by Shan Sze Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 145-166). / Abstracts in English and Chinese. / Title --- p.i / Abstract --- p.ii / 摘要 --- p.v / Acknowledgements --- p.vii / Table of Contents --- p.viii / List of Abbreviations --- p.xiv / List of Figures --- p.xvii / List of Tables --- p.xx / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- What are the general functions of cytokines? --- p.2 / Chapter 1.2 --- What is TNP-α? --- p.4 / Chapter 1.3 --- Actions of TNF-α --- p.5 / Chapter 1.4 --- General functions of TNF-α in astrocytes --- p.6 / Chapter 1.5 --- TNF-α receptors (TNF-Rs) --- p.8 / Chapter 1.6 --- Second messengers induced by TNP-α --- p.10 / Chapter 1.7 --- Glial Cells --- p.11 / Chapter 1.7.1 --- Oligodendroglia --- p.12 / Chapter 1.7.2 --- Brain Macrophages (Microglia) --- p.12 / Chapter 1.7.3 --- Astrocytes --- p.14 / Chapter 1.7.3.1 --- Functions of astrocytes --- p.15 / Chapter 1.8 --- "Brain injury, astrogliosis and scar formation" --- p.20 / Chapter 1.9 --- β-Adrenergic receptors (β-ARs) --- p.21 / Chapter 1.9.1 --- The active functional unit: the receptor complex --- p.22 / Chapter 1.9.2 --- General functions and distribution of β-ARs --- p.22 / Chapter 1.10 --- Functions of β-ARs in astrocytes --- p.24 / Chapter 1.10.1 --- Regulations of astrogliosis by β-ARs --- p.24 / Chapter 1.10.1.1 --- β-ARs are expressed in normal optic nerves and up-regulated after nerve crush --- p.24 / Chapter 1.10.1.2 --- Injury-induced alterations in endogenous catecholamine leads to enhanced β-AR activation --- p.25 / Chapter 1.10.1.3 --- β-AR blockade suppresses glial scar formation --- p.25 / Chapter 1.10.1.4 --- β-AR agonists affect the proliferation of astrocytes in normal brain --- p.26 / Chapter 1.11 --- Manganese Superoxide Dismutase (MnSOD) --- p.27 / Chapter 1.11.1 --- MnSOD is the target gene of NF-kB --- p.29 / Chapter 1.11.2 --- Induction of MnSOD by proinflammatory cytokines in rat primary astrocytes --- p.29 / Chapter 1.11.3 --- SMase and ceramides induce MnSOD in various cell types --- p.30 / Chapter 1.12 --- Why do we use C6 glioma cells? --- p.31 / Chapter 1.13 --- Aims and Scopes of this project --- p.32 / Chapter Chapter 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.36 / Chapter 2.1.1 --- Cell Line --- p.36 / Chapter 2.1.2 --- Cell Culture Reagents --- p.36 / Chapter 2.1.2.1 --- Complete Dulbecco´ةs modified Eagle medium (CDMEM) --- p.36 / Chapter 2.1.2.2 --- Rosewell Park Memorial Institute (RPMI) medium --- p.37 / Chapter 2.1.2.3 --- Phosphate buffered saline (PBS) --- p.37 / Chapter 2.1.3 --- Recombinant cytokines --- p.38 / Chapter 2.1.4 --- Chemicals for signal transduction study --- p.38 / Chapter 2.1.4.1 --- Modulators of protein kinase C (PKC) --- p.38 / Chapter 2.1.4.2 --- Modulator of protein kinase A (PKA) --- p.39 / Chapter 2.1.4.3 --- β-Adrenergic agonist and antagonist --- p.39 / Chapter 2.1.5 --- Antibodies --- p.40 / Chapter 2.1.5.1 --- Anti-TNF-receptor type 1 (TNF-R1) antibody --- p.40 / Chapter 2.1.5.2 --- Anti-TNF-receptor type 2 (TNF-R2) antibody --- p.41 / Chapter 2.1.5.3 --- Anti-βi-adrenergic receptor (βl-AR) antibody --- p.42 / Chapter 2.1.5.4 --- Anti-β2-adrenergic receptor (β2-AR) antibody --- p.42 / Chapter 2.1.5.5 --- Antibody conjugates --- p.43 / Chapter 2.1.6 --- Reagents for RNA isolation --- p.43 / Chapter 2.1.7 --- Reagents for reverse transcription-polymerase chain reaction (RT-PCR) --- p.43 / Chapter 2.1.8 --- Reagents for electrophoresis --- p.45 / Chapter 2.1.9 --- Reagents and buffers for Western blot --- p.45 / Chapter 2.1.10 --- Other chemicals and reagents --- p.47 / Chapter 2.2 --- Maintenance of rat C6 glioma cell line --- p.47 / Chapter 2.3 --- RNA isolation --- p.48 / Chapter 2.3.1 --- Measurement of RNA yield --- p.49 / Chapter 2.4 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.50 / Chapter 2.5 --- Western blot analysis --- p.52 / Chapter Chapter 3 --- RESULTS / Chapter 3.1 --- Effect of TNF-α on the expression of TNF-receptors (TNFRs) in C6 glioma cells --- p.55 / Chapter 3.1.1 --- Effect of TNF-α on TNF-R1 and -R2 mRNA expression in C6 cells --- p.56 / Chapter 3.1.2 --- The signaling systems mediating TNP-α-induced TNF-R2 expression in C6 cells --- p.57 / Chapter 3.1.2.1 --- The involvement of PKC in TNF-α-induced TNF-R2 expression in C6 cells --- p.57 / Chapter 3.1.2.2 --- Effect of PMA on the TNF-R protein levels in C6 cells --- p.63 / Chapter 3.1.2.3 --- Effect of Ro31 on the TNF-α-induced TNF-R protein level in C6 cells --- p.65 / Chapter 3.1.2.4 --- Effect of PKA activator on the level of TNF-R2 mRNA in C6 cells --- p.67 / Chapter 3.2 --- Effect of TNP-α on the expression of β1- and β2-adrenergic receptors (β1- and β2-ARs) in C6 glioma cells --- p.69 / Chapter 3.2.1 --- Effect of TNF-α on β1- and β2-ARs mRNA expression in C6 cells --- p.70 / Chapter 3.2.2 --- The signaling systems mediating TNF-α-induced β1- and β2-AR expression in C6 cells --- p.70 / Chapter 3.2.2.1 --- The involvement of PKC mechanism between TNF-α and β-ARs in C6 cells --- p.71 / Chapter 3.2.2.2 --- Effect of PMA on the β1- and β2-ARs protein level in C6 cells --- p.76 / Chapter 3.2.2.3 --- Effect of Ro31 on the TNF-α-induced β1- and β2-AR protein levels in C6 cells --- p.78 / Chapter 3.2.2.4 --- Effect of dbcAMP on the levels of βl- and β2-ARs mRNA in C6 cells --- p.80 / Chapter 3.3 --- Relationship between TN1F-R2 and β-adrenergic mechanism in C6 cells --- p.82 / Chapter 3.3.1 --- Effects of isproterenol and propranolol on endogenous TNF-α mRNA levels in C6 cells --- p.82 / Chapter 3.3.2 --- Effects of