3.691 2.581 CDC7 Cell division cycle 7 homolog (S. Cerevisiae) AY585721 Nucleus Kinase 1.7 CDH16 Cadherin 16, KSP cadherin XM_546890 Plasma Membrane Enzyme 2.226 1.086 CDK5R2 Cyclin dependent kinase 5, regulatory subunit 2 (p39) XM_848027 Nucleus Other 1.264 CDX2 Caudal type homeobox 2 XM_522747 Nucleus Transcription regulator 1.514 CEACAM1 Carcinoembryonic antigen related cell adhesion molecule 1 (biliary glycoprotein) DQ989182 Plasma Membrane Transmembra ne receptor 2.647 3.391 3.295 CHIT1 Chitinase 1 (chitotriosidase) XM_514112 Extracellular Space Enzyme 2.153 2.046 1.383 CHORDC1 Cysteine and histidine rich domain (CHORD) containing 1 NM_001045912 unknown Other 1.046 CLCF1 Cardiotrophin like cytokine factor 1 XM_540818 Extracellular Space Cytokine 1.818 CLDN10 Claudin 10 XM_509702 Plasma Membrane Other 1.065 1.342 CLIC1 Chloride intracellular channel 1 BC102103 Nucleus Ion channel 1.863 CLIC4 Chloride intracellular channel 4 XM_001168510 Cytoplasm Ion channel 1.354 CMPK2 Cytidine monophosphate (UMP CMP) kinase 2, mitochondrial NM_001108017 Cytoplasm Kinase 2.476 3.001 2.022 CMTM7 CKLF like MARVEL transmembrane domain containing 7 AK055554 Extracellular Space Cytokine 1.862 CNRIP1 Cannabinoid receptor interacting protein 1 XM_419337 unknown Other 1.256 CNTN4 Contactin 4 AC026882 Plasma Membrane Enzyme 1.218 COBLL1 COBL like 1 NM_014900 unknown Other 1.494 CPEB3 Cytoplasmic polyadenylation element binding protein 3 NM_014912 unknown Other 1.338 CPNE3 Copine III XM_544163 Cytoplasm Kinase 1.002 CPNE5 Copine V XM_518438 unknown Other 1.743 CRTAM Cytotoxic and regulatory T cell molecule XM_001136009 Plasma Membrane Other 1.253 1.317 CRYBG3 Beta gamma crystallin domain containing 3 XM_594681 unknown Other 1.972 CSDA Cold shock domain protein A NM_003651 Nucleus Transcription regulator 2.243 3.216 1.808

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182 Table B 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation CSMD3 (includes EG:114788) CUB and Sushi multiple domains 3 AC104380 unknown Enzyme 1.069 CSNK1A1 Casein kinase 1, alpha 1 XM_001163993 Cytoplasm Kinase 1.048 CSNK1D Casein kinase 1, delta NM_001102080 Cytoplasm Kinase 1.244 1.184 CTDSPL2 CTD (carboxy terminal domain, RNA polymerase II, polypeptide A) small phosphatase like 2 XM_001161626 unknown Other 1.422 CTNNA3 Catenin (cadherin associated protein), alpha 3 AC018979 Plasma Membrane Other 1.636 CTNNB1 Catenin (cadherin associated protein), beta 1, 88kda XM_845101 Nucleus Transcription regulator 1.126 CTNND1 Catenin (cadherin associated protein), delta 1 XM_853917 Nucleus Other 1.319 1.25 1.033 CTNND2 Catenin (cadherin associated protein), delta 2 (neural plakophilin related arm repeat protein) AC003954 Plasma Membrane Other 5.619 5.741 2.321 CTTN Cortactin NM_005231 Plasma Membrane Other 1.671 CUGBP2 CUG triplet repeat, RNA binding protein 2 XM_507653 Nucleus Other 1.236 1.197 CX3CL1 Chemokine (C X3 C motif) ligand 1 XM_544391 Extracellular Space Cytokine 1.151 CXCR6 (includes EG:10663) Chemokine (C X C motif) receptor 6 XM_846798 Plasma Membrane G protein coupled receptor 1.062 CYBB Cytochrome b 245, beta polypeptide XM_001136243 Cytoplasm Enzyme 1.303 1.351 CYBRD1 Cytochrome b reductase 1 XM_001142821 Cytoplasm Enzyme 1.154 CYP4F22 Cytochrome P450, family 4, subfamily F, polypeptide 22 XM_541984 unknown Enzyme 1.166 CYTIP Cytohesin 1 interacting protein NM_001102243 Cytoplasm Other 2.434 3.185 2.773 DAGLB Diacylglycerol lipase, beta XM_536885 unknown Enzyme 1.295 1.213 1.072 DCAF7 DDB1 and CUL4 associated factor 7 XM_511593 Cytoplasm Other 1.522 1.068 DDX54 DEAD (Asp Glu Ala Asp) box polypeptide 54 XM_001152033 Nucleus Transcription regulator 1.207 DDX58 DEAD (Asp Glu Ala Asp) box polypeptide 58 NM_014314 Cytoplasm Enzyme 5.135 5.684 2.393 DDX60 DEAD (Asp Glu Ala Asp) box polypeptide 60 XM_532716 unknown Enzyme 5.189 DENND1B DENN/MADD domain containing 1B AK091207 unknown Other 1.071 DGKH Diacylglycerol kinase, eta XM_534133 Cytoplasm Kinase 1.292 DGKI Diacylglycerol kinase, iota XM_539825 Cytoplasm Kinase 1.332 DHX15 DEAH (Asp Glu Ala His) box polypeptide 15 AC102739 Nucleus Enzyme 1.453 DIO2 Deiodinase, iodothyronine, type II NM_001007023 Cytoplasm Enzyme 1.908 DNAH10 Dynein, axonemal, heavy chain 10 NM_001083900 unknown Other 1.382 DNAH11 Dynein, axonemal, heavy chain 11 XM_539463 Cytoplasm Enzyme 1.555 1.768

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183 Table B 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation DNAH6 Dynein, axonemal, heavy chain 6 XM_532984 unknown Other 3.114 DNAJA1 Dnaj (Hsp40) homolog, subfamily A, member 1 XM_860318 Nucleus Other 1.195 1.182 DNAJB1 Dnaj (Hsp40) homolog, subfamily B, member 1 XM_847807 Nucleus Other 1.173 1.126 DNAJC13 Dnaj (Hsp40) homolog, subfamily C, member 13 AC026374 unknown Other 2.391 DOT1L DOT1 like, histone H3 methyltransferase (S. Cerevisiae) XM_542191 Nucleus Phosphatase 1.027 DPCR1 Diffuse panbronchiolitis critical region 1 NM_080870 unknown Other 2.376 DUOXA1 Dual oxidase maturation factor 1 XM_544660 unknown Other 1.564 DYNLT1 Dynein, light chain, Tctex type 1 XM_001147088 Cytoplasm Other 1.008 DYRK4 Dual specificity tyrosine (Y) phosphorylation regulated kinase 4 XM_534917 Nucleus Kinase 1.331 EDNRB Endothelin receptor type B XM_001141717 Plasma Membrane G protein coupled receptor 1.473 EFEMP1 EGF containing fibulin like extracellular matrix protein 1 NM_001081717 Extracellular Space Other 2.501 1.322 EIF2C2 Eukaryotic translation initiation factor 2C, 2 XM_532338 Cytoplasm Translation regulator 1.112 EIF2S3 Eukaryotic translation initiation factor 2, subunit 3 gamma, 52kda XM_001149353 Cytoplasm Translation regulator 1.326 EIF5 Eukaryotic translation initiation factor 5 XM_863525 Cytoplasm Translation regulator 1.063 ELF1 E74 like factor 1 (ets domain transcription factor) XM_852043 Nucleus Transcription regulator 1.163 1.872 ELK1 ELK1, member of ETS oncogene family XM_548979 Nucleus Transcription regulator 1.242 ELK3 ELK3, ETS domain protein (SRF accessory protein 2) XM_001146216 Nucleus Transcription regulator 1.871 1.151 1.465 EMILIN2 Elastin microfibril interfacer 2 XM_592120 Extracellular Space Other 3.313 3.091 1.685 EML6 Echinoderm microtubule associated protein like 6 NM_146016 unknown Other 1.035 ENPP2 Ectonucleotide pyrophosphatase/phospho diesterase 2 XM_856150 Plasma Membrane Enzyme 1.185 ENPP4 Ectonucleotide pyrophosphatase/phospho diesterase 4 (putative function) NM_001081535 unknown Enzyme 1.468 2.123 EOMES Eomesodermin homolog (Xenopus laevis) XM_001165845 Nucleus Transcription regulator 3.849 3.344 EPHA10 EPH receptor A10 XM_539588 Extracellular Space Kinase 1.764 ERBB3 V erb b2 erythroblastic leukemia viral oncogene homolog 3 (avian) NM_001103105 Plasma Membrane Kinase 1.359 ERI1 Exoribonuclease 1 XM_539997 unknown Enzyme 1.042 ERMN Ermin, ERM like protein NM_001009959 Extracellular Space Other 1.421 ETHE1 Ethylmalonic encephalopathy 1 XM_850148 Cytoplasm Other 1.582

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184 Table B 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation ETV6 Ets variant 6 NM_001987 Nucleus Transcription regulator 1.07 ETV7 Ets variant 7 XM_001172937 Nucleus Transcription regulator 5.863 6.179 2.475 EYA3 Eyes absent homolog 3 (Drosophila) NM_001990 Nucleus Phosphatase 1.465 1.617 F10 Coagulation factor X XM_534191 Extracellular Space Peptidase 1.867 2.495 1.283 F12 Coagulation factor XII (Hageman factor) XM_546206 Extracellular Space Peptidase 3.041 2.435 1.489 F3 Coagulation factor III (thromboplastin, tissue factor) XM_001252442 Plasma Membrane Transmembra ne receptor 1.301 1.561 1.025 FAM107A Family with sequence similarity 107, member A XM_844019 Nucleus Other 1.823 FAM107B Family with sequence similarity 107, member B NM_031453 unknown Other 1.24 1.561 FAM126A Family with sequence similarity 126, member A XM_532489 Cytoplasm Other 1.307 FAM129B Family with sequence similarity 129, member B XM_864533 unknown Other 1.187 FAM92B Family with sequence similarity 92, member B BC111944 unknown Other 1.689 FCGR3A Fc fragment of igg, low affinity iiia, receptor (CD16a) XM_001174057 Plasma Membrane Transmembra ne receptor 4.213 5.193 2.557 FGFRL1 Fibroblast growth factor receptor like 1 XM_610839 Plasma Membrane Transmembra ne receptor 1.245 FKBP5 FK506 binding protein 5 XM_518427 Nucleus Enzyme 1.982 2.208 FLJ32810 Rho type gtpase activating protein FLJ32810 XM_001127597 unknown Other 1.482 FMN1 Formin 1 XM_535422 Nucleus Transporter 1.218 FMN2 Formin 2 XM_001155137 unknown Other 1.279 1.806 FOXP2 Forkhead box P2 NM_014491 Nucleus Transcription regulator 1.598 FRMD4B FERM domain containing 4B NM_001102099 Cytoplasm Other 1.332 FRY Furry homolog (Drosophila) XM_001477828 unknown Other 1.187 FUSIP1 Splicing factor, arginine/serine rich 13A XM_001166490 Nucleus Other 1.043 FYB FYN binding protein (FYB 120/130) NM_001105414 Nucleus Other 1.522 GAB1 GRB2 associated binding protein 1 NM_002039 Cytoplasm Other 1.541 GADD45A Growth arrest and DNA damage inducible, alpha NM_001924 Nucleus Other 1.792 1.407 1.324 GALNT13 UDP N acetyl alpha D galactosamine:polypeptide N acetylgalactosaminyltransf erase 13 (galnac T13) AC009227 Cytoplasm Enzyme 1.543 GALNT7 UDP N acetyl alpha D galactosamine:polypeptide N acetylgalactosaminyltransf erase 7 (galnac T7) NM_017423 Cytoplasm Enzyme 1.187 GALNT8 UDP N acetyl alpha D galactosamine:polypeptide N acetylgalactosaminyltransf NM_017417 Cytoplasm Enzyme 3.1 4.034 2.983

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185 Table B 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation erase 8 (galnac T8) GBP1 (includes EG:2633) Guanylate binding protein 1, interferon inducible, 67kda XM_590008 Cytoplasm Enzyme 3.061 4.381 2.033 GBP5 Guanylate binding protein 5 NM_001075746 Plasma Membrane Enzyme 3.2 3.467 2.283 GBP6 Guanylate binding protein family, member 6 XM_617067 unknown Enzyme 3.829 4.739 GBX2 Gastrulation brain homeobox 2 XM_543300 Nucleus Transcription regulator 1.721 3.717 GCH1 GTP cyclohydrolase 1 XM_846790 Cytoplasm Enzyme 1.296 GFPT1 Glutamine fructose 6 phosphate transaminase 1 XM_515528 Cytoplasm Enzyme 1.124 1.212 GFRA4 GDNF family receptor alpha 4 XM_846396 Plasma Membrane Transmembra ne receptor 1.134 GHR Growth hormone receptor XM_526938 Plasma Membrane Transmembra ne receptor 1.171 1.025 GHRHR Growth hormone releasing hormone receptor NM_000823 Plasma Membrane G protein coupled receptor 2.169 GLDC Glycine dehydrogenase (decarboxylating) XM_538655 Cytoplasm Enzyme 1.621 1.326 GLDN Gliomedin XM_510405 Cytoplasm Other 2.456 GLRA3 Glycine receptor, alpha 3 AC093868 Plasma Membrane Ion channel 1.04 GNG12 Guanine nucleotide binding protein (G protein), gamma 12 NM_018841 Plasma Membrane Enzyme 1.789 1.925 2.023 GNG5 Guanine nucleotide binding protein (G protein), gamma 5 BC003563 Plasma Membrane Enzyme 1.359 GRIA4 Glutamate receptor, ionotrophic, AMPA 4 AP001561 Plasma Membrane Ion channel 1.253 GTF2E1 General transcription factor IIE, polypeptide 1, alpha 56kda NM_001103294 Nucleus Transcription regulator 1.51 1.872 GUCY1A3 Guanylate cyclase 1, soluble, alpha 3 BX649180 Cytoplasm Enzyme 1.55 1.024 HAVCR2 Hepatitis A virus cellular receptor 2 NM_001077105 Plasma Membrane Transmembra ne receptor 1.367 1.647 1.574 HBEGF Heparin binding EGF like growth factor XM_001137119 Extracellular Space Growth factor 2.064 2.899 2.64 HELB Helicase (DNA) B XM_866120 Nucleus Enzyme 3.483 2.251 HLA DQA1 Major histocompatibility complex, class II, DQ alpha 1 BC125044 Plasma Membrane Transmembra ne receptor 1.767 3.555 2.095 HMMR Hyaluronan mediated motility receptor (RHAMM) XM_849558 Plasma Membrane Other 3.501 4.141 3.2 HN1 Hematological and neurological expressed 1 NM_001002032 Nucleus Other 1.181 1.039 HN1L Hematological and neurological expressed 1 like NM_001081546 Cytoplasm Other 1.548 1.276 HNRNPA3 Heterogeneous nuclear ribonucleoprotein A3 XM_857035 Nucleus Other 1.169 HRASLS5 HRAS like suppressor family, member 5 NM_054108 unknown Other 1.587 HRH2 Histamine receptor H2 AY136744 Plasma Membrane G protein coupled 1.793 1.418

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186 Table B 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation receptor HSPBAP1 HSPB (heat shock 27kda) associated protein 1 NM_001014911 unknown Other 2.137 HSPG2 (includes EG:3339) Heparan sulfate proteoglycan 2 M85289 Plasma Membrane Other 1.658 1.116 ICK Intestinal cell (MAK like) kinase NM_016513 Cytoplasm Kinase 1.125 ID4 Inhibitor of DNA binding 4, dominant negative helix loop helix protein XM_001170946 Nucleus Transcription regulator 1.363 IFIT5 Interferon induced protein with tetratricopeptide repeats 5 XM_521554 unknown Other 4.908 IGJ Immunoglobulin J polypeptide, linker protein for immunoglobulin alpha and mu polypeptides XM_532398 Extracellular Space Other 1.838 2.228 2.031 IGSF9 Immunoglobulin superfamily, member 9 AB037776 Plasma Membrane Other 1.509 IKZF2 IKAROS family zinc finger 2 (Helios) NM_016260 Nucleus Transcription regulator 1.414 IL10RA Interleukin 10 receptor, alpha XM_591164 Plasma Membrane Transmembra ne receptor 1.358 IL15 Interleukin 15 AK290619 Extracellular Space Cytokine 2.369 2.29 2.004 IL1RAP Interleukin 1 receptor accessory protein XM_001161189 Plasma Membrane Transmembra ne receptor 1.618 IL4I1 Interleukin 4 induced 1 AY358933 Cytoplasm Enzyme 3.176 3.265 1.405 IL7 Interleukin 7 X64540 Extracellular Space Cytokine 2.952 2.844 3.148 IMPACT Impact homolog (mouse) NM_018439 unknown Other 1.436 INADL Inad like (Drosophila) NM_176877 Plasma Membrane Other 2.169 INPPL1 Inositol polyphosphate phosphatase like 1 XM_001251422 Cytoplasm Phosphatase 1.312 IRF2 Interferon regulatory factor 2 XM_532847 Nucleus Transcription regulator 2.61 1.797 IRF3 Interferon regulatory factor 3 AK292027 Nucleus Transcription regulator 1.31 1.935 1.086 IRX1 Iroquois homeobox 1 XM_001251876 Nucleus Transcription regulator 5.101 IRX3 Iroquois homeobox 3 NM_001104996 Nucleus Transcription regulator 3.398 IRX4 Iroquois homeobox 4 NM_001098466 Nucleus Transcription regulator 1.02 ISL1 ISL LIM homeobox 1 XM_001150633 Nucleus Transcription regulator 2.327 ITGAD Integrin, alpha D XM_547050 Plasma Membrane Other 1.457 1.697 ITGAV Integrin, alpha V (vitronectin receptor, alpha polypeptide, antigen CD51) XM_545559 Plasma Membrane Other 1.132 ITGB1 Integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12) XM_507735 Plasma Membrane Transmembra ne receptor 1.569 ITGB2 Integrin, beta 2 (complement component 3 receptor 3 and 4 subunit) NM_000211 Plasma Membrane Other 2.26 1.71

PAGE 187

187 Table B 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation ITIH2 Inter alpha (globulin) inhibitor H2 XM_535195 Extracellular Space Other 1.026 ITPR1 Inositol 1,4,5 triphosphate receptor, type 1 NM_001099952 Cytoplasm Ion channel 1.31 ITSN2 Intersectin 2 NM_006277 Cytoplasm Other 1.327 JAK1 Janus kinase 1 XM_001161295 Cytoplasm Kinase 1.079 JUNB Jun B proto oncogene NM_001075656 Nucleus Transcription regulator 2.201 2.017 KANK2 KN motif and ankyrin repeat domains 2 AY639929 Nucleus Transcription regulator 1.115 KCNA4 Potassium voltage gated channel, shaker related subfamily, member 4 AC124657 Plasma Membrane Ion channel 3.018 2.528 1.645 KHDRBS1 KH domain containing, RNA binding, signal transduction associated 1 CU210913 Nucleus Transcription regulator 1.116 KIAA0101 Kiaa0101 AK290748 Nucleus Other 2.014 1.988 1.464 KIAA0174 Kiaa0174 XM_857726 unknown Other 1.017 KIAA0494 Kiaa0494 XM_524573 unknown Other 1.435 KIAA1244 Kiaa1244 XM_518767 unknown Other 1.108 KIAA1486 Kiaa1486 NM_020864 unknown Other 1.631 KLF6 Kruppel like factor 6 AK151769 Nucleus Transcription regulator 1.746 KPTN Kaptin (actin binding protein) XM_849863 Cytoplasm Other 1.627 LAMP2 Lysosomal associated membrane protein 2 XM_859449 Plasma Membrane Enzyme 2.575 LASS2 LAG1 homolog, ceramide synthase 2 NM_001034667 Nucleus Transcription regulator 1.029 LBH Limb bud and heart development homolog (mouse) NM_001099152 Nucleus Transcription regulator 2.376 2.11 1.208 LCP1 Lymphocyte cytosolic protein 1 (L plastin) XM_001157284 Cytoplasm Other 1.791 2.476 1.668 LHFP Lipoma HMGIC fusion partner XM_001147653 unknown Other 2.318 3.616 1.866 LHFPL2 Lipoma HMGIC fusion partner like 2 NM_001099151 unknown Enzyme 1.432 2.356 LMNB2 Lamin B2 XM_542188 Nucleus Other 1.496 LRCH1 Leucine rich repeats and calponin homology (CH) domain containing 1 BC117472 unknown Other 1.011 LRRC16A Leucine rich repeat containing 16A NM_017640 unknown Enzyme 1.008 1.111 LRRFIP1 Leucine rich repeat (in FLII) interacting protein 1 BC083492 Nucleus Transcription regulator 1.42 1.516 LSP1 Lymphocyte specific protein 1 NM_001075374 Cytoplasm Other 1.954 3.078 2.579 LYPD1 LY6/PLAUR domain containing 1 AC153655 Plasma Membrane G protein coupled receptor 1.645 MACF1 Microtubule actin crosslinking factor 1 XM_001476185 Cytoplasm Other 1.048 MAN1A1 Mannosidase, alpha, class 1A, member 1 AK154820 Cytoplasm Enzyme 1.243 MAP3K1 Mitogen activated protein kinase kinase kinase 1 NM_005921 Cytoplasm Kinase 1.335 1.281 MAX MYC associated factor X XM_847808 Nucleus Transcription regulator 1.087 1.382

