Indiana University-Purdue University Indianapolis (IUPUI) / Human cord blood (CB) derived circulating endothelial colony forming cells (ECFCs) display a hierarchy of clonogenic proliferative potential and possess de novo vessel forming ability upon implantation in immunodeficient mice. Since survival of ECFC post-implantation is a critical variable that limits in vivo vasculogenesis, we tested the hypothesis that activation of Notch signaling or co-implantation of ECFC with human platelet lysate (HPL) would enhance cultured ECFC vasculogenic abilities in vitro and in vivo. Co-implantation of ECFCs with Notch ligand Delta-like 1 (DL1) expressing OP9 stromal cells (OP9-DL1) decreased apoptosis of ECFC in vitro and increased vasculogenesis of ECFC in vivo. The co-culture of ECFC with HPL diminished apoptosis of ECFC by altering the expression of pro-survival molecules (pAkt, pBad and Bcl-xL) in vitro and increased vasculogenesis of human EC-derived vessels both in vitro and in vivo. Thus, activation of the Notch pathway by OP9-DL1 stromal cells or co-implantation of ECFC with HPL enhances vasculogenesis and augments blood vessel formation by diminishing apoptosis of the implanted ECFC. The results from this study will provide critical information for the development of a cell therapy for limb and organ re-vascularization that can be applied to recovery of ischemic tissues in human subjects.

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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

Kam Man Fai Afia. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves [195]-224). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgement --- p.v / Publications --- p.vi / Abbreviations --- p.vii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- The Mast Cell --- p.2 / Chapter 1.1.1 --- Historical Perspective --- p.2 / Chapter 1.1.2 --- Mast Cell Origin and Development --- p.3 / Chapter 1.1.3 --- Mast Cell Heterogeneity --- p.5 / Chapter 1.1.3.1 --- Rodent Mast Cell Heterogeneity --- p.5 / Chapter 1.1.3.2 --- Human Mast Cell Heterogeneity --- p.7 / Chapter 1.1.4 --- Mast Cell Mediators --- p.10 / Chapter 1.1.4.1 --- Preformed Mediators --- p.11 / Chapter 1.1.4.2 --- Newly Synthesized Lipid Mediators --- p.14 / Chapter 1.1.4.3 --- Cytokines --- p.16 / Chapter 1.1.5 --- Mast Cell Activation --- p.17 / Chapter 1.1.5.1 --- Immunological Activation --- p.19 / Chapter 1.1.5.1.1 --- FcεIR Activation and Protein Tyrosine Phosphorylation --- p.19 / Chapter 1.1.5.1.2 --- Activation of Phospholipases --- p.20 / Chapter 1.1.5.1.3 --- The Role of Calcium --- p.22 / Chapter 1.1.5.1.3.1 --- Intracellular Calcium Mobilization --- p.23 / Chapter 1.1.5.1.3.2 --- Calcium Influx --- p.24 / Chapter 1.1.5.1.3.3 --- Mechanisms of Action of Calcium in Mast Cells --- p.28 / Chapter 1.1.5.1.4 --- The Role of G-proteins --- p.30 / Chapter 1.1.5.1.5. --- The Role of Cylic AMP --- p.33 / Chapter 1.1.5.1.2.1 --- Mechanisms of Action of Cyclic AMP in Mast Cells --- p.36 / Chapter 1.1.5.1.2.2 --- Implications for the Inhibitory Role of Cyclic AMP in Mast Cell Activation --- p.37 / Chapter 1.2 --- The Cyclic Nucleotide Phosphodiesterases --- p.39 / Chapter 1.2.1 --- Introduction --- p.39 / Chapter 1.2.2 --- Classification and Structure --- p.41 / Chapter 1.2.3 --- Distribution and Physiological Functions of the Different PDE Families --- p.45 / Chapter 1.2.4 --- Phosphodiesterase Inhibitors --- p.49 / Chapter 1.2.4.1 --- Non-selective PDE Inhibitors --- p.50 / Chapter 1.2.4.2 --- Selective PDE Inhibitors --- p.52 / Chapter 1.2.4.2.1 --- PDE1 and PDE2 Inhibitors --- p.52 / Chapter 1.2.4.2.2 --- PDE3 Inhibitors --- p.53 / Chapter 1.2.4.2.3 --- PDE4 Inhibitors --- p.54 / Chapter 1.2.4.2.4.1 --- PDE5 Inhibitors --- p.56 / Chapter 2. --- Materials and Methods --- p.59 / Chapter 2.1 --- Materials --- p.60 / Chapter 2.1.1 --- Drugs --- p.60 / Chapter 2.1.1.1 --- Phosphodiesterase Inhibitors --- p.60 / Chapter 2.1.1.2 --- Mast Cell Secretagogues --- p.61 / Chapter 2.1.2 --- Materials for Rat Peritoneal Mast Cell Experiments --- p.61 / Chapter 2.1.2.1 --- Materials for Rat Sensitization --- p.61 / Chapter 2.1.2.2 --- Materials for Buffers --- p.62 / Chapter 2.1.2.3 --- Materials for Histamine Assay --- p.62 / Chapter 2.1.2.4 --- Miscellaneous --- p.63 / Chapter 2.1.3 --- Materials for RBL-2H3 Cell Line Experiments --- p.63 / Chapter 2.1.3.1 --- Materials for Cell Culture --- p.63 / Chapter 2.1.3.2 --- Materials for Cell Sensitization and Enzyme Release --- p.64 / Chapter 2.1.3.3 --- Materials for β-Hexosaminidase Assay --- p.64 / Chapter 2.1.3.4 --- Miscellaneous --- p.64 / Chapter 2.2 --- Rat Peritoneal Mast Cell Experiments --- p.65 / Chapter 2.2.1 --- Preparation of Buffers --- p.65 / Chapter 2.2.2 --- Preparation of Stock Solutions --- p.66 / Chapter 2.2.2.1 --- Mast Cell Secretagogue Stock Solutions --- p.66 / Chapter 2.2.2.2 --- Phosphodiesterase Inhibitor Stock Solutions --- p.66 / Chapter 2.2.3 --- Animals and Cell Isolation --- p.71 / Chapter 2.2.3.1 --- Animals --- p.71 / Chapter 2.2.3.2 --- Sensitization of Animals --- p.71 / Chapter 2.2.3.3 --- Cell Isolation --- p.71 / Chapter 2.2.3.4 --- Cell Purification --- p.72 / Chapter 2.2.3.5 --- Determination of Cell Number and Viability --- p.73 / Chapter 2.2.4 --- General Protocol for Histamine Release and Histamine Measurement --- p.75 / Chapter 2.2.4.1 --- Histamine Release --- p.75 / Chapter 2.2.4.2 --- Spectrofluorometric Determination of Histamine Content --- p.76 / Chapter 2.2.4.2.1 --- Manual Histamine Assay --- p.76 / Chapter 2.2.4.2.2 --- Automated Histamine Assay --- p.78 / Chapter 2.2.4.3 --- Calculation of Histamine Levels --- p.78 / Chapter 2.2.4.4 --- Presentation and Statistics --- p.79 / Chapter 2.3 --- RBL-2H3 Cell Line Experiments --- p.80 / Chapter 2.3.1 --- Preparation of Stock Solutions --- p.80 / Chapter 2.3.2 --- Preparation of Materials for Enzyme Release and Assay --- p.81 / Chapter 2.3.2.1 --- Cell Culture --- p.81 / Chapter 2.3.2.2 --- Preparation of Cells for β-Hexosaminidase Release Experiments --- p.82 / Chapter 2.3.2.3 --- β-Hexosaminidase Release --- p.82 / Chapter 2.3.2.4 --- β-Hexosaminidase Assay --- p.83 / Chapter 3. --- Effects of Phosphodiesterase Inhibitors on Mediator Release from Rat Mast Cells --- p.84 / Chapter 3.1 --- Introduction --- p.85 / Chapter 3.2 --- Materials and Methods --- p.87 / Chapter 3.2.1 --- Rat Peritoneal Mast Cells --- p.87 / Chapter 3.2.1.1 --- Experiments Employing Immunological Stimulus in RPMCs --- p.87 / Chapter 3.2.1.2 --- Experiments Employing Non-Immunological Stimuli in RPMCs --- p.88 / Chapter 3.2.2 --- Rat Basophilic Leukemia Cells --- p.88 / Chapter 3.3 --- Results --- p.89 / Chapter 3.3.1 --- Rat Peritoneal Mast Cells --- p.89 / Chapter 3.3.1.1 --- Immunologically Activated Rat Peritoneal Mast Cells --- p.89 / Chapter 3.3.1.1.1 --- Effects of Non-Selective PDE Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.89 / Chapter 3.3.1.1.2 --- Effects of Selective PDE1 and PDE2 Inhibitors on Anti-IgE- Mediated Histamine Release from RPMCs --- p.90 / Chapter 3.3.1.1.3 --- Effects of Selective PDE3 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.90 / Chapter 3.3.1.1.4 --- Effects of Selective PDE4 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.91 / Chapter 