No description available.

Epidemiological data indicate that children conceived in vitro have a greater relative risk of low birth-weight, major and minor birth defects, and rare disorders involving imprinted genes, suggesting that epigenetic changes may be associated with assisted reproduction. DNA methylation and gene expression differences have been found in cord blood and placenta comparing children conceived in vitro using assisted reproductive technology (ART) and children conceived in vivo. The source of these differences (the effect of ART versus underlying infertility) has never been identified in humans. In order to determine what fraction of the DNA methylation and gene expression difference is attributable to the ART procedure and what fraction is attributable to underlying infertility, quasi-transcriptome-wide DNA methylation profiles were compared between (1) in vitro ART children of mothers who are infertile as a result of a physical impediment to fertilization (tubal blockage) or children conceived with the aid of donor oocytes as a group (tubal and donor egg group), and (2) children of parent(s) who have idiopathic infertility (infertility group). Both groups were compared to children conceived in vivo. Our data suggest strongly that many of the DNA methylation and gene expression differences observed between the in vitro and in vivo conceptions are associated with some aspect of ART procedure, rather than underlying infertility. / Molecular Biology and Genetics

DNA hypermethylation is known to contribute to the formation of cancer and this process has been widely studied. However, DNA hypomethylation has received far less attention and the factors controlling the balance between hypo and hypermethylation and its impact on tumorigenesis remains unclear. TET1 is a DNA demethylase that regulates DNA methylation, hydroxymethylation and gene expression. Full length TET1 (TET1FL) has a CXXC domain that binds to un-methylated CG islands (CGIs). This CXXC domain allows TET1 to protect CGIs from aberrant methylation but it also limits its ability to regulate genes outside of CGIs. This dissertation reports a novel isoform of TET1 (TET1ALT) that has a unique transcription start site from an alternate promoter in intron 2, yielding a protein with a unique translation start site. Importantly, TET1ALT lacks the CXXC domain but retains the catalytic domain. TET1ALT is repressed in ESCs but becomes activated in embryonic and adult tissues while TET1FL is ex / Biomedical Sciences

Blast-induced neurotrauma (BINT) is a prevalent brain injury within both military and civilian populations due to current engagement in overseas conflict and ongoing terrorist events worldwide. In the early 2000s, 78% of injuries were attributable to an explosive mechanism during overseas conflicts, which has led to increased incidences of BINT [1a]. Clinical manifestations of BINT include long-term psychological impairments, which are driven by the underlying cellular and molecular sequelae of the injury. Development of effective treatment strategies is limited by the lack of understanding on the cellular and molecular level [2a]. The overall hypothesis of this work is that epigenetic regulatory mechanisms contribute to the progression of the BINT pathology and neurological impairments. Epigenetic mechanisms, including DNA methylation and histone acetylation, are important processes by which cells coordinate neurological and cellular response to environmental stimuli. To date, the role of epigenetics in BINT remains largely unknown. To test this hypothesis, an established rodent model of BINT was employed [3a]. Analysis of DNA methylation, which is involved in memory processes, showed decreased levels one week following injury, which was accompanied by decreased expression of the enzyme responsible for facilitating the addition of methyl groups to DNA. The one week time point also showed dramatic decreases in histone acetylation which correlated to decline in memory. This change was observed in astrocytes and may provide a mechanistic understanding for a hallmark characteristic of the injury. Treatment with a specific enzyme inhibitor was able to mitigate some of the histone acetylation changes. This corresponded with reduced astrocyte activation and an altered behavioral phenotype, which was characterized by high response to novelty. The diagnostic efficacy of epigenetic changes following blast was elucidated by the accumulation of cell-free nucleic acids in cerebrospinal fluid one month after injury. Concentrations of these molecules shows promise in discriminating between injured and non-injured individuals. To date, the diagnostic and therapeutic efforts of BINT have been limited by the lack of a mechanistic understanding of the injury. This work provides novel diagnostic and therapeutic targets. The clinical potential impact on diagnosis and therapeutic intervention has been demonstrated. / Ph. D. / Blast-induced neurotrauma (BINT) is a prevalent brain injury within both military and civilian