isoproterenol and propranolol on TNF-R2 mRNA levels in C6 cells --- p.83 / Chapter 3.3.3 --- Effects of β1-agonist and antagonist on endogenous TNF-α mRNA expression in C6 cells --- p.87 / Chapter 3.3.4 --- Effects of β1-agonist and antagonist on TNF-R2 mRNA expression in C6 cells --- p.91 / Chapter 3.3.5 --- Effects of β2-agonist and antagonist on endogenous TNF-α mRNA in C6 cells --- p.93 / Chapter 3.3.6 --- Effects of β2-agonist and antagonist on TNF-R2 mRNA in C6 cells --- p.100 / Chapter 3.4 --- Effect ofTNF-α on the expression of a transcriptional factor nuclear factor kappa B (NF-kB) in C6 glioma cells --- p.102 / Chapter 3.4.1 --- Effect ofTNF-α on NF-kB (p50) mRNA expression in C6 cells --- p.106 / Chapter 3.4.2 --- Effect of β-agonist and antagonist on NF-kB (p50) mRNA expression in C6 cells --- p.108 / Chapter 3.4.3 --- Effect of PMA and Ro31 on the levels of NF-kB mRNA in C6 cells --- p.109 / Chapter 3.5 --- Effects of TNF-α on the expression of manganese superoxide dismutase (MnSOD) in C6 glioma cells --- p.111 / Chapter 3.5.1 --- Effects of TNF-α on MnSOD and Cu-ZnSOD mRNAs expression in C6 cells --- p.114 / Chapter 3.5.2 --- Effects of β-agonist and β-antagonist on MnSOD mRNA expression in C6 cells --- p.115 / Chapter 3.5.3 --- Effects of PKC activator and inhibitor on the levels of MnSOD mRNA in C6 cells --- p.117 / Chapter Chapter 4 --- DISCUSSION AND CONCLUSION / Chapter 4.1 --- Effects of TNF-α on the expression of TNF-receptors (TNFRs) in C6 glioma cells --- p.122 / Chapter 4.2 --- Effects of TNF-a on the expression of β1- and β2-adrenergic receptors (β1 and β2-ARs) in C6 glioma cells --- p.126 / Chapter 4.3 --- Relationship between TNF-α and β-adrenergic mechanism in C6 cells --- p.128 / Chapter 4.4 --- Effects of TNF-α on the expression of a transcriptional factor nuclear factor kappa B (NF-kB) in C6 glioma cells --- p.131 / Chapter 4.5 --- Effects of TNF-α on the expression of manganese superoxide dismutase (MnSOD) in C6 glioma cells --- p.133 / Chapter 4.6 --- Possible sources of β-agonists --- p.136 / Chapter 4.7 --- Conclusions --- p.137 / Appendix A --- p.143 / References --- p.145

by Ricardo Wong Chi Ho. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 128-138). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Table of Contents --- p.vi / List of Figures --- p.xi / List of Tables --- p.xiii / List of Abbreviations --- p.xiv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Overview of Serine Protease Inhibitors --- p.1 / Chapter 1.2 --- Classification of Serine Protease Inhibitors --- p.2 / Chapter 1.2.1 --- Kunitz Type Serine Protease Inhibitors --- p.7 / Chapter 1.2.2 --- Bowman-Birk Type Serine Protease Inhibitors --- p.11 / Chapter 1.2.3 --- Squash Type Serine Protease Inhibitors --- p.16 / Chapter 1.3 --- Role of Serine Protease Inhibitors in Plants --- p.20 / Chapter 1.4 --- Nutritional Fact of Serine Protease Inhibitors --- p.22 / Chapter 1.5 --- Possible Applications of Serine Protease Inhibitors --- p.25 / Chapter 1.5.1 --- Medical Applications --- p.25 / Chapter 1.5.2 --- Agricultural Applications --- p.29 / Chapter 1.6 --- Rationale of the Present Study --- p.31 / Chapter Chapter 2 --- Screening of Seeds for Inhibitory Activities Against Serine Proteases --- p.33 / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.2 --- Materials and Methods --- p.37 / Chapter 2.2.1 --- Materials --- p.37 / Chapter 2.2.2 --- Extraction Method --- p.37 / Chapter 2.2.3 --- Assays for Proteases Inhibitory Activities --- p.38 / Chapter 2.2.3.1 --- Assay for Chymotrypsin Activity --- p.38 / Chapter 2.2.3.2 --- Assay for Trypsin Activity --- p.38 / Chapter 2.2.3.3 --- Assay for Elastase Activity --- p.39 / Chapter 2.2.3.4 --- Assay for Subtilisin Activity --- p.39 / Chapter 2.2.3.5 --- Assays for Protease Inhibitory Activities --- p.40 / Chapter 2.2.4 --- Determination of Protein Concentration --- p.41 / Chapter 2.3 --- Results --- p.42 / Chapter 2.3.1 --- Extraction --- p.42 / Chapter 2.3.2 --- Serine Proteases Inhibitory Activities --- p.42 / Chapter 2.4 --- Discussion --- p.47 / Chapter Chapter 3 --- Isolation of Chymotrypsin Inhibitor and Trypsin Inhibitors from Momordica cochinchinensis Seeds --- p.49 / Chapter 3.1 --- Introduction --- p.49 / Chapter 3.2 --- Materials and Methods --- p.56 / Chapter 3.2.1 --- Materials --- p.56 / Chapter 3.2.2 --- Protein Extraction --- p.57 / Chapter 3.2.3 --- SP-Sepharose Chromatography --- p.57 / Chapter 3.2.4 --- Reversed Phase High Pressure Liquid Chromatography --- p.58 / Chapter 3.2.5 --- Assays for Chymotrypsin and Trypsin Inhibitory Activities --- p.60 / Chapter 3.2.6 --- Titration of Chymotrypsin --- p.61 / Chapter 3.2.7 --- Tricine Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.62 / Chapter 3.2.8 --- Coupling of Trypsin-Sepharose 4B Affinity Column --- p.63 / Chapter 3.2.9 --- Affinity Chromatography on Trypsin-Sepharose 4B --- p.64 / Chapter 3.3 --- Results --- p.65 / Chapter 3.3.1 --- SP-Sepharose Chromatography --- p.65 / Chapter 3.3.2 --- Reversed Phase High Pressure Liquid Chromatography --- p.67 / Chapter 3.3.3 --- Summary of Purification --- p.71 / Chapter 3.3.4 --- Titration of Chymotrypsin --- p.74 / Chapter 3.3.5 --- Tricine Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.74 / Chapter 3.3.6 --- Affinity Chromatography on Trypsin-Sepharose 4B --- p.78 / Chapter 3.4 --- Discussion --- p.81 / Chapter Chapter 4 --- Characterization of Chymotrypsin Inhibitor and Trypsin Inhibitors --- p.88 / Chapter 4.1 --- Introduction --- p.88 / Chapter 4.2 --- Materials and Methods --- p.90 / Chapter 4.2.1 --- Materials --- p.90 / Chapter 4.2.2 --- Determination of Molecular Weight --- p.90 / Chapter 4.2.3 --- Amino Acid Sequence Analysis --- p.91 / Chapter 4.2.4 --- Surface Plasmon Resonance Measurement --- p.92 / Chapter 4.2.4.1 --- Immobilization of Ligands on the Surface of Optical Biosensors --- p.92 / Chapter 4.2.4.2 --- Determination of Kinetics Constants --- p.93 / Chapter 4.2.4.3 --- pH Dependence of the Inhibition by Chymotrypsin Inhibitor --- p.93 / Chapter 4.2.4.4 --- Data Analysis --- p.94 / Chapter 4.2.5 --- Effect of Chymotrypsin Inhibitor on the Estereolytic Activity and Proteolytic Activity of Chymotrypsin --- p.95 / Chapter 4.2.6 --- Specificities of the Inhibitors % --- p.96 / Chapter 4.2.7 --- Binding Ratio of CI to Different Proteases --- p.97 / Chapter 4.2.8 --- Effects of the Proteases on Their Corresponding Inhibitors --- p.97 / Chapter 4.2.8.1 --- Tricine Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.97 / Chapter 4.2.8.2 --- Assay for Chymotrypsin Inhibitory Activity --- p.98 / Chapter 4.3 --- Results --- p.99 / Chapter 4.3.1 --- Molecular Weight of the Inhibitors --- p.99 / Chapter 4.3.2 --- N-terminal Amino Acid Sequence --- p.99 / Chapter 4.3.3 --- Surface Plasmon Resonance Measurement --- p.102 / Chapter 4.3.3.1 --- Kinetics of Chymotrypsin Inhibitor --- p.102 / Chapter 4.3.3.2 --- Kinetics of Trypsin Inhibitors --- p.106 / Chapter 4.3.3.3 --- pH Dependence of the Inhibition by Chymotrypsin Inhibitor --- p.106 / Chapter 4.3.4 --- Effect of Chymotrypsin Inhibitor on the Estereolytic Activity and Proteolytic Activity of Chymotrypsin --- p.106 / Chapter 4.3.5 --- Specificities of the Inhibitors --- p.110 / Chapter 4.3.6 --- Binding Ratio of CI to Different Proteases --- p.112 / Chapter 4.3.7 --- Effects of the Proteases on Their Corresponding Inhibitors --- p.112 / Chapter 4.4 --- Discussion --- p.119 / Chapter Chapter 5 --- Conclusion --- p.125 / References --- p.128

Wong Yue Hang Albert. / Thesis submitted in: December 2005. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 108-119). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.v / List of Figures --- p.x / List of Tables --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.4 / Chapter 2.1 --- Nasopharyngeal Carcinoma (NPC) --- p.4 / Chapter 2.1.1 --- Anatomy of Nasopharynx --- p.4 / Chapter 2.1.2 --- Histopathology of Nasopharyngeal Carcinoma --- p.5 / Chapter 2.1.3 --- Epidemiology and Etiology of Nasopharyngeal Carcinoma --- p.5 / Chapter 2.1.3.1 --- Endemic Regions of Nasopharyngeal Carcinoma --- p.5 / Chapter 2.1.3.2 --- Gender and Age Bias --- p.6 / Chapter 2.1.3.3 --- Nasopharyngeal Carcinoma in Hong Kong --- p.6 / Chapter 2.1.3.4 --- Environmental Factors and Diet --- p.7 / Chapter 2.1.3.5 --- HLA Haplotypes and Nasopharyngeal Carcinoma --- p.9 / Chapter 2.1.4 --- Epstein-Barr Virus (EBV) and Nasopharyngeal Carcinoma --- p.10 / Chapter 2.1.4.1 --- EBV and Human Cacners --- p.10 / Chapter 2.1.4.2 --- EBV Infection --- p.10 / Chapter 2.1.4.3 --- "Latent, Clonal EBV Infection" --- p.11 / Chapter 2.1.4.4 --- EBV Latency Form --- p.11 / Chapter 2.1.4.5 --- Reactivation of EBV --- p.12 / Chapter 2.1.5 --- Molecular Pathogenesis of Nasopharyngeal Carcinoma --- p.13 / Chapter 2.1.5.1 --- Genetic Changes --- p.13 / Chapter 2.1.5.2 --- Epigenetic Changes --- p.13 / Chapter 2.1.6 --- Therapy of Nasopharyngeal Carcinoma and its Deficiency --- p.14 / Chapter 2.1.6.1 --- Radiotherapy --- p.14 / Chapter 2.1.6.2 --- Concurrent Chemoradiotherapy --- p.16 / Chapter 2.1.6.3 --- Adjuvant and Neo-adjuvant Chemotherapy --- p.17 / Chapter 2.1.6.4 --- Chemotherapy in Metastatic Nasopharyngeal Carcinoma --- p.18 / Chapter 2.1.6.5 --- Novel Therapeutic Agents and Approach --- p.19 / Chapter 2.2 --- Histone Modification and Cancer --- p.20 / Chapter 2.2.1 --- Histone Modification and Transcription Regulation --- p.20 / Chapter 2.2.2 --- Carcinogenic Effect of Aberrant HAT and HDAC Activities --- p.21 / Chapter 2.2.3 --- Structural Classes of HDAC Inhibitors --- p.24 / Chapter 2.2.4 --- Anti-Cancer Mechanisms of HDAC Inhibitors --- p.25 / Chapter 2.3 --- Suberoylanilide Hydroxamic Acid (SAHA) --- p.27 / Chapter 2.3.1 --- Anti-tumor Effect of SAHA in Various Cancer Cell Lines --- p.27 / Chapter 2.3.2 --- SAHA Mediated Non-apoptotic Programmed Cell Death --- p.29 / Chapter 2.3.3 --- Anti-tumor and Preventive Effect of SAHA in Animal Model --- p.29 / Chapter 2.3.4 --- Clinical Trials of SAHA --- p.30 / Chapter 2.4 --- FK228 (Depsipeptide or FR901228) --- p.31 / Chapter 2.4.1 --- Anti-malignancy mechanism of FK228 --- p.31 / Chapter 2.4.2 --- Anti-angiogenesis --- p.32 / Chapter 2.4.3 --- Drug Resistance and FK228 --- p.33 / Chapter 2.4.4 --- Studies of FK228 on Animal Models --- p.33 / Chapter 2.4.5 --- Clinical Trials --- p.34 / Chapter 2.5 --- Histone Modification and Nasopharyngeal Carcinoma --- p.34 / Chapter Chapter 3 --- Materials and Methods --- p.36 / Chapter 3.1 --- Cell Lines --- p.36 / Chapter 3.2 --- EBER ish Hybridization (EBER ISH) --- p.37 / Chapter 3.3 --- HDAC Inhibitors --- p.38 / Chapter 3.4 --- Cellular Sensitivity of NPC Cell Lines to HDAC Inhibitors --- p.38 / Chapter 3.4.1 --- Drug Treatment --- p.38 / Chapter 3.4.2 --- Determining Relative Amount of Survival Cells (WST-1 Assay) --- p.39 / Chapter 3.5 --- Flow Cytometry Analysis --- p.40 / Chapter 3.5.1 --- Collecting Cells and Fixation --- p.40 / Chapter 3.5.2 --- Staining --- p.41 / Chapter 3.5.3 --- Flow Cytometry Analysis --- p.41 / Chapter 3.6 --- Protein Extraction --- p.41 / Chapter 3.6.1 --- Harvesting Samples --- p.41 / Chapter 