上皮間充質轉化（EMT），作為重要的生理和病理事件，廣泛的參與胚胎發育、組織纖維化病變及腫瘤轉移的過程。這一顯著的細胞表型變化包括上皮細胞失去緊密連接和極性，上皮細胞呈現纖維細胞形態以及增強的細胞移動性。囊性纖維變性跨膜電導調節器（CFTR）是一種廣泛表達於上皮細胞的氯離子和碳酸根離子通道。研究證實，CFTR 的蛋白轉運與上皮連接的形成和功能有關，同時 CFTR 的表達受到 EMT 誘導因子 HIF-1 和 TGF-β 的反向調節。另外，CFTR 的表達和功能被證實參與 EMT 相關信號分子 Wnt 和 NF-κB活性的調節。基於上述發現，本研究旨在闡述 CFTR 與 EMT 的相關性。 / CFTR 參與的腎上皮 EMT 以及後續的腎纖維化首先被關注。實驗表明，在腎上皮細胞（MDCK）中，小 RNA 介導的 CFTR 基因敲降或抑製劑引起的CFTR 通道功能缺陷均引起間充質細胞特徵的出現，包括纖維狀細胞形態、細胞連接分子 E-cadherin, ZO-1 和 Occludin 表達下調和間充質細胞標誌分子 Vimentin 和 N-cadherin 上調、上皮細胞跨膜電阻減低以及細胞遷徙能力的增強。有趣的是，在單側尿道結紮的腎纖維化模型中，CFTR 表達被顯著下調。同時，動物實驗證實一個最常見的 CFTR 分子突變（deltaF508 -/-）增加了單側尿道結紮導致的腎纖維化的程度。另外，在缺氧引起的 EMT 過程中CFTR 的表達顯著下調；同時，腎纖維化模型中，HIF-1 和 CFTR 的表達呈現負相關。結果提示，生理及病理的條件下，氧氣的調節可能作為 CFTR 下調及其後續事件的誘因。進一步實驗發現，CFTR 功能抑製或基因突變可以引起Wnt 的富集和 β-catenin 的細胞核轉移。基於以上的實驗結果，在腎纖維化的過程中，CFTR 參與了缺氧引起的 EMT 過程，並通過激活 Wnt/β-catenin 信號調節相關的下游因子。 / 第二部分集中探究了 CFTR 在癌細胞EMT 及腫瘤轉移中的作用及機制。實驗證實，在 TGF-β 誘導的腫瘤細胞 EMT 過程中，CFTR 表達被抑制。TGF-β 可能作為病理狀態下的調節因子，引起腫瘤細胞中 CFTR 表達下調及EMT。抑制 CFTR 通道功能或敲降其蛋白表達導致明顯的間充質細胞特徵，這一變化在不同來源的腫瘤細胞系中呈均一性。相對地，過表達 CFTR 引起細胞遷移和侵潤能力地顯著下降。在體實驗顯示，CFTR 表達與腫瘤的轉移能力呈現負相關。進一步機制研究證明，CFTR 通過調節多重的通路參與 EMT的過程。首先，uPA 的表達和活性受到 CFTR 的反向調節，並且這一調節作用是由激活的 NF-κB 介導的。其次，抑制 CFTR 通道功能引起 β-catenin 的細胞核轉移。 / 綜上所述，研究發現 CFTR 通過調節多重信號參與腎上皮及腫瘤細胞的 EMT。同時，研究顯示 CFTR 的表達和功能與腎纖維化及腫瘤轉移有關。此研究對相關疾病的診斷和預後具有潛在的提示作用。 / Epithelial-Mesenchymal Transition (EMT) is an intricate process by which epithelial cells lose their epithelial characteristics and acquire a mesenchymal-like phenotype. It is essential for numerous physiological and pathological processes, such as embryonic development, tissue fibrosis and cancer metastasis. The dramatic phenotype changes of EMT include loss of tight junctions and polarity, acquisition of a fibroblastic morphology and increased motility. The cystic fibrosis transmembrane regulator (CFTR) is known as an anion channel and extensively expressed in a variety of epithelial cells. Interestingly, the apical membrane expression of CFTR is reported to be required for the normal organization and function of epithelial junctions. Moreover, EMT inducers, such as HIF-1 and TGF-β, are known to suppress the expression of CFTR in epithelial cells. In addition, CFTR has been reported to be associated with expression and/or activity of Wnt and NF- κB, key factors known to be involved in EMT. Thus, we hypothesized that CFTR might play an important role in EMT. / In the first part of the study, the involvement of CFTR in EMT of kidney epithelial cells and renal fibrosis was investigated. Our experiments revealed that suppression of CFTR by either inhibitor or knockdown induced EMT in Madin- Darby canine kidney epithelial cells (MDCK). This was accompanied by the appearance of fibroblastic morphology, with reduced expression of epithelial junction proteins E-cadherin, ZO-1 and occludin and accumulated expression of the mensenchymal markers vimentin and N-cadherin, as well as reduced transepithelial resistance (TER) and enhanced migratory ability. Interestingly, the expression of CFTR was found significantly down-regulated in unilateral urethral obstruction (UUO) kidney. In addition, CFTR mutant (deltaF508 -/-), the most common mutation found in CF patients, increased the risk of renal fibrosis in UUO model. Our results showed that the expression of CFTR down-regulated in hypoxia induced-EMT in MDCK, and the expression of hypoxia-sensitive transcription factor, HIF-1, is inversely correlated with CFTR in UUO kidney. Accumulation of Wnt and translocation of β-catenin were also observed in CFTR inhibitors-treated MDCK and deltaF508 -/- UUO mice. Taken together, these findings suggest that CFTR may be involved in mediating hypoxia-induced EMT by influencing the Wnt/β-catenin signaling contributing to renal fibrosis. / In the second part of the study, the role of CFTR in EMT during cancer metastasis and the underlying