PAGE 188

188 Table B 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation MCL1 Myeloid cell leukemia sequence 1 (BCL2 related) NM_001003016 Cytoplasm Transporter 1.113 MCM6 Minichromosome maintenance complex component 6 NM_001046234 Nucleus Enzyme 1.129 MED12L Mediator complex subunit 12 like NM_053002 unknown Other 1.118 MED21 Mediator complex subunit 21 XM_534858 Nucleus Transcription regulator 1.056 1.001 METTL8 Methyltransferase like 8 XM_001142517 unknown Enzyme 1.317 MFAP3L Microfibrillar associated protein 3 like XM_001154389 unknown Other 1.458 1.521 1.889 MFNG MFNG O fucosylpeptide 3 beta N acetylglucosaminyltransfer ase NM_001101051 Cytoplasm Enzyme 1.104 MGAT5B Mannosyl (alpha 1,6 ) glycoprotein beta 1,6 N acetyl glucosaminyltransferase, isozyme B XM_843834 unknown Other 2.844 MGST2 Microsomal glutathione S transferase 2 NM_001076382 Cytoplasm Enzyme 1.311 MLLT4 Myeloid/lymphoid or mixed lineage leukemia (trithorax homolog, Drosophila); translocated to, 4 XM_581038 Nucleus Other 1.426 MMP19 Matrix metallopeptidase 19 NM_001075983 Extracellular Space Peptidase 3.448 2.549 3.85 MNDA Myeloid cell nuclear differentiation antigen AK290392 Nucleus Other 1.392 1.928 1.541 MOXD1 Monooxygenase, DBH like 1 XM_864684 Cytoplasm Enzyme 1.051 MSI1 Musashi homolog 1 (Drosophila) XM_849159 Cytoplasm Other 1.205 MSN Moesin EF076770 Plasma Membrane Other 1.13 2.026 1.291 MTR 5 methyltetrahydrofolate homocysteine methyltransferase BC130616 Cytoplasm Enzyme 1.023 1.312 MUC3A (includes EG:4584) Mucin 3A, cell surface associated BC142358 Extracellular Space Other 1.988 1.055 MYB (includes EG:4602) V myb myeloblastosis viral oncogene homolog (avian) D26147 Nucleus Transcription regulator 1.557 1.081 MYBPC1 Myosin binding protein C, slow type XM_001076591 Cytoplasm Other 1.948 1.754 MYL2 Myosin, light chain 2, regulatory, cardiac, slow DQ896055 Cytoplasm Other 1.898 2.435 MYO1F Myosin IF XM_542129 unknown Other 1.051 MYST3 MYST histone acetyltransferase (monocytic leukemia) 3 NM_001099413 Nucleus Enzyme 1.431 1.297 NAAA N acylethanolamine acid amidase XM_001152628 Cytoplasm Enzyme 1.294 1.537 NCOA3 Nuclear receptor coactivator 3 XM_543039 Nucleus Transcription regulator 1.112 NEB Nebulin NM_004543 Cytoplasm Other 1.253 2.486 NECAP2 NECAP endocytosis associated 2 BC109915 Cytoplasm Other 1.494 1.16

PAGE 189

189 Table B 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation NEK7 NIMA (never in mitosis gene a) related kinase 7 XM_001139810 Nucleus Kinase 1.379 NELL2 NEL like 2 (chicken) AC163991 Extracellular Space Other 1.715 NEXN Nexilin (F actin binding protein) XM_547323 Plasma Membrane Other 3.083 NFATC2IP Nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 2 interacting protein XM_844795 Nucleus Other 1.331 NFE2L2 Nuclear factor (erythroid derived 2) like 2 XM_857112 Nucleus Transcription regulator 1.037 1.337 NFIC Nuclear factor I/C (CCAAT binding transcription factor) XM_542179 Nucleus Transcription regulator 1.626 NFIL3 Nuclear factor, interleukin 3 regulated NM_001075240 Nucleus Transcription regulator 1.802 2.263 1.836 NFKBIE Nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, epsilon XM_583214 Nucleus Transcription regulator 1.482 NHSL1 NHS like 1 AK025347 unknown Other 1.172 NIACR2 G protein coupled receptor 109B NM_006018 Plasma Membrane G protein coupled receptor 1.41 1.956 NKTR Natural killer tumor recognition sequence NM_005385 Plasma Membrane Other 1.02 1.619 NMUR2 Neuromedin U receptor 2 XM_546288 Plasma Membrane G protein coupled receptor 2.206 1.656 NOC3L Nucleolar complex associated 3 homolog (S. Cerevisiae) XM_534972 Nucleus Other 1.432 NOL10 Nucleolar protein 10 AK290680 Nucleus Other 1.012 NOX1 NADPH oxidase 1 XM_549136 Cytoplasm Ion channel 1.142 NPBWR1 Neuropeptides B/W receptor 1 XM_544078 Plasma Membrane G protein coupled receptor 1.472 NRG2 (includes EG:9542) Neuregulin 2 XM_843380 Extracellular Space Growth factor 1.874 NRG3 Neuregulin 3 AL096706 Extracellular Space Growth factor 1.348 NRK Nik related kinase NM_198465 unknown Kinase 2.136 1.275 NTS Neurotensin XM_849385 Extracellular Space Other 8.55 NUFIP2 Nuclear fragile X mental retardation protein interacting protein 2 XM_001250302 unknown Other 1.028 NUP62CL (includes EG:54830) Nucleoporin 62kda C terminal like BC016327 unknown Other 1.005 NUP98 Nucleoporin 98kda XM_856693 Nucleus Transporter 1.597 1.476 NXT2 Nuclear transport factor 2 like export factor 2 NM_001100353 Nucleus Transporter 1.451 OGFR Opioid growth factor receptor XM_543089 Plasma Membrane Other 1.153 1.271 OSBPL3 Oxysterol binding protein like 3 AY008372 Cytoplasm Other 1.154 OTUD4 OTU domain containing 4 NM_001102653 unknown Other 1.392

PAGE 190

190 Table B 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation OTUD7B OTU domain containing 7B XM_603932 Cytoplasm Peptidase 1.477 1.396 P2RY1 Purinergic receptor P2Y, G protein coupled, 1 U34041 Plasma Membrane G protein coupled receptor 1.112 2.513 PARP10 Poly (ADP ribose) polymerase family, member 10 NM_032789 Nucleus Other 1.783 2.145 PARP14 Poly (ADP ribose) polymerase family, member 14 NM_017554 Cytoplasm Other 4.851 5.43 2.281 PAX3 Paired box 3 AC118213 Nucleus Transcription regulator 2.534 PAX4 Paired box 4 NM_006193 Nucleus Transcription regulator 2.674 PCDHB11 (includes EG:56125) Protocadherin beta 11 XM_844111 Plasma Membrane Other 1.052 PCGF5 Polycomb analyse ring finger 5 NM_032373 unknown Other 1.937 1.547 PCP4 Purkinje cell protein 4 AK289964 Cytoplasm Other 2.807 PDE10A Phosphodiesterase 10A XM_518849 Cytoplasm Enzyme 1.442 1.071 PDE4B Phosphodiesterase 4B, camp specific (phosphodiesterase E4 dunce homolog, Drosophila) AL109926 Cytoplasm Enzyme 1.917 1.022 PDE4D Phosphodiesterase 4D, camp specific (phosphodiesterase E3 dunce homolog, Drosophila) AC008829 Cytoplasm Enzyme 1.166 PDHA2 Pyruvate dehydrogenase (lipoamide) alpha 2 AC100752 Cytoplasm Enzyme 1.408 PDK4 Pyruvate dehydrogenase kinase, isozyme 4 NM_001101883 Cytoplasm Kinase 2.442 5.548 4.063 PDPN Podoplanin XM_513046 Plasma Membrane Transporter 2.336 2.807 2.742 PGGT1B Protein geranylgeranyltransferase type I, beta subunit XM_526978 Cytoplasm Enzyme 1.826 1.127 PHC3 Polyhomeotic homolog 3 (Drosophila) XM_545282 Nucleus Other 1.557 PIK3R3 Phosphoinositide 3 kinase, regulatory subunit 3 (gamma) XM_856294 Cytoplasm Kinase 1.537 PLA2R1 Phospholipase A2 receptor 1, 180kda XM_545489 Plasma Membrane Transmembra ne receptor 1.328 1.125 2.103 PLK2 Polo like kinase 2 (Drosophila) XM_587229 Nucleus Kinase 1.022 PLN Phospholamban NM_001003332 Cytoplasm Other 1.618 1.2 1.615 PLOD1 Procollagen lysine 1, 2 oxoglutarate 5 dioxygenase 1 BT025353 Cytoplasm Enzyme 1.133 POMC Proopiomelanocortin XM_515334 Extracellular Space Other 1.168 PPIA (includes EG:5478) Peptidylprolyl isomerase A (cyclophilin A) XM_001252497 Cytoplasm Enzyme 1.694 PPP1R1C Protein phosphatase 1, regulatory (inhibitor) subunit 1C AC064837 Cytoplasm Phosphatase 1.133 1.293

PAGE 191

191 Table B 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation PPP1R2 Protein phosphatase 1, regulatory (inhibitor) subunit 2 NM_001035392 Cytoplasm Phosphatase 1.239 PPP2R2B Protein phosphatase 2 (formerly 2A), regulatory subunit B, beta isoform XM_001159292 Cytoplasm Phosphatase 1.14 PPPDE2 PPPDE peptidase domain containing 2 XM_847031 unknown Other 1.07 PRAM1 PML RARA regulated adaptor molecule 1 XM_849317 unknown Other 1.38 1.818 PREX2 (includes EG:80243) Phosphatidylinositol 3,4,5 trisphosphate dependent Rac exchange factor 2 XM_544113 unknown Other 1.026 PRKCH Protein kinase C, eta NM_006255 Cytoplasm Kinase 4.052 PRKCH Protein kinase C, eta NM_006255 Cytoplasm Kinase 1.49 PRMT3 Protein arginine methyltransferase 3 NM_005788 Nucleus Enzyme 1.257 PRRG1 Proline rich Gla (G carboxyglutamic acid) 1 NM_001105640 Plasma Membrane Other 1.229 PRRX1 Paired related homeobox 1 NM_006902 Nucleus Transcription regulator 1.319 1.421 PRTFDC1 Phosphoribosyl transferase domain containing 1 XM_001156975 unknown Enzyme 1.133 1.725 PRTN3 Proteinase 3 BC096186 Extracellular Space Peptidase 1.732 PRUNE2 Prune homolog 2 (Drosophila) AC147026 unknown Other 1.306 1.568 1.125 PSEN1 Presenilin 1 BC151458 Plasma Membrane Peptidase 1.271 PSTPIP2 Proline serine threonine phosphatase interacting protein 2 NM_001101112 Cytoplasm Other 2.616 2.166 1.336 PTGFRN Prostaglandin F2 receptor negative regulator BC152454 Plasma Membrane Other 2.266 1.653 1.657 PTPN22 Protein tyrosine phosphatase, non receptor type 22 (lymphoid) XM_597200 Cytoplasm Phosphatase 1.713 2.03 1.957 PTPRC Protein tyrosine phosphatase, receptor type, C XM_547374 Plasma Membrane Phosphatase 2.333 3.479 2.52 PTPRJ Protein tyrosine phosphatase, receptor type, J NM_002843 Plasma Membrane Phosphatase 1.089 PTX3 Pentraxin related gene, rapidly induced by IL 1 beta DQ207368 Extracellular Space Other 9.021 7.746 4.277 PURA Purine rich element binding protein A XM_001251355 Nucleus Transcription regulator 1.225 PXK PX domain containing serine/threonine kinase NM_001099132 Cytoplasm Kinase 1.223 1.162 QKI Quaking homolog, KH domain RNA binding (mouse) AK055085 Nucleus Other 1.08 QRICH2 Glutamine rich 2 XM_533125 unknown Other 1.205 RAB11FIP1 RAB11 family interacting protein 1 (class I) XM_852408 Cytoplasm Other 2.075 2.035 1.231 RARB Retinoic acid receptor, beta X04014 Nucleus Ligand dependent nuclear receptor 1.357

PAGE 192

192 Table B 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation RBBP6 (includes EG:5930) Retinoblastoma binding protein 6 BC029649 Nucleus Other 1.084 1.089 1.155 RBMS1 RNA binding motif, single stranded interacting protein 1 CR591760 Nucleus Other 1.39 RCAN1 Regulator of calcineurin 1 XM_844202 Nucleus Transcription regulator 1.378 1.506 RDX Radixin BC149864 Cytoplasm Other 1.068 REL V rel reticuloendotheliosis viral oncogene homolog (avian) XM_531836 Nucleus Transcription regulator 1.124 1.88 2.204 REV1 REV1 homolog (S. Cerevisiae) XM_001160176 Nucleus Enzyme 1.07 RFFL Ring finger and FYVE like domain containing 1 XM_867129 Cytoplasm Enzyme 1.175 RGL3 Ral guanine nucleotide dissociation stimulator like 3 XM_542058 Cytoplasm Other 1.478 RGS16 Regulator of G protein signaling 16 NM_002928 Cytoplasm Other 2.038 2.282 RGS18 Regulator of G protein signaling 18 NM_130782 Cytoplasm Other 1.669 1.971 RHOG Ras homolog gene family, member G (rho G) XM_542335 Cytoplasm Enzyme 1.048 RIMS1 Regulating synaptic membrane exocytosis 1 AL445256 Cytoplasm Enzyme 1.515 RIT1 Ras like without CAAX 1 NM_006912 Plasma Membrane Enzyme 1.611 1.659 RNASE4 Ribonuclease, rnase A family, 4 BC102072 Extracellular Space Enzyme 1.04 RNF152 Ring finger protein 152 AC105183 unknown Other 1.165 RNF213 Ring finger protein 213 XM_590465 unknown Other 2.08 2.057 1.228 ROPN1L Ropporin 1 like NM_001075717 unknown Kinase 1.293 RORA RAR related orphan receptor A AC012404 Nucleus Ligand dependent nuclear receptor 1.435 RP13 102H20.1 Hypothetical protein FLJ30058 XM_549258 unknown Other 2.879 RPGR (includes EG:6103) Retinitis pigmentosa gtpase regulator NM_001003126 Cytoplasm Other 1.128 RXRA Retinoid X receptor, alpha XM_881943 Nucleus Ligand dependent nuclear receptor 1.199 RYR3 Ryanodine receptor 3 NM_001036 Plasma Membrane Ion channel 1.288 S100A3 S100 calcium binding protein A3 BC012893 unknown Transporter 1.76 SAP30 Sin3A associated protein, 30kda XM_843990 Nucleus Transcription regulator 1.382 1.973 1.588 SASS6 Spindle assembly 6 homolog (C. Elegans) XM_001159651 Cytoplasm Other 1.064 SBNO2 Strawberry notch homolog 2 (Drosophila) XM_542207 unknown Other 1.756 1.141 SC5DL Sterol C5 desaturase (ERG3 delta 5 desaturase homolog, S. Cerevisiae) like XM_001167254 Cytoplasm Enzyme 1.005 1.416

PAGE 193

193 Table B 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation SCN7A Sodium channel, voltage gated, type VII, alpha AC092583 Plasma Membrane Ion channel 6.334 6.37 3.151 SDCBP Syndecan binding protein (syntenin) AK128645 Plasma Membrane Enzyme 2.306 1.202 SEMA3B Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3B XM_590757 Extracellular Space Other 1.309 SEMA5A Sema domain, seven thrombospondin repeats (type 1 and type 1 like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5A NM_003966 Plasma Membrane Transmembra ne receptor 1.663 SEPP1 Selenoprotein P, plasma, 1 XM_530812 Extracellular Space Other 1.463 SFRS18 Splicing factor, arginine/serine rich 18 XM_863061 Nucleus Other 1.012 SFRS2 Splicing factor, arginine/serine rich 2 XM_852679 Nucleus Transcription regulator 1.075 SFRS3 Splicing factor, arginine/serine rich 3 XM_532124 Nucleus Other 1.178 SFRS6 Splicing factor, arginine/serine rich 6 NM_001035272 Nucleus Other 1.26 SH2D1B SH2 domain containing 1B NM_053282 unknown Other 1.23 SHISA5 Shisa homolog 5 (Xenopus laevis) XM_846245 Nucleus Other 1.856 SHISA5 Shisa homolog 5 (Xenopus laevis) XM_846245 Nucleus Other 1.604 SHOX2 Short stature homeobox 2 AK145063 Nucleus Transcription regulator 3.957 SHROOM3 Shroom family member 3 XM_844910 Cytoplasm Other 2.249 SIM2 Single minded homolog 2 (Drosophila) XM_001169429 Nucleus Transcription regulator 1.408 SLAIN1 SLAIN motif family, member 1 NM_001040153 unknown Other 1.149 SLC11A2 Solute carrier family 11 (proton coupled divalent metal ion transporters), member 2 XM_509061 Plasma Membrane Transporter 1.663 SLC16A9 Solute carrier family 16, member 9 (monocarboxylic acid transporter 9) XM_507806 unknown Other 1.194 1.08 SLC24A4 Solute carrier family 24 (sodium/potassium/calciu m exchanger), member 4 AC130838 unknown Transporter 1.51 SLC26A5 Solute carrier family 26, member 5 (prestin) XM_616468 Plasma Membrane Transporter 1.651 1.288 SLC2A9 Solute carrier family 2 (facilitated glucose transporter), member 9 XM_536240 Plasma Membrane Transporter 1.01 1.525 SLC35F1 Solute carrier family 35, member F1 Z95326 unknown Other 1.878 1.644 SLC38A2 Solute carrier family 38, member 2 XM_543722 Plasma Membrane Transporter 1.173 SLC39A1 Solute carrier family 39 (zinc transporter), member 1 NM_001035381 Plasma Membrane Transporter 1.013 SLC44A3 Solute carrier family 44, member 3 NM_001098900 unknown Other 1.844 1.377

PAGE 194

194 Table B 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation SLC8A1 Solute carrier family 8 (sodium/calcium exchanger), member 1 AC007281 Plasma Membrane Transporter 1.393 SLC9A3R1 Solute carrier family 9 (sodium/hydrogen exchanger), member 3 regulator 1 XM_540418 Plasma Membrane Other 1.171 SLC9A7 Solute carrier family 9 (sodium/hydrogen exchanger), member 7 XM_857172 Cytoplasm Transporter 1.569 SLK STE20 like kinase (yeast) XM_858785 Nucleus Kinase 1.004 SLMO2 Slowmo homolog 2 (Drosophila) NM_016045 unknown Other 1.285 SMPD3 Sphingomyelin phosphodiesterase 3, neutral membrane (neutral sphingomyelinase II) XM_546863 Cytoplasm Enzyme 1.304 SMPX Small muscle protein, X linked NM_001037626 unknown Other 2.377 SOCS3 Suppressor of cytokine signaling 3 NM_174466 Cytoplasm Other 1.535 1.809 SORBS1 Sorbin and SH3 domain containing 1 XM_001154001 Plasma Membrane Other 2.506 1.88 1.515 SOX10 SRY (sex determining region Y) box 10 DQ896471 Nucleus Transcription regulator 1.049 SOX2 SRY (sex determining region Y) box 2 XM_516895 Nucleus Transcription regulator 1.016 SP1 Sp1 transcription factor XM_509098 Nucleus Transcription regulator 1.155 1.625 1.364 SPAG1 Sperm associated antigen 1 XM_843637 Cytoplasm Other 1.64 SPATA13 Spermatogenesis associated 13 XM_001152608 unknown Other 1.075 SPCS3 Signal peptidase complex subunit 3 homolog (S. Cerevisiae) NM_021928 Cytoplasm Peptidase 1.047 SPOCK3 Sparc/osteonectin, cwcv and kazal like domains proteoglycan (testican) 3 XM_517526 Extracellular Space Other 1.102 SSPO SCO spondin homolog (Bos taurus) NM_174706 unknown Other 1.333 ST18 Suppression of tumorigenicity 18 (breast carcinoma) (zinc finger protein) XM_001148965 Nucleus Transcription regulator 1.307 ST8SIA4 ST8 alpha N acetyl neuraminide alpha 2,8 sialyltransferase 4 NM_005668 Cytoplasm Enzyme 1.025 STARD13 Star related lipid transfer (START) domain containing 13 NM_178006 Cytoplasm Other 1.78 2.285 1.776 STAT1 Signal transducer and activator of transcription 1, 91kda BC151378 Nucleus Transcription regulator 3.021 3.763 2.384 STK38 Serine/threonine kinase 38 NM_007271 Cytoplasm Kinase 1.193 STRA8 Stimulated by retinoic acid gene 8 homolog (mouse) XM_847727 unknown Other 2.39 STRN Striatin, calmodulin binding protein XM_525732 Cytoplasm Other 1.118 SUDS3 Suppressor of defective silencing 3 homolog (S. Cerevisiae) XM_509415 Nucleus Other 1.026

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195 Table B 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation SUDS3 Suppressor of defective silencing 3 homolog (S. Cerevisiae) XM_509415 Nucleus Other 1.142 1.109 SYNPO Synaptopodin AM393443 Cytoplasm Other 1.201 SYNRG Synergin, gamma XM_001173273 Cytoplasm Other 1.134 SYPL1 Synaptophysin like 1 XM_533096 Plasma Membrane Transporter 1.263 TARBP1 (includes EG:6894) TAR (HIV 1) RNA binding protein 1 XM_514281 Nucleus Transcription regulator 1.041 TAX1BP1 Tax1 (human T cell leukemia virus type I) binding protein 1 XM_859846 unknown Other 1.046 TBK1 TANK binding kinase 1 XM_538266 Cytoplasm Kinase 1.427 TCF12 Transcription factor 12 NM_001077885 Nucleus Transcription regulator 1.157 TCIRG1 T cell, immune regulator 1, atpase, H+ transporting, lysosomal V0 subunit A3 XM_540812 Plasma Membrane Enzyme 1.44 1.781 TEAD1 TEA domain family member 1 (SV40 transcriptional enhancer factor) XM_001171565 Nucleus Transcription regulator 1.786 1.319 TEP1 Telomerase associated protein 1 XM_582150 Nucleus Enzyme 1.602 TGFBR1 Transforming growth factor, beta receptor 1 XM_001159150 Plasma Membrane Kinase 1.071 TGIF2 TGFB induced factor homeobox 2 NM_021809 Nucleus Transcription regulator 1.11 THEMIS Thymocyte selection associated XM_541237 unknown Other 3.534 3.277 2.199 TIAM2 (includes EG:26230) T cell lymphoma invasion and metastasis 2 NM_012454 Cytoplasm Enzyme 1.225 TICAM2 Toll like receptor adaptor molecule 2 NM_021649 Plasma Membrane Other 1.951 1.394 TINAG Tubulointerstitial nephritis antigen XM_518550 Extracellular Space Peptidase 1.653 TJP2 Tight junction protein 2 (zona occludens 2) NM_001003204 Plasma Membrane Kinase 1.098 TLL1 Tolloid like 1 AC097502 Extracellular Space Peptidase 2.082 TMBIM1 Transmembrane BAX inhibitor motif containing 1 BC142530 unknown Other 1.19 1.115 TMEM123 Transmembrane protein 123 XM_001152796 Plasma Membrane Other 1.409 TMEM27 Transmembrane protein 27 NM_020665 Plasma Membrane Other 1.549 TMEM43 Transmembrane protein 43 NM_001102480 Nucleus Other 1.126 TMOD4 Tropomodulin 4 (muscle) XM_540312 unknown Other 1.449 TMTC3 Transmembrane and tetratricopeptide repeat containing 3 XM_532644 unknown Other 1.154 1.561 TMX1 Thioredoxin related transmembrane protein 1 XM_848339 Cytoplasm Enzyme 1.077 TNFAIP8 Tumor necrosis factor, alpha induced protein 8 XM_001152429 Cytoplasm Other 1.015 1.897 TNFRSF11B Tumor necrosis factor receptor superfamily, member 11b NM_001098056 Plasma Membrane Transmembra ne receptor 1.832 1.25