3.6.2 --- Protein Extraction --- p.42 / Chapter 3.6.3 --- Protein Quantification --- p.42 / Chapter 3.7 --- Western Blotting --- p.43 / Chapter 3.7.1 --- SDS-Polyarcylamide Gel Electrophoresis (PAGE) (SDS-PAGE) --- p.43 / Chapter 3.7.2 --- Wet Transfer of Proteins --- p.43 / Chapter 3.7.3 --- Immunoblotting --- p.44 / Chapter 3.7.4 --- Signal Detection --- p.44 / Chapter 3.8 --- CodeLin´kёØ Oligonucleotide Microarray --- p.45 / Chapter 3.8.1 --- HDAC Inhibitor Treatment --- p.45 / Chapter 3.8.2 --- RNA Extraction --- p.45 / Chapter 3.8.3 --- Quality and Quantity Assessment of Total RNA Extracted --- p.46 / Chapter 3.8.4 --- CodeLinkIM Expression Bioarray System --- p.46 / Chapter 3.8.5 --- Data Analysis --- p.48 / Chapter 3.9 --- Real-time Reverse Transcription PCR (Real-time RT-PCR) --- p.48 / Chapter Chapter 4 --- Results --- p.50 / Chapter 4.1 --- Presence of EBV --- p.50 / Chapter 4.2 --- Anti-prolirative Effect of HDAC Inhibitors --- p.52 / Chapter 4.3 --- Histone Acetylation --- p.56 / Chapter 4.4 --- Induction of p21 Expression in NPC Cell Lines --- p.58 / Chapter 4.5 --- HDAC Inhibitors Induced Cell Cycle Arrest and Polyploidy Formation --- p.60 / Chapter 4.5.1 --- Trichostatin A Induced G2/M Arrest --- p.60 / Chapter 4.5.2 --- Suberoylanilide Hydroxamic Acid Induced G1 Arrest --- p.62 / Chapter 4.5.3 --- FK228 Mediated G2/M Arrest --- p.64 / Chapter 4.6 --- HDAC Inhibitors Altered the Expression of Cell Cycle Regulatory Proteins --- p.66 / Chapter 4.6.1 --- TSA Down-regulated Cyclin A and B --- p.66 / Chapter 4.6.2 --- Suppressed Expression of Cyclin D1 and B by SAHA --- p.69 / Chapter 4.6.3 --- Effect of FK228 on Expression of Different Cyclins in NPC Cell Lines --- p.71 / Chapter 4.7 --- Effect of HDAC Inhibitors on EBV Proteins --- p.73 / Chapter 4.8 --- HDAC Inhibitors Modulated Gene Expression Profile --- p.76 / Chapter 4.8.1 --- SAHA and FK228-Induced Gene Expression Profile --- p.76 / Chapter 4.8.2 --- Validation of Expression Profile of Selected Genes by Real-time RT-PCR --- p.83 / Chapter Chapter 5 --- Discussion --- p.87 / Chapter 5.1 --- Anti-proliferative Effect of SAHA and FK228 on NPC Cell Lines --- p.88 / Chapter 5.2 --- Resistance of SAHA or FK228 in NPC --- p.93 / Chapter 5.3 --- Growth Inhibitory Mechanism of SAHA and FK228 in NPC Cells --- p.94 / Chapter 5.4 --- Induction of Polyploidy Cells in NPC Cell Lines --- p.98 / Chapter 5.5 --- Does EBV play a Role in HDAC Inhibiotrs Induced Growth Arrest in NPC Cell Lines? --- p.99 / Chapter 5.6 --- Transcriptional Signature of SAHA and FK228 in NPC Cell Lines --- p.100 / Chapter 5.7 --- Combining SAHA or FK228 with other Anti-tumor Agents --- p.104 / Chapter 5.8 --- Future Prospectus --- p.105 / Chapter Chapter 6 --- Summary --- p.106 / References --- p.108 / Appendix 1 --- p.120 / Appendix 2 --- p.121

多發性骨髓瘤(Multiple myeloma) 為骨髓內漿細胞異常增生的惡性腫瘤，到目前為止仍然難以治癒。其發生發展是一個複雜的多步驟事件，涉及腫瘤細胞中遺傳和表觀遺傳的改變，以及骨髓微環境的支持。現已確定骨髓瘤細胞和骨髓微環境之間的相互作用對於骨髓瘤的病理發生，以及骨髓瘤細胞的生長，遷移和抗藥性起著關鍵作用。血小根因子四(Platelet factor 4, or PF4) 是一種抗血管生成的趨化因子。它不僅在體外抑制血管內皮細胞增殖和遷移，而且在體內抑制腫瘤的生長。此前，我們發現PF4 基因在多發性骨髓瘤中等位缺失以及DNA 高度甲基化，因而導致其在骨髓瘤病人及細胞系中的表達缺失或降低。在本研究中，我們利用體內和體外實驗鑒定了PF4 對骨髓瘤細胞以及血管生成的作用，並闡明了其作用機制。 / 首先，我們在體外鑒定了PF4 在骨髓瘤細胞中的功能。我們發現PF4 抑制骨髓瘤細胞系以及從病人骨髓中分離出來的骨髓瘤細胞的生長，以及促進其凋亡。其促凋亡活性與caspase-3 和PARP 的激活有關。我們也檢測了PF4 在骨髓瘤中對血管生成的作用。我們首先分離了病人骨髓中的內皮細胞。結果顯示PF4抑制骨髓瘤內皮細胞的生長和管狀物的形成。這些結果證明PF4 在骨髓瘤中可能是一個抑癌因子。 / 接下來我們進一步檢測了PF4 在體內的抑癌功能。在第一種模型中，骨髓瘤細胞被皮下移植到重症聯合兔疫缺陷型(NOD-SCID) 小鼠中。尾靜脈注射200ngPF4 明顯的抑制了腫瘤的生長，並延長了小鼠的成活率。第二種小鼠模型稱為兔鼠融合模型(SCID-rab model) 。在這一模型中，大白兔的腿骨先被皮下移植到(NOD-SCID) 小鼠中，再將骨髓瘤細胞注射入已植入的大白兔腿骨的骨腔中。兩周後，小鼠被尾靜脈注射入20 或200ng PF4 。結果顯示200ng PF4 顯著抑制了腫瘤的生長。通過兔疫組化分析大白兔腿骨切片，我們進一步證明了PF4 在腫瘤細胞中的增瘟，凋亡以及血管生成的作用。我們的發現因此證實了PF4 是骨髓瘤中的一個抑制因子。 / 為了鑒定PF4 在骨髓瘤中的作用機制，我們用Protein/DNA 微陣列(Protein/DNA array) 分析了PF4 參與的信號通路。結果顯示PF4 調節了若干個轉錄因子，其中包括STAT3 。凝膠遷移(EMSA) 和螢光素酪報告基因(luciferase reporter assay )檢測進一步證實PF4 抑制了STAT3 的DNA 結合能力以及轉錄活性。因此PF4 可能通過抑制STAT3 信號通路而抑制骨髓瘤的生長。我們進一步發現PF4 能抑制組成性的以及自介素6 (IL-6) 誘導的STAT3的激活。我們發現PF4 下調了STAT3 下游的靶基因，包括Mc1-1， Survivin 以及血管內皮細胞生長因子(VEGF)。而過表達組成性激活的STAT3 能逆轉PF4 所誘導的細胞凋亡。在兔鼠敵合模型中，通過兔疫組化分析大白兔腿骨切片，我們發現PF4 能抑制STAT3 的入核。SOCS3 是STAT3 其中的一個抑制因子，我們發現PF4 能誘導SOCS3 的表達。而干擾掉SOCS3 能使PF4 喪失其抑制STAT3 激活的能力。這些結果表明PF4 可能通過誘導SOCS3 的表達，從而抑制STAT3 信號通路，引起骨髓瘤的生長抑制以及抗血管生成。 / 總而言之，本研究表明PF4 是骨髓髓中一個重要調節因子。在體外和體內，PF4 通過抑制STAT3 信號通路，從而抑制腫瘤細胞的生長，促進凋亡以及抑制血管生成。本文為PF4 的臨床研究，作為一種新的治療骨髓瘤藥物，提高骨髓瘤病人的治療效果提供基礎。 / Multiple myeloma (MM) is an incurable hematological malignancy characterized by accumulation of clonal plasma cells in bone marrow (BM). The development and progression of MM is a complex multistep tumorigenic event involving both genetic and epigenetic changes in the tumor cell as well as the support by the BM microenvironment. It has been well established that the physical interaction of MM cells with the BM milieu are crucial for MM pathogenesis, MM cell growth, survival, migration and drug resistance. Platelet factor 4 (PF4), a potent antiangiogenic chemokine, not only inhibits endothelial cell proliferation and migration in vitro but also solid tumor growth in vivo. Our group previously demonstrated loss of PF4 expression in patient MM samples and MM cell lines due to concurrent allelic loss and DNA hypermethylation. In this study, we characterized the effects of PF4 on MM cells and angiogenesis in the BM milieu both in vitro and in vivo and elucidated the mechanism of PF4 effects on MM. / To characterize the effects of PF4 on MM cells in vitro, assays on cell growth, cell cycle arrest and apoptosis were performed and we found that PF4 inhibited growth and induced apoptosis in both MM cell lines