Cheung Shiu-fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 131-139). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.iii / 摘要 --- p.vi / TABLE OF CONTENTS --- p.ix / LIST OF FIGURES --- p.xvi / LIST OF TABLES --- p.xviii / ABBREVIATIONS --- p.xix / PUBLICATION --- p.xxi / AWARD --- p.xix / Chapter SECTION 1. --- INTRODUCTION OF HEPATOCELLULAR CARCINOMA --- p.1 / Chapter 1.1 --- Epidemiology of Hepatocellular Carcinoma --- p.1 / Chapter 1.2 --- Etiologies of HCC --- p.1 / Chapter 1.2.1 --- Hepatitis B Virus and Hepatitis C Virus --- p.2 / Chapter 1.2.2 --- Aflatoxins and Alcohol --- p.3 / Chapter 1.3 --- Major Diagnostic and Prognostic Markers of HCC --- p.4 / Chapter 1.3.1 --- Biochemical Tumor Markers --- p.5 / Chapter 1.3.2 --- Clinico-pathological Features of HCC --- p.6 / Chapter SECTION 2. --- THE MITOCHONDRION --- p.7 / Chapter 2.1 --- Structure of the Mitochondrial Genome --- p.9 / Chapter 2.1.1 --- Nicotinamide Adenine Dinucleotide Dehydrogenase --- p.10 / Chapter 2.1.2 --- Cytochrome b --- p.10 / Chapter 2.1.3 --- Cytochrome c Oxidase --- p.11 / Chapter 2.1.4 --- ATP Synthase --- p.11 / Chapter 2.1.5 --- Ribosomal RNA --- p.11 / Chapter 2.1.6 --- Transfer RNA --- p.12 / Chapter 2.1.7 --- Displacement Loop --- p.12 / Chapter 2.2 --- Replication of Mitochondrial DNA --- p.17 / Chapter 2.3 --- Transcription of Mitochondrial DNA --- p.17 / Chapter SECTION 3. --- PHYSIOLOGY OF MITOCHONDRIA --- p.19 / Chapter 3.1 --- Energy Production by Oxidative Phosphorylation (OXPHOS) --- p.19 / Chapter 3.2 --- Programmed Cell Death: Apoptosis --- p.22 / Chapter 3.3 --- Morphology of Mitochondria in Hepatocytes --- p.25 / Chapter SECTION 4. --- MUTATIONS OF MITOCHONDRIAL DNA --- p.26 / Chapter 4.1 --- Special Terms Used in This Study --- p.26 / Chapter 4.1.1 --- Somatic Mutations and Polymorphisms --- p.26 / Chapter 4.1.2 --- Homoplasmic and Heteroplasmic Mutations --- p.26 / Chapter 4.2 --- Factors Causing High Mutation Frequency in mtDNA --- p.27 / Chapter 4.2.1 --- Presence of Reactive Oxygen Species --- p.27 / Chapter 4.2.2 --- Lack of Protective Histories --- p.28 / Chapter 4.2.3 --- Limited DNA Repair Mechanism --- p.29 / Chapter 4.3 --- Theories of Homoplasmic Mutations --- p.31 / Chapter 4.3.1 --- Replicative Advantage on Mutated mtDNA Sequence Selection --- p.31 / Chapter 4.3.2 --- Random Mutagenesis and Segregation --- p.32 / Chapter 4.4 --- MtDNA Mutations in Mitochondrial Disease and Aging --- p.33 / Chapter 4.5 --- MtDNA Deletions in Cancer --- p.34 / Chapter 4.6 --- Somatic Mutations of MtDNA in Various Cancers --- p.35 / Chapter 4.6.1 --- Frequencies of Somatic Mutations --- p.35 / Chapter 4.6.2 --- Distribution of Somatic Mutations in mtDNA --- p.36 / Chapter 4.7 --- Somatic Mutations of Mitochondrial DNA in HCC --- p.37 / Chapter SECTION 5. --- OBJECTIVES OF THIS STUDY --- p.44 / Chapter SECTION 6. --- MATERIALS AND METHODS --- p.46 / Chapter 6.1 --- Patients and Samples Collection --- p.46 / Chapter 6.2 --- DNA Extraction from Liver Tissues --- p.46 / Chapter 6.3 --- Amplification of Mitochondrial DNA by Polymerase Chain Reaction --- p.51 / Chapter 6.3.1 --- Design of Primers --- p.51 / Chapter 6.3.2 --- PCR Conditions and Contents --- p.54 / Chapter 6.3.3 --- Assessment of PCR Products by Agarose Gel Electrophoresis --- p.54 / Chapter 6.4 --- Purification of PCR Products --- p.54 / Chapter 6.5 --- Cyclesequencing of Mitochondrial DNA --- p.55 / Chapter 6.5.1 --- Design of Primers --- p.55 / Chapter 6.5.2 --- PCR Contents and Cycle Sequencing Procedures --- p.56 / Chapter 6.6 --- Purification of Sequencing Products --- p.56 / Chapter 6.7 --- Sequence Analysis by Automated Sequencer --- p.57 / Chapter 6.7.1 --- Preparation of Polyacrylamide Gel --- p.57 / Chapter 6.7.2 --- Sequence Analysis by Automated Sequencer --- p.58 / Chapter 6.7.3 --- "Search for Sequence Variants, Polymorphisms and Somatic Mutations" --- p.58 / Chapter 6.8 --- Further Studies on mtDNA Mutations --- p.59 / Chapter 6.8.1 --- Sequence Analysis in Buffy Coat --- p.59 / Chapter 6.8.2 --- Detection of the Presence of Somatic mtDNA Mutations in Plasma --- p.59 / Chapter 6.8.3 --- Frequency of Mutations in Two Nucleotide Repeat Sequences --- p.60 / Chapter 6.9 --- Clinical Data and Statistical Analysis --- p.61 / Chapter 6.9.1 --- Clinical and Pathological Data --- p.61 / Chapter 6.9.2 --- Statistical Analysis --- p.61 / Chapter SECTION 7. --- RESULTS --- p.63 / Chapter 7.1 --- Sequence Analysis of the Entire Mitochondrial Genome --- p.63 / Chapter 7.1.1 --- Sequence Variants and Polymorphisms --- p.63 / Chapter 7.1.2 --- Somatic Mutations --- p.71 / Chapter 7.2 --- Study of Mitochondrial Sequence in Lymphocytes --- p.78 / Chapter 7.3 --- Detection of Tumor DNA in Serum --- p.78 / Chapter 7.4 --- Analysis of Nucleotide Repeat Sequences --- p.79 / Chapter 7.4.1 --- General Results --- p.79 / Chapter 7.4.2 --- Statistical Analysis --- p.84 / Chapter SECTION 8. --- DISCUSSION --- p.89 / Chapter 8.1 --- Comparative Analysis of mtDNA Mutations with Two Previous HCC Studies --- p.89 / Chapter 8.1.1 --- Number of Cases and Region Studied --- p.89 / Chapter 8.1.2 --- Number and Distribution of Mutations in Normal Controls --- p.89 / Chapter 8.1.3 --- Number of Somatic Mutations --- p.90 / Chapter 8.1.4 --- Distribution of Somatic Mutations --- p.91 / Chapter 8.2 --- Similarities of Somatic mtDNA Mutations in This Study with Other Cancer Types --- p.93 / Chapter 8.2.1 --- Frequency and Distribution of Somatic Mutations --- p.93 / Chapter 8.2.2 --- Number of Homoplasmic Mutations --- p.93 / Chapter 8.3 --- Evaluation of Somatic Mutations of mtDNA in This Study --- p.96 / Chapter 8.3.1 --- Specificity of Somatic Mutations in Tumor Proved by Sequence Analysis in Lymphocytes --- p.96 / Chapter 8.3.2 --- Importance of Conserved Amino Acid Sequences with Other Species to the Presence of Somatic Mutations in Tumor --- p.96 / Chapter 8.3.3 --- Four Somatic Mutation Sites Are Detected in More than One Cancer Type --- p.101 / Chapter 8.3.4 --- Presence of Homoplasmic and Heteroplasmic Mutations --- p.101 / Chapter 8.3.5 --- Absence of Large-scale Deletions in Tumor Tissues --- p.102 / Chapter 8.4 --- Mutation Hotspots Region: Hypervariable Displacement-loop --- p.103 / Chapter 8.5 --- D310 Mononucleotide Repeats --- p.106 / Chapter 8.5.1 --- Description of D310 Mononucleotide Repeats --- p.106 / Chapter 8.5.2 --- Possible Causes of Varied Sequences at D310 --- p.106 / Chapter 8.5.3 --- Appearance of Nucleotide Repeats at D310 in Tumors --- p.107 / Chapter 8.5.4 --- Possible Outcomes of D310 Aberrations in mtDNA Replication and Transcription --- p.108 / Chapter 8.5.5 --- Comparison of D310 Alternations in HCC with Other Cancers --- p.109 / Chapter 8.6 --- Other Nucleotide Repeat Sequences --- p.112 / Chapter 8.6.1 --- The CA Dinucleotide Repeats --- p.112 / Chapter 8.6.2 --- Other Nucleotide Repeat Sequences Showing Genome Instability --- p.112 / Chapter 8.7 --- Evaluation of Somatic mtDNA Mutations as a Cancer Diagnostic Marker --- p.114 / Chapter 8.7.1 --- Coding Region --- p.114 / Chapter 8.7.2 --- D-loop Region --- p.114 / Chapter 8.7.3 --- D310 Nucleotide Repeats --- p.115 / Chapter 8.7.4 --- Possibility of Detecting Somatic Mutations in Serum --- p.116 / Chapter 8.8 --- Somatic mtDNA Mutations May Be a Prognostic Marker in HCC --- p.117 / Chapter 8.8.1 --- Possible Problems in Current Prognostic Factors --- p.117 / Chapter 8.8.2 --- Interpretation of Results --- p.117 / Chapter 8.8.3 --- Prognostic Values of Somatic Mutations at D310 --- p.118 / Chapter 8.9 --- Hypothesis of Somatic MtDNA Mutations on Tumorigenesis and Tumor Progression --- p.119 / Chapter 8.9.1 --- Somatic mtDNA Mutations Decline OXPHOS and May Inactivate Apoptotic Pathways --- p.119 / Chapter 8.9.2 --- Moderate Reactive Oxygen Species Production May Promote Mitosis --- p.120 / Chapter 8.10 --- Possible Appearance of Somatic Mutations in HCC with Chronic HBV Infection --- p.123 / Chapter 8.11 --- Possibility of HBx Protein Integration to MtDNA Mutations --- p.123 / Chapter 8.12 --- Conclusions --- p.125 / Chapter SECTION 9. --- LIMITATIONS AND FURTHER STUDIES --- p.127 / Chapter 9.1 --- Limitations and Improvements of Study --- p.127 / Chapter 9.1.1 --- Small Sample Size --- p.127 / Chapter 9.1.2 --- Sequence Analysis Method --- p.127 / Chapter 9.1.3 --- Fidelity of PCR Reactions and Long-range PCR Fragments --- p.128 / Chapter 9.2 --- Further Studies --- p.129 / Chapter SECTION 10. --- REFERENCES --- p.131

by Cheung Yuk Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves xx-xxviii). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.iii / Table of Content --- p.iv / Abbreviations / for Amino Acids --- p.viii / for Standard Genetic Code --- p.ix / for Units --- p.x / for Prefixes --- p.xi / for Terms commonly used in the report --- p.xii / List of Figures --- p.xiii / List of Tables --- p.xiv / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Epidemiology of Hepatitis B Virus (HBV) --- p.1 / Chapter 1.2 --- Relationship between Hepatitis B Virus and Hepatocellular Carcinoma --- p.2 / Chapter 1.3 --- Brief Description of HBV Genome --- p.2 / Chapter 1.4 --- Possible Roles of HBx in Hepatocellular Carcinoma --- p.4 / Chapter 1.5 --- Novel Interacting Partner of HBx - HBx-lnteracting Protein (XIP) --- p.6 / Chapter 1.6 --- Objective --- p.6 / Chapter Chapter 2 --- Methodology / Chapter 2.1 --- Information of the HBx and XIP Clones --- p.7 / Chapter 2.2 --- "Information of the Expression Vectors (pRSETA, 6xHis-pRSETA and pET8C)" --- p.7 / Chapter 2.3 --- Sub-Cloning of HBx and XIP into Different Vectors --- p.9 / Chapter 2.3.1 --- Design of Primers for Cloning of HBx and XIP into Different Vectors --- p.9 / Chapter 2.3.2 --- Polymerase Chain Reaction (PCR) Protocol --- p.12 / Chapter 2.3.3 --- Enzyme Digestion Reaction Protocol --- p.14 / Chapter 2.3.4 --- Ligation Protocol --- p.16 / Chapter 2.3.5 --- Preparation of Competent Cells --- p.17 / Chapter 2.3.6 --- Transformation --- p.18 / Chapter 2.3.7 --- Gel Extraction Protocol --- p.19 / Chapter 2.3.7.1 --- Life Technologies CONCERT´ёØ Rapid Gel Extraction System --- p.19 / Chapter 2.3.7.2 --- QIAGEN Gel Extraction Kit --- p.20 / Chapter 2.3.8 --- Plasmid Preparation Protocol --- p.22 / Chapter 2.3.8.1 --- Life Technologies CONCERT´ёØ Rapid Plasmid Minipreps --- p.22 / Chapter 2.3.8.2 --- QIAGEN Plasmid Maxi Kit --- p.23 / Chapter 2.4 --- Expression of HBx and XIP in E. coli Strain C41 (DE3) --- p.25 / Chapter 2.4.1 --- Transformation --- p.25 / Chapter 2.4.2 --- Expression of HBx and 6xHis-HBx in E. coli Strain C41 (DE3) --- p.26 / Chapter 2.4.3 --- Expression of XIP in E. coli Strain C41 (DE3) --- p.27 / Chapter 2.5 --- Preparation of Buffers for Chromatography and Circular Dichroism Spectrum Measurement --- p.28 / Chapter 2.6 --- Purification and Refolding of HBx and His-Tagged HBx --- p.28 / Chapter 2.6.1 --- Washing of HBx and His-Tagged HBx Inclusion Bodies --- p.28 / Chapter 2.6.2 --- His-Tagged HBx Purification by Affinity Chromatography --- p.29 / Chapter 2.6.3 --- HBx Purification by Size Exclusion Chromatography --- p.30 / Chapter 2.6.4 --- Refolding of HBx and His-Tagged HBx by Oxidative Dialysis --- p.30 / Chapter 2.7 --- Purification of XIP --- p.33 / Chapter 2.7.1 --- Screening of Chromatographic Conditions for the Purification of XIP --- p.33 / Chapter 2.7.2 --- XIP 1st Step of Purification by Hydrophobic Interaction Chromatography --- p.34 / Chapter 2.7.3 --- XIP 2nd step of Purification by Size Exclusion Chromatography --- p.34 / Chapter 2.8 --- Chemical Denaturation Experiment of HBx and XIP --- p.36 / Chapter 2.8.1 --- Preparation of Urea Buffers for the Chemical Denaturation of HBx --- p.37 / Chapter 2.8.2 --- Preparation of Different GdnHCI Buffer for the Chemical Denaturation of XIP --- p.38 / Chapter 2.8.3 --- Calculation for Chemical Denaturation Experiment --- p.39 / Chapter 2.8.3.1 --- Protein Concentration Calculation --- p.39 / Chapter 2.8.3.2 --- Residual Molar Elipticity Calculation --- p.39 / Chapter 2.8.3.3 --- Free Energy Change (ΔGu) Calculation --- p.40 / Chapter 2.9 --- Two-dimensional Heteronuclear Nuclear Magnetic Resonance (NMR) Experiment --- p.41 / Chapter 2.10 --- Interaction Confirmation between HBx and XIP --- p.42 / Chapter 2.10.1 --- "Transfection of pEGFP, pEGFP-HBx and pEGFP-XIP into HepG2" --- p.42 / Chapter 2.10.2 --- Yeast Two Hybrid System for Confirmation of HBx and XIP Interaction --- p.44 / Chapter 2.10.2.1 --- Preparation of Y187 Competent Cells --- p.44 / Chapter 2.10.2.2 --- Transformation of pGBKT7-HBx and pACT2-XIP into Y187 --- p.45 / Chapter 2.10.2.3 --- β-galactosidase Colony Lift Assay --- p.46 / Chapter Chapter 3 --- "Expression, Purification and Characterization of Hepatitis B Virus X Protein (HBx)" / Chapter 3.1 --- Introduction --- p.47 / Chapter 3.2 --- Construction of Recombinant HBx-pRSETA and 6xHis-HBx-pRSETA Plasmids --- p.48 / Chapter 3.3 --- Expression of 6xHis-HBx in E. coli C41 (DE3) using M9ZB Medium --- p.52 / Chapter 3.4 --- Expression of HBx in E. coli C41 (DE3) using M9ZB Medium --- p.54 / Chapter 3.5 --- Purification and Refolding of 6xHis-HBx Fusion Proteins --- p.56 / Chapter 3.6 --- Purification and Refolding of HBx Proteins --- p.60 / Chapter 3.7 --- Structural Characterization of Refolded HBx --- p.65 / Chapter 3.7.1 --- Introduction --- p.55 / Chapter 3.7.2 --- Experimental Analysis of HBx Secondary Structure --- p.66 / Chapter 3.7.3 --- Chemical Unfolding Experiment of HBx --- p.68 / Chapter 3.8 --- Discussion --- p.70 / Chapter 3.8.1 --- "HBx was Expressed, Purified and Characterized instead of 6xHis-HBx" --- p.71 / Chapter 3.8.2 --- High Concentration of DTT was used to Minimize Formation of HBx Aggregates --- p.72 / Chapter 3.8.3 --- Oxidative Refolding to Ensure Proper Disulfide Bond Formation --- p.73 / Chapter 3.8.4 --- Computational Prediction and Experimental Prediction of Secondary Structure of HBx --- p.75 / Chapter 3.9 --- Concluding Remarks --- p.77 / Chapter Chapter 4 --- "Expression, Purification and Characterization of HBx-lnteracting Protein (XIP)" / Chapter 4.1 --- Introduction --- p.78 / Chapter 4.2 --- Construction of Recombinant XIP-pET8C --- p.78 / Chapter 4.3 --- Expression of XIP in E. coli C41 (DE3) using M9ZB and M9 Mediums --- p.82 / Chapter 4.4 --- Screening of Chromatographic Conditions for the Purification of XIP --- p.83 / Chapter 4.4.1 --- Introduction --- p.83 / Chapter 4.4.2 --- Purification Details --- p.83 / Chapter 4.5 --- Purification of XIP by HiTrap Phenyl HP 5-ml Column --- p.87 / Chapter 4.6 --- Purification of XIP by HiLoad 26/60 Superdex 75 Prep Grade --- p.89 / Chapter 4.7 --- Structural Characterization of XIP --- p.92 / Chapter 4.7.1 --- CD Spectrum --- p.92 / Chapter 4.7.2 --- Chemical Denaturation Experiment of XIP --- p.93 / Chapter 4.7.3 --- Two-Dimensional Heteronuclear Nuclear Magnetic Resonance (NMR) Spectrum of 15N Labeled XIP --- p.95 / Chapter 4.8 --- Discussion --- p.97 / Chapter 4.8.1 --- Purification Method Development --- p.97 / Chapter 4.8.2 --- "Do Different Protein Cosolutes, Protein Stabilizers and Detergents Help XIP to Adopt a Stable Conformation?" --- p.99 / Chapter 4.9 --- Concluding Remarks --- p.101 / Chapter Chapter 5 --- In vivo Studies of HBx and XIP Interactions / Chapter 5.1 --- Investigation of Sub-Cellular Localization of HBx and XIP in Liver Cells --- p.102 / Chapter 5.1.1 --- Introduction --- p.102 / Chapter 5.1.2 --- "Construction of Recombinant HBx-pECFP-C1, HBx-pEGFP-C1, HBx-pEYFP-C1 and XIP-pECFP-C1, XIP-pEGFP-C1, XIP-pEYFP-C1" --- p.103 / Chapter 5.1.3 --- Transfection of pEGFP-C1 HBx and pEGFP-C1 XIP into HepG2 to Find Out HBx and XIP Sub-Cellular Localization --- p.106 / Chapter 5.1.3.1 --- Introduction --- p.107 / Chapter 5.1.3.2 --- Investigation of EGFP Proteins Expression using the Confocal Microscope and the Leica TCS Software --- p.108 / Chapter 5.1.4 --- Discussion and Future Prospects --- p.111 / Chapter 5.2 --- Interaction of HBx and XIP Studied by Yeast Two-Hybrid System --- p.113 / Chapter 5.2.1 --- Introduction --- p.113 / Chapter 5.2.2 --- Construction of Recombinant HBx-pGBKT7 and XIP-pACT2 Plasmids --- p.114 / Chapter 5.2.3 --- Confirmation of HBx and XIP Interaction by Yeast Two-Hybrid System --- p.117 / Chapter 5.2.4 --- Discussion --- p.121 / Chapter Chapter 6 --- Conclusion --- p.123 / Appendix I Sequence of HBx and XIP --- p.I / Chapter II --- Vector Sequences --- p.II / Chapter III --- Vector Maps --- p.VI / Chapter IV --- Electrophoresis Markers --- p.XI / Chapter V --- Agarose Gel Electrophoresis --- p.XII / Chapter VI --- SDS-PAGE Eectrophoresis --- p.XIII / Chapter VII --- Medium for Bacterial Culture --- p.XV / Chapter VIII --- Medium for Cell Culture --- p.XVII / Chapter IX --- Medium for Yeast Culture --- p.XVIII / Chapter X --- Buffers for Yeast Transformation --- p.XIX / Reference --- p.XX

