Colorectal cancer (CRC), with an estimated 1.2 million new cases annually, is the third leading cause of cancer incidence and death worldwide. Generally, the majority of CRC patients are diagnosed at the advanced stages with poor prognosis and unfavorable response to multiple therapeutic drugs. In spite of increasing knowledge of the molecular mechanism for the tumorigenesis in CRC patients, the translation from basic science into clinical therapy has been limited for quite a long time. In order to develop novel treatment strategies against CRC, intensive and extensive attempts have been made in the past decades. / The epithelial to mesenchymal transition (EMT) is a multi-step process characterized by the loss of cell polarity, decreased cell-cell adhesion as well as enhanced migration and invasion capacity. It is well documented that EMT is essential for a variety of cellular biological events ranging from embryogenesis to tumor progression. The field of lncRNA is developing rapidly and currently it is one of the most intensively studied fields in the biomedical sciences. Emerging evidence indicates that the majority of human genome encodes thousands of non-protein-coding RNA transcripts, nevertheless, the function of long non-coding RNAs (lncRNAs) in orchestrating EMT progression remains elusive. Historically, the lncRNA H19 was the first identified imprinted non-coding RNA transcript in human, and the H19/IGF2 locus acted as an ideal paradigm for the investigation of genomic imprinting genes. In recent years, the expression profiling and functional characterization of the H19 gene in a variety of human diseases has been extensively studied. / In our studies, H19 was characterized as a novel regulator of EMT in colon cancer. We first observed significant mesenchymal characteristics in the methotrexate-resistant HT-29 cells. Interestingly, significant upregulation of H19 was observed in mesenchymal-like MTX resistant HT-29 cells. We subsequently demonstrated that after treatment of TGF-β1, one of the most widely used EMT inducers, H19 presented dramatic increase during the EMT progression. To further investigate the functional role of H19 in EMT, we generated the stable cell lines overexpressing H19 in colon cancer cells using retroviral infection. Stable overexpression of H19 significantly promoted EMT progression in two epithelial colon cancer cell lines HT-29 and HCT-116. However, overexpression of H19 did not affect cell proliferation as well as cell cycle progression. Further proteomics studies screened out that ectopic expression of H19 upregulated the protein level of Vimentin, a vital biomarker for mesenchymal cells. By using the bioinformatics study in combination with luciferase reporter assays, we demonstrated that H19 potentiated the expression of several core marker genes essential for mesenchymal cells by serving as a competing endogenous RNA(ceRNA), which builds up the missing link between the regulatory miRNA network and EMT progression. According to the results from xenograft tumor model and soft agar assay, stable expression of H19 reinforced the in vitro and in vivo tumor growth. Moreover, the investigation of clinical specimens verified that H19 RNA level was significantly increased in colon cancer tissues compared with corresponding adjacent normal tissues. Taken together, the above observations imply that the lncRNA H19, by acting as a competing endogenous RNA, is an important regulator which tightly modulated the expression of multiple important genes involved in EMT and it could probably serve as a novel therapeutic target against colon cancer. / 大腸癌每年有一百二十萬新增個案，是世界第三大癌症殺手。通常情況下，大部分大腸癌病人發現時已經處於晚期，該時期的癌症病人對多種臨床治療藥物已無法治愈。盡管關於大腸癌發病的分子生物學機制已經不斷完善，但如何從基礎研究轉化為臨床治療手段在很長一段時間內不可實現。為了進一步研究新的抗擊大腸癌治療手段，廣泛且深入的研究已經不斷開展。 / 上皮間充質轉化是一個多步驟的過程，該過程的典型特徵為失去細胞的極性，細胞間粘連減弱以及細胞爬行遷移能力的不斷加強。目前科學家已經知道上皮間充質轉化對於從胚胎發育到腫瘤發展都起著重要的作用。近年來，長非編碼RNA的研究不斷快速發展，已然成為醫學研究中最激烈的領域之一。眾多證據表明人體基因組編碼數以千計不編碼蛋白質的RNA轉錄體。然而，這些RNA轉錄體在上皮間充質轉化中的功能依然所知甚少。長非編碼RNA H19是人體內第一個被鑒別出來參與到基因印記的非編碼RNA。資料表明H19/IGF2位點是一個非常理想的研究基因印記的位點。近年來，H19在眾多癌症中的表達以及功能學研究已不斷湧現，同時也不斷取得令人鼓舞的研究成果。 / 在我們的研究中，H19被鑒定為大腸癌裏上皮間充質轉化過程中一個重要的參與者。通過研究甲氨蝶呤耐藥大腸癌HT-29細胞株，我們發現該HT-29耐藥細胞株有著顯著的間充質細胞特性。有趣的是，H19在該細胞株中有著顯著升高。我們隨後用經典的上皮間充質轉化誘導劑TGF-β1處理兩株大腸癌細胞，處理後H19亦有著顯著升高。為了進一步研究H19在上皮間充質轉化，通過使用逆轉錄病毒，我們建立H19的穩定表達細胞株。穩定表達H19顯著地促進了HT-29以及SW620大腸癌細胞株的上皮間充質轉化。然後，高水平表達(過表達)H19並不影響細胞的生長以及細胞周期的進程。進一步的蛋白質組學研究表明，過表達H19能促進間充質細胞一個重要標記基因Vimentin的表達。通過生物信息學以及熒光素酶報告基因實驗，我們證明了H19通過其競爭內源性RNA的作用，能夠促進間充質細胞所需的幾個重要基因的表達。該發現建立起了miRNA網絡以及上皮間充質轉化進程的交流網絡。通過異位移植以及軟瓊脂實驗，我們發現過表達H19能夠促進腫瘤細胞的生長。而在臨床大腸癌病人組織中，我們更發現H19在大腸癌病人組織中高表達。綜上所述，我們的結果證明H19這一長非編碼RNA，能夠通過其競爭內源性RNA的作用機制，從而調控上皮間充質轉化過程中的關鍵基因。同時H19亦有可能成為治療大腸癌的臨床新靶點。 / Liang, Weicheng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 95-124). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016). / Liang, Weicheng.

