by Li Pok Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 94-114). / Abstracts in English and Chinese. / Acknowledgements --- p.I / Abbreviations --- p.II / Abstract --- p.V / 摘要 --- p.VIII / Publications --- p.XI / Table of contents --- p.XII / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Human Th lymphocytes and their immunopathogenic roles --- p.1 / Chapter 1.1.1 --- Characteristics of Th lymphocytes --- p.1 / Chapter 1.1.2 --- Migration and activation --- p.1 / Chapter 1.1.3 --- Th cell differentiation --- p.2 / Chapter 1.1.4 --- Pathological roles --- p.4 / Chapter 1.2 --- Cytokines as modulator in Th lymphocyte activation --- p.6 / Chapter 1.2.1 --- IL-18 --- p.6 / Chapter 1.2.2 --- IL-25 --- p.7 / Chapter 1.3 --- Surface marker expression in Th lymphocytes --- p.8 / Chapter 1.3.1 --- Adhesion molecules --- p.8 / Chapter 1.3.2 --- Cytokine and chemokine receptors --- p.9 / Chapter 1.3.3 --- Costimulatory molecules --- p.11 / Chapter 1.4 --- Cytokine and chemokine release from Th lymphocytes / Chapter 1.4.1 --- Thl cytokines --- p.13 / Chapter 1.4.2 --- Th2 cytokines --- p.14 / Chapter 1.4.3 --- Chemokines --- p.15 / Chapter 1.5 --- Intracellular signaling pathways in Th lymphocytes --- p.19 / Chapter 1.5.1 --- p38 MAPK pathway --- p.19 / Chapter 1.5.2 --- ERK pathway --- p.20 / Chapter 1.5.3 --- JNK pathway --- p.20 / Chapter 1.5.4 --- NF- k B pathway --- p.21 / Chapter 1.6 --- Pharmacological intervention of signaling pathways --- p.22 / Chapter 1.7 --- Aims and scope of the study --- p.24 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.26 / Chapter 2.1.1 --- Blood samples --- p.26 / Chapter 2.1.2 --- Media and reagents for cell culture --- p.26 / Chapter 2.1.3 --- Antibodies for costimulation of Th cells --- p.28 / Chapter 2.1.4 --- Recombinant human cytokines --- p.28 / Chapter 2.1.5 --- "Signaling pathway inhibitors: SB203580, PD98035, SP600125 and BAY117082" --- p.28 / Chapter 2.1.6 --- Monoclonal antibodies and reagents for immunofluorescent staining --- p.29 / Chapter 2.1.7 --- Reagents and buffers for the purification of human Th lymphocytes --- p.31 / Chapter 2.1.8 --- Reagents and buffers for protein array --- p.32 / Chapter 2.1.9 --- Reagents and buffers for Thl/2 cytokine and chemokine detection --- p.32 / Chapter 2.1.10 --- Reagents and buffers for protein extraction --- p.32 / Chapter 2.1.11 --- Reagents and buffers for SDS-polyacrylamide gel electrophoresis --- p.33 / Chapter 2.1.12 --- Reagents and buffers for Western blot analysis --- p.35 / Chapter 2.1.13 --- Reagents and buffers for non-radioactive electromobility shift assay (EMSA) --- p.37 / Chapter 2.1.14 --- Reagents and buffers for cell viability and proliferation assay --- p.39 / Chapter 2.1.15 --- Reagent kit for endotoxin level assay --- p.39 / Chapter 2.1.16 --- Other reagent kits --- p.40 / Chapter 2.2 --- Methods --- p.41 / Chapter 2.2.1 --- Purification of human Th lymphocytes and cell culture --- p.41 / Chapter 2.2.2 --- Measurement of total and allergen-specific IgE concentrations --- p.41 / Chapter 2.2.3 --- Immunophenotyping of cells by flow cytometry --- p.42 / Chapter 2.2.4 --- Protein array --- p.42 / Chapter 2.2.5 --- Quantitative analysis of cytokines and chemokines by flow cytometry --- p.43 / Chapter 2.2.6 --- Quantitative analysis of IFN-γ by ELISA --- p.43 / Chapter 2.2.7 --- SDS-PAGE --- p.44 / Chapter 2.2.8 --- Western blot analysis --- p.44 / Chapter 2.2.9 --- EMSA / gel shift assay --- p.45 / Chapter 2.2.10 --- MTT assay --- p.46 / Chapter 2.2.11 --- Cell proliferation assay --- p.46 / Chapter 2.2.12 --- Endotoxin level assay --- p.47 / Chapter 2.2.13 --- Statistical analysis --- p.47 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Effects of IL-18 and IL-25 on the induction of Thl/2 cytokine and chemokine release from costimulated Th lymphocytes --- p.48 / Chapter 3.1.1 --- IL-18 and IL-25 could up-regulate the protein expression of cytokines and chemokines --- p.48 / Chapter 3.1.2 --- IL-18 but not IL-25 induced the release of IFN-γ and TNF-α --- p.48 / Chapter 3.1.3 --- "IL-18 and IL-25 induced the release of IL-5, IL-6 and IL-10" --- p.49 / Chapter 3.1.4 --- "IL-18 induced the release of IP-10, MIG, RANTES, MlP-lα and IL-8" --- p.49 / Chapter 3.1.5 --- "IL-25 induced the release of IP-10, MIG and RANTES" --- p.49 / Chapter 3.1.6 --- IL-18 and IL-25 did not enhance the proliferation of costimulated Th cells --- p.49 / Chapter 3.2 --- "Effects of IL-18 and IL-25 on the activation of p38 MAPK, ERK, JNK and NF- k B" --- p.58 / Chapter 3.2.1 --- "Costimulation with or without IL-18 and IL-25 could activate p38 MAPK, ERK and JNK" --- p.58 / Chapter 3.2.2 --- Costimulation with or without IL-18 and IL-25 could induce NF- k B activity --- p.58 / Chapter 3.3 --- Effects of inhibitors on the IL-18 and IL-25-induced release of Thl/2 cytokines and chemokines --- p.63 / Chapter 3.3.1 --- "Optimal dosage of SB203580, PD98035, SP600125 and BAY117082" --- p.63 / Chapter 3.3.2 --- "SB203580, PD98035 and BAY 117082 but not SP600125 suppressed the IL-18 and IL-25-induced release of Thl/2 cytokines" --- p.63 / Chapter 3.3.3 --- SP600125 suppressed the IL-18 and IL-25-induced release of chemokines --- p.64 / Chapter 3.4 --- Effects of inhibitors on the cell surface expression of IL-18 and IL-25 receptors --- p.72 / Chapter 3.4.1 --- "SB203580, PD98035, BAY 117082 but not SP600125 could suppress IL-18 receptor on costimulated Th cells" --- p.72 / Chapter 3.4.2 --- "SB203580, SP600125, PD98035 and BAY 117082 could not suppress IL-25 receptor on costimulated Th cells" --- p.72 / Chapter 3.5 --- Effects of costimulation on the expression of cell surface markers on Th lymphocytes --- p.75 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Effects of IL-18 and IL-25 on the release of Th1/2 cytokines and chemokines --- p.80 / Chapter 4.2 --- "Regulation of Thl/2 cytokines and chemokines through intracellular p38 MAPK, ERK, JNKand NF-kB" --- p.83 / Chapter 4.3 --- Effects of costimulation on different surface markers in Th cells --- p.87 / Chapter 4.4 --- Concluding remarks and future perspectives --- p.90 / References --- p.94

Gho Wai-Man. / Thesis submitted in: August 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 114-123). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 摘要 --- p.IV / ACKNOWLEDGEMENT --- p.VI / TABLE OF CONTENT --- p.VII / LIST OF TABLES --- p.XII / LIST OF FIGURES --- p.XIII / ABBREVIATIONS --- p.XVI / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Epidemiology --- p.2 / Chapter 1.3 --- Etiological factors --- p.6 / Chapter 1.3.1 --- Viral Hepatitis Infection --- p.6 / Chapter 1.3.1.1 --- Hepatitis B Virus (HBV) --- p.7 / Chapter 1.3.1.2 --- Hepatitis C Virus (HCV) --- p.9 / Chapter 1.3.2 --- Aflatoxin Exposure --- p.10 / Chapter 1.3.3 --- Alcohol Abuse --- p.11 / Chapter 1.3.4 --- Liver Cirrhosis --- p.12 / Chapter 1.4 --- Genetic alterations in hcc --- p.16 / Chapter 1.4.1 --- Chromosomal Gain --- p.16 / Chapter 1.4.2 --- Chromosomal Loss --- p.17 / Chapter 1.5 --- Discovery of common activating transcription factor 5 (atf5) down-regulations in hcc --- p.19 / Chapter 1.5.1 --- Chromosome 19 Aberration in HCC --- p.19 / Chapter 1.5.2 --- Discovery of High Frequency of　ATF5 Down-regulations --- p.19 / Chapter 1.5.3 --- Activating Transcription Factor Family --- p.20 / Chapter 1.6 --- Aim of thesis --- p.28 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.29 / Chapter 2.1 --- Materials --- p.30 / Chapter 2.1.1 --- Chemicals --- p.30 / Chapter 2.1.2 --- Buffers --- p.31 / Chapter 2.1.3 --- Cell culture --- p.31 / Chapter 2.1.4 --- Nucleic acids --- p.32 / Chapter 2.1.5 --- Enzymes --- p.32 / Chapter 2.1.6 --- Equipment --- p.32 / Chapter 2.1.7 --- Kits --- p.33 / Chapter 2.1.8 --- Software and Web Resource --- p.33 / Chapter 2.2 --- Dna extraction --- p.34 / Chapter 2.2.1 --- Cell Lines --- p.34 / Chapter 2.2.2 --- Primary HCC --- p.34 / Chapter 2.2.3 --- Lymphocytic DNA --- p.35 / Chapter 2.3 --- Rna extraction --- p.36 / Chapter 2.4 --- Dna sequencing --- p.38 / Chapter 2.4.1 --- Polymerase Chain