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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

Hui Wai Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 158-189). / Abstracts in English and Chinese. / Abstract / Acknowledgements / List of Tables / List of Figures / Table of Contents / Table of Contents / Chapter Chapter I: --- Introduction --- p.1 / Chapter I. --- Aim of Study --- p.1 / Chapter II. --- Literature Review --- p.3 / Chapter 1. --- Background --- p.3 / Chapter A. --- Epidemiology --- p.3 / Chapter B. --- Histopathology --- p.3 / Chapter C. --- Etiology --- p.4 / Chapter i. --- Environmental Factors --- p.5 / Chapter ii. --- Epstein-Barr Virus (EBV) Infection --- p.6 / Chapter iii. --- Genetic Factors --- p.9 / Chapter 2. --- Molecular Genetics of NPC --- p.11 / Chapter A. --- Genome-Wide Studies --- p.11 / Chapter i. --- Comparative Genomic Hybridization (CGH) --- p.11 / Chapter ii. --- Loss of Heterozygosity (LOH) Studies --- p.12 / Chapter iii. --- Homozygous Deletion Study --- p.12 / Chapter B. --- NPC-related Oncogenes and Tumor Suppressor Genes --- p.13 / Chapter i. --- Oncogenes --- p.13 / Chapter ii. --- Tumor Suppressor Genes --- p.14 / Chapter 3. --- Chromosome 14q and NPC --- p.19 / Chapter A. --- Tumor Suppressor Loci and Cancer-Related Genes on Chromosome14q --- p.20 / Chapter i. --- Tumor Suppressor Loci on Chromosome 14q --- p.20 / Chapter ii. --- Cancer-Related Genes on Chromosome 14q --- p.26 / Chapter 4. --- Chromosome 16q and NPC --- p.28 / Chapter A. --- Tumor Suppressor Loci and Candidate Tumor Suppressor genes on Chromosome16q --- p.28 / Chapter i. --- Tumor Suppressor Loci on Chromosome 16q --- p.28 / Chapter ii. --- Metastasis Suppressor Loci on Chromosome 16q --- p.34 / Chapter iii. --- Candidate Tumor Suppressor Genes on Chromosome 16q --- p.34 / Chapter Chapter II: --- Materials and Methods --- p.40 / Chapter I. --- Cell Lines and Xenografts --- p.40 / Chapter 1. --- Cell Lines --- p.40 / Chapter 2. --- Xenografts --- p.41 / Chapter 3. --- DNA Extraction --- p.42 / Chapter II. --- Patients and Biopsy Specimens --- p.44 / Chapter 1. --- Manual Microdissection --- p.44 / Chapter 2. --- Laser Captured Microdissection (LCM) --- p.46 / Chapter 3. --- DNA Extraction --- p.46 / Chapter III. --- Comprehensive Screening for Homozygous Deletion Regions on Chromosomes 14q32.12-32.33 and 16q23.1-24.3 in Human Cancers --- p.48 / Chapter 1. --- DNA of Human Cancer Cell Lines --- p.48 / Chapter 2. --- Sequence-Tagged Sites (STS) Markers --- p.48 / Chapter 3. --- Polymerase Chain Reaction (PCR) --- p.49 / Chapter IV --- . Investigation of Inactivation of Potential Tumor Suppressor Genes on Chromosome 14q32.12-32.33 and 16q23.1-24.3 --- p.58 / Chapter 1. --- Detection of Homozygous Deletion --- p.58 / Chapter 2. --- Expression Analysis --- p.58 / Chapter A. --- RNA Extraction --- p.58 / Chapter B. --- Reverse-Transcription (RT) PCR --- p.61 / Chapter i. --- DNase I Digestion --- p.62 / Chapter ii. --- First-strand cDNA Synthesis and RNase Digestion --- p.62 / Chapter iii. --- Reverse-Transcription (RT)-PCR --- p.63 / Chapter C. --- Real-Time RT PCR --- p.63 / Chapter 3. --- Methylation Analysis --- p.68 / Chapter A. --- Sodium Bisulfite Modification --- p.68 / Chapter B. --- Methylation-Specific PCR (MSP) --- p.69 / Chapter C. --- Bisulfite Sequencing --- p.70 / Chapter D. --- Combined Bisulfite Restriction Analysis (COBRA) --- p.75 / Chapter E. --- 5 -aza-2' -deoxycytidine Treatment --- p.76 / Chapter ChapterIII： --- Results --- p.78 / Chapter I. --- Comprehensive Screening for Homozygous Deletion Regions in Human Cancers --- p.78 / Chapter 1. --- Chromosome 14q32.12-3233 --- p.78 / Chapter 2. --- Chromosome 16q23.1-243 --- p.79 / Chapter II. --- Investigation of Inactivation of Potential Tumor Suppressor Genes in NPC --- p.86 / Chapter 1. --- Chromosome 14q --- p.86 / Chapter A. --- "WW Domain-Containing Protein, 45-kD （WW45)" --- p.86 / Chapter B. --- Apoptosis Stimulating Protein of p53（ASPP1) --- p.88 / Chapter 2. --- Chromosome 16q --- p.92 / Chapter A. --- WW Domain-Containing Oxidoreductase （WWOX) --- p.92 / Chapter i. --- Homozygous Deletion