Cheung, Wing Ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 100-112). / Abstracts in English and Chinese. / Acknowledgments / Abbreviations / Abstract 一 English --- p.i / - Chinese --- p.iii / Chapter Chapter 1 --- General Introduction / Chapter 1.1. --- Colon Cancer / Chapter 1.1.1. --- Colon cancer statistic in Hong Kong --- p.1 / Chapter 1.1.2. --- Development of Colon cancer --- p.1 / Chapter 1.1.3. --- Treatment --- p.2 / Chapter 1.2. --- Chemistry of ascorbates / Chapter 1.2.1. --- Sodium-L-ascorbate --- p.3 / Chapter 1.2.2. --- "Sodium 5,6-benazylidene-L-ascorbate" --- p.4 / Chapter 1.3. --- "Reactive oxygen species and reactive nitrogen species, and their biological consequences" --- p.5 / Chapter 1.4. --- Cell cycle --- p.7 / Chapter 1.5. --- Autophagy --- p.8 / Chapter 1.6. --- Human colon cancer HT29 cells for anti-tumor study --- p.9 / Chapter 1.7 --- Aim of study --- p.10 / Chapter Chapter 2 --- Comparative studies of cytotoxicity of SAA and SBA in short term treatment / Chapter 2.1. --- Introduction --- p.11 / Chapter 2.2. --- Materials and Methods --- p.14 / Chapter 2.3. --- Results --- p.17 / Chapter 2.4. --- Discussion --- p.26 / Chapter Chapter 3 --- Comparative studies of SAA and SBA in oxidative stress induction and their corresponding ROS inhibitors / Chapter 3.1. --- Introduction --- p.28 / Chapter 3.2. --- Materials and Methods --- p.31 / Chapter 3.3. --- Results --- p.35 / Chapter 3.4. --- Discussion --- p.42 / Chapter Chapter 4 --- "Effects of SAA and SBA treatments on cell cycle regulatory proteins and the induction of transient cell cycle arrests in Gl, S and G2 phases Cell Cycle" / Chapter 4.1. --- Introduction --- p.45 / Chapter 4.2. --- Materials and Methods --- p.49 / Chapter 4.3. --- Results --- p.53 / Chapter 4.4. --- Discussion --- p.69 / Chapter Chapter 5 --- Autophagy induction during SBA treatment and autophagy inhibition during SAA treatment / Chapter 5.1. --- Introduction --- p.72 / Chapter 5.2. --- Materials and Methods --- p.74 / Chapter 5.3. --- Results --- p.77 / Chapter 5.4. --- Discussion --- p.91 / Chapter Chapter 6 --- General Discussion --- p.93 / References --- p.100

Lectins (hemagglutinins) are a type of proteins that could recognize different sugar structures and specifically initiate reversible binding with them. Though they have been universally found in a variety of organisms, they are exceptionally abundant in legumes. From the initial finding of agglutinating red blood cells to the discovery of recognizing carbohydrates on cell membranes, multiple functions of lectins have been gradually unveiled by numerous researchers across a century. Based on its carbohydrate-binding property, lectins have found great value in the study of glycomics. Many lectin-based biological tools, like lectin affinity chromatography, lectin blotting, lectin histochemistry, lectin microarray and lectin-based biosensor have been developed and applied to the study of glycoproteins. Besides, lectins are also reported to be potential agents for anti-insect, anti-fungi, anti-HIV, anti-bacterial and anti-tumor applications. / The present study focuses on the isolation of a new hemagglutinin from an edible legume, exploration of its anti-colorectal cancer effect and mechanisms, its cytokine inducing function and anti-HIV activities. The protein was purified by liquid chromatography techniques which entailed affinity chromatography on Affi-Gel Blue Gel, ion exchange chromatography on Mono Q and gel filtration on Superdex 75 with an FPLC system. The hemagglutinating activity of this hemagglutinin was demonstrated to be ion-dependent and stable over a wide range of temperatures (20-60℃) and pH (2-11) values. Like most of the lectins or hemagglutinins, this novel hemagglutinin could also attenuate the activity of HIV-1 reverse transcriptase. / This hemagglutinin could potently suppress the proliferation of colorectal carcinoma HCT116 cells and colorectal adenocarcinoma HT29 cells. It induced cell