Colorectal cancer (CRC) is the one of the commonest causes of cancer and cancer related death worldwide. Its aetiology is linked to a number of reversible and irreversible genetic and environmental factors, including age, sex, genetics, smoking and diet. There has been a drive in recent years for non-invasive biomarkers for many malignant and non-malignant diseases across multiple medical specialties. One of the areas of interest is the detection of volatile organic compounds (VOCs) in various bodily substances by means such as mass spectrometry and electronic noses. CRC patients have been shown to be distinguishable from healthy controls using urinary VOC detection in several studies, including two published by the research group at UHCW and the University of Warwick. There has also been much interest in recent years into the role that the intestinal microbiome plays in health and disease in humans. The aim of this thesis was to characterise the urinary VOC and stool microbiome profiles of CRC patients, their spouses and first degree relatives. The aim being to determine whether the urinary VOC profiles could be distinguished using this technology and to try and better understand the underlying mechanism which lead to CRC carcinogenesis. The first degree relatives and spouses were selected as “common gene pool” and “shared environment” control groups respectively. This work was done using an LC-FAIMS-MS hybrid machine to detect urinary VOCs and 16s RNA sequencing using an Illumina Miseq platform. Comparisons were also made between pre-treatment and post-treatment CRC samples to try and determine if there was any change in either VOC or microbiome profiles after CRC treatment. The urinary VOC profiles of CRC subjects could be distinguished from both sets of healthy controls using a 5-fold cross validation and sparse logistics regression and Random Forrest statistical classifiers, achieving sensitivities of 63-69%, specificities of 64-69% and AUC 0.71-0.72. No statistically significant differences could be found in the urinary VOC profiles of pre-operative and post operative samples. Microbiome analysis revealed over 1300 operational taxonomic units (OTUs), with a similarity of >93% between the CRC samples and the control groups, with significantly different bacterial abundances identified in only 82 OTUs (6.2%), mainly Clostridiales bacteria. Pre-treatment and post-treatment sample analysis revealed differences of 17 (3%) and 22 (4%) OTUs at 3 and 6 months respectively, again principally clostridiales. This thesis provides further data on the microbiome composition in CRC. It also provides further proof of the utility of urinary VOCs, for the first time here using LC-FAIMS-MS technology, a variant of the previously utilised FAIMS technology, as a non-invasive biomarker for CRC.

Central chondroid bone tumours are one of the most common primary bone tumours. Benign central chondroid tumours are termed enchondromas and its malignant counterpart are called chondrosarcomas. Enchondromas are frequently observed on routine imaging. Similarly, chondrosarcomas are the second most common primary bone tumour after osteosarcoma. Imaging is crucial in the diagnosis of central chondroid tumours and in the differentiation of enchondromas from chondrosarcomas. Furthermore, imaging plays a vital role in the staging of chondrosarcomas. In this thesis, the published scientific literature on the role of imaging in the diagnosis of benign chondroid tumours and chondrosarcomas and the role of imaging in the staging of chondrosarcomas is reviewed and summarised. Furthermore, the contribution of the authors’ published work is highlighted in the thesis. The first two articles are review articles which discuss the clinical and imaging features of benign and malignant chondrogenic tumours and the significance of imaging in the diagnosis of these tumours. The third article is an original article which investigates the theory of the pathogenesis of enchondromas. It is widely believed that enchondromas arise from cartilage islands which are displaced from the growth plate during the process of skeletal maturation. However, this theory is unproven, and the origin of this theory was forgotten prior to the authors’ study. Based on the incidental prevalence of enchondromas of the knee in the adult population of 2.9%, the study assesses the prevalence of cartilage islands/enchondromas in skeletally immature patients. In this study, no cartilage islands/enchondromas in skeletally immature patients were identified. The study therefore shows the rarity of enchondromas in skeletally immature individuals which is in contrast to the adult population. Furthermore, in view of the absence of cartilage islands in this study, the study raises doubts about the validity of the unproven theory. Lastly, the very origin of this theory is rediscovered in this thesis which has been forgotten in modern medicine. The fourth article is an original article which evaluates the role of diffusion-weighted MRI (DWI) in the diagnosis of central cartilage tumours. Prior to the authors’ study the role of DWI in the diagnosis of central cartilage tumours was uncertain. The authors’ study demonstrates that DWI cannot be used to differentiate between enchondromas and chondrosarcomas and that DWI does not aid in the distinction of low-grade chondroid tumours from high-grade chondrosarcomas. This is a finding which was not known prior to the study. The fifth article is an original article which assesses the utility of conventional MRI in the differentiation of low-grade from high-grade chondrosarcomas of long bone. Prior to the authors’ study the role of conventional MRI in the differentiation of low- grade from high-grade chondrosarcomas of long bone was unknown. The authors’ study shows that bone expansion, active periostitis, soft tissue mass and tumour length can be used to differentiate high-grade from low-grade chondral lesions of long bone on conventional MRI. Furthermore, the presence of these four MRI features shows a diagnostic accuracy of 95.6%. These findings were not known prior to the study and have significantly furthered the knowledge about the role of conventional MRI in the grading of chondrosarcoma of long bone. The sixth article is an original article which evaluates the role of bone scintigraphy and Computed Tomography of the chest in the staging of chondrosarcoma of bone. Whilst guidelines regarding the staging of bone sarcomas state that bone scintigraphy should be performed to assess for the presence of skeletal metastases and that Computed Tomography (CT) of the chest should be performed to evaluate for possible pulmonary metastases, there has been no research on the utility of bone scintigraphy in chondrosarcoma of bone and on the role of CT-chest in the staging of chondrosarcomas. Furthermore, the prevalence of skeletal and pulmonary metastases of chondrosarcoma at presentation was unknown prior to this study. The authors’ study demonstrated no skeletal metastases on bone scintigraphy in chondrosarcoma of bone at presentation. In contrast, pulmonary metastases were observed in approximately 5% of all patients with chondrosarcoma at presentation on CT-chest. The finding therefore demonstrates the rarity of skeletal metastases in chondrosarcoma of bone at presentation which is in contrast to osteosarcoma and Ewing sarcoma. The study therefore concludes that there is little role for skeletal scintigraphy in the surgical staging of chondrosarcoma. In contrast, the study shows that there is a role for CT-chest in the staging of chondrosarcoma. These above described findings are important new findings and represent a significant contribution to the knowledge base regarding metastatic behaviour of chondrosarcomas at presentation and regarding the staging of chondrosarcoma of bone. In summary, the authors’ publications have significantly enhanced and furthered the understanding of the pathogenesis of enchondromas, the role of functional MRI in the differentiation of enchondromas from chondrosarcomas, the utility of MRI in the grading of chondrosarcomas and the role of skeletal scintigraphy in the staging of chondrosarcomas.

