Wayne State University Wayne State University Dissertations 1-1-2016 Genome Wide Analysis Identifies phinS golipid Metabolism As A New Target Of Valproic Acid Shyamalagauri Jadhav Jadhav Wayne State University, Follow this and additional works at: https://digitalcommons.wayne.edu/oa_dissertations Part of the Biology Commons, and the Molecular Biology Commons Recommended Citation Jadhav, Shyamalagauri Jadhav, "Genome Wide Analysis Identifies Sphingolipid Metabolism As A New Target Of Valproic Acid" (2016). Wayne State University Dissertations. 1545. https://digitalcommons.wayne.edu/oa_dissertations/1545 This Open Access Dissertation is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Dissertations by an authorized administrator of DigitalCommons@WayneState. GENOME WIDE ANALYSIS IDENTIFIES SPHINGOLIPID METABOLISM AS A NEW TARGET OF VALPROIC ACID by SHYAMALAGAURI JADHAV DISSERTATION Submitted to the Graduate School of Wayne State University, Detroit, Michigan In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY 2016 MAJOR: BIOLOGICAL SCIENCES Approved By: _____________________________ Advisor Date _____________________________ _____________________________ _____________________________ ©COPYRIGHT BY SHYAMALAGAURI JADHAV 2016 All Rights Reserved DEDICATION I would like to dedicate my dissertation to my late mother Chandrakala Jadhav who was a constant support and inspiration through the years. ii ACKNOWLEDGEMENTS First and foremost I would like to thank my advisor, Dr. Miriam L. Greenberg for her intellectual, moral, financial, and emotional support throughout my graduate journey. The realization of this thesis would have been impossible without her constant encouragement and strong support. I would like to thank her for the confidence she had in my ability to think and design the project through; and for her guidance when needed. She stood by my side and gave me the scientific freedom to explore my research endeavors. This has helped me to develop into a scientist with the ability to think critically and independently. I have seen her not just as my dissertation advisor, but a mentor whose guidance and advice I shall cherish forever. I am grateful for all the time and effort she has invested in educating and training me towards achieving my professional goals. I would also like to extend my sincere gratitude to my committee members, Dr. Lori Pile, Dr. David L. Njus, and Dr. Menq-Jer Lee for their continuous support and valuable suggestions. Their constructive evaluation of my work helped me stay focused and productive. I would like to thank Dr. Ashley Cowart at Medical University of South Carolina (MUSC) for sphingolipid quantification and Jeff Landgraf at Michigan State University for performing the microarray analysis. I would like to thank the past and current members of the Greenberg laboratory for their support, assistance, and for being such wonderful colleagues. I would especially like to thank Dr. Vinay Patil, Dr. Amit Joshi, Dr. Rania Deranieh, Dr. Cunqi Ye, Dr. Zheni Shen, Janani Ganesh, Vaishnavi Raja, Yiran Li, Jajia Ji, Wenxi Yu, Wenjia Lou, Keanna McCain, Dr. Christian Reynolds, and Michael Salsaa, with whom I spent a wonderful time in the laboratory. I thank each and every one for their scientific contribution and all iii the fun experiences and making the lab environment both intellectually stimulating and enjoyable. I would like to specially thank Vaishnavi Raja for being a great friend and support in lab. I would also like to thank all the past and present biological science department faculty and staff for their assistance throughout my Ph.D. and for making graduate student life in the department a smooth sail. Special thanks to Dr. Victoria Meller, Dr. James Tucker, Dr. Edward Golenberg, Dr. Philip Cunningham, Dr. Choong-Min Kang, Krystyn Purvis and Charles Hogan for making my life as a graduate teaching assistant, a comfortable one. I acknowledge the Graduate School and the department for financial support in the form of graduate teaching assistantships and graduate travel awards. I would not have made it this far without the unconditional love and support from my parents, Chandrakant and Chandrakala Jadhav. They have always encouraged me to do my best and invested all their energy and time towards the realization of my dreams and ambitions. I would like to thank my little brother, Mayur Jadhav, for his immense love and support. He has helped me get through the toughest of days by being my stress buster. Thank you for always being my side. I would like to acknowledge my aunt, Dr. Dhanashree Jagtap, for being my source of inspiration throughout. I would also like to acknowledge my parents-in-law, Jayant and Jyoti Supnekar, for being so loving and caring. Lastly, I am indebted to my husband, Vivek Supnekar, for his love, patience and support. He stood by my side through the tough times and good times and instilled in me a positive attitude and a sense of confidence. I am grateful to my friends Dr. Rohan Kulkarni and Dr. Rajalakshmi S. Asur for being my guardians throughout my graduate school days. I feel immensely privileged to iv have received your guidance, love and support. I would like to specially thank my roommate and great friend Sonal Joshi for being there with me through all the ups and downs throughout graduate school days. I will cherish all the good times and memories forever. I am grateful to my friends Dr. Amit Joshi, Dr. Manasi Apte, Dr. Debashish Menon, Dr. Shalini Menon, Dr. Vinay Patil, Rohini Hiremath, Vaishnavi Raja, Ashlesha Chaubal, and Nikita Deshpande, for their love, care and kindness. They have been my family away from home. All of you have given me wonderful memories that I will treasure my whole life. I appreciate constant love and encouragement from my school and college friends all over the world without whom life would have been much less interesting. I shall be eternally grateful for the valuable service of my teachers, especially the entire faculty and staff at the Dept. of Microbiology, Ruia College, Mumbai, whose efforts have inspired me to follow my passion in science and research. v TABLE OF CONTENTS Dedication ....................................................................................................................... ii Acknowledgements ........................................................................................................ iii List of Tables ................................................................................................................. viii List of Figures ................................................................................................................. ix CHAPTER 1- INTRODUCTION ...................................................................................... 1 CHAPTER 2- IDENTIFICATION OF GENES AND CELLULAR PATHWAYS AFFECTED BY ACUTE AND CHRONIC TREATMENT WITH VALPROIC ACID Introduction ......................................................................................................... 40 Materials and Methods ....................................................................................... 41 Results and Discussion ...................................................................................... 43 CHAPTER 3- VALPROIC ACID INDUCES THE UNFOLDED RESPONSE INCREASING CERAMIDE LEVELS Introduction ........................................................................................................ 143 Materials and Methods ...................................................................................... 145 Results ............................................................................................................... 148 Discussion ......................................................................................................... 162 CHAPTER 4- ACUTE VALPROIC ACID TREATMENT INCREASES LEVELS OF PHYTOSPHINGOSINE VIA INOSITOL DEPLETION Introduction ........................................................................................................ 169 Material and Methods ........................................................................................ 173 Results ............................................................................................................... 176 Discussion ......................................................................................................... 182 CHAPTER 5- FUTURE DIRECTIONS ......................................................................... 194 Appendix- IDENTIFICATION OF SYNTHETIC GENETIC INTERACTIONS OF LCB4198 References ................................................................................................................... 222 vi Abstract ........................................................................................................................ 260 Autobiographical Statement ......................................................................................... 263 vii LIST OF TABLES Table 1.1. Yeast and mammalian genes involved in sphingolipid metabolism .............. 19 Table 2.1. Fold changes of genes that show more than 2 fold change in response to acute VPA treatment ........................................................................................... 54 Table 2.2. Fold changes of genes that show more that 2 fold change in response to chronic VPA treatment ...................................................................................... 107 Table