3.3.1.1.5 --- Effects of Selective PDE5 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.91 / Chapter 3.3.1.2 --- Non-Immunologically Activated Rat Peritoneal Mast Cells --- p.92 / Chapter 3.3.1.2.1 --- Effects of Selective PDE Inhibitors on Compound 48/80- Mediated Histamine Release from RPMCs --- p.92 / Chapter 3.3.1.2.2 --- Effects of Selective PDE Inhibitors on Histamine Release from RPMCs Stimulated by Calcium Ionophores --- p.93 / Chapter 3.3.2 --- Rat Basophilic Leukemia Cells --- p.93 / Chapter 3.3.2.1 --- Effects of Non-Selective PDE Inhibitors on Antigen-Mediated β-Hexosaminidase Release from RBL-2H3 Cells --- p.93 / Chapter 3.3.2.2 --- Effects of Selective PDE Inhibitors on Antigen-Mediated β-Hexosaminidase Release from RBL-2H3 Cells --- p.94 / Chapter 3.4 --- Discussion --- p.95 / Chapter 3.4.1 --- Rat Peritoneal Mast Cells --- p.95 / Chapter 3.4.1.1 --- Immunologically Activated RPMCs --- p.95 / Chapter 3.4.1.2 --- Non-Immunologically Activated RPMCs --- p.99 / Chapter 3.4.2 --- Rat Basophilic Leukemia Cells --- p.103 / Chapter 4. --- Combined Effects of Selective Phosphodiesterase Inhibitors on Immunologically Induced Histamine from Rat Mast Cells --- p.143 / Chapter 4.1 --- Introduction --- p.144 / Chapter 4.2 --- Materials and Methods --- p.144 / Chapter 4.2.1 --- Simultaneous Addition of PDE3 and PDE4 Inhibitors --- p.145 / Chapter 4.2.2 --- Sequential Addition of PDE3 and PDE4 Inhibitors --- p.145 / Chapter 4.3 --- Results --- p.146 / Chapter 4.3.1 --- Effects of the Selective Inhibitors for PDE3 and PDE4 Alone: Calculation of the Expected Inhibition Curve --- p.146 / Chapter 4.3.2 --- Effects of the Simultaneous Addition of PDE3 and PDE4 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.148 / Chapter 4.3.2.1 --- Rolipram and Siguazodan --- p.148 / Chapter 4.3.2.2 --- Ro 20-1724 and Siguazodan --- p.149 / Chapter 4.3.2.3 --- Rolipram and Quazinone --- p.149 / Chapter 4.3.2.4 --- Ro 20-1724 and Quazinone --- p.150 / Chapter 4.3.3 --- Effects of the Sequential Addition of PDE3 and PDE4 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.150 / Chapter 4.3.3.1 --- Rolipram and Siguazodan --- p.150 / Chapter 4.3.3.2 --- Ro 20-1724 and Siguazodan --- p.151 / Chapter 4.3.3.3 --- Rolipram and Quazinone --- p.151 / Chapter 4.3.3.4 --- Ro 20-1724 and Quazinone --- p.152 / Chapter 4.4 --- Discussion --- p.153 / Chapter 5. --- Future Directions --- p.191 / Chapter 5.1 --- Future Directions --- p.192 / References --- p.195

Tse, Chi Lok. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 116-131). / Abstracts in English and Chinese. / Declaration --- p.i / Abstract --- p.iii / Abstract in Chinese --- p.v / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Figures --- p.X / List of Tables --- p.xii / List of Abbreviations --- p.xiii / Chapter CHAPTER 1 --- General Introduction / Chapter 1.1 --- Regenerative Medicine --- p.1 / Chapter 1.2 --- Embryonic Stem Cells and Reprogramming --- p.3 / Chapter 1.3 --- Transdifferentiation --- p.6 / Chapter 1.4 --- The Cerebellum --- p.7 / Chapter 1.4.1 --- Functions of the cerebellum --- p.7 / Chapter 1.4.2 --- Structure and organization of the cerebellum --- p.8 / Chapter 1.4.3 --- Principle cellular components in the cerebellum --- p.12 / Chapter 1.4.3.1 --- Purkinje cells --- p.12 / Chapter 1.4.3.2 --- Granule cells --- p.12 / Chapter 1.4.3.3 --- Mossy fibres --- p.13 / Chapter 1.4.3.4 --- Climbing fibres --- p.13 / Chapter 1.4.3.5 --- Deep cerebellar nuclei --- p.13 / Chapter 1.4.3.6 --- Other cerebellar neurons --- p.14 / Chapter 1.4.3.7 --- Neuroglia of the cerebellum --- p.16 / Chapter 1.4.4 --- Circuitry of the