Lam Wai-Chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves [64]-[73]). / Abstracts in English and Chinese. / Abstract (in English) --- p.i-ii / Abstract (in Chinese) --- p.iii -iv / Acknowledgements --- p.v / Table of contents --- p.vi -ix / List of Figures --- p.x / List of Tables --- p.x / Abbreviations --- p.xi -xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Hepatocellular Carcinoma (HCC) --- p.1-2 / Chapter 1.2 --- Major risk factors of HCC / Chapter (1) --- Hepatitis B Virus (HBV) --- p.2-4 / Chapter (2) --- Hepatitis C Virus (HCV) --- p.5-6 / Chapter (3) --- Cirrhosis --- p.6 / Chapter (4) --- Dietary alfatoxin B1 (AFB1) --- p.6 -7 / Chapter (5) --- Alcoholic consumption --- p.7 / Chapter (6) --- Iron overload --- p.8 / Chapter 1.3 --- Genetic aberrations in HCC --- p.8-9 / Chapter (1) --- Chromosomal loss --- p.10-13 / Chapter (2) --- Chromosomal gains --- p.13-15 / Chapter 1.4 --- roposed study --- p.15 / Chapter (1) --- Hypomethylation of heterochromatin in chromosome 1q copy number gain. --- p.16 / Chapter (2) --- ositional mapping on 1q21 - q22 by interphase cytogenetics. --- p.16-17 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Southern Blot Analysis for Satellite DNA Hypomethylation. --- p.18-19 / Chapter 2.1.2 --- ositional Mapping by Interphase Cytogenetics. --- p.19 -24 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Southern Blot Analysis for Satellite DNA Hypomethylation / Chapter (1) --- Extraction of high molecular weight DNA --- p.25 / Chapter (2) --- DNA digestion with methyl-sensitive restriction enzyme --- p.25 -26 / Chapter (3) --- Control for the complete DNA digestion. --- p.26 / Chapter (4) --- Southern Blotting. --- p.26 -27 / Chapter 2.2.2 --- ositional Mapping by Interphase Cytogenetics / Chapter (1) --- Yeast Artificial Chromosome (YAC) --- p.28 -29 / Chapter (i) --- YAC culturing --- p.29 -30 / Chapter (ii) --- YAC DNA extraction --- p.30 -31 / Chapter (iii) --- Inter-Alu-Polymerase Chain Reaction --- p.32 -33 / Chapter (2) --- -1 derived Bacterial Artificial Chromosome (PAC) --- p.34 / Chapter (i) --- AC culturing and DNA extraction --- p.34 -35 / Chapter (3) --- FISHrobe labeling by nick translation. --- p.35 / Chapter (4) --- FISHrobereparation --- p.36 / Chapter (5) --- Dot-blot analysis. --- p.36 -37 / Chapter (6) --- Verification of the YAC andACrobes by metaphase FISH --- p.37 / Chapter (7) --- Hybridization efficiency test --- p.38 / Chapter Chapter 3 --- Southern Blot Analysis for Satellite DNA Hypomethylation / Chapter 3.1 --- Introduction --- p.39 -40 / Chapter 3.2 --- Materials and Methods / Chapter (1) --- atients --- p.41 / Chapter (2) --- Mathyl-sensitive restriction enzyme digestion. --- p.42 / Chapter (3) --- Classical satellite 2 DNArobe labeling and hybridization. --- p.42 -43 / Chapter (4) --- Membrane washing and signal detection. --- p.43 / Chapter (5) --- Signal detection and reference ratio determination. --- p.43 -44 / Chapter (6) --- Comparative Genomic Hybridization (CGH) --- p.44 -45 / Chapter 3.3 --- Results / Chapter (1) --- Heterochromatin hypomethylation and 1q12 breakpoint. --- p.45 / Chapter (2) --- Heterochromatin hypomethylation in adjacent hepatitis Infected liver tissue. --- p.46 / Chapter 3.4 --- Discussion --- p.47-51 / Chapter Chapter4 --- ositional Mapping of 1q21 - q22 by Interphase Cytogenetics / Chapter 4.1 --- Introduction --- p.52-53 / Chapter 4.2 --- Materials and Methods / Chapter (1) --- atients --- p.53 / Chapter (2) --- YAC clones --- p.53 -54 / Chapter (3) --- AC clones --- p.55 / Chapter (4) --- Formalin-fixedaraffin-embedded tissue sections pretreatment. --- p.55 / Chapter (5) --- Hybridization --- p.56 / Chapter (6) --- Signal detection --- p.56 -57 / Chapter 4.3 --- Results / Chapter (1) --- Relative copy number gain on YAC examined. --- p.57 -59 / Chapter (2) --- AC findings --- p.60 / Chapter 4.4 --- Discussion --- p.60 -63 / References

Li Ching-wan. / Thesis submitted in: November 2001. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 104-130). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACTS (ENGLISH/CHINESE) --- p.iii / LIST OF FIGURES --- p.xi / LIST OF TABLES --- p.xiii / LIST OF ABBREVIATIONS --- p.xiv / Chapter CHAPTER1 --- INTRODUCTION / Chapter 1.1. --- Liver Cancer --- p.1 / Chapter 1.2. --- Hepatocellular Carcinoma --- p.1 / Chapter 1.2.1. --- Types of Liver Cancer --- p.1 / Chapter 1.2.2. --- Epidemiology --- p.4 / Chapter 1.2.2.1. --- Geographical Distribution --- p.4 / Chapter 1.2.2.2. --- Age and Gender Distribution --- p.8 / Chapter 1.2.3. --- Etiologic Factors --- p.9 / Chapter 1.2.3.1. --- Chronic Infection with Hepatitis B (HBV) and C (HCV) Viruses --- p.9 / Chapter 1.2.3.2. --- Aflatoxin B1 --- p.11 / Chapter 1.2.3.3. --- Alcohol --- p.12 / Chapter 1.2.3.4. --- Summary --- p.12 / Chapter 1.3. --- HCC in Hong Kong --- p.14 / Chapter 1.4. --- Role of Viral Hepatitis B in HCC --- p.17 / Chapter 1.4.1. --- HBV Genome --- p.17 / Chapter 1.4.2. --- Consequences of HBV DNA Integration --- p.17 / Chapter 1.4.2.1. --- HBV Integration --- p.17 / Chapter 1.4.2.2. --- Transactivation of Cellular Genes by HBV DNA --- p.19 / Chapter 1.4.2.3. --- Chromosomal DNA Instability --- p.20 / Chapter 1.5. --- Genetic Alterations in HCC --- p.21 / Chapter 1.5.1. --- Tumor Suppressor Gene --- p.21 / Chapter 1.5.2. --- Proto-oncogene --- p.23 / Chapter 1.5.3. --- Genetic Studies in HCC --- p.23 / Chapter 1.5.3.1. --- Loss of Heterozygosity (LOH) --- p.25 / Chapter 1.5.3.2. --- Comparative Genomic Hybridization (CGH) --- p.26 / Chapter 1.5.3.3. --- Array CGH --- p.26 / Chapter 1.5.4. --- Large-Scale Genetic Analysis in HCC --- p.27 / Chapter CHAPTER2 --- RATIONALE IN THIS STUDY --- p.35 / Chapter CHAPTER3 --- MATERIALS AND METHODS / Chapter 3.1. --- Patients and Materials --- p.38 / Chapter 3.1.1. --- DNA Extraction --- p.40 / Chapter 3.2. --- Loss of Heterozygosity Analysis on Chromosome 4q --- p.40 / Chapter 3.2.1. --- Microsatellite Markers --- p.41 / Chapter 3.2.2. --- Amplification of Target Sequences by PCR --- p.42 / Chapter 3.2.2.1. --- 5-end Labeling Primers --- p.42 / Chapter 3.2.2.2. --- Amplification of Target Sequences --- p.42 / Chapter 3.2.3. --- Denaturing Polyacrylamide Gel --- p.44 / Chapter 3.2.3.1. --- Electrophoresis --- p.44 / Chapter 3.2.4. --- Detection of Loss of Heterozygosity (LOH) --- p.45 / Chapter 3.2.5. --- Duplex PCR Analysis of Homozygous Deletion --- p.45 / Chapter 3.3. --- Amplification Analysis by Array-CGH --- p.46 / Chapter 3.3.1. --- Nick-Translation --- p.49 / Chapter 3.3.2. --- Hybridization --- p.49 / Chapter 3.3.3. --- Imaging and Data Analysis --- p.50 / Chapter 3.3.4. --- Determination of Normal Range for All Cases --- p.51 / Chapter 3.3.5. --- Assessment of Data Quality --- p.51 / Chapter 3.4. --- Statistical Analysis --- p.52 / Chapter CHAPTER4 --- RESULTS / Chapter 4.1. --- Loss of Heterozygosity Analysis on Chromosome 4q --- p.53 / Chapter 4.1.1. --- Region I of Smallest Common Deletion Region --- p.54 / Chapter 4.1.2. --- Region II of Smallest Common Deletion Region --- p.54 / Chapter 4.2. --- Amplification Analysis by Array-CGH --- p.62 / Chapter CHAPTER5 --- DISCUSSION / Chapter 5.1. --- LOH Analysis on Chromosome 4q --- p.73 / Chapter 5.1.1. --- LOH of Chromosome 4q in Various Cancers --- p.74 / Chapter 5.1.1.1. --- Hepatocellular Carcinomas --- p.74 / Chapter 5.1.1.2. --- Other Neoplasia --- p.76 / Chapter 5.1.2. --- Functional Studies on Chromosome 4 --- p.76 / Chapter 5.1.3. --- Putative Tumor Suppressors on Chromosome 4q --- p.80 / Chapter 5.1.3.1. --- Region I (4q27-q28.1) --- p.80 / Chapter 5.1.3.1.1. --- MAD2L1 (4q27) --- p.80 / Chapter 5.1.3.2. --- Region II (4q35.2) --- p.81 / Chapter 5.1.3.2.1. --- INGlL(4q35.1) --- p.81 / Chapter 5.1.3.2.2. --- FAT (4q34-q35) --- p.81 / Chapter 5.1.3.2.3. --- Caspase 3 (4q35) --- p.82 / Chapter 5.1.4. --- Limitation of this Study --- p.83 / Chapter 5.1.4.1. --- Markers --- p.83 / Chapter 5.1.4.1.1. --- Limitation of the Markers --- p.83 / Chapter 5.1.4.1.2. --- Location of the Microsatellite Markers --- p.83 / Chapter 5.1.4.2. --- Tissue Samples --- p.84 / Chapter 5.1.4.2.1. --- Normal Reference --- p.84 / Chapter 5.1.4.2.2. --- Pathologic Characterization --- p.85 / Chapter 5.1.5. --- Future Studies --- p.85 / Chapter 5.1.5.1. --- Improvement of the Experiment --- p.85 / Chapter 5.1.5.2. --- Extension of the Present Study --- p.86 / Chapter 5.2. --- Amplification Analysis by Array-CGH --- p.88 / Chapter 5.2.1. --- Amplicons Showing Amplification in HCC --- p.89 / Chapter 5.2.1.1. --- Locus of 17q23 --- p.89 / Chapter 5.2.1.1.1. --- D17S1670 --- p.89 / Chapter 5.2.1.1.2. --- RPS6KB1 --- p.91 / Chapter 5.2.1.2. --- Locus of 1q25-q31 --- p.92 / Chapter 5.2.1.2.1. --- LAMC2 --- p.92 / Chapter 5.2.1.3. --- Locus of 3q26.3 --- p.93 / Chapter 5.2.1.3.1. --- PIK3CA --- p.93 / Chapter 5.2.1.4. --- Locus of 8p22 --- p.94 / Chapter 5.2.1.4.1. --- CTSB --- p.94 / Chapter 5.2.1.5. --- Locus of 6q22 --- p.95 / Chapter 5.2.1.5.1. --- MYB --- p.95 / Chapter 5.2.1.6. --- Locus of 20ql3 --- p.96 / Chapter 5.2.1.6.1. --- CSE1L --- p.96 / Chapter 5.2.1.7. --- Locus of Ip36.2-p35.1 --- p.97 / Chapter 5.2.1.7.1. --- FGR --- p.97 / Chapter 5.2.1.8. --- Locus of 7q21.1 --- p.98 / Chapter 5.2.1.8.1. --- PGY1 --- p.98 / Chapter 5.2.2. --- Amplicons Showing Deletion in HCC --- p.99 / Chapter 5.2.2.1. --- Loss at 11ql3 and 14q32.3 --- p.99 / Chapter 5.2.3. --- Limitation of the Study --- p.100 / Chapter 5.2.3.1. --- Samples and Materials --- p.100 / Chapter 5.2.4. --- Further Study --- p.101 / Chapter 5.2.4.1. --- Confirmation of the Result in Various Levels --- p.101 / Chapter 5.2.4.2. --- Assessment of the Significant Losses on Chromosomes 11ql3 and 14ql3 --- p.101 / Chapter 5.2.5. --- Application of Microarray in Genetic Studies --- p.102 / Chapter 5.2.5.1. --- Deletion Analysis --- p.102 / Chapter 5.2.5.2 --- Tissue Microarray --- p.103 / Chapter 5.2.5.3. --- cDNA Microarray --- p.103 / Chapter chapter6 --- references --- p.104

by Ray, Kit Ng. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 112-121). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgments --- p.iv / Abbreviations --- p.x / List of Figures --- p.xii / List of Tables --- p.xiv / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Hepatitis B Virus (HBV) --- p.1 / Chapter 1.2 --- Hepatitis B Virus X Protein (HBx) --- p.5 / Chapter 1.2.1 --- The Genomic Structure of HBx --- p.5 / Chapter 1.2.2 --- The HBx Protein Structure --- p.6 / Chapter 1.2.3 --- Subcellular Localization of HBx --- p.7 / Chapter 1.2.4 --- Possible Functions of HBx --- p.8 / Chapter 1.3 --- Etiology of Hepatocellular Carcinoma (HCC) --- p.12 / Chapter 1.4 --- Relationship between HCC and HBx --- p.13 / Chapter 1.5 --- Aims of Study --- p.14 / Chapter 1.6 --- The Basis of Tet-On System --- p.15 / Chapter 1.7 --- The Basis of DNA Microarray --- p.18 / Chapter 1.8 --- The Basis of Two-Dimensional Electrophoresis --- p.20 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Construction of a Tet-On HBx Expressing Cell Model --- p.22 / Chapter 2.1.1 --- Cloning of HBx Gene into pTRE2 Vector --- p.22 / Chapter 2.1.1.1 --- PCR of HBx Gene --- p.22 / Chapter 2.1.1.2 --- Purification of the PCR Product --- p.23 / Chapter 2.1.1.3 --- Restriction Enzyme Digestion --- p.23 / Chapter 2.1.1.4 --- Ligation of HBx into pTRE Vector --- p.24 / Chapter 2.1.1.5 --- Transformation of the Ligation Product into Competent Cells --- p.24 / Chapter 2.1.2 --- Preparation of the Plasmid DNA --- p.24 / Chapter 2.1.2.1 --- DNA Sequencing of the Cloned Plasmid DNA --- p.25 / Chapter 2.1.3 --- Cell Culture of AML12 Cell Line --- p.26 / Chapter 2.1.4 --- Transfection of pTet-On Vector into AML12 Cells --- p.26 / Chapter 2.1.5 --- Selection of the Transfected AML12 Cells by G418 --- p.27 / Chapter 2.1.6 --- Single Clone Isolation --- p.27 / Chapter 2.1.6.1 --- Luciferase Assay for Selection of Highly Inducible Clones --- p.28 / Chapter 2.1.7 --- Second Transfection of pTRE-HBx Plasmid --- p.28 / Chapter 2.1.8 --- Selection of the Transfected Cells by Hygromycin --- p.29 / Chapter 2.1.9 --- Second Single Clone Isolation --- p.29 / Chapter 2.1.10 --- Total RNA Isolation --- p.29 / Chapter 2.1.11 --- DNase I Digestion --- p.30 / Chapter 2.1.12 --- First-Strand cDNA Synthesis --- p.31 / Chapter 2.1.13 --- RT-PCR of HBx Gene --- p.31 / Chapter 2.1.14 --- Northern Blotting --- p.32 / Chapter 2.1.15 --- Preparation of the Probe --- p.33 / Chapter 2.1.16 --- Northern Blot Hybridization --- p.33 / Chapter 2.1.17 --- 3H-Thymidine Incorporation Assay --- p.34 / Chapter 2.1.18 --- Analysis of Cell Cycle by Flow Cytometry --- p.35 / Chapter 2.2 --- Microarray Analysis of Differential Gene Expression upon HBx Induction --- p.35 / Chapter 2.2.1 --- Sample Preparation for Microarray Analysis --- p.35 / Chapter 2.2.2 --- Probe Labelling --- p.36 / Chapter 2.2.3 --- Microarray Hybridization --- p.37 / Chapter 2.2.4 --- RT-PCR of the Candidate Genes --- p.38 / Chapter 2.2.5 --- Northern Blot Analysis of the Candidate Genes --- p.39 / Chapter 2.3 --- Two-Dimensional (2D) Gel Electrophoretic Analysis --- p.40 / Chapter 2.3.1 --- Protein Sample Preparation for 2D Gel Electrophoresis --- p.40 / Chapter 2.3.2 --- First-Dimension Isoelectric Focusing (IEF) --- p.40 / Chapter 2.3.3 --- Second-Dimension SDS-PAGE --- p.41 / Chapter 2.3.4 --- Silver Stain of 2D Gel --- p.42 / Chapter 2.3.5 --- Mass Spectroscopic Analysis --- p.43 / Chapter 2.4 --- Subcellular Localization of HBx --- p.44 / Chapter 2.4.1 --- Cloning of HBx into Green Fluorescent Protein (GFP) Expression Vector --- p.44 / Chapter 2.4.2 --- Transfection of GFP-HBx --- p.44 / Chapter 2.4.3 --- Propidium Iodide (PI) Staining --- p.45 / Chapter 2.4.4 --- Mitochondria Staining --- p.45 / Chapter 2.4.5 --- Subcellular Localization Study using Epi-Fluorescent Microscopy --- p.45 / Chapter 2.5 --- Analysis of Mitochondrial Transmembrane Potential --- p.46 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Construction of Tet-On AML12 Cell Line of HBx Gene --- p.47 / Chapter 3.2 --- Characterization of the HBx-Expressing Cell Model --- p.53 / Chapter 3.2.1 --- 3H-Thymidine Proliferation Assay --- p.53 / Chapter 3.2.2 --- Cell Cycle Analysis --- p.55 / Chapter 3.3 --- Microarray Analysis of Differential Gene Expression Pattern upon HBx Induction --- p.57 / Chapter 3.4 --- Northern Blot Analysis and RT-PCR of the Candidate Genes --- p.65 / Chapter 3.5 --- Differential Protein Expression Pattern under HBx Induction --- p.70 / Chapter 3.6 --- Subcellular Localization of HBx --- p.77 / Chapter 3.7 --- Analysis of Mitochondrial Transmembrane Potential --- p.83 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Conditional HBx-Expressing Cell Model --- p.84 / Chapter 4.2 --- The Effects of HBx in Clone X18 --- p.86 / Chapter 4.2.1 --- Proliferative Effect of HBx --- p.86 / Chapter 4.2.2 --- Deregulation of G2/M Checkpoint by HBx --- p.86 / Chapter 4.3 --- Early Differential Gene Expression due to HBx Induction --- p.88 / Chapter 4.4 --- The Relationship of the Potential Candidate Genes and Cancer Development --- p.90 / Chapter 4.5 --- The Protein Expression Pattern due to HBx Induction --- p.93 / Chapter 4.6 --- The Subcellular Localization of HBx --- p.96 / Chapter 4.7 --- The Possible Involvement of HBx in Mitochondrial Transmembrane Potential --- p.98 / Chapter 4.8 --- Conclusions --- p.101 / Chapter 4.9 --- Future Prospects --- p.104 / Appendix --- p.107 / References --- p.112

Au Chi Chuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 157-169). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Table of Contents --- p.ii / Abstract --- p.v / 論文摘要 --- p.vii / Abbreviations --- p.viii / List of Figures --- p.ix / List of Tables --- p.x / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Hepatitis B virus and Hepatocellular carcinoma --- p.3 / Chapter 1.3 --- Pathogenesis of HBV related HCC --- p.6 / Chapter 1.4 --- Current screening test and tumor markers --- p.10 / Chapter 1.5 --- Expressed sequence tag (EST) sequencing --- p.13 / Chapter 1.6 --- Aim of the present study --- p.15 / Chapter 1.7 --- Characterization of KIAA0022 --- p.16 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Construction of liver HCC and normal counterpart libraries --- p.19 / Chapter 2.2 --- Plating out the human liver cDNA libraries --- p.19 / Chapter 2.3 --- PCR amplification of clones human liver cancer and the normal counterpart cDNA libraries --- p.21 / Chapter 2.4 --- Cycle sequencing of cloned human liver cancer and the normal counterpart cDNA libraries --- p.21 / Chapter 2.4.1 --- Dye-primer cycle sequencing (Pharmacia) --- p.21 / Chapter 2.4.1.1 --- Using Pharmacia LBKA.L.F. DNA sequencer --- p.21 / Chapter 2.4.1.2 --- Using Li-Cor 4200 Automated DNA sequencer --- p.22 / Chapter 2.4.2 --- Dye-terminator cycle sequencing (Pharmacia) --- p.22 / Chapter 2.5 --- Sequences analysis --- p.23 / Chapter 2.6 --- Cloning of full-length cDNA of KIAA0022 --- p.24 / Chapter 2.6.1 --- Amplification of KIAA0022 gene using PCR --- p.24 / Chapter 2.6.2 --- Purification of the PCR product --- p.25 / Chapter 2.6.3 --- Ligation --- p.25 / Chapter 2.6.4 --- One Shot® TOP 10 Chemical Transformation --- p.25 / Chapter 2.6.5 --- Small-scale preparation of the plasmid DNA --- p.26 / Chapter 2.6.6 --- Large-scale preparation of the plasmid DNA Table of Contents (continued) --- p.26 / Chapter 2.6.7 --- DNA sequencing of the full-length cDNA of KIAA0022 --- p.28 / Chapter 2.7 --- Northern Hybridization --- p.29 / Chapter 2.7.1 --- The Human multiple tissue Northern Blot --- p.29 / Chapter 2.7.2 --- Synthesis of the radiolabeled DNA probe --- p.29 / Chapter 2.7.3 --- Hybridization of the Northern blot --- p.30 / Chapter 2.8 --- Subcellular localization of KIAA0022 by