Screening of WWOX --- p.92 / Chapter ii. --- Expression of Aberrant Splicing Transcripts of WWOX in NPC --- p.94 / Chapter iii. --- Sequencing of WWOX Aberrant Transcripts --- p.95 / Chapter iv. --- Quantitative Analysis of WWOX Transcripts in NPC --- p.95 / Chapter v. --- Methylation Analysis --- p.99 / Chapter B. --- H-Cadherin (CDH13) --- p.102 / Chapter i. --- Analysis of H-cadherin Deletion on Cancer Cell Lines and Xenografts --- p.102 / Chapter ii. --- Expression Analysis of H-Cadherin by RT-PCR and Real-Time RT-PCR --- p.102 / Chapter iii. --- Analysis of Promoter Hypermethylation by Methylation-Specific PCR (MSP) and Bisulfite Sequencing in NPC Cell Lines and Xenografts --- p.104 / Chapter iv. --- Demethylation Study of H-Cadherin in C666-1 Cell Line --- p.105 / Chapter v. --- Methylation Analysis of H-Cadherin in Primary Tumors --- p.105 / Chapter vi. --- Methylation Analysis of H-Cadherin in Human Cancer Cell Lines --- p.106 / Chapter C. --- Myeloid Translocation Gene on Chromosome 16 (MTG16) --- p.113 / Chapter i. --- Deletion Analysis of MTG16 in Cancer Cell Lines and Xenografts --- p.113 / Chapter ii. --- Differential Expression of MTG16a and MTG16b Transcripts in NPC Cell Lines and Xenografts --- p.113 / Chapter iii. --- Methylation Analysis of MTG16b in NPC Cell Lines and Xenografts --- p.118 / Chapter iv. --- Sequencing of MTGl 6b RT-PCR Products --- p.119 / Chapter v. --- Demethylation Study of MTG16b in HK-1 Cell Line --- p.119 / Chapter vi. --- Promoter Methylation Analysis of MTG16b by MSP in Primary NPC and Cancer Cell Lines --- p.120 / Chapter Chapter IV: --- Discussion --- p.124 / Chapter I. --- Comprehensive Homozygous Deletion Screening of Chromosomes 14q32.12-32.33 and 16q23.1-24.3 in Human Cancer Cell Lines and Xenografts --- p.124 / Chapter II. --- Investigation of Candidate Tumor Suppressor Genes on Chromosome 14q in NPC --- p.128 / Chapter III. --- Alterations of Candidate Tumor Suppressor Genes on Chromosome 16q in NPC --- p.133 / Chapter 1. --- Expression of Aberrant Transcripts of WWOX in NPC --- p.133 / Chapter 2. --- Methylation-Associated Silencing of H-Cadherin and MTG16b in NPC --- p.140 / Chapter Chapter V: --- Conclusion --- p.154 / Chapter Chapter VI: --- References --- p.158

Ng Yeung Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 180-209). / Abstracts in English and Chinese. / acknowledgements --- p.i / abstract --- p.ii / abstract in chinese --- p.vi / table of contents --- p.ix / list of tables --- p.xiii / list of figures --- p.xi v / list of abbreviations --- p.xvi / Chapter 1 --- chapter1 introduction and literature review --- p.1 / Chapter 1.1 --- Introduction of brain tumors --- p.1 / Chapter 1.2 --- Oligodendroglial tumors (OTs) --- p.3 / Chapter 1.2.1 --- Oligodendroglioma (OD) and anaplastic oligodendroglioma (AOD) --- p.3 / Chapter 1.2.1.1 --- WHO's definition and grading --- p.3 / Chapter 1.2.1.2 --- "Incidence, age, sex distribution, tumor location and survival rate" --- p.3 / Chapter 1.2.1.3 --- Clinical presentation --- p.4 / Chapter 1.2.1.4 --- Macroscopy and histopathology --- p.4 / Chapter 1.2.1.5 --- Immunohistochemistry --- p.5 / Chapter 1.2.1.6 --- Treatment --- p.6 / Chapter 1.2.2 --- Oligoastrocytoma (OA) and anaplastic oligoastrocytoma (AOA) --- p.11 / Chapter 1.2.2.1 --- WHO's definition and grading --- p.11 / Chapter 1.2.2.2 --- "Incidence, age, sex distribution, tumor location and survival rate" --- p.12 / Chapter 1.2.2.3 --- Clinical features --- p.12 / Chapter 1.2.2.4 --- Macroscopy and histopathology --- p.12 / Chapter 1.3 --- Overview of Genetic and Epigenetic Aberrations of OTs --- p.14 / Chapter 1.3.1 --- Chromosomal and genetic aberrations in OTs --- p.14 / Chapter 1.3.2 --- Candidate regions and genes on 1 p --- p.15 / Chapter 1.3.3 --- Candidate regions and genes on 19q --- p.20 / Chapter 1.3.4 --- Other aberrations in WHO grade II OTs --- p.24 / Chapter 1.3.5 --- Progression-associated aberrations in ODs --- p.25 / Chapter 1.3.6 --- Chromosomal and genetic aberrations in OAs --- p.29 / Chapter 1.4 --- Correlation of genetic alterations with response to therapy and survival --- p.31 / Chapter 1.4.1 --- Response to PCV chemotherapy correlates with lp and combined lp/19q status in patients with AODs --- p.31 / Chapter 1.4.2 --- Survival of patients with AODs correlates with lp/19q status --- p.32 / Chapter 1.4.3 --- WHO grade II ODs behavior and lp/19q status --- p.32 / Chapter 1.4.4 --- Response to other therapies (temozolomide and radiotherapy) and lp/19q status in patients with ODs --- p.33 / Chapter 1.4.5 --- lp and 19q loss in OAs and diffuse astrocytomas --- p.34 / Chapter 1.5 --- Microarray-based expression profiling of OTs --- p.35 / Chapter 1.6 --- Description of p73 protein --- p.37 / Chapter 1.6.1 --- Introduction of p73 --- p.37 / Chapter 1.6.2 --- p73: gene structure and splicing variants --- p.37 / Chapter 1.6.3 --- Signaling in p73 --- p.40 / Chapter 1.6.4 --- Regulation ofp73 protein stability and transcriptional activity --- p.43 / Chapter 1.6.4.1 --- Regulation by DNA damage --- p.43 / Chapter 1.6.4.2 --- Regulation by oncogenes --- p.44 / Chapter 1.6.4.3 --- Interaction with viral proteins --- p.44 / Chapter 1.6.5 --- Role of p73 in the nervous system --- p.45 / Chapter 1.6.6 --- p73 in cancer --- p.45 / Chapter 1.6.6.1 --- p73 knockout mice --- p.45 / Chapter 1.6.6.2 --- Alteration of p73 expression in human cancers --- p.46 / Chapter 1.6.7 --- p73 and chemosensitivity --- p.50 / Chapter CHAPTER2 --- AIMS OF STUDY --- p.51 / Chapter CHAPTER3 --- MATERIALS AND METHODS --- p.53 / Chapter 3.1 --- Tumor and blood samples --- p.53 / Chapter 3.2 --- Cell culture --- p.53 / Chapter 3.3 --- DNA extraction from frozen tissues and blood samples --- p.54 / Chapter 3.4 --- Detection of allelic loss of chromosome lp --- p.58 / Chapter 3.4.1 --- LOH analysis --- p.58 / Chapter 3.4.2 --- Fluorescence in situ Hybridization (FISH) analysis on Paraffin and Frozen Sections --- p.60 / Chapter 3.6 --- DNA sequencing analysis --- p.62 / Chapter 3.7 --- Analysis of Methylation --- p.63 / Chapter 3.7.1 --- Bisulfite sequencing --- p.63 / Chapter 3.7.2 --- Methylation-specific polymerase chain reaction (MSP) --- p.66 / Chapter 3.8 --- Northern Blot analysis --- p.68 / Chapter 3.9 --- RNA isolation and cDNA preparation --- p.70 / Chapter 3.10 --- Laser microdissection and RNA extraction from microdissected tumor cells --- p.71 / Chapter 3.10.1 --- Conventional RT-PCR --- p.71 / Chapter 3.11 --- Primer design for TP73 and its isoforms --- p.74 / Chapter 3.12 --- Real-time RT-PCR --- p.77 / Chapter 3.12.1 --- Real-time RT-PCR for TP73 and its isoforms --- p.78 / Chapter 3.12.2 --- Real-time RT-PCR for KIAA0495 --- p.79 / Chapter 3.13 --- Statistical analyses --- p.81 / Chapter CHAPTER4 --- RESULTS --- p.82 / Chapter 4.1 --- Genes annotated in the minimally deleted regions --- p.82 / Chapter 4.2 --- Expression analyses of TP73 and its isoforms in ODs by quantitative real-time RT-PCR --- p.85 / Chapter 4.3 --- Methylation analysis of TP73 in ODs by methylation sensitive PCR (MSP) --- p.97 / Chapter 4.4 --- A rapid screen of candidate genes for aberrant expression in microdissected tumors --- p.100 / Chapter 4.5 --- Quantitative real-time RT-PCR of KIAA0495 gene --- p.103 / Chapter 4.6 --- Mutation analysis of KIAA0495 gene --- p.110 / Chapter 4.7 --- Methylation analysis of KIAA0495 in ODs by bisulfite sequencing…… --- p.112 / Chapter 4.8 --- Detection of allelic loss of lp by LOH analysis and interphase FISH --- p.121 / Chapter 4.9 --- Two-hit inactivation of KIAA0495 gene in ODs --- p.126 / Chapter 4.10 --- Tissue distribution of KIAA0495 gene --- p.130 / Chapter 4.11 --- Bioinformatics of KIAA0495 --- p.133 / Chapter CHAPTER5 --- DISCUSSION --- p.146 / Chapter 5.1 --- Expression analysis of TP73 and its isoforms in ODs by isoform-specific RT-PCR --- p.148 / Chapter 5.2 --- Methylation status ofTP73 in ODs --- p.153 / Chapter 5.3 --- A rapid screening of candidate genes for aberrant expressionin microdissected tumors --- p.156 / Chapter 5.4 --- Expression pattern of KIAA0495 mRNA in a large cohort of ODs --- p.157 / Chapter 5.5 --- No somatic mutation in coding region of KIAA0495 --- p.158 / Chapter 5.6 --- Methylation status of putative promoter region of KIAA0495 in ODs --- p.159 / Chapter 5.7 --- Status of chromosome lp in ODs --- p.161 / Chapter 5.8 --- Two-hit inactivation of KIAA0495 gene in ODs by promoter hypermethylation and allelic loss of lp --- p.162 / Chapter 5.9 --- Evaluation of expression of KIAA0495 gene as a marker for the response to chemotherapy and prognostic marker in patients with OTs --- p.164 / Chapter 5.10 --- Tissue distribution of KIAA0495 --- p.166 / Chapter 5.11 --- "KIAA0495 cDNA sequence, protein sequence and potential functional features" --- p.167 / Chapter 5.12 --- Candidate tumor suppressor genes on lp in other type of tumors with loss of lp --- p.171 / Chapter CHAPTER6 --- CONCLUSIONS --- p.174 / Chapter CHAPTER7 --- FUTURE STUDIES --- p.177 / Chapter CHAPTER8 --- REFERENCES --- p.180

全球人類健康的頭號殺手--腫瘤目前仍是難以攻克的醫學難題。腫瘤的發生是一個復雜的過程，主要由促癌基因的異常增多或激活及抑癌基因（TSG）的缺失或功能喪失的累積效果導致。近年來基於非基因序列改變所致基因表達水平變化的表觀遺傳學的研究進展表明，啟動子區CpG島甲基化所致的表觀遺傳沉默是抑癌基因轉錄失活的重要機制。Ras和Wnt信號轉導通路在癌病的發生和發展過程中均起到重要的作用，因此針對該兩種信號通路的拮抗因子的表觀遺傳調控及功能學研究將為我們提供有研究及應用前景的候選抑癌基因。 / 作為一種重要的原癌基因，Ras家族基因具有致癌活性的點突變及其導致的過度激活的Ras信號通路被發現廣泛存在於大約30%的人類腫瘤中。然而在一些缺乏Ras基因突變的腫瘤類型中，持續激活的Ras信號通路仍然普遍存在並具有重要作用，昭示著除了Ras基因點突變以外的信號轉導異常激活的機制。與GTP的結合可激活Ras，而RasGAP家族蛋白可通過水解GTP達到使Ras失活的作用。通過采用微陣列比較基因組雜交(aCGH)的實驗手段我們發現6p21.3染色體區具有半接合子缺失， 並於此區域發現了候選抑癌基因RASA5。在以往的研究報道中，RASA5被命名為SynGAP且其功能研究僅限於神經系統。我們的研究發現不同於RasGAP家族的其它基因RASA2-4，RASA5廣泛表達於人類正常器官組織中，並特異性地在腫瘤細胞，特別是鼻咽癌(NPC)，食管鱗狀上皮細胞癌(ESCC)和乳腺癌這些具有野生型Ras基因但Ras信號通路仍被過度激活的細胞中被表觀遺傳沉默。RASA5的異位表達可有效促進腫瘤細胞的雕亡，抑制腫瘤細胞的生長、遷移及“幹性（stemness）“。同時，使用siRNA敲除內源性RASA5可以激發細胞的克隆形成及上皮-間質(EMT)轉化。RASA5的抑癌功能是通過調低Ras-GTP水平並進而抑制其下遊信號通路的活性實現的。過量表達具有致癌活性的點突變的Ras或RasGAP結構域缺失均可部分逆轉這種抑癌作用。此項研究首次證明了RASA5的抑癌功能。 / Wnt/Dvl/β-catenin信號轉導通路在人類腫瘤中存在廣泛的異常激活。我們發現DACT (Dpr/Frodo)家族成員TUSC-T2的表觀遺傳沉默是一種普遍存在於人類腫瘤中的現象。TUSC-T2編碼一種胞質蛋白，外源性表達TUSC-T2可促進腫瘤細胞雕亡並導致腫瘤細胞的克隆形成能力下降。TUSC-T2可與Dvl蛋白結合並下調其活化水平，從而保護GSK-3β蛋白不被Dvl蛋白抑制。GSK-3β可與Axin及APC蛋白形成蛋白質復合物，該復合物可捕捉並降解細胞內信號分子β-catenin。TUSC-T2的過量表達可以抑制β-catenin的激活及其向細胞核內的富集，並進一步阻止β-catenin在細胞核內與Lef/Tcf轉錄因子家族的作用及下遊特定原癌基因，例如c-Myc, CCND1及Fibronectin的表達。因此TUSC-T2具有抑制腫瘤細胞增殖、遷移及上皮-間質(EMT)轉化的作用。 / 綜上所述，我們的研究結果表明RASA5及TUSC-T2是具有抑癌功能的Ras或Wnt/Dvl/β-catenin信號轉導通路抑制因子，其表觀遺傳沉默導致的轉錄失活對於腫瘤的發生發展具有重要意義。同時，針對這兩種抑癌基因的進一步研究將為我們提供富有應用前景的腫瘤標記物。值得註意的是，RASA5課題的研究開創性地闡明了Ras信號通路的拮抗因子的表觀遺傳沉默是一種Ras信號轉導通路於腫瘤細胞中異常激活的新機制。 / Cancer is the top killer of the world, as well as the medical problem difficult to overcome. The conversion of a normal cell to a cancer cell is usually caused by upregulation of oncogenes and downregulation of tumor suppressor genes (TSGs). Epigenetic silencing has been proved to be important in TSGs inactivation, often through methylation of CpG-rich promoter regions. Ras and Wnt signaling pathways are both important for the tumorigenesis, epigenetic and functional studies of antagonists to Ras and Wnt signaling would provide us with candidate TSGs. / Ras is a well-known oncogene. Aberrant mutations of Ras genes occur in approximately 30% of human tumors, causing constitutively activated Ras signaling. However, in certain types of tumors with wild type Ras genes, abnormally activated Ras signaling is still a common and critical event, suggesting alternative mechanisms for Ras signaling hyperactivation. Ras is active when it is bound to GTP, while the hydrolysis of bound GTP and inactivation of Ras is catalyzed by Ras GTPase activating proteins (RasGAPs). Using 1-Mb array CGH (aCGH), we refined a small hemizygous deletion at the 6p21.3 chromosome region that contains a RasGAP family member gene RASA5, which used to be named as SynGAP and studied only in the neuron systems. We demonstrated that RASA5, rather than other RasGAP family members RASA2-4, is broadly expressed in human normal tissues while frequently epigenetically silenced in multiple tumors, especially in certain tumor types such as nasopharyngeal (NPC), esophageal (ESCC) and breast carcinomas (BrCa) with wild-type Ras while Ras cascade is still constitutively active. Ectopic expression of RASA5 led to apoptosis, growth and migration inhibition, as well as ‘stemness’ repression of tumor cells. Meanwhile, knockdown of RASA5 by siRNA promoted the tumor cell colony formation as well as epithelial-mesenchymal transition (EMT). The tumor-suppressive