Reaction (PCR) --- p.38 / Chapter 2.4.2 --- Cycle Sequencing --- p.39 / Chapter 2.5 --- Dual-labeled fluirescence in situ hybridization (fish) --- p.41 / Chapter 2.5.1 --- FISH Probe Preparation --- p.41 / Chapter 2.5.1.1 --- Preparation of Human Bacterial Artificial Chromosome (BAC) --- p.41 / Chapter 2.5.1.2 --- Nick Translation --- p.41 / Chapter 2.5.2 --- FISH --- p.42 / Chapter 2.6 --- 5-aza-2'-deoxycytidine & trichostatin a treatment on cell lines --- p.43 / Chapter 2.7 --- Bisulfite modificaiton of dna --- p.43 / Chapter 2.8 --- Methylation-specific pcr (msp) --- p.44 / Chapter 2.9 --- Bisulfite dna sequencing --- p.44 / Chapter 2.10 --- Quantitative reverse transcription pcr (qrt-pcr) --- p.46 / Chapter 2.11 --- In-vitro and in-vivo functinal examination --- p.49 / Chapter 2.11.1 --- ATF5 Transfection --- p.49 / Chapter 2.11.2 --- Cell Growth Assay --- p.50 / Chapter 2.11.3 --- Xenograft Development --- p.51 / Chapter 2.12 --- codelink expression microarray --- p.51 / Chapter 2.13 --- Statistical analysis --- p.53 / Chapter CHAPTER 3 --- INACTIVATION OF MECHANISMS UNDERLYING ATF5 DOWN-REGULATION --- p.54 / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Materials and methods --- p.58 / Chapter 3.2.1 --- Cell Lines --- p.58 / Chapter 3.2.2 --- Mutational Analysis --- p.58 / Chapter 3.2.3 --- Copy Number Loss --- p.59 / Chapter 3.2.4 --- Epigenetic Control --- p.59 / Chapter 3.3 --- Results --- p.67 / Chapter 3.3.1 --- Sequencing Analysis of A TF5 Gene --- p.67 / Chapter 3.3.2 --- FISH Analysis of ATF5 Copy Number --- p.73 / Chapter 3.3.3 --- Epigenetic Control of A TF5 Expression --- p.73 / Chapter 3.4 --- Discussion --- p.82 / Chapter CHAPTER 4 --- FUNCTIONAL EXAMINATION AND INVESTIGATION OF DOWNSTREAM TARGETS MODULATED BY ATF5 --- p.85 / Chapter 4.1 --- Introduction --- p.86 / Chapter 4.2 --- Materials and methods --- p.88 / Chapter 4.2.1 --- Cell Lines --- p.88 / Chapter 4.2.2 --- Plasmids and Transfection --- p.88 / Chapter 4.2.3 --- Cell Growth Assay --- p.88 / Chapter 4.2.4 --- Xenograft Development --- p.88 / Chapter 4.2.5 --- CodeLink Expression Microarray --- p.89 / Chapter 4.2.6 --- Quantitative RT-PCR --- p.90 / Chapter 4.2.7 --- Statistical analysis --- p.90 / Chapter 4.3 --- Results --- p.91 / Chapter 4.3.1 --- Cell Proliferation --- p.91 / Chapter 4.3.1.1 --- In-Vitro Examination --- p.91 / Chapter 4.3.1.2 --- In-Vivo Examination --- p.91 / Chapter 4.3.2 --- Microarray A nalysis --- p.91 / Chapter 4.3.3 --- Correlation of A TF5 with Id-1 Expression --- p.103 / Chapter 4.4 --- Discussion --- p.106 / Chapter CHAPTER 5 --- PROPOSED FUTURE INVESTIGATIONS --- p.110 / Chapter 5.1 --- inactivation mechanisms of atf5 gene --- p.111 / Chapter 5.2 --- Molecular pathways modulated by atf5 --- p.112 / Chapter CHAPTER 6 --- REFERENCES --- p.114

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

Wong, Siu Lun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 129-141). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese version) --- p.iii / Acknowledgements --- p.iv / List of Abbreviations --- p.v / List of Tables --- p.vii / List of Figures --- p.viii / Chapter 1. --- INTRODUCTION / Chapter 1.1 --- Neurodegenerative disorders - a brief overview --- p.1 / Chapter 1.2 --- Polyglutamine diseases --- p.2 / Chapter 1.3 --- Microscopically visible polyglutamine protein aggregates and its relation to toxicity --- p.7 / Chapter 1.4 --- Polyglutamine protein conformers and their relation to toxicity --- p.10 / Chapter 1.5 --- Modeling polyglutamine diseases in Drosophila / Chapter 1.5.1 --- GAL4/UAS spatial transgene expression system in Drosophila --- p.14 / Chapter 1.5.2 --- Temporal control of GAL4/UAS transgene expression system in Drosophila --- p.16 / Chapter 1.5.3 --- Drosophila as a model to study human pathologies --- p.19 / Chapter 1.5.4 --- Drosophila as a model to study polyglutamine diseases --- p.21 / Chapter 1.6 --- Aims of study --- p.26 / Chapter 2. --- MATERIALS AND METHODS / Chapter 2.1 --- Drosophila culture and manipulation / Chapter 2.1.1 --- Drosophila culture --- p.27 / Chapter 2.1.2 --- Phenotypic examination of adult external eye degeneration --- p.27 / Chapter 2.1.3 --- Pseudopupil assay of adult retinal degeneration and observation of green fluorescent protein in adult eyes --- p.28 / Chapter 2.2 --- Semi-quantitative Reverse Transcription-Polymerase Chain Reaction / Chapter 2.2.1 --- RNA extraction from adult Drosophila heads --- p.30 / Chapter 2.2.2 --- DNase treatment of extracted RNA --- p.31 / Chapter 2.2.3 --- Reverse transcription-Polymerase Chain Reaction (RT-PCR) --- p.31 / Chapter 2.2.4 --- Agarose gel electrophoresis --- p.33 / Chapter 2.3 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) / Chapter 2.3.1 --- Protein extraction from adult Drosophila heads --- p.33 / Chapter 2.3.2 --- Preparation of SDS-polyacrylamide gel and electrophoresis --- p.34 / Chapter 2.3.3 --- Western blotting --- p.35 / Chapter 2.3.4 --- Immunodetection --- p.36 / Chapter 2.4 --- Immunoprecipitation --- p.38 / Chapter 2.5 --- Filter retardation assay --- p.39 / Chapter 2.6 --- Isolation and solubilization of SDS-insoluble protein --- p.40 / Chapter 2.7 --- Sucrose gradient sedimentation --- p.41 / Chapter 2.8 --- Preparation of Drosophila tissues for immunofluorescence analysis / Chapter 2.8.1 --- Dissection and immunostaining of Drosophila larval imaginal eye discs --- p.42 / Chapter 2.8.2 --- Cryosectioning and immunostaining of adult Drosophila heads --- p.44 / Chapter 2.9 --- Atomic force microscopy --- p.47 / Chapter 2.10 --- Reagents and buffers / Chapter 2.10.1 --- Reagents for Drosophila culture --- p.48 / Chapter 2.10.2 --- Reagents for RT-PCR --- p.52 / Chapter 2.10.3 --- Reagents for SDS-PAGE --- p.54 / Chapter 2.10.4 --- Reagents for immunoprecipitation --- p.57 / Chapter 2.10.5 --- Reagents for filter retardation assay --- p.57 / Chapter 2.10.6 --- Reagents for isolation and solubilization of SDS-insoluble protein --- p.58 / Chapter 2.10.7 --- Reagents for sucrose gradient sedimentation --- p.58 / Chapter 2.10.8 --- Reagents for immunofluorescence --- p.59 / Chapter 3. --- RESULTS / Chapter 3.1 --- Establishment of an inducible transgenic Drosophila model of polyglutamine diseases / Chapter 3.1.1 --- Introduction --- p.60 / Chapter 3.1.2 --- Results / Chapter 3.1.2.1 --- GAL80ts-mediated inducible expression of expanded polyglutamine protein in Drosophila / Chapter 3.1.2.1.1 --- GAL80ts controls GAL4/UAS-mediated polyQ protein expression --- p.61 / Chapter 3.1.2.1.2 --- Inducible expression of SDS-soluble expanded polyglutamine protein --- p.64 / Chapter 3.1.2.1.3 --- Inducible expression of expanded polyglutamine protein accumulates gradually in form of SDS-insoluble protein --- p.66 / Chapter 3.1.2.1.4 --- Inducible expression of expanded polyglutamine protein results in progressive accumulation of microscopically visible aggregates --- p.68 / Chapter 3.1.2.2 --- Inducible expression of expanded polyglutamine protein causes late-onset progressive neuronal degeneration in Drosophila / Chapter 3.1.2.2.1 --- Inducible expression of expanded polyglutamine protein leads to late-onset progressive deterioration of photoreceptor neurons --- p.68 / Chapter 3.1.2.2.2 --- Inducible expression of expanded polyglutamine protein neither causes external eye degenerative phenotype nor disrupts gross retinal morphology despite deterioration of photoreceptor neurons --- p.72 / Chapter 3.1.2.3 --- Co-expression of caspase inhibitor P35 suppresses polyglutamine-induced neuronal degeneration --- p.72 / Chapter 3.1.2.4 --- Co-expression of molecular chaperone Hsp70 suppresses polyglutamine-induced neuronal degeneration --- p.74 / Chapter 3.1.2.5 --- Inducible expression of expanded polyglutamine protein results in biphasic expression of molecular chaperone Hsp70 in Drosophila --- p.76 / Chapter 3.1.3 --- Discussion --- p.76 / Chapter 3.2 --- Involvement of microscopically visible polyglutamine aggregates in neurodegeneration / Chapter 3.2.1 --- Introduction --- p.83 / Chapter 3.2.2 --- Results / Chapter 3.2.2.1 --- Effect