mechanisms were investigated. Recent studies have demonstrated that cancer cells may reinstitute properties of developmental EMT including enhanced migration and invasion. On the other hand, the reverse process, known as mesenchymal-to-epithelial transition (MET), has been implicated in forming a secondary metastatic tumor. Using various tissue-derived cancer cell lines including human colorectal cancer cell line LIM1863, human lung carcinoma cell line A549, and human breast cancer cell lines MCF7 and MDA-MB-231, we report that induction of EMT by TGF-β sharply reduces CFTR expression in various tissue derived cancer cell lines, while overexpression of CFTR can reverse the TGF-β- induced EMT phenyotype. Interfering with CFTR function either by its specific inhibitor or lentiviral miRNA-mediated knockdown mimicks TGF-β-induced EMT and enhances cell migration and invasion. Ectopic overexpression of CFTR in a highly metastatic cancer cell lines downregulates EMT markers and suppresses cell invasion and migration in vitro, as well as the ability of the cells to metastasize to the lung in vivo. The EMT-suppressing effect of CFTR is found to be associated with its ability to alter NF-κB targeting urokinase-type plasminogen activator (uPA) and the nuclear translocation of β-catenin. Taken together, the present study has demonstrated a previously undefined role of CFTR as an EMT suppressor in cancer. / In summary, our findings have demonstrated a regulatory role of CFTR in EMT in both normal kidney epithelial cell line and various cancer cell lines. We conclude that CFTR plays important roles in renal fibrosis and cancer progression/metastasis by modulating EMT process through multiple pathways. The insights afforded by these studies will provide critical new information about the function of CFTR as a suppressor of EMT, which may have potential application in diagnosis and prognosis of fibrosis and 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. / Zhang, Jieting. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 136-150). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Epithelial-Mesenchymal Transition --- p.1 / Chapter 1.1.1 --- Concept and features of EMT --- p.2 / Chapter 1.1.2 --- Roles of EMT in development and diseases --- p.10 / Chapter 1.1.3 --- The Regulators of EMT --- p.13 / Chapter 1.2 --- Structure and function of CFTR --- p.18 / Chapter 1.2.1 --- General structure and channel functions of CFTR --- p.18 / Chapter 1.2.2 --- Gene mutations and CF --- p.18 / Chapter 1.3 --- Potential role of CFTR in EMT --- p.20 / Chapter 1.3.1 --- CFTR in formation of cell-cell junction and membrane polarity --- p.20 / Chapter 1.3.2 --- CFTR and EMT inducers --- p.21 / Chapter 1.3.3 --- CFTR and EMT related pathways and factors --- p.22 / Chapter 1.4 --- Hypothesis and aim of the study --- p.22 / Chapter Chapter 2 --- CFTR involves in hypoxia induced EMT in renal fibrosis --- p.24 / Chapter 2.1 --- Abstract --- p.24 / Chapter 2.2 --- Introduction --- p.25 / Chapter 2.3 --- Results --- p.30 / Chapter 2.3.1 --- Knockdown of CFTR induces EMT in MDCK --- p.30 / Chapter 2.3.2 --- Inhibition of CFTR channel function induces EMT in MDCK --- p.30 / Chapter 2.3.3 --- CFTR is downregulated during the process of renal fibrosis --- p.36 / Chapter 2.3.4 --- CFTR defect increases the risk of renal fibrosis --- p.39 / Chapter 2.3.5 --- Hypoxia/HIF-1α rather than TGF-β as the inducer of CFTR repression during EMT and renal fibrosis --- p.44 / Chapter 2.3.6 --- CFTR as a negative regulator of Wnt/β-catenin signaling in renal epithelium --- p.51 / Chapter 2.4 --- Discussion --- p.57 / Chapter 2.5 --- Conclusion --- p.61 / Chapter 2.6 --- Materials and Methods --- p.61 / Chapter 2.6.1 --- Cell culture and treatments --- p.61 / Chapter 2.6.2 --- Plasmids and transient transfection --- p.62 / Chapter 2.6.3 --- Western blot analysis --- p.62 / Chapter 2.6.4 --- Measurement of trans epithelial electric resistance --- p.64 / Chapter 2.6.5 --- Wound-healing migration assay --- p.64 / Chapter 2.6.6 --- Animals and Obstructive model --- p.64 / Chapter 2.6.7 --- HE and Masson's trichrome stain --- p.65 / Chapter 2.6.8 --- Immunofluorescent and immunohistochemistry staining --- p.65 / Chapter 2.6.9 --- Statistical analysis --- p.66 / Chapter Chapter 3 --- CFTR down-regulation mediates EMT during cancer metastasis --- p.67 / Chapter 3.1 --- Abstract --- p.67 / Chapter 3.2 --- Introduction --- p.67 / Chapter 3.3 --- Results --- p.73 / Chapter 3.3.1 --- Repression of CFTR during TGF-β induced EMT in cancer cells --- p.73 / Chapter 3.3.2 --- Hypoxia does not have significant effect on CFTR expression --- p.78 / Chapter 3.3.3 --- Repression of CFTR channel function induces EMT in cancer cells --- p.81 / Chapter 3.3.4 --- Knockdown/overexpression of CFTR induces/inhibits EMT and malignant phenotypes --- p.84 / Chapter 3.3.5 --- CFTR inhibits lung metastasis in vivo --- p.94 / Chapter 3.3.6 --- Anti-metastatic effect of CFTR involves NF-κB targeting uPA --- p.104 / Chapter 3.3.7 --- Correlation between CFTR and β-catenin --- p.112 / Chapter 3.4 --- Discussion --- p.116 / Chapter 3.5 --- Conclusion --- p.122 / Chapter 3.6 --- Materials and methods --- p.122 / Chapter 3.6.1 --- Cell culture and treatments --- p.122 / Chapter 3.6.2 --- Lentiviral production and transduction --- p.123 / Chapter 3.6.3 --- Plasmids and stable transfection --- p.124 / Chapter 3.6.4 --- RT-PCR analysis --- p.124 / Chapter 3.6.5 --- Western blot analysis --- p.126 / Chapter 3.6.6 --- Immunofluorescence staining --- p.126 / Chapter 3.6.7 --- Cell growth assay --- p.127 / Chapter 3.6.8 --- Migration assay --- p.127 / Chapter 3.6.9 --- Invasion assay --- p.128 / Chapter 3.6.10 --- In vivo tumor growth assay --- p.128 / Chapter 3.6.11 --- In vivo metastasis assay --- p.128 / Chapter 3.6.12 --- Human EMT PCR array --- p.129 / Chapter 3.6.13 --- uPA activity assay --- p.129 / Chapter 3.6.14 --- Statistical analysis --- p.129 / Chapter Chapter 4 --- General discussion --- p.130 / Chapter 4.1 --- Normal function of CFTR in epithelial polarity and barrier function --- p.130 / Chapter 4.2 --- Down-regulation of CFTR is associated with EMT-related diseases --- p.131 / Chapter 4.3 --- CFTR functions as a central mediator of different EMT signals --- p.132 / Chapter 4.4 --- Future directions --- p.134 / Chapter 4.5 --- Conclusion --- p.135 / References --- p.136 / Declaration --- p.151

卵巢是女性生殖系統中一個重要的器官，負責為受精提供卵子，以及合成生殖過程中所必需，同時也在其他生理過程中起重要作用的各種激素。大約有30%的不育源於卵巢的問題，包括無排卵，無月經，月經週期不規律和激素水平異常等。雄激素:雌激素比例過高，卵泡發育異常，無排卵等卵巢功能障礙常見於各種疾病中，例如多囊性卵巢綜合征(PCOS)--一種影響5~10%育齡婦女的內分泌疾病，以及囊性纖維化( CF)--一種由囊性纖維化跨膜電導調節器(CFTR) 基因突變引起的遺傳疾病。然而引起這些卵巢功能障礙的確切機制並不清楚。 / 雌激素是在卵泡雌激素(FSH) 的調節下，在卵巢顆粒細胞中通過芳香化臨的住激素轉化而生成的。在論文第一部分的研究中，我們旨在證明CFTR 在卵巢顆粒細胞中的表達，以及它參與雌激素生成的過程。實驗結果證實了CFTR 在小鼠和人顆粒細胞中的表達，同時表明CFTR 通過一種碳酸氫根離子(HC0₃⁻) 敏感的可溶性腺苦酸環化梅(sAC) ，放大FSH 所刺激的雌激素生成過程。實驗結果顯示，在原代小鼠顆粒細胞中， HC0₃⁻能夠增強FSH 所引起的CREB 磷酸化，芳香化晦表達，以及雌激素的生成，而在抑制CFTR 的情況下，或在CFTR 敲除/DeltaF508 突變小鼠的顆粒細胞中， HCO3-的放大作用顯著降低。CFTR 和芳香化醋的表達水準在人顆粒細胞中具有正相關性，進一步支持CFTR 對雌激素生成的調節作用。在PCOS 患者的顆粒細胞和大鼠PCOS 模型的卵巢中， CFTR 和芳香化醋的表達水準顯著下調。這些結果提示， CFTR 對雌激素生成調節這一機制的缺陷可能參與了CF 和PCOS 中卵巢功能障礙的發病機理。 / 卵泡發育很大程度上依賴於顆粒細胞的增殖'生存和凋亡，這些過程在PCOS 中都會出現異常。論文的第二部分冒在研究顆粒細胞的CFTR 在PCOS 的卵泡發育異常中的作用。實驗結果表明， CFTR 在PCOS 大鼠的囊，性卵泡的顆粒細胞中表達降低，同時伴隨著PCNA 和Bcl-2 的下調，而Bax 和cleaved caspase-3則沒有變化，提示顆粒細胞的增殖和生存/抗凋亡能力降低。敲減或抑制顆粒細胞中的CFTR 導致細胞存活降低， PCNA 和Bcl-2 表達下調，以及細胞凋亡增加，提示CFTR 對顆粒細胞增殖和生存的調節作用。CFTR 通過HC0₃⁻/ sAC/PKA 信號通路，調節基礎及FSH 刺激引起的ERK I!2 磷酸化，及其下游的CyclinD2 和PCNA表達，從而促進顆樹圍胞的增殖。顆粒細胞CFTR 的下調可能通過抑制細胞增殖和降低細胞生存能力，參與了PCOS 中的囊性卵泡的形成過程。 / 綜上所述，本論文證明了CFTR 在卵巢顆粒細胞上的表達，並且參與調節顆粒細胞雌激素生成和細胞的增殖和生存。CFTR 的缺陷或表達下調可能是導致CF和PCOS 的卵巢功能障礙的發病機理。 / The ovary is the female reproductive organ, which produces female gametes, oocytes for fertilization and sex hormones essential to reproduction and important to a wide range of physiological and pathological events as well. About 30% of infertility cases arise from ovarian problems, including anovulation, amenorrhea, irregular menstrual cycle and abnormal hormone levels. Ovarian disorders, such as high androgen to estrogen ratio, abnormal folliculogenesis and anovulation, are often seen in diseases, including polycystic ovarian syndrome (PCOS) and cystic fibrosis (CF). The former is an endocrine disorder affecting 5~10% women of reproductive age, and the latter is a common genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). However, the exact mechanisms underlying the ovarian disorders seen in these diseases are not well understood. / Estrogen biosynthesis is profoundly influenced by follicle-stimulating hormone (FSH) that regulates the conversion of androgen to estrogen in ovarian granulosa cells by the rate-limiting enzyme aromatase. The first part of the study aims to investigate the expression of CFTR in granulosa cells and its involvement in regulating estrogen production. The results demonstrate the expression of CFTR in both mouse and human granulosa cells, and provide evidence demonstrating a previously unsuspected role of CFTR in amplification of FSH-stimulated ovarian estrogen biosynthesis and the involvement of a HC0₃⁻ sensor, the soluble adenylyl cyclase (sAC) in this synthesis. FSH-stimulated CREB phosphorylation, aromatae expression, as well as estradiol production are enhanced by HC0₃⁻ and sAC, which could be significantly reduced by CFTR inhibition or in ovaries or granulosa cells of cftr knockout/deltaF508 mutant mice. The fact that CFTR expression is found positively correlated with aromatase expression in human granulosa cells supports its role in regulating estrogen production in humans. Reduced CFTR and aromatase expression is also found in polycystic ovarian syndrome (PCOS) rodent models and human patients. These findings suggest that defective CFTR-dependent regulation of estrogen production may underline the ovarian disorders seen in CF and PCOS. / Folliculogenesis largely depends on the proliferation, survival and apoptosis of granulosa cells in the follicles and alteration in which has been found in PCOS. The second part of the study aims to investigate the possible involvement of granulosa cell CFTR in the impaired folliculogenesis in PCOS. The results show that downregulation of CFTR is found in the cystic follicles, which is accompanied by reduced expression of PCNA and Bcl-2, but not Bax and cleaved caspase-3, in the ovaries of PCOS rat models, indicating reduced cell proliferation and survival/anti-apoptotic ability. Knockdown or inhibition of CFTR in granulosa cell culture results in reduced cell viability, downregulation of PCNA and Bcl-2 and increase of apoptosis, supporting a role of CFTR in regulating granulosa cell proliferation and survival. CFTR exerts its effect on granuloa cell proliferation by modulating basal and FSH-stimulated ERKl/2 phosphorylation and the expression of its downstream target CyclinD2 and PCNA through the HC0₃⁻/sAC/PKA pathway. These findings suggest that downregulation of CFTR may play a role in the formation of cystic follicles by inhibiting granulosa cell proliferation and reducing cell survival ability, therefore providing a possible mechanism for the abnormal folliculogenesis in PCOS. / In conclusion, the present study has demonstrated the expression of CFTR in the ovarian granulosa cell and its role in regulation of granulosa cell proliferation, survival and estrogen production. Defect of CFTR in CF and downregulation of CFTR in PCOS may contribute to the abnonnal honnone profile and impaired folliculogenesis in both disease conditions. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Hui. / "October 2011." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 124-137). / 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 / 摘要 --- p.iv / ACKNOWLEDGEMENT --- p.vi / LIST OF PUBLICATIONS --- p.viii / ABBREVIATIONS --- p.xiii / LIST OF FIGURES AND TABLES --- p.xvi / Chapter 1 --- CHAPTER I: Introduction --- p.1 / Chapter 1.1 --- The ovary --- p.1 / Chapter 1.1.1 --- Structure and function of the ovary --- p.1 / Chapter 1.1.2 --- Follicle development --- p.5 / Chapter 1.1.3 --- Ovulation and luteinization --- p.7 / Chapter 1.1.4 --- Ovarian hormone biosynthesis --- p.10 / Chapter 1.2 --- Diseases with ovarian dysfunction --- p.14 / Chapter 1.2.1 --- Polycystic ovarian syndrome (PCOS) --- p.14 / Chapter 1.2.1.1 --- Introduction to PCOS --- p.14 / Chapter 1.2.1.2 --- Diagnostic criteria --- p.14 / Chapter 1.2.1.3 --- Abnormal hormone profile in PCOS --- p.16 / Chapter 1.2.1.4 --- Abnormal folliculogenesis in PCOS --- p.18 / Chapter 1.2.1.5 --- Etiology --- p.22 / Chapter 1.2.2 --- Cystic Fibrosis (CF) --- p.24 / Chapter 1.2.2.1 --- Introduction to CF --- p.24 / Chapter 1.2.2.2 --- Cause and pathogenesis of CF --- p.25 / Chapter 1.2.2.3 --- Ovarian disorder in CF --- p.27 / Chapter 1.3 --- CFTR in reproduction --- p.29 / Chapter 1.3.1 --- Introduction to CFTR --- p.29 / Chapter 1.3.2 --- Channel function --- p.30 / Chapter 1.3.3 --- Protein regulator function --- p.32 / Chapter 1.3.4 --- Regulation of CFTR expression --- p.34 / Chapter 1.3.5 --- Role of CFTR in reproduction --- p.35 / Chapter 1.3.6 --- CFTR in the ovary --- p.39 / Chapter 1.4 --- General hypothesis and aims --- p.39 / Chapter 1.4.1 --- General hypothesis --- p.39 / Chapter 1.4.2 --- Aims of the study --- p.40 / Chapter 2 --- CHAPTER II: General Methods --- p.42 / Chapter 2.1 --- Meterials --- p.42 / Chapter 2.1.1 --- Animals --- p.42 / Chapter 2.1.2 --- Chemicals and reagents --- p.42 / Chapter 2.1.3 --- Antibodies --- p.44 / Chapter 2.1.4 --- Primers --- p.45 / Chapter 2.2 --- Methods --- p.45 / Chapter 2.2.1 --- Determination of estrous cycle --- p.45 / Chapter 2.2.2 --- Granulosa cell culture --- p.46 / Chapter 2.2.3 --- PCGS rat model --- p.47 / Chapter 2.2.4 --- Collection of human granulosa cells --- p.47 / Chapter 2.2.5 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.48 / Chapter 2.2.6 --- Western blot --- p.50 / Chapter 2.2.7 --- Histological studies --- p.53 / Chapter 2.2.8 --- siRNA transfection --- p.55 / Chapter 2.2.9 --- Intracellular pH measurement --- p.56 / Chapter 2.2.10 --- Whole-cell patch clamp recording --- p.57 / Chapter 2.2.11 --- Statistics --- p.57 / Chapter 3 --- CHAPTER III: Result I - The Role of CFTR in FSH-stimulated Estrogen Production: Implication in Cystic Fibrosis and PCGS --- p.59 / Chapter 3.1 --- Summary --- p.59 / Chapter 3.2 --- Introduction --- p.60 / Chapter 3.3 --- Methods --- p.63 / Chapter 3.3.1 --- Intracellular cAMP assay --- p.63 / Chapter 3.3.2 --- Nuclei isolation and nuclear cAMP measurement --- p.63 / Chapter 3.3.3 --- CREB phosphorylation assay --- p.64 / Chapter 3.3.4 --- Estradiol enzyme immunoassay --- p.64 / Chapter 3.4 --- Results --- p.64 / Chapter 3.4.1 --- Functional expression of CFTR in granulosa cells --- p.64 / Chapter 3.4.2 --- Expression and localization of sAC in granulosa cells and its involvement in BC03f CFTR-dependent cAMP production --- p.66 / Chapter 3.4.3 --- Effect of CFTR and HC0₃⁻ on basal and FSB-stimulated CREB phosphorylation --- p.67 / Chapter 3.4.4 --- Effect of CFTR and HC0₃⁻ on basal and FSB-stimulated aromatase expression and estradiol production --- p.68 / Chapter 3.4.5 --- Impaired CREB phosphorylation aromatase expression and estradiol production by granulosa cells from CFTR-deficient mice --- p.70 / Chapter 3.4.6 --- Reduced CFTR and