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Investigation of Immunity Related Genes in a Disease Host Using Applied Bioinformatics

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Investigation of Immunity Related Genes in a Disease Host Using Applied Bioinformatics Investigation of Immunity Related Genes in a Disease Host Using Applied Bioinformatics By: Ashraf Mohamed Ahmed A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy Under the supervision of Professor David Hornby The University of Sheffield Faculty of Science Department of Molecular Biology and Biotechnology June 2017 Acknowledgement I would like to express my sincere gratitude to my supervisor Prof. David Hornby for the continuous support of my Ph.D. study and related research, for his patience, motivation, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis. I could not have imagined having a better advisor and mentor for my Ph.D. study. I am grateful to Dr. Qaiser Sheikh for enlightening me the first glance of research and for the support, advice and guidance throughout my entire course of study especially in the first year. My sincere thanks also goes to Prof. Richard Pleass for the provision of the badger’s raw data, Liverpool life science UTC for supporting mealworm genome sequencing and Dr. Roy Chaudhuri, who provided me an opportunity to access the bioinformatics research facilities. Without their precious support it would not be possible to conduct this research. I thank my fellow lab colleagues in for the stimulating discussions, for the sleepless nights we were working together before deadlines, and for all the fun we have had in the last four years. Last but not the least, I would like to thank my family: my wife and to my children for supporting me spiritually throughout writing this thesis and my life in general. II Summary The economic burden and the health risks of bovine tuberculosis have led to an ongoing political and scientific debate on the control of the disease in badgers, perceived to be the main carrier, responsible for spreading the infection among cattle. Although culling of badgers has already been introduced in some parts of Britain, its efficacy remains unclear. Moreover, the implementation of alternative strategies, such as vaccination illustrate the need for a deeper understanding of the badger’s immune system. In addition, there is also a need to develop additional models and systems for studying the complexity of the immunological response in host organisms: simple organisms, including many flies and beetles are becoming increasingly popular in this respect. The first aim of this thesis is to obtain the nucleotide sequence of the badger transcriptome from peripheral blood cells, and to profile the immunity related genes, through critical evaluation of bioinformatics data extracted from public domain databases. In the second part of the thesis, the introduction of the yellow mealworm beetle, Tenebrio molitor, is developed through initiation of a genome sequencing project. It is hoped that this simple organism will provide insight into immune challenge and support the annotation of immune-related genes from more complex organisms, including the badger as well as providing an accessible model organism that is easy to manipulate in simpler laboratory environment such as schools. The sequencing of both the badger transcriptome from peripheral blood cells and the T. molitor genome generated large data sets. The transcriptome analysis resulted in the identification of 15967 transcripts related to 698 known immunity genes in different mammals. 1825 transcripts were found to match genes involved in tuberculosis pathogenesis. III It is believed that, these findings will improve our understanding of future attempts to both prevent and treat bovine tuberculosis. The determination of the T. molitor genome will facilitate and improve its use as a model organism to study infections. The genome data have been deposited and assembly of an annotated genome, although incomplete, is currently best described as “work in progress”. IV Abbreviations AHVLA Animal Health and Veterinary Laboratories Agency APCs Antigen presenting cells BCG Bacillus Calmette–Guérin vaccine BGI Beijing Genomics Institute BLAST NCBI Basic Local Alignment Search Tool Blastn BLAST nucleotide sequences Blastp BLAST protein sequences bp Base-pair bTB Bovine Tuberculosis cDNA Complementary DNA CMI Cell-mediated immunity COG Clusters of Orthologous Groups database DCs Dendritic cells DEFRA Department for Environment, Food & Rural Affairs dNTPs Deoxynucleotides ESTs Expressed sequence tags GB Gigabyte GO Gene Ontology IFN-γ interferon gamma IKB Immunome Knowledge Base IL interleukin KEGG Kyoto Encyclopaedia of Genes and Genomes LPS Lipid polysaccharide LTT Lymphocyte transformation test MHC Major histocompatibility complex mRNA Messenger RNA N50 A statistical measure of average length of a set of sequences NCBI National Centre for Biotechnology Information ncRNA Non-coding RNA NGS Next generation sequencing technology NK Natural killer cells NR (nr) Non-redundant database nt Nucleotide NT NCBI nucleotide database RBCT Randomised badger culling trail RNA Ribonucleic acid RNA-Seq RNA sequencing rpm Revolutions per minute snRNA small nuclear RNA SRA Sequence Read Archive of NCBI TNF Tumour necrosis factor tRNA transfer RNA V Table of Contents Acknowledgement ................................................................................................................................ II Summary .............................................................................................................................................. III Abbreviations ........................................................................................................................................ V 1 Introduction .................................................................................................................................. 2 1.1 The economic burden of bTB ................................................................................................ 2 1.2 Badgers as bTB carriers ......................................................................................................... 3 Badger Biology .............................................................................................................. 3 Tuberculosis in Badgers ................................................................................................. 4 Routes of bTB transmission ........................................................................................... 5 1.3 An overview of the mammalian immune system: ................................................................. 6 Pathogenesis: Cellular progression of infection ............................................................ 6 Innate immunity ............................................................................................................ 8 Adaptive immunity ...................................................................................................... 10 Major histocompatibility complex class I and II........................................................... 11 Humoral Immune Response ........................................................................................ 12 Cellular immune response ........................................................................................... 13 1.4 Immune response to TB and bTB ........................................................................................ 14 Intradermal route ........................................................................................................ 14 Endobronchial route ................................................................................................... 14 Response to vaccination .............................................................................................. 15 Mycobacterium and Macrophages.............................................................................. 15 Cell-mediated immune response in mycobacterial infection ...................................... 16 1.5 Current control strategies of bTB in badgers ...................................................................... 17 Badger culling .............................................................................................................. 17 Badger and cattle vaccination ..................................................................................... 18 1.6 Contemporary experimental and computational approaches to understanding the transmission of TB .......................................................................................................................... 20 Gene transcription ...................................................................................................... 21 Transcriptome analysis ................................................................................................ 27 Advantages of transcriptome analysis ......................................................................... 29 Limitations of transcriptome analysis.......................................................................... 31 The principles of RNA-Seq technology ........................................................................ 32 VI Advantages of RNA-Seq technology ............................................................................ 33 1.7 Transcriptome assembly and annotation ............................................................................ 34 Assembly ..................................................................................................................... 34 Annotation .................................................................................................................
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