of Hsc70-K71S on microscopically visible polyglutamine aggregates and neuronal degeneration / Chapter 3.2.2.1.1 --- Co-expression of Hsc70-K71S reduces the level of microscopically visible polyglutamine aggregates --- p.83 / Chapter 3.2.2.1.2 --- Co-expression of Hsc70-K71S does not alter polyglutamine transgene expression --- p.84 / Chapter 3.2.2.1.3 --- Co-expression of Hsc70-K71S does not modify polyglutamine-induced neuronal degeneration --- p.87 / Chapter 3.2.2.2 --- Microscopically visible polyglutamine aggregates do not correlate with neuronal degeneration --- p.90 / Chapter 3.2.3 --- Discussion --- p.93 / Chapter 3.3 --- Detection of small SDS-insoluble expanded polyglutamine protein species and its association with neurodegeneration / Chapter 3.3.1 --- Introduction --- p.97 / Chapter 3.3.2 --- Results / Chapter 3.3.2.1 --- Accumulation of SDS-soluble expanded polyglutamine protein does not correlate with neuronal degeneration --- p.98 / Chapter 3.3.2.2 --- Identification of small SDS-insoluble expanded polyglutamine protein species / Chapter 3.3.2.2.1 --- Accumulation of total SDS-insoluble expanded polyglutamine protein positively correlates with progressive neuronal degeneration --- p.99 / Chapter 3.3.2.2.2 --- Accumulation of large SDS-insoluble expanded polyglutamine protein does not correlate with neuronal degeneration --- p.99 / Chapter 3.3.2.2.3 --- Accumulation of small SDS-insoluble expanded polyglutamine protein correlates with neuronal degeneration --- p.104 / Chapter 3.3.3 --- Discussion --- p.107 / Chapter 3.4 --- Biophysical characterization of small SDS-insoluble expanded polyglutamine protein species / Chapter 3.4.1 --- Introduction --- p.109 / Chapter 3.4.2 --- Results / Chapter 3.4.2.1 --- Separation of expanded polyglutamine protein species by sucrose gradient sedimentation --- p.110 / Chapter 3.4.2.2 --- Morphological studies of small SDS-insoluble expanded polyglutamine protein species by atomic force microscopy --- p.112 / Chapter 3.4.3 --- Discussion --- p.118 / Chapter 4. --- GENERAL DISCUSSION --- p.124 / Chapter 5. --- CONCLUSION --- p.127 / Chapter 6. --- REFERENCES --- p.129

A proteína dissulfeto isomerase (PDI) tem ganhado destaque em processos de sinalização celular. O objetivo deste trabalho foi investigar o papel da PDI na sinalização redox induzida por TNF-a em células endoteliais e por Angiotensina II (Ang II) em células musculares lisas vasculares (CMLV). Em cultura de células endoteliais isoladas da veia umbilical humana (HUVECs) os dados demonstraram que a PDI e a ERp46 regulam especificamente a fosforilação da ERK 1/2 induzida por TNF-a, possivelmente via alterações redox sobre a GTPase Ras e participa da angiogenese induzida por TNF-a. Em CMLV de artérias de resistência, os dados sugerem a participação da PDI na contração induzida por Ang II, bem como na disfunção vascular associada à hipertensão arterial, através da regulação da expressão e atividade da Nox1. Desta forma, podemos concluir que a PDI apresenta um papel na regulação da sinalização redox induzida por TNF-a e Ang II em células endoteliais e CMLV, respectivamente. Tais resultados apontam para um novo papel da PDI na fisiopatologia do sistema cardiovascular. / Protein disulfide isomerase (PDI), an oxidoreductase of endoplasmic reticulum, has emerged as a key player in cell signaling. The aim of the present study was to investigate the role of PDI in redox signaling induced by TNF-a in endothelial cells and by Angiotensin II (Ang II) in vascular smooth muscle cells (VSMCs). In human umbilical vein endothelial cells (HUVECs) ERp46 or PDI inhibition reduced specifically TNF-a-induced ERK1/2 phosphorylation, possibly via redox modifications in Ras GTPase and TNF-a-induced angiogenesis. In VSMCs from resistance arteries, our results suggest that PDI positively regulates Nox1 dependent signaling and expression in VSMCs from resistance arteries and could be a new player in the oxidative stress and vascular dysfunction observed in hypertension. Altogether, the results provide evidence for a role for PDI in cardiovascular pathophysiology.