aromatase expression in human PCOS granulosa cells and rat PCOS ovaries --- p.71 / Chapter 3.5 --- Discussion --- p.87 / Chapter 4 --- CHAPTER IV: Result II - The Role of CFTR in Granulosa Cell Proliferation and survival in PCOS --- p.91 / Chapter 4.1 --- Summary --- p.91 / Chapter 4.2 --- Introduction --- p.92 / Chapter 4.3 --- Methods --- p.95 / Chapter 4.3.1 --- Cell viability assay (MTT and MTS assay) --- p.95 / Chapter 4.3.2 --- ERKI/2 phosphorylation assay --- p.95 / Chapter 4.4 --- Results --- p.96 / Chapter 4.4.1 --- Reduced CFTR expression in PCOS rat models --- p.96 / Chapter 4.4.2 --- Downregulation of genes related to proliferation and survival in PCOS --- p.96 / Chapter 4.4.3 --- CFTR affect viability of granulosa cells --- p.97 / Chapter 4.4.4 --- CFTR regulate cell cycle protein and promote proliferation via HC0₃⁻/sAC/PKA and ERK pathway --- p.98 / Chapter 4.4.5 --- CFTR regulates apoptosis-related protein expression --- p.100 / Chapter 4.5 --- Discussion --- p.114 / Chapter 5 --- CHAPTER V: General Discussion --- p.119 / Chapter 5.1 --- Role of CFTR in ovarian function --- p.119 / Chapter 5.2 --- Role of CFTR/HC0₃⁻/sAC in modulating FSH signaling in the ovary --- p.120 / Chapter 5.3 --- CFTR/HC0₃⁻/sAC as a general modulator in receptor-mediated signaling cascades --- p.122 / Chapter 5.4 --- Concluding remarks --- p.123 / REFERENCES --- p.124 / APPENDICES --- p.138

The E3 ubiquitin ligase RNF43 (RING finger protein 43) is an important negative modulator of the WNT signalling pathway that acts at the plasma membrane by targeting Frizzled and its co-receptor LRP for degradation. In the small intestine, this prevents uncontrolled expansion of the stem cell compartment and so it is essential to the maintenance of normal tissue homeostasis. However, despite its crucial role in fine-tuning the WNT pathway and its role as a tumour suppressor, it is unclear whether RNF43 has further binding partners and what their functional relevance is to the modulation of WNT signalling. Here, I describe the development of a new screening strategy which combines CRISPR/Cas9 technology with 3D-intestinal organoid culture for the identification of novel molecular interactors of RNF43. Overall, this study and the technology developed provide a tool to enable the detailed description of the mechanism of action of RNF43, which is important not only in order to increase our understanding of WNT pathway regulation but also to gain potential new insights into RNF43 paralogs, by analogy. The investigation of paralogs is crucial as RNF43 belongs to a newly identified family of E3 ubiquitin ligases, named the PA-RING family, whose members are still poorly characterised. The majority of PA-RING family members have not been linked to any signalling pathway, most of their targets are still unknown and in many cases their in vivo function has not been addressed. In this context, my work has specifically focused on the investigation of the potential involvement of additional PA-RING family members in WNT pathway modulation and also on target identification for selected members. The results summarised in this dissertation show that no other PA-RING family member plays a prominent role in WNT pathway modulation aside from Rnf43 and its homologue Znrf3, however, different classes of adhesion molecules are likely to be regulated by certain of these E3 ligases. In conclusion, my work has contributed to unravelling previously unexplored aspects of this protein family, with particular regard to RNF43 and its mechanism of action. Thanks to this original approach, it was possible to identify potential new players involved either in membrane clearance of Frizzled or in RNF43 maturation. In particular, my thesis focuses on the characterisation of the role of DAAM in RNF43-mediated Frizzled internalisation.

