Fu Zhenming. / Thesis submitted in: December 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 138-162). / Abstracts in English and Chinese. / List of abbreviations --- p.i / Abstract (in English) --- p.ii / 摘要（中文） --- p.iii / Acknowledgement --- p.iv / Chapter Chapter 1. --- Introduction and Objectives of Study --- p.1 / Chapter 1.1 --- Hepatocellular carcinoma in general --- p.2 / Chapter 1.1.1 --- "Epidemiology, risk factors" --- p.2 / Chapter 1.1.2 --- Pathology and staging --- p.4 / Chapter 1.1.3 --- Treatment --- p.6 / Chapter 1.1.4 --- Improvement of early detection and treatment of HCC --- p.7 / Chapter 1.2 --- General aspects of mitochondria and mitochondrial DNA (mtDNA) --- p.10 / Chapter 1.2.1 --- Structure and dynamics of mitochondria --- p.10 / Chapter 1.2.1.1 --- General introduction of mitochondria --- p.10 / Chapter 1.2.1.2 --- Respiration chain of mitochondria --- p.11 / Chapter 1.2.2 --- The mitochondrial genome --- p.14 / Chapter 1.2.2.1 --- Strucure --- p.14 / Chapter 1.2.2.2 --- Genes for structure proteins --- p.16 / Chapter 1.2.2.3 --- Genes for translation --- p.17 / Chapter 1.2.2.4 --- Imported proteins and RNAs --- p.17 / Chapter 1.2.3 --- Mitochondrial DNA maintenance --- p.19 / Chapter 1.2.4 --- Mitochondrial DNA replication --- p.25 / Chapter 1.2.5 --- Mitochondrial DNA transcription --- p.30 / Chapter 1.2.6 --- Mitochondrial DNA translation --- p.32 / Chapter 1.3 --- MtDNA diseases --- p.35 / Chapter 1.4 --- MtDNA mutation and HCC --- p.35 / Chapter 1.5 --- Aims of the study --- p.39 / Chapter Chapter 2. --- Materials and Methods --- p.41 / Chapter 2.1 --- Materials --- p.42 / Chapter 2.1.1 --- Chemicals --- p.42 / Chapter 2.1.2 --- Primers --- p.42 / Chapter 2.1.3 --- Enzymes --- p.45 / Chapter 2.1.4 --- Cell line --- p.45 / Chapter 2.1.5 --- Collection of specimens --- p.46 / Chapter 2.2 --- Methodology --- p.47 / Chapter 2.2.1 --- "DNA extraction from hcc tissues, cell line Hep3B and PBMCs" --- p.47 / Chapter 2.2.1.1 --- DNA extraction from HCC tissues --- p.47 / Chapter 2.2.1.2 --- DNA extraction from cell line Hep3B --- p.49 / Chapter 2.2.1.3 --- DNA extraction from and PBMCs --- p.50 / Chapter 2.2.1.3.1 --- Preparation of PBMCs --- p.50 / Chapter 2.2.1.3.2 --- DNA extraction from and PBMCs --- p.51 / Chapter 2.2.2 --- Detection of mt whole genome mutation by direct sequencing --- p.51 / Chapter 2.2.2.1 --- Design of mtDNA primers --- p.51 / Chapter 2.2.2.2 --- PCR amplification of the whole mt genome --- p.51 / Chapter 2.2.2.3 --- Direct sequencing of the whole mt genome --- p.52 / Chapter 2.2.2.3.1 --- Primer used in sequencing --- p.52 / Chapter 2.2.2.3.2 --- Purification of the PCR products of the whole mt genome --- p.53 / Chapter 2.2.2.3.3 --- Dye terminator cycle sequencing reaction --- p.53 / Chapter 2.2.2.3.4 --- Purification of extension products --- p.54 / Chapter 2.2.3 --- Detection of mtDNA control region mutation --- p.55 / Chapter 2.2.3.1 --- PCR amplification of D310 in the mtDNA control region --- p.55 / Chapter 2.2.3.2 --- Screening of D310 mutation by PFLDA --- p.55 / Chapter 2.2.3.2.1 --- Making 8% denatured gel mixture --- p.55 / Chapter 2.2.3.2.2 --- Setting up and Pouring the denatured gel --- p.56 / Chapter 2.2.3.2.4 --- Preparing and Loading the PCR products --- p.57 / Chapter 2.2.3.2.5 --- Electrophoresis --- p.57 / Chapter 2.2.3.2.6 --- "Gel fixing, silver staining and color development " --- p.58 / Chapter 2.2.3.3 --- Direct sequencing of D310 in the mtDNA control region --- p.59 / Chapter 2.2.4 --- Detection of mt DNA coding region mutation --- p.60 / Chapter 2.2.4.1 --- PCR amplification of the 5 respiratory chain subunit genes --- p.60 / Chapter 2.2.4.2 --- Restriction enzyme digestion of 5 genes in mtDNA coding region --- p.60 / Chapter 2.2.4.3 --- Screening of mtDNA coding region mutation by SSCP --- p.61 / Chapter 2.2.4.3.1 --- Making 6% 49:1 acrylamide/Bis SSCP gel mixture --- p.61 / Chapter 2.2.4.3.2 --- "Setting up the SSCP gel, loading sample, fixing, staining and developing of the gel " --- p.62 / Chapter 2.2.4.4 --- Sequencing conformation of the mtDNA coding region mutation --- p.62 / Chapter 2.2.5 --- Statistics --- p.63 / Chapter 2.2.5.1 --- The chi-square test --- p.63 / Chapter 2.2.5.2 --- The Friedman test --- p.63 / Chapter 2.2.5.3 --- Wilcoxon signed ranks test --- p.63 / Chapter Chapter 3. --- Results --- p.64 / Chapter 3.1 --- Detection mt DNA whole genome mutation --- p.65 / Chapter 3.1.1 --- Identification of mtDNA whole genome by direct sequencing --- p.65 / Chapter 3.2 --- Detection mt DNA D-loop mutation --- p.76 / Chapter 3.2.1 --- Screening of C-tract alteration in HCC tissus by PCR fragments length detection assay (PFLDA) --- p.76 / Chapter 3.2.2 --- Screening of coding region alteration in HCC tissues by SSCP --- p.77 / Chapter 3.2.2.1 --- Identification of C-tract alterations in HCC and non-tumorous tissues by direct sequencing --- p.77 / Chapter 3.2.3 --- Identification of C-tract alterations by direct sequencing --- p.82 / Chapter 3.2.3.1 --- Identification of C-tract alterations in HCC tissues by direct sequencing --- p.82 / Chapter 3.2.3.2 --- Identification of C-tract alteration in PBMC of normal subjects by direct sequencing --- p.82 / Chapter 3.2.3.3 --- Identification of C-tract alteration in PBMC of HCC patients by direct sequencing --- p.82 / Chapter 3.2.4 --- Statistics of the analysis of C-tract alterations --- p.82 / Chapter 3.3 --- Detection mt DNA mutation in the coding region --- p.87 / Chapter Chapter 4. --- Discussion --- p.98 / Chapter 4.1 --- Detection mtDNA whole genome mutation --- p.99 / Chapter 4.2 --- Detection mtDNA D-loop mutation --- p.107 / Chapter 4.3 --- Detection mtDNA mutation in the coding region --- p.119 / Chapter 4.4 --- Possible mechanisms of mtDNA mutation in HCC carcinogenesis --- p.125 / Chapter 4.5 --- Proposals for prospective studies --- p.126 / Chapter 4.5.1 --- Function of C7 in D310 --- p.128 / Chapter 4.5.2 --- Function changes of mtDNA coding region mutation --- p.130 / Chapter 4.5.3 --- Detection of D310 C-tract mutation in patients' plasma --- p.131 / Chapter 4.5.4 --- Relationship between nMSl and mtMSI --- p.132 / Chapter 4.6 --- Summary --- p.134 / References --- p.137
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324741 |
| Date | January 2004 |
| Contributors | Fu, Zhenming., Chinese University of Hong Kong Graduate School. Division of Medical Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, vii, 162 leaves : ill. (some col.) ; 30 cm. |
| Coverage | China, China |
| 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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