cerebellum --- p.17 / Chapter 1.5 --- Development of the Cerebellum --- p.21 / Chapter 1.5.1 --- Anatomical changes during cerebellar development --- p.21 / Chapter 1.5.2 --- Molecular control of cerebellar development --- p.25 / Chapter 1.5.2.1 --- Specification of the cerebellar region --- p.25 / Chapter 1.5.2.2 --- Neurogenesis from the ventricular zone --- p.26 / Chapter 1.5.2.3 --- Neurogenesis from rhombic lip --- p.29 / Chapter 1.6 --- Scope of the Thesis --- p.33 / Chapter CHAPTER 2 --- Materials and General Methods / Chapter 2.1 --- Materials for Molecular Biological Work --- p.35 / Chapter 2.1.1 --- Enzymes --- p.35 / Chapter 2.1.2 --- Chemicals and others --- p.35 / Chapter 2.1.3 --- Plasmid vectors and plasmid --- p.36 / Chapter 2.1.4 --- Solutions and media --- p.36 / Chapter 2.2 --- Materials for Tissue/Cell Culture --- p.38 / Chapter 2.2.1 --- Chemicals --- p.38 / Chapter 2.2.2 --- Culture media and solutions --- p.38 / Chapter 2.2.3 --- Culture cells --- p.39 / Chapter 2.3 --- Animals --- p.40 / Chapter 2.4 --- Materials for Immunocytochemistry --- p.40 / Chapter 2.5 --- Oligonucleotide Primers --- p.41 / Chapter 2.6 --- RNA Extraction --- p.44 / Chapter 2.7 --- Generation of cDNA from mRNA --- p.44 / Chapter 2.8 --- Preparation of Recombinant Plasmid DNA --- p.45 / Chapter 2.8.1 --- Small scale preparation of DNA --- p.45 / Chapter 2.8.2 --- QLAGEN plasmid midiprep kit method --- p.46 / Chapter 2.9 --- Preparation of Specific DNA Fragment from Agarose Gel --- p.46 / Chapter 2.10 --- Subcloning of DNA Fragments --- p.47 / Chapter 2.10.1 --- Preparation of cloning vectors --- p.47 / Chapter 2.10.2 --- Subcloning of DNA fragment --- p.48 / Chapter 2.10.3 --- Transformation of DNA into competent cells --- p.48 / Chapter 2.11 --- Preparation of Competent Cells --- p.48 / Chapter CHAPTER 3 --- Generation and Characterization of Induced Neurons / Chapter 3.1 --- Introduction --- p.50 / Chapter 3.2 --- Experimental Procedures --- p.51 / Chapter 3.2.1 --- Construction of expression vector --- p.51 / Chapter 3.2.1.1 --- Preparation of insert DNA --- p.51 / Chapter 3.2.1.2 --- Construction of entry vector --- p.52 / Chapter 3.2.1.3 --- Construction of destination vector --- p.52 / Chapter 3.2.1.4 --- Construction of expression vector --- p.52 / Chapter 3.2.2 --- Generation of induced neural cells --- p.57 / Chapter 3.2.2.1 --- Culture of mouse embryonic fibroblasts (MEF) --- p.57 / Chapter 3.2.2.2 --- Production of expression vector containing retroviruses --- p.57 / Chapter 3.2.2.3 --- Transfection and induction of neural fate of MEF --- p.57 / Chapter 3.2.3 --- Immunocytochemcial analysis --- p.58 / Chapter 3.2.4 --- Efficiency calculation --- p.59 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.1 --- A screen for cerebellar Purkinje and granule cell fate-inducing factors --- p.62 / Chapter 3.3.2 --- Characterization of the induced neurons --- p.67 / Chapter 3.3.2.1 --- Granule cell induction --- p.67 / Chapter 3.3.2.2 --- Purkinje cell induction --- p.71 / Chapter 3.4 --- Discussion --- p.102 / Chapter 3.4.1 --- Roles of inducing factors in Purkinje cells and granule cells development --- p.102 / Chapter 3.4.2 --- Mechanism of neural transdifferentiation --- p.107 / Chapter CHAPTER 4 --- Future Directions / Chapter 4.1 --- Complete Induction of Purkinje Cell Fate --- p.111 / Chapter 4.2 --- Induced Neurons of Different Subtypes --- p.112 / Chapter 4.3 --- Mechanism of Transdifferentiation --- p.114 / Chapter 4.4 --- Transdifferentiation and Regenerative Medicine --- p.114 / Bibliography --- p.116