Further studies were conducted to define an indispensable role of uterine bicarbonate secretion at pre-implantation for the success of blastocyst implantation. The in vitro implantation experiments showed that only when cultured in bicarbonate-containing medium, the blastocysts exhibited normal rate of attachment and outgrowth level. The forskolin-induced endometrial bicarbonate secretion measured by the Isc on pregnant day 4 was almost abolished by CA inhibitor acetazolamide. The efflux of intracellular bicarbonate, measured by intracellular pH-sensitive dye, was blocked by CFTR inhibitor, NPPB, and SLC26a6 inhibitor, DIDS, indicating their involvement in mediating uterine bicarbonate secretion. / In conclusion, the present findings have demonstrated an important role of CFTR in formation of optimal uterine fluid, in terms of both volume and composition, which is crucial for various reproductive events occurring in the uterus. Deviation from the normal uterine fluid composition and volume due to defects in CFTR function or abnormal regulation under pathological conditions, such as CF and genital bacteria infection, probably leads to infertility. The information obtained may provide insight into regulatory mechanism underlying fertility and infertility, as well as the rationale for development of treatment methods for female infertility and new strategies for female contraception. (Abstract shortened by UMI.) / The last part of the study was to demonstrate possible cause of infertility by disturbance of uterine fluid dynamic due to abnormal expression of CFTR using a model of uterine Chlamydia (C.) trachomatis infection, the most common infection-related sterility with the underlying cause unexplained. Uterine C. trachomatis infection induced up-regulated expression of CFTR with enhanced electrolyte and fluid transport as demonstrated by the increase in the cAMP-dependent Isc and uterine wet weight with obvious fluid accumulation in the lumen at diestrus stage, during which the endometrium normally undergoes a series of changes preparing for blastocyst implantation with minimum CFTR expression and uterine fluid volume. The abnormal uterine fluid accumulation upon uterine C. trachomatis infection significantly reduced implantation rate in uterine C. trachomatis infection mouse model. / The present study was aimed to elucidate the cellular and molecular mechanisms underlying the CFTR-related reproductive events in physiological and pathological conditions by using a variety of techniques, including RT-PCR, Western blot, intracellular and extracellular pH measurements, and the short-circuit current (Isc) measurement, in conjunction with mouse primary culture of endometrial cells and blastocyst, as well as several animal models including CF mouse, mouse uterine infectious model and overyectomized (OVX) mouse, etc. / We first examined dynamic changes in uterine bicarbonate secretion, as indicated by bicarbonate-dependent forskolin-induced Isc and epithelial surface pH measurement, and the expression profile of candidate genes and proteins known to be involved in bicarbonate secretion throughout the estrous cycle in mouse uterus. The results showed that the maximum mRNA and protein levels of CFTR, SLC26a6, carbonic anhydrase (CA)2 and CA12 were observed at proestrus stage and/or estrus stages. Luminal surface pH measured by 5-N-hexadecanoyl-aminofluorescein (HAF) showed that the basal endometrial epithelial surface pH at estrus stage was significantly higher than that in diestrus, which could be reduced significantly by CFTR inhibitor DPC, SLC26a6 inhibitor 4',4'-Diisothiocyanostilbene-2',2' Disulfonic Acid (DIDS) and CA suppressor acetazolamide. In the ovariectimized (OVX) mice and primary culture of endometrial cells, estrogen could induce up-regulation of CFTR, SLC26a6, CA2 and CA12 expression with corresponding increase in the bicarbonate-dependent Isc, suggesting a novel role of estrogen in regulating uterine bicarbonate secretion. / He, Qiong. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3247. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 163-176). / 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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Cheung King Ho. / "August 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 140-158). / 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.

Mollusc shells are unique adaptations that serve to protect the organisms that make them, and are a defining feature of the phylum. However the molecular underpinnings of shell forming processes are still largely unexplored. To further understand mollusc shell formation, I studied three bivalve species in this project: the blue mussel Mytilus edulis, the Pacific oyster Crassostrea gigas, and the king scallop Pecten maximus. While previous analyses of the shell proteomes showed species specificity, transcriptomes of the mantle tissues revealed more commonalities. To reconcile these differences, I studied differential gene expression in shell damage-repair experiments and during the formation of the first larval shell, to produce a comprehensive overview of shell formation processes. Expression data showed large biological variability between individuals, requiring matched-pair experimental designs to detect differential gene expression during shell repair. Loci differentially expressed during shell repair and in the larvae encoded shell matrix proteins, transmembrane transporters, and novel transcripts. A large number of shell matrix proteins, encoded in differentially expressed loci, were common in all three species during shell formation, indicating that shell forming proteins between different species may be more common than previously thought. Differential expression of transmembrane transporters during shell repair indicated that the animals may be regulating bicarbonate ions during shell formation. Finally, the experiments revealed novel transcripts, with unknown annotations to public datasets, that may putatively be involved in shell formation.