and MM cells from patients. The proapoptotic activity of PF4 is associated with activation of caspase-3 and poly (ADP) ribose polymerase (PARP). We also investigated the effects of PF4 on angiogenesis in MM using endothelial cells isolated from patient's BM aspirates (MMECs). Our results showed that PF4 suppressed MMECs growth and tube formation on matrigel in a dose-dependent manner. / Given the ability of PF4 to suppress MM cell growth and angiogenesis in vitro, we evaluated its tumor suppressive function in vivo. In human subcutaneously matrigel xenograft mouse model, tail vein injection of 200ng PF4 significantly reduced MM tumor growth and prolonged survival. We next used the SCID-rab mouse model which recapitulates the human BM milieu in vivo. In this model, MM cells were directly injected into the rabbit bone which was subcutaneously implanted into the NOD-SCID mice. Two weeks after injection, SCID mice were treated with various dose of PF4 (20 or 200ng per injection, three times per week) or PBS by tail vein injection. ELISA assay for hIg (lambda) showed that tumor growth in 200ng PF4-treated mice was markedly reduced by 58% compared with the control group, which was further confirmed by immunohistochemistry analysis of CD 138 staining on rabbit bone section. Consistent with the in vitro results, induction of apoptosis in MM cells and inhibition of angiogenesis by PF4 could also be demonstrated in vivo, as evidenced by the findings on ki67, Cleaved caspase-3, CD31 and VEGF staining on rabbit bone sections from treated versus control mice. Our findings thus confirmed that PF4 is a novel tumor suppressor in MM. / However, the molecular mechanism of how PF4 inhibits MM tumorigenesis is still unclear. To identify the signal pathway PF4 involved in MM, Protein/DNA array was performed. We found that PF4 regulated several transcription factors including STAT3 in U266 cells. EMSA and luciferase reporter assay further confirmed that PF4 suppressed STAT3 DNA binding and transcriptional activity. So it is possible that PF4 mediates its tumor suppressive function, through suppressing STAT3 pathway in MM cells. We further found that pre-treatment of PF4 blocked both constitutive and interleukin-6-induced STAT3 activation in a time-dependent manner in human MM cells. PF4 could also down-regulate the STAT3-regulated gene products including Mcl-I, Survivin and vascular endothelial growth factor (VEGF). Moreover, enforced expression of constitutively active STAT3 rescued cells from PF4-induced apoptosis. In SCID-rab mouse model, we also found that PF4 inhibited STAT3 nuclear translocation by immunostaining of rabbit bone sections. When examined further, we found that PF4 induced the expression of one of the STAT3 inhibitor SOCS3, and gene silencing of SOCS3 by small interfering RNA abolished the ability of PF4 to inhibit STAT3 activation, suggesting a critical role of SOCS3 in the action of PF4. Our findings therefore suggest that by inducing SOCS3 expression, PF4 abrogates STAT3 activity, thus induces tumor growth inhibition and anti-angiogenesis. / Together, these novel studies have shown that PF4 is an important regulator of MM tumorigenesis. By abrogating STAT3 signaling it targets cell growth, induces apoptosis, suppresses angiogenesis both in vitro and in vivo in MM. These scientific observations provide the framework for clinical studies of this chemokine, as a novel drug for treatment of MM to improve patient outcome in MM. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liang, Pei. / "November 2011." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 139-161). / 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 in English --- p.I / Abstract in Chinese --- p.IV / List of Publications --- p.VI / Acknowledgements --- p.VII / List of abbreviations --- p.IX / List of Tables --- p.XII / List of Figures --- p.xm / Table of Contents --- p.XV / Chapter Chapter1 --- Introduction and Literature Review --- p.1 / Chapter 1.1 --- Multiple myeloma-General description --- p.1 / Chapter 1.1.1 --- Epidemiology of MM --- p.1 / Chapter 1.1.2 --- Stages of MM --- p.1 / Chapter 1.2 --- The bone marrow (BM) microenvironment in MM --- p.3 / Chapter 1.3 --- Signal pathways in MM cells --- p.5 / Chapter 1.3.1 --- JAK/STAT3 in cancers and MM --- p.5 / Chapter 1.3.1.1 --- IL-6 and its receptor --- p.7 / Chapter 1.3.1.2 --- Activation of downstream signals-The "on" signals --- p.9 / Chapter 1.3.1.3 --- Inactivation of downstream signaling --- p.11 / Chapter 1.3.1.3.1 --- Phosphatases --- p.12 / Chapter 1.3.1.3.2 --- SOCS family --- p.13 / Chapter 1.3.1.3.3 --- The PIAS family --- p.14 / Chapter 1.3.2. --- NF-κB pathway --- p.15 / Chapter 1.3.3 --- RAS-MAPK pathway --- p.17 / Chapter 1.3.4 --- Phosphatidyl inositol-3 kinase (PI3K)/AKT --- p.18 / Chapter 1.4 --- Angiogenesis in MM --- p.18 / Chapter 1.4.1 --- The process of angiogenesis --- p.18 / Chapter 1.4.2 --- Angiogenesis in caner --- p.20 / Chapter 1.4.3 --- Angiogenesis in MM --- p.22 / Chapter 1.5 --- Animal models in MM --- p.24 / Chapter 1.6 --- Treatment of MM --- p.27 / Chapter 1.6.1 --- Chemotherapy --- p.27 / Chapter 1.6.2 --- Autologous stem cell transplantation --- p.28 / Chapter 1.6.3 --- Biologically based therapies --- p.28 / Chapter 1.7 --- Platelet factor 4 (PF4) --- p.30 / Chapter 1.8 --- Structure of PF 4 --- p.30 / Chapter 1.9 --- Role of PF4 in physiological process --- p.32 / Chapter 1.9.1 --- Inhibition of megakaryocytopoiesis --- p.32 / Chapter 1.9.2 --- PF4 and coagulation --- p.33 / Chapter 1.10 --- Role of PF4 in pathological