Fetal pancreatic tissues can be a promising source for pancreatic progenitor cells (PPCs). In this regard, we have successfully isolated and characterized a population of fetal PPCs from first trimester human fetal pancreas using a previously established basic protocol. Upon exposure to a cocktail of conventional growth factors, these PPCs are amenable to differentiate into insulin-secreting islet-like cell clusters (ICCs); however, these ICCs have yet to exert additional efforts to direct to glucose-responsive cells. To address this issue, we have proposed two novel morphogenic factors in the present study, namely PDZ-domain-containing 2 (PDZD2) and angiotensin II (Ang II), a physiologically active peptide of the renin-angiotensin system (RAS), that potentially promote the differentiation and maturation of PPCs/ICCs. / In light of these findings, we conclude that we have discovered two novel mechanisms, the PDZD2 and Ang II/AT2 receptor signaling pathways, in the regulation of the development of PPCs/ICCs, thus implying their novel roles during islet development in vivo. The present study provides a "proof-of-principle" that a local RAS is critically involved in governing islet cell development. This work may contribute to devising protocols for maturation of pancreatic progenitors for clinical islet transplantation. / Local RASs have been reported to regulate the differentiation of tissue progenitor cells. It has yet to be confirmed whether such systems exist and govern the PPC development. To address this issue, we herein provided evidence that expression of RAS components was highly regulated throughout PPC differentiation. Locally generated Ang II was found to maintain PPC growth and differentiation via mediation of the Ang II type 1 and type 2 (AT1 and AT 2) receptors. We found that the AT2, but not AT1, receptor was a key mediator of Ang II-induced upregulation of beta-cell transcription factors. Transplantation of AT2 receptor-depleted ICCs into immune-privileged diabetic mice failed to ameliorate hyperglycemia, implying that AT2 receptors are indispensable during ICC maturation in vivo. / PDZD2 and its secreted form (sPDZD2) have been found to express in our fetal PPCs. We first evaluated the potential role of sPDZD2 in stimulating PPC differentiation and established an optimal concentration for such stimulation. We found that 10-9 M sPDZD2 promoted PPC differentiation, as evidenced by the up-regulation of the pancreatic endocrine markers and C-peptide content in the ICCs. It enhanced their expression of the L-type voltage-gated calcium ion channel (Cav1.2) and conferred an ability to secrete insulin in response to membrane depolarization. Yet these ICCs remained glucose-unresponsive because of the minimal expression of GLUT-2. We thus attempted to study another potential morphogenic candidate, Ang II. / To further test whether a functional RAS is present and if so, whether it regulates islet development in vivo, we employed a mouse embryo model at different embryonic days and reported a stronger AT2 receptor expression during the 2nd developmental transition of pancreas development. AT2 receptor blockade from e8.0 resulted in abnormalities in fetal pancreatic development. Neonates from these mother mice displayed destructed pancreas/islet architecture, a hampered ability in glucose-stimulated insulin-secretion possibly attributed to a decreased ratio of beta-cell to alpha-cell, and an impaired glucose tolerance at 4-wk old. / Leung, Kwan Keung. / Adviser: Po Sing Leung. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 254-284). / 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.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.