泛基因组的概念来源于比较分析同一微生物物种的多个基因组。泛基因组分析已经被用于研究病原微生物基因组的变化，并且揭示了与菌株进化和宿主适应相关的特异基因。目前泛基因组研究主要集中在估计不同物种的泛基因组大小。但是对泛基因组的结构进行进化分析的生物信息方法还有待开发，以便研究不同基因在不同菌株的进化，比如基因的获得或者丢失，或者共同进化的基因簇。这样的分析方法可以把基因和菌株之间的表型关联起来，为进一步的生物学实验提供线索。 / 为了研究结核分枝杆菌种和分枝杆菌属泛基因组进化的规律并揭示其生物学意义，本论文开发了两种泛基因组数据分析的生物信息学方法。第一个是基于局部最大简约计算的祖先状态重构算法。它被用来分析结核分枝杆菌北京型全基因组水平插入／缺失序列（indels）的进化。分析表明基因组退化不仅塑造了该物种不同亚种的形成，并且也塑造了同一亚种不同亚型的分化，比如北京型。该分析还找出了北京型全基因组水平的RD区域和各种被中断的基因，这些基因可能同北京型的毒力进化相关。同时，该算法提供了另一个理解简约分析的视角；该视角可以把统计分析引入到该算法中。本论文提出的第二个模型是基于泛基因组进化的基因聚类模型。通过计算泛基因组中不同基因家族的分布频率，结合基于图论的聚类算法，该模型可以找出泛基因组中共同进化的基因聚类。对分枝杆菌属的泛基因组进行聚类分析发现了不同类别的基因簇，它们与不同分枝杆菌种的表型进化相关。这些结果说明了，一方面结核分枝杆菌在进化过程中丢失大量环境相关的基因；另一方面，它可能通过水平基因转移获得一些基因，特别是PE/PPE基因家族。因此，结核分枝杆菌可能是通过不断的基因组收缩，从一个环境菌种进化为与宿主共进化的病原菌。 / 总地来说，上面的两种方法能够被有效地用于结核分枝杆菌种和分枝杆菌属的泛基因组分析。 将来的工作可以考虑进一步引进随机模型；同时需要建立分枝杆菌的泛基因组数据库，以面对大规模测序的需求。 / Comparative analysis of multiple genomes of the same microbial species has led to the concept of pangenome to characterize the variations of gene content in different strains and to study their relationship to strain phenotype variations. Pangenome studies of microbial pathogens have identified strain-specific genes that may play roles in the evolution and adaptation of the pathogens. In previous studies, much attention was paid to estimate the size of the pangenome of different microbial species. But it is also important to develop bioinformatic methods for analyzing the evolution of the pangenome of a species, such as gene gain and loss or coevolution of clusters of genes, which may help to associate genotype variations with phenotype variations of a microbial species, and thus provides biological insights for further studies. / In this thesis, to analyze the pangenome consisting of complete mycobacterial genomes from public database and additional five Mycobacterium tuberculosis (MTB) Beijing genotype genomes sequenced by our own project, two bioinformatic approaches have been developed. The first is a local parsimony ancestral state reconstruction method, which was used to analyze genome-wide indels evolution of the MTB Beijing genotype. The key finding was that reductive evolution shaped the formation of not only different MTB species, but also different subspecies or genotypes, such as the Beijing genotype, for which genome-wide deletions of large RDs and disruption of individual genes were identified. This finding might have implications for the virulence evolution of the Beijing genotype. The method also provides an alternative perspective to understand parsimony analysis in phylogenetics, which can be used to incorporate statistical analysis into the method. / The second approach developed is a pangenome phyletic model for analyzing the coevolution of genes in the pangenome of a microbial species. This phyletic model calculates coevolution scores of gene frequencies in a pangenome. And graph-based clustering is used to identify coevolved clusters of genes. Applying this method to the genus Mycobacterium helped us to identify various gene clusters, from conserved core clusters of housekeeping genes to species-specific clusters, including genes related to pathogenesis. The key finding was that different MTB species have arose from their mycobacterial ancestor mainly by loss of many environmental related genes. On the other hand, gain of genes has also occurred within the MTB genomes, especially the clusters of the PE/PPE genes. This finding implied that the MTB species have undergone reductive evolution from an environmental species to adapt to and coevolve with their specific hosts. / In conclusion, the two methods were shown to be powerful in analyzing the pangenome of the MTB species and also of the Mycobacterium genus, and have provided useful insights into their genome and virulence evolution for further studies, including both pathogenesis related genes and genotyping genetic markers. Future works in that direction is to introduce stochastic models of gene evolution into these two methods. Finally, this work indicated that pangenome modeling is critical and can provide a good starting point for comprehensive pangenome sequencing of mycobacteria. Therefore, a database of Mycobacterial genomes for integrative pangenome annotation and evolutionary analysis should be developed. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhou, Haokui. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 121-135). / Abstracts also in Chinese.