function of RASA5 was exerted through downregulating Ras-GTP level and further inactivating Ras signaling. Such an inhibitory effect could be partially abrogated in the presence of mutated, activated Ras or by deletion of the RasGAP domain. For the first time, our study refined the role of RASA5 as a tumor suppressor. / Wnt/DVL/β-catenin signaling pathway is aberrantly activated in a wide range of human cancers. We identified a DACT (Dpr/Frodo) family member TUSC-T2 as an epigenetically downregulated gene in human tumors. TUSC-T2 encodes a punctate cytoplasmic protein. Ectopic expression of TUSC-T2 dramatically inhibited tumor cell colony formation in silenced tumor cell lines, mainly through inducing apoptosis. TUSC-T2 interacts and downregulates Dishevelled (Dvl) protein, thus protecting glycogen synthase kinase 3β (GSK-3β) from inactivation by Wnt/Dvl and allowing GSK-3β to form a complex with Axin and APC to promote the phosphorylation and proteasomal degradation of β-catenin. Overexpression of TUSC-T2 disrupted β-catenin activation and accumulation in nuclei, thus preventing its binding to transcription factors of the Lef/Tcf family. This caused the downregulation of β-catenin target oncogenes such as c-Myc, CCND1 and Fibronectin as well as the inhibition of tumor cell proliferation and migration. We also observed that TUSC-T2 could inhibit tumor cell EMT. / Taken together, our data demonstrate that RASA5 and TUSC-T2 are functional tumor suppressors epigenetically silenced in multiple tumors through acting as negative regulators of the Ras or Wnt/Dvl/β-catenin cancer pathways, and could be developed as promising biomarkers for human tumors. Of note, our study reveals that epigenetic silencing of the Ras antagonist represents a new mechanism responsible for Ras aberrant activation in cancers with wild-type Ras. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Fan, Yichao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 184-216). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / List of abbreviations --- p.ii-iii / List of tables --- p.iv / List of Figures --- p.v-vii / List of Publications --- p.viii-ix / Abstract in English --- p.x-xii / Abstract in Chinese --- p.xiii-xiv / Table of Contents --- p.xv / Chapter Chapter 1 --- Introduction and Literature Review --- p.1 / Chapter 1.1 --- Cancer epigenetics --- p.4 / Chapter 1.1.1 --- Epigenetic modifications --- p.5 / Chapter 1.1.1.1 --- DNA Methylation --- p.5 / Chapter 1.1.1.2 --- Histone modifications --- p.10 / Chapter 1.1.1.3 --- RNA interference --- p.14 / Chapter 1.1.1.4 --- Nucleosome positioning --- p.15 / Chapter 1.1.2 --- Epigenetic alteration induced Tumor suppressor genes (TSGs) silencing during carcinogenesis --- p.17 / Chapter 1.2 --- Epigenetic alterations in cancer pathways --- p.23 / Chapter 1.2.1 --- Brief introduction of cancer pathways --- p.23 / Chapter 1.2.2 --- Ras pathway --- p.25 / Chapter 1.2.2.1 --- Ras pathway and carcinogenesis --- p.25 / Chapter 1.2.2.2 --- Epigenetic regulation of RasGAP proteins in carcinogenesis --- p.28 / Chapter 1.2.2.3 --- Epigenetic silencing of other negative regulators of Ras signaling --- p.30 / RAS association domain family (RASSF) proteins --- p.30 / PTEN --- p.32 / Sprouty (SPRY) proteins --- p.33 / Chapter 1.2.2.4 --- Hypomethylation induced Ras oncogenes activation --- p.35 / Chapter 1.2.2.5 --- Ras mediates epigenetic regulation through feedback loop --- p.36 / Chapter 1.2.3 --- Wnt pathway --- p.43 / Chapter 1.2.3.1 --- Wnt signaling pathway and carcinogenesis --- p.43 / Chapter 1.2.3.2 --- Epigenetic silencing of negative regulators of Wnt signaling --- p.45 / Chapter 1.2.3.3 --- DACT family proteins and carcinogenesis --- p.48 / Chapter 1.3 --- Application of tumor specific epigenetic alterations as tumor biomarkers and therapeutic targets --- p.49 / Chapter 1.3.1 --- The potential and advantage of tumor specific epigenetic alterations used as tumor biomarkers and therapeutic targets --- p.49 / Chapter 1.3.2 --- Epigenetic-disrupted regulators of Ras signaling as tumor biomarkers and therapeutic targets --- p.50 / Chapter 1.3.3 --- Epigenetic-disrupted regulators