Expression studies have implicated Fibronectin Leucine Rich Transmembrane protein 2 (FLRT2) in cranial neural crest cell migration and pre-chondrogenic cell condensation during craniofacial skeletogenesis. This aim of this study was to characterize the expression of FLRT2 and its relationship to the extracellular matrix (ECM) in ATDC5 chondroprogenitor cells. Immunofluorescence studies localized FLRT2 to the cell membrane as well as exracellularly, where it colocalized with fibronectin. FLRT2 was identified in the ATDC5-derived ECM after cell extraction. Further to its colocalization with fibronectin, FLRT2 associated with fibronectin-coated beads in cell cultures. Co-immunoprecipitation confirmed that FLRT2 and fibronectin interact, either directly or indirectly. Blocking fibronectin fibril formation in ATDC5 cell cultures demonstrated a concomitant decrease in extracellular FLRT2 accumulation. It appears that FLRT2 may exist in both a membrane-bound and a shed form. Either or both of these forms may participate in cell-ECM interactions in cooperation with fibronectin or other ECM proteins.

Expression studies have implicated Fibronectin Leucine Rich Transmembrane protein 2 (FLRT2) in cranial neural crest cell migration and pre-chondrogenic cell condensation during craniofacial skeletogenesis. This aim of this study was to characterize the expression of FLRT2 and its relationship to the extracellular matrix (ECM) in ATDC5 chondroprogenitor cells. Immunofluorescence studies localized FLRT2 to the cell membrane as well as exracellularly, where it colocalized with fibronectin. FLRT2 was identified in the ATDC5-derived ECM after cell extraction. Further to its colocalization with fibronectin, FLRT2 associated with fibronectin-coated beads in cell cultures. Co-immunoprecipitation confirmed that FLRT2 and fibronectin interact, either directly or indirectly. Blocking fibronectin fibril formation in ATDC5 cell cultures demonstrated a concomitant decrease in extracellular FLRT2 accumulation. It appears that FLRT2 may exist in both a membrane-bound and a shed form. Either or both of these forms may participate in cell-ECM interactions in cooperation with fibronectin or other ECM proteins.

ATP binding cassette (ABC) transporters constitute one of the largest families of transport proteins. The occurrence of multidrug resistance (MDR) in human cancer cells has been correlated with the over expression of human ABC, P-glycoprotein (Pgp). Streptomyces peucetius produces two anticancer agents, doxorubicin and daunorubicin, that belong to the anthracycline family of antibiotics. The organism is self-resistant to the potent effects of the antibiotics it produces due to the action of an efflux pump, DrrAB. Both Pgp and DrrAB carry out similar functions, but in two different cell types. An understanding of the bacterial drug transporter DrrAB is thus expected to help in obtaining a better understanding of the function and evolution of the multidrug transporter P-glycoprotein. In DrrAB, the catalytic and membrane domains are present on separate subunits, DrrA and DrrB respectively. How the catalytic ATP-binding domains and the membrane domains in transporters interact with each other, or how energy is transduced between them, is not well understood. We introduced several single cysteine substitutions in DrrB and then by using a cysteine to amine hetero-bifunctional cross-linker showed that DrrA interacts predominantly with the N-terminal cytoplasmic tail of DrrB. Within this region of DrrB, we also identified a sequence with similarities to the EAA motif found in importers of the ABC family of proteins, thus leading to the proposal that the EAA or the EAA-like motif may be involved in forming a generalized interface between the ABC and the TMD of both uptake and export systems. By using a combination of approaches, including point mutations and disulfide cross-linking analysis, we show here that the Q-loop region of DrrA plays an important role in dimerization of DrrA as well as in interactions with DrrB. Furthermore, we also show that the interaction of the Q-loop with the N-terminus of DrrB is involved in transmitting conformational changes between DrrA and DrrB. The scope of the present study further extends into identifying the factors involved in the biogenesis of the DrrAB pump. We have identified two accessory proteins namely, FtsH and GroEL that may be involved in proper folding and assembly of the transporter.

With the efforts to understand the protein structure, many computational approaches have been made recently. Among them, the Support Vector Machine (SVM) methods have been recently applied and showed successful performance compared with other machine learning schemes. However, despite the high performance, the SVM approaches suffer from the problem of understandability since it is a black-box model; the predictions made by SVM cannot be interpreted as biologically meaningful way. To overcome this limitation, a new association rule based classifier PCPAR was devised based on the existing classifier, CPAR to handle the sequential data. The performance of the PCPAR was improved more by designing the following two hybrid schemes. The PCPAR/SVM method is a parallel combination of the PCPAR and the SVM and the PCPAR_SVM method is a sequential combination of the PCPAR and the SVM. To understand the SVM prediction, the SVM_PCPAR scheme was developed. The experimental result presents that the PCPAR scheme shows better performance with respect to the accuracy and the number of generated patterns than CPAR method. The PCPAR/SVM scheme presents better performance than the PCPAR, PCPAR_SVM or the SVM_PCPAR and almost equal performance to the SVM. The generated patterns are easily understandable and biologically meaningful. The system sturdiness evaluation and the ROC curve analysis proved that this new scheme is robust and competent.