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Hon, Ching Yi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 144-157). / Abstracts in English and Chinese. / Examination Committee List --- p.i / Acknowledgements --- p.ii / Abstract --- p.iv / 摘要 --- p.vi / Table of Contents --- p.viii / List of Tables --- p.xiv / List of Figures --- p.xv / List of Abbreviations --- p.xvii / List of Chemicals --- p.xx / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Mycobacterium tuberculosis --- p.1 / Chapter 1.2 --- Tuberculosis --- p.2 / Chapter 1.3 --- Pathogenesis of TB --- p.3 / Chapter 1.3.1 --- Mycobacterial entry to the host macrophage --- p.3 / Chapter 1.3.2 --- Modulation of the host endocytic pathway --- p.4 / Chapter 1.3.2.1 --- The fusion between lysosome and mycobacterial phagosomeis blocked --- p.4 / Chapter 1.3.2.2 --- The endosomal pH of mycobacterial phagosome is preserved --- p.5 / Chapter 1.3.2.3 --- Mtb successfully mediates host cell apoptosis inhibition --- p.6 / Chapter 1.3.3 --- Cell migration and granuloma formation --- p.7 / Chapter 1.4 --- Insights from the complete genome sequence of Mtb H37Rv --- p.9 / Chapter 1.4.1 --- PE protein family --- p.9 / Chapter 1.4.2 --- PPE protein family --- p.10 / Chapter 1.5 --- Interaction between PE and PPE proteins --- p.11 / Chapter 1.5.1 --- Integrated bioinformatics prediction --- p.11 / Chapter 1.5.2 --- In vitro interaction studies of PE25/PPE41 protein complex --- p.12 / Chapter 1.5.3 --- Structural characterization of PE/PPE complex as revealed by PE25/PPE41 crystal structure --- p.14 / Chapter 1.6 --- Biological roles of PE and PPE proteins --- p.15 / Chapter 1.6.1 --- Immunological roles of PE family proteins --- p.15 / Chapter 1.6.2 --- Immunological roles of PPE family proteins --- p.16 / Chapter 1.6.3 --- Immunological roles of PE/PPE protein complex --- p.16 / Chapter 1.7 --- Sub-cellular localization of PE and PPE proteins --- p.17 / Chapter 1.8 --- PE and PPE as exported proteins --- p.18 / Chapter 1.8.1 --- Association of Mtb secreted proteins to pathogenesis of tuberculosis --- p.18 / Chapter 1.8.2 --- Exported PE and PPE proteins --- p.19 / Chapter 1.9 --- Differential expression of PE and PPE genes --- p.20 / Chapter 1.10 --- Objective of project --- p.21 / Chapter CHAPTER 2 --- "CLONING, EXPRESSION, PURIFICATION AND VERIFICATION OF PE15 AND PPE20 AS COMPLEX" --- p.24 / Chapter 2.1 --- Materials --- p.24 / Chapter 2.1.1 --- Bacterial expression plasmids for expression of PE or PPE proteins --- p.24 / Chapter 2.1.2 --- E. coli strains --- p.27 / Chapter 2.1.3 --- Reagents and buffers for molecular cloning --- p.27 / Chapter 2.1.4 --- Reagents and buffers for E. coli protein expression --- p.29 / Chapter 2.1.5 --- Buffers for protein purification --- p.30 / Chapter 2.2 --- Methods --- p.31 / Chapter 2.2.1 --- Molecular cloning --- p.31 / Chapter 2.2.1.1 --- Primers --- p.31 / Chapter 2.2.1.2 --- Gene Amplification by Polymerase Chain Reaction (PCR) --- p.33 / Chapter 2.2.1.3 --- Agarose gel electrophoresis --- p.34 / Chapter 2.2.1.4 --- Extraction and purification of DNA from agarose gel by QIAquick Gel Extraction Kit --- p.34 / Chapter 2.2.1.5 --- Restriction digestion of DNA --- p.35 / Chapter 2.2.1.6 --- Purification of DNA by QIAquick PCR Purification Kit --- p.36 / Chapter 2.2.1.7 --- Ligation of DNA and expression vector to produce