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196 Table B 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation TNFRSF17 Tumor necrosis factor receptor superfamily, member 17 BC058291 Plasma Membrane Other 1.217 TNFRSF25 Tumor necrosis factor receptor superfamily, member 25 XM_546752 Plasma Membrane Transmembra ne receptor 1.636 1.26 1.454 TNPO1 Transportin 1 NM_002270 Nucleus Transporter 1.053 1.25 TOX Thymocyte selection associated high mobility group box AC105150 Nucleus Other 1.27 1.419 TP73 Tumor protein p73 XM_593064 Nucleus Transcription regulator 1.171 TRIB2 Tribbles homolog 2 (Drosophila) XM_001161050 Plasma Membrane Kinase 1.166 TRIP6 Thyroid hormone receptor interactor 6 NM_001035469 Extracellular Space Cytokine 1.109 TRPC3 Transient receptor potential cation channel, subfamily C, member 3 XM_540964 Plasma Membrane Ion channel 1.943 TRPV2 Transient receptor potential cation channel, subfamily V, member 2 XM_546641 Plasma Membrane Ion channel 1.619 TSPAN2 Tetraspanin 2 NM_005725 unknown Other 1.31 TSPAN8 Tetraspanin 8 XM_531678 Plasma Membrane Other 2.49 TTC9C Tetratricopeptide repeat domain 9C NM_001083792 unknown Other 1.169 TTF2 Transcription termination factor, RNA polymerase II XM_513683 Nucleus Transcription regulator 1.17 1.74 TYK2 Tyrosine kinase 2 XM_590006 Plasma Membrane Kinase 1.504 UBA6 Ubiquitin like modifier activating enzyme 6 NM_018227 Cytoplasm Enzyme 1.302 1.461 1.327 UBE2C Ubiquitin conjugating enzyme E2C NM_007019 Cytoplasm Enzyme 3.688 2.958 2.15 UBE2G1 Ubiquitin conjugating enzyme E2G 1 (UBC7 homolog, yeast) XM_001174528 Cytoplasm Enzyme 1.004 1.464 UBE2L3 Ubiquitin conjugating enzyme E2L 3 XM_855294 Cytoplasm Enzyme 1.188 UBR1 Ubiquitin protein ligase E3 component n recognin 1 XM_510341 Cytoplasm Enzyme 1.061 UHMK1 U2AF homology motif (UHM) kinase 1 AL359699 Nucleus Kinase 1.383 UNC119B Unc 119 homolog B (C. Elegans) XM_849209 unknown Other 1.343 UNC13A Unc 13 homolog A (C. Elegans) NM_001080421 Plasma Membrane Other 1.44 USP13 (includes EG:8975) Ubiquitin specific peptidase 13 (isopeptidase T 3) XM_535813 unknown Peptidase 1.272 USP15 Ubiquitin specific peptidase 15 XM_001166186 Cytoplasm Peptidase 1.399 USP25 Ubiquitin specific peptidase 25 BS000022 unknown Peptidase 1.331 1.324 USP43 Ubiquitin specific peptidase 43 XM_511843 unknown Peptidase 1.134 USP53 Ubiquitin specific peptidase 53 XM_545046 unknown Enzyme 2.099 2.066 1.8 VAT1L Vesicle amine transport protein 1 homolog (T. Californica) like XM_001139539 unknown Enzyme 1.367 VGLL2 Vestigial like 2 BC118622 Nucleus Transcription 2.693

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197 Table B 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation (Drosophila) regulator VRK2 Vaccinia related kinase 2 AC073215 Nucleus Kinase 1.755 WDR33 WD repeat domain 33 NM_001006623 Nucleus Other 1.14 WIPF1 WAS/WASL interacting protein family, member 1 NM_001076923 Cytoplasm Other 1.753 WISP3 WNT1 inducible signaling pathway protein 3 BC105941 Extracellular Space Growth factor 1.354 1.421 WWTR1 WW domain containing transcription regulator 1 XM_847454 Nucleus Transcription regulator 3.107 2.939 2.33 XRN1 5' 3' exoribonuclease 1 XM_847344 Cytoplasm Enzyme 1.002 ZAP70 Zeta chain (TCR) associated protein kinase 70kda BC142505 Plasma Membrane Kinase 4.244 2.8 1.177 ZFP57 Zinc finger protein 57 homolog (mouse) NM_001109809 Nucleus Transcription regulator 1.025 ZIC1 Zic family member 1 (odd paired homolog, Drosophila) XM_516806 Nucleus Transcription regulator 1.836 ZNF185 Zinc finger protein 185 (LIM domain) XM_549348 Nucleus Other 1.092 ZNF503 Zinc finger protein 503 XM_001256479 Nucleus Other 1.454 ZNFX1 Zinc finger, NFX1 type containing 1 XM_534452 Nucleus Transcription regulator 3.531 3.311 1.255

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198 APPENDIX C LIST OF HIGHLY DOWNR EGULATED TRANSCRIPTS RECOGNIZED BY IPA Table C 1 Transcripts decreased in expression r ecognized by IPA Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation AASDHPPT Aminoadipate semialdehyde dehydrogenase phosphopantetheinyl transferase XM_508734 Cytoplasm Enzyme 1.423 ABCA2 ATP binding cassette, sub family A (ABC1), member 2 NM_001606 Plasma Membrane Transporter 2.244 2.019 ABCA2 ATP binding cassette, sub family A (ABC1), member 2 NM_001606 Plasma Membrane Transporter ABCB7 ATP binding cassette, sub family B (MDR/TAP), member 7 AL359545 Cytoplasm Transporter 1.402 ABCD2 ATP binding cassette, sub family D (ALD), member 2 XM_001168647 Cytoplasm Transporter 1.286 ABCD3 ATP binding cassette, sub family D (ALD), member 3 NM_001105396 Cytoplasm Transporter 1.917 ABI2 Abl interactor 2 XM_001173163 Cytoplasm Other 1.132 ABL1 C abl oncogene 1, receptor tyrosine kinase NM_005157 Nucleus Kinase 1.181 ABLIM1 Actin binding LIM protein 1 XM_535022 Cytoplasm Other 1.463 ABLIM2 Actin binding LIM protein family, member 2 XM_847882 Cytoplasm Other 1.632 ACADS Acyl Coenzyme A dehydrogenase, C 2 to C 3 short chain XM_534712 Cytoplasm Enzyme 1.033 ACCN1 Amiloride sensitive cation channel 1, neuronal XM_548270 Plasma Membrane Ion channel 2.229 2.002 2.603 ACCN2 Amiloride sensitive cation channel 2, neuronal XM_001155207 Plasma Membrane Ion channel 1.194 ACER3 Alkaline ceramidase 3 NM_001102285 Cytoplasm Enzyme 1.388 ACSBG1 Acyl coa synthetase bubblegum family member 1 XM_510525 Cytoplasm Enzyme 1.886 ACSL3 Acyl coa synthetase long chain family member 3 XM_516118 Cytoplasm Enzyme 1.027 ACSS2 Acyl coa synthetase short chain family member 2 NM_001076552 Cytoplasm Enzyme 1.169 ACTR1A ARP1 actin related protein 1 homolog A, centractin alpha (yeast) NM_001003164 Cytoplasm Other 1.031 ADAM22 ADAM metallopeptidase domain 22 XM_001164160 Plasma Membrane Peptidase 1.062 1.138 ADAMTS2 ADAM metallopeptidase with thrombospondin type 1 motif, 2 XM_538574 Extracellular Space Peptidase 1.031 ADCK1 Aarf domain containing kinase 1 XM_547933 unknown Kinase 1.443 ADCY1 Adenylate cyclase 1 (brain) NM_174229 Plasma Membrane Enzyme 1.789 1.898 ADCY2 Adenylate cyclase 2 (brain) XM_851103 Plasma Membrane Enzyme 1.683 ADCY2 Adenylate cyclase 2 (brain) XM_535798 Plasma Membrane Enzyme 1.223 2.027 ADCY5 Adenylate cyclase 5 NM_183357 Plasma Membrane Enzyme 1.258 1.379 ADCY8 Adenylate cyclase 8 (brain) XM_539166 Plasma Membrane Enzyme 1.021 1.954 ADCY9 Adenylate cyclase 9 BC151229 Plasma Membrane Enzyme 1.46 ADHFE1 Alcohol dehydrogenase, iron containing, 1 XM_844355 unknown Enzyme 1.32 1.415 ADORA3 Adenosine A3 receptor BC029831 Plasma Membrane G protein coupled receptor 1.966

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199 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation ADRA1B Adrenergic, alpha 1B receptor XM_001250068 Plasma Membrane G protein coupled receptor 1.222 ADRA2A Adrenergic, alpha 2A receptor BC035047 Plasma Membrane G protein coupled receptor 1.285 ADRBK2 Adrenergic, beta, receptor kinase 2 NM_005160 Cytoplasm Kinase 1.806 1.882 1.792 AFF3 AF4/FMR2 family, member 3 XM_001161010 Nucleus Transcription regulator 1.068 1.474 AFG3L2 AFG3 atpase family gene 3 like 2 (yeast) XM_547682 Cytoplasm Peptidase 1.007 AGAP2 Arfgap with gtpase domain, ankyrin repeat and PH domain 2 XM_581013 Nucleus Enzyme 1.881 1.544 AGPAT4 1 acylglycerol 3 phosphate O acyltransferase 4 (lysophosphatidic acid acyltransferase, delta) XM_001153359 Cytoplasm Enzyme 1.087 AHI1 Abelson helper integration site 1 BC151742 unknown Other 2.099 1.644 AHNAK AHNAK nucleoprotein NM_024060 Nucleus Other 1.841 1.088 AK5 Adenylate kinase 5 XM_547325 Cytoplasm Kinase 1.29 1.044 2.248 AKAP11 A kinase (PRKA) anchor protein 11 XM_001151850 Cytoplasm Other 1.148 1.084 ALDH4A1 Aldehyde dehydrogenase 4 family, member A1 XM_850179 Cytoplasm Enzyme 1.27 ALDH6A1 Aldehyde dehydrogenase 6 family, member A1 XM_001152670 Cytoplasm Enzyme 1.852 1.52 ALOX15B Arachidonate 15 lipoxygenase, type B XM_588924 Cytoplasm Enzyme 1.511 1.032 ALOX5 Arachidonate 5 lipoxygenase XM_613515 Cytoplasm Enzyme 1.761 1.274 ALOX5AP Arachidonate 5 lipoxygenase activating protein XM_534516 Plasma Membrane Other 2.782 1.019 ALS2 Amyotrophic lateral sclerosis 2 (juvenile) NM_001079920 Cytoplasm Other 1.074 1.067 AMT (includes EG:275) Aminomethyltransferase NM_001033993 Cytoplasm Enzyme 2.11 1.193 1.126 AMY2A Amylase, alpha 2A (pancreatic) NM_001035016 Extracellular Space Enzyme 1.2 1.429 ANKH Ankylosis, progressive homolog (mouse) NM_001109793 Plasma Membrane Transporter 1.75 1.213 1.383 ANKIB1 Ankyrin repeat and IBR domain containing 1 XM_844926 Nucleus Transcription regulator 1.194 ANKRD11 Ankyrin repeat domain 11 XM_546778 Nucleus Other 1.235 ANKRD54 Ankyrin repeat domain 54 XM_538382 Nucleus Transcription regulator 1.217 1.074 ANKS1B Ankyrin repeat and sterile alpha motif domain containing 1B AC084374 Nucleus Other 2.279 ANTXR1 Anthrax toxin receptor 1 AF090095 Plasma Membrane Other 1.132 ANTXR2 Anthrax toxin receptor 2 AC109518 Plasma Membrane Other 1.881 ANXA3 Annexin A3 XM_535624 Cytoplasm Enzyme 1.341 ANXA9 Annexin A9 NM_001035373 Plasma Membrane Transmembrane receptor 2.76 AP2B1 Adaptor related protein complex 2, beta 1 subunit XM_001174101 Cytoplasm Transporter 1.024 1.219 AP2S1 Adaptor related protein complex 2, sigma 1 subunit XM_533634 Cytoplasm Transporter 1.137 AP3M2 Adaptor related protein complex 3, mu 2 subunit XM_877825 Cytoplasm Transporter 2.167 2.199 1.352 APBA2 Amyloid beta (A4) precursor protein binding, family A, member 2 XM_843605 Cytoplasm Transporter 1.356 1.543

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200 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation APH1B Anterior pharynx defective 1 homolog B (C. Elegans) XM_001173827 Plasma Membrane Peptidase 1.106 APLNR Apelin receptor AK097232 Plasma Membrane G protein coupled receptor 2.857 2.059 2.364 APOLD1 Apolipoprotein L domain containing 1 BC126435 unknown Other 2.369 1.406 APPL2 Adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 2 AY113704 Cytoplasm Other 1.205 ARF2 ADP ribosylation factor 2 XM_537606 Cytoplasm Transporter 1.591 ARF3 ADP ribosylation factor 3 BC007647 Cytoplasm Enzyme 1.333 1.284 ARHGAP22 Rho gtpase activating protein 22 NM_021226 Cytoplasm Other 1.124 1.139 ARHGAP6 Rho gtpase activating protein 6 XM_548858 Cytoplasm Other 1.058 ARHGDIG Rho GDP dissociation inhibitor (GDI) gamma XM_849221 Cytoplasm Other 1.27 ARHGEF2 Rho/Rac guanine nucleotide exchange factor (GEF) 2 BC020567 Cytoplasm Other 1.13 ARID4A AT rich interactive domain 4A (RBP1 like) XM_859819 Nucleus Transcription regulator 1.76 2.145 2.296 ARL4C ADP ribosylation factor like 4C NM_001102348 Nucleus Enzyme 1.246 ARL5A ADP ribosylation factor like 5A XM_001136344 unknown Enzyme 1.576 ARL5B ADP ribosylation factor like 5B XM_843199 unknown Enzyme 1.099 ARMCX2 Armadillo repeat containing, X linked 2 AK291342 unknown Other 1.529 ARNT2 Aryl hydrocarbon receptor nuclear translocator 2 AC101776 Nucleus Transcription regulator 2.328 1.231 1.274 ARX Aristaless related homeobox XM_854885 Nucleus Transcription regulator 1.721 AS3MT Arsenic (+3 oxidation state) methyltransferase AY817668 Cytoplasm Enzyme 1.253 ASB1 Ankyrin repeat and SOCS box containing 1 XM_516189 Nucleus Transcription regulator 2.439 1.24 ASB13 Ankyrin repeat and SOCS box containing 13 XM_001145055 unknown Other 1.765 ASB5 Ankyrin repeat and SOCS box containing 5 NM_001075744 Nucleus Transcription regulator 1.292 ASPA Aspartoacylase (Canavan disease) XM_849422 unknown Enzyme 2.156 ASTN2 (includes EG:23245) Astrotactin 2 NM_014010 unknown Other 1.039 ATMIN ATM interactor XM_001249565 Nucleus Other 1.835 ATP1A2 Atpase, Na+/K+ transporting, alpha 2 (+) polypeptide XM_513921 Plasma Membrane Transporter 1.752 ATP2B2 Atpase, Ca++ transporting, plasma membrane 2 NM_001001331 Plasma Membrane Transporter 1.796 1.967 ATP2B3 Atpase, Ca++ transporting, plasma membrane 3 XM_001251087 Plasma Membrane Transporter 1.379 1.784 ATP8A1 (includes EG:10396) Atpase, aminophospholipid transporter (APLT), class I, type 8A, member 1 XM_517167 Cytoplasm Transporter 1.641 1.144 ATP9B Atpase, class II, type 9B NM_198531 Cytoplasm Transporter 1.098 ATRNL1 Attractin like 1 NM_207303 unknown Other 1.085 ATXN1 Ataxin 1 NM_000332 Nucleus Other 1.01 AUH AU RNA binding protein/enoyl Coenzyme A hydratase XM_533549 Cytoplasm Enzyme 1.123 AVP Arginine vasopressin BC102897 Extracellular Space Other 2.246

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201 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation AVPR1A Arginine vasopressin receptor 1A NM_000706 Plasma Membrane G protein coupled receptor 2.362 3.53 AXIN2 Axin 2 XM_001163124 Cytoplasm Other 2.657 1.554 B3GALNT1 Beta 1,3 N acetylgalactosaminyltransferase 1 (globoside blood group ) NM_001076963 Cytoplasm Enzyme 1.157 B3GALT2 UDP Gal:betaglcnac beta 1,3 galactosyltransferase, polypeptide 2 NM_001076188 Cytoplasm Enzyme 1.823 B3GAT1 Beta 1,3 glucuronyltransferase 1 (glucuronosyltransferase P) NM_054025 Cytoplasm Enzyme 1.507 1.53 1.451 B3GAT2 Beta 1,3 glucuronyltransferase 2 (glucuronosyltransferase S) BC113995 Cytoplasm Enzyme 1.777 B3GNT1 UDP glcnac:betagal beta 1,3 N acetylglucosaminyltransferase 1 XM_533222 Cytoplasm Enzyme 1.121 B4GALT6 UDP Gal:betaglcnac beta 1,4 galactosyltransferase, polypeptide 6 XM_523901 Cytoplasm Enzyme 1.323 BAALC Brain and acute leukemia, cytoplasmic AK093819 Cytoplasm Other 2.305 1.263 1.561 BACE1 Beta site APP cleaving enzyme 1 XM_001158264 Cytoplasm Peptidase 1.546 BAG4 BCL2 associated athanogene 4 XM_519710 Cytoplasm Other 1.167 BAG5 BCL2 associated athanogene 5 NM_001015049 unknown Other 1.278 BASP1 Brain abundant, membrane attached signal protein 1 XM_001175409 Plasma Membrane Other 1.276 BAT2L HLA B associated transcript 2 like XM_520327 unknown Other 1.331 BBOX1 Butyrobetaine (gamma), 2 oxoglutarate dioxygenase (gamma butyrobetaine hydroxylase) 1 BC011034 Cytoplasm Enzyme 2.586 2.935 2.068 BCHE Butyrylcholinesterase AC009811 Plasma Membrane Enzyme 1.653 BCL11A B cell CLL/lymphoma 11A (zinc finger protein) NM_022893 Nucleus Transcription regulator 1.539 BCLAF1 BCL2 associated transcription factor 1 XM_855478 Nucleus Transcription regulator 1.121 BICD1 Bicaudal D homolog 1 (Drosophila) AC087245 Cytoplasm Other 1.079 BLOC1S3 Biogenesis of lysosomal organelles complex 1, subunit 3 NM_001099086 Cytoplasm Other 2.57 BMP2K (includes EG:55589) BMP2 inducible kinase XM_843801 Nucleus Kinase 1.151 BMP3 Bone morphogenetic protein 3 XM_001144027 Extracellular Space Growth factor 1.048 1.662 1.235 BNIP3L BCL2/adenovirus E1B 19kda interacting protein 3 like XM_001162226 Cytoplasm Other 1.032 BRP44 Brain protein 44 Z97876 Plasma Membrane Other 1.134 BRUNOL4 Bruno like 4, RNA binding protein (Drosophila) NM_001099068 Nucleus Translation regulator 1.272 1.335 BSN Bassoon (presynaptic cytomatrix protein) NM_003458 Plasma Membrane Other 1.911 1.478 2.518 C10ORF10 Chromosome 10 open reading frame 10 XM_534951 Cytoplasm Other 2.06 C10ORF72 Chromosome 10 open reading frame 72 NM_001031746 unknown Other 1.385 C11ORF41 Chromosome 11 open reading frame 41 NM_012194 unknown Other 1.967 C12ORF51 Chromosome 12 open reading frame 51 NM_001109662 unknown Other 1.229 1.086 C17ORF28 Chromosome 17 open reading frame 28 AK074401 unknown Other 1.713 1.894 1.452

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202 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation C17ORF46 Chromosome 17 open reading frame 46 XM_843189 unknown Other 1.173 C18ORF1 Chromosome 18 open reading frame 1 NM_001003675 unknown Other 1.499 1.305 C1ORF96 Chromosome 1 open reading frame 96 XM_611981 unknown Other 1.797 C1QL3 Complement component 1, q subcomponent like 3 XM_865369 Extracellular Space Other 3.055 C1QTNF4 C1q and tumor necrosis factor related protein 4 XM_540740 Extracellular Space Other 1.766 C20ORF103 Chromosome 20 open reading frame 103 XM_845541 Cytoplasm Other 2.458 3.139 C20ORF194 Chromosome 20 open reading frame 194 NM_001009984 Nucleus Other 1.099 C21ORF33 Chromosome 21 open reading frame 33 NM_198155 Cytoplasm Other 1.718 C2CD2L C2CD2 like BC022219 unknown Other 1.032 C3ORF10 Chromosome 3 open reading frame 10 XM_591430 unknown Other 1.267 C5ORF44 Chromosome 5 open reading frame 44 XM_001163559 unknown Other 1.141 C6ORF142 Chromosome 6 open reading frame 142 NM_138569 unknown Other 2.615 C6ORF168 Chromosome 6 open reading frame 168 AK055101 unknown Other 1.148 C6ORF222 Chromosome 6 open reading frame 222 XM_845363 unknown Other 1.041 C7ORF42 Chromosome 7 open reading frame 42 XM_536835 unknown Other 1.586 C8ORF46 Chromosome 8 open reading frame 46 NM_001076475 unknown Other 1.568 C8ORF79 Chromosome 8 open reading frame 79 NM_001099677 unknown Other 1.119 2.078 1.123 C9ORF93 Chromosome 9 open reading frame 93 XM_531940 unknown Other 1.367 1.215 CA14 Carbonic anhydrase XIV XM_001167925 Plasma Membrane Enzyme 1.655 1.654 CA5B Carbonic anhydrase VB, mitochondrial XM_001139130 Cytoplasm Enzyme 1.757 1.393 CA7 Carbonic anhydrase VII XM_598644 Cytoplasm Enzyme 2.335 CA8 Carbonic anhydrase VIII NM_001083690 Cytoplasm Enzyme 1.453 CAB39 Calcium binding protein 39 NM_001046087 Cytoplasm Other 1.115 CACNA1B Calcium channel, voltage dependent, N type, alpha 1B subunit BC033060 Plasma Membrane Ion channel 1.579 CACNA1E Calcium channel, voltage dependent, R type, alpha 1E subunit L27745 Plasma Membrane Ion channel 1.778 CACNA1G Calcium channel, voltage dependent, T type, alpha 1G subunit XM_001252666 Plasma Membrane Ion channel 1.257 CACNG2 Calcium channel, voltage dependent, gamma subunit 2 NM_007583 Plasma Membrane Ion channel 1.513 CADPS2 Ca++ dependent secretion activator 2 NM_001102055 Plasma Membrane Other 3.008 CALB1 Calbindin 1, 28kda NM_001076195 Cytoplasm Other 1.27 CALCRL Calcitonin receptor like NM_005795 Plasma Membrane G protein coupled receptor 1.833 CALN1 Calneuron 1 NM_001017440 unknown Other 1.579 1.806 CAMK2A Calcium/calmodulin dependent protein kinase II alpha NM_001075938 Cytoplasm Kinase 1.408 3.062