process --- p.34 / Chapter 1.10.1 --- PF4 and cancer --- p.34 / Chapter 1.10.2 --- PF4 is an angiogenic inhibitor --- p.35 / Chapter 1.11 --- Clinical applications of PF4 --- p.37 / Chapter 1.12 --- Summary and project aims --- p.37 / Chapter Chapter 2 --- Materials and Methods --- p.40 / Chapter 2.1 --- Reagents and antibodies --- p.40 / Chapter 2.2 --- MM Cell lines --- p.40 / Chapter 2.3 --- CD138⁺ primary MM cells --- p.41 / Chapter 2.4 --- CD31⁺ MM endothelial cells (MMECs) --- p.42 / Chapter 2.5 --- WST-1 assay --- p.43 / Chapter 2.6 --- Trypan blue exclusion --- p.43 / Chapter 2.7 --- Cell cycle analysis --- p.44 / Chapter 2.8 --- Apoptosis analysis --- p.44 / Chapter 2.9 --- In vitro tube formation assay --- p.45 / Chapter 2.10 --- SCID-rab mice model --- p.45 / Chapter 2.10.1 --- Construction of SCID-rab mice --- p.45 / Chapter 2.10.2 --- Establishment and monitoring of myeloma in SCID-rab mice --- p.46 / Chapter 2.10.3 --- Enzyme-linked immunosorbent assay (ELISA) --- p.46 / Chapter 2.10.4 --- PF4 treatment --- p.47 / Chapter 2.10.5 --- Immunohistochemistry --- p.48 / Chapter 2.11 --- Protein/DNA arrays --- p.49 / Chapter 2.12 --- Electrophoretic mobility shift assay (EMSA) --- p.50 / Chapter 2.13 --- Luciferase reporter assay --- p.52 / Chapter 2.14 --- Western blotting --- p.53 / Chapter 2.15 --- RNA extraction --- p.54 / Chapter 2.16 --- Real-time Polymerase Chain Reaction (Real-time PCR) --- p.54 / Chapter 2.17 --- Nuclear transfection --- p.55 / Chapter 2.18 --- Statistical analysis --- p.55 / Chapter Chapter3 --- The role of PF4 in MM: in vitro studies --- p.58 / Chapter 3.1 --- Results --- p.58 / Chapter 3.1.1 --- PF4 inhibited growth of human MM cell lines --- p.58 / Chapter 3.1.2 --- PF4 did not cause cell cycle arrest --- p.59 / Chapter 3.1.3 --- PF4 induced apoptosis of myeloma cell lines --- p.63 / Chapter 3.1.4 --- PF4 caused cell apoptosis in primary MM cells cultured in vitro --- p.64 / Chapter 3.1.5 --- PF4 suppressed MMECs growth --- p.69 / Chapter 3.1.6 --- PF4 suppressed MMECs tube formation --- p.69 / Chapter 3.2 --- Discussion --- p.73 / Chapter 3.2.1 --- Negative regulation of PF4 in MM cells growth in vitro --- p.73 / Chapter 3.2.2 --- PF4 induces apoptosis in MM cell lines and primary MM cells --- p.74 / Chapter 3.2.3 --- PF4 inhibits angiogenesis in MM in vitro --- p.76 / Chapter 3.3 --- Summary --- p.79 / Chapter Chapter4 --- The role ofPF4 in MM tumorigenesis: in vivo studies --- p.82 / Chapter 4.1 --- Results --- p.82 / Chapter 4.1.1 --- PF4 inhibited MM tumor growth and prolonged survival in subcutaneous matrigel xenograft model --- p.82 / Chapter 4.1.2 --- PF4 inhibited MM tumor growth and prolonged survival in SCID-rab mouse model --- p.85 / Chapter 4.1.3 --- PF4 reduced human MM cell proliferation, angiogenesis and induced apoptosis in SCID-rab mice --- p.88 / Chapter 4.2 --- Discussion --- p.91 / Chapter 4.2.1 --- PF4 inhibited human tumor growth in subcutaneous matrigel xenograft mouse model --- p.91 / Chapter 4.2.2 --- SCID-rab mouse model was successfully established and PF4 inhibited human MM turnor growth in this model --- p.92 / Chapter 4.2.3 --- PF4 inhibited human MM cell proliferation, angiogenesis and induced apoptosis in SCID-rab mice --- p.95 / Chapter 4.3 --- Summary --- p.96 / Chapter Chapter 5 --- The molecular mechanisms of PF4 in MM tumorigenesis --- p.98 / Chapter 5.1 --- Results --- p.98 / Chapter 5.1.1 --- ProteinlDNA array hybridization and Quantification of protein/DNA array spots --- p.98 / Chapter 5.1.2 --- PF4 suppressed DNA binding and transcriptional activity of STAT3 --- p.102 / Chapter 5.1.3 --- PF4 inhibited constitutive STAT3 phosphorylation in MM cells --- p.104 / Chapter 5.1.4 --- PF4 inhibited IL-6-induced STAT3 activation --- p.105 / Chapter 5.1.5 --- PF4 suppressed STAT3 regulated gene expression --- p.107 / Chapter 5.1.6 --- Enforced expression of constitutively active STAT3 rescued cells from PF4-induced apoptosis --- p.109 / Chapter 5.1.7 --- PF4 induced the expression of SOCS3 --- p.111 / Chapter 5.1.8 --- PF4-induced inhibition of STAT3 activation was reversed by gene silencing of SOCS3 --- p.111 / Chapter 5.1.9 --- PF4 inhibited nuclear accumulation of STAT3 and induced expression of SOCS3 in vivo --- p.114 / Chapter 5.2 --- Discussion --- p.115 / Chapter 5.2.1 --- PF4 regulated several TFs --- p.115 / Chapter 5.2.2 --- PF4 inhibited constitutive activation of STAT3 --- p.118 / Chapter 5.2.3 --- PF4 inhibited IL-6 induced activation of STAT3 --- p.120 / Chapter 5.2.4 --- PF4 suppressed STAT3 regulated gene expression --- p.121 / Chapter 5.2.5 --- PF4 induced the expression of SOCS3 --- p.124 / Chapter 5.3 --- Summary --- p.125 / Chapter Chapter 6 --- Conclusion and future studies --- p.128 / Chapter 6.1 --- Conclusion --- p.128 / Chapter 6.2 --- Future studies --- p.135 / Appendices --- p.137 / References list --- p.139

by Ho Wai Chan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (p. [254]-271). / 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. / Abstract in Chinese.