Cheung Ka Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 83-93). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Publications --- p.ii / Awards --- p.iii / List of abbreviations --- p.iv / List of figures --- p.vi / List of tables --- p.vii / Abstract in English --- p.viii / Abstract in Chinese --- p.ix / Table of Contents / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- literature Review / Chapter 2.1 --- Anatomy and Physiology of Endometrium --- p.2 / Chapter 2.2 --- Endometrial cancer --- p.5 / Chapter 2.2.1 --- Epidemiology --- p.6 / Chapter 2.2.2 --- Etiologies and Risk Factors --- p.7 / Chapter 2.3 --- Pathology --- p.11 / Chapter 2.3.1 --- Grading of endometrial cancer --- p.14 / Chapter 2.3.2 --- Staging of endometrial cancer --- p.14 / Chapter 2.4 --- Prevention and Treatment --- p.16 / Chapter 2.5 --- Molecular alterations in endometrial cancer --- p.17 / Chapter 2.5.1 --- Genetic alterations in endometrial cancer --- p.18 / Chapter 2.5.1.1 --- Oncogene activation --- p.19 / Chapter 2.5.1.2 --- Tumor suppressor gene inactivation --- p.20 / Chapter 2.5.1.2.1 --- Mutation and loss of heterozygosity of tumor suppressor genes in endometrial cancer --- p.22 / Chapter 2.5.2 --- Epigenetic alterations --- p.27 / Chapter 2.5.2.1 --- CpG islands methylation --- p.29 / Chapter 2.5.2.2 --- de novo methylation --- p.29 / Chapter 2.5.2.3 --- Detection of gene promoter hypermethylation --- p.31 / Chapter 2.5.2.4 --- Epigenetic alteration in endometrial cancer --- p.31 / Chapter 2.5.2.5 --- Promoter hypermethylation of tumor suppressor genes in other cancers --- p.39 / Chapter 2.5.3 --- Microsatellite instability --- p.42 / Chapter Chapter 3 --- The objectives of study --- p.45 / Chapter Chapter 4 --- Materials and Methods --- p.46 / Chapter 4.1 --- Samples --- p.46 / Chapter 4.1.1 --- Formalin fixed paraffin embedded tissues --- p.46 / Chapter 4.1.2 --- Cell lines --- p.46 / Chapter 4.2 --- Histological grading and staging of samples --- p.47 / Chapter 4.3 --- Microdissection on tissue sections --- p.47 / Chapter 4.4 --- Extraction of nucleic acid / Chapter 4.4.1 --- Extraction of DNA from paraffin-embedded tissues --- p.48 / Chapter 4.4.2 --- Extraction of DNA from cell lines --- p.49 / Chapter 4.5 --- DNA methylation analysis --- p.49 / Chapter 4.5.1 --- Overview of Methylation-Specific PCR (MSP) --- p.49 / Chapter 4.5.2 --- Bisulfite modification of DNA --- p.50 / Chapter 4.5.3 --- Methylation specific PCR (MSP) --- p.51 / Chapter 4.6 --- Microsatellite Analysis --- p.53 / Chapter 4.7 --- Statistical analysis --- p.56 / Chapter Chapter 5 --- Results / Chapter 5.1 --- Clinical-pathological features of endometrioid adenocarcinoma --- p.57 / Chapter 5.2 --- Promoter hypermethylation in endometrial cancer --- p.57 / Chapter 5.3 --- Microsatellite status (MSI) analysis --- p.65 / Chapter Chapter 6 --- Discussion / Chapter 6.1 --- Promoter hypermethylation in endometrial cancer --- p.71 / Chapter 6.1.1 --- Concurrent hypermethylation of multiple genesin endometrioid adenocarcinoma and its precursor lesions --- p.72 / Chapter 6.1.1.1 --- Promoter hypermethylation of E-cad --- p.73 / Chapter 6.1.1.2 --- Promoter hypermethylation of APC --- p.73 / Chapter 6.1.1.3 --- Promoter hypermethylation of MGMT --- p.74 / Chapter 6.1.1.4 --- Promoter hypermethylation of RASSF1A --- p.75 / Chapter 6.1.1.5 --- Promoter hypermethylation of hMLH-1 --- p.76 / Chapter 6.1.1.6 --- Promoter hypermethylation in ECA coexisting with hyperplasia and not coexisting with hyperplasia --- p.77 / Chapter 6.1.2 --- Promoter hypermethylation in SCA --- p.77 / Chapter 6.2 --- Microsatellite status analysis --- p.78 / Chapter 6.2.1 --- MSI in endometrial cancer --- p.78 / Chapter 6.2.2 --- MSI and concurrent promoter hypermethylation --- p.79 / Chapter 6.2.3 --- MSI and promoter hypermethylation of hMLH-1 --- p.80 / Chapter Chapter 7 --- Conclusion --- p.81 / Further studies --- p.82 / References --- p.83

Lee Mei Lan May. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 48-56). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 中文摘要 --- p.III / ACKNOWLEDGEMENTS --- p.IV / PUBLICATION --- p.V / LIST OF FIGURES --- p.VI / LIST OF TABLES --- p.VII / ABBREVIATION --- p.VIII / CONTENTS --- p.IX / Chapter CHAPTER ONE: --- INTRODUCTION AND LITERATURE / Chapter 1.1 --- THYROID CANCER AND ITS EPIDEMIOLOGY --- p.1 / Chapter 1.1.1 --- Histology --- p.1 / Chapter 1.1.2 --- Gender comparison --- p.3 / Chapter 1.1.3 --- Female hormone 一 risk factor --- p.6 / Chapter 1.2 --- BIOLOGICAL BACKGROUND OF HORMONE´-´ؤؤ --- p.7 / Chapter 1.2.1 --- Estrogen --- p.8 / Chapter 1.2.2 --- Estrogen antagonist --- p.9 / Chapter 1.2.3 --- Estrogen receptors --- p.10 / Chapter 1.2.4 --- Estrogen receptor and thyroid cancer --- p.12 / Chapter 1.3 --- THE ROLE OF APOPTOSIS --- p.13 / Chapter 1.3.1 --- Bcl-family protein --- p.13 / Chapter 1.3.2 --- Bcl-family protein and cancer --- p.14 / Chapter 1.3.3 --- Estrogen