populations due to current engagement in overseas conflict and ongoing terrorist events worldwide. In the early 2000s, 78% of injuries were attributable to an explosive mechanism during overseas conflicts which has led to increased incidences of BINT [1a]. Clinical manifestations of BINT include long-term psychological impairments which are driven by the underlying cellular and molecular sequelae of the injury. To date, the development of effective treatment strategies has been unsuccessful. The work described herein seeks to evaluate the specific cellular mechanisms that contribute to the progression of the BINT pathology and neurological impairments. Epigenetic mechanisms are regulatory mechanisms that coordinate DNA modifications and DNA storage to facilitate altered cellular phenotypes. DNA modifications often involves DNA methylation, which is the addition of methyl groups to the DNA backbone. DNA storage is regulated by specific modifications to histone proteins. Histone acetylation is a well-studied modification process that is capable inciting either chromatin relaxation or compaction. Both DNA methylation and histone acetylation are important processes by which cells coordinate neurological and cellular response to environmental stimuli. To date, the role of epigenetics in BINT remains largely unknown. An established rodent model of BINT was employed [3a]. Analysis of DNA methylation, which is involved in memory processes, showed decreased levels one week following injury which was accompanied by decreased expression of one of the enzymes responsible for facilitating the addition of methyl groups to DNA. The one week time point also showed dramatic decreases in histone acetylation which correlated to memory impairment. This change was observed in astrocytes which are support cells in the brain and are particularly vulnerable to blast-induced aberrations. Drug administration, targeting the histone acetylation equilibrium, successfully mitigated astrocyte activation and altered the behavioral phenotype. Diagnosis of BINT remains clinically challenging. An accumulation of cell-free nucleic acids was observed the in cerebrospinal fluid one month after injury. Concentrations of these molecules shows promise in discriminating between injured and non-injured individuals. These nucleic acids are susceptible to DNA methylation and may provide a platform for studying epigenetic biomarkers. To date, the diagnostic and therapeutic efforts of BINT have been limited by the lack of a mechanistic understanding of the injury. This work provides novel diagnostic and therapeutic targets. The potential clinical impact on diagnosis and therapeutic intervention has been demonstrated.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第25151号 / 理博第5058号 / 新制||理||1721(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 村山 美穂, 教授 平田 聡, 教授 三谷 曜子 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

"Epigenetic" refers to a heritable change in the gene expression pattern that is not mediated by any alterations in the primary nucleotide sequence of a gene in the genome. This change involves methylation of DNA in the gene promoter regions, modification of histone residues and chromatin remodeling. Among them, methylation of DNA promoter region is an essential step in epigenetic gene silencing and is known to be closely related to carcinogenesis and cancer progression. / Our preliminary study on effect of treatments of some potential anti-cancer drug candidates, namely Pheophorbide A (Pa), Pa combining with photodynamic therapy, Polyphyllin D (designated as HK-18), and its derivative designated as HK-27 on human breast cancer cell lines MCF-7 and MDA-MB-231 showed that the promoter methylation of CDH1 was decreased in response to treatments of Pa, HK-18, and HK-27 in MDA-MB-231 cells. / The aim of this study was to explore whether any methylation of DNA promoters mechanism is involved in drug resistance of a doxorubicin-induced human multidrug resistant hepatocellular carcinoma sub-linage R-HepG2 which was established from the doxorubicin sensitive HepG2 cell line in our laboratory. In this project, it was observed that the DNA promoter methylations of ESR1, Rassf2A, CDH1 and MDR1 in R-HepG2 were higher than those in HepG2 cells respectively by methylation specific polymerase chain reaction method. Bisulfite sequencing showed that the total 32 CpGs of CDH1 promoter region in R-HepG2 cells were hypermethylated while they were hypomethylated in HepG2 cells. CDH1 is the encoding gene of E-cadherin. The promoter hypermethylation induced CDH1 silencing in R-HepG2 cells was confirmed by reverse transcription polymerase chain reaction and Western blotting that CDH1 transcription and E-cadherin expression were maintained in HepG2 cells but both were lost in R-HepG2 cells. RT-PCR of 10 multidrug resistant related genes revealed that transcription of MDR1 was obviously increased in R-HepG2 cells, transcription of MRP1 and MRP5 were slightly increased in R-HepG2 cells, transcription of MRP6 and BCRP were slightly