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12-Lipoxygenase Promotes Macrophage Infiltration and Pancreatic

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12-Lipoxygenase Promotes Macrophage Infiltration and Pancreatic 12-LIPOXYGENASE PROMOTES MACROPHAGE INFILTRATION AND PANCREATIC ISLET DYSFUNCTION IN THE VERTEBRATE MODELS OF DIABETES PATHOGENESIS Abhishek Anant Kulkarni Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Biochemistry and Molecular Biology, Indiana University May 2020 Accepted by the Graduate Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Doctoral Committee ______________________________________ Maureen Harrington, Ph.D., Chair ______________________________________ Raghavendra Mirmira, MD, Ph.D. February 27, 2020 ______________________________________ Ryan Anderson, Ph.D. ______________________________________ Mark Goebl, Ph.D. ______________________________________ Amber Mosley, Ph.D. ______________________________________ James Marrs, Ph.D. ii © 2020 Abhishek Anant Kulkarni iii DEDICATION To my parents, I am eternally indebted to you for all your sacrifices, support and encouragement as I pursue my dreams. iv ACKNOWLEDGEMENT As famously said by John F. Kennedy. "We must find time to stop and thank the people who make a difference in our lives". I would like to take this opportunity to thank everyone for being there for me on this journey. This dissertation would not have been possible without the guidance and the help of several individuals who contributed and extended their valuable assistance in the preparation and completion of this study. Ph.D. in Biomedical Sciences was not just a degree, but a dream. Firstly, I would like to thank my parents for always being there by my side and making sure that I turn this dream into reality. You have sacrificed a lot for me and defied innumerable odds to make sure that I could fly high and achieve something worthy like this. I am forever indebted to you. Whatever is good in me, I owe it to you. I would also like to especially thank my uncle Sameet Kaka and his family, who were the backbone of my journey. Research journey can be tough, and it is critical to have a good mentor. However, I was blessed not to just have a good, but a great mentor. I would like to thank my idol Dr. Raghavendra Mirmira, for accepting me in his lab. It was very supportive of you for assigning me a project based on my research interest and really giving me the freedom to explore it. Scientific dialogs, as well as data discussions with you, were so informative, always making me realize how I should interpret data and present my story. More importantly, I cannot thank you enough for providing me with ample opportunities and encouragement to present my study at so many conferences and seminars. You have inspired me in many ways; your passion for diabetes research, multi-tasking abilities, your intelligence, etc. I will consider myself successful if I could possess even a fraction of your skills and abilities. v I was lucky enough not to have just one, but two amazing mentors. I would like to thank my co-mentor, Dr. Ryan Anderson, for literally being there for me, right from the first day in the lab. Your unparalleled expertise in the zebrafish model and the ability to simplify the most complex ideas and theories made it easier for me to work on this model system and end up loving it. I got to learn a lot from you. Right from your insights into experimentation and research to your suggestions on how I should be thinking about the progression of my career, it has helped me to navigate smoothly throughout this journey. Whatever success I have had during this Ph.D. tenure, I owe a lot of that to you. Thank you for being so patient with me as you were my go-to person for any issues. Lastly, I would just say that you have simply set pretty high standards of how a mentor should be. I was also privileged to have a team of great scientists as my committee members. I would like to thank Dr. Maureen Harrington, Dr. Mark Goebl, Dr. Amber Mosley, and Dr. James Marrs, who played a crucial role in shaping my project. Whatever improvement I have made with respect to thinking like a researcher is only due to your challenging questions and important feedback on my project. I was able to be on track with my coursework as well as research work because of your directions. Your expertise in the respective fields helped me view my project from various perspectives and helped me develop as a researcher. I would also like to specially mention and thank my rotation advisors, Dr. Mark Kaplan and Dr. Haitao Guo. It was a great learning experience in your respective labs. My tenure in your lab was terrific. I would like to thank the lab-mates as well, who helped me during the rotation