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Forensic Taphonomy: Investigating the Post Mortem Biochemical Properties of Cartilage and Fungal Succession As Forensic Taphonomy: Investigating the Post Mortem Biochemical Properties of Cartilage and Fungal Succession as Potential Forensic Tools A thesis presented for the degree of Doctor of Philosophy Shawna N. Bolton, B.A. (Hons.), M.Sc. University of Wolverhampton Faculty of Science and Engineering PhD Supervisors: Dr Michael Whitehead and Dr Raul Sutton ii iii Abstract Post mortem interval (PMI – the time elapsed since death and discovery) is important to medicolegal investigations. It helps to construct crucial time lines and assists with the identification of unknown persons by inclusion or exclusion of a suspect’s known movements. Accurate methodologies for establishing PMI are limited to about 48-hours. Such methods involve use of increasing levels of potassium in vitreous humour, and algor mortis. This study is two-fold. Firstly, it explores the biomolecular changes in degrading porcine cartilage buried in soil environments and its potential to determine PMI in the crucial two days to two months period. Trotters were interred in a number of graves at two distinct locations exhibiting dissimilar soil environments. Weekly disinterments (for 6 weeks) resulted in dissection for cartilage samples which were processed for protein immunoblot analyses and cell vitality assays. Results demonstrate that aggrecan, a major structural proteoglycan, produces high (230kDa) and low (38kDa) molecular weight cross-reactive polypeptides (CRPs) within cartilage extracellular matrix. The 230kDa CRP degrades in a reproducible manner irrespective of the different soil environments utilised. As PMI increases, aggrecan diminishes and degrades forming heterogeneous subpopulations with time. Immunodetection of aggrecan ceases when joint exposure to the soil environment occurs. At this time, aggrecan is metabolised by soil microbes. The molecular breakdown of cartilage proteoglycans has potential for use as a reliable indicator of PMI, irrespective of differing soil environments, beyond the 48-hours period. Likewise, vitality assays also demonstrated viable chondrocytes for as long as 35 PM days. The second component of this study examined the fungal activity associated with trotters buried below ground. Results indicate that fungal growth was considerably influenced by soil chemistry and changes in the environment. Fungal colonisation did not demonstrate temporal patterns of succession. The results of this study indicate that cartilage has the potential to prolong PMI determination iv well beyond the current 48- and 100-hour limitations posed by various other soft tissue methods. Moreover, the long-term post mortem viability of chondrocytes presents an opportunity to explore DNA extraction from these cells for the purpose of establishing a positive identification for unidentified remains. On the contrary, the growth and colonisation patterns of post putrefactive fungi in relation to decomposing porcine trotters proved to be futile for estimating PMI. Therefore, fungi may not be a suitable candidate for evaluating PMI during the early phase fungal activity. v Acknowledgements When I first embarked on this new chapter of my life, I had no idea where the road ahead would lead me. However, the past four years have been such a blessing. This is owed to the many wonderful staff members and friends who have made my experience far richer than I could have ever imagined. Without their loving support, I could not have made it this far. Firstly, I would like to express my deepest gratitude to my supervisory team, Drs Michael Whitehead and Raul Sutton for their guidance and support. Thank you for giving me the opportunity to take control of my research and for the constructive criticisms you both have provided during the course of my studies. In doing so, I have learned so much and a great deal about my self. To Dr Jayesh Dudhia of the Royal Veterinary College for providing me with training on how to prepare cartilage samples and perform Western blots, for gifting me the MAb 2-B-6 monoclonal antibody to perform my analyses, and for offering me his advice and support on related subject matter. Thank you to Dr Tim Baldwin for providing me with SDS-PAGE training and for allowing me to use your chemicals and equipment. To the many staff members who have assisted me in more ways than one, Dr Terry Bartlett and Professor John Howl for allowing me to audit their lectures, Drs Andrew Black and Dave Townrow for providing me with transportation to Hilton and data for soil and weather analyses, Mr Robert Hooton for maintaining the burial plot at Compton Park and for constructing the plot at Hilton. Thanks to Dr Angel Armesilla for advice on Western blotting and for gifting the anti-α-tubulin, Dr Mark Morris for his helpful suggestions and black India ink for staining Western blot membranes, Dr Ian Nichol for gifting protease inhibitors, Dr Wei Wang for gifting monoclonal antibodies, anti-vinculin and anti-vimentin, to Professor Craig Williams for assisting me with the interpretations for my soil analysis. Thank you, Drs vi Angie Williams and Andrew Black for your assistance with processing orders. Thank you, Ms. Raman Kaur, for taking care of all my research administrative work. A warm thank you to the lab technicians, Andy Brooke, Bal Bains, Ann Dawson, Nick and Jo Skidmore, Clare Murcott and Henrik for their continued technical assistance with various matters. To Wilma, Jen, and Kate, some of the most wonderful cleaning technicians I have met (thanks for the heart-to-heart conversations), Drs Wera Schmerer, Martin Khechara, and Steve Saffrany for providing me with teaching opportunities. Dr Malcolm Inman, thank you for your help with microscopy, for providing me with photographic images of my fungal plates, and for our much welcomed discussions about life in general, including faith. Thank you Dr Alison McCrea for assisting me with much needed help for statistics and to Rachael Walker, Stephanie Yeoman and Emily Schofield for providing me with additional cartilage samples to assess from their research projects. Thank you to my “sister-friend,” Tinesha Jones, for reminding me about what it means to have faith during difficult times. To my family away from home, Anushree Singh, Nikita Mehta, Tania Pereira, Shaymaa Al-dabagh, who have kept me laughing every single day for four years. And last, but not least, to my wonderful fiancé who has been extremely supportive and for putting up with my extended periods of absence. To anyone whose name I may have forgotten to mention, my sincerest apologies. Thank you for the years of steadfast support! vii Dedication To my parents, Patricia and Garfield Bolton, who have sacrificed a great deal so that I could follow my heart. To my siblings, Sheldon and Daniella, and my cousin, Kisrene McKenzie, for keeping me grounded. Thank you for believing in me, when at times, I did not believe in myself and for encouraging me to pursue my dreams. This thesis is dedicated to Partricia, Garfield, Sheldon, Daniella and Kisrene viii ix Table of Contents Abstract ............................................................................................................................................ iv Acknowledgements .......................................................................................................................... vi Dedication ...................................................................................................................................... viii List of Abbreviations ....................................................................................................................... xx Chapter 1 ........................................................................................................................................... 1 1.1 Death and forensic taphonomy: An introduction to forensic investigations ............................... 2 1.1.1 Post mortem interval versus post burial interval ................................................................ 4 1.1.2 Corporal changes and determination of PMI ..................................................................... 5 1.1.3 Issues with determining PMI ............................................................................................. 9 1.1.4 Recent research studies exploring new methods for ascertaining late stage PMI .............. 11 1.1.5 The application of accumulated degree days for PMI determination ................................ 13 1.2 A research gap in the field of forensic taphonomy .................................................................. 15 1.2.1 Cartilage ......................................................................................................................... 17 1.2.2 The basic structure and function of hyaline articular cartilage and its components .......... 19 1.2.3 Type II collagen and aggrecan ........................................................................................ 23 1.2.4 Chondrocytes and cartilage homeostasis ......................................................................... 28 1.2.5 Cartilage degradation: Loss of homeostasis, roles of proteinases and their mechanisms .. 31 1.2.6 Post mortem cartilage degradation and forensic research ............................................... 35 1.2.7 Research goals for investigation of post mortem articular cartilage ................................. 36 1.3 Forensic mycology ................................................................................................................. 37 1.3.1 Forensic investigations
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