recombinant plasmid --- p.37 / Chapter 2.2.1.8 --- Transformation of plasmid into E. coli competent cell --- p.38 / Chapter 2.2.1.9 --- PCR Screening of recombinant clones --- p.39 / Chapter 2.2.1.10 --- Plasmid DNA purification using the QIAprep Spin Miniprep Kit --- p.40 / Chapter 2.2.1.11 --- DNA sequencing --- p.41 / Chapter 2.2.2 --- Expression of PE15 and PPE20 proteins --- p.41 / Chapter 2.2.2.1 --- Small scale protein expression of PE 15 and PPE20 proteins --- p.41 / Chapter 2.2.2.2 --- Small scale co-expression of PE 15 and PPE20 proteins --- p.42 / Chapter 2.2.2.3 --- Protein solubility analysis --- p.43 / Chapter 2.2.2.4 --- Large scale expression of PE and PPE proteins --- p.43 / Chapter 2.2.3 --- SDS-PAGE --- p.44 / Chapter 2.2.4 --- Bradford assay --- p.45 / Chapter 2.2.5 --- Protein purification of PE15 --- p.46 / Chapter 2.2.5.1 --- Sonication and extraction of proteins --- p.46 / Chapter 2.2.5.2 --- Protein purification of PE15 by gutathione-sepharose affinity chromatography --- p.46 / Chapter 2.2.5.3 --- Protein purification of PE15 by re-binding to glutathione-sepharose --- p.47 / Chapter 2.2.5.4 --- Protein purification of PE15 by size exclusion chromatography --- p.47 / Chapter 2.2.5.5 --- Protein purification of GST-PE15 --- p.48 / Chapter 2.2.6 --- Protein purification of PPE20(PPE) --- p.49 / Chapter 2.2.6.1 --- Sonication and extraction of proteins --- p.49 / Chapter 2.2.6.2 --- Protein purification of PPE20(PPE) by glutathione-sepharose affinity chromatography --- p.49 / Chapter 2.2.6.3 --- Protein purification of PPE20(PPE) by re-binding to glutathione-sepharose --- p.49 / Chapter 2.2.6.4 --- Protein purification of PPE20(PPE) by size exclusion chromatography --- p.50 / Chapter 2.2.6.5 --- Protein purification of GST-PPE20(PPE) --- p.50 / Chapter 2.2.7 --- Verification of PEPPE protein complex --- p.50 / Chapter 2.2.7.1 --- Size exclusion chromatography --- p.50 / Chapter 2.2.7.2 --- Cross-linking --- p.50 / Chapter 2.2.7.3 --- Pull-down assay --- p.51 / Chapter 2.3 --- Results --- p.52 / Chapter 2.3.1 --- Construction of bacterial expression plasmids for PE15 and PPE20 genes --- p.52 / Chapter 2.3.2 --- Expression of PE15 and PPE20 proteins --- p.54 / Chapter 2.3.2.1 --- Small scale protein expression of PE15 --- p.55 / Chapter 2.3.2.2 --- Small scale protein expression of PPE20 --- p.56 / Chapter 2.3.2.3 --- Small scale co-expression of PE15 and PPE20 proteins --- p.60 / Chapter 2.3.3 --- Large scale purification of PE15 and PPE20(PPE) proteins --- p.66 / Chapter 2.3.3.1 --- Large scale purification of PE15 --- p.66 / Chapter 2.3.3.2 --- Large scale purification of GST-PE15 --- p.68 / Chapter 2.3.3.3 --- Large scale purification of PPE20(PPE) --- p.69 / Chapter 2.3.3.4 --- Large scale purification of GST-PPE20(PPE) --- p.70 / Chapter 2.3.4 --- Verification of PE15/PPE20 complex --- p.71 / Chapter 2.3.4.1 --- Size exclusion chromatography --- p.71 / Chapter 2.3.4.2 --- Cross-linking study --- p.74 / Chapter 2.3.4.3 --- Pull-down assay --- p.77 / Chapter 2.4 --- Discussion --- p.79 / Chapter 2.4.1 --- Expression of PE15 and PPE20 proteins --- p.79 / Chapter 2.4.2 --- Co-expression of PE15 and PPE20 proteins --- p.81 / Chapter 2.4.3 --- Prediction of PE/PPE interaction --- p.82 / Chapter 2.4.4 --- Study ofPE15 and PPE20(PPE) interaction --- p.83 / Chapter CHAPTER 3 --- PRELIMINARY X-RAY ANALYSIS