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Establishment, Immortalization and Toxicological Applications of Primary Skin Fibroblasts from Three Sea Turtle Species

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Establishment, Immortalization and Toxicological Applications of Primary Skin Fibroblasts from Three Sea Turtle Species Establishment, Immortalization and Toxicological Applications of Primary Skin Fibroblasts from Three Sea Turtle Species by Sarah J. Webb, B.S., M.S. A DISSERTATION IN ENVIRONMENTAL TOXICOLOGY Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for The Degree of DOCTOR OF PHILOSOPHY Approved Dr. Céline A. J. Godard-Codding (Chair of the Committee) Dr. Christine A. Bishop Dr. Michael J. Hooper Dr. Christopher J. Salice Dr. Kamaleshwar P. Singh Dr. Mark Sheridan Dean of the Graduate School December, 2014 © 2014, Sarah J. Webb Texas Tech University, Sarah J. Webb, December 2014 Acknowledgements This work was only possible through contributions of the time and knowledge of many people, without whom I would never have been able to complete my doctoral studies. Dr. Céline Godard-Codding has been a supportive and encouraging mentor who has provided me with excellent guidance throughout the formation and completion of my work. The opportunity to work with endangered species is rare, and I appreciate greatly the chance I had in her laboratory at Texas Tech. I am also deeply grateful for the experiences she provided, including travel to scientific conferences and to do hands on work with real, live sea turtles. I would also like to thank my doctoral committee: Dr. Christine Bishop, Dr. Michael Hooper, Dr. Christopher Salice, and Dr. Kamaleshwar Singh have all been extremely helpful by answering any questions I may have. Dr. Singh in particular has answered many of my cell culture questions as I refined my techniques and deepened my knowledge of cell culture theory and practice. In addition to my committee, many of the faculty at the Institute of Environmental and Human Health (TIEHH) have provided valuable insight and support. I especially appreciate Dr. Todd Anderson, Dr. Jaclyn Cañas-Carrell, Dr. Weimin Gao, Dr. Jonathan Maul, and Dr. Greg Mayer, all of whom I have gone to with questions at some point. They graciously answered my many questions thoroughly and without complaint when I appeared at their office doors with random questions. In addition to the faculty, the staff at TIEHH has been extremely helpful and I greatly appreciate their help with paperwork, etc. over the years. Stephanie White, Allyson Smith, Karinne Truelock, Stephanie Presley, and Stephanie Franco Alexander all provided invaluable assistance. I would also like to thank our collaborators for their hard work and advice on the various aspects of my project. Without Benjamin Higgins and Lyndsey Howell at the NOAA/NMFS sea turtle facility in Galveston, Texas my work would not have been possible. I thoroughly enjoyed the time Ben spent teaching me about the husbandry of sea turtles in captivity, and appreciate all of the times he answered questions for me about sea turtles. I greatly appreciate Kathryn Furr at the Texas Tech Health Sciences Center, who was instrumental in getting flow cytometry data and kindly devoted several days of her time to analyzing my data. ii Texas Tech University, Sarah J. Webb, December 2014 Dr. Patricia McClellan-Green was a role model to me, as a toxicologist who had worked on sea turtles during her career as well as being a mother who had successfully raised children while maintaining a career in science. She was kind enough to open her home to me for a week while I worked with sea turtle hatchlings in North Carolina, and I thoroughly enjoyed the time I got to spend visiting with her and talking about her work. Without the support of my labmates I would not have been able to complete my work. I would especially like to thank those students who trained me in the lab, Jennifer Cole, Sandy Wiggins, and Greg Zychowski. Becca Pezdek is amazing and awesome and I’m so glad we were able to work together. Jan Yacabucci and I spent many enjoyable hours sitting in the dark working with samples. Subsequent lab members have provided invaluable help in the laboratory and company along the way: Kayla Campasino, Lauren McGeorge, Micah Thal, Kimberly Smelker, Andrew East, and Mary Gendron. Best wishes to our new lab members, Jocylin Pierro and Courtney Alexander as they begin their graduate work at Texas Tech. The friends I have made at Texas Tech have been wonderful, and without them I never would have survived. Darcy Chase, Kristyn Urban, Kim Wooten, Kaylyn Germ, and Anna Hoffarth are some of the best friends I have had and I’m so glad I had the opportunity to meet them while in Lubbock. They provided excellent advice and comic relief throughout my time here and kept my spirits up along the way. I appreciate the food, drink, babysitting, and company they provided over the years. Long live WW! I never would have been able to complete my graduate studies without the help and support of my family. My very best friends, Kara Allen and Amanda White have helped to keep me sane. My parents and sister have been incredibly supportive over the many years I have spent on graduate studies. I’d like to thank my husband, Ryan Webb, who has supported my efforts to earn my degrees. He has been amazing: cooking, cleaning and caring for our daughter and essentially being a single parent many nights. Last, I dedicate all of my work to my daughter, Lilah Webb. I love you more than anything. iii Texas Tech University, Sarah J. Webb, December 2014 Table of Contents Acknowledgements ............................................................................................................. ii Abstract ............................................................................................................................... v List of Tables .................................................................................................................... vii List of Figures .................................................................................................................... ix Chapter 1 ............................................................................................................................. 1 1.1 Introduction and Historical Perspective ............................................................... 1 1.2 Culture Methods ................................................................................................... 5 1.3 Suggestions for Successful Culture of Chelonian Cells ....................................... 7 1.4 References .......................................................................................................... 14 Chapter 2 ........................................................................................................................... 19 2.1 Introduction ........................................................................................................ 19 2.2 Experimental Methods ....................................................................................... 22 2.3 Results ................................................................................................................ 28 2.4 Discussion .......................................................................................................... 29 2.6 References .......................................................................................................... 44 Chapter 3 ........................................................................................................................... 53 3.1 Introduction ........................................................................................................ 53 3.2 Methods .............................................................................................................. 55 3.3 Results ................................................................................................................ 56 3.4 Discussion .......................................................................................................... 56 3.4 References .......................................................................................................... 61 Chapter 4 ........................................................................................................................... 65 4.1 Introduction ........................................................................................................ 65 4.2 Methods .............................................................................................................. 68 4.3 Results ................................................................................................................ 72 4.4 Discussion .......................................................................................................... 75 4.5 References ........................................................................................................ 100 Chapter 5 ......................................................................................................................... 111 5.1 Introduction ...................................................................................................... 111 5.2 Methods ............................................................................................................ 113 5.3 Results .............................................................................................................. 115 5.4 Discussion ........................................................................................................ 115 5.4 References ........................................................................................................ 121 iv Texas Tech University, Sarah J. Webb, December 2014
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