decreased in R-HepG2 cells comparing to those in the parental HepG2 cells. This result suggests that up-regulation of P-glycoprotein expression which is the protein product of MDR1 may be one of the major causes of multidrug resistance in R-HepG2 cells. Transient transfection of CDH1 cDNA increased the CDH1 transcription and E-cadherin expression in R-HepG2 cells. I also found that the CDH1 transfected R-HepG2-CDH1 cells showed increased amount of doxorubicin uptake, increased apoptotic population of cells exposed to doxorubicin, suppressed cell migration, and decreased P-glycoprotein expression comparing to those in R-HepG2 cells. It was also found that the transcription levels of SNAI2, TWIST1, ASNA1 and FYN were obviously higher in R-HepG2 cells than those in HepG2 cells. The transcription of FYN and TWIST1 were obviously decreased in CDH1 cDNA transfected R-HepG2-CDH1 cells which displayed a negative correlation with the transcription level of CDH1 and these results imply a suppressive role of CDH1 in regulating these genes which were involved in cancer metastasis and multidrug resistance. / Jiang, Lei. / Adviser: Kwok-Pui, Fang. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 144-171). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

La méthylation anormale de l'ADN est l'une des principales caractéristiques du cancer. La nature dynamique et réversible de cette modification épigénétique en a fait une cible potentielle pour le traitement du cancer. UHRF1, une protéine essentielle dans la maintenance de la méthylation de l'ADN, est également impliquée dans la tumorogenèse. UHRF1 est surexprimée dans une variété de cancers et est liée à l’inhibition des TSGs et à la prolifération cellulaire. Dans ce contexte, le but de ma thèse est d’identifier de potentiels inhibiteurs d’UHRF1 qui pourront être efficaces en clinique comme thérapie anti-cancéreuse. Pour atteindre cet objectif, une approche diversifiée a été adoptée qui inclue le criblage virtuel, des techniques biophysiques et biologiques qui permettent à caractériser l'activité inhibitrice des molécules actives et à comprendre leur mécanisme d'action. Nous avons identifié un composé positif de la famille des anthraquinones qui inhibe UHRF1 en se liant à son domaine SRA et perturbe son interaction avec DNMT1, l'enzyme responsable du maintien de la méthylation de l'ADN. Ce composé présente une activité antiproliférative dans différentes lignées cancéreuses. / Abnormal DNA methylation is one of the major hallmarks of cancer. The dynamic and reversible nature of this epigenetic modification has made it a potential target for cancer treatment. UHRF1, a pivotal DNA methylation maintenance protein, is also strongly involved in tumorogenesis. It isoverexpressed in a wide array of cancers and leads to silencing of TSGs and tumor growth. In this context, the aim of the thesis is to develop potential UHRF1 inhibitors that may be clinically effective for anti-cancer therapy. To reach this objective, a diverse approach was adopted including virtual screening, biophysical and biological techniques that helped to characterize the inhibitory activity of active molecules and understand their mechanism of action. The tests revealed one positive compound from the anthraquinone family that inhibited UHRF1 by binding to its SRA domain and impairing its interaction with DNMT1, the enzyme responsible for DNA methylation maintenance. This compound showed an anti-proliferative activity in various cancer cells.

Astrocytoma is defined as the neoplasia of astrocytes, the most abundant non-neuronal glial cells in brain. According to the WHO classification, the different grades of astrocytoma are- gradeI/pilocytic astrocytoma (benign form), grade II/diffuse astrocytoma (DA), grade III/anaplastic astrocytoma (AA) and grade IV/Glioblastoma (GBM). Patients with grade II astrocytoma have median survival time of 6-8 years after surgical intervention. While the more aggressive grade III has a median survival of 2-3 years. Grade IV is the most malignant form and has a median survival of 15 months approximately. In spite of all the progress in the fields of diagnosis and therapy, the prognosis of GBM still remains very poor. The aggressiveness and poor survival of GBM is due to the recurrence which is primarily because of intratumoral heterogeneity, presence of glioma stem cells and infiltration of the tumor cells into the normal brain parenchyma. Apart from the role of genetic mechanisms in triggering tumorigenesis, epigenetic modifications particularly the DNA methylation and histone modifications, are now recognized as frequent alterations playing a crucial role in the development and progression of human malignancies. There are two distinct DNA methylation abnormalities. The first is the reduction in genome-wide DNA methylation levels (global hypomethylation) and the second is the hypermethylation in the CpG island of specific gene promoters. Hypomethylation is believed to induce proto-oncogene activation and chromosomal instability, whereas hypermethylation