OF PPE20(PPE) PROTEIN CRYSTAL --- p.86 / Chapter 3.1 --- Materials --- p.86 / Chapter 3.1.1 --- Crystallization screening kits --- p.86 / Chapter 3.1.2 --- Crystallization chemicals --- p.86 / Chapter 3.2 --- Methods --- p.87 / Chapter 3.2.1 --- Crystallization screening using Phoenix´ёØ RE --- p.87 / Chapter 3.2.2 --- Optimization of PPE20(PPE) crystals by grid screening --- p.88 / Chapter 3.2.3 --- Optimization using Additive screen for PPE20(PPE) --- p.88 / Chapter 3.2.4 --- X-ray diffraction and data collection --- p.88 / Chapter 3.3 --- Results --- p.89 / Chapter 3.3.1 --- Crystallization screening --- p.89 / Chapter 3.3.2 --- Crystallization optimization --- p.90 / Chapter 3.3.3 --- Preliminary X-ray diffraction analysis --- p.92 / Chapter 3.3.4 --- Attempts to solve the phase by molecular replacement --- p.96 / Chapter 3.4 --- Discussion --- p.97 / Chapter CHAPTER 4 --- ISOLATION OF INTERACTING PARTNERS OF PE15 AND PPE20(PPE) FROM HUMAN MACROPHAGE U-937 --- p.99 / Chapter 4.1 --- Materials --- p.99 / Chapter 4.1.1 --- Mammalian cell line --- p.99 / Chapter 4.1.2 --- Mammalian cell growth medium --- p.99 / Chapter 4.1.3 --- Reagents and buffers for mammalian cell culture --- p.100 / Chapter 4.1.4 --- Reagents and buffers for mass spectrometry sample preparation --- p.100 / Chapter 4.2 --- Methods --- p.101 / Chapter 4.2.1 --- U-937 cell culturing --- p.101 / Chapter 4.2.1.1 --- Thawing U-937 cells --- p.101 / Chapter 4.2.1.2 --- Monitoring cell growth --- p.102 / Chapter 4.2.1.3 --- Cell differentiation --- p.102 / Chapter 4.2.1.4 --- Cell Harvesting --- p.103 / Chapter 4.2.2 --- In-vitro pull-down to identify interacting partners of PE15 or PPE20(PPE) --- p.103 / Chapter 4.2.2.1 --- Preparation of cellular proteins from U-937 cells --- p.103 / Chapter 4.2.2.2 --- Pre-clearing of U-937 supernatant --- p.104 / Chapter 4.2.2.3 --- Pull-down of U-937 cellular proteins with immobilized GST-PE15 --- p.104 / Chapter 4.2.2.4 --- Pull-down of U-937 cellular proteins with immobilized GST-PPE20(PPE) --- p.106 / Chapter 4.2.2.5 --- SDS-PAGE analysis --- p.106 / Chapter 4.2.2.6 --- Silver staining --- p.106 / Chapter 4.2.3 --- Mass- Spectrometry --- p.107 / Chapter 4.2.3.1 --- De-staining of silver stained gel spots --- p.107 / Chapter 4.2.3.2 --- Trypsin digestion --- p.108 / Chapter 4.2.3.3 --- Peptide Extraction --- p.108 / Chapter 4.2.3.4 --- Desalting and concentration of peptide mixture --- p.109 / Chapter 4.3 --- Results --- p.110 / Chapter 4.3.1 --- U-937 differentiation --- p.110 / Chapter 4.3.2 --- In-vitro pull-down --- p.113 / Chapter 4.3.2.1 --- Pull-down with immobilized GST-PE15 --- p.113 / Chapter 4.3.2.2 --- Pull-down with immobilized GST-PPE20(PPE) --- p.116 / Chapter 4.3.2.3 --- Mass spectrometry identification of protein --- p.120 / Chapter 4.4 --- Discussion --- p.122 / Chapter 4.4.1 --- Differentiation of U-937 --- p.122 / Chapter 4.4.2 --- Isolation of PE15 and PPE20(PPE) interacting partners from U-937 --- p.125 / Chapter CHAPTER 5 --- CONCLUSION AND FUTURE PERSPECTIVES --- p.133 / Chapter 5.1 --- Conclusion --- p.133 / Chapter 5.2 --- Future perspectives --- p.137

Leung, Siu Sing. / Thesis M.Phil. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 92-97). / Abstracts also in Chinese. / Title from PDF title page (viewed on 25, October, 2016).

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