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203 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation CAMK2G Calcium/calmodulin dependent protein kinase II gamma XM_846444 Cytoplasm Kinase 2.489 1.146 1.625 CAMK2N1 Calcium/calmodulin dependent protein kinase II inhibitor 1 BC151544 Plasma Membrane Kinase 1.096 1.297 CAMK4 Calcium/calmodulin dependent protein kinase IV XM_517873 Nucleus Kinase 1.976 CAMKK2 Calcium/calmodulin dependent protein kinase kinase 2, beta NM_172214 Cytoplasm Kinase 1.087 1.495 CAMTA1 (includes EG:23261) Calmodulin binding transcription activator 1 BC151835 unknown Other 1.335 1.397 CAND1 Cullin associated and neddylation dissociated 1 XM_531667 Cytoplasm Transcription regulator 1.104 CAPN2 Calpain 2, (m/II) large subunit NM_001748 Cytoplasm Peptidase 1.194 CASK Calcium/calmodulin dependent serine protein kinase (MAGUK family) BC029936 Plasma Membrane Kinase 1.144 CASKIN1 CASK interacting protein 1 XM_848538 Nucleus Transcription regulator 1.253 1.474 CAST Calpastatin BC148894 Cytoplasm Peptidase 1.055 CBFA2T2 Core binding factor, runt domain, alpha subunit 2; translocated to, 2 XM_606138 Nucleus Transcription regulator 1.097 1.196 CBFA2T3 Core binding factor, runt domain, alpha subunit 2; translocated to, 3 XM_546780 Nucleus Transcription regulator 1.112 CBLC Cas Br M (murine) ecotropic retroviral transforming sequence c NM_001101248 Nucleus Enzyme 1.145 CBLN1 Cerebellin 1 precursor XM_001163762 Cytoplasm Other 2.19 CCDC132 Coiled coil domain containing 132 AC027655 unknown Other 1.062 CCND2 Cyclin D2 XM_849493 Nucleus Other 1.035 1.658 CD109 CD109 molecule NM_133493 Plasma Membrane Other 2.211 CD163L1 (includes EG:283316) CD163 molecule like 1 NM_174941 Plasma Membrane Transmembrane receptor 1.024 CDH10 Cadherin 10, type 2 (T2 cadherin) NM_006727 Plasma Membrane Other 2.094 CDH4 (includes EG:1002) Cadherin 4, type 1, R cadherin (retinal) NM_001794 Plasma Membrane Other 1.934 CDH9 Cadherin 9, type 2 (T1 cadherin) XM_001135234 Plasma Membrane Other 1.244 1.892 CDK5R1 Cyclin dependent kinase 5, regulatory subunit 1 (p35) NM_003885 Nucleus Kinase 1.567 CDR2L Cerebellar degeneration related protein 2 like XM_540425 unknown Other 1.774 CENPB Centromere protein B, 80kda NM_001810 Nucleus Other 1.027 CEP170 Centrosomal protein 170kda XM_537218 Nucleus Other 1.239 CETN2 Centrin, EF hand protein, 2 NM_001038515 Nucleus Enzyme 1.679 CHD3 Chromodomain helicase DNA binding protein 3 NM_005852 Nucleus Enzyme 1.367 1.852 1.585 CHGB Chromogranin B (secretogranin 1) XM_534354 Extracellular Space Other 1.184 1.449 CHN1 (includes EG:1123) Chimerin (chimaerin) 1 XM_856276 Cytoplasm Other 1.943 2.319 CHN2 Chimerin (chimaerin) 2 NM_001045963 Cytoplasm Other 1.174 CHP Calcium binding protein P22 BT030715 Cytoplasm Transporter 1.276 CHP2 Calcineurin B homologous protein 2 XM_547089 Cytoplasm Other 1.921

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204 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation CHRNA4 Cholinergic receptor, nicotinic, alpha 4 XM_543097 Plasma Membrane Transmembrane receptor 2.705 CHRNB4 Cholinergic receptor, nicotinic, beta 4 NM_174517 Plasma Membrane Transmembrane receptor 1.908 CLASP2 Cytoplasmic linker associated protein 2 XM_609911 Cytoplasm Other 1.072 CLCN3 Chloride channel 3 XM_001154165 Plasma Membrane Ion channel 1.767 CLCN4 Chloride channel 4 NM_001830 Plasma Membrane Ion channel 1.249 CLIP2 CAP GLY domain containing linker protein 2 XM_583422 Cytoplasm Transcription regulator 1.084 1.056 1.116 CLIP4 CAP GLY domain containing linker protein family, member 4 XM_001162138 unknown Other 1.274 CLTB Clathrin, light chain (Lcb) XM_546220 Plasma Membrane Other 1.069 CMTM4 CKLF like MARVEL transmembrane domain containing 4 CR933620 Extracellular Space Cytokine 1.37 1.28 CNP 2',3' cyclic nucleotide 3' phosphodiesterase XM_511496 Cytoplasm Enzyme 1.785 CNTN1 Contactin 1 XM_001168019 Plasma Membrane Enzyme 1.716 1.624 CNTN2 Contactin 2 (axonal) X67734 Plasma Membrane Other 1.453 CNTN4 Contactin 4 AC026882 Plasma Membrane Enzyme 1.608 COBRA1 Cofactor of BRCA1 BC114764 Nucleus Other 1.374 1.247 COCH Coagulation factor C homolog, cochlin (Limulus polyphemus) XM_509886 Extracellular Space Other 2.996 COL13A1 Collagen, type XIII, alpha 1 XM_001170115 Plasma Membrane Other 1.322 2.04 COL5A1 Collagen, type V, alpha 1 NM_000093 Extracellular Space Other 1.01 COL5A2 Collagen, type V, alpha 2 AC064833 Extracellular Space Other 1.02 1.038 COL6A6 Collagen, type VI, alpha 6 NM_001102608 unknown Other 2.26 5.583 COPG2 Coatomer protein complex, subunit gamma 2 AC144863 Cytoplasm Transporter 2.578 2.591 1.047 CORO6 Coronin 6 XM_001137660 unknown Other 1.23 CORO7 Coronin 7 NM_001075903 Cytoplasm Other 1.715 1.899 COX6B1 Cytochrome c oxidase subunit Vib polypeptide 1 (ubiquitous) XM_850376 Cytoplasm Enzyme 1.349 COX7A2 Cytochrome c oxidase subunit viia polypeptide 2 (liver) XM_848778 Cytoplasm Enzyme 1.382 COX7C (includes EG:1350) Cytochrome c oxidase subunit viic AC108110 Cytoplasm Enzyme 1.447 1.366 CPEB1 Cytoplasmic polyadenylation element binding protein 1 AF329402 Cytoplasm Other 1.013 CRAT Carnitine acetyltransferase BT030711 Cytoplasm Enzyme 1.258 1.009 CREBL2 Camp responsive element binding protein like 2 XM_001153386 Nucleus Transcription regulator 1.359 CREG1 Cellular repressor of E1A stimulated genes 1 NM_001075942 Nucleus Transcription regulator 1.883 CRLF1 Cytokine receptor like factor 1 XM_588353 Extracellular Other 2.126 1.456

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205 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation Space CRTC1 CREB regulated transcription coactivator 1 XM_866768 Nucleus Transcription regulator 1.268 1.098 CRTC3 CREB regulated transcription coactivator 3 NM_173863 unknown Other 1.561 CRYZ Crystallin, zeta (quinone reductase) AK128794 Cytoplasm Enzyme 1.422 CSMD1 CUB and Sushi multiple domains 1 AC087367 Plasma Membrane Other 1.6 2.328 2.461 CSMD3 (includes EG:114788) CUB and Sushi multiple domains 3 AK095111 unknown Enzyme 1.334 CSNK1A1 Casein kinase 1, alpha 1 XM_862545 Cytoplasm Kinase 1.211 CSRP1 Cysteine and glycine rich protein 1 XM_843516 Nucleus Other 1.226 CTDSPL CTD (carboxy terminal domain, RNA polymerase II, polypeptide A) small phosphatase like XM_001170981 Nucleus Other 1.15 CTNNA3 Catenin (cadherin associated protein), alpha 3 AC018979 Plasma Membrane Other 1.221 CUX2 Cut like homeobox 2 BC151245 Nucleus Transcription regulator 2.455 CX3CL1 Chemokine (C X3 C motif) ligand 1 XM_544391 Extracellular Space Cytokine 1.015 CYB5B Cytochrome b5 type B (outer mitochondrial membrane) XM_582806 Cytoplasm Enzyme 2.232 1.636 1.143 CYFIP2 (includes EG:26999) Cytoplasmic FMR1 interacting protein 2 XM_597034 Cytoplasm Other 1.441 1.381 1.25 CYTSB Cytospin B AC004702 Nucleus Other 1.156 DAB1 Disabled homolog 1 (Drosophila) XM_847827 Cytoplasm Other 1.181 1.156 DAP3 Death associated protein 3 XM_580421 Cytoplasm Other 1.526 DARC Duffy blood group chemokine receptor XM_001170629 Plasma Membrane G protein coupled receptor 1.009 DCAF7 DDB1 and CUL4 associated factor 7 XM_511593 Cytoplasm Other 2.092 DCLK1 Doublecortin like kinase 1 NM_004734 Cytoplasm Kinase 1.902 3.317 DCN Decorin AK291309 Extracellular Space Other 1.224 1.917 3.633 DDAH1 Dimethylarginine dimethylaminohydrolase 1 NM_001102201 Cytoplasm Enzyme 2.404 DDO D aspartate oxidase BC103184 Cytoplasm Enzyme 1.003 DDX17 DEAD (Asp Glu Ala Asp) box polypeptide 17 XM_855709 Nucleus Enzyme 1.44 1.557 DEPDC6 (includes EG:64798) DEP domain containing 6 AC091563 unknown Other 1.699 1.14 DFFA DNA fragmentation factor, 45kda, alpha polypeptide NM_001075342 Nucleus Enzyme 1.811 DGKG Diacylglycerol kinase, gamma 90kda NM_001080745 Cytoplasm Kinase 1.447 1.309 1.244 DGKQ Diacylglycerol kinase, theta 110kda XM_872918 Cytoplasm Kinase 2.06 1.188 DHCR24 24 dehydrocholesterol reductase XM_001153810 Cytoplasm Enzyme 1.463 DIP2B DIP2 disco interacting protein 2 homolog B (Drosophila) NM_173602 unknown Other 1.847 DIRAS1 DIRAS family, GTP binding RAS like 1 XM_542186 Plasma Membrane Enzyme 1.889 1.41 1.262 DIRAS2 DIRAS family, GTP binding RAS like 2 XM_001142598 Plasma Membrane Enzyme 1.116 1.501 DIXDC1 DIX domain containing 1 NM_033425 unknown Other 1.909 1.299

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206 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation DLAT Dihydrolipoamide S acetyltransferase XM_590291 Cytoplasm Enzyme 1.106 DLC1 Deleted in liver cancer 1 XM_001139043 Cytoplasm Other 1.422 DLG3 Discs, large homolog 3 (Drosophila) NM_021120 Plasma Membrane Kinase 1.19 1.423 DLGAP1 Discs, large (Drosophila) homolog associated protein 1 XM_852594 Plasma Membrane Other 2.262 DLK1 Delta like 1 homolog (Drosophila) XM_547982 Extracellular Space Other 2.824 DMXL2 Dmx like 2 NM_015263 Cytoplasm Other 1.096 DNAH8 Dynein, axonemal, heavy chain 8 XM_001173791 Cytoplasm Enzyme 1.418 1.052 DNAJA4 Dnaj (Hsp40) homolog, subfamily A, member 4 XM_510526 Nucleus Other 1.42 1.668 DNAJB14 Dnaj (Hsp40) homolog, subfamily B, member 14 XM_001167856 unknown Enzyme 1.04 DNAJC10 Dnaj (Hsp40) homolog, subfamily C, member 10 XM_001159760 Cytoplasm Enzyme 1.308 DNAJC9 Dnaj (Hsp40) homolog, subfamily C, member 9 XM_588123 unknown Other 1.206 1.27 1.515 DNASE1L1 Deoxyribonuclease I like 1 XM_862570 Extracellular Space Enzyme 1.076 DNM3 Dynamin 3 NM_015569 Cytoplasm Enzyme 1.073 DOCK3 Dedicator of cytokinesis 3 XM_533813 Cytoplasm Other 1.078 1.671 1.025 DPEP3 Dipeptidase 3 XM_546868 unknown Peptidase 1.448 DPP6 Dipeptidyl peptidase 6 BC150304 Plasma Membrane Peptidase 1.502 1.325 DPT Dermatopontin XM_547476 Extracellular Space Other 2.949 2.518 DPYS Dihydropyrimidinase BC034395 Cytoplasm Enzyme 1.202 DPYSL2 Dihydropyrimidinase like 2 XM_519672 Cytoplasm Enzyme 1.717 1.062 DPYSL3 Dihydropyrimidinase like 3 NM_001387 Cytoplasm Enzyme 1.045 1.434 DRD5 Dopamine receptor D5 XM_604584 Plasma Membrane G protein coupled receptor 1.097 DSCAML1 Down syndrome cell adhesion molecule like 1 XM_508782 Plasma Membrane Other 1.199 DSPP Dentin sialophosphoprotein XM_544971 Extracellular Space Other 2.079 DTNA Dystrobrevin, alpha NM_001392 Plasma Membrane Other 1.35 DUSP3 Dual specificity phosphatase 3 BC151264 Cytoplasm Phosphatase 1.252 DUSP8 Dual specificity phosphatase 8 NM_004420 Nucleus Phosphatase 1.453 1.534 DUSP9 Dual specificity phosphatase 9 XM_549360 Nucleus Phosphatase 1.112 DYNLT3 Dynein, light chain, Tctex type 3 XM_001136823 Cytoplasm Other 1.069 DYRK2 Dual specificity tyrosine (Y) phosphorylation regulated kinase 2 XM_538273 Cytoplasm Kinase 1.065 1.139 DYSF Dysferlin, limb girdle muscular dystrophy 2B (autosomal recessive) NM_001102490 Plasma Membrane Other 1.322 EBF1 Early B cell factor 1 CU012046 Nucleus Transcription regulator 1.014 ECE2 Endothelin converting enzyme 2 NM_177956 Plasma Membrane Peptidase 1.443 EDIL3 EGF like repeats and discoidin I like domains 3 XM_001146613 Extracellular Other 1.12

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207 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation Space EDN1 Endothelin 1 AC154635 Extracellular Space Other 2.635 EDNRB Endothelin receptor type B XM_001141717 Plasma Membrane G protein coupled receptor 2.271 EEF1A2 Eukaryotic translation elongation factor 1 alpha 2 NM_001037464 Cytoplasm Translation regulator 1.356 1.323 1.533 EFCAB2 EF hand calcium binding domain 2 NM_001101247 unknown Other 1.19 EFHD1 EF hand domain family, member D1 NM_001109310 unknown Other 1.794 EFNA5 Ephrin A5 AK025909 Plasma Membrane Kinase 1.457 EGF Epidermal growth factor (beta urogastrone) NM_001003094 Extracellular Space Growth factor 1.06 EGLN3 Egl nine homolog 3 (C. Elegans) NM_001101164 Cytoplasm Enzyme 1.805 EGR4 Early growth response 4 XM_540228 Nucleus Transcription regulator 2.667 3.288 EHD3 EH domain containing 3 NM_014600 Cytoplasm Other 1.178 1.638 2.038 EIF2AK3 Eukaryotic translation initiation factor 2 alpha kinase 3 AF110146 Cytoplasm Kinase 1.16 ELOVL2 Elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast) like 2 XM_001175069 Cytoplasm Enzyme 1.877 1.846 ELOVL6 ELOVL family member 6, elongation of long chain fatty acids (FEN1/Elo2, SUR4/Elo3 like, yeast) XM_545023 Cytoplasm Enzyme 2.336 ELP3 (includes EG:55140) Elongation protein 3 homolog (S. Cerevisiae) NM_018091 Nucleus Enzyme 1.606 EMID1 EMI domain containing 1 AJ416090 Extracellular Space Other 2.515 ENAH Enabled homolog (Drosophila) NM_018212 Cytoplasm Other 1.983 1.337 ENC1 Ectodermal neural cortex (with BTB like domain) NM_001078067 Nucleus Peptidase 1.978 3.011 ENDOD1 Endonuclease domain containing 1 NM_001102519 Extracellular Space Enzyme 1.924 1.053 ENPEP Glutamyl aminopeptidase (aminopeptidase A) AC113992 Plasma Membrane Peptidase 2.44 ENPP7 Ectonucleotide pyrophosphatase/phosphodiesterase 7 XM_845707 unknown Enzyme 2.059 EPB41L3 Erythrocyte membrane protein band 4.1 like 3 XM_853634 Plasma Membrane Other 1.6 EPDR1 Ependymin related protein 1 (zebrafish) NM_001102288 Nucleus Other 1.674 EPHA4 EPH receptor A4 NM_004438 Plasma Membrane Kinase 1.129 EPHA7 EPH receptor A7 AL354857 Plasma Membrane Kinase 1.455 1.297 EPHB1 EPH receptor B1 AC109247 Plasma Membrane Kinase 1.482 EPM2A (includes EG:7957) Epilepsy, progressive myoclonus type 2A, Lafora disease (laforin) NM_001099709 Cytoplasm Phosphatase 1.149 EPN1 Epsin 1 AK022454 Plasma Membrane Other 1.124 ERMN Ermin, ERM like protein Other 1.138

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208 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation XM_001144090 Extracellular Space ETV5 Ets variant 5 NM_004454 Nucleus Transcription regulator 1.36 1.263 EVI5L Ecotropic viral integration site 5 like NM_145245 unknown Other 1.276 EVL Enah/Vasp like AK289720 Cytoplasm Other 2.224 EXOC2 Exocyst complex component 2 NM_018303 Cytoplasm Transporter 1.46 EXTL2 Exostoses (multiple) like 2 NM_001076224 Cytoplasm Enzyme 1.161 FA2H Fatty acid 2 hydroxylase XM_847921 unknown Enzyme 2.089 1.133 FAAH Fatty acid amide hydrolase XM_539627 Plasma Membrane Enzyme 1.022 1.287 FABP7 Fatty acid binding protein 7, brain XM_533484 Cytoplasm Transporter 1.647 FADS1 Fatty acid desaturase 1 NM_013402 Plasma Membrane Enzyme 1.027 FAF2 Fas associated factor family member 2 NM_014613 unknown Other 1.999 FAM117B Family with sequence similarity 117, member B XM_516038 unknown Other 1.004 FAM120C Family with sequence similarity 120C NM_017848 unknown Other 1.147 FAM135A Family with sequence similarity 135, member A XM_848166 unknown Enzyme 1.166 FAM135B Family with sequence similarity 135, member B AC103777 unknown Enzyme 1.06 FAM158A Family with sequence similarity 158, member A XM_586913 Plasma Membrane Other 1.257 FAM162A Family with sequence similarity 162, member A XM_526284 Cytoplasm Other 1.245 FAM168A Family with sequence similarity 168, member A XM_508631 unknown Other 1.408 FAM171A1 Family with sequence similarity 171, member A1 NM_001102180 unknown Other 1.924 1.712 FAM190B Family with sequence similarity 190, member B XM_001155519 unknown Other 1.157 1.113 FAM198B Family with sequence similarity 198, member B AK095474 Cytoplasm Other 1.14 FAM19A5 Family with sequence similarity 19 (chemokine (C C motif) like), member A5 Z83837 Extracellular Space Other 2.342 2.147 1.615 FAM5C Family with sequence similarity 5, member C XM_536117 Cytoplasm Other 1.373 FAM81A Family with sequence similarity 81, member A NM_152450 unknown Other 1.415 2.4 1.966 FANCM Fanconi anemia, complementation group M XM_537429 Nucleus Enzyme 1.674 FARP1 FERM, rhogef (ARHGEF) and pleckstrin domain protein 1 (chondrocyte derived) AF339817 unknown Other 1.778 1.011 1.698 FAT3 FAT tumor suppressor homolog 3 (Drosophila) NM_001008781 unknown Other 1.244 FBN2 (includes EG:2201) Fibrillin 2 NM_001999 Extracellular Space Other 1.283 FBXL16 F box and leucine rich repeat protein 16 XM_547211 unknown Other 1.637 FBXL17 F box and leucine rich repeat protein 17 BC018548 unknown Other 1.427 1.36 FBXO34 F box protein 34 XM_509963 unknown Other 1.456 FBXW7 F box and WD repeat domain containing 7 NM_018315 Nucleus Transcription regulator 2.149 FCGBP Fc fragment of igg binding protein NM_003890 Extracellular Other 1.656

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209 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation Space FCHO1 FCH domain only 1 AK291410 unknown Other 1.365 1.063 1.09 FDX1 Ferredoxin 1 XM_508877 Cytoplasm Transporter 1.339 FEZF1 FEZ family zinc finger 1 BC127714 unknown Other 2.104 FGD4 FYVE, rhogef and PH domain containing 4 XM_001135948 Cytoplasm Other 1.466 FGFR1 Fibroblast growth factor receptor 1 XM_001171010 Plasma Membrane Kinase 1.24 FGGY FGGY carbohydrate kinase domain containing AC093424 unknown Other 1.037 FHL1 Four and a half LIM domains 1 AK122708 Cytoplasm Other 1.341 FHOD3 (includes EG:80206) Formin homology 2 domain containing 3 XM_537280 unknown Other 1.134 FITM2 Fat storage inducing transmembrane protein 2 BC029662 unknown Other 1.216 FLJ10357 Hypothetical protein FLJ10357 XM_605753 unknown Other 1.261 FNDC3B Fibronectin type III domain containing 3B AC069259 unknown Other 1.37 FOXG1 Forkhead box G1 NM_005249 Nucleus Transcription regulator 3.475 5.43 FOXO1 Forkhead box O1 NM_002015 Nucleus Transcription regulator 1.012 FOXO4 Forkhead box O4 XM_529032 Nucleus Transcription regulator 1.942 1.496 FRMD5 FERM domain containing 5 AC090513 unknown Other 1.239 1.482 FRMPD3 FERM and PDZ domain containing 3 XM_937007 unknown Other 1.165 FRS2 Fibroblast growth factor receptor substrate 2 NM_001042555 Plasma Membrane Other 1.015 FSTL5 Follistatin like 5 XM_001147682 Extracellular Space Other 1.283 FUT9 Fucosyltransferase 9 (alpha (1,3) fucosyltransferase) NM_006581 Cytoplasm Enzyme 2.355 1.387 1.928 FYB FYN binding protein (FYB 120/130) NM_001465 Nucleus Other 1.493 FZD2 Frizzled homolog 2 (Drosophila) XM_580783 Plasma Membrane G protein coupled receptor 1.248 G3BP1 Gtpase activating protein (SH3 domain) binding protein 1 XM_001168937 Nucleus Enzyme 1.666 GAB1 GRB2 associated binding protein 1 AK022142 Cytoplasm Other 2.077 GABBR1 Gamma aminobutyric acid (GABA) B receptor, 1 BC149396 Plasma Membrane G protein coupled receptor 1.653 1.642 1.479 GABBR2 Gamma aminobutyric acid (GABA) B receptor, 2 XM_538749 Plasma Membrane G protein coupled receptor 2.458 1.167 2.305 GABRA1 Gamma aminobutyric acid (GABA) A receptor, alpha 1 XM_001145178 Plasma Membrane Ion channel 1.625 GABRA2 Gamma aminobutyric acid (GABA) A receptor, alpha 2 AC104072 Plasma Membrane Ion channel 1.807 GABRB1 Gamma aminobutyric acid (GABA) A receptor, beta 1 AC097712 Plasma Membrane Ion channel 1.985 2.339 1.995 GABRB2 Gamma aminobutyric acid (GABA) A receptor, beta 2 XM_518078 Plasma Membrane Ion channel 1.213 GABRG2 Gamma aminobutyric acid (GABA) A receptor, gamma 2 NM_198904 Plasma Membrane Ion channel 1.043 GALNTL6 (includes EG:442117) UDP N acetyl alpha D galactosamine:polypeptide N acetylgalactosaminyltransferase like 6 NM_001034845 unknown Other 1.05