cycle arrest in G0/G1 phase, downregulated the expression of Cyclin D1 and upregulated P21expression. The protein initially bound on the cell membranes most probably through glycoproteins and subsequently entered the cytoplasm, which was achieved as early as 3h post treatment. The hemagglutinin was found to be preferentially localized in Golgi apparatus and initiated aggregation of the Golgi apparatus, which may possibly attenuate its protein processing capacity by reducing total superficial area or even partially blocking the transportation of proteins from the endoplasmic reticulum (ER). The impaired protein reception ability of Golgi apparatus may lead to the protein accumulation in the ER and induce cell apoptosis. Accordingly, two ER stress sensors (IRE1α and ATF6) and one late product of ER stress (CHOP) were found to up-regulated. Apoptosis-inducing effect of this hemagglutinin on HT29 and HCT116 cells were further confirmed using methods based on different principles. Cells treated with the hemagglutinin were observed to undergo obvious chromatin condensation, mitochondrial membrane depolarization and phosphatidylserine exposure. An apoptosis initiator (Apaf-1) and one important indicator (cleaved PARP) of cell apoptosis were accordingly detected. Besides, intraperitoneal administration of this hemagglutinin to colorectal tumor bearing nude mice could slow down the growth of tumors. / At last, this hemagglutinin exerted an immunomodulatory function on splenocytes by stimulating the mRNA expression level of interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), interferon- gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α). Secretion of IL-1β and IL-2 from splenocytes also increased with the concentration of this hemagglutinin. / In a short conclusion, we have isolated a new hemagglutinin with anti-HIV RT, anti-colorectal cancer and immunomodulatory activities. / 凝集素（血凝素）是一类能够识别不同糖结构并能和它们发生可逆性结合的蛋白。虽然他们在许多生物体内均有发现，但这类蛋白在豆科植物中的含量尤其丰富。经过一个多世纪来众多研究者的努力，从最初认识到其具有红血细胞凝集功能到糖类识别作用，凝集素的诸多功能已被逐步挖掘。基于其独特的糖结构识别特性，凝集素在糖组学的研究中具有重大意义。许多基于凝集素的生物方法，如凝集素亲和层析法，凝集素印迹法，凝集素组织化学，凝集素生物芯片以及基于凝集素的生物传感器已被研究出来, 并用于研究糖蛋白。除此之外，研究表明，凝集素还具有抗虫，抗真菌，抗HIV，抗细菌和抗癌等活性。 / 该凝集素可以极大抑制结肠直肠癌HCT116细胞和结直肠腺癌HT29细胞增殖，引发细胞周期停滞，分别下调和上调Cyclin D1和P21的表达。该蛋白极有可能首先通过和细胞表面的糖蛋白结合而附在细胞膜上，然后进入细胞内。该过程可在往细胞培养液内加入该蛋白后的3小时内完成。该凝集素优先与细胞内的高尔基体结合，随后引发高尔基体聚集。该聚集作用可能会通过减少高尔基体总表面积甚至阻塞内质网和高尔基体间的蛋白运输，进而减弱高尔基体处理蛋白质的能力。当高尔基体接受蛋白的能力降低时，蛋白可能会堆积在内质网上并进一步引发细胞程序性死亡。相应地，两个内质网应激感受蛋白IRE1α和 ATF6以及内质网应激后期产物CHOP均被发现上调。该凝集素对HT29细胞和HCT116细胞的凋亡诱导作用采用不同的方法进行了进一步的确认，这些方法都是基于不同检测原理进行的。结果表明，该凝集素可导致细胞产生明显的染色质凝缩，线粒体膜电位去极化和磷脂酰丝氨酸外翻。与此相应地，凋亡启动蛋白Apaf-1和凋亡后期蛋白(被剪切的PARP)可在处理后的细胞中检测到。通过腹腔注射的方法给接种大肠癌细胞的裸鼠给药可降低肿瘤的生长速度。 / 本研究的工作包括：从一种可食用豆类中提取一种新的凝集素；检测其抗大肠癌的作用和机制；研究其细胞素诱导作用以及抗HIV活性。该蛋白采用液相色谱法分离提纯，其中包括亲和层析柱Affi-Gel Blue Gel, 离子交换层析柱Mono Q 和凝胶层析柱Superdex 75，后两种层析法在FPLC系统上操作。该蛋白的红血细胞凝集作用具有金属阳离子依赖性，并在20-60℃和pH2-11范围内保持活性稳定。像许多其它的凝集素一样，该蛋白也可以削弱HIV逆转录酶活性。 / 最后，该蛋白还具有免疫调节作用，它可促进白细胞介素-2，白细胞介素-6，白细胞介素-1β，干扰素-γ和肿瘤坏死因数-α在mRNA水平上的表达并刺激白细胞介素-2和细胞介素-1β的分泌。 / 综上所诉，本研究分离提纯了一种新凝集素，它具有抗HIV，抗大肠癌和免疫调节作用。 / Dan, Xiuli. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 153-170). / Abstracts also in Chinese. / Title from PDF title page (viewed on 05, October, 2016). / 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.

Includes bibliographical references. / xiii, 260 leaves ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examins Fecal Occult Blood Test (FOBT) screening as a possible means of reducing mortality from colorectal cancer in Australia. Consists of an evaluation of a FOBT screening program in South Australia (in terms of numbers of cancers detected, accuracy of the test used, costs of the program and characteristics of participants) and surveys of the general population and of South Australian general practitioners, providing information on knowledge, attitudes and practices in relation to colorectal cancer and its prevention. / Thesis (Ph.D.)--University of Adelaide, Dept. of Community Medicine, 1995?