of Wnt signaling as tumor biomarkers and therapeutic targets --- p.52 / Chapter Chapter 2 --- Aims of this study --- p.54 / Chapter 2.1 --- To identify epigenetically silenced candidate TSGs as antagonists to Ras or Wnt signaling --- p.55 / Chapter 2.2 --- To elucidate the functional of candidate TSGs --- p.56 / Chapter Chapter 3 --- Materials and Methods --- p.57 / Chapter 3.1 --- Cell lines, tumor samples and routine cell line maintenance --- p.57 / Chapter 3.2 --- Drug and stress treatments --- p.59 / Chapter 3.3 --- DNA and RNA extraction --- p.59 / Chapter 3.4 --- Semi-quantitative RT-PCR and Real time PCR --- p.60 / Chapter 3.5 --- Direct sequencing of PCR products --- p.67 / Chapter 3.6 --- CpG island analysis --- p.67 / Chapter 3.7 --- Bisulfite treatment --- p.67 / Chapter 3.8 --- Methylation-specific PCR (MSP) and bisulfite genomic sequencing --- p.68 / Chapter 3.9 --- Plasmid extraction --- p.69 / Chapter 3.9.1 --- Bacteria culture --- p.69 / Chapter 3.9.2 --- Mini-scale preparation of plasmid DNA --- p.70 / Chapter 3.9.3 --- Large-scale endotoxin-free plasmids extraction --- p.71 / Chapter 3.10 --- Construction of expression plasmids --- p.71 / Chapter 3.10.1 --- Gene cloning and plasmids construction of RASA5 --- p.71 / Chapter 3.10.2 --- Gene cloning and plasmids construction of TUSC-T2 --- p.74 / Chapter 3.11 --- Immunofluorescence Staining --- p.74 / Chapter 3.12 --- Colony formation assay --- p.76 / Chapter 3.13 --- Apoptosis assay --- p.77 / Chapter 3.14 --- Luciferase reporter assay --- p.78 / Chapter 3.15 --- Protein preparation and Western blot --- p.79 / Chapter 3.16 --- Ras Activity Assay --- p.80 / Chapter 3.17 --- Wound healing assay --- p.81 / Chapter 3.18 --- Matrigel invasion assay --- p.81 / Chapter 3.19 --- RNA Interference --- p.81 / Chapter 3.20 --- Statistical analysis --- p.82 / Chapter Chapter 4: --- Epigenetic disruption of Ras signaling through silencing of a Ras GTPase-activating protein RASA5 in human cancers --- p.83 / Chapter 4.1 --- Identification of RASA5 as a downregulated gene residing in the 6p21.3 deletion region --- p.86 / Chapter 4.2 --- RASA5 is widely expressed in human normal tissues but downregulated in tumor cell lines --- p.91 / Chapter 4.3 --- The tumor-specific downregulation pattern of RASA5 is unique in the RASA family genes --- p.95 / Chapter 4.4 --- RASA5 promoter CpG methylation resulted in its transcription inactivation --- p.96 / Chapter 4.5 --- Frequent methylation of RASA5 promoter in multiple primary tumors --- p.101 / Chapter 4.6 --- Cloning and characterization of human RASA5 --- p.104 / Chapter 4.7 --- RASA5 inhibits tumor cell clonogenicity through inducing apoptosis --- p.108 / Chapter 4.8 --- RasGAP domain is required for the tumor suppressive function of RASA5 --- p.111 / Chapter 4.9 --- Certain cancer types harbor wild type Ras but active Ras signaling, with RASA5 epigenetically silenced --- p.114 / Chapter 4.10 --- RASA5 antagonizes Ras signaling pathway --- p.117 / Chapter 4.10.1 --- RASA5 represses Ras signaling through downregulating Ras-GTP level --- p.117 / Chapter 4.10.2 --- Oncogenic mutant form of Ras abrogated colony formation inhibitory effect of RASA5 on tumor cells --- p.120 / Chapter 4.10.3 --- Knockdown of RASA5 promoted the tumor cell colony formation and Ras signaling activation --- p.122 / Chapter 4.10.4 --- RASA5 inhibits ERK1/2 nuclei translocation and activation --- p.123 / Chapter 4.10.5 --- RASA5 negatively regulates Ras target gene expression --- p.125 / Chapter 4.11 --- RASA5 inhibits tumor cell migration and invasion through the Ras/Rac/cofilin signaling --- p.127 / Chapter 4.12 --- RASA5 suppresses tumor cell epithelial-mesenchymal transition (EMT) and stemness --- p.133 / Chapter 4.13 --- RASA5 appears in the cellcell interaction region nanotubes --- p.139 / Chapter 4.14 --- Discussion --- p.141 / Chapter Chapter 5: --- The Wnt/Dvl signaling antagonist TUSC-T2 is a pro-apoptotic tumor suppressor epigenetically silenced in tumors and inhibits tumor cell proliferation and migration --- p.150 / Chapter 5.1 --- Expression of TUSC-T2 is downregulated in human tumors --- p.150 / Chapter 