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210 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation GARNL1 Ral gtpase activating protein, alpha subunit 1 (catalytic) XM_001140817 Cytoplasm Other 1.848 GBE1 Glucan (1,4 alpha ), branching enzyme 1 AC099049 Cytoplasm Enzyme 1.06 GBX2 Gastrulation brain homeobox 2 XM_543300 Nucleus Transcription regulator 1.602 GCNT4 (includes EG:51301) Glucosaminyl (N acetyl) transferase 4, core 2 (beta 1,6 N acetylglucosaminyltransferase) XM_546063 unknown Enzyme 1.239 GCSH Glycine cleavage system protein H (aminomethyl carrier) BC009065 Cytoplasm Enzyme 1.08 GDF6 Growth differentiation factor 6 XM_867875 Extracellular Space Growth factor 1.271 GFRA1 GDNF family receptor alpha 1 AC102457 Plasma Membrane Transmembrane receptor 2.657 1.53 GFRA2 GDNF family receptor alpha 2 XM_846385 Plasma Membrane Transmembrane receptor 1.235 1.738 2.787 GINS3 GINS complex subunit 3 (Psf3 homolog) XM_001152113 unknown Other 1.757 GJB6 Gap junction protein, beta 6, 30kda NM_001110219 Plasma Membrane Transporter 1.841 1.465 1.21 GLRA3 Glycine receptor, alpha 3 AC093868 Plasma Membrane Ion channel 2.423 GLS Glutaminase AC005540 Cytoplasm Enzyme 1.009 GNA11 Guanine nucleotide binding protein (G protein), alpha 11 (Gq class) NM_174322 Plasma Membrane Enzyme 1.216 GNAI1 Guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 1 BC105419 Plasma Membrane Enzyme 1.304 1.367 1.388 GNAO1 Guanine nucleotide binding protein (G protein), alpha activating activity polypeptide O AK056008 Plasma Membrane Enzyme 1.715 1.372 1.254 GNAS GNAS complex locus BC149250 Plasma Membrane Enzyme 1.751 GNB1 Guanine nucleotide binding protein (G protein), beta polypeptide 1 BC004186 Plasma Membrane Enzyme 1.638 GNPTAB N acetylglucosamine 1 phosphate transferase, alpha and beta subunits NM_024312 unknown Enzyme 1.252 GNS Glucosamine (N acetyl) 6 sulfatase NM_001075562 Cytoplasm Enzyme 1.292 GORASP1 Golgi reassembly stacking protein 1, 65kda XM_542713 Cytoplasm Other 1.954 GOSR1 Golgi SNAP receptor complex member 1 NM_001007025 Cytoplasm Transporter 1.219 GPC2 Glypican 2 XM_870336 Plasma Membrane Other 1.128 GPC5 Glypican 5 NM_001102070 Plasma Membrane Other 1.345 GPR12 G protein coupled receptor 12 XM_001157564 Plasma Membrane G protein coupled receptor 1.429 GPR126 G protein coupled receptor 126 XM_518772 Plasma Membrane G protein coupled receptor 1.017 GPR22 G protein coupled receptor 22 XM_843786 Plasma Membrane G protein coupled receptor 2.962 GPR98 G protein coupled receptor 98 AC034215 Plasma Membrane G protein coupled receptor 1.539 GPRASP1 G protein coupled receptor associated sorting protein 1 XM_538117 Cytoplasm Transporter 1.475 1.244

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211 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation GRASP GRP1 (general receptor for phosphoinositides 1) associated scaffold protein XM_583750 Plasma Membrane Other 1.999 1.645 GRIA1 Glutamate receptor, ionotropic, AMPA 1 AB209094 Plasma Membrane Ion channel 1.398 1.057 1.498 GRIA2 Glutamate receptor, ionotropic, AMPA 2 NM_000826 Plasma Membrane Ion channel 1.505 1.643 GRIA3 Glutamate receptor, ionotrophic, AMPA 3 NM_007325 Plasma Membrane Ion channel 2.33 2.719 GRIA4 Glutamate receptor, ionotrophic, AMPA 4 NM_000829 Plasma Membrane Ion channel 1.039 GRID2 Glutamate receptor, ionotropic, delta 2 AC022317 Plasma Membrane Ion channel 1.581 GRIK1 Glutamate receptor, ionotropic, kainate 1 XM_544843 Plasma Membrane Ion channel 1.098 1.933 2.626 GRIK2 Glutamate receptor, ionotropic, kainate 2 XM_866973 Plasma Membrane Ion channel 1.533 GRIN1 Glutamate receptor, ionotropic, N methyl D aspartate 1 AF015731 Plasma Membrane Ion channel 1.949 1.487 GRIN2A Glutamate receptor, ionotropic, N methyl D aspartate 2A NM_001034189 Plasma Membrane Ion channel 2.836 1.631 2.369 GRIN2B Glutamate receptor, ionotropic, N methyl D aspartate 2B AC007535 Plasma Membrane Ion channel 1.563 1.989 GRIN3A Glutamate receptor, ionotropic, N methyl D aspartate 3A XM_862276 Plasma Membrane Ion channel 1.032 GRIP1 Glutamate receptor interacting protein 1 XM_001162097 Plasma Membrane Other 1.23 GRLF1 Glucocorticoid receptor DNA binding factor 1 NM_004491 Nucleus Transcription regulator 1.153 GRM8 Glutamate receptor, metabotropic 8 AC079957 Plasma Membrane G protein coupled receptor 1.856 GSTT3 Glutathione S transferase, theta 3 XM_534750 unknown Enzyme 1.067 GYG2 Glycogenin 2 XM_548837 unknown Enzyme 1.267 HAP1 Huntingtin associated protein 1 XM_844535 Cytoplasm Other 1.373 HCN1 Hyperpolarization activated cyclic nucleotide gated potassium channel 1 NM_021072 Plasma Membrane Ion channel 1.318 2.128 HDLBP High density lipoprotein binding protein NM_005336 Nucleus Transporter 1.026 HECW2 HECT, C2 and WW domain containing E3 ubiquitin protein ligase 2 AC020571 unknown Enzyme 1.121 HERC1 Hect (homologous to the E6 AP (UBE3A) carboxyl terminus) domain and RCC1 (CHC1) like domain (RLD) 1 XM_001477062 Cytoplasm Other 1.862 HGSNAT Heparan alpha glucosaminide N acetyltransferase XM_519741 unknown Other 1.504 1.405 HISPPD2A Histidine acid phosphatase domain containing 2A AF502588 Nucleus Phosphatase 1.29 HIVEP2 Human immunodeficiency virus type I enhancer binding protein 2 XM_518773 Nucleus Transcription regulator 1.097 HMGB1 (includes EG:3146) High mobility group box 1 AK291494 Nucleus Other 1.432 HNRPDL Heterogeneous nuclear ribonucleoprotein D like BC105386 Nucleus Other 1.519 HOMER1 Homer homolog 1 (Drosophila) XM_001139767 Plasma Membrane Other 1.084 HOMER3 Homer homolog 3 (Drosophila) XM_541929 Plasma Membrane Other 1.718 1.134 HSPA12A Heat shock 70kda protein 12A NM_025015 unknown Other 1.821 1.305

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212 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation HTATSF1 HIV 1 Tat specific factor 1 EU176345 Nucleus Transcription regulator 1.341 HTR5B 5 hydroxytryptamine (serotonin) receptor 5B XM_601784 Plasma Membrane G protein coupled receptor 1.877 2.428 ICAM5 Intercellular adhesion molecule 5, telencephalin NM_003259 Plasma Membrane Other 4.261 ID4 Inhibitor of DNA binding 4, dominant negative helix loop helix protein XM_001170946 Nucleus Transcription regulator 1.647 IDS Iduronate 2 sulfatase NM_000202 Cytoplasm Enzyme 1.173 IGBP1 Immunoglobulin (CD79A) binding protein 1 XM_843517 Cytoplasm Phosphatase 1.027 IGF1R Insulin like growth factor 1 receptor NM_000875 Plasma Membrane Transmembrane receptor 1.173 IKBKB Inhibitor of kappa light polypeptide gene enhancer in B cells, kinase beta NM_174353 Cytoplasm Kinase 1.558 IL25 Interleukin 25 XM_605190 Extracellular Space Cytokine 1.135 IL31 Interleukin 31 BC132998 unknown Other 1.503 IL33 Interleukin 33 NM_001075297 Extracellular Space Cytokine 1.848 INA Internexin neuronal intermediate filament protein, alpha NM_001075958 Cytoplasm Other 1.367 INSR Insulin receptor XM_590552 Plasma Membrane Kinase 1.359 1.259 IPCEF1 Interaction protein for cytohesin exchange factors 1 XM_527543 Cytoplasm Enzyme 1.696 IPP Intracisternal A particle promoted polypeptide XM_001252535 Cytoplasm Other 2.089 IPPK Inositol 1,3,4,5,6 pentakisphosphate 2 kinase BC026154 Cytoplasm Kinase 1.6 IQCE IQ motif containing E XM_547007 unknown Other 1.219 IQSEC1 IQ motif and Sec7 domain 1 XM_516294 Cytoplasm Other 1.129 1.328 ITFG1 Integrin alpha FG GAP repeat containing 1 XM_846378 Plasma Membrane Other 1.022 ITGAM Integrin, alpha M (complement component 3 receptor 3 subunit) XM_510949 Plasma Membrane Other 2.852 1.789 ITGB5 Integrin, beta 5 XM_516706 Plasma Membrane Other 2.332 1.676 ITIH2 Inter alpha (globulin) inhibitor H2 XM_535195 Extracellular Space Other 1.605 ITPKA Inositol 1,4,5 trisphosphate 3 kinase A XM_544631 Cytoplasm Kinase 1.889 2.504 ITPR3 Inositol 1,4,5 triphosphate receptor, type 3 NM_174370 Cytoplasm Ion channel 1.639 JMY Junction mediating and regulatory protein, p53 cofactor NM_152405 Nucleus Transcription regulator 1.662 JPH4 Junctophilin 4 XM_547737 Cytoplasm Other 1.069 KAL1 Kallmann syndrome 1 sequence NM_000216 Extracellular Space Other 1.371 1.349 KBTBD7 Kelch repeat and BTB (POZ) domain containing 7 XM_522666 unknown Other 1.284 KCNH1 Potassium voltage gated channel, subfamily H (eag related), member 1 AC158791 Plasma Membrane Ion channel 1.576 KCNH4 Potassium voltage gated channel, subfamily H (eag related), member 4 XM_844412 Plasma Membrane Ion channel 1.552 1.097 KCNJ3 Potassium inwardly rectifying Plasma Ion channel 2.406 2.02

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213 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation channel, subfamily J, member 3 XM_001141809 Membrane KCNJ4 Potassium inwardly rectifying channel, subfamily J, member 4 XM_538374 Plasma Membrane Ion channel 1.962 2.195 KCNK1 Potassium channel, subfamily K, member 1 XM_525096 Plasma Membrane Ion channel 1.675 KCNK9 Potassium channel, subfamily K, member 9 XM_519977 Plasma Membrane Ion channel 1.535 KCNMA1 Potassium large conductance calcium activated channel, subfamily M, alpha member 1 U09383 Plasma Membrane Ion channel 1.102 1.832 KCNN1 Potassium intermediate/small conductance calcium activated channel, subfamily N, member 1 XM_541945 Plasma Membrane Ion channel 1.008 1.317 KCNQ3 Potassium voltage gated channel, KQT like subfamily, member 3 NM_031597 Plasma Membrane Ion channel 1.775 KCNS3 Potassium voltage gated channel, delayed rectifier, subfamily S, member 3 XM_001137103 Plasma Membrane Ion channel 1.547 3.49 KDM6A Lysine (K) specific demethylase 6A XM_548964 Nucleus Other 1.153 KIAA0319 Kiaa0319 NM_014809 unknown Other 1.877 KIAA0494 Kiaa0494 XM_524573 unknown Other 1.579 KIAA1045 Kiaa1045 NM_015297 unknown Other 1.745 KIAA1543 Kiaa1543 XM_869459 unknown Other 1.801 KIAA1671 Kiaa1671 XM_531255 unknown Other 1.229 KIDINS220 Kinase D interacting substrate, 220kda XM_532865 Nucleus Transcription regulator 1.676 1.505 1.016 KIF13B Kinesin family member 13B AC105979 Cytoplasm Other 1.633 KIF1A Kinesin family member 1A NM_004321 Cytoplasm Other 1.039 1.237 KIF1B Kinesin family member 1B NM_015074 Cytoplasm Transporter 1.322 1.254 KIF26A Kinesin family member 26A NM_015656 unknown Other 1.574 1.207 KIF26B Kinesin family member 26B NM_018012 unknown Other 1.065 1.014 KIF5A Kinesin family member 5A XM_531648 Cytoplasm Transporter 1.793 KIF5C Kinesin family member 5C AK126689 Cytoplasm Other 1.666 KIF5C Kinesin family member 5C AK126689 Cytoplasm Other 1.282 KIFC2 Kinesin family member C2 XM_532358 Cytoplasm Other 1.694 KLHDC5 Kelch domain containing 5 XM_520814 unknown Other 1.046 KLHL35 Kelch like 35 (Drosophila) BC132710 unknown Other 1.574 1.116 KRT72 Keratin 72 XM_522393 unknown Other 1.414 1.017 KSR2 Kinase suppressor of ras 2 NM_173598 Cytoplasm Kinase 1.602 LAMB3 Laminin, beta 3 NM_000228 Extracellular Space Transporter 1.07 LANCL1 Lanc lantibiotic synthetase component C like 1 (bacterial) NM_001076227 Plasma Membrane Other 1.406 LARP1 La ribonucleoprotein domain family, member 1 XM_582017 unknown Other 1.019 LCOR Ligand dependent nuclear receptor corepressor XM_584325 Nucleus Transcription regulator 1.23 LENG8 Leukocyte receptor cluster (LRC) member 8 NM_052925 unknown Other 1.299 LHX5 LIM homeobox 5 NM_001102061 Nucleus Transcription regulator 1.487 LIMCH1 LIM and calponin homology domains 1 CR936664 unknown Other 1.574 LMBR1 Limb region 1 homolog (mouse) NM_022458 Plasma Membrane Transmembrane receptor 1.04 1.047

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214 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation LMO3 LIM domain only 3 (rhombotin like 2) XM_520773 Nucleus Other 2.642 LMTK2 Lemur tyrosine kinase 2 XM_846103 unknown Kinase 1.011 LOC285696 Hypothetical LOC285696 AC026790 unknown Other 1.036 LOR Loricrin NM_000427 Cytoplasm Other 1.795 LOXL1 Lysyl oxidase like 1 BC126600 Extracellular Space Enzyme 2.189 LPAR1 Lysophosphatidic acid receptor 1 XM_001146627 Plasma Membrane G protein coupled receptor 1.117 LPAR3 Lysophosphatidic acid receptor 3 XM_612024 Plasma Membrane G protein coupled receptor 1.942 LPHN1 Latrophilin 1 XM_001254045 Plasma Membrane G protein coupled receptor 1.799 2.19 1.402 LPHN2 Latrophilin 2 AC113949 Plasma Membrane G protein coupled receptor 1.081 LPHN3 Latrophilin 3 BC039452 Plasma Membrane G protein coupled receptor 1.375 1.136 LRPPRC Leucine rich PPR motif containing XM_531800 Cytoplasm Other 1.242 LTBP3 Latent transforming growth factor beta binding protein 3 XM_611940 Extracellular Space Other 2.047 2.524 2.134 LUZP2 Leucine zipper protein 2 BC151234 unknown Other 2.011 MACF1 Microtubule actin crosslinking factor 1 XM_583217 Cytoplasm Other 2.19 MACROD1 MACRO domain containing 1 NM_001046509 Cytoplasm Other 1.489 1.547 1.158 MADD MAP kinase activating death domain XM_855777 Cytoplasm Other 1.092 1.507 MAL2 Mal, T cell differentiation protein 2 NM_001081719 Plasma Membrane Transporter 1.391 1.662 MAP1B Microtubule associated protein 1B XM_857079 Cytoplasm Other 1.636 1.067 MAP2 Microtubule associated protein 2 XM_001144480 Cytoplasm Other 1.233 1.369 MAP2K1 Mitogen activated protein kinase kinase 1 XM_612526 Cytoplasm Kinase 1.4 MAP2K6 Mitogen activated protein kinase kinase 6 AC002546 Cytoplasm Kinase 1.534 1.134 MAP3K5 (includes EG:4217) Mitogen activated protein kinase kinase kinase 5 XM_001171211 Cytoplasm Kinase 1.842 MAP4 Microtubule associated protein 4 BC051843 Cytoplasm Other 1.24 MAP4K3 Mitogen activated protein kinase kinase kinase kinase 3 AC007684 unknown Kinase 1.849 MAP4K4 Mitogen activated protein kinase kinase kinase kinase 4 XM_515665 Cytoplasm Kinase 1.041 MAP4K5 Mitogen activated protein kinase kinase kinase kinase 5 NM_198794 Cytoplasm Kinase 2.021 MAP7 Microtubule associated protein 7 NM_001101874 Cytoplasm Other 1.091 MAP7D1 MAP7 domain containing 1 NM_018067 unknown Other 1.006 MAPK1 Mitogen activated protein kinase 1 NM_002745 Cytoplasm Kinase 1.095 1.061 MAPK1IP1L Mitogen activated protein kinase 1 interacting protein 1 like XM_001148351 Nucleus Other 1.056 MAPK4 Mitogen activated protein kinase 4 NM_002747 Cytoplasm Kinase 1.941 1.391

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215 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation MAPK8IP1 Mitogen activated protein kinase 8 interacting protein 1 XM_540760 Cytoplasm Other 1.322 1.528 1.173 MAPRE2 Microtubule associated protein, RP/EB family, member 2 XM_512089 Cytoplasm Other 1.349 1.004 MATK Megakaryocyte associated tyrosine kinase XM_849722 Cytoplasm Kinase 1.207 2.265 MBOAT1 Membrane bound O acyltransferase domain containing 1 AL450321 unknown Other 1.178 MBP Myelin basic protein AK122594 Extracellular Space Other 1.927 MCAM Melanoma cell adhesion molecule AK291571 Plasma Membrane Other 1.919 MDGA1 MAM domain containing glycosylphosphatidylinositol anchor 1 XM_616613 Plasma Membrane Other 1.117 2.233 MDGA2 MAM domain containing glycosylphosphatidylinositol anchor 2 AL079306 unknown Other 1.739 MED16 Mediator complex subunit 16 XM_849586 Nucleus Transcription regulator 1.48 MEF2B Myocyte enhancer factor 2B NM_001103231 Nucleus Transcription regulator 2.336 MEGF10 Multiple EGF like domains 10 AK021631 unknown Other 1.34 1.139 MEIS2 Meis homeobox 2 NM_170674 Nucleus Transcription regulator 2.279 MELK Maternal embryonic leucine zipper kinase AL354932 Cytoplasm Kinase 1.049 MEOX2 Mesenchyme homeobox 2 NM_001098045 Nucleus Transcription regulator 1.714 2.945 METTL8 Methyltransferase like 8 XM_001142517 unknown Enzyme 1.23 MFHAS1 Malignant fibrous histiocytoma amplified sequence 1 AC090567 Cytoplasm Other 1.742 MGST2 Microsomal glutathione S transferase 2 NM_001076382 Cytoplasm Enzyme 1.811 MID1IP1 MID1 interacting protein 1 (gastrulation specific G12 homolog (zebrafish)) XM_001137047 Cytoplasm Other 1.515 MINK1 Misshapen like kinase 1 (zebrafish) NM_170663 Cytoplasm Kinase 1.204 MKX Mohawk homeobox NM_177595 unknown Other 2.207 MLL2 Myeloid/lymphoid or mixed lineage leukemia 2 XM_543684 Nucleus Transcription regulator 1.004 MMAA Methylmalonic aciduria (cobalamin deficiency) cbla type NM_001105112 Cytoplasm Other 1.33 MMD Monocyte to macrophage differentiation associated NM_012329 Plasma Membrane Other 1.911 2.287 MMP11 Matrix metallopeptidase 11 (stromelysin 3) XM_584877 Extracellular Space Peptidase 1.211 MMP28 Matrix metallopeptidase 28 NM_024302 Extracellular Space Peptidase 1.229 MOBKL1A MOB1, Mps One Binder kinase activator like 1A (yeast) XM_001159188 Cytoplasm Other 1.417 MOBP Myelin associated oligodendrocyte basic protein XM_845077 Cytoplasm Other 2.232 MPP2 Membrane protein, palmitoylated 2 (MAGUK p55 subfamily member 2) XM_867199 Plasma Membrane Kinase 1.029 1.439 1.138 MPRIP Myosin phosphatase Rho interacting protein XM_536669 Cytoplasm Other 1.983 1.545 MRC2 Mannose receptor, C type 2 XM_607489 Plasma Membrane Transmembrane receptor 3.218 2.436 2.226

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216 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation MTCH2 Mitochondrial carrier homolog 2 (C. Elegans) BC150080 Cytoplasm Other 1.623 MTM1 Myotubularin 1 BC030779 Cytoplasm Phosphatase 1.773 MTMR10 Myotubularin related protein 10 XM_867245 unknown Other 1.146 MTMR6 Myotubularin related protein 6 NM_004685 Cytoplasm Phosphatase 1.432 MTSS1L Metastasis suppressor 1 like XM_600126 unknown Other 1.22 MTUS1 Microtubule associated tumor suppressor 1 BC142971 unknown Other 1.392 MTUS2 Microtubule associated tumor suppressor candidate 2 NM_015233 unknown Other 1.172 MYBPC1 Myosin binding protein C, slow type XM_861573 Cytoplasm Other 1.849 MYCN V myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian) XM_540091 Nucleus Transcription regulator 1.166 1.441 1.682 MYH11 Myosin, heavy chain 11, smooth muscle XM_845671 Cytoplasm Other 1.213 1.542 MYL2 Myosin, light chain 2, regulatory, cardiac, slow DQ896055 Cytoplasm Other 1.441 MYLK Myosin light chain kinase NM_053027 Cytoplasm Kinase 1.224 MYO10 Myosin X XM_001175408 Cytoplasm Other 1.679 2.012 1.365 MYO6 Myosin VI XM_001145098 Cytoplasm Other 1.29 MYOCD Myocardin AL669846 Nucleus Transcription regulator 2.41 1.62 MYT1L Myelin transcription factor 1 like NM_001093776 Nucleus Transcription regulator 2.568 NAB1 NGFI A binding protein 1 (EGR1 binding protein 1) AC006460 Nucleus Transcription regulator 1.929 1.809 NAP1L5 Nucleosome assembly protein 1 like 5 XM_845030 unknown Other 2.09 NAPB N ethylmaleimide sensitive factor attachment protein, beta AK124876 Cytoplasm Transporter 1.12 1.318 NBEA (includes EG:26960) Neurobeachin XM_844120 Cytoplasm Other 1.321 NCALD Neurocalcin delta NM_001040630 Cytoplasm Other 2.335 1.635 NCAN Neurocan BC154393 Extracellular Space Other 1.586 2.117 1.601 NCKIPSD NCK interacting protein with SH3 domain XM_595101 Nucleus Other 1.177 1.164 NDFIP2 Nedd4 family interacting protein 2 XM_522688 Cytoplasm Other 1.312 2.57 NDST3 N deacetylase/N sulfotransferase (heparan glucosaminyl) 3 XM_001147455 Cytoplasm Enzyme 1.231 NDUFB5 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5, 16kda NM_002492 Cytoplasm Enzyme 1.964 NEBL Nebulette AL157398 Cytoplasm Other 2.473 NEDD4L Neural precursor cell expressed, developmentally down regulated 4 like XM_001140823 Cytoplasm Enzyme 1.112 1.83 NEFH Neurofilament, heavy polypeptide NM_001003352 Cytoplasm Other 2.788 1.045 NEFL Neurofilament, light polypeptide XM_534572 Cytoplasm Other 1.744 NEFM Neurofilament, medium polypeptide XM_543237 Cytoplasm Other 1.092 NEGR1 Neuronal growth regulator 1 XM_001167096 Extracellular Space Other 1.409 NELF Nasal embryonic LHRH factor XM_868817 Extracellular Other 1.805 1.816 1.999