and Bcl-family protein --- p.15 / Chapter 1.4 --- Objectives --- p.16 / Chapter CHAPTER TWO: --- GENERAL MATERIALS AND METHODS / Chapter 2.1 --- MATERIALS --- p.17 / Chapter 2.1.1 --- Culture media and treatment reagents --- p.17 / Chapter 2.1.2 --- Reagents for Western blot assay --- p.18 / Chapter 2.1.3 --- Antibodies --- p.19 / Chapter 2.1.4 --- Materials for RT-PCR --- p.19 / Chapter 2.1.5 --- Kits --- p.20 / Chapter 2.1.6 --- Instrumentations --- p.20 / Chapter 2.2 --- Methods --- p.21 / Chapter 2.2.1 --- Cell culture and treatment --- p.21 / Chapter 2.2.2 --- MTT assay --- p.22 / Chapter 2.2.3 --- Western blot analysis --- p.23 / Chapter 2.2.3.1 --- Protein extraction --- p.23 / Chapter 2.2.3.2 --- SDS-PAGE and protein transfer --- p.23 / Chapter 2.2.3.3 --- Immunoblotting analysis --- p.24 / Chapter 2.2.4 --- RNA extraction and RT-PCR --- p.25 / Chapter 2.2.4.1 --- RNA extraction --- p.25 / Chapter 2.2.4.2 --- cDNA synthesis --- p.26 / Chapter 2.2.4.3 --- Polymerase Chain Reaction (PCR) --- p.27 / Chapter 2.2.5 --- Statistical analysis --- p.28 / Chapter CHAPTER THREE: --- RESULTS / Chapter 3.1 --- Effect of E2 and tamoxifen on proliferation --- p.29 / Chapter 3.2 --- Comparison of effects of E and testosterone on Proliferation --- p.31 / Chapter 3.3 --- Differential Bcl-xL expression in response to E2 and testosterone stimulation --- p.33 / Chapter 3.4 --- Expression of ERα and ERβ in response to E2 stimulation --- p.35 / Chapter 3.5 --- Bcl-xL and Bax protein expression in response to E2 stimulation --- p.36 / Chapter 3.6 --- Expression of Bcl-xL and Bax mRNA in response to E2 stimulation --- p.39 / Chapter CHAPTER FOUR: --- DISCUSSION --- p.41 / Chapter CHAPTER FIVE: --- CONCLUSION --- p.47 / REFERENCES --- p.48

Tam Wai-Kwan Karen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 109-128). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgements --- p.vi / Abbreviations --- p.vii / Table of Contents --- p.ix / List of Tables --- p.xii / List of Figures --- p.xiv / Chapter 1. --- Introduction --- p.1 / Chapter 2. --- Literature Review --- p.2 / Chapter 2.1 --- Introduction of Cordyceps --- p.2 / Chapter 2.2 --- Pharmacological functions of Cordyceps --- p.3 / Chapter 2.2.1 --- Functions in respiratory system --- p.3 / Chapter 2.2.2 --- Functions in renal system --- p.7 / Chapter 2.2.3 --- Functions in hepatic system --- p.8 / Chapter 2.2.4 --- Functions in cardiovascular system --- p.9 / Chapter 2.2.5 --- Functions in endocrine and steroid system --- p.10 / Chapter 2.2.6 --- Functions in the immune system --- p.11 / Chapter 2.2.7 --- Functions in nervous system --- p.15 / Chapter 2.2.8 --- Controls in glucose metabolism --- p.15 / Chapter 2.2.9 --- Anti-oxidation activity --- p.16 / Chapter 2.2.10 --- Anti-tumor activity --- p.18 / Chapter 2.3 --- Active ingredients of Cordyceps and their related biological activities --- p.20 / Chapter 2.3.1 --- Polysaccharides --- p.20 / Chapter 2.3.2 --- Nucleosides --- p.21 / Chapter 2.3.2.1 --- Adenosine --- p.21 / Chapter 2.3.2.2 --- Cordycepin --- p.24 / Chapter 2.4 --- Proteomic tools in studies of the change in protein expression --- p.25 / Chapter 2.4.1 --- Two-dimensional electrophoresis --- p.27 / Chapter 2.4.2 --- Mass Spectrometry --- p.28 / Chapter 3. --- Methods and Materials --- p.30 / Chapter 3.1 --- Cell lines and culture conditions --- p.30 / Chapter 3.2 --- Trypan blue exclusion method --- p.30 / Chapter 3.3 --- Cell counting --- p.31 / Chapter 3.4 --- Anti-proliferation assay --- p.31 / Chapter 3.5 --- Anti-proliferation assay of normal cell line --- p.32 / Chapter 3.6 --- Determination of ic50 --- p.33 / Chapter 3.7 --- Sample preparation for proteins studies --- p.33 / Chapter 3.8 --- Protein quantitation --- p.34 / Chapter 3.9 --- Gel electrophoresis --- p.36 / Chapter 3.10 --- Image analysis --- p.37 / Chapter 3.11 --- In-gel digestion and MALDI-ToF MS --- p.37 / Chapter 3.12 --- Statistical Analysis --- p.39 / Chapter 3.13 --- Chemicals --- p.39 / Chapter 4. --- Results --- p.41 / Chapter 4.1 --- MTT assay --- p.41 / Chapter 4.1.1 --- The anti-proliferating activity of adenosine against cancer cell lines (HepG2 and SV7tert) and normal cell line (Hs68) --- p.41 / Chapter 4.1.2 --- The anti-proliferating activity of cordycepin against cancer cell lines (HepG2 and SV7tert) and normal cell line (Hs68) --- p.42 / Chapter 4.1.3 --- The anti-proliferation effects of adenosine and cordycepin --- p.42 / Chapter 4.2 --- Changes in protein expression --- p.50 / Chapter 4.2.1 --- "Corresponding drug treatment of cell lines (HepG2, SV7tert and Hs68)" --- p.50 / Chapter 4.2.2 --- "Comparison of protein profiles from cells (HepG2, SV7tert or Hs68) under the normal and drug treated (with either adenosine or cordycepin) conditions" --- p.51 / Chapter 4.2.2.1 --- HepG2 study --- p.51 / Chapter 4.2.2.2 --- SV7tert study --- p.52 / Chapter 4.2.2.3 --- Hs68 study --- p.52 / Chapter 4.2.3 --- Protein identification --- p.53 / Chapter 4.2.3.1 --- HepG2 cell line --- p.53 / Chapter 4.2.3.2 --- HepG2-changes in protein expressions after adenosine treatment --- p.54 / Chapter 4.2.3.3 --- HepG2-changes in protein expressions after cordycepin treatment --- p.54 / Chapter 4.2.3.4 --- SV7tert cell line --- p.54 / Chapter 4.2.3.5 --- SV7tert-changes in protein expressions after cordycepin treatment --- p.55 / Chapter 4.2.3.6 --- Hs68 cell line --- p.55 / Chapter 4.2.3.7 --- Hs68-changes in protein expressions after cordycepin treatment --- p.56 / Chapter 5. --- Discussion --- p.89 / Chapter 5.1 --- anti-proliferation assays --- p.89 / Chapter 5.2 --- changes in protein expression: --- p.90 / Chapter 5.2.1 --- Protein alterations in HepG2 --- p.91 / Chapter 5.2.1.1 --- Changes in protein expression (membrane protein and transport: Trimethyllysine hydroxylase) --- p.91 / Chapter 5.2.1.2 --- Changes in protein expression (protein synthesis and folding: carboxypeptidase E) --- p.92 / Chapter 5.2.1.3 --- Changes in protein expression (membrane proteins and transport: calumenin and electron transfer flavoproteins) --- p.93 / Chapter 5.2.2 --- Protein alterations in SV7tert --- p.94 / Chapter 5.2.2.1 --- Changes in protein expression (protein synthesis and folding: BiP(GRP78)) --- p.94 / Chapter 5.2.2.2 --- Changes in protein expression (cell defense and tolerance: Hsp60 (chaperonin); TANK binding kinase-1) --- p.96 / Chapter 5.2.2.3 --- Changes in protein expression (metabolism: prolyl 4-hydroxylase; aldolase A; glyceraldehyde-3-phosphate dehydrogenase) --- p.97 / Chapter 5.2.2.4 --- Changes in protein expression (cell growth and division: βII tubulin; HnRNP Al) --- p.100 / Chapter 5.2.3 --- Protein alterations in Hs68 --- p.101 / Chapter 5.2.3.1 --- Changes in protein expression (metabolism: triosephosphate isomerse 1) --- p.101 / Chapter 6. --- Discussion --- p.103 / Chapter 6.1 --- The antiproliferating activities of adenosine and cordycepin --- p.103 / Chapter 6.2 --- "Effects of adenosine and cordycepin on the changes in protein expressions in HepG2, SV7tert and Hs68" --- p.104 / Chapter 6.3 --- Problems and improvements in two-dimensional gel electrophoresis --- p.105 / Chapter 7. --- Conclusion and future prospectives --- p.107 / References --- p.109

Le cancer du sein reste la principale cause de décès par cancer chez les femmes et est connu pour la divergence des comportements cliniques et des résultats pour les patientes, malgré les caractéristiques histopathologiques courantes au moment du diagnostic. Cela s'explique par la grande hétérogénéité histologique et moléculaire de la maladie, qui rend difficile le choix d'une thérapie adaptée à chaque patient.L'épigénétique se rapporte aux modifications du phénotype et de l'expression génique. Les modifications épigénétiques du génome peuvent être acquises de novo et sont potentiellement héritées. Les mécanismes épigénétiques agissent pour modifier l'accessibilité de la chromatine à la régulation de la transcription localement et globalement via des modifications de l'ADN et par des modifications ou des réarrangements de nucléosomes. L'épigénétique consiste en plusieurs mécanismes moléculaires: modifications de l'histone, petits ARN non codants ou anti-sens et méthylation de l'ADN étroitement interconnectés.L'incidence et la mortalité par cancer du sein sont plus élevées (incidence environ trois fois supérieure) dans le monde occidental que dans les pays d'Asie avec des différences régionales dans les pays occidentaux. Plusieurs études impliquant des immigrés des pays occidentaux suggèrent que le mode de vie et l'alimentation sont deux des causes principales de ces différences. Un apport alimentaire élevé en phytoestrogènes, principalement sous forme de produits à base de soja, peut produire des taux circulants de phytoestrogènes à effets œstrogéniques. Des études épidémiologiques et expérimentales suggèrent qu'un régime alimentaire riche en phytoestrogènes pouvait avoir des effets protecteurs contre sur les affections liées aux œstrogènes, telles que le cancer du sein.Sur la base de ces informations, nous avons étudié les effets du traitement par les phytoestrogènes; génistéine, daidzéine et 17-β-estradiol sur la modification post-traductionnelle des histones, telles que la méthylation de la lysine et l’acétylation des histones H3 et H4 dans des lignées cellulaires du cancer du sein. Nous avons ensuite étudié les effets de l'inhibiteur de la méthylation de l'histone et de l'inhibiteur de l'histone désacétylase sur la triméthylation et l'acétylation de l'histone-lysine dans les lignées cellulaires du cancer du sein. Nous avons utilisé deux lignées cellulaires de cancer du sein, MCF-7 et MDA-MB-231, chacune traitée respectivement avec de l'hydrochlorure de 3-désazanule (DZNep) [5 µM] (HMTi), du butyrate de sodium (NaBu) [2 mM] (HDACi) et de l'acide de type Suberoylanilide Hydroxamic (SAHA) [1 µM] (HDACi). Enfin, nous avons mené des