La voie de signalisation Hedgehog (Hh) joue un rôle fondamental au cours du développement embryonnaire et participe au maintien des niches neurogéniques dans le cerveau adulte (Ruat et al, 2015). Son activation requiert la liaison d’un peptide Hh sur le récepteur Patched (Ptc) qui réprime l’activité constitutive de Smoothened (Smo), un membre de la classe F des récepteurs couplés aux protéines G (Wang et al, 2013). La dérégulation de la voie Hh peut conduire au développement de tumeurs, comme les médulloblastomes ou les carcinomes basocellulaires. Des agonistes et des antagonistes de Smo ont été développés et présentent un intérêt thérapeutique dans le traitement des maladies neurodégénératives et des tumeurs Hh-dépendantes, respectivement (Ruat and Hoch, 2015). Les études cristallographiques du Smo humain (hSmo) complexé à différents ligands ont identifié deux types d’antagonistes ; ceux se liant principalement aux boucles extracellulaires de Smo (site 1, comme le LY2940680) et ceux se liant plus profondément dans la cavité transmembranaire (site 2, comme le SANT 1) (Ruat et al, 2014).Mes travaux de thèse ont conduit à la caractérisation du composé acylguanidine MRT-92, l’un des plus puissants antagonistes du récepteur Smo. Le MRT-92 inhibe différentes réponses biologiques induites par l’activation de la voie Hh, notamment la prolifération des précurseurs des cellules granulaires du cervelet de rat avec une affinité sub-nanomolaire. Le MRT 92 bloque aussi la translocation de Smo dans le cil primaire induite par l’activation de la voie Hh. Le développement de sa forme tritiée [3H]MRT-92 (Kd = 0.3 nM pour hSmo) a permis d’étudier les interactions des modulateurs avec le récepteur hSmo et d’analyser les résistances associées aux mutations de hSmo. Le composé MRT 92 se lie au récepteur hSmoD473H, résistant au traitement par le GDC-0449, suggérant son intérêt thérapeutique pour le traitement de cette résistance. Par une modélisation moléculaire et une étude de mutagenèse, j’ai identifié que le MRT-92 se lie sur un nouveau site au niveau du domaine transmembranaire de Smo qui se superpose aux sites 1 et 2 préalablement décrits. Le développement d’un modèle pharmacophorique des agonistes de Smo a permis le criblage virtuel d’une banque de molécules et l’identification du composé quinolone GSA-10. Le GSA-10 stimule une voie Hh non canonique qui permet la différenciation des cellules mésenchymateuses C3H10T1/2 en ostéoblastes en se fixant au récepteur Smo. Contrairement au composé SAG, agoniste de référence de Smo, le GSA-10 n’induit pas de prolifération cellulaire des cellules granulaires du cervelet de rat et n’augmente pas la transcription des gènes cibles de la voie tels que Gli1 et Ptc. Le GSA-10 est la première molécule agoniste de Smo qui n’induit pas sa translocation au cil primaire. De plus, nous avons observé que la forskoline, un inhibiteur de l’adénylate cyclase, est un régulateur positif et négatif de la différenciation ostéoblastique induite par le GSA-10 et le SAG, respectivement. Le GSA-10 nous a également permis de mettre en évidence deux formes conformationnelles de Smo, SmoSAG et SmoGSA-10, pouvant être discriminées pharmacologiquement par les antagonistes de Smo. Plusieurs antagonistes comme le GDC-0449, le CUR61414, la cyclopamine ou le MRT-92 perdent leur sensibilité pour inhiber le SmoGSA-10. L’ensemble de ce travail a conduit au développement de nouveaux outils pharmacologiques qui pourraient permettre d’améliorer notre compréhension des mécanismes impliqués dans la modulation de l’activité du récepteur Smo et permettre le développement de nouvelles molécules en clinique pour le traitement des tumeurs du cerveau Hh-dépendantes. / The Hedgehog (Hh) signaling pathway plays a critical role during embryogenesis and participates to the maintenance of neural stem cells in the adult brain (Ruat et al, 2015). Its activation requires the binding of a Hh peptide to its receptor Patched (Ptc) which represses the constitutive activity of Smoothened (Smo), a member of class F G-protein-coupled receptors (Wang et al, 2013). Deregulation of the Hh pathway is associated with the development of tumors, such as medulloblastoma and basal cell carcinoma. Agonists and antagonists of Smo are candidates for the treatment of degenerative diseases and Hh-linked tumors, respectively (Ruat and Hoch, 2015). Crystallization studies of human Smo (hSmo) bound to different ligands have identified two types of 7 transmembrane-directed antagonists : those binding mostly to extracellular loops (site 1, e.g., LY2940680) and those penetrating deeply in the 7-transmembrane cavity (site 2, e.g., SANT-1) (Ruat et al, 2014).The present work allowed the caracterization of the acylguanidine MRT-92, one of the most potent Smo antagonist. MRT-92 inhibits Smo induced-responses in different cell-based assays, notably the proliferation of rat cerebellar granule cell with nanomolar potency. We developed its tritiated derivative [3H]MRT-92 (Kd= 0.3 nM for hSmo) for creating a comprehensive framework for the interaction of small molecule modulators with hSmo and for understanding chemoresistance linked to hSmo mutations. MRT-92 binds to the mutated hSmoD473H receptor resistant to GDC-0449 treatment, suggesting its therapeutic interest for the treatment of this resistance. Guided by molecular docking and site-directed mutagenesis data, we demonstrated the existence of a third type of Smo antagonists represented by MRT 92 that simultaneously recognized and occupied both sites 1 and 2.The development of a pharmacophoric model of Smo agonists allowed a virtual screening strategy to identify the GSA-10 compound, a quinolinecarboxamide. GSA-10 stimulates a non-canonical Hh pathway allowing C3H10T1/2 mesenchymal cells differentiation into osteoblasts. However, GSA-10 does not induce Gli-dependent reporter gene transcription nor rat cerebellar granule cell proliferation, and it does not regulate the subcellular localization of Smo at the primary cilium. Moreover, we observed that forskolin, a known activator of adenylate cyclase, is a positive and negative regulator of GSA-10 and SAG-mediated cell differentiation, respectively. Our data provide also evidences for two different conformational forms of Smo named SmoSAG and SmoGSA-10, which can be pharmacologically discriminate by Smo antagonists. Different antagonists including GDC 0449, CUR61414, Cyclopamine and MRT-92 loose their sensibility to inhibit SmoGSA-10.The present work allowed the identification of new pharmacological tools which should be useful for understanding the mechanisms underlying the resistance of Smo inhibitors in cancer cells and may help to design new therapies with improved pharmacological properties for treating Hh-linked brain tumors.