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217 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation Space NETO1 Neuropilin (NRP) and tolloid (TLL) like 1 NM_138966 Extracellular Space Other 1.529 1.668 NFIA Nuclear factor I/A XM_536691 Nucleus Transcription regulator 2.799 1.243 NFIB Nuclear factor I/B XM_531936 Nucleus Transcription regulator 1.524 1.168 NFIX Nuclear factor I/X (CCAAT binding transcription factor) XM_862151 Nucleus Transcription regulator 1.043 1.16 1.198 NGFR Nerve growth factor receptor (TNFR superfamily, member 16) XM_511950 Plasma Membrane Transmembrane receptor 1.625 NGRN Neugrin, neurite outgrowth associated XM_001168693 unknown Other 1.416 NHEJ1 Nonhomologous end joining factor 1 XM_848099 Nucleus Other 1.338 NIPA1 Non imprinted in Prader Willi/Angelman syndrome 1 NM_144599 Plasma Membrane Other 1.666 1.112 NKAIN2 Na+/K+ transporting atpase interacting 2 AL109842 unknown Other 2.393 NKAIN3 Na+/K+ transporting atpase interacting 3 AC023095 unknown Other 1.271 1.272 NKX2 8 NK2 homeobox 8 XM_584660 Nucleus Transcription regulator 2.093 NLRP10 NLR family, pyrin domain containing 10 XM_848981 unknown Other 1.895 NLRP5 NLR family, pyrin domain containing 5 XM_533576 Cytoplasm Other 3.204 NMNAT3 (includes EG:349565) Nicotinamide nucleotide adenylyltransferase 3 XM_534286 Cytoplasm Enzyme 1.717 1.746 NMT2 N myristoyltransferase 2 NM_174456 Cytoplasm Enzyme 1.566 1.287 NNAT Neuronatin XM_530290 Plasma Membrane Transporter 1.941 NOV Nephroblastoma overexpressed gene NM_001102382 Extracellular Space Growth factor 1.495 3.622 NOVA2 Neuro oncological ventral antigen 2 XM_849950 Nucleus Other 1.792 2.137 NPAS3 Neuronal PAS domain protein 3 XM_509895 Nucleus Other 1.44 NPAS4 Neuronal PAS domain protein 4 XM_540832 Nucleus Transcription regulator 1.881 2.893 NPHS1 Nephrosis 1, congenital, Finnish type (nephrin) XM_597931 Plasma Membrane Other 1.704 1.269 2.549 NPL N acetylneuraminate pyruvate lyase (dihydrodipicolinate synthase) XM_001161861 unknown Enzyme 1.6 NPR1 Natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A) XM_612318 Plasma Membrane Enzyme 1.661 2.209 1.886 NPTX1 Neuronal pentraxin I NM_002522 Extracellular Space Other 2.131 4.515 NPY Neuropeptide Y NM_001014845 Extracellular Space Other 2.532 3.728 NR1D2 Nuclear receptor subfamily 1, group D, member 2 XM_857692 Nucleus Ligand dependent nuclear receptor 1.232 NR2F1 Nuclear receptor subfamily 2, group F, member 1 XM_852342 Nucleus Ligand dependent nuclear receptor 1.395 NRG3 Neuregulin 3 DQ857894 Extracellular Space Growth factor 1.069 1.582

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218 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation NRGN Neurogranin (protein kinase C substrate, RC3) NM_006176 Cytoplasm Other 1.731 3.458 NRIP3 Nuclear receptor interacting protein 3 NM_001102218 unknown Other 1.276 NRXN1 Neurexin 1 NM_174404 Plasma Membrane Transporter 1.387 2.591 1.66 NRXN3 Neurexin 3 XM_001165759 Plasma Membrane Transporter 1.128 1.279 NT5C1A 5' nucleotidase, cytosolic IA XM_580585 Cytoplasm Phosphatase 2.508 1.501 NTM Neurotrimin NM_017354 Plasma Membrane Other 1.137 1.218 NTN3 Netrin 3 XM_537003 Extracellular Space Other 1.243 1.624 NTN4 Netrin 4 XM_532655 Extracellular Space Other 1.041 NTNG2 Netrin G2 AB058760 Plasma Membrane Enzyme 2.318 1.377 NTRK2 Neurotrophic tyrosine kinase, receptor, type 2 AL445532 Plasma Membrane Kinase 1.354 1.559 NXT2 Nuclear transport factor 2 like export factor 2 NM_001100353 Nucleus Transporter 1.003 OBSCN (includes EG:84033) Obscurin, cytoskeletal calmodulin and titin interacting rhogef NM_001098623 Cytoplasm Kinase 2.173 1.772 OCIAD1 OCIA domain containing 1 AC079927 unknown Other 1.737 OGN Osteoglycin NM_008760 Extracellular Space Growth factor 2.024 1.648 OLFM1 Olfactomedin 1 BC008763 Cytoplasm Other 1.222 1.931 OPCML Opioid binding protein/cell adhesion molecule like AC154803 Plasma Membrane Transmembrane receptor 1.682 1.929 1.373 OPN3 Opsin 3 NM_014322 Plasma Membrane G protein coupled receptor 2.557 OSBPL3 Oxysterol binding protein like 3 AY008372 Cytoplasm Other 1.06 OTUD3 OTU domain containing 3 NM_015207 unknown Other 2.618 OXA1L Oxidase (cytochrome c) assembly 1 like XM_537362 Cytoplasm Enzyme 1.845 OXR1 Oxidation resistance 1 XM_539119 Cytoplasm Other 1.341 P2RY12 Purinergic receptor P2Y, G protein coupled, 12 NM_001003365 Plasma Membrane G protein coupled receptor 3.352 2.293 P2RY2 Purinergic receptor P2Y, G protein coupled, 2 XM_001174775 Plasma Membrane G protein coupled receptor 1.147 PADI2 Peptidyl arginine deiminase, type II NM_007365 Cytoplasm Enzyme 1.742 1.282 PAFAH1B1 Platelet activating factor acetylhydrolase, isoform Ib, subunit 1 (45kda) NM_174663 Cytoplasm Enzyme 1.015 PAK1 P21 protein (Cdc42/Rac) activated kinase 1 XM_844558 Cytoplasm Kinase 1.661 PANK1 Pantothenate kinase 1 XM_001143021 Cytoplasm Kinase 2.432 1.305 PBX1 Pre B cell leukemia homeobox 1 XM_001174513 Nucleus Transcription regulator 2.117 1.794 1.64 PCDH10 Protocadherin 10 AC105383 Plasma Membrane Other 1.482 PCDH11X Protocadherin 11 X linked AF332219 Plasma Membrane Other 1.567

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219 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation PCDH19 Protocadherin 19 XM_602429 unknown Other 1.52 PCDH20 Protocadherin 20 AL833830 unknown Other 1.153 PCDH7 Protocadherin 7 XM_001134665 Plasma Membrane Other 1.365 1.553 1.393 PCDH8 Protocadherin 8 XM_542588 Plasma Membrane Other 2.661 PCDH9 Protocadherin 9 AL160254 Plasma Membrane Other 2.885 2.025 PCDHA1 Protocadherin alpha 1 XM_843778 Plasma Membrane Other 1.075 1.124 1.359 PCDHA3 Protocadherin alpha 3 XM_843795 Plasma Membrane Other 1.272 1.362 1.593 PCDHAC2 Protocadherin alpha subfamily C, 2 XM_852697 Plasma Membrane Other 1.584 1.687 PCLO Piccolo (presynaptic cytomatrix protein) XM_001160582 Cytoplasm Transporter 1.339 1.355 PCM1 Pericentriolar material 1 NM_006197 Cytoplasm Other 1.416 PCNX Pecanex homolog (Drosophila) NM_014982 Plasma Membrane Other 1.302 1.071 1.128 PCSK1 Proprotein convertase subtilisin/kexin type 1 XM_001134900 Extracellular Space Peptidase 2.352 PCYT1B Phosphate cytidylyltransferase 1, choline, beta EU181262 Cytoplasm Enzyme 1.4 1.787 PDE10A Phosphodiesterase 10A XM_518849 Cytoplasm Enzyme 1.989 PDE1A Phosphodiesterase 1A, calmodulin dependent AL110263 Cytoplasm Enzyme 1.095 1.456 3.065 PDE4DIP Phosphodiesterase 4D interacting protein NM_014644 Cytoplasm Enzyme 1.322 PDXDC1 Pyridoxal dependent decarboxylase domain containing 1 NM_001101859 unknown Other 1.57 PEA15 Phosphoprotein enriched in astrocytes 15 AK095879 Cytoplasm Transporter 1.639 1.297 PERP PERP, TP53 apoptosis effector AK097958 Plasma Membrane Other 2.971 PFKFB2 6 phosphofructo 2 kinase/fructose 2,6 biphosphatase 2 BC147889 Cytoplasm Kinase 1.072 PFN2 Profilin 2 AK132651 Cytoplasm Other 1.042 1.153 PGM2L1 Phosphoglucomutase 2 like 1 XM_508639 unknown Enzyme 1.213 PGM5 Phosphoglucomutase 5 NM_001102335 Cytoplasm Enzyme 1.13 1.166 PHACTR2 Phosphatase and actin regulator 2 NM_014721 unknown Other 1.593 PHYHIPL Phytanoyl coa 2 hydroxylase interacting protein like XM_001164280 Cytoplasm Other 2.107 2.399 PIAS2 Protein inhibitor of activated STAT, 2 XM_612798 Nucleus Transcription regulator 1.014 PIK3IP1 Phosphoinositide 3 kinase interacting protein 1 XM_543490 unknown Other 2.794 1.663 PIK3R1 Phosphoinositide 3 kinase, regulatory subunit 1 (alpha) NM_181504 Cytoplasm Kinase 1.062 1.339 PIK3R2 Phosphoinositide 3 kinase, regulatory subunit 2 (beta) XM_847313 Cytoplasm Kinase 2.44 2.341 PIP4K2A Phosphatidylinositol 5 phosphate 4 kinase, type II, alpha XM_845439 Cytoplasm Kinase 1.595 PITPNM1 Phosphatidylinositol transfer protein, membrane associated 1 XM_846736 Cytoplasm Transporter 1.378 1.211 PKM2 Pyruvate kinase, muscle BT030503 Cytoplasm Kinase 1.005 PKP2 Plakophilin 2 NM_004572 Plasma Membrane Other 1.098 1.273 PLA2G12A Phospholipase A2, group XIIA CR591422 Extracellular Space Enzyme 1.523 1.233 1.13

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220 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation PLCB2 Phospholipase C, beta 2 XM_510305 Cytoplasm Enzyme 1.285 PLCL1 Phospholipase C like 1 XM_001169525 Cytoplasm Enzyme 2.849 PLEKHG3 Pleckstrin homology domain containing, family G (with rhogef domain) member 3 BC129953 unknown Other 1.488 PLEKHG3 Pleckstrin homology domain containing, family G (with rhogef domain) member 3 BC129953 unknown Other 1.085 PLEKHG5 Pleckstrin homology domain containing, family G (with rhogef domain) member 5 NM_001042665 Cytoplasm Other 1.939 PLK1 Polo like kinase 1 (Drosophila) XM_510879 Nucleus Kinase 1.285 PLK2 Polo like kinase 2 (Drosophila) XM_587229 Nucleus Kinase 1.33 2.329 PLSCR4 Phospholipid scramblase 4 AK128442 Plasma Membrane Enzyme 1.202 1.316 PLXNB1 Plexin B1 XM_533841 Plasma Membrane Transmembrane receptor 2.001 1.596 1.201 PLXNB3 Plexin B3 NM_005393 Plasma Membrane Other 1.395 PODXL2 Podocalyxin like 2 DQ202369 Plasma Membrane Other 1.109 1.014 1.46 POGK Pogo transposable element with KRAB domain NM_001099100 Nucleus Other 1.665 POLE Polymerase (DNA directed), epsilon XM_543348 Nucleus Enzyme 1.374 POLE3 Polymerase (DNA directed), epsilon 3 (p17 subunit) BT030577 Nucleus Enzyme 2.567 PPAP2B Phosphatidic acid phosphatase type 2B XM_536696 Plasma Membrane Phosphatase 2.486 2.111 1.577 PPFIA2 Protein tyrosine phosphatase, receptor type, f polypeptide (PTPRF), interacting protein (liprin), alpha 2 AB210009 Plasma Membrane Phosphatase 1.129 PPFIA3 (includes EG:8541) Protein tyrosine phosphatase, receptor type, f polypeptide (PTPRF), interacting protein (liprin), alpha 3 AK289757 Plasma Membrane Phosphatase 2.359 2.532 PPP1R12C Protein phosphatase 1, regulatory (inhibitor) subunit 12C BC010628 Cytoplasm Phosphatase 1.656 PPP1R14A Protein phosphatase 1, regulatory (inhibitor) subunit 14A XM_867134 Cytoplasm Other 2.083 PPP1R3C Protein phosphatase 1, regulatory (inhibitor) subunit 3C BT030698 Cytoplasm Phosphatase 2.27 1.746 1.686 PPP1R3F Protein phosphatase 1, regulatory (inhibitor) subunit 3F XM_548997 unknown Other 1.243 PPP1R9A Protein phosphatase 1, regulatory (inhibitor) subunit 9A XM_001169436 Cytoplasm Other 1.279 PPP2R2B Protein phosphatase 2 (formerly 2A), regulatory subunit B, beta isoform XM_001159292 Cytoplasm Phosphatase 2.348 PPP2R2C Protein phosphatase 2 (formerly 2A), regulatory subunit B, gamma isoform XM_001250700 unknown Phosphatase 1.243 1.378 1.446 PPP3CB Protein phosphatase 3 (formerly 2B), catalytic subunit, beta isoform XM_857434 unknown Phosphatase 1.636 PRDX2 Peroxiredoxin 2 AK289485 Cytoplasm Enzyme 1.059 PRICKLE2 Prickle homolog 2 (Drosophila) NM_198859 Nucleus Other 1.497 PRKAA2 Protein kinase, AMP activated, alpha 2 catalytic subunit NM_006252 Cytoplasm Kinase 1.591 1.394 PRKACB Protein kinase, camp dependent, catalytic, beta XM_862471 Cytoplasm Kinase 2.225 PRKAR2B Protein kinase, camp dependent, Cytoplasm Kinase 1.627

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221 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation regulatory, type II, beta XM_001148361 PRKCA Protein kinase C, alpha BC071767 Cytoplasm Kinase 1.002 1.513 PRKCE Protein kinase C, epsilon XM_583587 Cytoplasm Kinase 1.979 PRKG2 Protein kinase, cgmp dependent, type II NM_006259 Cytoplasm Kinase 1.047 PRKRA Protein kinase, interferon inducible double stranded RNA dependent activator AK290601 Cytoplasm Other 1.093 1.031 PRMT3 Protein arginine methyltransferase 3 NM_005788 Nucleus Enzyme 1.166 PROX1 Prospero homeobox 1 BX928753 Nucleus Transcription regulator 1.367 PRR5 Proline rich 5 (renal) NM_001101305 unknown Other 1.063 1.099 PRRT1 Proline rich transmembrane protein 1 NM_030651 unknown Other 1.024 1.364 PRRT2 (includes EG:112476) Proline rich transmembrane protein 2 NM_145239 unknown Other 1.559 2.515 PRUNE Prune homolog (Drosophila) BC142289 Nucleus Enzyme 1.759 PSAT1 Phosphoserine aminotransferase 1 NM_001102150 Cytoplasm Enzyme 3.321 1.734 PSD Pleckstrin and Sec7 domain containing XM_543989 unknown Other 2.275 2.953 PSD3 Pleckstrin and Sec7 domain containing 3 NM_015310 unknown Other 1.527 1.851 1.444 PTGS1 Prostaglandin endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase) XM_001136739 Cytoplasm Enzyme 2.728 1.491 PTK2B PTK2B protein tyrosine kinase 2 beta XM_543228 Cytoplasm Kinase 1.029 1.545 PTP4A1 Protein tyrosine phosphatase type IVA, member 1 NM_003463 Nucleus Phosphatase 1.484 PTPRD Protein tyrosine phosphatase, receptor type, D AL135790 Plasma Membrane Phosphatase 1.668 PTPRF Protein tyrosine phosphatase, receptor type, F NM_001101079 Plasma Membrane Phosphatase 1.019 1.611 2.088 PTPRG Protein tyrosine phosphatase, receptor type, G XM_001174413 Plasma Membrane Phosphatase 1.045 PTPRK Protein tyrosine phosphatase, receptor type, K XM_001167645 Plasma Membrane Phosphatase 1.074 1.07 PTPRN Protein tyrosine phosphatase, receptor type, N XM_536080 Plasma Membrane Phosphatase 1.123 1.582 PTPRN2 Protein tyrosine phosphatase, receptor type, N polypeptide 2 AC159625 Plasma Membrane Phosphatase 2.299 PTPRO Protein tyrosine phosphatase, receptor type, O NM_030667 Plasma Membrane Phosphatase 1.829 PTPRT Protein tyrosine phosphatase, receptor type, T XM_543002 Plasma Membrane Phosphatase 1.352 1.488 PVALB Parvalbumin NM_001076114 Cytoplasm Other 1.515 1.932 PVRL3 Poliovirus receptor related 3 AC133477 Plasma Membrane Other 1.281 PXMP3 Peroxisomal membrane protein 3, 35kda NM_001079867 Cytoplasm Other 1.621 QKI Quaking homolog, KH domain RNA binding (mouse) NM_206853 Nucleus Other 1.717 RAB4A RAB4A, member RAS oncogene family AY585832 Cytoplasm Enzyme 1.107 RAB5B RAB5B, member RAS oncogene family XM_866612 Cytoplasm Enzyme 1.903 RAB6B RAB6B, member RAS oncogene family XM_001147918 Cytoplasm Enzyme 1.869 1.013 1.345 RABEP1 Rabaptin, RAB gtpase binding XM_869715 Cytoplasm Transporter 2.06

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222 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation effector protein 1 RAI14 Retinoic acid induced 14 XM_001151240 Nucleus Transcription regulator 1.081 RALGPS1 Ral GEF with PH domain and SH3 binding motif 1 NM_014636 Cytoplasm Other 1.174 RANBP3L RAN binding protein 3 like XM_546345 unknown Other 1.594 RAP1GAP2 RAP1 gtpase activating protein 2 NM_001100398 unknown Other 1.16 1.45 1.834 RAP2A RAP2A, member of RAS oncogene family XM_509705 Cytoplasm Enzyme 1.03 RAPGEF5 Rap guanine nucleotide exchange factor (GEF) 5 NM_012294 Nucleus Other 1.285 RASAL1 RAS protein activator like 1 (GAP1 like) XM_590469 unknown Other 1.957 RASGRF1 Ras protein specific guanine nucleotide releasing factor 1 XM_510534 Cytoplasm Other 1.177 1.272 2.325 RASGRF2 Ras protein specific guanine nucleotide releasing factor 2 BC041953 Cytoplasm Other 1.341 RASGRP2 RAS guanyl releasing protein 2 (calcium and DAG regulated) XM_508531 Cytoplasm Other 1.236 RASL10A RAS like, family 10, member A XM_001173334 Nucleus Enzyme 3.098 5.486 RAVER2 Ribonucleoprotein, PTB binding 2 XM_546676 Nucleus Other 1.333 RBM9 RNA binding motif protein 9 NM_001082579 Nucleus Transcription regulator 1.303 RECK Reversion inducing cysteine rich protein with kazal motifs XM_001168411 Plasma Membrane Other 1.225 RECQL5 Recq protein like 5 XM_540436 Nucleus Enzyme 1.368 RELT RELT tumor necrosis factor receptor XM_542318 Plasma Membrane Transmembrane receptor 1.496 RERE Arginine glutamic acid dipeptide (RE) repeats XM_536734 Nucleus Transcription regulator 1.17 RFC1 Replication factor C (activator 1) 1, 145kda AY600371 Nucleus Transcription regulator 2.124 RFFL Ring finger and FYVE like domain containing 1 XM_867129 Cytoplasm Enzyme 1.233 RG9MTD3 RNA (guanine 9 ) methyltransferase domain containing 3 NM_001076859 unknown Other 1.279 RGNEF Rho guanine nucleotide exchange factor NM_012026 unknown Other 1.22 RGS12 Regulator of G protein signaling 12 XM_845461 Nucleus Other 1.489 1.676 2.104 RGS4 Regulator of G protein signaling 4 XM_001174415 Cytoplasm Other 1 RGS5 Regulator of G protein signaling 5 XM_001174428 Plasma Membrane Other 1.569 1.617 RHOBTB3 Rho related BTB domain containing 3 NM_014899 unknown Enzyme 1.27 1.054 RHOU Ras homolog gene family, member U DQ384425 Cytoplasm Enzyme 1.815 1.937 RICS Rho gtpase activating protein XM_546401 Cytoplasm Other 1.15 1.345 RIMKLB Ribosomal modification protein rimk like family member B BC015879 unknown Other 1.203 RIMS1 Regulating synaptic membrane exocytosis 1 NM_014989 Cytoplasm Enzyme 1.497 RIN2 Ras and Rab interactor 2 XM_843542 Cytoplasm Other 1.513 RLTPR RGD motif, leucine rich repeats, tropomodulin domain and proline rich containing BC115830 unknown Other 2.351 RND2 Rho family gtpase 2 XM_587874 Cytoplasm Enzyme 1.537 RNF112 Ring finger protein 112 XM_546649 Nucleus Transcription regulator 2.082 1.936