études sur toutes les lignées cellulaires atteintes de tumeurs du sein afin d'évaluer l'immunoprécipitation de la chromatine (PIP) de certaines modifications de l'histone dans le cancer. Les niveaux relatifs de trois histones modifiées, y compris H3K27me3 (histone 3 Lysine 27 méthylation), H3K9ac (Histone 3 Lysine 9 acétylation) et H3K4ac (Histone 3 Lysine 4 acétylation) seront déterminés dans les tumeurs du sein par rapport au tissu normal correspondant selon le classement de Saint-Gall. Aujourd'hui, ChIP a été couplé à des puces à ADN de promoteur afin d'évaluer les mécanismes de régulation du gène humain à l'échelle du génome. La technologie de la puce sur puce pourrait être utilisée pour étudier les altérations de l'expression globale des gènes dans la tumorigenèse. Ici, nous avons étudié les gènes exprimés de manière différentielle associés aux histones modifiées H3K27me3, H3K9ac et H3K4ac dans les tumeurs du sein à l'aide de microréseaux Agilent SurePrint G3 400kX2 contenant environ 21 000 transcrits humains. Nous analyserons les régions enrichies de chaque promoteur de gène dans trente tumeurs du sein par rapport à des échantillons de tissus normaux. Les échantillons de tumeurs du sein seront classés en fonction de leur profil clinique, en particulier du statut des récepteurs hormonaux. / Breast cancer remains the leading cause of cancer-related deaths in women, and is noted for conflicting clinical behaviors and patient outcomes, despite common histopathological features at diagnosis. This can be explained by the high histological and molecular heterogeneity of the disease, making it hard to choose a therapy adapted uniquely to each patient. Epigenetics refer to changes in phenotype and gene expression. Epigenetic modifications of the genome can be acquired de novo and are potentially inherited. Epigenetic mechanisms work to change the accessibility of chromatin to transcriptional regulation locally and globally via modifications of the DNA and by modifications or rearrangements of nucleosomes. Epigenetics consist in several molecular mechanisms: histone modifications, small non-coding or antisense RNAs and DNA methylation that are closely interconnected. The incidence and mortality of breast cancer is high in the Western world as compared with countries in Asia. There are also differences in the regional cancer incidence rates in Western countries. Several studies involving immigrants to Western countries suggest that lifestyle and diet are two of the main causes of these differences. In Eastern countries, the incidence of breast cancer is approximately one-third that of Western countries, whilst their high dietary intake of phytoestrogens, mainly in the form of soy products, can produce circulating levels of phytoestrogens that are known experimentally to have estrogenic effects. An increasing number of epidemiological and experimental studies have suggested that the consumption of a 4 phytoestrogen-rich diet may have protective effects on estrogen-related conditions, such as breast cancer.Based upon this information, we studied the effects of treatment phytoestrogens; genistein, daidzein and 17-β-estradiol on the post-translational modification of histones such as lysine methylation and acetylation of histones H3 and H4 in breast cancer cell lines. Subsequently, we studied the effects of histone methylation inhibitor and histone deacetylase inhibitor on histone lysine trimethylation and acetylation in breast cancer cell lines. For this study, we used two breast cancer cell lines MCF-7 and MDA-MB-231. Each cell line was treated respectively with 3-Deazaneplanocin A hydrochloride (DZNep) [5 μM] (HMTi), Sodium Butyrate (NaBu) [2 mM] (HDACi) and Suberoylanilide Hydroxamic acid (SAHA) [1 μM] (HDACi) for 48 hours. Finally, we completed studies in all cell lines with breast tumors to assess Chromatin ImmunoPrecipitation (ChIP) of selected histone modifications in cancer. The relative levels of three modified histones, including H3K27me3 (Histone 3 Lysine 27 Methylation), H3K9ac (Histone 3 Lysine 9 Acetylation), and H3K4ac (Histone 3 Lysine 4 Acetylation) will be determined in breast tumors compared to matched normal tissue according to the classification of Saint Gallen. Today, ChIP has been coupled with promoter DNA microarrays to evaluate the mechanisms of human gene regulation on a genome-wide scale. ChIP-on-chip technology could be used to investigate the alterations of global gene expression in tumorigenesis. Here, we investigated differentially expressed genes associated with modified histones H3K27me3, H3K9ac and H3K4ac in breast tumors by Agilent SurePrint G3 400kX2 microarrays containing approximately 21,000 of human transcripts. We will scan the enriched regions at each gene promoter in thirty breast tumors compared with normal tissue samples. Breast tumor samples will be classified according to their clinical profiles, especially hormone receptor status.