is strongly associated with silencing of tumor suppressor genes. Thus, DNA methylation can function as a “switch” to activate or repress gene transcription, providing an essential mechanism for overexpressed or silenced genes involved in the regulation of cell cycle, DNA repair, growth signalling, angiogenesis, apoptosis, migration, invasion and thus in the initiation and progression of astrocytoma. Recent studies have identified biomarkers with prognostic impact which would include promoter methylation of O⁶-methyl guanine-DNA methyltransferase methylation (MGMT), isocitrate dehydrogenase 1(IDH1) mutation and a glioma CpG-island methylator (G-CIMP) phenotype. In the current study, we have characterized the DNA methylation profile for the different grades of astrocytoma and analysed the significance of methylation events occurring commonly in all the grades or uniquely only in grade IV. One of the GBM-specific hypomethylated and upregulated genes, Fibromodulin (FMOD), was extensively investigated in terms of its role in glioma pathogenesis and its regulation. FMOD was found to induce F-actin stress fibre formation and promote glioma cell migration. We also found that FMOD-mediated glioma cell migration is dependent on Integrin/FAK/Src/Small Rho GTPases signalling cascade. We further found that TGFβ pathway regulates FMOD expression through a process involving active demethylation and chromatin state transitions on FMOD promoter. This work has been divided into three parts: Part I: Characterization of DNA methylome during progression of Astrocytoma To investigate the aberrant methylation pattern on a genome-wide scale, 17 Grade II, 16 Grade III and 36 Grade IV tumor samples as well as 9 control brain tissues were analysed using Infinium Human Methylation 450K Bead Array on Illumina platform. The analysis was carried out in two parts. Firstly, we validated the dataset with already existing TCGA dataset. Upon comparison, the methylation profile of our dataset was highly correlated to the TCGA dataset with correlation coefficient of 0.99. In addition, we also checked the methylation status of few known hypermethylated and hypomethylated genes which showed the similar type of differential methylation. Then, we characterized the differentially methylated CpGs based on their spatial distribution in the human genome, for different grades of astrocytoma. CpG-rich regions show more of hypermethylation while the non-CpG rich regions, like open sea or gene body, are observed to be hypomethylated. Secondly, we also analysed the differentially methylated genes which contribute to physiological events in gliomagenesis. We hypothesized that the methylation specific events that occur in grade II and remain similarly methylated in grade IV are the ones probably contributing to the initial astrocyte transformation. However, the methylation specific events responsible for the aggressive nature of grade IV may occur as differentially methylated genes only in grade IV (and not in grade II). In this analysis, we have identified differentially methylated genes that play a role in initial transformation process (293 genes hypermethylated and downregulated while 23 genes were hypomethylated and upregulated) and also those that play a role in tumor aggressiveness (459 genes hypermethylated and downregulated while 350 genes were hypomethylated and upregulated). The differentially methylated genes that were common in both grade II and grade IV showed an enrichment of cell proliferation pathways while the differentially methylated genes uniquely present in grade IV showed enrichment in pathways related to the aggressiveness phenotype of tumorigenesis like cell motility and angiogenesis. Part II: Fibromodulin (FMOD), a GBM-specific hypomethylated and upregulated gene, is essential for glioma cell migration Among differentially methylated genes specifically in GBM, fibromodulin (FMOD) is one of the top most hypomethylated genes. FMOD is a member of leucine – rich repeat proteoglycan that is widely distributed in interstitial connective tissues. We found that FMOD is hypomethylated and upregulated only in grade IV/GBM, not in the grade II. FMOD promoter methylation status is significantly negatively correlated to its transcript levels.Towards identifying functions of FMOD in glioma cells, total RNA derived from U251 cells transfected with either non-targeting siRNA or FMOD siRNA was subjected to transcriptome profiling. There were 872 genes upregulated and 299 genes downregulated in FMOD silenced cells than in control cells. PANTHER pathway analysis using the differentially regulated genes identified several pathways to be associated with FMOD. Cytoskeleton regulation by Rho GTPase, which is known to be involved in cell motility and migration, is enriched with highest significance. In coherence with the pathway analysis, modulating FMOD levels in glioma cells affected in glioma cell migration. Upon FMOD overexpression, there was significant increase in migration than in control cells. Conversely, when FMOD is