Increasing number of people is diagnosed with colorectal neoplasms (CRN) in the form of polyps or cancers. Colorectal cancer (CRC) is one of the most common causes of cancer deaths among Chinese in Hong Kong. Advances in diagnosis and treatments have increased the survival rate of CRN patients, resulting in a large group of cancer survivors. Preserving and maintaining health-related quality of life (HRQOL) has become an important direction in research and clinical care of patients with CRN. This research aimed to evaluate the HRQOL and its association with socio-demographics and disease-related characteristics among patients with CRN, and illustrate how HRQOL data could be converted to preference for the estimation of quality adjustment of life years in health economic evaluations. This research comprised of three parts. First was the establishment of the validity and reliability of the traditional Chinese version of Functional Assessment of Cancer Therapy-Colorectal (FACT-C) as a HRQOL measure for Chinese patients by a cross-sectional sample of 536 adult patients with CRN. Psychometric testing and concurrent validation of the FACT-C with European Organization for Research and Treatment of Cancer Core Questionnaire plus Colorectal-specific Module Questionnaire and the Short-Form 12-item Health Survey_Version2 (SF-12V2) were carried out. Second was a longitudinal study on the HRQOL of 554 CRN patients at baseline, six (n=479) and twelve (n=414) months of recruitment. The associations of HRQOL with socio-demographics and disease-related factors, and change of HRQOL over time were explored. Comparisons of HRQOL between CRN patients and the general population and among different CRN groups were made. Cross-sectional data were used to develop mapping functions to estimate SF-6D preference scores from FACT-C subscale scores. Third was the application of the health preference scores by CRN stages collected at baseline of the longitudinal study, in combination with survival data extracted from the literature in a Markov model on the cost-effectiveness of different CRC screening strategies (colonoscopy, guaiac and immunochemical fecal occult blood tests) in comparison to no screening in terms of quality-adjusted life-years gained. This research comprised of three parts. First was the establishment of the validity and reliability of the traditional Chinese version of Functional Assessment of Cancer Therapy-Colorectal (FACT-C) as a HRQOL measure for Chinese patients by a cross-sectional sample of 536 adult patients with CRN. Psychometric testing and concurrent validation of the FACT-C with European Organization for Research and Treatment of Cancer Core Questionnaire plus Colorectal-specific Module Questionnaire and the Short-Form 12-item Health Survey_Version2 (SF-12V2) were carried out. Second was a longitudinal study on the HRQOL of 554 CRN patients at baseline, six (n=479) and twelve (n=414) months of recruitment. The associations of HRQOL with socio-demographics and disease-related factors, and change of HRQOL over time were explored. Comparisons of HRQOL between CRN patients and the general population and among different CRN groups were made. Cross-sectional data were used to develop mapping functions to estimate SF-6D preference scores from FACT-C subscale scores. Third was the application of the health preference scores by CRN stages collected at baseline of the longitudinal study, in combination with survival data extracted from the literature in a Markov model on the cost-effectiveness of different CRC screening strategies (colonoscopy, guaiac and immunochemical fecal occult blood tests) in comparison to no screening in terms of quality-adjusted life-years gained. Psychometric analysis confirmed that FACT-C had satisfactory reliability, construct validity and responsiveness in Chinese patients Patients with CRN reported worse physical HRQOL but better mental HRQOL and similar health preference score compared to the general population. Disease severity indicated by tumor stage at initial diagnosis was the most significant determinant of HRQOL of CRN patients. Rectal cancer also significantly associated with a decrease in physical HRQOL and health preference scores. Markov modelling showed that immunochemical fecal occult blood (I-FOBT) yearly was the most effective and two-yearly was the most cost-effective screening strategy compared to no screening. / published_or_final_version / Family Medicine and Primary Care / Doctoral / Doctor of Philosophy

Background Colorectal cancer is becoming the commonest cancer in Hong Kong in 2011. Colorectal cancer screening is becoming a hot topic of discussion after the proposal of a local pilot screening program of colorectal cancer in the Policy Address 2014. Colorectal screening is traditionally performed by faecal occult blood test and optical colonoscopy. Computed tomographic colonography is a new imaging technology, with high sensitivity and specificity for clinically significant colonic lesions and polyps. It is therefore emerging as a new method for the colorectal cancer screening. Method This project aims to systemically review the literature, try to explore the potential utility and cost effectiveness of using computed tomographic colonography as one of the screening modality for asymptomatic patients aged 50 years or older. Results and Conclusion The results of the review are presented and conclusion is made. The limitation of the systematic review and its implications of local policy making are also discussed. / published_or_final_version / Public Health / Master / Master of Public Health