腫瘤發生過程中，遺傳和/或表觀遺傳異常都可導致抑癌基因(TSG)的失活。新TSG的鑒定和研究對理解癌症發展至關重要，并能提供潛在的治療靶點和腫瘤標誌物。本研究的目標是在人類腫瘤中鑒定新的被表觀遺傳異常沉默的TSG并進一步研究其抑癌的分子機理。 / 表觀遺傳修飾因子是重要的腫瘤相關基因。這裡，我研究了兩個作為功能性TSG的表觀遺傳修飾基因。首先，我發現一個在腫瘤中被甲基化和下調的候選TSG, PRDM5, 可通過抑制TCF/LEF依賴的轉錄和引起多個癌基因的表觀遺傳下調而抑制癌細胞增殖。其次，通過檢測24個表觀遺傳修飾基因在癌細胞系中的表達，我發現一個頻繁下調的新候選TSG，TUSC12。TUSC12在正常組織中普遍表達，但在腫瘤細胞系中被啟動子區甲基化下調，且原發腫瘤中也存在高頻TUSC12甲基化。TUSC12顯著抑制癌細胞克隆形成能力，但這種抑制效果因其PHD功能域的失活被部份削弱。TUSC12與轉錄抑制因子KAP1在細胞核中共定位，并可能通過招募HDAC相關複合體而抑制基因轉錄。 / 另外，我研究了一個通過以前鼻咽癌(NPC) aCGH鑒定得到的新3p14.2 TSG，ZNF312。啟動子區甲基化導致ZNF312在NPC細胞系和原發癌中的沉默。恢復ZNF312表達可抑制NPC細胞生長，并引起細胞週期阻滯和凋亡。作為一個轉錄抑制因子，ZNF312靶向EZH2和MDM2的啟動子區并下調它們的表達。 / 之前的基因數字表達分析已篩選出一些可能在腫瘤中低表達的基因。我研究了一個通過這種方法分離出的新TSG，TUSC45。相比正常組織，TUSC45表達在癌組織中顯著下降，而且TUSC45低表達與病人的低存活相關。TUSC45在多個腫瘤細胞系中也被下調，但它的基因組缺失卻不多見。啟動子區甲基化導致TUSC45在多數細胞系中的沉默，而且藥物或遺傳去甲基化能顯著激活它的表達。特別的，TUSC45在腫瘤組織而不在正常組織中被高頻率甲基化。誘導TUSC45表達可抑制細胞增殖，引起細胞凋亡、週期阻滯、和衰老，並上調一個關鍵抑癌基因p53。TUSC45以p53依賴的方式激活p53的靶基因，而TUSC45對p53缺失的H1299和HCT116/p53KO細胞沒有生長抑制作用。TUSC45與p53/MDM2複合物結合并正向調節p53蛋白穩定性。 TUSC45表達導致MDM2蛋白半衰期縮短，提示一種可能的TUSC45調節p53通路的機制。 / 本研究表明癌變過程中四個抑癌基因腫瘤特異的甲基化和沉默引起了多個細胞信號通路紊亂，並可作為潛在的腫瘤檢測標誌物。 / Tumor suppressor genes (TSGs) can be inactivated by genetic and/or epigenetic alterations during carcinogenesis. Identification and characterization of novel TSGs are critical for the understanding of cancer development, and provide potential targets for clinical treatment and biomarkers for tumor diagnosis. In this study, I aimed to identify novel TSGs epigenetically silenced in human cancers and further characterize the molecular basis of their anti-tumorigenic functions. / Emerging evidence highlights the importance of epigenetic modifiers as cancer genes. Here, I characterized two epigenetic modifier genes as functional TSGs. First, I found PRDM5, a candidate TSG methylated and downregulated in multiple cancers, suppressed tumor cell proliferation through inhibiting TCF/LEF-dependent transcription and inducing epigenetic repression of multiple oncogenes. Second, through expression profiling of 24 epigenetic modifiers, I identified a novel candidate TSG, TUSC12, showing frequent silencing in tumor cell lines. TUSC12 was broadly expressed in human normal tissues, but downregulated by promoter CpG methylation in tumor cell lines. Frequent TUSC12 methylation was detected in primary tumors as well. TUSC12 dramatically inhibited tumor cell clonogenicity, but this growth inhibitory effect was partially impaired by disrupting its PHD domain. TUSC12 colocalized with the transcription corepressor KAP1 in the nucleus and is likely to repress gene transcription through recruit HDAC-associated complex. / I also studied a novel 3p14.2 TSG, ZNF312, identified from previous aCGH profiling of nasopharyngeal carcinoma (NPC). Frequent promoter methylation silenced ZNF312 in NPC cell lines and tissues. Restored ZNF312 expression strongly suppressed NPC cell growth through inducing cell cycle arrest and apoptosis. Further, ZNF312 acted as a transcription repressor targeting the promoter regions of EZH2 and MDM2 and downregulating their expression. / Moreover, previous digital gene expression subtraction from cDNA libraries revealed a list of genes possibly downregulated in tumors compared to normal tissues. I characterized a novel TSG, TUSC45, initially isolated from this strategy. The expression of TUSC45 was significantly reduced in tumor tissues compared to normal tissues, with lower TUSC45 expression associated with poorer patient survival. Downregulation of TUSC45 in multiple tumor cell lines was also observed, while only infrequent genomic deletion was detected. In contrast, promoter methylation was responsible for TUSC45 silencing in most cell lines, in which pharmacologic or genetic demethylation can dramatically restore its expression. Remarkably, TUSC45 was frequently methylated in primary tumors but not in normal tissues. Further, TUSC45 suppressed anchorage-dependent and -independent tumor cell growth. Induced TUSC45 expression inhibited cell proliferation, induced apoptosis, cell cycle arrest and senescence, and lead to the upregulation of a key tumor suppressor