silenced, there was delay in migration than in control cells and the delayed migration was rescued by the addition of recombinant purified FMOD protein. Prior neutralization with FMOD specific antibody inhibited cell migration suggesting that secreted FMOD promotes glioma cell migration. Overexpression of FMOD in glioma cells induced actin stress fibre formation required for the migration of cells. On the contrary, FMOD silencing resulted in the loss of F-actin stress fibres which was restored upon addition of FMOD purified protein exogenously to the media. To investigate further the role and the requirement of specific Rho GTPase in FMOD-mediated migration, each of members of Rho GTPase family was silenced and their effect on FMOD-induced silencing was studied. FMOD mediated glioma cell migration was delayed when RhoA, Rac1 and Cdc42 were silenced. In order to understand whether FMOD activates Integrin mediated signalling pathway, we performed western blot analysis to check the levels of phospho-FAK in either FMOD overexpressing or knockdown condition. We observed phospho-FAK levels increased upon FMOD overexpression and decreased upon FMOD silencing compared to the respective controls. Additional experiments revealed that inhibitors to Integrin, FAK and Src were able to abrogate the FMOD induced glioma cell migration. These results suggest that FMOD utilizes a pathway that involves Integrins, FAK, Src and Rho GTPases in promoting glioma cell migration. To comprehend the effect of FMOD promoter methylation status and its expression in GBM patient scenario, we stratified the patients into either high or low FMOD expression and promoter hypermethylation or hypomethylation. The GBM patients with low FMOD transcript levels and promoter hypermethylation showed better survival than the other group. Part III: Regulation of FMOD expression through TGFβ-dependent epigenetic remodelling in glioma To study how FMOD is regulated in glioma, we investigated the promoter sequence of FMOD by MatInspector. Several Smad-binding sites were located in FMOD promoter which indicated that FMOD might be regulated via TGFβ signalling pathway. Firstly, we checked active TGFβ signalling in glioma cell lines – LN229, U87 and U251. TGFβ-dependent signalling was active in U251 and U87 cells compared to LN229 cells as seen by the levels of phospho-Smad2. Moreover, FMOD transcript level was found to be high in U251 compared to LN229 cells. Further, TGFβ treatment increased FMOD promoter luciferase activity as well as FMOD transcript level in LN229 cells. In contrast, U251 cells that were treated with TGFβ RI inhibitor showed a significant decrease in FMOD promoter luciferase activity as well as FMOD transcript level. We correlated these findings with Smad2 occupancy at FMOD promoter by chromatin immunoprecipitation (ChIP). Smad2 association at FMOD promoter is found to be relatively higher in U251 cells than in LN229 cells which suggested that TGFβ induced transcription factor, Smad2, drives FMOD expression in U251 cells. Next, we investigated the role of TGFβ in FMOD promoter demethylation and chromatin state transition. Upon TGFβ treatment in LN229 cells, we found that there was gradual demethylation of FMOD promoter in a time-dependent manner. TGFβ treatment also altered the chromatin state by increasing the active marks (H3K4me3 and H3K9Ac) and decreasing the repressive mark (H3K27me3) with a simultaneous increase in Smad2 occupancy in the FMOD promoter. In contrast, TGFβ RI inhibitor treatment of U251 cells resulted in methylation of FMOD promoter in a time-dependent manner. Further, we observed a significant enrichment of repressive histone marks (H3K27me3) and loss of active chromatin marks (H3K4me3 and H3K9Ac) with a concomitant decrease in Smad2 occupancy at FMOD promoter. DNMT3A/B and EZH2 enzymes play a key role in DNA methylation and H3K27 trimethylation respectively. Accordingly, we examined the transcript levels of DNMT3A/B and EZH2 in LN229 cells treated with TGFβ as well as U251 cells treated with TGFβ RI inhibitor. In presence of TGFβ, DNMT3A/B and EZH2 transcript levels were significantly downregulated than in untreated cells in a time-dependent manner. Conversely, in U251 cells treated with TGFβ RI inhibitor, there was a significant increase in DNMT3A/B and EZH2 transcript levels when compared to untreated cells. TGFβ is known to promote glioma cell migration. In order to understand whether TGFβ-mediated glioma cell migration occurs via FMOD, we performed migration assay in U251 cells with or without TGFβ RI inhibitor followed by addition of either BSA control or FMOD purified protein. Upon TGFβ RI inhibitor treatment, there was delay in the migration of U251 cells than in untreated control cells which was rescued when purified FMOD protein was added, indicating that FMOD is essential for TGFβ signalling cascade to induce glioma cell migration. Therefore, we conclude from these results that epigenetically regulated FMOD is essential for TGFβ mediated glioma cell migration.

No description available.

