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The Role of Stress-Derived Vesicles in the Bystander Effect and Cancer Related Cachexia

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The Role of Stress-Derived Vesicles in the Bystander Effect and Cancer Related Cachexia THE ROLE OF STRESS-DERIVED VESICLES IN THE BYSTANDER EFFECT AND CANCER RELATED CACHEXIA Thesis is submitted in partial fulfilment of the requirements of the award of Doctor of Philosophy FINDLAY REDVERS BEWICKE-COPLEY Department of Biological and Medical Sciences Degree awarded by Oxford Brookes University First submitted for examination: January 2018 ACKNOWLEDGEMENTS Thank you to Oxford Brookes for providing funding for my research, and especially to Professor Nigel Groome whose research and generosity has allowed so many to complete their PhDs. I would also like to say thank you to the Cancer and Polio trust for providing some of the funding for my PhD I would like to thank my Supervisors Dave and Ryan for their support throughout my PhD and for only occasionally saddling me with unrelated projects. Without your guidance and support I would have curled up into a ball in the corner of the office and gently sobbed to myself for the last 5 years. Thanks to Priya for her tireless work ensuring the lab functions correctly and supporting all other members of the lab. I’d also like to thank past members of the lab, Laura Jacobs and Laura Mulcahy for their support throughout both my MSc and my PhD. Sunny Vijen for chatting with me whilst he smoked and making me leave lunch early, so he could have another smoke before going back to work. Thank you to Robbie Crickley for all the lunch time chats. To Lia I would like to say χασμουριέμαι! Thanks to Bianca for all her help getting to know the world of immunocytochemistry. To all members of the lab, thank you for your help, for your camaraderie and for putting up with me mashing the return key with all my might whilst running code. Thanks to Phoebe for bingeing Psychology and Louie Theroux documentaries on the days I was writing, as well as for her support through the hardships and breakdowns during the writing up phase. To Mum and Dad for reluctantly having their own live-in tech support and “getting things off tall shelves” support for the last 5 years. Sparks, Scruff, Gren, Jester, Jen, Jez and Es thanks for helping me destress by getting mad at video games, and to Babs for all the games of Talisman that ended in network trouble. Thank you also to the members of the Jiu Jitsu Foundation for trying to break my limbs and dump me into the floor as hard as possible. Especially Edward Mearns, Mark Ham, Natalie Lockyer and Jake Gately for all their help over the past 5 years. I would also like to thank Big Finish Audio productions, the podcasts: My Dad Wrote a Porno, The Dollop, The Guilty Feminist, Sit Down and Shut Up and many others for the many hours of audio that got me through the long hours in the lab. Oh, and cheers to Dr. Millard for telling me never to get into academia. Findlay Bewicke-Copley Page | i PhD Thesis ABSTRACT Extracellular vesicles are small, lipid bound structures that are involved in intercellular signalling. They are known to be involved in numerous processes within the body, including in disease. One interesting function of EVs appears to be the induction of the bystander effect. The bystander effect refers to the non-targeted effects of stress, whereby stressed cells induce damage in neighbouring cells. EVs released from cells following irradiation have previously been shown to induce the bystander effect. EVs have also been implicated in the induction of cancer-cachexia, a muscle wasting disease. This disease is common in patients with cancer and is often linked to poor prognosis. In this project the ability of EVs released from heat shocked cells to induce bystander effects has been assessed. EVs released from cancer cells following 45°C treatment induced the bystander effect and the bystander cells were shown to be more resistant to subsequent stress treatment. EVs retained this functionality for up to two weeks when stored at -80°C. EVs released following short, 70°C treatment were also able to induce bystander effects. The ability of EVs from both stressed (cisplatin) and unstressed cancer cells to induce cachexia was also examined. Cancer EVs were able to reduce differentiation in vitro, but no effects were observed when these EVs were injected into mice. The proteome of these EVs and their parent cells was also identified via liquid chromatography-mass spectrometry and pathway analysis was carried out on these proteins. These data suggest possible roles for EVs in cell-cell communication during stress and disease, with EVs being able to induce bystander effects and alter muscle development in vitro. Findlay Bewicke-Copley Page | ii PhD Thesis PUBLICATION ARISING FROM THIS WORK Bewicke-Copley F, Mulcahy LA, Jacobs LA, Samuel P, Akbar N, Pink RC and Carter DRF (2017) Extracellular vesicles released following heat stress induce bystander effect in unstressed populations. Journal of Extracellular Vesicles. 