5.2 --- TUSC-T2 promoter methylation results in its transcriptional inactivation --- p.151 / Chapter 5.3 --- Cloning and characterization of TUSC-T2 --- p.155 / Chapter 5.4 --- TUSC-T2 inhibits tumor cell clonogenicity through inducing apoptosis --- p.157 / Chapter 5.5 --- TUSC-T2 inhibits Wnt/Dvl/β-catenin pathway --- p.161 / Chapter 5.6 --- TUSC-T2 suppresses cell migration and EMT through upregulating E-cadherin --- p.165 / Chapter 5.7 --- Discussion --- p.171 / Chapter Chapter 6: --- Conclusions --- p.176 / Chapter 6.1. --- RasGAP family member RASA5 is epigenetically silenced in human cancers, acting as a tumor suppressor through negatively regulating Ras signaling --- p.177 / Chapter 6.2. --- DACT family member TUSC-T2 functions as a candidate TSG silenced by promoter methylation and inhibits Wnt/Dvl/β-catenin pathway --- p.178 / Chapter Chapter 7: --- Future Studies --- p.181 / Chapter 7.1. --- Further functional study of RASA5 and TUSC-T2 --- p.181 / Chapter 7.2. --- Clinical application of epigenetic silenced candidate TSGs --- p.182 / Chapter 7.3. --- Further screening of candidate TSGs as antagonists to cancer pathways --- p.183 / Reference list --- p.184

Zhong, Lan. / Thesis Ph.D. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 122-154). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016). / Zhong, Lan.

Yuen, Ka Chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 160-185). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 摘要 --- p.IV / ACKNOWLEDGEMENTS --- p.VI / LIST OF ABBREVIATIONS --- p.VII / TABLE OF CONTENTS --- p.VIII / LIST OF TABLES --- p.XII / LIST OF FIGURES --- p.XIII / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- Pseudomalignant nature of the placenta --- p.2 / Chapter 1.1 --- Overview --- p.2 / Chapter 1.2 --- "Proliferation, migration and invasion behaviour" --- p.3 / Chapter 1.3 --- Gene expression --- p.4 / Chapter 1.3.1 --- Angiogenic factors --- p.5 / Chapter 1.3.2 --- Growth factors --- p.5 / Chapter 1.3.3 --- Proto-oncogenes --- p.6 / Chapter 1.3.4 --- Tumour suppressor genes --- p.8 / Chapter CHAPTER 2: --- Epigenetics --- p.10 / Chapter 2.1 --- Overview --- p.10 / Chapter 2.2 --- DNA methylation in mammals --- p.11 / Chapter 2.3 --- Regulation of DNA methylation machinery --- p.12 / Chapter 2.4 --- Role of DNA methylation --- p.13 / Chapter 2.5 --- Aberrant DNA methylation --- p.16 / Chapter 2.6 --- DNA methylation in normal cells --- p.17 / Chapter 2.6.1 --- X-chromosome inactivation --- p.17 / Chapter 2.6.2 --- Genomic imprinting --- p.18 / Chapter 2.6.3 --- Cell-type-specific methylation --- p.19 / Chapter 2.6.4 --- Placental-specific methylation --- p.20 / Chapter 2.7 --- Aim of Thesis --- p.21 / Chapter SECTION II: --- MATERIALS AND METHODOLOGY --- p.23 / Chapter CHAPTER 3: --- Materials and methods --- p.24 / Chapter 3.1 --- Preparation of samples --- p.24 / Chapter 3.1.1 --- Collection of placental tissues --- p.24 / Chapter 3.1.2 --- Preparation of blood cells --- p.25 / Chapter 3.1.3 --- Preparation of cell lines --- p.25 / Chapter 3.1.4 --- Treatment of JAR and JEG3 with 5-aza-2'-deoxycytidine (5-aza-CdR) and Trichostatin A (TSA) --- p.26 / Chapter 3.2 --- Nucleic acid extraction --- p.26 / Chapter 3.2.1 --- DNA extraction from tissue samples --- p.26 / Chapter 3.2.2 --- DNA extraction from blood cells --- p.29 / Chapter 3.2.3 --- RNA extraction from cell lines --- p.30 / Chapter 3.3 --- Methylation analysis --- p.31 / Chapter 3.3.1 --- Principles of bisulfite modification --- p.31 / Chapter 3.3.2 --- Bisulfite Conversion --- p.32 / Chapter 3.3.3 --- Primer design for methylation-specific polymerase chain reaction / Chapter 3.3.4 --- Methylation-specific polymerase chain reaction (MSP) --- p.33 / Chapter 3.3.5 --- Primer design for bisulfite sequencing --- p.34 / Chapter 3.3.6 --- Cloning and bisulfite genomic sequencing --- p.35 / Chapter 3.4 --- Quantitative measurements of nucleic acids --- p.39 / Chapter 3.4.1 --- Principles of real-time quantitative PCR --- p.39 / Chapter 3.4.2 --- Real-time quantitative MSP --- p.42 / Chapter 3.4.3 --- Real-time reverse transcriptase (RT)-PCR --- p.42 / Chapter 3.5 --- MALDI-TOF