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FUNCTIONAL ANALYSES OF THE MOLECULAR MECHANISMS UNDERLYING TWO EQUINE RESPIRATORY DISEASES: RECURRENT AIRWAY OBSTRUCTION AND RHODOCOCCUS EQUI PNEUMONIA A Dissertation by PRIYANKA KACHROO Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 2012 Major Subject: Biomedical Sciences Functional Analyses of the Molecular Mechanisms Underlying Two Equine Respiratory Diseases: Recurrent Airway Obstruction and Rhodococcus equi Pneumonia Copyright 2012 Priyanka Kachroo FUNCTIONAL ANALYSES OF THE MOLECULAR MECHANISMS UNDERLYING TWO EQUINE RESPIRATORY DISEASES: RECURRENT AIRWAY OBSTRUCTION AND RHODOCOCCUS EQUI PNEUMONIA A Dissertation by PRIYANKA KACHROO Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Chair of Committee, Bhanu P. Chowdhary Committee Members, Christopher M. Seabury Huaijun Zhou Ivan Ivanov Head of Department, Evelyn Tiffany-Castiglioni May 2012 Major Subject: Biomedical Sciences iii ABSTRACT Functional Analyses of the Molecular Mechanisms Underlying Two Equine Respiratory Diseases: Recurrent Airway Obstruction and Rhodococcus equi Pneumonia. (May 2012) Priyanka Kachroo, B.E., PES Institute of Technology Chair of Advisory Committee: Dr. Bhanu P. Chowdhary Recurrent airway obstruction (RAO) and Rhodococcus equi (R. equi) pneumonia are two equine respiratory diseases. RAO is an allergic asthma like disease of the middle-aged horses while the R. equi pneumonia affects only young foals. Respiratory disease is considered among the major causes of economic loss to the equine industry and tops the priority list for research that will focus on preventative and diagnostic facets of such disease. The objective of this research was to investigate the effect of antigen exposure and remission (via allergen avoidance and/or drug) on chronically affected RAO horses. Additionally, we also wanted to understand the changes in equine neonatal immune system due to R. equi exposure and identify molecular biomarkers for early disease screening. Various biological samples (lung tissue for the RAO study and blood leukocytes and nasal epithelial cells for the R. equi study) were used to extract ribonucleic acid (RNA). Complimentary deoxyribonucleic acid (cDNA) obtained from RNA was used to perform microarray hybridization experiments. iv Our findings suggest that compared to control horses allergen exposure leads to an elevated protein synthesis and inflammation that contributes to aggravation of symptoms and airway changes. We found that allergen avoidance controls inflammation and causes an improvement in lung function and other chronic features of RAO. The drug administration led to an accelerated remission in the chronic RAO features; a complete remission could however not be achieved. Hence it appears that although not a complete resolution, but allergen avoidance and drugs will help in a better management of chronic RAO symptoms. Our results suggest that the neonatal immune system is capable of initiating a protective immune response through birth up to 8 weeks of age. However there are also processes present that may be counter-productive to the host. Induction of such suppressive mechanisms may be a result of bacterial modulation of the host immune response or a result of immature host immune system. We also identified molecular biomarkers that will have the potential to screen foals for R. equi pneumonia soon after birth and before the onset of clinical symptoms. The research findings of this study will improve the current understanding of the two equine diseases. v DEDICATION I would like to dedicate this work to my parents, Mrs. Asha Kachroo and Mr K. L. Kachroo , my sister Prerna Kachroo and my fiancé Arun Rao . Their love and support throughout this period has made it possible to accomplish this goal. Soon after completing college, pursuing a doctoral degree was nowhere in my plans and it just happened. When I did decide to start a Ph.D., my parents were highly supportive of my decision. I have troubled them with late night calls and nervous breakdowns during dissertation writing and defense. They gave me strength to overcome all hurdles and tackle every situation head on. My Dad is my role model and I always look up to him. His achievements and dedication towards work have taught me that everything is possible once you put your mind to it. My mother is a friend and guardian. I derive my strength from her and thank her for her unconditional love. I continue to learn from my parents and I truly dedicate this achievement to them. My heartfelt gratitude to my fiancé Arun Rao for being my rock. His love and belief in my abilities made it easier to sail through this period. He believed in my capabilities and stood by me no matter what. He made sure nothing led me astray from my goal, no matter how difficult the circumstance. I feel lucky to have him in my life. I owe this degree to him and my family. Finally I thank the almighty god for the blessings and showing me the way. vi ACKNOWLEDGEMENTS I would like to thank my advisor Dr. Bhanu Chowdhary for his guidance and support. His suggestions, comments and critiques have helped me immensely in improving as a researcher. I thank him for believing in me and giving me an opportunity to pursue a Ph.D. under his supervision. I am sincerely thankful to my committee members for their immense help throughout the course of my research. I would like to thank Dr. Ivan Ivanov for always being there whenever I have needed any help with my research or when I just needed to talk. I am, and continue to be, grateful for his help, support and belief in me. I have learnt a lot from him and always look up to him for any advice. I would also like to thank Dr. Christopher Seabury for being