Chung, Po Yin. / Thesis submitted in: December 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 128-155). / Abstracts in English and Chinese. / Thesis Abstract --- p.i / 論文摘要 --- p.iv / Acknowledgements --- p.vi / Abbreviations --- p.vii / Thesis Content --- p.xi / List of Figures --- p.xv / List of Tables --- p.xvii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1. --- Normal Hematopoiesis --- p.1 / Chapter 1.2. --- Hematological Malignancy and the Aberrant Development of Blood Cells --- p.2 / Chapter 1.3. --- Leukemia and Its Classification --- p.3 / Chapter 1.4. --- Childhood Acute Lymphoblastic Leukemia (ALL) --- p.5 / Chapter 1.4.1. --- Epidem iology --- p.5 / Chapter 1.4.2. --- Causes and Risk Factors --- p.6 / Chapter 1.4.3. --- Molecular Pathophysiology --- p.7 / Chapter 1.4.4. --- Clinical Presentation --- p.9 / Chapter 1.4.5. --- Classification --- p.10 / Chapter 1.4.5.1. --- Immunophenotyping --- p.10 / Chapter 1.4.5.2. --- French-American-British (FAB) Classification --- p.12 / Chapter 1.4.6. --- Diagnosis and Prognosis --- p.14 / Chapter 1.4.6.1. --- Morphological and Cytochemical Analysis --- p.15 / Chapter 1.4.6.2. --- Cytogenetic and Molecular Genetic Characterizations --- p.16 / Chapter 1.4.7. --- Treatment --- p.19 / Chapter 1.5. --- Overview of Epigenetics --- p.21 / Chapter 1.6. --- Concepts ofDNA Methylation --- p.23 / Chapter 1.6.1. --- CpG Islands --- p.23 / Chapter 1.6.2 --- Mechanisms of DNA Methylation --- p.24 / Chapter 1.6.3 --- Physiological Roles of DNA Methylation --- p.28 / Chapter 1.6.4 --- Initiation of Aberrant DNA Methylation --- p.30 / Chapter 1.7. --- DNA Methylation in Tumorigenesis --- p.31 / Chapter 1.7.1. --- Regional Hypermethylation --- p.33 / Chapter 1.7.2 --- Global and Regional Hypomethylation --- p.34 / Chapter 1.7.3 --- Microatellite Instability and Oncogeneic Mutation --- p.35 / Chapter Chapter 2 --- Literature Review --- p.37 / Chapter 2.1. --- Aberrant DNA Methylation in Childhood ALL --- p.37 / Chapter 2.1.1. --- Cell Cycle --- p.39 / Chapter 2.1.2. --- Apoptosis --- p.41 / Chapter 2.1.3. --- Tissue Invasion and Metastasis --- p.42 / Chapter 2.1.4. --- Transcription Factors and Metabolic Enzymes --- p.44 / Chapter 2.1.5. --- Putative Tumor Suppressor Genes --- p.44 / Chapter 2.1.6. --- Chromosome Instability --- p.46 / Chapter 2.2. --- Methodologies in DNA Methylation Analysis --- p.50 / Chapter 2.2.1. --- Principle of Methylation-sensitive Arbitrarily Primed PCR (MS-AP PCR) --- p.50 / Chapter 2.2.2. --- Combined Bisulfite Restriction Analysis (COBRA) --- p.53 / Chapter 2.2.3. --- Cloned Bisulfite Sequencing --- p.55 / Chapter 2.2.4. --- Experimental Use of Demethylating Agents --- p.55 / Chapter Chapter 3 --- Background of Research --- p.58 / Chapter 3.1. --- Current Methylation Studies in Childhood ALL --- p.58 / Chapter 3.2. --- Objectives of Research --- p.60 / Chapter 3.3. --- Study Approach and Experimental Design --- p.61 / Chapter Chapter 4 --- Materials and Methods --- p.63 / Chapter 4.1. --- Clinical Samples and ALL Cell Lines --- p.63 / Chapter 4.1.1. --- Clinical Samples from Pediatric Patients with ALL and Normal Healthy Donors --- p.63 / Chapter 4.1.2. --- ALL Cell Lines --- p.63 / Chapter 4.2. --- Genomic DNA Isolation from Clinical Samples and Cell Lines --- p.64 / Chapter 4.2.1. --- Ficoll Gradient Centrifugation --- p.64 / Chapter 4.2.2. --- DNA Extraction --- p.64 / Chapter 4.3. --- MS-AP PCR --- p.65 / Chapter 4.3.1. --- Methylation-sensitive Restriction Enzyme Digestion of Genomic DNA --- p.65 / Chapter 4.3.2. --- Arbitrarily Primed Polymerase Chain Reaction --- p.66 / Chapter 4.3.3. --- Isolation of Differentially Methylated DNA Fragments --- p.69 / Chapter 4.4. --- Cloning of Differentially Methylated DNA Fragments --- p.70 / Chapter 4.4.1. --- TA Cloning --- p.70 / Chapter 4.4.2. --- Screening of Positive Clones --- p.71 / Chapter 4.4.3. --- Preparation of Plasmid DNA by Alkaline Lysis Method --- p.72 / Chapter 4.5. --- DNA Sequence Analysis of Differentially Methylated DNA Fragments --- p.72 / Chapter 4.5.1. --- Dye-terminator Cycle Sequencing --- p.72 / Chapter 4.5.2. --- CpG islands Analysis of Differentially Methylated Sequences --- p.73 / Chapter 4.6. --- DNA Methylation Analysis --- p.74 / Chapter 4.6.1. --- Sodium Bisulfite Modification --- p.74 / Chapter 4.6.2. --- Combined Bisulfite Restriction Analysis --- p.75 / Chapter 4.6.3. --- Cloned Bisulfite Genomic Sequencing --- p.76 / Chapter 4.7 --- Gene Expression Study --- p.76 / Chapter 4.7.1. --- RNA Extraction from Clinical Samples and ALL Cell Lines --- p.76 / Chapter 4.1.2. --- Reverse Transcription PCR --- p.77 / Chapter 4.7.3. --- Semi-quantitative RT-PCR --- p.78 / Chapter 4.7.4. --- 5-aza-2 '-deoxycytidine Demethylation Treatment --- p.79 / Chapter Chapter 5 --- Results --- p.80 / Chapter 5.1. --- Generation of DNA Methylation Pattern by MS-AP PCR --- p.80 / Chapter 5.1.1. --- Differential Methylation Patterns of Childhood ALL --- p.84 / Chapter 5.1.2. --- Methylation Patterns of B and T lineages Childhood ALL --- p.86 / Chapter 5.2. --- UCSC BLAT Analysis of Differential Methylated DNA Sequences / Chapter 5.3. --- Identification of Candidate Gene --- p.89 / Chapter 5.4. --- Fibrillin 2 --- p.90 / Chapter 5.4.1. --- FBN2 CpG Islands: UCSC BLAT Search Analysis --- p.90 / Chapter 5.4.2. --- Verification ofFBN2 by ALL Cell Lines --- p.91 / Chapter 5.4.2.1. --- Semi-quantitative RT-PCR --- p.91 / Chapter 5.4.2.2. --- COBRA --- p.92 / Chapter 5.4.2.3. --- Cloned Bisulfite Sequencing --- p.94 / Chapter 5.4.2.4. --- Demethylation Treatment Resorted FBN2 mRNA Expression in ALL Cell Lines --- p.98 / Chapter 5.4.3. --- Studies ofFBN2 in Childhood ALL --- p.99 / Chapter 5.4.3.1. --- Methylation Analysis --- p.99 / Chapter 5.4.3.2. --- Semi-quantitative RT-PCR --- p.105 / Chapter Chapter 6 --- Discussion --- p.107 / Chapter 6.1. --- Genome-wide Screening Approach: MS-AP PCR --- p.107 / Chapter 6.2. --- Sample Selection in this Study --- p.109 / Chapter 6.2.1. --- MS-AP PCR --- p.109 / Chapter 6.2.2. --- Methylation Studies --- p.109 / Chapter 6.2.3. --- Studies in ALL Cell Lines --- p.110 / Chapter 6.3. --- Methylation Patterns in Childhood ALL --- p.111 / Chapter 6.4. --- Candidate Genes Selection Strategies in MS-AP PCR --- p.112 / Chapter 6.5. --- Fibrillin 2: mRNA Expression and Methylation Studies --- p.113 / Chapter 6.5.1 --- ALL Cell Lines --- p.113 / Chapter 6.5.2 --- Childhood ALL --- p.113 / Chapter 6.5.2.1 --- mRNA Expression and Methylation Studies --- p.113 / Chapter 6.5.2.2 --- Statistical Analysis --- p.115 / Chapter 6.5.3. --- Possible Roles of FBN2 in Leukemogenesis --- p.116 / Chapter 6.6. --- Clinical Application of FBN2 Aberrant Methylation --- p.119 / Chapter 6.6.1. --- Tumor Markers --- p.119 / Chapter 6.6.2. --- Use of Demethylating Drugs in Chemotherapy --- p.121 / Chapter 6.7. --- Limitations of Methylation Studies --- p.122 / Chapter 6.7.1. --- MS-AP PCR --- p.122 / Chapter 6.7.2. --- Techniques Used in Methylation Study --- p.122 / Chapter 6.7.3. --- Problems in Methylation Study --- p.123 / Chapter 6.8. --- Future Studies --- p.125 / Chapter Chapter 7 --- Conclusion --- p.127 / References --- p.128 / Appendix --- p.155
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325852 |
| Date | January 2007 |
| Contributors | Chung, Po Yin., Chinese University of Hong Kong Graduate School. Division of Anatomical and Cellular Pathology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xvii, 155 leaves, [1] fold leaf : 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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