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Chan, King Lun Michel. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 111-121). / Abstracts in English and Chinese. / THESIS ASSESSMENT COMMITTEE --- p.i / ACKNOWLEDGEMENTS --- p.ii / 摘要 --- p.iv / ABSTRACT --- p.v / LIST OF ABBREVIATIONS --- p.xi / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Aldehyde Dehydrogenase Superfamily / Chapter 1.1.1 --- Classification and Substrate Specificities of Aldehyde Dehydrogenases --- p.1 / Chapter 1.1.2 --- Multiple Functions of Aldehyde Dehydrogenases and their Roles in Metabolism --- p.4 / Chapter 1.1.3 --- Structural Organization of Aldehyde Dehydrogenases in view of their Catalytic Mechanism --- p.7 / Chapter 1.2 --- Antiquitin / Chapter 1.2.1 --- Discovery and Plant Antiquitins --- p.15 / Chapter 1.2.2 --- Animal Antiquitins --- p.17 / Chapter 1.2.3 --- Human Antiquitin Gene Mutations and Pyridoxine-dependent Seizures --- p.19 / Chapter 1.2.4 --- Previous Findings on Seabream Antiquitin --- p.20 / Chapter 1.3 --- Aims of Study --- p.22 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Materials / Chapter 2.1.1 --- "Subcloning, Expression and Purification of Human Antiquitin" --- p.24 / Chapter 2.1.2 --- Characterization of Human Antiquitin --- p.25 / Chapter 2.1.3 --- "Crystallization of Human Antiquitin, Diffraction Data Collection and Structure Determination" --- p.26 / Chapter 2.1.4 --- Mutational Studies of Human Antiquitin --- p.26 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- "Subcloning, Expression and Purification of Human Antiquitin" / Chapter 2.2.1.1 --- Subcloning of the Full-length Human Antiquitin cDNA --- p.27 / Chapter 2.2.1.2 --- Bacterial Expression of Recombinant Human Antiquitin --- p.29 / Chapter 2.2.1.3 --- Purification of Human Antiquitin --- p.30 / Chapter 2.2.2 --- Characterization of Human Antiquitin / Chapter 2.2.2.1 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.31 / Chapter 2.2.2.2 --- Size-exclusion Chromatography - Multi-angle Light Scattering --- p.32 / Chapter 2.2.2.3 --- Isoelectric Focusing --- p.33 / Chapter 2.2.2.4 --- pH-rate Profile --- p.33 / Chapter 2.2.2.5 --- Stability Studies --- p.34 / Chapter 2.2.2.6 --- Substrate Specificity Study --- p.35 / Chapter 2.2.3 --- "Crystallization of Human Antiquitin, Diffraction Data Collection and Structure Determination" / Chapter 2.2.3.1 --- Crystallization of Human Antiquitin --- p.36 / Chapter 2.2.3.2 --- Diffraction Data Collection and Model Building --- p.37 / Chapter 2.2.4 --- Mutational Studies of Human Antiquitin / Chapter 2.2.4.1 --- Preparation of Mutant Plasmids --- p.39 / Chapter 2.2.4.2 --- "Expression, Purification and Kinetics Studies of Mutants" --- p.39 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- "Subcloning, Expression and Purification of Human Antiquitin" --- p.43 / Chapter 3.2 --- Characterization of Human Antiquitin --- p.49 / Chapter 3.3 --- "Crystallization of Human Antiquitin, Diffraction Data Collection and Structure Determination" / Chapter 3.3.1 --- "Crystallization, Data Collection and Refinement" --- p.59 / Chapter 3.3.2 --- Human Antiquitin Structure --- p.63 / Chapter 3.4 --- Mutational Studies of Human Antiquitin --- p.75 / Chapter CHAPTER 4 --- DISCUSSION / Chapter 4.1 --- Characterization and Substrate Specificity of Recombinant Human Antiquitin --- p.83 / Chapter 4.2 --- Crystallization and Crystal Structure of Human Antiquitin / Chapter 4.2.1 --- Crystallization --- p.86 / Chapter 4.2.2 --- Overall structure --- p.86 / Chapter 4.2.3 --- Cofactor binding --- p.88 / Chapter 4.2.4 --- Substrate Binding and Catalysis --- p.91 / Chapter 4.3 --- Mutations in Human Antiquitin Gene and their Relationship with Pyridoxine-dependent Seizures --- p.95 / Chapter 4.4 --- Comparison of Antiquitin Gene and Other Aldehyde Dehydrogenases --- p.104 / Chapter CHAPTER 5 --- FUTURE PROSPECTS --- p.106 / LIST OF REFERENCES --- p.111 / APPENDIX --- p.122

Viruses that infect thermophilic Archaea are unique in both their structure and genetic makeup. The lemon-shaped fuselloviruses - which infect members of the order Sulfolobales, growing optimally at 80º C and pH 3 - are some of the most ubiquitous and best studied viruses of the thermoacidophilic Archaea. They provide a malleable and useful genetic tool for probing into the functions of their host, as well as the host responses to infection. Nonetheless, much about these viruses remains to be learned to further understand their morphological, genetic, and life cycle characteristics. In order to investigate these aspects of these Fuselloviridae, as well as their evolution, this work reports the isolation and characterization of a novel fusellovirus, Sulfolobus Spindle-shaped virus 10 (formerly SSV-L1). Genetic and genomic analyses highlight significant homology with both SSV8 and SSV9, as well as conservation of promoter elements within the Fuselloviridae. SSV10 encodes five ORFs with no homology within or outside of the Fuselloviridae, as well as a putatively functional Cas4-like ORF which may play a role in anti-CRISPR host evasion. Moreover, we demonstrate the ability of SSV10 to withstand mutation in a fashion consistent with mutagenesis in SSV1. Lastly, analysis of predicted protein structures from SSV10 provide new insights into virus-host interactions. These analyses help to expand our understanding of the viral life cycle while contextualizing the mutagenesis data presented in the following chapters.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 144-157). Also available online.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 144-157). Also available in print.

Thesis (M. Sc.)--York University, 1998. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 144-164). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pMQ39236.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Botany / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy