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 --- p.160
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326018 |
| Date | January 2007 |
| Contributors | Yuen, Ka Chun., Chinese University of Hong Kong Graduate School. Division of Chemical Pathology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xiv, 185 leaves : ill. (some col.) ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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