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Environmental Influences on Endothelial Gene Expression

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Environmental Influences on Endothelial Gene Expression ENDOTHELIAL CELL GENE EXPRESSION John Matthew Jeff Herbert Supervisors: Prof. Roy Bicknell and Dr. Victoria Heath PhD thesis University of Birmingham August 2012 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT Tumour angiogenesis is a vital process in the pathology of tumour development and metastasis. Targeting markers of tumour endothelium provide a means of targeted destruction of a tumours oxygen and nutrient supply via destruction of tumour vasculature, which in turn ultimately leads to beneficial consequences to patients. Although current anti -angiogenic and vascular targeting strategies help patients, more potently in combination with chemo therapy, there is still a need for more tumour endothelial marker discoveries as current treatments have cardiovascular and other side effects. For the first time, the analyses of in-vivo biotinylation of an embryonic system is performed to obtain putative vascular targets. Also for the first time, deep sequencing is applied to freshly isolated tumour and normal endothelial cells from lung, colon and bladder tissues for the identification of pan-vascular-targets. Integration of the proteomic, deep sequencing, public cDNA libraries and microarrays, delivers 5,892 putative vascular targets to the science community. These analyses identify Endothelial Specific Molecule 1 as a pan vascular target and lysyl oxidase-like 2 as putative novel vascular target. It is envisioned vascular targets and angiogenesis genes in this data will destroy or inhibit tumour vessel growth without the side effects manifest with current clinical regimens. Dedicated to Robin and Wendy ACKNOWLEDGEMENTS A big thank you to everyone who has helped me along the way. Obviously thanks for the support and advice given from my supervisor Roy Bicknell and Vicky Heath (co- supervisor). Thank you to several people and groups who have given me the data to analyse through their hard work and great experimentation. Particularly Andreas Bikfalvi and his group for providing embryonic proteomic data, Patricia Lalor for liver endothelial cells (and Ben James), Xiaodong Zhuang and Forhad Ahmed for providing endothelial cell isolated RNA for deep sequencing, qPCRs and to Peter Noy and Vicky Heath for other experimental assistance. Thank you to the Plate-forme Protéomique de la Génopole Toulouse Midi-Pyrénées group for proteomics data, Genepool Edinburgh for 454 and Illumina sequencing data and the University of Birmingham technology hub for SOLiD4 sequencing data. Thank you to Miss Teresa Smithers, B.Sc. PGCE MIfL, for professionally proof reading, checking grammar and spelling (www.c2btuition.co.uk/). Thank you to Dov Stekel and Francesco Falciani for early inspiration and opportunity. Thank you for kind support from family, Olga Kolomeitz and Molly Herbert. Contents CHAPTER 1; INTRODUCTION ....................................................................................... 1 1.1 Introduction ............................................................................................................... 1 1.2 The endothelial cell and angiogenesis ...................................................................... 1 1.3 Selective barrier and inflammation ........................................................................... 1 1.4 Blood vessel formation ............................................................................................. 2 Figure 1.4.1 Overview of tumour angiogenesis process ............................................. 4 Figure 1.4.2 Sprouting and intussusceptive forms of angiogenesis ............................ 5 1.5 Tumour angiogenesis ................................................................................................ 6 1.6 bFGF and VEGF signalling, the most potent angiogenesis initiators and important growth factors for tumour growth ................................................................................... 6 1.7 Treatment of cancer and anti-angiogenics ................................................................ 7 Table 1.7.1 A list of potential anti-angiogenic therapy targets ................................. 11 1.8 Tumour micro-environment and endothelial cell gene expression repertoires ....... 13 1.9 Treatment of cancer and vascular targeting ............................................................ 14 1.10 Reasons for vascular targeting .............................................................................. 15 1.11 A recent history of vascular target discovery ....................................................... 15 1.12 Silica pellicle ......................................................................................................... 15 1.13 Chemically coupling membrane and extracellular proteins to biotin esters ......... 16 1.14 Serial Analyses of Gene Expression (SAGE) ....................................................... 16 1.15 cDNA or Expressed Sequence Tag (EST) gene discovery ................................... 18 1.16 Differentiating physiological and pathological angiogenesis ............................... 19 1.17 Phage display technology and vascular zip codes ................................................ 22 1.18 DNA and sequencing technologies ....................................................................... 24 1.19 Sequencing of DNA/RNA .................................................................................... 24 1.20 Quick history of DNA ........................................................................................... 24 1.21 Maxam-Gilbert's early method of sequencing (1st generation) ............................ 26 Figure 1.21.1 Overview of Maxam-Gilbert's sequencing process ............................ 27 1.22 Sanger method of sequencing (1st generation) ..................................................... 28 Figure 1.22.1 Sanger sequencing overview .............................................................. 29 1.23 First generation to second generation ................................................................... 30 1.24 Illumina (Solexa) .................................................................................................. 30 Figure 1.24.1 Illumina sequencing, bridge amplification ......................................... 31 Figure 1.24.2 Illumina, sequencing by synthesis using dye terminators .................. 32 1.25 Roche 454 titanium ............................................................................................... 33 1.26 Applied Biosystems SOLiD sequencing and RNA-seq ........................................ 33 Figure 1.26.1 Overview of RNAseq protocol (A); poly-A mRNA isolation............ 35 Figure 1.26.2 Overview of RNAseq protocol (B); fragmentation, cDNA synthesis 36 Figure 1.26.3 Overview of RNAseq protocol (C); emulsion PCR and sequencing . 37 Figure 1.26.4 Overview of SOLiD colour-space sequencing process ...................... 38 Figure 1.26.5 SOLiD colour space decoding ............................................................ 39 1.27 Transcriptome RNA-seq or deep sequencing ....................................................... 39 1.28 Need for new targets is the driving force behind work ......................................... 41 Figure 1.28.1 Newspaper article relating to the expense of some current treatments ................................................................................................................................... 41 1.29 Chapter 2, Overview; characterisation and identification of vascular targets in an embryonic system. ........................................................................................................ 42 1.30 Chapter 3 combining 1st and 2nd generation sequenced cDNA libraries to identify vascular targets.............................................................................................................. 42 1.31 Chapter 4; using RNA-seq deep sequencing to find putative pan-vascular targets ....................................................................................................................................... 42 1.32 Chapter 5; identifying vascular targets in lung cancer using microarray data from multiple patient tumour endothelium ............................................................................ 42 CHAPTER 2; CHARACTERISATION AND IDENTIFICATION OF VASCULAR TARGETS IN AN EMBRYONIC SYSTEM ................................................................... 43 2.1 Introduction ............................................................................................................. 43 2.2 Principle component analyses ................................................................................. 44 Figure 2.2.1 Principle Component Analyses of normalized protein counts for CIKL and CTW ................................................................................................................... 46 Figure 2.2.2 Principle Component Analyses of the CIKL pool ...............................
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