TCP-1是一种新型的定向于肿瘤血管的多肽，通过小鼠体内的噬菌体展示技术筛选得到。在之前的研究中，我们已证明TCP-1具有定向于肿瘤血管并有效靶向运输抗肿瘤药物和显像剂的特性。本研究的目的是进一步研究在原位结肠癌模型中定向运输抗肿瘤药物肿瘤坏死因子(TNFα)，以及在结肠癌临床样本中运输显像剂异硫氰酸荧光素(FITC)的能力。并对TCP-1与肿瘤坏死因子的融合蛋白TCP-1/TNFα的抗肿瘤机制进行阐述。 / 本研究中，我们首先尝试用TCP-1作为载体，将增强绿色荧光蛋白靶向运输至肿瘤血管。结果证明TCP-1可以成功将蛋白运输到在肿瘤血管而非其它正常的组织器官上。TCP-1还可以靶向运输肿瘤坏死因子并增强其抗肿瘤作用。和肿瘤坏死因子比较，融合蛋白TCP-1/TNFα处理组的凋亡细胞数量增多，肿瘤微血管数目减少，并且无明显毒副作用。与结肠癌的一线化疗药物5-氟尿嘧啶(5-FU)联合给药后，与TNFα与5-FU联合给药相比较，融合蛋白TCP-1/TNFα联合5-FU在以下方面具有更明显的作用：抑制肿瘤生长，增加肿瘤细胞凋亡和抑制肿瘤细胞增殖，促进肿瘤血管正常化，升高瘤内免疫细胞以及减轻骨髓和脾内的免疫抑制反应。经检测TCP-1的靶向运输增加了瘤内的TNFα以及5-FU的浓度。这些都表明TCP-1不但可以靶向运输TCP-1/TNFα至肿瘤血管，还可以增加CD8+细胞的浸润增加瘤内免疫反应以及增加血管对抗肿瘤药物的通透性。以上都对抗肿瘤起到重要作用。 / 在临床的结肠癌样本中，TCP-1对肿瘤血管的结合能力也得到了证实。48.98%的结肠癌样本对TCP-1的结合为阳性。统计学分析显示TCP-1的结合与结肠癌的分期和肿瘤位置有关，对于N2期，位于乙状结肠的肿瘤的结合尤为明显。本研究的主要目的是将分离鉴定出的TCP-1发展成为结肠癌的生物标记，并且作为运输抗肿瘤药物和显像剂的载体应用于结肠癌的诊断和治疗中。鉴于TCP-1的靶向运输特点，将会有机会研发更多的抗肿瘤药物，同时增强传统化疗药的抗肿瘤作用。这些都可以优化肿瘤治疗的方案。综上所述，TCP-1是一种在结肠癌治疗诊断中具有广阔前景的多肽。 / TCP-1 is a novel vasculature-targeting peptide. It was discovered through the in vivo phage library selection in mice. It was demonstrated that TCP-1 peptide exhibited a homing ability to the neovasculature of colon tumors and was capable of efficiently delivering imaging agents and chemotherapeutic drugs to this target site. The current study is to further investigate the targeting ability of TCP-1 to deliver a known immunomodulator, tumor necrosis factor α (TNFα) as an example of anti-cancer drug in an orthotopic colorectal cancer (CRC) model and fluorescein isothiocyanate (FITC) as imaging agent for testing the binding capacity for tumors in colorectal cancer patients. The mechanisms for the action of this novel biologic TCP-1/TNFα in the treatment of colon cancer in mice were also defined. / In this study, we observed that TCP-1 peptide delivered enhanced green fluorescent protein (EGFP) only to tumor blood vessel other than normal organs after TCP- 1/EGFP injection. This was not observed after EGFP injection. This finding showed that TCP-1 can deliver biologic protein to the tumor blood vessels. Furthermore, results from TNFα or TCP-1/TNFα targeted delivery experiments showed that TCP- 1/TNFα displayed stronger anti-cancer effects than TNFα alone on the induction of apoptosis and reduction in number of microvessels in the tumors, without significant effect in systemic toxicity. In the combined therapy with 5-fluorouracil (5-FU), a standard drug for colon cancer treatment, pretreatment with low dose (1 ng TNFα /mouse) of TNFα or TCP-1/TNFα potentiated the anti-cancer action of 5-FU. In this regard, TCP-1/TNFα could significantly reduce tumor size and weight, increase number of apoptotic cells, inhibit tumor cell proliferation, normalize tumor blood vessels, facilitate infiltration of immune cells to tumor mass and attenuate immunosuppression in bone marrow and spleen. Moreover, TCP-1 could significantly increase intratumoral levels of TNFα and 5-FU. It was also suggested that TCP-1 could selectively deliver TNFα to the tumor blood vessels and modulate the immune response by increasing CD8+ cells infiltration to tumors and increase vascular permeability to 5-FU. These observations may be the key actions to reduce tumor growth. / The binding ability of TCP-1 was also detected in clinical samples from colorectal cancer patients in which 24/49 (48.98%) tumor tissues were positive with TCP-1 binding signal. Statistical analysis showed that TCP-1 had a strong and significant binding with colorectal cancer at the N2 stage among the different colorectal cancer stages (P=0.028) and location in the colon at the sigmoid (P<0.001). / Our study also focused on the isolation and identification of the binding molecule of TCP-1 in order to develop it into a biomarker for CRC and using TCP-1 as a carrier in delivering anti-cancer drugs and imaging agents to colon tumors for cancer therapy and diagnosis. With the homing property of TCP-1 on colon tumor blood vessels, new types of anti-cancer drugs will be developed and their combinations with conventional chemotherapy drugs will optimize the therapeutic outcome and improve regimen of treatment for CRC. Taken together, TCP-1 peptide appears to be a promising agent in molecular imaging and drug delivery for CRC diagnosis and therapy. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lu, Lan. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 157-177). / Abstracts also in Chinese. / Lu, Lan.