Further studies of Stk31 in spermatogenesis in vivo would allow the identification of the asymmetry machinery of GSCs and the signaling mechanism underlying cell fate determination. Further studies of STK31 in cancer stem cells would allow the development of new diagnostic and therapeutic approaches. / In the first part of the experiment, the expression and cellular localization of STK31 were investigated. RT-PCR results showed that STK31 was reactivated in 47 -- 86% of multiple cancers. Immunofluorescent study and GFP tagging experiment showed that STK31 was localized in the cytoplasm and formed aggregated granules that divide asymmetrically during mitosis. Further study by co-staining with E-cadherin demonstrated that the mouse homolog, Stk31, was expressed in the transition state between undifferentiated and differentiated spermatogonia. These data suggest the possible involvement of STK31 in mouse spermatogonia and cancer development. / In the second part of the experiment, the function of Stk31 in mouse spermatogonia was investigated- A GSC culture on an STO feeder layer was established. Studies on growing properties, expression of molecular markers and germ cell transplantation showed that GSC culture maintained spermatogonial stem cell activity. Retinoic acid was then used to induce differentiation of GSC. The differentiation status was confirmed by monitoring the expression of molecular markers. RT-PCR and immunofluorescent study showed that the expression of Stk31 was induced in RA-induced differentiation and Stk31 proteins were asymmetrically distributed during GSC division. Overexpression of Stk31 in GSCs using retroviral transduction induced the differentiation phenotypes. These data indicate the involvement of Stk31 in mouse spermatogonia cell fate determination. / In the third part of the experiment, the function of STK31 in human colon cancer was investigated. A stable STK31 knock-down Caco2 cells were established by stably transfecting two miR RNAi designs with different efficiency into Caco2 cells. Flow cytometry analysis showed that knock-down of STK31 resulted in G1 phase arrest. Cell counts and MTS assays suggested that knock-down of STK31 decreased cell proliferation in confluent cultures. Knock-down of STK31 also enhanced cell attachment to several ECM proteins and decreases cell migration as suggested by attachment assays and migration assays. Moreover, knock-down of STK31 enhanced enterocytic differentiation and inhibited tumorigenicity both in vitro and in vivo as indicated by colony formation assays and xenograft assays. Date obtained from whole genome microarray studies indicate that STK31 regulates these "stemness" properties through altering the expression of key players in various pathways including KIT, SMAD1 and Cyclin D2. These results suggest the involvement of STK31 in colon cancer as a regulator of "sternness". / Spermatogenesis is a complicated process involving mitosis, meiosis and post-meiotic differentiation. Due to the lack of in vitro models, genes that are involved in mammalian spermatogenesis are largely unknown. Spermatogenesis and tumorigenesis share important biological similarities. This co-relation can be signified by a special group of genes called cancer/testis (CT) antigens, which are only expressed in the testes and cancer. Although cancer biology has been extensively studied for decades, promising therapeutic methods are not available for every type of cancer. Recent discovery of cancer stem cells and functional genomics studies have shed light on the development of new diagnostic and therapeutic approaches. This thesis describes the expression, cellular localization and function of a novel CT gene, STK31, in spermatogonia and cancer development. / Fok, Kin Lam Ellis. / "December 2009." / Adviser: H.C. Chan. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 143-169). / 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. / Abstract also in Chinese.