ever so helpful and kind. He has proactively shown interest in my research and my progress. I am really thankful for his guidance during this period. I thank Dr. Huaijun Zhou for always taking time to discuss my research and helping me with various technical difficulties. I feel lucky to have great professionals on board as my committee members and feel I have learnt a lot from them. I would like to acknowledge all the collaborators in my research projects. To begin with, I would like to thank Dr. Jean-Pierre Lavoie and Ms. Josiane Lavoie from University of Montreal, for kindly providing us with samples for the RAO related projects. I also thank Dr. Noah Cohen for his valuable help with foal pneumonia related projects. He has vii been very instrumental in all stages of our research projects and continues to provide guidance and support. I would also like to thank Dr. Glenn Blodgett and Ms. Cassie DeFiori from Ranch 6666, Guthrie, Texas, for their assistance in sample collection in the foal pneumonia project. I would like to thank other faculty members for their help with various research related questions. Dr. Michael Criscitiello took his precious time to read and critique one of my manuscripts. He provided valuable insights and significant suggests towards improving the manuscript. I thank Dr. Terje Raudepp for supervising and teaching me various laboratory techniques when I was new in the lab. I would also like to thank the office staff of Veterinary Integrative Biological Sciences department -Ms. Dana Parks, Ms. Deborah Daniels, Ms. Sandra Reyes, Ms. Gay Perry and the rest. Their valuable service and help towards managing several administrative tasks has smoothened my graduate career. I would finally like to thank all my lab colleagues. I would like to thank Dr. Jan Janecka for being very instrumental with revisions of all my manuscripts. He has been very kind and understanding and I truly appreciate his help. He has been there whenever I needed any kind of help or guidance. I would like to thank Dr. Samantha Steelman and Dr. Pranab Jyoti Das for teaching me various lab techniques and for providing valuable suggestions regarding the manuscripts. I would like to thank Sharmila Ghosh, Felipe viii Avila and Jana Caldwell for all their help and for simply being wonderful people. I would like to thank Dr. Ashley Seabury for mentoring me when I first joined the lab. She is a wonderful person and had been immensely helpful. I would also like to thank Dr. Nandina Paria for being a great colleague and an ever so wonderful friend. I feel grateful for all the things I learnt from her and for making the lab a wonderful place to work. ix TABLE OF CONTENTS Page ABSTRACT .............................................................................................................. iii DEDICATION .......................................................................................................... v ACKNOWLEDGEMENTS ...................................................................................... vi TABLE OF CONTENTS .......................................................................................... ix LIST OF FIGURES ................................................................................................... xii LIST OF TABLES .................................................................................................... xiv CHAPTER I INTRODUCTION AND LITERATURE REVIEW ............................ 1 Introduction to respiratory diseases in the horse ............................ 1 Recurrent airway obstruction (RAO)............................................. 2 Rhodococcus equi pneumonia........................................................
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  • Technical Note, Appendix: an Analysis of Blood Processing Methods to Prepare Samples for Genechip® Expression Profiling (Pdf, 1

    Technical Note, Appendix: an Analysis of Blood Processing Methods to Prepare Samples for Genechip® Expression Profiling (Pdf, 1

    Appendix 1: Signature genes for different blood cell types. Blood Cell Type Source Probe Set Description Symbol Blood Cell Type Source Probe Set Description Symbol Fraction ID Fraction ID Mono- Lympho- GSK 203547_at CD4 antigen (p55) CD4 Whitney et al. 209813_x_at T cell receptor TRG nuclear cytes gamma locus cells Whitney et al. 209995_s_at T-cell leukemia/ TCL1A Whitney et al. 203104_at colony stimulating CSF1R lymphoma 1A factor 1 receptor, Whitney et al. 210164_at granzyme B GZMB formerly McDonough (granzyme 2, feline sarcoma viral cytotoxic T-lymphocyte- (v-fms) oncogene associated serine homolog esterase 1) Whitney et al. 203290_at major histocompatibility HLA-DQA1 Whitney et al. 210321_at similar to granzyme B CTLA1 complex, class II, (granzyme 2, cytotoxic DQ alpha 1 T-lymphocyte-associated Whitney et al. 203413_at NEL-like 2 (chicken) NELL2 serine esterase 1) Whitney et al. 203828_s_at natural killer cell NK4 (H. sapiens) transcript 4 Whitney et al. 212827_at immunoglobulin heavy IGHM Whitney et al. 203932_at major histocompatibility HLA-DMB constant mu complex, class II, Whitney et al. 212998_x_at major histocompatibility HLA-DQB1 DM beta complex, class II, Whitney et al. 204655_at chemokine (C-C motif) CCL5 DQ beta 1 ligand 5 Whitney et al. 212999_x_at major histocompatibility HLA-DQB Whitney et al. 204661_at CDW52 antigen CDW52 complex, class II, (CAMPATH-1 antigen) DQ beta 1 Whitney et al. 205049_s_at CD79A antigen CD79A Whitney et al. 213193_x_at T cell receptor beta locus TRB (immunoglobulin- Whitney et al. 213425_at Homo sapiens cDNA associated alpha) FLJ11441 fis, clone Whitney et al. 205291_at interleukin 2 receptor, IL2RB HEMBA1001323, beta mRNA sequence Whitney et al.