mass spectrometry (MS) --- p.43 / Chapter 3.5.1 --- Principle of homogeneous MassEXTEND assay and MALDI-TOF MS --- p.43 / Chapter 3.5.2 --- Methylation-sensitive restriction enzyme digestion and homogeneous MassEXTEND assay for APC and H19 --- p.46 / Chapter SECTION III: --- A SEARCH FOR HYPERMETHYLATED TUMOUR SUPPRESSOR GENES IN THE HUMAN PLACENTA --- p.48 / Chapter CHAPTER 4: --- Screening on TSGs and non TSGs --- p.49 / Chapter 4.1 --- Introduction --- p.49 / Chapter 4.2 --- Materials and methods --- p.50 / Chapter 4.2.1 --- Sample collection --- p.50 / Chapter 4.2.2 --- Sample processing and DNA extraction --- p.50 / Chapter 4.2.3 --- Experimental Design --- p.51 / Chapter 4.3 --- Results --- p.63 / Chapter 4.3.1 --- Identification of hypermethylated TSGs by methylation-specific PCR screening --- p.63 / Chapter 4.3.2 --- Validation of hypermethylated TSGs by bisulfite sequencing --- p.69 / Chapter 4.4 --- Discussion --- p.77 / Chapter CHAPTER 5: --- Methylation status of TSGs in different tissues --- p.80 / Chapter 5.1 --- Introduction --- p.80 / Chapter 5.2 --- Materials and methods --- p.81 / Chapter 5.2.1 --- Sample collection --- p.81 / Chapter 5.2.2 --- Sample processing and DNA extraction --- p.81 / Chapter 5.2.3 --- Experimental design --- p.81 / Chapter 5.3 --- Results --- p.86 / Chapter 5.3.1 --- Methylation patterns of TSGs in non-placental fetal tissues --- p.86 / Chapter 5.4 --- Discussion --- p.90 / Chapter SECTION IV: --- FUNCTIONAL IMPLICATION OF HYPERMETHYLATED TUMOUR SUPPRESSOR GENES IN THE PLACENTA --- p.94 / Chapter CHAPTER 6: --- Imprinting checking --- p.95 / Chapter 6.1 --- Introduction --- p.95 / Chapter 6.2 --- Materials and methods --- p.96 / Chapter 6.2.1 --- Sample collection --- p.96 / Chapter 6.2.2 --- Sample processing and DNA extraction --- p.97 / Chapter 6.2.3 --- Experimental design --- p.97 / Chapter 6.3 --- Results --- p.100 / Chapter 6.3.1 --- Imprinting checking of H19 by enzyme digestion on placental tissues --- p.100 / Chapter 6.3.2 --- Imprinting checking of　APC by enzyme digestion on placental tissues --- p.101 / Chapter CHAPTER 7: --- CORRELATION OF HYPERMETHYLATION AND GENE EXPRESSION --- p.107 / Chapter 7.1 --- Introduction --- p.107 / Chapter 7.2 --- Materials and methods --- p.108 / Chapter 7.2.1 --- Sample preparation and processing --- p.108 / Chapter 7.2.2 --- DNA and RNA extraction from cell lines --- p.108 / Chapter 7.2.3 --- Experimental design --- p.108 / Chapter 7.3 --- Results --- p.111 / Chapter 7.3.1 --- Methylation status of APC in choriocarcinoma cell lines --- p.111 / Chapter 7.3.2 --- Demethylation of APC in choriocarcinoma cell lines --- p.114 / Chapter 7.4 --- Discussion --- p.115 / Chapter SECTION V: --- CONSERVATION OF METHYLATION IN PLACENTA ACROSS DIFFERENT SPECIES --- p.118 / Chapter CHAPTER 8: --- Methylation analysis of hypermethylated TSG homologues in the placentas of the mouse and rhesus monkey --- p.119 / Chapter 8.1 --- Introduction --- p.119 / Chapter 8.2 --- Materials and methods --- p.120 / Chapter 8.2.1 --- Sample collection --- p.120 / Chapter 8.2.2 --- Sample processing and DNA extraction --- p.120 / Chapter 8.2.3 --- Experimental design --- p.120 / Chapter 8.3 --- Results --- p.124 / Chapter 8.3.1 --- Methylation status of TSGs in rhesus monkey and murine placental tissues --- p.124 / Chapter 8.4 --- Discussion --- p.136 / Chapter SECTION VI: --- CONCLUDING REMARKS --- p.138 / Chapter CHAPTER 9: --- Conclusion and future perspectives --- p.139 / Chapter 9.1 --- Pseudomalignant nature of placenta at the epigenetic level --- p.139 / Chapter 9.2 --- Functional implication of TSG hypermethylation --- p.140 / Chapter 9.3 --- Significance of hypermethylated TSGs in the placental evolution --- p.142 / Chapter 9.4 --- Clinical implication of TSG hypermethylation --- p.143 / Chapter 9.5 --- Future perspectives --- p.145 / APPENDIX I COMPLETE BISULFITE SEQUENCING DATA FOR HYPERMETHYLATED TSGS --- p.147 / APPENDIX II BISULFITE SEQUENCING DATA FOR PTEN --- p.156 / APPENDIX III BISULFITE SEQUENCING DATA OF LOCI NOT SHOWING HYPERMETHYLATION --- p.158 / REFERENCES 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