p53. Moreover, TUSC45 activated p53 target genes in a p53-dependent manner. Forced TUSC45 expression in p53-null H1299 and HCT116/p53KO (knock out) cells showed no inhibitory effect on cell growth. Finally, TUSC45 interacted with p53/MDM2 complex and positively regulated p53 protein stability. The protein half-life of MDM2 was shortened by TUSC45, indicating a possible mechanism for TUSC45 modulation on p53 signaling. / In conclusion, this study showed the tumor-specific methylation and silencing of the four TSGs lead to the epigenetic disruption of multiple cell signalings during tumorigenesis and could potentially be used as biomarkers for cancer detection. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Shu, Xingsheng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 121-140). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Tables --- p.xi / List of Figures --- p.xii / List of Publications --- p.xiv / Abbreviations --- p.xv / Chapter Chapter 1 --- Introduction and Literature Reviews --- p.1 / Chapter 1.1 --- Tumor suppressor genes (TSGs) and the pathways they control --- p.2 / Chapter 1.1.1 --- Basic concepts about TSG --- p.2 / Chapter 1.1.1.1 --- The discovery of TSG --- p.2 / Chapter 1.1.1.2 --- Knudson’s “two-hit“ hypothesis --- p.3 / Chapter 1.1.1.3 --- New insights of the “two-hit“ model --- p.4 / Chapter 1.1.2 --- Key TSGs and their cellular pathways --- p.5 / Chapter 1.1.2.1 --- p53 pathway --- p.6 / Chapter 1.1.2.2 --- Rb pathway --- p.9 / Chapter 1.1.2.3 --- APC and Wnt/β-catenin pathway --- p.10 / Chapter 1.1.2.4 --- Chromatin regulators with tumor suppressive properties --- p.12 / Chapter 1.1.3 --- Methods for TSG identification --- p.17 / Chapter 1.2 --- The epigenetic abnormalities of TSGs in cancer --- p.19 / Chapter 1.2.1 --- Promoter CpG methylation --- p.20 / Chapter 1.2.1.1 --- Molecular basis of DNA methylation --- p.20 / Chapter 1.2.1.2 --- Silencing of TSGs by promoter methylation --- p.22 / Chapter 1.2.1.3 --- Mechanism of methylation-induced transcription repression --- p.23 / Chapter 1.2.1.4 --- Abnormal DNA methylation contributes to early stages of tumorigenesis --- p.25 / Chapter 1.2.2 --- Aberrant histone modification and chromatin remodeling --- p.26 / Chapter 1.2.3 --- Clinical applications of epigenetic biomarkers and therapeutic targets --- p.28 / Chapter 1.2.3.1 --- DNA methylation and histone modification as biomarkers for cancer diagnosis and prognosis --- p.28 / Chapter 1.2.3.2 --- Epigenetic targets for cancer treatment --- p.29 / Chapter Chapter 2 --- Aims and Design of This Study --- p.32 / Chapter Chapter 3 --- Materials and Methods --- p.34 / Chapter 3.1 --- Cell lines --- p.34 / Chapter 3.2 --- Human normal and cancer tissues --- p.35 / Chapter 3.3 --- DNA and RNA extraction --- p.35 / Chapter 3.4 --- Semi-quantitative and quantitative RT-PCR --- p.36 / Chapter 3.5 --- DNA methylation analysis --- p.37 / Chapter 3.5.1 --- Bisulfite modification of genomic DNA --- p.37 / Chapter 3.5.2 --- CpG island analysis --- p.37 / Chapter 3.5.3 --- Methylation-specific PCR (MSP) --- p.38 / Chapter 3.5.4 --- Bisulfite genomic sequencing (BGS) --- p.38 / Chapter 3.5.5 --- Pharmacologic demethylation treatment of cell lines --- p.38 / Chapter 3.6 --- Luciferase assay of gene promoter activity --- p.39 / Chapter 3.7 --- Multiplex genomic-DNA PCR --- p.39 / Chapter 3.8 --- Construction of expression plasmids and PCR-mediated mutagenesis --- p.40 / Chapter 3.9 --- Plasmid mini- and midi-preparation --- p.41 / Chapter 3.10 --- Creation of stable cell line for inducible gene expression --- p.42 / Chapter 3.11 --- Monolayer-culture and soft-agar colony formation assay --- p.42 / Chapter 3.12 --- Cell proliferation assay --- p.43 / Chapter 3.13 --- Flow cytometry analysis of cell cycle --- p.43 / Chapter 3.14 --- Apoptosis assay --- p.44 / Chapter 3.15 --- Senescence cell staining --- p.44 / Chapter 3.16 --- Western blotting --- p.45 / Chapter 3.17 --- Co-immunoprecipitation (Co-IP) --- p.46 / Chapter 3.18 --- Transcription factor activity assay --- p.46 / Chapter 3.19 --- Immunofluorescence