tagging with green fluorescence protein (GFP) --- p.30 / Chapter 2.8.1 --- Amplification and purification of the KIAA0022 gene product --- p.30 / Chapter 2.8.2 --- Restriction enzymes digestion --- p.31 / Chapter 2.8.3 --- DNA ligation --- p.31 / Chapter 2.8.4 --- Preparation of the Escherichia coli competent cells for transformation --- p.31 / Chapter 2.8.5 --- Transformation of the plasmid DNA into competent Escherichia coli cells --- p.32 / Chapter 2.8.6 --- Small-scale preparation of the plasmid DNA --- p.32 / Chapter 2.8.7 --- Large-scale preparation of the plasmid DNA --- p.32 / Chapter 2.8.8 --- DNA sequencing of the cloned plasmid DNA --- p.33 / Chapter 2.8.9 --- Transfection --- p.33 / Chapter 2.8.10 --- Fluorescence microscopy examination --- p.33 / Chapter 2.9 --- Yeast two-hybrid screening assay --- p.34 / Chapter 2.9.1 --- "Cloning of the KIAA0022 gene into the yeast two-hybrid DNA-BD vector, pGBKT7" --- p.34 / Chapter 2.9.2 --- Small-scale transformation of pGBKT7-KIAA0022 plasmid --- p.34 / Chapter 2.9.2.1 --- Preparation of yeast competent cells --- p.34 / Chapter 2.9.2.2 --- Transformation of the pGBKT7-KIAA 0022 plasmid into the yeast strain PJ69-2A --- p.35 / Chapter 2.9.3 --- Screening a pretransformed library by yeast mating --- p.35 / Chapter 2.9.4 --- β -Galactosidase analysis - colony lift filter assay --- p.36 / Chapter 2.9.5 --- Analysis of yeast plasmid inserts using PCR and DNA sequencing --- p.37 / Chapter 2.9.5.1 --- PCR --- p.37 / Chapter 2.9.5.2 --- DNA sequencing --- p.37 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Results of ESTs sequencing in normal counterpart and HCC libraries --- p.38 / Chapter 3.1.1 --- The sequencing results of the normal counterpart cDNA clones --- p.38 / Chapter 3.1.2 --- Sequencing results of the human liver cancer cDNA clones --- p.41 / Chapter 3.1.3 --- The accuracy of the automated sequencing technique --- p.41 / Chapter 3.1.4 --- Catalogue of normal counterpart ESTs --- p.45 / Chapter 3.1.5 --- Catalogue of liver cancer ESTs --- p.47 / Chapter 3.2 --- Identification of genes differentially expressed in HCC using in silico method --- p.115 / Chapter 3.3 --- Sequence analysis of KIAA0022 --- p.121 / Chapter 3.3.1 --- Structural analysis of KIAA0022 --- p.121 / Chapter 3.3.2 --- Homology alignment --- p.122 / Chapter 3.4 --- Tissue distribution and expression profile of KIAA0022 using Northern blot analysis --- p.132 / Chapter 3.5 --- Subcellular localization of the KIAA0022 tagging by green fluorescence protein --- p.134 / Chapter 3.6 --- Yeast two-hybrid screening assay --- p.136 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Large-scale partial cDNA sequencing --- p.138 / Chapter 4.2 --- Characterization of ESTs --- p.139 / Chapter 4.3 --- Identification of genes differentially expressed in liver cancer using Poisson probability --- p.143 / Chapter 4.4 --- Characterization of KIAA0022 --- p.154 / Reference --- p.157 / Appendix --- p.170

Zhang, Bin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 154-187). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Xu, Jiaying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 98-107). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Leung Ho-yin. / Thesis submitted in: July 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 104-118). / Abstracts in English and Chinese. / Acknowlegements --- p.i / Abstract (English) --- p.ii / Abstract (Chinese) --- p.iv / "Table of Contents," --- p.vi / List of Figures --- p.xi / List of Tables --- p.xii / Abbreviation --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 . --- Cancer Incidences in Hong Kong --- p.2 / Chapter 1.2. --- Hepatocellular Carcinoma (HCC) --- p.2 / Chapter 1.3. --- "Etiological Risk Factors," --- p.7 / Chapter 1.3.1. --- Liver Cirrhosis / Chapter 1.3.2. --- Chronic Viral Hepatitis / Chapter 1.3.2.1. --- Hepatitis B Virus (HBV) / Chapter 1.3.2.2. --- Hepatitis C Virus (HCV) / Chapter 1.3.3. --- Dietary Aflatoxin B1 exposure / Chapter 1.3.4. --- Heavy Alcohol Consumption / Chapter 1.3.5. --- Hemochromatosis / Chapter 1.4. --- Genetic Aberration in HCC --- p.12 / Chapter 1.4.1. --- Chromosomal Gains / Chapter 1.4.2. --- Chromosome Losses / Chapter 1.5. --- Epigenetic Changes --- p.18 / Chapter 1.6. --- Aims of Thesis --- p.20 / Chapter Chapter 2 --- Materials and Methods --- p.22 / Chapter 2.1. --- Materials --- p.23 / Chapter 2.1.1. --- Culture of Cell Lines / Chapter 2.1.2. --- Preparation of Normal Human Metaphase / Chapter 2.1.3. --- DNA Extraction from Cell Lines / Chapter 2.1.4. --- DNA Extraction from Tissues / Chapter 2.1.5. --- DNA Extraction from Blood / Chapter 2.1.6. --- Nick Translation / Chapter 2.1.7. --- Dot Blot / Chapter 2.1.8. --- Probe Preparation / Chapter 2.1.9. --- Fluorochrome-conjugated antibodies / Chapter 2.1.10. --- Fluorescence Microscopy and Image Analysis / Chapter 2.1.11. --- Primer Labeling / Chapter 2.1.12. --- Polymerase Chain Reaction / Chapter 2.1.13. --- Gel Preparation / Chapter 2.1.14. --- Gel Electrophoresis / Chapter 2.2. --- Sample --- p.28 / Chapter 2.2.1. --- Patients / Chapter 2.2.2. --- Cell Lines / Chapter 2.3. --- Comparative Genomic Hybridization --- p.30 / Chapter 2.3.1. --- Method / Chapter 2.3.1.1. --- Preparation of Normal Human Metaphase / Chapter 2.3.1.2. --- DNA Extraction / Chapter 2.3.1.3. --- Nick Translation / Chapter 2.3.1.4. --- Labeling Efficiency / Chapter 2.3.1.5. --- Probe Preparation / Chapter 2.3.1.6. --- Slide Preparation / Chapter 2.3.1.7. --- Hybridization / Chapter 2.3.1.8. --- Post Hybridization Wash / Chapter 2.3.1.9. --- Image Capturing and Analysis / Chapter 2.3.1.10. --- Control Experiment / Chapter 2.4. --- Microsatellite Analysis --- p.46 / Chapter 2.4.1. --- Method / Chapter 2.4.1.1. --- Fluorescent-Labeled