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PDF (Ph.D.Thesis) The biological and clinical characterisation and validation of novel biomarkers in colorectal cancer Seán Fitzgerald B.Sc., M.Sc. This thesis is submitted to Dublin City University for the degree of Ph.D. July 2015 Based on research carried out at School of Biotechnology, Dublin City University, Dublin 9, Ireland. Supervisors: Professor Richard O’Kennedy Dr. Gregor Kijanka External Supervisor: Professor Elaine Kay Department of Pathology, RCSI, Beaumont Hospital. i Declaration I hereby certify that this material, which I now submit for assessment on the programme of study leading to the award of Ph.D. is entirely my own work, that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge breach any law of copyright, and has not been taken from the work of others save and to the extent that such work has been cited and acknowledged within the text of my work. Signed: ____________ ID No.: ___________ Date: _______ ii Acknowledgements Firstly, I would like to express my sincere gratitude and appreciation to my supervisors Prof. Richard O’Kennedy, Prof. Elaine Kay and Dr. Gregor Kijanka. This thesis would not have been possible without the expert advice and guidance that I received from each of you, both on an academic and personal level. I am especially grateful to Dr. Gregor Kijanka for his endless guidance, wisdom and friendship throughout my PhD. I would like to thank all the members of the Applied Biochemistry Group and the School of Biotechnology in DCU for their help, support and friendship over the last few years. In addition, I would like to thank all of my colleagues in the Biomedical Diagnostics Institute who funded and supported this work. I would like to acknowledge and thank Dr. Katherine Sheehan, Dr. Tony O’Grady, Mr. Robert Cummins, Dr. Joanna Fay, Ms. Etáin Daly, Ms. Deirdre Hyland, Ms. Joan Kehoe and all of the staff at the RCSI Histopathology Department of Beaumont Hospital for their help and support with my research. I would also like to thank Prof. Lance Liotta, Dr. Virginia Espina and all at the Centre for Applied Proteomics and Molecular Medicine, George Mason University, Virginia, USA, for allowing me to visit their laboratory and for their valued contribution in a very successful collaboration. Finally, I would like to thank my parents, Mike and Olive, my family and friends, and especially my girlfriend Aoife, for their love, support and advice during the course of my 10-year stay in university. iii Table of Contents: Declaration ....................................................................................................................................... ii Acknowledgements.......................................................................................................................... iii Table of Contents ............................................................................................................................. iv Abstract ........................................................................................................................................ ....ix List of Abbreviations: ....................................................................................................................... ix Units ................................................................................................................................................ xv List of Figures: ............................................................................................................................... xvii List of Tables: .................................................................................................................................. xx Publications, Awards, Presentations and Patent applications: .................................................... xxii Chapter 1 Introduction and Aims of the Study ............................................................................... 1 1.1 The Colon and Rectum .......................................................................................................... 2 1.2 Colorectal Cancer .................................................................................................................. 4 1.2.1 Epidemiology of Colorectal Cancer ................................................................................ 8 1.2.2 Causes, symptoms and diagnosis of colorectal cancer ................................................ 11 1.2.3 Histopathology of Colorectal Cancer ........................................................................... 13 1.2.4 The Dukes and TNM Staging Systems .......................................................................... 16 1.2.5 Prevention, Prognosis and Treatment of Colorectal Cancer........................................ 19 1.3 Biomarkers in Colorectal Cancer ......................................................................................... 23 1.4 CerS5, Ceramide Synthases and Sphingolipid Metabolism in CRC ..................................... 26 1.5 TRIM28 and the TRIM Family .............................................................................................. 31 1.6 The Tumour Microenvironment ......................................................................................... 36 1.6.1 Fibroblasts and Cancer-Associated Fibroblasts ........................................................... 39 1.7 Project Aims ........................................................................................................................ 41 iv Chapter 2 Materials and Methods ................................................................................................ 42 2.1 Introduction ........................................................................................................................ 43 2.2 Materials ............................................................................................................................. 43 2.2.1 Equipment List ............................................................................................................. 43 2.2.2 General Buffers ............................................................................................................ 45 2.2.3 Buffers for Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis ................ 46 2.2.4 Buffers for Western Blotting ........................................................................................ 47 2.2.5 Buffers for protein purification under denaturing conditions: .................................... 47 2.2.6 Cell Lysis Buffer ............................................................................................................ 49 2.2.7 General Reagents ......................................................................................................... 49 2.2.8 Commercial Antibodies ................................................................................................ 50 2.2.9 E.coli Protein Purification ............................................................................................. 52 2.3 TRIM28 and CerS5 Protein Expression and Antibody Validation Methods ........................ 53 2.3.1 Protein Expression ....................................................................................................... 53 2.3.2 Protocol for Expression of TRIM28 and CerS5 ............................................................. 54 2.3.3 Protein Purification using Immobilized metal ion affinity chromatography (IMAC) ... 55 2.3.4 SDS-PAGE ..................................................................................................................... 57 2.3.5 Western Blotting .......................................................................................................... 58 2.3.6 Lowry Assay .................................................................................................................. 59 2.4 Histological Methods .......................................................................................................... 60 2.4.1 Ethical Approval, Study Cohort and Sample Collection ............................................... 60 2.4.2 Tissue Microarrays (TMA) ............................................................................................ 61 2.4.3 Immunohistochemical Staining .................................................................................... 63 2.4.4 Immunohistochemical Analysis and Assessment......................................................... 64 2.4.5 Frozen Tissue Sectioning .............................................................................................. 65 2.4.6 Haematoxylin Staining for LCM .................................................................................... 66 v 2.4.7 Laser Capture Microdissection ..................................................................................... 67 2.4.8 Protein Extraction of Microdissected Material for Downstream Analysis .................. 71 2.5 Reverse-Phase Protein Microarrays .................................................................................... 72 2.5.1 RPPA immunostaining, image acquisition and data analysis ....................................... 74 2.6 Cell Culture Techniques and Protein Analysis ..................................................................... 77 2.7 Proteomic Network Analysis ............................................................................................... 83 2.8 Statistical Analysis ..............................................................................................................
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