丹参是一种著名的传统中药，富含丹酚酸和丹参酮。其中，丹参酮的潜在抗肿瘤作用近年来引起众多关注。本研究评价了主要的丹参酮及其衍生物对结肠癌细胞的细胞毒性。结果显示DHTS具有最强的抗结肠癌活性和显著的肿瘤特异性细胞毒性，其细胞毒性主要由于凋亡诱导而不是引起坏死。初步的构效关系分析提示丹参酮母环结构中的A环和B环增加的离域性有助于提高其对结肠癌细胞的细胞毒性，而非二维结构及较小的D环也是进行结构改造的可能方向。 / 基于以上发现，本研究进一步探讨了DHTS的体内外抗肿瘤活性及内在机制。本研究发现DHTS的促凋亡活性并不依赖于p53的表达，而依赖于caspase活性及线粒体介导的细胞质中氧自由基 ROS及钙离子的聚集。DHTS可引起浓度及时间依赖caspase-9/-3/-7的活化而并未显著引起caspase-8的活化，这一现象发生于同样以浓度及时间依赖方式进行的线粒体中cytochrome c及AIF转位之后。在DHTS诱导的结肠癌细胞凋亡中，cytochrome c及caspase介导的凋亡通路及AIF介导的凋亡通路均被激活并显示出两条通路之间的交叉调控。 / 此外，线粒体在DHTS的促凋亡活性中的作用在本研究中被深入探讨。本研究发现线粒体可能是DHTS的一个直接靶点, 而氧化磷酸化复合体III则更可能是其作用位点。DHTS可以引起迅速而明显的线粒体功能障碍，随之引起细胞质中大量的氧自由基及钙离子聚集，诱导凋亡的产生。 / 与体外结果一致，本研究证实了DHTS对免疫缺陷小鼠中的结肠癌移植廇也具有明显的抗肿瘤作用。与溶媒对照组比较，DHTS治疗组中移植廇的增长显著被减缓，在治疗终点时的廇体积与重量也显著被降低。TUNEL检测确认DHTS诱导移植廇中癌细胞的显著凋亡。免疫荧光检测也发现DHTS诱导caspase-3及caspase-7在移植廇中癌细胞的明显活化。 / 综上所述，本研究提供了丹参酮抗结肠癌活性的一些初步构效关系的信息，为提高丹参酮抗结肠癌活性的结构改造提供一定的参考。更重要的是，本研究证明了DHTS的体内外抗结肠癌活性并探讨了其作用机制及可能靶点，为DHTS作为新的应用于抗结肠癌药物或辅助治疗用药提供了临床前研究证据。 / Salvia miltiorrhiza Bunge, also known as Danshen, rich in phenolic acid and tanshinones, has been widely used to treat various kinds of diseases including heart diseases and hepatitis in China with minimal side effects. Among the tanshinones, tanshinone I, tanshinone IIA, cryptotanshinone and dihydrotanshinone I are the major bioactive constituents in this herb. In this study, the anti-colon cancer potential of five tanshinones and six derivatives of tanshinone IIA were evaluated in several colon cancer cell lines. It was found that apoptosis but not necrosis contributed significantly to the cytotoxicity of the tanshinones. Dihydrotanshinone I (DHTS) was confirmed to be the most potent and selective anti-cancer compound among the tanshinones tested in this study. Preliminary SAR (structure activity relationship) of tanshinones reveals that the increase of delocalizability of A and B rings in the chemical structure of the tanshinones enhances their cytotoxicity on cancer cells, while compounds with a non-planar and small sized D ring region are better choices for anti-cancer effect. / The underlying mechanisms of the anti-colon cancer activity of DHTS were further studied. It was found that apoptosis induced by DHTS was p53 independent but caspase dependent, which was closely related to intracellular accumulation of ROS (reactive oxidant stress) and calcium mediated by mitochondria. A concentration- and time-dependent activation of caspase-9,-3,-7 but not caspase-8 by DHTS in HCT116 cells was detected after the translocation of cytochrome c and AIF (apoptosis inducing factor) from mitochondria. In this process, the crosstalk between the caspase-dependent and caspase-independent pathways was firstly shown in the apoptotic mechanism of DHTS. To this end, the release of cytochrome c happened first and the translocation of apoptosis inducing factor (AIF) was prevented by a pan caspase inhibitor. In the meantime, the release of cytochrome c and activation of caspase-9 and PARP (poly-ADP-ribose polymerase) cleavage were decreased after AIF knockdown. Especially, mitochondrion was suggested to be the direct target of DHTS and OXPHOS complex III but not OXPHOS complex I was probably the acting site of DHTS. / In accordance with the results obtained in vitro, the potential anti-colon cancer activity of DHTS was also observed in nude mice with xenograft tumors and the compound did not produce any observable systemic toxicity. DHTS efficiently delayed tumor growth by decreasing the tumor size and weight through the induction of apoptosis in cancer cells but not by inhibition of cell proliferation. In the same tissues, a distinct activation of caspase-3 and caspase-7 in tumor cells was also detected by immunofluorescence assay. / Collectively, the present study provides preliminary information about the SAR of the anti-colon cancer activity for tanshinones. It also confirms that DHTS is a promising compound for anti-cancer action both in vitro and in vivo. In addition, this study gives us a better understanding regarding the mechanisms of how DHTS induces apoptosis in cancer cells. All these findings could provide solid pre-clinical evidence to propel the development and application of DHTS and perhaps its derivatives as novel therapeutic or adjuvant agents for the treatment of colon cancer. / 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. / Wang, Lin. / Thesis (Ph.D.) Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 122-132). / Abstracts also in Chinese. / Wang, Lin.

CFTR基因突變或者功能缺失是否導致包括胃腸道在內的各種組織惡性腫瘤的發生風險增加目前仍然是一個充滿爭議的問題。同時，眾所周知，緊密連接分子在腫瘤發生和轉移的過程發揮了關鍵的作用。本論文首次發現了CFTR基因與一種緊密連接分子AF-6/afadin的在人類結直腸腫瘤中的表達水平呈高度相關，并研究了CFTR和AF-6/afadin之間潛在的相互作用及其在結直腸腫瘤轉移中的功能。 / 論文的第一部份首先用實時定量PCR和免疫組織化學的方法比較了CFTR在結直腸腫瘤和正常組織的表達情況，發現CFTR表達水平在腫瘤組織中有顯著的下降。令人感興趣的是，我們同時發現CFTR和AF-6/afadin在腫瘤組織中的表達呈高度正相關，并由此展開了後續的體外實驗，研究對CFTR與AF-6/afadin之間可能的相互聯繫。利用免疫螢光染色和免疫共沉澱的方法，我們發現了這兩種蛋白分子共表達在結直腸腫瘤細胞的接觸面，并存在相互作用。用CFTR突變蛋白的免疫共沉澱實驗進一步發現，這種相互作用需要CFTR分子在細胞膜表面的正確定位及其PDZ結構域結合位點。實驗還發現與CFTR的相互作用加強了AF-6/afadin與細胞骨架蛋白系統的結合。在結直腸腫瘤細胞中CFTR基因敲减导致了AF-6/afadin蛋白定位混亂，從細胞連接位點轉移到細胞漿內，并因此破壞了上皮細胞的緊密性。極性生長細胞的跨上皮電阻降低而滲透性增強的實驗結果證實了CFTR基因敲減導致的上皮細胞緊密性的破壞。同時，AF-6/afadin蛋白水平也隨著CFTR基因敲減而降低，但mRNA水平未發生明顯的改變。蛋白降解系統的抑製劑逆轉了CFTR基因敲減細胞中AF-6/afadin蛋白的減少，提示CFTR基因敲減增加了AF-6/afadin的蛋白降解。這些實驗結果揭示了通過與細胞連接分子AF-6/afadin的相互作用以及調節，CFTR可能在上皮細胞極性的調節以及腫瘤發展過程中起重要作用。 / 論文的第二部份研究了CFTR和AF-6/afadin在結直腸腫瘤細胞上皮細胞間充質化（EMT）和轉移過程中的功能及機制。