Polymorphic Markers / Chapter 2.4.1.1.1. --- Polymerase Chain Reaction / Chapter 2.4.1.1.2. --- Gel Preparation / Chapter 2.4.1.1.3. --- Gel Electrophoresis / Chapter 2.4.1.1.4. --- Data Analysis / Chapter 2.4.1.2. --- Radioisotope-Labeled Polymorphic Markers / Chapter 2.4.1.2.1. --- Primer Labeling / Chapter 2.4.1.2.2. --- Polymerase Chain Reaction / Chapter 2.4.1.2.3. --- Gel Preparation / Chapter 2.4.1.2.4. --- Gel Electrophoresis / Chapter 2.4.1.2.5. --- Autoradiography and Data Analysis / Chapter 3. --- Chapter 3 Genetic Imbalances on Chromosome 1 --- p.55 / Chapter 3.1. --- Introduction --- p.56 / Chapter 3.2. --- Methods --- p.57 / Chapter 3.2.1. --- Patients and Cell Lines / Chapter 3.2.2. --- CGH / Chapter 3.2.3. --- MSA with Fluorescent-labeled Polymorphic Markers / Chapter 3.2.4. --- Refinement of lp36 loss / Chapter 3.2.5. --- Investigation of Homozygous Deletion in lp36 / Chapter 3.3. --- Results --- p.63 / Chapter 3.3.1. --- CGH / Chapter 3.3.2. --- MSA on Primary HCC Cases / Chapter 3.3.3. --- Refinement of lp36 loss / Chapter 3.3.4. --- Investigation of Homozygous Deletion in lp36 / Chapter 3.3.5. --- CGH vs MSA / Chapter 3.4. --- Discussion --- p.74 / Chapter 4. --- Chapter 4 Genetic Imbalances on Chromosome 4q --- p.78 / Chapter 4.1. --- Introduction --- p.79 / Chapter 4.2. --- Methods --- p.82 / Chapter 4.2.1. --- Patients and Cell Lines / Chapter 4.2.2. --- CGH / Chapter 4.2.3. --- MSA with Radioisotope-labeled Polymorphic Markers / Chapter 4.3. --- Results --- p.86 / Chapter 4.3.1. --- CGH / Chapter 4.3.2. --- MSA / Chapter 4.3.2.1. --- MSA on Primary HCC cases / Chapter 4.3.2.2. --- MSA on In-house developed HCC cell lines / Chapter 4.3.2.3. --- Combined MSA Results / Chapter 4.4. --- Discussion --- p.94 / Chapter 5. --- Chapter 5 Proposed Future Studies --- p.99 / Chapter 5.1. --- "Microarray Analysis," --- p.101 / Chapter 5.2. --- Functional Studies --- p.102 / Chapter 6. --- Bibliography --- p.104

Hu Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 95-122). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract (in English) --- p.ii / Abstract (in Chinese) --- p.iv / Table of contents --- p.vi / List of figures --- p.x / List of tables --- p.xi / Abbreviations --- p.vii / Chapter CHAPTER ONE: --- INTRODUCATION --- p.1 / Chapter 1.1 --- Hepatocellular Carcinoma --- p.2 / Chapter 1.1.1. --- Epidemiology of HCC --- p.2 / Chapter 1.1.2. --- The major risk factors --- p.2 / Chapter 1.1.3. --- Management of HCC --- p.3 / Chapter 1.2 --- Mechanisms of multidrug resistance (MDR) in cancer cells --- p.4 / Chapter 1.2.1. --- Major mechanisms in reduced drug accumulation --- p.5 / Chapter 1.2.1.1. --- P-glycoprotein (P-gp) --- p.6 / Chapter 1.2.1.2. --- Multidrug Resistance-associated Protein (MRP) --- p.7 / Chapter 1.2.1.3. --- Other effluxes --- p.8 / Chapter 1.2.2. --- Inhibition of apoptotic signaling pathways --- p.11 / Chapter 1.2.2.1. --- TP53 and multidrug resistance --- p.11 / Chapter 1.2.2.2. --- Anti-oncogene PTEN and drug resistance --- p.13 / Chapter 1.2.2.3. --- Influence of BCL2 family on drug resistance --- p.14 / Chapter 1.3 --- The chemotherapeutic agent of doxorubicin --- p.15 / Chapter 1.4 --- Aims of study --- p.18 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.20 / Chapter 2.1 --- Cell culture --- p.21 / Chapter 2.1.1 --- Cell lines and cell culture --- p.21 / Chapter 2.1.2 --- Subculture --- p.23 / Chapter 2.1.3 --- Cryopreservation --- p.23 / Chapter 2.1.4 --- Recovery of cryopreserved culture --- p.24 / Chapter 2.1.5 --- Cell number counting --- p.24 / Chapter 2.2 --- MTT experiments --- p.26 / Chapter 2.2.1 --- Determination of cell seeding density --- p.26 / Chapter 2.2.2 --- Cytotoxic assay --- p.27 / Chapter 2.3 --- Spectral Karytyping (SKY) --- p.27 / Chapter 2.3.1 --- Pretreatment of chromosome slides for SKY --- p.28 / Chapter 2.3.2 --- Hybridization --- p.28 / Chapter 2.3.3 --- Detection --- p.29 / Chapter 2.4 --- Positional expression profiling --- p.30 / Chapter 2.4.1 --- RNA extraction --- p.32 / Chapter 2.4.2 --- Reverse transcription and cDNA labling --- p.34 / Chapter 2.4.3 --- Probe purification and hybridization --- p.34 / Chapter 2.4.4 --- Image acquisition and data analysis --- p.35 / Chapter 2. 5 --- Quantitative RT-PCR --- p.37 / Chapter 2.5.1 --- RNA extraction --- p.37 / Chapter 2.5.2 --- Primer design --- p.37 / Chapter 2.5.3 --- Reverse transcription --- p.37 / Chapter 2.5.4 --- Quantitative PCR --- p.39 / Chapter 2.6. --- Statistical analysis --- p.40 / Chapter CHAPTER 3 --- RESULTS --- p.43 / Introduction --- p.44 / Chapter 3.1 --- Doxorubicin resistance in HCC cell lines --- p.44 / Chapter 3.2 --- Candidate drug resistance genes --- p.56 / Chapter 3.3 --- The roles of chromosomal instability --- p.58 / Chapter 3.4 --- Candidate resistance genes identified in chromosome 10 --- p.69 / Chapter CHAPTER 4 --- DISCUSSION --- p.75 / Introduction --- p.76 / Chapter 4.1 --- In vitro cell models facilitate drug resistance investigations --- p.11 / Chapter 4.2 --- Aneuploidy and DX resistance --- p.78 / Chapter 4.3 --- The role of known resistance genes on chromosome 10 --- p.79 / Chapter 4.4 --- Identification of novel DX resistance genes on chromosome 10 --- p.80 / Chapter 4.5 --- Common drug resistance genes --- p.83 / Chapter 4.5.1. --- The roles of classical drug resistance --- p.85 / Chapter 4.5.2. --- Inhibition of apoptosis and deregulation of cell cycle --- p.86 / Chapter CHAPTER 5 --- PROPOSED FUTURE STUDIES --- p.90 / Chapter 5.1. --- Validate significant in vitro findings by clinical trials --- p.91 / Chapter 5.2. --- Molecular mechanisms in inactivation of ECHS1 in resistant cells --- p.92 / Chapter 5.3. --- Future utilization of cDNA microarray data --- p.93 / REFERENCES --- p.95 / PUBLICATION --- p.122