University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 5-2016 EVALUATING DIFFERENTIAL NUCLEAR DNA YIELD RATES AMONG HUMAN BONE TISSUE TYPES: A SYNCHROTRON MICRO-CT APPROACH Janna Michelle Andronowski University of Tennessee - Knoxville, [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Biological and Physical Anthropology Commons Recommended Citation Andronowski, Janna Michelle, "EVALUATING DIFFERENTIAL NUCLEAR DNA YIELD RATES AMONG HUMAN BONE TISSUE TYPES: A SYNCHROTRON MICRO-CT APPROACH. " PhD diss., University of Tennessee, 2016. https://trace.tennessee.edu/utk_graddiss/3646 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Janna Michelle Andronowski entitled "EVALUATING DIFFERENTIAL NUCLEAR DNA YIELD RATES AMONG HUMAN BONE TISSUE TYPES: A SYNCHROTRON MICRO-CT APPROACH." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Anthropology. Amy Z. Mundorff, Major Professor We have read this dissertation and recommend its acceptance: Dawnie W. Steadman, Benjamin M. Auerbach, Yong C. Bradley, Christian M. Crowder, David M. L. Cooper Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) EVALUATING DIFFERENTIAL NUCLEAR DNA YIELD RATES AMONG HUMAN BONE TISSUE TYPES: A SYNCHROTRON MICRO-CT APPROACH A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Janna Michelle Andronowski May 2016 Copyright © 2016 by Janna Michelle Andronowski. All rights reserved. ii DEDICATION For Daniel C. Harvey (Harvey M. Harvey), my longest and truest friend. I could not have successfully completed this endeavor (or learned to play the tuba) without your love and support. iii ACKNOWLEDGEMENTS I could not have accomplished this goal without the contributions and support of many colleagues and friends. First and foremost, I would like to express my deepest thanks to my advisor Dr. Amy Z. Mundorff for her role in shaping this work. Her support and direction over the past (almost) four years has allowed me to excel in my studies. I am indebted to my longtime mentor, committee member, and friend, Dr. Christian Crowder. I greatly appreciate all of the time he spent teaching me how to prepare bone thin- sections, and proficiency in histological analysis. Without his mentorship and friendship, I certainly would not be where I am today. I am grateful to have had the privilege of working with Dr. David Cooper on this project. His knowledge and expertise in synchrotron science and 3D bone imaging have been invaluable, and without his support and encouragement this work certainly would not have been possible. I also wish to thank Dr. Cooper for the remote use of his laboratory computing facilities for data processing. I would like to thank the remaining members of my committee: Drs. Dawnie Steadman, Benjamin Auerbach, and Yong Bradley for providing direction, insightful comments, and advice regarding this research. I especially want to offer my gratitude to Dr. Dawnie Steadman who granted me permission for the use of skeletal material from the William M. Bass Donated Skeletal Collection. I feel privileged to have worked with this valuable collection. Thanks to Dr. Bradley for providing access to the clinical CT scanner at the University of Tennessee Medical Center, and to Shelley Acuff for operating the CT system and assisting with bone set-up prior to scanning. I am also very grateful to Josh Price who provided helpful statistical advice regarding data analysis and interpretation. iv I wish to thank Dr. Graciela Cabana for providing space in the Molecular Anthropology Laboratory for me to prepare my skeletal samples. Thank you also to Drs. Yasmin Carter, Alix Black, and Steve Thomopouloss who offered helpful advice regarding bone sectioning for SR micro-CT. And many thanks to Hannah Johnson who helped to prepare bone specimens for 3D imaging over her summer break. Financial resources for this research have come from a number of sources. I would like to acknowledge the Canadian Light Source (CLS) in Saskatoon, Saskatchewan, Canada, and the Forensic Anthropology Center and Department of Anthropology at the University of Tennessee, Knoxville for funding a portion of this project through the Walter Leitner and Kneberg-Lewis Doctoral Completion Awards. I extend my deepest appreciation to the Biomedical Imaging and Therapy beamline (BMIT) staff at the CLS. To Dr. George Belev, thank you for providing endless user support and beam line troubleshooting during my many experimental shifts. My experiments would not have been possible without your extensive knowledge and dedication to their success at the beam line. I also want to sincerely thank Drs. Tomasz Wysokinski, Adam Webb, and Ning Zhu for providing additional user support and advice regarding my experimental set-up. I am forever indebted to my fantastic beam team, Isaac Pratt, Danielle Kabatoff, Kim Harrison, and Dr. David Cooper. Without your assistance, companionship, and camaraderie during many long continuous days of beam time I would have surely gone mad. I am especially indebted to Isaac Pratt for teaching me the ropes at the CLS, running paperwork around for me, providing late-night software teaching sessions, and both intellectual and emotional support at the beam line during data collection. This research would not have been possible without your help and v dedication. Thank you to Tracey MacDonald, Mylene Tham, and Kelly Summers for the ice cream trips, board game playing, and overall friendship and company during long stints of beam time. I cannot wait to work and spend time with you all in the upcoming years in Saskatoon. I wish to sincerely thank two other scholars, colleagues, and mentors who helped shape my interest in bone histology. To Dr. Lynne Bell, who first sparked my interest in bone histology and provided me with many invaluable learning opportunities during my undergraduate career. Thank you for starting it all. Your fire in the belly was contagious! And to my Master’s advisor, Dr. Susan Pfeiffer, you have perfected the art of mentorship and your enthusiasm for research continues to be an inspiration to me. I am immensely indebted to my family and friends who have supported me through my entire tenure in graduate school. I extend a special thank you to my beautiful Mom, Karen Andronowski, and Dad, Max Andronowski, for their unconditional love, support, and encouragement during my graduate career. There are no words to express how appreciative I am of their undying love and support. They have championed my academic career goals from the very beginning and have always supported me in my endeavors, no matter how far away from home they took me. They have made it a top priority to help me achieve my goals and I truly strive to make them proud. To my brother, John, thank you for not allowing me to take myself too seriously. I always greatly enjoy our visits in the Motherland and have fond (yet painful) memories from when you ventured to the Knoxville area. Thank you for always being there for me. To my best friends in Western Canada who have been by my side throughout my undergraduate and graduate school years, Heather Broski, Ashley Turrie, and Nicole vi Scott. Thank you for being so understanding and supportive of my career goals (and for the endless fun times, of course!). I know that no matter how far apart we are, we will always stay in touch and remain the best of friends. To Alexs Hermans, thank you for being my rock and platonic Knoxville life partner. Your home cooked meals allowed me to finish my dissertation without going hungry. You have truly made my life a little sweeter over this past year and I am greatly appreciative for that. I also want to thank Chris Agee for standing by me throughout the writing process. Thank you for listening to my endless ramblings about bone biology and forensic anthropology. To my anthropology peers and friends who have commiserated with me both near and far, especially Ashley Kendall, Michelle Cameron, Dr. Catherine Merritt, Hilary Duke, Guy Duke, Dr. Sarah M. Rowe, Jonathon Eaton, Sean Tallman, Dr. Audrey Scott, Dr. Giovanna Vidoli, Dr. Cate Bird, Christina Fojas, and Katie Corcoran. You have all given me a wealth of personal and academic advice and enriched my life. vii ABSTRACT Molecular human identification has conventionally focused on DNA sampling from dense, weight-bearing cortical bone tissue from femora or tibiae. A comparison of skeletal elements from three contemporary individuals demonstrated that elements with high quantities of cancellous bone yielded nuclear DNA at the highest rates, suggesting that preferentially sampling cortical bone is suboptimal (Mundorff & Davoren, 2014). Despite these findings, the reason for the differential DNA yields between cortical and cancellous bone tissues remains unknown. The primary goal of this research is to ascertain whether differences in bone microstructure can be used to explain differential nuclear DNA yield among bone tissue types, with a focus on osteocytes and the 3D quantification of their associated lacunae. Osteocytes and other bone cells are recognized to house DNA in bone tissue, thus examining the density of their lacunae may explain why nuclear DNA yield rates differ among bone tissue types. Methods included: (1) quantifying cortical and cancellous bone volume from each bone-sampling site using Computed Tomography (CT), and (2) visualizing and quantifying osteocyte lacunae using synchrotron radiation micro-Computed Tomographic imaging (SR micro-CT).