我們之前的工作已經揭示抑制CFTR的功能可以誘導結直腸腫瘤LIM1863細胞的EMT過程。本研究在另外三株不同的結直腸腫瘤細胞（SW480，SW1116和HRT-18）中進一步證實了抑制CFTR誘導的EMT過程。細胞形態轉變，上皮細胞標誌物的下調，間充質細胞標誌物的上調以及受損的上皮細胞緊密性均證實了對CFTR的抑制可以在這三種細胞中成功誘導EMT的發生。我們發現在以上所有細胞EMT的過程中，AF-6/afadin的蛋白表達水平都發生了顯著的下調。在HRT-18細胞中過表達AF-6/afadin，可以逆轉由CFTR抑製劑誘導的上皮細胞標誌分子的下調和間充質標誌分子的上調，表明抑制CFTR誘導的EMT過程是由AF-6/afadin參與介導的。此外，CFTR基因敲減導致結直腸腫瘤細胞的惡性表型強化，包括減弱的細胞粘附性，增強的貼壁依賴性生長、侵襲和遷移。另外，CFTR基因敲減激活了ERK的磷酸化，過表達AF-6/afadin可以阻斷ERK途徑的激活。CFTR基因敲減而增強的細胞侵襲性也可以被外源性AF-6/afadin或者ERK途徑的抑製劑U0126完全逆轉，提示作為AF-6/afadin的下游靶信號，ERK介導了CFTR在腫瘤侵襲中的作用。更重要的是，我們分析了CFTR和AF-6/afadin的表達水平與結直腸癌病人腫瘤進展的關係，發現在嚴重TNM腫瘤分期或者有腫瘤遠處轉移的病人中CFTR的表達水平顯著低於輕型分期或未发生转移的病人中的水平，而且CFTR和/或AF-6/afadin低表達的病人的預後更差。這些實驗結果顯示CFTR的缺失可能通過抑制AF-6/afadin和激活ERK通路而與EMT和結直腸癌癥轉移的過程高度相關。 / 綜上所述，本研究揭示了以往未報道過的CFTR在結直腸腫瘤發病機理中的功能，提示CFTR可以用作一種新的腫瘤的潛在預後指標。 / The question whether mutation or dysfunction of CFTR increases the risk of malignancies in various tissues, including the gastrointestinal tract, remains highly controversial. Meanwhile, it is well-known that adherens junctions play critical roles in the process of cancer development and metastasis. In this thesis we found for the first time a highly correlation between expression levels of CFTR and an adherens junction molecule AF-6/afadin in human colorectal tumours, and investigated the potential interaction between CFTR and AF-6/afadin and their functional roles in the metastasis of colorectal cancer. / In the first section of this thesis, we started our studies with comparing the expression of CFTR between human colorectal tumours and normal colorectal tissues. Real time quantitative PCR and immunohistochemistry results revealed a dramatically reduced CFTR level in the cancer tissues. Intriguingly, we noticed a highly positive correlation between CFTR and AF-6/afadin expression in tumours, which prompted the further in vitro investigation of possible interaction between CFTR and AF-6/afadin. Using immunofluoresent staining and co-immunoprecipitation, we found that the two proteins were colocalized at cell-cell junctions and interacted with each other in colorectal cancer cell lines. Further Co-IP experiments performed with CFTR mutations revealed that this protein interaction requires the proper localization of CFTR in cell membrane and its PDZ-interacting domain. Moreover the interaction with CFTR strengthens the binding of AF-6/afadin to the cytoskeleton system. Knockdown of CFTR in colorectal cancer cells resulted in the disorganized localization of AF-6/afadin protein from junctional sites to the cytoplasm and impaired epithelial tightness, which was confirmed by significantly reduced transepithelial resistance and increased permeability of polarized cells. Meanwhile, the protein level of AF-6/afadin was down-regulated in CFTR-knockdown cells, while no significant changes were detected at the mRNA level. Protein degradation inhibitor reversed the repression of AF-6/afadin protein in CFTR knockdown cells, suggesting the protein degradation of AF-6/afadin was increased by CFTR knockdown. These data revealed that CFTR interacts with and regulates the cell adhesion molecular AF-6/afadin in colorectal cells, which may be important in the regulation of epithelial cell polarity and cancer development. / In the second section of this thesis, we studied the functional roles and mechanisms of CFTR and AF-6/afadin in the epithelial-mesenchymal transition (EMT) and metastasis of human colorectal cancer cells. Our previous work has revealed inhibition of CFTR can induce EMT in a colorectal cancer cell line, LIM1863. This study further confirmed the induction of EMT by inhibiting CFTR in several other colorectal cancer cell lines (SW480, SW1116 and HRT-18), which was evaluated by morphological changes, down-regulation of epithelial markers or up-regulation of mesenchymal markers, and impaired epithelial cell tightness. In all these cell lines, we found that the protein levels of AF-6/afadin were significantly reduced. Over-expression of AF-6/afadin in HRT-18 cells reversed the down-regulated epithelial markers and up-regulated mesenchymal markers induced by CFTR inhibition, indicating that the CFTR inhibition-induced EMT is mediated by AF-6/afadin. Moreover, knockdown of CFTR in HRT-18 or RKO cells resulted in enhanced malignant phenotypes, including decreased cell adhesion, increased anchorage-independent cell growth, invasion, and migration. In addition, extracellular signal-regulated kinase (ERK) phosphorylation was activated by CFTR knockdown, which was abolished by over-expression of AF-6/afadin. The enhanced invasiveness of CFTR knockdown cells was also completely inhibited by either exogenous AF-6/afadin or ERK inhibitor, U0126, suggesting that ERK, the downstream target of AF-6/afadin, is involved in mediating the effect of CFTR in cancer invasion. More importantly, we analyzed the association of CFTR and AF-6/afadin expression levels with tumour progression of patients with colorectal cancer, and revealed that CFTR expression was significantly lower in patients with more severe TNM stage or with metastasis to distant organs than those with milder stage or with no metastasis. The prognosis was poorer in patients with lower expression of CFTR and/or AF-6/afadin than those with higher expressions. These data showed that dysfunction of CFTR is highly associated with EMT and colorectal cancer metastasis, probably via repression of AF-6/afadin and activation of ERK pathways. / In summary, the present study has revealed a previously undefined role of CFTR in the pathogenesis of colorectal cancer and indicated its potential as a new prognostic indicator. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Sun, Tingting. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 113-127). / 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 / 中文摘要 --- p.iv / Publications --- p.vi / Conference Abstract --- p.vii / Declaration --- p.viii / Acknowledgements --- p.x / List of Figures --- p.xi / List of Tables --- p.xiii / List of Abbreviations --- p.xiv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1. --- Colorectal Cancer --- p.1 / Chapter 1.1.1. --- Structure of Human Normal Colon and Rectum Epithelium --- p.1 / Chapter 1.1.2. --- Staging of Colorectal Cancer --- p.3 / Chapter 1.1.3. --- Metastasis of Colorectal Cancer --- p.3 / Chapter 1.1.4. --- K-Ras mutation and It Downstream Pathways in Colorectal Cancer Metastasis --- p.11 / Chapter 1.1.5. --- Prognosis of Colorectal Cancer --- p.14 / Chapter 1.2. --- Epithelial Cell Junctional Complexes --- p.14 / Chapter 1.2.1. --- Junctional Complexes and Epithelial Cell Polarity --- p.15 / Chapter 1.2.2. --- Classic Cadherin-catenin Complex --- p.17 / Chapter 1.2.3. --- Novel Nectin-afadin Complex --- p.19 / Chapter 1.2.4. --- Cell Polarity and Cancer Progression --- p.23 / Chapter 1.3. --- Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) --- p.24 / Chapter 1.3.1. --- Structure of CFTR --- p.24 / Chapter 1.3.2. --- Mutations of CFTR --- p.24 / Chapter 1.3.3. --- Functions of CFTR --- p.26 / Chapter 1.3.4. --- Cancer Risk of CF Patients --- p.33 / Chapter 1.4. --- Hypothesis and Aims --- p.34 / Chapter Chapter 2 --- Materials and Methods --- p.35 / Chapter 2.1. --- Materials --- p.35 / Chapter 2.1.1. --- Reagents and Chemicals --- p.35 / Chapter 2.1.2. --- Antibodies --- p.35 / Chapter 2.1.3. --- Primers --- p.35 / Chapter 2.1.4. --- Solutions and Buffers --- p.35 / Chapter 2.1.5. --- Human Specimens --- p.36 / Chapter 2.2. --- Methods --- p.36 / Chapter 2.2.1. --- Cell Culture --- p.36 / Chapter 2.2.2. --- Transfection --- p.36 / Chapter 2.2.3. --- Selection of Stable Clones --- p.40 / Chapter 2.2.4. --- RNA Extraction and RT-PCR --- p.40 / Chapter 2.2.5. --- Quantitative Real Time PCR --- p.41 / Chapter 2.2.6. --- Protein Extraction and Western Blotting --- p.42 / Chapter 2.2.7. --- Immunostaining --- p.45 / Chapter 2.2.8. --- In vitro Cell Functional Assays --- p.46 / Chapter 2.2.9. --- Epithelial Tightness Measurement --- p.48 / Chapter 2.2.10. --- Statistical Analysis --- p.49 / Chapter Chapter 3 --- Interaction of CFTR with AF-6/afadin and Its Importance in Maintaining Colorectal Epithelial Cell Polarity --- p.50 / Chapter 3.1. --- Introduction --- p.50 / Chapter 3.2. --- Objectives --- p.53 / Chapter 3.3. --- Experimental plan --- p.54 / Chapter 3.4. --- Results --- p.55 / Chapter 3.4.1. --- The expression of CFTR and AF-6/afadin is decreased and positively correlated in human colorectal cancer --- p.55 / Chapter 3.4.2. --- CFTR colocalizes and interacts with AF-6/afadin in human colorectal cancer cells --- p.58 / Chapter 3.4.3. --- PDZ binding motif and membrane localization of CFTR are necessary for the interaction between CFTR and AF-6/afadin --- p.64 / Chapter 3.4.4. --- Knockdown of CFTR interferes with cell junction formation in colorectal cancer cells --- p.66 / Chapter 3.5. --- Discussion --- p.71 / Chapter Chapter 4 --- CFTR as a Suppressor and Prognosis Indicator of Metastasis in Human Colorectal Cancer --- p.77 / Chapter 4.1. --- Introduction --- p.77 / Chapter 4.2. --- Objectives --- p.80 / Chapter 4.3. --- Experimental plan --- p.81 / Chapter 4.4. --- Results --- p.82 / Chapter 4.4.1. --- CFTR inhibition-induced EMT in colorectal cancer cells involves AF-6/afadin --- p.82 / Chapter 4.4.2. --- Knockdown of CFTR aggravates malignant phenotype of colorectal cancer cells --- p.86 / Chapter 4.4.3. --- AF-6/afadin mediates the effect of CFTR on cell invasion in colon cancer through ERK --- p.91 / Chapter 4.4.4. --- CFTR and AF-6/afadin expression is correlated with the prognosis of colorectal cancer --- p.97 / Chapter 4.5. --- Discussion --- p.100 / Chapter Chapter 5 --- General Discussion and Conclusion --- p.105 / Chapter 5.1. --- The diversified roles of CFTR in epithelial cells --- p.105 / Chapter 5.2. --- The unfolding relationship between CFTR and cancer development --- p.107 / Chapter 5.3. --- Future studies --- p.109 / Chapter 5.4. --- Conclusions --- p.112 / Reference List --- p.113 / Chapter Appendix A --- Reagents and Chemicals --- p.128 / Chapter Appendix B --- Antibody List --- p.131 / Chapter Appendix C --- Primer List --- p.132 / Chapter Appendix D --- Solution Recipe --- p.133

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