Bai Hsing Chen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 89-94). / Abstracts in English and Chinese. / Acknowledgments --- p.ii / Publication --- p.iii / List of Abbreviations --- p.iv / List of Tables --- p.v / List of Figures --- p.vi / Abstract --- p.vii / 摘要 --- p.x / Table of Contents --- p.xii / Chapter Chapter 1 --- INTRODUCTION / Chapter 1.1 --- Molecular Biology in Cancer Development --- p.2 / Chapter 1.1.1 --- Cell Cycle and Cancer --- p.2 / Chapter 1.1.2 --- Oncogenes and Tumor Suppressor Genes --- p.4 / Chapter 1.1.3 --- Epigenetic Alteration in Tumor Cells --- p.6 / Chapter 1.2 --- Colorectal Cancer Development --- p.9 / Chapter 1.2.1 --- Epidemiology of Colorectal Cancer --- p.9 / Chapter 1.2.2 --- Adenoma-Carcinoma Sequence --- p.11 / Chapter 1.2.2.1 --- Hyperplastic (metaplastic) Polyps --- p.11 / Chapter 1.2.2.2 --- Aberrant Crypt Foci (ACF) --- p.13 / Chapter 1.2.2.3 --- Adenomas --- p.13 / Chapter 1.2.2.4 --- Serrated adenomas --- p.15 / Chapter 1.2.2.5 --- Colorectal Carcinomas --- p.16 / Chapter 1.2.3 --- Genetic alterations in CRC --- p.18 / Chapter 1.2.4 --- Epigenetic alterations in CRC --- p.21 / Chapter 1.2.5 --- Staging of Colorectal Cancer --- p.23 / Chapter 1.3 --- Hypothesis --- p.25 / Chapter 1.4 --- Aim of Study --- p.26 / Chapter Chapter 2 --- MATERIALS and METHODES / Chapter 2.1 --- Patient Populations --- p.28 / Chapter 2.2 --- Microdissection and Immunohistochemistry --- p.29 / Chapter 2.3 --- DNA Isolation and Modification --- p.31 / Chapter 2.3.1 --- DNA Extraction from Microdissected Tissues --- p.31 / Chapter 2.3.2 --- DNA Extraction from Frozen Biopsy --- p.31 / Chapter 2.3.3 --- Bisulfite Modification of DNA --- p.32 / Chapter 2.4 --- Detection of K-ras Mutation --- p.33 / Chapter 2.5 --- Methylation-specific PCR (MSP) --- p.36 / Chapter 2.6 --- Bisulfite DNA Sequencing --- p.42 / Chapter 2.7 --- Statistical analysis --- p.44 / Chapter Chapter 3 --- RESULTS / Chapter 3.1 --- Promoter Hypermethylation of Tumor Related Genes in the Progression of Colorectal Neoplasia --- p.46 / Chapter 3.1.1 --- Clinico-Pathological parameters --- p.46 / Chapter 3.1.2 --- "Frequencies of Promoter Hypermethylation in Colorectal Cancers, Adenomas and Normal Colonic Tissues" --- p.47 / Chapter 3.1.3 --- Promoter Hypermethylation in Multiple Genes --- p.50 / Chapter 3.1.4 --- Promoter Hypermethylation in Advanced vs. Non-advanced Adenoma --- p.50 / Chapter 3.1.5 --- Methylation Patterns in Paired Adjacent Tissues from Cancer Patients --- p.53 / Chapter 3.1.6 --- Immunohistochemistry --- p.55 / Chapter 3.1.7 --- K-ras mutation --- p.61 / Chapter 3.1.8 --- Clinicopathological Correlations with Promoter Hypermethylation --- p.64 / Chapter 3.2 --- DNA Methylation Spread within HLTF CpG Island in Colorectal neoplasia --- p.67 / Chapter Chapter 4 --- DISCUSSION / Chapter 4.1 --- Methylation is an early event in Colorectal Carcinogenesis --- p.72 / Chapter 4.1.1 --- Methylation is frequently detected in both adenoma and carcinoma --- p.74 / Chapter 4.1.2 --- Concurrent methylation in multiple genes --- p.76 / Chapter 4.1.3 --- Methylation in advanced and non-advanced colorectal adenomas --- p.76 / Chapter 4.1.4 --- Relationship between K-ras mutation and methylation --- p.78 / Chapter 4.1.5 --- Methylation in adjacent tissues --- p.80 / Chapter 4.2 --- DNA Methylation Spread in HLTF gene --- p.81 / Chapter 4.2.1 --- HLTF is Frequently Methylated in Gastrointestinal Neoplasm --- p.82 / Chapter 4.2.2 --- Methylation Spread Patterns in Cancers and Adenomas --- p.83 / Chapter 4.2.3 --- Age Dependent Methylation Spread --- p.85 / Chapter Chapter 5 --- CONCLUSION --- p.87 / References --- p.89