Ng Sin Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 120-135). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abbreviations --- p.iii / Abstract --- p.vi / 撮要 --- p.ix / Publications --- p.xi / Table of contents --- p.xiii / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Allergic Diseases and Allergic Inflammation --- p.1 / Chapter 1.1.1 --- Prevalence of Allergic Diseases --- p.1 / Chapter 1.1.2 --- Common Allergic Diseases: Allergic Asthma --- p.1 / Chapter 1.1.3 --- Common Allergic Diseases: Atopic Dermatitis --- p.2 / Chapter 1.1.4 --- Allergic Inflammation --- p.3 / Chapter 1.2 --- The Inflammatory Leukocytes: Mast Cells and Eosinophils --- p.6 / Chapter 1.2.1 --- Characteristics of Mast Cells --- p.6 / Chapter 1.2.2 --- Mast Cells Distribution --- p.8 / Chapter 1.2.3 --- Mast Cells Subtypes --- p.8 / Chapter 1.2.4 --- HMC-1 Cells --- p.9 / Chapter 1.2.5 --- Characteristics of Eosinophils --- p.12 / Chapter 1.3 --- Adhesion Molecules in Allergic Diseases --- p.15 / Chapter 1.3.1 --- Adhesion Molecules and Leukocyte Migration --- p.15 / Chapter 1.3.2 --- Selectin --- p.17 / Chapter 1.3.3 --- Intermolecular Adhesion Molecules --- p.17 / Chapter 1.3.4 --- Integrin --- p.18 / Chapter 1.4 --- Cytokines and Chemokines in Allergic Diseases --- p.18 / Chapter 1.4.1 --- IL-6 --- p.20 / Chapter 1.4.2 --- CXCL1 --- p.21 / Chapter 1.4.3 --- CXCL8 --- p.21 / Chapter 1.4.3 --- CCL2 --- p.22 / Chapter 1.5 --- Intercellular Signal Transduction Pathways in Inflammation --- p.24 / Chapter 1.5.1 --- RAS-RAF-mitogen-activated Protein Kinases --- p.24 / Chapter 1.5.2 --- Janus Kinase/ Signal Transducers and Activators of Transcriptions Pathway --- p.27 / Chapter 1.5.3 --- Nuclear Factor-KB Pathway --- p.29 / Chapter 1.5.4 --- Phosphoinositide 3-Kinase Pathway --- p.31 / Chapter 1.6 --- Aims and Scope of the Study --- p.33 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- HMC-1 Cell Line --- p.35 / Chapter 2.1.2 --- Human Buffer Coat --- p.35 / Chapter 2.1.3 --- Human Mast Cell Chymase and TLR ligands --- p.35 / Chapter 2.1.4 --- Media and Reagents for Cell Culture --- p.36 / Chapter 2.1.5 --- Reagents and Buffers for Purification of Human Eosinophils --- p.37 / Chapter 2.1.6 --- Reagents and Buffers for Flow Cytmetry --- p.38 / Chapter 2.1.7 --- Reagents and Buffers for Total RNA Extraction --- p.41 / Chapter 2.1.8 --- Reagents and Buffers for Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.42 / Chapter 2.1.9 --- Reagents and Buffers for Agarose Gel Electrophoresis --- p.45 / Chapter 2.1.10 --- Reagents and Buffers for Sodium Dodecyl Sulfate -polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.46 / Chapter 2.1.11 --- Reagents and Buffers for Western Blot Analysis --- p.48 / Chapter 2.1.12 --- Chemotactic Migration --- p.51 / Chapter 2.1.13 --- Signaling Transduction Inhibitors and Protein Synthesis Inhibitors --- p.51 / Chapter 2.2 --- Methods --- p.52 / Chapter 2.2.1 --- HMC-1 Cell Cultures --- p.52 / Chapter 2.2.2 --- Purification of Buffy Coat Eosinophils by MACS and Eosinophil Culture --- p.52 / Chapter 2.2.3 --- Total Cellular RNA Extraction --- p.53 / Chapter 2.2.4 --- RT-PCR --- p.54 / Chapter 2.2.5 --- Agarose Gel Electrophoresis --- p.55 / Chapter 2.2.6 --- Flow Cytometry Analysis --- p.55 / Chapter 2.2.7 --- Protein Array Analysis of Cytokine Release --- p.57 / Chapter 2.2.8 --- Quantitative Analysis ofCXCLl --- p.58 / Chapter 2.2.9 --- Total Protein Extraction --- p.58 / Chapter 2.2.10 --- SDS-PAGE --- p.58 / Chapter 2.2.11 --- Western Blot Analysis --- p.59 / Chapter 2.2.12 --- Chemotactic Migration Analysis --- p.60 / Chapter 2.2.13 --- Statistical Analysis --- p.60 / Chapter Chapter 3 --- Effects of Mast Cell Derived Chymase on Human Eosinophils and the Signaling Mechanisms: Implication in Allergic Inflammation / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.2 --- Results --- p.65 / Chapter 3.2.1 --- Effects of Chymase on Eosinophil Survival --- p.65 / Chapter 3.2.2 --- Effects of Chymase on the Adhesion Molecule Expression of Eosinophils --- p.68 / Chapter 3.2.3 --- Effects of Chymase on the Chemokinetic Properties on Eosinophils --- p.71 / Chapter 3.2.4 --- Effects of Chymase on the Release of Chemokines and IL-6 from Eosinophils --- p.73 / Chapter 3.2.5 --- Signal Transduction Mechanism Involved in Regulating Chymase-induced Effects on Eosinophils --- p.78 / Chapter 3.3 --- Discussion --- p.71 / Chapter Chapter 4 --- TLR-mediated Effects and Signal Transduction Mechanism of HMC-1 Cells / Chapter 4.1 --- Introduction --- p.92 / Chapter 4.2 --- Results --- p.97 / Chapter 4.2.1 --- Expression of Adhesion Molecules on HMC-1 Cells --- p.95 / Chapter 4.2.2 --- TLR Expression Profile on HMC-1 Cells --- p.97 / Chapter 4.2.3 --- Effects of TLR ligands on HMC-1 Cell Adhesion Molecule Expressions --- p.99 / Chapter 4.2.4 --- TLR7-induced Phosphorylation of ERK and Effects of PD98059 on TLR7-induced ERK Phosphorylation --- p.104 / Chapter 4.2.5 --- Effect of TLR7 Ligand on HMC-1 Cells Cytokine Release --- p.108 / Chapter 4.3 --- Discussion --- p.110 / Chapter Chapter 5 --- Conclusions and Future Perspectives / Chapter 5.1 --- Conclusions --- p.115 / Chapter 5.2 --- Future Perspectives --- p.117 / References --- p.120 / Appendix --- p.136

Abstract not available

Includes list of publications arising from this thesis / Erratum attached to inside back cover. / "25/03/2004." / Includes bibliographical references (leaves 185-257) / xii, 257 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine and Royal Adelaide Hospital, Rheumatology Unit, 2004

Les connaissances actuelles de la physiopathologie de la migraine ne permettent pas encore de déterminer avec exactitude le processus pathologique à lorigine de lactivation du système trigémino-vasculaire conduisant à la céphalée migraineuse. Certains arguments sont en faveur danomalies fonctionnelles du cortex cérébral à même denclencher le processus de la crise de migraine. Parmi ces anomalies, une hypersensibilité sensorielle a été décrite de longue date et a conduit à un important travail en neurophysiologie clinique. Ainsi lutilisation des techniques de potentiels évoqués a abouti à divers résultats dont le plus reproductible consiste en un déficit dhabituation des réponses évoquées corticales lors de stimulations sensorielles répétées chez les migraineux en période intercritique. Comprendre lorigine du déficit dhabituation retrouvé chez les migraineux devrait permettre de préciser le rôle du cortex cérébral dans la cascade dactivation menant à la crise de migraine. Nous avons utilisé la stimulation magnétique transcrânienne répétitive (SMTr) chez les migraineux afin de moduler lexcitabilité des cortex visuel et moteur, en enregistrant ses effets respectivement sur les potentiels évoqués visuels et moteurs. La SMTr a ainsi permis détudier lorigine du déficit dhabituation des potentiels évoqués corticaux retrouvé en période intercritique chez les migraineux. Ce déficit dhabituation des réponses corticales semble provenir dun niveau réduit de préactivation corticale mais également dune activité réduite des interneurones inhibiteurs corticaux. Par ailleurs, ce déficit dhabituation ne semble pas être un élément pathogénique prépondérant dans la mesure où sa seule présence chez les sujets sains ne permet pas den faire des migraineux. Il pourrait correspondre soit à un épiphénomène, soit à un des éléments étiopathogéniques de la migraine, au même titre que la prédisposition génétique, les troubles du métabolisme mitochondrial, Il est vraisemblable que le poids relatif de chacun de ces éléments étiopathogéniques participe à lhétérogénéité des migraines.