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223 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation RNF125 Ring finger protein 125 NM_017831 unknown Other 1.845 RNF14 Ring finger protein 14 NM_001081540 Nucleus Transcription regulator 1.112 RNF144A Ring finger protein 144A NM_014746 Nucleus Other 1.198 RNF165 Ring finger protein 165 XM_547590 unknown Other 1.702 1.622 RNF220 Ring finger protein 220 XM_845722 unknown Other 1.524 1.186 RNLS Renalase, FAD dependent amine oxidase AL353149 Extracellular Space Other 1.041 ROBO3 Roundabout, axon guidance receptor, homolog 3 (Drosophila) XM_546425 Plasma Membrane Other 1.068 ROPN1L Ropporin 1 like NM_001075717 unknown Kinase 1.473 RP11 307F22.3 Notch 5 like XM_001126083 unknown Other 2.403 RPH3A Rabphilin 3A homolog (mouse) BC150131 Plasma Membrane Transporter 1.354 2.134 RTN1 Reticulon 1 NM_001075966 Cytoplasm Other 1.152 RYK RYK receptor like tyrosine kinase NM_002958 Plasma Membrane Kinase 1.22 RYR2 Ryanodine receptor 2 (cardiac) XM_514296 Plasma Membrane Ion channel 2.312 3.405 RYR3 Ryanodine receptor 3 NM_001036 Plasma Membrane Ion channel 1.367 S100B S100 calcium binding protein B NM_009115 Cytoplasm Other 1.647 SAMD11 Sterile alpha motif domain containing 11 XM_536715 Nucleus Other 1.395 SAR1B SAR1 homolog B (S. Cerevisiae) XM_001167398 Cytoplasm Enzyme 1.405 1.184 SBF1 SET binding factor 1 NM_002972 Plasma Membrane Phosphatase 1.349 SBF2 SET binding factor 2 XM_534052 Cytoplasm Other 1.248 SC5DL Sterol C5 desaturase (ERG3 delta 5 desaturase homolog, S. Cerevisiae) like XM_001167254 Cytoplasm Enzyme 1.592 SCARA5 Scavenger receptor class A, member 5 (putative) XM_543223 Cytoplasm Other 1.807 SCARB2 Scavenger receptor class B, member 2 NM_001102153 Plasma Membrane Other 1.229 SCD Stearoyl coa desaturase (delta 9 desaturase) XM_543968 Cytoplasm Enzyme 1.392 SCD Stearoyl coa desaturase (delta 9 desaturase) XM_543968 Cytoplasm Enzyme 1.148 SCN2B Sodium channel, voltage gated, type II, beta XM_522196 Plasma Membrane Ion channel 1.206 SCN3B Sodium channel, voltage gated, type III, beta BC126265 Plasma Membrane Ion channel 1.389 2.12 SCN8A Sodium channel, voltage gated, type VIII, alpha subunit XM_845041 Plasma Membrane Ion channel 1.934 2.209 SCNN1D Sodium channel, nonvoltage gated 1, delta XM_546718 Plasma Membrane Ion channel 1.243 SCRIB Scribbled homolog (Drosophila) BC146321 Cytoplasm Other 1.129 SDHD Succinate dehydrogenase complex, subunit D, integral membrane protein XM_536573 Cytoplasm Enzyme 1.579 SEL1L Sel 1 suppressor of lin 12 like (C. Elegans) BC040498 Cytoplasm Other 1.254 SEMA3B Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3B XM_590757 Extracellular Space Other 1.167 SEMA3C Sema domain, immunoglobulin domain (Ig), short basic domain, XM_001159821 Extracellular Other 1.167

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224 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation secreted, (semaphorin) 3C Space SEMA6A Sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6A NM_020796 Plasma Membrane Other 1.135 SEMA6D Sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6D AC018900 Plasma Membrane Other 2.368 1.326 SENP5 (includes EG:205564) SUMO1/sentrin specific peptidase 5 NM_152699 unknown Peptidase 1.617 SEPT7 Septin 7 AK290545 Cytoplasm Other 1.239 1.19 1.07 SEPW1 Selenoprotein W, 1 NM_003009 Cytoplasm Enzyme 2.598 SERP1 Stress associated endoplasmic reticulum protein 1 XM_606511 Cytoplasm Other 1.714 SERTAD4 SERTA domain containing 4 AK021425 unknown Other 2.597 2.669 SESTD1 SEC14 and spectrin domains 1 BC061918 unknown Other 1.213 SEZ6 Seizure related 6 homolog (mouse) NM_001098635 unknown Other 1.353 SEZ6L Seizure related 6 homolog (mouse) like XM_515042 Plasma Membrane Other 2.079 1.91 SFXN5 Sideroflexin 5 NM_001075914 Cytoplasm Transporter 1.632 SGCB Sarcoglycan, beta (43kda dystrophin associated glycoprotein) NM_000232 Plasma Membrane Other 1.433 SGCD Sarcoglycan, delta (35kda dystrophin associated glycoprotein) XM_001134904 Cytoplasm Other 1.205 SGK1 Serum/glucocorticoid regulated kinase 1 AL135839 Cytoplasm Kinase 1.643 SGSM2 Small G protein signaling modulator 2 NM_014853 unknown Other 1.652 SGTB Small glutamine rich tetratricopeptide repeat (TPR) containing, beta XM_535258 unknown Other 1.313 1.655 SH3BP5 SH3 domain binding protein 5 (BTK associated) XM_542777 Cytoplasm Other 1.467 SH3GL2 SH3 domain GRB2 like 2 XM_848878 Plasma Membrane Enzyme 1.154 SH3GL3 SH3 domain GRB2 like 3 XM_847205 Cytoplasm Other 1.587 1.328 1.459 SH3GLB2 (includes EG:56904) SH3 domain GRB2 like endophilin B2 NM_020145 Cytoplasm Other 1.133 1.45 2.083 SHANK1 SH3 and multiple ankyrin repeat domains 1 AF102855 Cytoplasm Other 2.026 1.564 SHANK2 SH3 and multiple ankyrin repeat domains 2 XM_522093 Cytoplasm Other 1.379 SHB Src homology 2 domain containing adaptor protein B XM_538741 unknown Other 1.093 1.159 SHF Src homology 2 domain containing F XM_590296 unknown Other 1.219 SHROOM2 Shroom family member 2 NM_001649 Plasma Membrane Ion channel 2.02 1.381 SIN3B SIN3 homolog B, transcription regulator (yeast) XM_847635 Nucleus Transcription regulator 1.559 SIPA1L2 Signal induced proliferation associated 1 like 2 XM_867577 unknown Other 1.882 SLC12A5 Solute carrier family 12 (potassium chloride transporter), member 5 BC154376 Plasma Membrane Transporter 1.032 1.303 SLC16A12 Solute carrier family 16, member 12 (monocarboxylic acid transporter 12) XM_543918 unknown Other 1.678 SLC17A7 Solute carrier family 17 (sodium dependent inorganic phosphate cotransporter), member 7 NM_001098046 Plasma Membrane Transporter 1.598 2.652 8.147 SLC19A3 Solute carrier family 19, member 3 Plasma Transporter 1.365 1.436

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225 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation NM_001102198 Membrane SLC1A2 Solute carrier family 1 (glial high affinity glutamate transporter), member 2 NM_004171 Plasma Membrane Transporter 1.639 2.869 2.278 SLC24A2 Solute carrier family 24 (sodium/potassium/calcium exchanger), member 2 AL133281 Plasma Membrane Transporter 1.047 SLC25A23 Solute carrier family 25 (mitochondrial carrier; phosphate carrier), member 23 XM_542138 unknown Transporter 1.718 SLC25A36 Solute carrier family 25, member 36 XM_001159385 unknown Transporter 1.247 SLC25A46 Solute carrier family 25, member 46 AC008650 unknown Other 1.247 SLC25A5 Solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5 BC102950 Cytoplasm Transporter 1.274 SLC27A1 Solute carrier family 27 (fatty acid transporter), member 1 NM_001033625 Plasma Membrane Transporter 1.125 SLC2A12 Solute carrier family 2 (facilitated glucose transporter), member 12 XM_527510 unknown Transporter 1.736 SLC30A7 Solute carrier family 30 (zinc transporter), member 7 XM_001136030 Cytoplasm Transporter 1.311 1.098 SLC35F1 Solute carrier family 35, member F1 XM_527490 unknown Other 1.532 SLC38A2 Solute carrier family 38, member 2 XM_543722 Plasma Membrane Transporter 1.416 SLC39A10 Solute carrier family 39 (zinc transporter), member 10 XM_599261 unknown Transporter 1.197 SLC39A3 Solute carrier family 39 (zinc transporter), member 3 XM_849855 Plasma Membrane Transporter 1.728 1.39 SLC44A5 Solute carrier family 44, member 5 AC093156 unknown Other 2.683 1.762 SLC4A4 Solute carrier family 4, sodium bicarbonate cotransporter, member 4 NM_003759 Plasma Membrane Transporter 1.063 SLC6A1 Solute carrier family 6 (neurotransmitter transporter, GABA), member 1 XM_001152302 Plasma Membrane Transporter 1.367 1.04 1.192 SLC6A11 Solute carrier family 6 (neurotransmitter transporter, GABA), member 11 XM_533741 Plasma Membrane Transporter 1.122 SLC6A7 Solute carrier family 6 (neurotransmitter transporter, L proline), member 7 AK096607 Plasma Membrane Transporter 1.184 SLC6A9 Solute carrier family 6 (neurotransmitter transporter, glycine), member 9 NM_006934 Plasma Membrane Transporter 2.471 1.459 SLC7A11 Solute carrier family 7, (cationic amino acid transporter, y+ system) member 11 XM_001136486 Plasma Membrane Transporter 1.882 2.607 1.082 SLC8A2 Solute carrier family 8 (sodium/calcium exchanger), member 2 XM_615995 Cytoplasm Transporter 1.366 1.958 SLC9A9 (includes EG:285195) Solute carrier family 9 (sodium/hydrogen exchanger), member 9 XM_001162839 Cytoplasm Other 2.054 SLIT1 Slit homolog 1 (Drosophila) BC146761 Extracellular Space Other 1.656 3.085 4.752 SLIT3 Slit homolog 3 (Drosophila) AY358884 Extracellular Space Other 1.558 SLITRK4 SLIT and NTRK like family, member 4 XM_609417 unknown Other 2.469 1.725 SLITRK5 SLIT and NTRK like family, member 5 XM_542632 unknown Other 1.107 1.654

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226 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation SMAD4 SMAD family member 4 AC091551 Nucleus Transcription regulator 1.72 1.399 SMAD7 SMAD family member 7 XM_512124 Nucleus Transcription regulator 1.416 SMAD9 SMAD family member 9 XM_001144071 Nucleus Transcription regulator 1.053 SMARCAD1 SWI/SNF related, matrix associated actin dependent regulator of chromatin, subfamily a, containing DEAD/H box 1 NM_020159 Nucleus Enzyme 2.057 SNED1 Sushi, nidogen and EGF like domains 1 NM_001080437 Plasma Membrane Other 2.091 1.477 SNIP SNAP25 interacting protein XM_869703 Cytoplasm Other 1.497 SNIP1 Smad nuclear interacting protein 1 XM_532557 Nucleus Other 1.082 SNTG1 Syntrophin, gamma 1 XM_001066932 Nucleus Other 1.157 SNX30 Sorting nexin family member 30 NM_001012994 unknown Other 1.57 1.186 SNX6 Sorting nexin 6 AL445883 Cytoplasm Transporter 1.062 SNX9 (includes EG:51429) Sorting nexin 9 XM_582637 Cytoplasm Transporter 2.203 SOBP Sine oculis binding protein homolog (Drosophila) NM_001101170 unknown Other 1.544 1.551 SOCS7 Suppressor of cytokine signaling 7 AC124789 Cytoplasm Other 1.705 SORBS2 Sorbin and SH3 domain containing 2 AC108472 Nucleus Other 1.519 SOX10 SRY (sex determining region Y) box 10 DQ896471 Nucleus Transcription regulator 1.251 SOX6 SRY (sex determining region Y) box 6 AC068405 Nucleus Transcription regulator 1.351 SP4 Sp4 transcription factor XM_527679 Nucleus Transcription regulator 1.065 SPAG9 Sperm associated antigen 9 AC005920 Plasma Membrane Other 1.319 1.19 SPAM1 (includes EG:6677) Sperm adhesion molecule 1 (PH 20 hyaluronidase, zona pellucida binding) AC127559 Plasma Membrane Enzyme 2.199 SPEN Spen homolog, transcriptional regulator (Drosophila) XM_591419 Nucleus Transcription regulator 1.019 SPHKAP SPHK1 interactor, AKAP domain containing XM_001137938 unknown Other 1.692 SPOCK1 Sparc/osteonectin, cwcv and kazal like domains proteoglycan (testican) 1 XM_517947 Extracellular Space Other 1.111 SPON1 Spondin 1, extracellular matrix protein NM_174743 Extracellular Space Other 1.116 SPRYD3 SPRY domain containing 3 AK074694 unknown Other 1.392 SPTBN2 Spectrin, beta, non erythrocytic 2 XM_540827 Cytoplasm Other 2.253 2.573 SPTBN4 Spectrin, beta, non erythrocytic 4 XM_541613 Cytoplasm Other 1.656 1.955 2.401 SRF Serum response factor (c fos serum response element binding transcription factor) XM_847209 Nucleus Transcription regulator 1.177 SRY Sex determining region Y AC146189 Nucleus Transcription regulator 1.397 SS18L1 Synovial sarcoma translocation gene on chromosome 18 like 1 NM_001078095 Nucleus Transcription regulator 1.89 1.064 1.138 SSX2IP Synovial sarcoma, X breakpoint 2 interacting protein XM_001139311 Plasma Membrane Other 1.042 1.951 ST3GAL3 ST3 beta galactoside alpha 2,3 sialyltransferase 3 NM_001037299 Cytoplasm Enzyme 1.254

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227 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation ST6GAL2 ST6 beta galactosamide alpha 2,6 sialyltranferase 2 AK095049 Cytoplasm Enzyme 1.572 1.146 1.669 STAU2 Staufen, RNA binding protein, homolog 2 (Drosophila) XM_001165689 Cytoplasm Other 1.346 STIM1 Stromal interaction molecule 1 BC021300 Plasma Membrane Other 1.471 STMN1 Stathmin 1 XM_535349 Cytoplasm Other 1.944 STON2 Stonin 2 AK094799 Cytoplasm Other 1.631 1.239 STRA6 Stimulated by retinoic acid gene 6 homolog (mouse) BC142342 Plasma Membrane Other 1.78 STX2 Syntaxin 2 NM_001980 Cytoplasm Transporter 1.394 STXBP3 Syntaxin binding protein 3 NM_001083415 Plasma Membrane Transporter 1.081 STXBP5 Syntaxin binding protein 5 (tomosyn) BC113382 Plasma Membrane Other 1.241 STXBP6 Syntaxin binding protein 6 (amisyn) AL834346 Cytoplasm Other 2.01 SUB1 SUB1 homolog (S. Cerevisiae) NM_001105407 Nucleus Transcription regulator 3.347 2.235 2.353 SV2B Synaptic vesicle glycoprotein 2B AC123784 Plasma Membrane Transporter 2.218 SVEP1 Sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 XM_532030 unknown Other 1.282 SVOP SV2 related protein homolog (rat) BC033587 Cytoplasm Transporter 1.608 2.243 2.577 SYN1 Synapsin I BC149033 Plasma Membrane Transporter 1.824 SYN2 Synapsin II XM_001171832 Plasma Membrane Other 1.876 1.99 SYNC Syncoilin, intermediate filament protein XM_544429 Cytoplasm Other 1.294 SYNPR Synaptoporin XM_516566 Plasma Membrane Transporter 1.126 SYNRG Synergin, gamma XM_001173273 Cytoplasm Other 1.478 SYT11 Synaptotagmin XI NM_001099171 Cytoplasm Transporter 1.698 SYT13 Synaptotagmin XIII NM_001098115 unknown Transporter 1.613 1.25 SYT14 Synaptotagmin XIV AL513263 unknown Transporter 1.028 SYT9 Synaptotagmin IX XM_521824 Plasma Membrane Transporter 1.964 TADA1L Transcriptional adaptor 1 AK291922 unknown Other 1.309 TBC1D30 TBC1 domain family, member 30 XM_939476 Cytoplasm Other 1.293 TCEA2 Transcription elongation factor A (SII), 2 XM_001152936 Nucleus Transcription regulator 1.104 1.116 TCF4 Transcription factor 4 NM_003199 Nucleus Transcription regulator 1.122 1.255 TCF7L2 (includes EG:6934) Transcription factor 7 like 2 (T cell specific, HMG box) AL158212 Nucleus Transcription regulator 1.027 TEKT5 Tektin 5 XM_536976 unknown Other 1.154 TEX15 Testis expressed 15 NM_031271 unknown Other 1.731 TFRC Transferrin receptor (p90, CD71) XM_580860 Plasma Membrane Transporter 1.619 TGOLN2 (includes EG:10618) Trans golgi network protein 2 XM_001165520 Cytoplasm Other 1.354 TH Tyrosine hydroxylase BC149072 Cytoplasm Enzyme 2.857 THADA Thyroid adenoma associated AC092838 unknown Other 2.026 2.126 THRB Thyroid hormone receptor, beta (erythroblastic leukemia viral (v erb XM_001163770 Nucleus Ligand dependent 1.657 2.777

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228 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation a) oncogene homolog 2, avian) nuclear receptor TIPRL TIP41, TOR signaling pathway regulator like (S. Cerevisiae) NM_152902 unknown Other 1.029 TLE2 Transducin like enhancer of split 2 (E(sp1) homolog, Drosophila) NM_003260 Nucleus Transcription regulator 1.051 1.182 TM6SF1 Transmembrane 6 superfamily member 1 NM_001102295 Plasma Membrane Other 1.315 TMEM106C Transmembrane protein 106C XM_509025 unknown Other 1.397 TMEM116 Transmembrane protein 116 XM_509382 unknown Other 1.917 1.589 TMEM132A Transmembrane protein 132A XM_001142704 Cytoplasm Other 1.942 1.514 TMEM132C (includes EG:92293) Transmembrane protein 132C XM_522557 unknown Other 1.572 TMEM178 Transmembrane protein 178 AY358773 unknown Other 2.685 TMEM35 Transmembrane protein 35 BC122649 unknown Other 2.458 1.131 TMEM47 Transmembrane protein 47 NM_001003045 Plasma Membrane Other 1.427 TMEM8B Transmembrane protein 8B NM_001042589 Plasma Membrane Other 1.665 TMOD2 Tropomodulin 2 (neuronal) XM_001169950 Cytoplasm Other 1.26 1.542 1.483 TMX1 Thioredoxin related transmembrane protein 1 DQ786761 Cytoplasm Enzyme 1.343 TNFRSF17 Tumor necrosis factor receptor superfamily, member 17 BC058291 Plasma Membrane Other 1.274 TNIK TRAF2 and NCK interacting kinase XM_001164224 Cytoplasm Kinase 1.794 TNNC2 Troponin C type 2 (fast) AK291323 unknown Other 1.719 TNR Tenascin R (restrictin, janusin) Z94057 Plasma Membrane Other 1.864 1.734 1.426 TNRC6B Trinucleotide repeat containing 6B NM_015088 unknown Other 1.231 TNS1 Tensin 1 AC116419 Plasma Membrane Other 1.667 TOR1AIP1 Torsin A interacting protein 1 AL050126 Nucleus Other 1.013 TRAK1 Trafficking protein, kinesin binding 1 NM_001042646 Nucleus Other 1.216 1.274 TRAK2 Trafficking protein, kinesin binding 2 AB038964 Plasma Membrane Transporter 1.985 TRAPPC10 Trafficking protein particle complex 10 XM_544914 Cytoplasm Transporter 1.903 1.3 TRIM15 Tripartite motif containing 15 XM_591350 unknown Other 1.34 TRIM44 Tripartite motif containing 44 XM_508888 Cytoplasm Other 1.067 TRIM72 Tripartite motif containing 72 XM_547047 unknown Other 1.166 TRIM9 Tripartite motif containing 9 XM_001156808 Cytoplasm Other 1.251 TRPM3 Transient receptor potential cation channel, subfamily M, member 3 AL442645 Plasma Membrane Ion channel 1.229 1.814 TSHZ1 Teashirt zinc finger homeobox 1 XM_533368 Nucleus Transcription regulator 1.13 TSNARE1 T SNARE domain containing 1 XM_539185 unknown Other 1.362 TSPAN5 Tetraspanin 5 XM_001165628 Plasma Membrane Other 1.341 TTC13 Tetratricopeptide repeat domain 13 AL591292 unknown Other 1.299 TTC22 Tetratricopeptide repeat domain 22 NM_001098055 unknown Other 1.762 TTC9 Tetratricopeptide repeat domain 9 NM_015351 unknown Other 1.621 TTLL7 Tubulin tyrosine ligase like family, member 7 XM_001134864 Plasma Membrane Other 1.022 1.266

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229 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation TUBA1B Tubulin, alpha 1b BC146060 Cytoplasm Other 1.856 TULP3 Tubby like protein 3 NM_011657 unknown Other 1.195 TULP4 Tubby like protein 4 BC152476 Cytoplasm Transcription regulator 1.67 TUSC3 Tumor suppressor candidate 3 AC091559 Extracellular Space Enzyme 1.788 TYROBP TYRO protein tyrosine kinase binding protein XM_533687 Plasma Membrane Other 1.177 1.413 UBE4B Ubiquitination factor E4B (UFD2 homolog, yeast) XM_854670 Cytoplasm Enzyme 1.009 UBOX5 U box domain containing 5 XM_001160362 Nucleus Enzyme 1.206 UGT8 UDP glycosyltransferase 8 NM_001083635 Cytoplasm Enzyme 1.905 ULK2 Unc 51 like kinase 2 (C. Elegans) NM_014683 Cytoplasm Kinase 2.093 1.14 UNC119B Unc 119 homolog B (C. Elegans) XM_870226 unknown Other 1.593 UNC50 Unc 50 homolog (C. Elegans) BC103305 Cytoplasm Other 1.546 UNC5B Unc 5 homolog B (C. Elegans) XM_856306 Plasma Membrane Transmembrane receptor 1.511 UNG Uracil DNA glycosylase XM_543441 Nucleus Enzyme 1.301 1.062 UNQ1887 Signal peptide peptidase 3 XM_543427 Plasma Membrane Peptidase 1.315 USHBP1 Usher syndrome 1C binding protein 1 NM_001077137 unknown Other 1.583 USP31 Ubiquitin specific peptidase 31 NM_020718 unknown Peptidase 1.077 USP46 Ubiquitin specific peptidase 46 XM_001148401 unknown Peptidase 1.019 USP5 Ubiquitin specific peptidase 5 (isopeptidase T) NM_001098536 Cytoplasm Peptidase 1.152 1.021 VAMP1 Vesicle associated membrane protein 1 (synaptobrevin 1) NM_199245 Plasma Membrane Transporter 1.406 VCL Vinculin XM_507854 Plasma Membrane Enzyme 1.08 VDAC3 Voltage dependent anion channel 3 XM_001138480 Cytoplasm Ion channel 1.637 VHL Von Hippel Lindau tumor suppressor NM_001008552 Nucleus Other 1.063 VIP Vasoactive intestinal peptide NM_173970 Extracellular Space Other 2.367 6.831 VSTM2L V set and transmembrane domain containing 2 like NM_080607 unknown Other 2.516 1.966 WAC WW domain containing adaptor with coiled coil XM_611722 unknown Other 1.305 WAPAL Wings apart like homolog (Drosophila) XM_846909 Nucleus Other 1.021 WDR66 WD repeat domain 66 XM_854906 unknown Other 1.615 1.384 WIPF3 WAS/WASL interacting protein family, member 3 XM_001254241 Plasma Membrane Other 2.291 WNK2 WNK lysine deficient protein kinase 2 XM_582977 unknown Kinase 1.442 1.508 XIST X (inactive) specific transcript (non protein coding) AK054860 Nucleus Other 1.669 XYLT1 Xylosyltransferase I AC122836 Cytoplasm Enzyme 1.334 2.381 YY1 YY1 transcription factor XM_510162 Nucleus Transcription regulator 1.186 1.383 1.206 ZAK Sterile alpha motif and leucine zipper containing kinase AZK AF480462 Cytoplasm Kinase 1.618 1.052 ZCCHC24 Zinc finger, CCHC domain containing 24 XM_507867 unknown Other 1.097

