University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2017-08-22 Platelet Transcriptome Heterogeneity: A Role for RNA Uptake in Vascular Health and Disease Lauren R. Clancy University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Biology Commons, Cell Biology Commons, and the Translational Medical Research Commons Repository Citation Clancy LR. (2017). Platelet Transcriptome Heterogeneity: A Role for RNA Uptake in Vascular Health and Disease. GSBS Dissertations and Theses. https://doi.org/10.13028/M2468G. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/922 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. PLATELET TRANSCRIPTOME HETEROGENEITY: A ROLE FOR RNA UPTAKE IN VASCULAR HEALTH AND DISEASE A Dissertation Presented By LAUREN RUTH CLANCY Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSPHY August 22, 2017 Translational Sciences iii REVIEWER PAGE PLATELET TRANSCRIPTOME HETEROGENEITY: A ROLE FOR RNA UPTAKE IN VASCULAR HEALTH AND DISEASE A Dissertation Presented By LAUREN RUTH CLANCY This work was undertaken in the Graduate School of Biomedical Sciences Translational Sciences Program Under the mentorship of Jane E. Freedman, MD, Thesis Advisor Eric Mick ScD, Member of Committee Anastasia Khvorova, PhD, Member of Committee Evelyn Kurt-Jones, PhD, Member of Committee Robert Flaumenhaft, MD, PhD, External Member of Committee John Keaney, MD, Chair of Committee Anthony Carruthers, Ph.D., Dean of the Graduate School of Biomedical Sciences August 22, 2017 iv DEDICATION I would like to dedicate this work to my friends and family. I love you all more than words can say. You are and will always be what I am most grateful for in my life. In loving memory of my grandfather, Ferdinand “Freddie” Martino, 1927-2015 v ACKNOWLEDGMENTS There are many people I would like to thank, who have contributed to the success of this work and my growth throughout graduate school. First I would like to thank the Student Counseling Center at UMMS. The guidance and support I have gained from them has changed the type of person I am profoundly. The lessons I have learned through them have made me a better scientist and better person and I am sure without their services I would not have successfully finished my degree. I would like to thank the members of all my advisory committees, including Dr. Mary Munson, Dr. John Keaney, Dr. Anastasia Khvorova, Dr. Eric Mick, Dr. Evelyn Kurt-Jones and Dr. Robert Flaumenhaft. I would especially like to thank Drs. Keaney, Khvorova and Mick for serving on all three of my advisory committees and acting as constant guides throughout this entire process. I would like to thank all the members of my department, the Cardiovascular Medicine division. Though we would have been colleagues no matter what, I am happy to include many of them as friends and deeply appreciate their scientific interest, support and friendship. I have greatly enjoyed working alongside them and look forward to keeping in touch in the future. I would like to thank all the members of the Freedman lab, past and present, who have given me a home over the last five years. I would specifically like to thank several members: Antonina Risitano, under whom I first began work on the platelet RNA project; Kahraman Tanriverdi, for first educating me in RNA vi and for producing the microarray, high throughput gene expression and sequencing data presented here; Heather Corkrey, for being the best baymate a person could ask for - she has not only helped me scientifically but welcomed my loud, often crazy personality into her space for the last two years, and I am thankful for her constant support, upbeat personality and friendship; and Lea Beaulieu, who led the project presented in Chapter 2 and performed the initial platelet subpopulation sorting in Chapter 3 - she first convinced me to rotate in the lab, taught me that “platelets are powerful” and became not only a friend but a true mentor. Finally, Milka Koupenova-Zamor, who is my constant support, sounding board and extra pair of hands. She helped extensively with the in vivo experiments outlined in Chapter 3. When I didn’t think I was good enough, she always reminded me I was and then bought me a cookie or spinach “thingie” to cheer me up. Without many of my mornings starting with her and coffee, I would not have survived the last few years. She is and will always be a dear mentor and friend. A lab is a type of family and ours has a wonderful leader in Jane Freedman. Since the beginning she accepted that I was not your typical “academia-oriented” graduate student. She listened to what I wanted out of my time here and allowed me the freedom to take my career in the direction I chose. She gave me opportunities to grow whenever possible and always listened when I needed her. She has always been a steady presence on this project, calming me when there were unexpected results or mistakes and kicking me in the butt vii when I needed it. She has always made lab fun. Whether letting me lock our lab in puzzle rooms or hosting celebratory meals or just treating us to something special, Jane made sure our lab was a home. I want to thank her for being my biggest advocate, my constant source of support, my mentor and my friend. I have the most wonderful friends and family. Despite the fact that many of them don’t have science backgrounds, they have spent the last six years constantly interested in what I do and supporting me any way they can. I have never wanted for a shoulder to cry on or a good laugh and am deeply aware how lucky I am to have each and every one of them. I’d like to thank my brother specifically, for always supporting me any way he can, always knowing how to make me smile and always being willing to be silly and nerdy with me. I’d also like to especially thank Ryan Genga, who decided to go back to grad school at UMMS the same time I did. We have been together in this since day one and through it all he has constantly supported me, whether I needed last minute reagents, a pep talk or a beer. Without his constant support each day, I could not have done this, and without his friendship, I would not be the person I am. There is no better person I could have asked to have beside me. Finally, I would like to thank my Mom and Dad. They have supported me my whole life and encouraged me to always be who I am and strive for whatever I want. They made sure that throughout this entire experience I never felt alone and have always reminded me to smile, even when I really didn’t want to. I am truly grateful to have parents like them. They have always put my brother and me viii first and without their constant support, love and direction, I would not be the scientist or person I am today. To all those above and to anyone I missed who has someone helped me along this path, thank you, from the bottom of my heart. ix ABSTRACT As our understanding of the platelet’s systemic role continues to expand beyond hemostasis and thrombosis, interrogation of the platelet’s ability to affect diverse biological processes is required. Studies of the platelet’s non-traditional roles have focused on developing our understanding of the platelet’s relation to specific disease phenotypes as well as elucidation of platelet characteristics, content, and function. The generic content, traditional function and heterogeneity of platelets have long been accepted; more ambiguous and controversial has been how these factors are interrelated. Investigation of platelet content revealed the presence of biologically functional RNA in anucleated platelets, the correlation of platelet RNA to distinct phenotypes, and the ability of platelets to transfer RNA to other vascular cells; however how these processes occur is unclear. To further interrogate platelet RNA processes, we utilized sorting and RNA sequencing to develop platelet subpopulation transcriptome profiles. We found that platelet heterogeneity extends to the platelet transcriptome: distinct RNA profiles exist dependent on platelet size. We hypothesized that this RNA heterogeneity is the result of RNA transfer between platelets and vascular cells. Using in vitro and in vivo modeling, we were able to show the novel ability of platelets to take up RNA from vascular cells, correlating to the unique functional profile associated with small platelet transcriptomes. These findings reveal a role for platelet RNA transfer in platelet RNA heterogeneity, with potential correlation to platelet functional diversity x previously proposed. The ability of the platelet to bidirectionally transfer RNA within circulation has implications for vascular health and beyond. xi TABLE OF CONTENTS Title Page ........................................................................................................... ii Reviewer Page .................................................................................................. iii Dedication ......................................................................................................... iv Acknowledgements ...........................................................................................