microscopy --- p.47 / Chapter 3.20 --- Chromatin Immunoprecipitation (ChIP) --- p.47 / Chapter 3.21 --- Gene expression and copy number analysis using Oncomine database --- p.48 / Chapter 3.22 --- Protein half-life assay --- p.48 / Chapter 3.23 --- In vivo ubiquitination assay --- p.48 / Chapter 3.24 --- Statistical analysis --- p.49 / Chapter Chapter 4 --- Two Epigenetic Modifying Genes, PRDM5 and TUSC12, Suppress Tumor Cell Growth and are Silenced by Promoter Methylation in Human Cancers --- p.50 / Chapter 4.1 --- PRDM5 --- p.50 / Chapter 4.1.1 --- PRDM5 is a candidate TSG downregulated and methylated in multiple carcinomas --- p.50 / Chapter 4.1.2 --- PRDM5 is a stress responsive gene but its response is disrupted by promoter methylation --- p.53 / Chapter 4.1.3 --- PRDM5 suppresses cancer cell growth and proliferation --- p.54 / Chapter 4.1.4 --- PRDM5 inhibits TCF/LEF-dependent transcription and induced epigenetic repression of multiple oncogenes --- p.55 / Chapter 4.2 --- TUSC12 --- p.59 / Chapter 4.2.1 --- mRNA expression profiling of epigenetic modifying genes in tumor cell lines --- p.59 / Chapter 4.2.2 --- Promoter CpG methylation contributes to TUSC12 silencing in tumor cells --- p.61 / Chapter 4.2.3 --- Demethylation of TUSC12 promoter restores its expression --- p.63 / Chapter 4.2.4 --- TUSC12 methylation in primary tumor tissues --- p.65 / Chapter 4.2.5 --- TUSC12 expression inhibits anchorage-dependent and -independent tumor cell growth --- p.66 / Chapter 4.2.6 --- TUSC12 is an epigenetic modifier repressing transcription --- p.68 / Chapter 4.3 --- Discussion --- p.69 / Chapter Chapter 5 --- The Human Zinc Finger Protein 312 is a Novel Tumor Suppressor for Nasopharyngeal Carcinoma --- p.73 / Chapter 5.1 --- Identification of ZNF312 as a candidate 3p14.2 TSG --- p.74 / Chapter 5.2 --- Silencing of ZNF312 by promoter methylation in NPC cell lines --- p.75 / Chapter 5.3 --- ZNF312 is frequently downregulated and methylated in primary NPC tissues --- p.78 / Chapter 5.4 --- ZNF312 suppresses tumor cell clonogenicity --- p.79 / Chapter 5.5 --- ZNF312 is a transcription repressor --- p.80 / Chapter 5.6 --- ZNF312 regulates cell cycle progression, induces apoptosis, and inhibits cell stemness --- p.83 / Chapter 5.7 --- ZNF312 represses oncogene expression --- p.85 / Chapter 5.8 --- Discussion --- p.87 / Chapter Chapter 6 --- Identification of a Novel Tumor Suppressor Regulating p53 Signaling and Frequently Methylated in Multiple Tumors --- p.91 / Chapter 6.1 --- TUSC45 is broadly expressed in human normal tissues but frequently downregulated in tumor cell lines --- p.91 / Chapter 6.2 --- Reduced TUSC45 expression in primary tumors is associated with poor survival of patients --- p.94 / Chapter 6.3 --- TUSC45 is mainly silenced by promoter CpG methylation in tumor cell lines --- p.96 / Chapter 6.4 --- Pharmacologic or genetic demethylation reactivates TUSC45 in silenced cell lines --- p.98 / Chapter 6.5 --- TUSC45 is frequently methylated in primary tumors --- p.99 / Chapter 6.6 --- TUSC45 suppresses anchorage-dependent and -independent tumor cell growth --- p.101 / Chapter 6.7 --- Induction of TUSC45 expression inhibits tumor cell growth through inducing apoptosis, cell cycle arrest and senescence --- p.104 / Chapter 6.8 --- TUSC45 tumor-suppressive function is dependent on p53 signaling --- p.108 / Chapter 6.9 --- TUSC45 positively regulates p53 protein stability --- p.110 / Chapter 6.10 --- Discussion --- p.112 / Chapter Chapter 7 --- General Discussion --- p.116 / Chapter 7.1 --- General discussion --- p.116 / Chapter 7.2 --- Future perspectives --- p.118 / Reference List --- p.121

by Chan Chi Wai, Michael. / "July 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 238-272). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Lee Tin Lap. / "January 2001." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 161-185). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Lo Kwok-Wai. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (p. 171-199). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.