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230 Table C 1. Continued Symbol Entrez g ene n ame GenBank Location Type(s) Fold change e xposure Fold change s urvival Fold change l ocation ZDHHC20 Zinc finger, DHHC type containing 20 XM_509571 unknown Other 1.399 ZEB1 Zinc finger E box binding homeobox 1 XM_615192 Nucleus Transcription regulator 1.093 ZEB2 Zinc finger E box binding homeobox 2 AY029472 Nucleus Transcription regulator 2.033 1.285 ZFP161 Zinc finger protein 161 homolog (mouse) XM_512037 Nucleus Other 1.234 ZFP57 Zinc finger protein 57 homolog (mouse) NM_001109809 Nucleus Transcription regulator 2.323 ZFYVE26 Zinc finger, FYVE domain containing 26 AK055455 unknown Other 1.093 ZIC1 Zic family member 1 (odd paired homolog, Drosophila) XM_516806 Nucleus Transcription regulator 2.372 ZIC5 Zic family member 5 (odd paired homolog, Drosophila) NM_033132 Nucleus Other 1.412 ZMIZ1 Zinc finger, MIZ type containing 1 NM_020338 Nucleus Other 1.105 ZMYM2 Zinc finger, MYM type 2 NM_003453 Nucleus Other 1.582 ZMYND8 Zinc finger, MYND type containing 8 XM_866938 Nucleus Transcription regulator 1.082 ZNF219 Zinc finger protein 219 XM_867319 Nucleus Transcription regulator 1.162 ZNF267 Zinc finger protein 267 AC165088 Nucleus Other 1.397 1.126 ZNF331 Zinc finger protein 331 NM_001103251 Nucleus Other 2.17 ZNF395 Zinc finger protein 395 NM_018660 Cytoplasm Other 2.826 ZNF398 Zinc finger protein 398 AK290499 Nucleus Transcription regulator 1.446 ZNF407 Zinc finger protein 407 XM_533370 Nucleus Other 1.084 ZNF451 Zinc finger protein 451 XM_518562 Nucleus Other 1.01 ZNF532 Zinc finger protein 532 XM_613386 unknown Other 1.099 ZNF594 Zinc finger protein 594 NM_032530 unknown Other 1.853 ZNF653 Zinc finger protein 653 XM_848511 unknown Other 1.163 ZNF664 Zinc finger protein 664 AK023009 Nucleus Other 1.331 ZNF827 Zinc finger protein 827 XM_862289 unknown Other 1.346 ZYG11B Zyg 11 homolog B (C. Elegans) XM_001139134 unknown Other 1.009 ZZEF1 Zinc finger, ZZ type with EF hand domain 1 AC067815 unknown Other 1.162

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231 LIST OF REFERENCES 1. [< http://www.aphis.usda.gov/vs/nahss/equine/wnv/wnv_distribution_maps.htm> ] 2. Centers for Disease Control and Prevention WNS: 2010. 3. Farajollahi A, Crans WJ, Bryant P, Wolf B, Burkhalter KL, Godsey MS, Aspen SE, Nasci RS: Detection of West Nile viral RNA from an overwintering pool of Culex pipens pipiens (Diptera: Culicidae) in New Jersey, 2003 J Med Entomol 2005, 42 (3):490 494. 4. Gi rard YA, Klingler KA, Higgs S: West Nile virus dissemination and tissue tropisms in orally infected Culex pipiens quinquefasciatus Vector Borne Zoonotic Dis 2004, 4 (2):109 122. 5. CDC: Outbreak of West Nile like viral encephalitis -New York, 1999 MMWR M orb Mortal Wkly Rep 1999, 48 (38):845 849. 6. Lanciotti RS, Roehrig JT, Deubel V, Smith J, Parker M, Steele K, Crise B, Volpe KE, Crabtree MB, Scherret JH et al : Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States Science 1999, 286 (5448):2333 2337. 7. Beasley DW, Whiteman MC, Zhang S, Huang CY, Schneider BS, Smith DR, Gromowski GD, Higgs S, Kinney RM, Barrett AD: Envelope protein glycosylation status influences mouse neuroinvasion phenotype of geneti c lineage 1 West Nile virus strains J Virol 2005, 79 (13):8339 8347. 8. Botha EM, Markotter W, Wolfaardt M, Paweska JT, Swanepoel R, Palacios G, Nel LH, Venter M: Genetic determinants of virulence in pathogenic lineage 2 West Nile virus strains Emerg Inf ect Dis 2008, 14 (2):222 230. 9. Brault AC, Powers AM, Holmes EC, Woelk CH, Weaver SC: Positively charged amino acid substitutions in the e2 envelope glycoprotein are associated with the emergence of venezuelan equine encephalitis virus J Virol 2002, 76 (4 ):1718 1730. 10. Long MT: Equine Infectious Diseases In: Equine Infectious Diseases. Edited by Sellon DCL, M.T. St. Louis, MO: Elsevier; 2007: 198 206. 11. Nasci RS, Savage HM, White DJ, Miller JR, Cropp BC, Godsey MS, Kerst AJ, Bennett P, Gottfried K, Lanciotti RS: West Nile virus in overwintering Culex mosquitoes, New York City, 2000 Emerg Infect Dis 2001, 7 (4):742 744.

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232 12. Reisen W, Brault AC: West Nile virus in North America: perspectives on epidemiology and intervention Pest Manag Sci 2007, 63 (7):641 646. 13. Sardelis MR, Turell MJ, Dohm DJ, O'Guinn ML: Vector competence of selected North American Culex and Coquillettidia mosquitoes for West Nile virus Emerg Infect Dis 2001, 7 (6):1018 1022. 14. Turell MJ, Dohm DJ, Sardelis MR, Oguinn ML, And readis TG, Blow JA: An update on the potential of north American mosquitoes (Diptera: Culicidae) to transmit West Nile Virus J Med Entomol 2005, 42 (1):57 62. 15. Turell MJ, O'Guinn ML, Dohm DJ, Jones JW: Vector competence of North American mosquitoes (Diptera: Culicidae) for West Nile virus J Med Entomol 2001, 38 (2):130 134. 16. Turell MJ, Sardelis MR, Dohm DJ, O'Guinn ML: Potential North American vectors of West Nile virus Ann N Y Acad Sci 2001, 951 :317 324. 17. Tesh RB, Parsons R, Siirin M, Randl e Y, Sargent C, Guzman H, Wuithiranyagool T, Higgs S, Vanlandingham DL, Bala AA et al : Year round West Nile virus activity, Gulf Coast region, Texas and Louisiana Emerg Infect Dis 2004, 10 (9):1649 1652. 18. Reisen WK, Fang Y, Martinez VM: Avian host and mosquito (Diptera: Culicidae) vector competence determine the efficiency of West Nile and St. Louis encephalitis virus transmission J Med Entomol 2005, 42 (3):367 375. 19. Savage HM, Aggarwal D, Apperson CS, Katholi CR, Gordon E, Hassan HK, Anderson M, Ch arnetzky D, McMillen L, Unnasch EA et al : Host choice and West Nile virus infection rates in blood fed mosquitoes, including members of the Culex pipiens complex, from Memphis and Shelby County, Tennessee, 2002 2003 Vector Borne Zoonotic Dis 2007, 7 (3):36 5 386. 20. Sardelis MR, Turell MJ, O'Guinn ML, Andre RG, Roberts DR: Vector competence of three North American strains of Aedes albopictus for West Nile virus J Am Mosq Control Assoc 2002, 18 (4):284 289. 21. Goddard LB, Roth AE, Reisen WK, Scott TW: Vertical transmission of West Nile Virus by three California Culex (Diptera: Culicidae) species J Med Entomol 2003, 40 (6):743 746. 22. Hayes EB, Komar N, Nasci RS, Montgomery SP, O'Leary DR, Campbell GL: Epidemiology and transmission dynamics of West Nil e virus disease Emerg Infect Dis 2005, 11 (8):1167 1173.

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233 23. Weingartl HM, Neufeld JL, Copps J, Marszal P: Experimental West Nile virus infection in blue jays (Cyanocitta cristata) and crows (Corvus brachyrhynchos) Vet Pathol 2004, 41 (4):362 370. 24. Ko mar N, Langevin S, Hinten S, Nemeth N, Edwards E, Hettler D, Davis B, Bowen R, Bunning M: Experimental infection of North American birds with the New York 1999 strain of West Nile virus Emerg Infect Dis 2003, 9 (3):311 322. 25. Kilpatrick AM, Daszak P, Jo nes MJ, Marra PP, Kramer LD: Host heterogeneity dominates West Nile virus transmission Proc Biol Sci 2006, 273 (1599):2327 2333. 26. McLean RG, Francy DB, Campos EG: Experimental studies of St. Louis encephalitis virus in vertebrates J Wildl Dis 1985, 21 (2):85 93. 27. McLean RG, Kirk LJ, Shriner RB, Townsend M: Avian hosts of St. Louis encephalitis virus in Pine Bluff, Arkansas, 1991 Am J Trop Med Hyg 1993, 49 (1):46 52. 28. Austgen LE, Bowen RA, Bunning ML, Davis BS, Mitchell CJ, Chang GJ: Experimental infection of cats and dogs with West Nile virus Emerg Infect Dis 2004, 10 (1):82 86. 29. Davis A, Bunning M, Gordy P, Panella N, Blitvich B, Bowen R: Experimental and natural infection of North American bats with West Nile virus Am J Trop Med Hyg 2005, 73 (2):467 469. 30. Teehee ML, Bunning ML, Stevens S, Bowen RA: Experimental infection of pigs with West Nile virus Arch Virol 2005, 150 (6):1249 1256. 31. Platt KB, Tucker BJ, Halbur PG, Blitvich BJ, Fabiosa FG, Mullin K, Parikh GR, Kitikoon P, Bartholomay LC, Rowley WA: Fox Squirrels (Sciurus niger) Develop West Nile Virus Viremias Sufficient for Infecting Select Mosquito Species Vector Borne Zoonotic Dis 2008. 32. Platt KB, Tucker BJ, Halbur PG, Tiawsirisup S, Blitvich BJ, Fabiosa FG, Bartholomay LC, Rowley WA: West Nile virus viremia in eastern chipmunks (Tamias striatus) sufficient for infecting different mosquitoes Emerg Infect Dis 2007, 13 (6):831 837 33. Klenk K, Snow J, Morgan K, Bowen R, Stephens M, Foster F, Gordy P, Beckett S, Komar N, Gubler D et al : Alligators as West Nile virus amplifiers Emerg Infect Dis 2004, 10 (12):2150 2155.

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234 34. Jacobson ER, Ginn PE, Troutman JM, Farina L, Stark L, Klen k K, Burkhalter KL, Komar N: West Nile virus infection in farmed American alligators (Alligator mississippiensis) in Florida J Wildl Dis 2005, 41 (1):96 106. 35. Beasley DW, Davis CT, Whiteman M, Granwehr B, Kinney RM, Barrett AD: Molecular determinants o f virulence of West Nile virus in North America Arch Virol Suppl 2004(18):35 41. 36. Chambers TJ, Halevy M, Nestorowicz A, Rice CM, Lustig S: West Nile virus envelope proteins: nucleotide sequence analysis of strains differing in mouse neuroinvasiveness J Gen Virol 1998, 79 ( Pt 10) :2375 2380. 37. Beasley DW, Davis CT, Estrada Franco J, Navarro Lopez R, Campomanes Cortes A, Tesh RB, Weaver SC, Barrett AD: Genome sequence and attenuating mutations in West Nile virus isolate from Mexico Emerg Infect Dis 2004, 10 (12):2221 2224. 38. Beasley DW, Davis CT, Guzman H, Vanlandingham DL, Travassos da Rosa AP, Parsons RE, Higgs S, Tesh RB, Barrett AD: Limited evolution of West Nile virus has occurred during its southwesterly spread in the United States Virology 2003, 309 (2):190 195. 39. Davis CT, Li L, May FJ, Bueno R, Jr., Dennett JA, Bala AA, Guzman H, Quiroga Elizondo D, Tesh RB, Barrett AD: Genetic stasis of dominant West Nile virus genotype, Houston, Texas Emerg Infect Dis 2007, 13 (4):601 604. 40. Davis CT, Beasley DW, Guzman H, Siirin M, Parsons RE, Tesh RB, Barrett AD: Emergence of attenuated West Nile virus variants in Texas, 2003 Virology 2004, 330 (1):342 350. 41. Beasley DW: Recent advances in the molecular biology of west nile virus Curr Mo l Med 2005, 5 (8):835 850. 42. Yuan F, Lou Z, Li X, Chen YW, Bell JI, Rao Z, Gao GF: Refolding, crystallization and preliminary X ray structural studies of the West Nile virus envelope (E) protein domain III Acta Crystallogr Sect F Struct Biol Cryst Commu n 2005, 61 (Pt 4):421 423. 43. Zhou H, Singh NJ, Kim KS: Homology modeling and molecular dynamics study of West Nile virus NS3 protease: a molecular basis for the catalytic activity increased by the NS2B cofactor Proteins 2006, 65 (3):692 701. 44. Chernov AV, Shiryaev SA, Aleshin AE, Ratnikov BI, Smith JW, Liddington RC, Strongin AY: The two component NS2B NS3 proteinase represses DNA unwinding activity of the West Nile virus NS3 helicase J Biol Chem 2008, 283 (25):17270 17278.

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235 45. Tilgner M, Shi PY : Structure and function of the 3' terminal six nucleotides of the west nile virus genome in viral replication J Virol 2004, 78 (15):8159 8171. 46. Li W, Brinton MA: The 3' stem loop of the West Nile virus genomic RNA can suppress translation of chimeric mRNAs Virology 2001, 287 (1):49 61. 47. Shiryaev SA, Chernov AV, Aleshin AE, Shiryaeva TN, Strongin AY: NS4A regulates the ATPase activity of the NS3 helicase: a novel cofactor role of the non structural protein NS4A from West Nile virus J Gen Virol 2009 90 (Pt 9):2081 2085. 48. Yu L, Nomaguchi M, Padmanabhan R, Markoff L: Specific requirements for elements of the 5' and 3' terminal regions in flavivirus RNA synthesis and viral replication Virology 2008, 374 (1):170 185. 49. Li XF, Jiang T, Yu XD, Deng YQ, Zhao H, Zhu QY, Qin ED, Qin CF: RNA elements within the 5' UTR of West Nile virus genome are critical for RNA synthesis and viral replication J Gen Virol 2009. 50. Davis WG, Blackwell JL, Shi PY, Brinton MA: Interaction be tween the cellular protein eEF1A and the 3' terminal stem loop of West Nile virus genomic RNA facilitates viral minus strand RNA synthesis J Virol 2007, 81 (18):10172 10187. 51. Ray D, Shah A, Tilgner M, Guo Y, Zhao Y, Dong H, Deas TS, Zhou Y, Li H, Shi P Y: West Nile virus 5' cap structure is formed by sequential guanine N 7 and ribose 2' O methylations by nonstructural protein 5 J Virol 2006, 80 (17):8362 8370. 52. Leung JY, Pijlman GP, Kondratieva N, Hyde J, Mackenzie JM, Khromykh AA: Role of nonstructu ral protein NS2A in flavivirus assembly J Virol 2008, 82 (10):4731 4741. 53. Brinton MA: The molecular biology of West Nile Virus: a new invader of the western hemisphere Annu Rev Microbiol 2002, 56 :371 402. 54. Bunning ML, Bowen RA, Cropp CB, Sullivan KG, Davis BS, Komar N, Godsey MS, Baker D, Hettler DL, Holmes DA et al : Experimental infection of horses with West Nile virus Emerg Infect Dis 2002, 8 (4):380 386. 55. Nemeth N, Gould D, Bowen R, Komar N: Natural and experimental West Nile virus infection in five raptor species J Wildl Dis 2006, 42 (1):1 13.

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236 56. Tesh RB, Siirin M, Guzman H, Travassos da Rosa AP, Wu X, Duan T, Lei H, Nunes MR, Xiao SY: Persistent West Nile virus infection in the golden hamster: studies on its mechanism and possible implic ations for other flavivirus infections J Infect Dis 2005, 192 (2):287 295. 57. Styer LM, Bernard KA, Kramer LD: Enhanced early West Nile virus infection in young chickens infected by mosquito bite: effect of viral dose Am J Trop Med Hyg 2006, 75 (2):337 345. 58. Long MT, Gibbs EP, Mellencamp MW, Bowen RA, Seino KK, Zhang S, Beachboard SE, Humphrey PP: Efficacy, duration, and onset of immunogenicity of a West Nile virus vaccine, live Flavivirus chimera, in horses with a clinical disease challenge model Equine Vet J 2007, 39 (6):491 497. 59. Seino KK, Long, M.T., Gibbs, E.P.J., Abbott, J.R., Beachboard, S.E., Bourgeois, M.A., Fanta, D.K.: Protective Immune Responses to West Nile Virus (WNV) of 28 Day Vaccinated Horses In ; 2008. 60. Osorio JE, Go dsey MS, Defoliart GR, Yuill TM: La Crosse viremias in white tailed deer and chipmunks exposed by injection or mosquito bite Am J Trop Med Hyg 1996, 54 (4):338 342. 61. Nemeth NM, Bowen RA: Dynamics of passive immunity to West Nile virus in domestic chick ens (Gallus gallus domesticus) Am J Trop Med Hyg 2007, 76 (2):310 317. 62. Phipps LP, Gough RE, Ceeraz V, Cox WJ, Brown IH: Detection of West Nile virus in the tissues of specific pathogen free chickens and serological response to laboratory infection: a comparative study Avian Pathol 2007, 36 (4):301 305. 63. Joyner PH, Kelly S, Shreve AA, Snead SE, Sleeman JM, Pettit DA: West Nile virus in raptors from Virginia during 2003: clinical, diagnostic, and epidemiologic findings J Wildl Dis 2006, 42 (2):335 34 4. 64. Mashimo T, Lucas M, Simon Chazottes D, Frenkiel MP, Montagutelli X, Ceccaldi PE, Deubel V, Guenet JL, Despres P: A nonsense mutation in the gene encoding 2' 5' oligoadenylate synthetase/L1 isoform is associated with West Nile virus susceptibility i n laboratory mice Proc Natl Acad Sci U S A 2002, 99 (17):11311 11316. 65. Lucas M, Mashimo T, Frenkiel MP, Simon Chazottes D, Montagutelli X, Ceccaldi PE, Guenet JL, Despres P: Infection of mouse neurones by West Nile virus is modulated by the interferon inducible 2' 5' oligoadenylate synthetase 1b protein Immunol Cell Biol 2003, 81 (3):230 236.

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237 66. Perelygin AA, Scherbik SV, Zhulin IB, Stockman BM, Li Y, Brinton MA: Positional cloning of the murine flavivirus resistance gene Proc Natl Acad Sci U S A 2002 99 (14):9322 9327. 67. Bunning ML, Bowen RA, Cropp B, Sullivan K, Davis B, Komar N, Godsey M, Baker D, Hettler D, Holmes D et al : Experimental infection of horses with West Nile virus and their potential to infect mosquitoes and serve as amplifying hosts Ann N Y Acad Sci 2001, 951 :338 339. 68. Minke JM, Siger L, Karaca K, Austgen L, Gordy P, Bowen R, Renshaw RW, Loosmore S, Audonnet JC, Nordgren B: Recombinant canarypoxvirus vaccine carrying the prM/E genes of West Nile virus protects horses against a W est Nile virus mosquito challenge Arch Virol Suppl 2004(18):221 230. 69. Snook CS, Hyman SS, Del Piero F, Palmer JE, Ostlund EN, Barr BS, Desrochers AM, Reilly LK: West Nile virus encephalomyelitis in eight horses J Am Vet Med Assoc 2001, 218 (10):1576 1 579. 70. Porter MB, Long MT, Getman LM, Giguere S, MacKay RJ, Lester GD, Alleman AR, Wamsley HL, Franklin RP, Jacks S et al : West Nile virus encephalomyelitis in horses: 46 cases (2001) J Am Vet Med Assoc 2003, 222 (9):1241 1247. 71. IG M: Large Animal N eurology : In: Lea & Febiger; 1989. 72. Hayes EB, O'Leary DR: West Nile virus infection: a pediatric perspective Pediatrics 2004, 113 (5):1375 1381. 73. Kramer LD, Li J, Shi PY: West Nile virus Lancet Neurol 2007, 6 (2):171 181. 74. Wang T, Town T, Alexopoulou L, Anderson JF, Fikrig E, Flavell RA: Toll like receptor 3 mediates West Nile virus entry into the brain causing lethal encephalitis Nat Med 2004, 10 (12):1366 1373. 75. Daffis S, Samuel MA, Suthar MS, Gale M, Jr., Diamond MS: Toll like receptor 3 has a protective role against West Nile virus infection J Virol 2008, 82 (21):10349 10358. 76. Shrestha B, Samuel MA, Diamond MS: CD8+ T cells require perforin to clear West Nile virus from infected neurons J Virol 2006, 80 (1):119 129. 77. Shrestha B, Diamond MS: Role of CD8+ T cells in control of West Nile virus infection J Virol 2004, 78 (15):8312 8321.

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247 BIOGRAPHICAL SKETCH Melissa Bourgeois was born in Millington, Tennessee to Robert and Pamela Bourgeois. The youngest of two children, she sp ent most of her childhood in St. Albans, Maine and Ft. Myers, Florida. She graduated from the University of Florida with her B achelor of Science in zoology in 2002. She began veterinary school at the University of Florida that same year and enrolled duri ng her second year of veterinary school into a combined DVM/PhD program. She graduated with her D octorate in Veterinary Medicine from the Unversity of Florida in 2007 In 2010, she received her Doctor of Philosophy in veterinary medicine. Upon completio n of her PhD, she began work at the Centers for Disease Control and Prevention in Atlanta focusing on influenza research