6(1): 1340746. Findlay Bewicke-Copley Page | iii PhD Thesis ABBREVIATIONS AIDS Acquired immune deficiency syndrome AP-site Apurinic/Aprimidinic Site ARF6 ADP-ribosylation Factor 6 ATF Activating Translation Factor ATPase Adenosinetriphosphatase BAK Bcl-2 Homologous Antagonist Killer BAX Bcl-2-associated X Protein BiP Immunoglobulin heavy-chain binding protein BE Bystander effect Bcl-2 B-cell Lymphoma-2 BER Base Excision Repair BID BH3-interating Domain Death Agonist BLM Bloom Syndrome Protein BMI Body Mass Index CAD Caspase-activated DNase cAMP Cyclic adenosine monophosphate C/EBPβ CCAAT-enhancer-binding protein β CCM Cell Conditioned Media CD9,63,81 Cluster of Differentiation 9, 63, 81 DISC Death Induced Signalling Complex DSB Double Strand Break ER Endoplasmic Reticulum ESCRT Endosomal Sorting Complexes Required for Transport EV Extracellular Vesicle EXO1 Exonuclease 1 FADD Fas-associated Death Domain hnRNPs Heterogeneous Nuclear Ribonucleoproteins HR Homologous Recombination Hsps Heat shock Proteins ICAD Inhibitor of caspase-activated DNase LCMS Liquid chromatography-mass spectrometry Findlay Bewicke-Copley Page | iv PhD Thesis IGF Insulin-like Growth Factor IGFBP-3 Insulin-like Growth Factor Binding Protein 3 IL-1β Interleukin-1β ILV Intraluminal Vesicle IRE1α Inositol-requiring protein 1α LFQ Label Free Quantification LLC Lewis Lung Carcinoma lncRNA Long Non-Coding RNA MAC Membrane attack complex MAPK Mitogen-Activated Protein Kinases MMR Mismatch Repair MRN Mre11-Rad50-Nbs1 MST1 Mammalian Sterile-20 Kinase MVB Multivesicular Body MyHC Myosin Heavy Chain NCO Non-crossover NER Nucleotide Excision Repair NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells NHEJ Non-Homologous End Joining PCNA Proliferating Cell Nuclear Antigen PERK PRKR-like ER Kinase RFC Replication Factor C RIDD Regulated IRE1-dependent decay of mRNA ROCK 1 Rho-associated coiled-coil-containing protein kinase 1 ROS Reactive Oxygen Species RPA Replication Protein A SCE Sister chromatid exchange SEC Size-Exclusion Chromatography SSB Single Strand Break TFIIH Transcription Factor II Human TNF Tumour Necrosis Factor TRAIL TNF-related Apoptosis Inducing Ligand TSG101 Tumour Susceptibility Gene 101 UPR Unfolded Protein Response Findlay Bewicke-Copley Page | v PhD Thesis VSP4 Variant-specific surface protein 4 XP(A-G) Xeroderma Pigmentosum Group (A-G)-Complementing Protein XRCC4 X-ray Repair Cross-Complementing Protein 4 ZFAS1 Zinc finger antisense 1 Findlay Bewicke-Copley Page | vi PhD Thesis TABLE OF CONTENTS Acknowledgements ................................................................................................................. i Abstract .................................................................................................................................. ii Publication arising from this work ........................................................................................ iii Abbreviations ........................................................................................................................ iv Table of Contents ................................................................................................................. vii Table of Figures ..................................................................................................................... xi Table of Tables .....................................................................................................................xiii 1 Introduction..................................................................................................................... 1 1.1 Stress Response .................................................................................................................. 1 1.1.1 The cellular response to stress ............................................................................................ 1 1.1.2 The heat shock response ..................................................................................................... 5 1.1.3 Unfolded protein response .................................................................................................. 9 1.1.4 DNA damage response ...................................................................................................... 10 1.1.5 Apoptosis ........................................................................................................................... 12 1.2 The Bystander effect ......................................................................................................... 17 1.2.1 Non-targeted effects of stress ........................................................................................... 17 1.3 Cachexia ...........................................................................................................................
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