and trained me well. A significant part of the Ph.D. tenure is spent in the lab. I was happy to be in a lab with so many talented members. First, I would like to thank Dr. Sarah Tersey for her help vi in designing mouse experiments throughout my tenure. Your brilliance in murine genetics and physiology, as well as your experience working with transgenics, is unmatched. More importantly, thank you for patiently editing my documents. Next, I would like to thank Dr. Annie Pineros for teaching me everything about macrophages as well as handling the mice. Working with you has been a great privilege. I would also like to other lab members, Dr. Marimar Hernandez-Perez, Dr. Chris Reissaus, Dr. Farooq Sayed and Dr. Kentaro Yamada for their help in zebrafish studies, microscopy and molecular biology related queries. Thanks to Kara and Karishma, for helping me with those complex islet isolations. Special thanks to Jen for making sure that the reagents were there on time. Without your timely help, I would have been short of many experiments. The diabetes center is a conglomerate of many brilliant scientists who have always encouraged a collaborative environment. I would especially like to thank Linnemann lab and Sims lab for letting me use their space and reagents for a few experiments. I would also like to thank their lab members, especially Dr. Abass, Dr. Michelle, Charanya, and Sara, for teaching me various techniques, solving my queries, and helping me with presentations. I cannot forget the fun we had during conference trips. You made lab-work more enjoyable and made me want to come to the lab every day. I would also like to thank Molina lab, Ren lab, Spaeth lab, and Felton lab for all the scientific discussions and their availability whenever I needed them during my tenure. I would like to this opportunity also to thank all the members of the IBMG graduate office as well. A special mention to Tara and Brandy for being receptive to all my queries since my first day in the U.S., especially related to the administrative work. You make it seem so simple, but I know what length of effort you go through to make it easier for us. I vii would also like to thank all the members of the Biochemistry and Molecular Biology department for making sure that all the administrative work is done on time. Next, I would like to thank my teachers, right from my playschool to this day at IU, who molded me in a way to ensure that I retain my virtues and always behold the value of education. I am nothing without you all. You taught me that "Knowledge is everything". I am eternally indebted to you. Whatever research aptitude and technical expertise I have in me is all because of my excellent research mentors from my past institutes, Dr. Suvarna Sharma, Dr. Ikram Khatkhatay, Dr. Vikrant Bhor, and Dr. Indraneel Mittra. Before I joined my Ph.D. program, I was terrified as I was told most of the students lose it by the end of their program. However, I had many souls around me who assured me that they will continue loving me even after I go completely crazy. I would like to thank Ronak, Sameer, and Manasi, for being with me since my undergrad days. It was your encouragement that made me apply for a Ph.D. program in the U.S. Ironically, you guys dreamed of this day even before I did. In Indy, I was lucky to have a home away from home. Parth, Rakshin, Safa, and everyone from my amazing cohort, thank you for making it easier for me not to miss India with movie nights, birthdays, festivals, and amazing trips. I don’t have enough words to thank Siddhi, the love of my life, who patiently stood by and supported me in all the good and not-so-good times. Your unconditional love and mere presence gave me all the strength to face any challenge that came my way. Last but not least, thank you God, for the wisdom and perseverance that You have bestowed upon me during this research project, and indeed, throughout my life. Your blessings have helped me to sail through this journey successfully! viii Abhishek Anant Kulkarni 12-LIPOXYGENASE PROMOTES MACROPHAGE INFILTRATION AND PANCREATIC ISLET DYSFUNCTION IN THE VERTEBRATE MODELS OF DIABETES PATHOGENESIS Diabetes is a morbid metabolic disorder that affects almost 500 million people worldwide. Although multiple factors contribute to diabetes pathogenesis, pancreatic islet inflammation and dysfunction are shared characteristics of its major forms. 12- lipoxygenase (12-LOX), an enzyme involved in lipid metabolism, has been implicated in islet inflammation. 12-LOX generates reactive oxygen species (ROS) that activate inflammation and serve as major contributors to islet dysfunction. Importantly, since ROS are transient moieties, they are challenging to study in vivo.
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