Made available in DSpace on 2014-06-11T19:22:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-05Bitstream added on 2014-06-13T19:07:30Z : No. of bitstreams: 1 chagas_ha_me_botfca.pdf: 1461839 bytes, checksum: e3cb655599f9ae29650f6b80ac395c35 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A mamoneira (Ricinus communis L.) é uma espécie oleaginosa tropical, sendo o óleo extraído de suas sementes um dos mais versáteis da natureza e com inúmeras aplicações industriais. Embora ainda seja uma espécie rústica, ela está sujeita a diversas doenças, dentre elas o mofo-cinzento, causada pelo fungo Amphobotrys ricini. O melhoramento genético seria a melhor alternativa para o controle da doença, mas demanda tempo para se obter cultivares resistentes. Dessa Maneira, o uso de métodos de controle baseado em métodos químicos, alternativos ou biológicos mostra-se viável em curto prazo. O objetivo do trabalho foi estudar a eficiência de controle do mofocinzento, na cultura da mamoneira, utilizando-se de métodos químico, alternativo e biológico. Para tanto, foram verificados, in vitro, a eficiência de diferentes meios de cultura na esporulação do patógeno e do antagonista C. rosea. Verificou-se, também, a produção de escleródios do patógeno nos meios. Quanto à eficiência dos métodos, verificou-se, in vitro, a eficiência deles na inibição do crescimento micelial e da germinação dos conídios do patógeno. Após desenvolver e validar uma escala diagramática de avaliação de severidade da doença em racemos de mamoneira, verificou-se a eficiência de aplicação do antagonista C. rosea em frutos destacados da mamona inoculados com o patógeno. Em plantas submetidas a condições de estufa e em campo, verificou-se a eficiência dos métodos no controle da doença causada por A. ricini Quanto à esporulação, o melhor meio de cultura para o patógeno foi V8-20%, obtendo 5,7 x 106 conídios/mL. Para o antagonista C. rosea, o melhor meio foi o TJ- 5% (Suco de Tomate), produzindo 4,41 x 106 conídios/mL. O único meio que propiciou abundante produção de escleródios de A. ricini foi o aveia-ágar. Quanto a inibição do crescimento micelial do patógeno... / The castor bean (Ricinus communis L.) is a tropical oily species, and the oil extracted of its seeds is one of most versatile of the nature, with many industrial applications. Even being a rustic species, it still subjects to several diseases, between them the gray mold, caused by the fungus Amphobotrys ricini. The genetic breeding would be the best alternative for the disease control, but spend time to get a resistant cultivar. In this way, the use of methods of control based on chemical, alternative or biological methods shows viable in short time. The aim of this work was to study the efficiency of the control of gray mold, on castor bean crop, using chemical, alternative and biological methods. Therefore, they had been verified, in vitro, the efficiency of different culture medium in the pathogen sporulation and the antagonist C. rosea. The sclerotia production in the culture medium can be also verified. About the efficiency of the methods, in vitro, the inhibition of the mycelial growth and germination of conidia was verified. After to develop and to validate a diagrammatic scale of evaluation of severity of the disease in racemes of castor bean, the efficiency of application of the antagonist C. rosea in inoculated fruits detached of castor bean with the pathogen was verified. In plants submitted to greenhouse and field conditions, the efficiency of the methods in the control of the disease caused by A. ricini was verified About the sporulation, the optimum culture medium for the pathogen was V8-20%, getting 5,7 x 106 conidia/mL. For the antagonist... (Complete abstract click electronic access below)

Cheung Wai-chit. / "July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004 / Includes bibliographical references (p. 135-140) / 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.

Five traditional Chinese medicines were screened for their antiangiogenic activities through zebrafish angiogenic assay. Two of them, Tripterygium wilfordii and Rheum palmatum showed potential in the primary screening. T. wilfordii was selected in further study. / In the further investigation of antiangiogenic activity of triptolide on mammal systems, triptolide showed potent activity in human umbilical vein endothelial cells (HUVECs) assays including proliferation, migration and tube formation assay. It inhibited HUVEC proliferation with IC50 as low as 34 nM. It also showed more potency in HUVEC migration and tube formation assay at as low concentration as nanomolar level than SU5416, a putative VEGFR-2 inhibitor currently in clinic trials. RT-PCR and western blotting analysis showed that the underlying mechanism of triptolide correlated with down-regulation of VEGFR-2 and Tie2 expression and production. Tie2 inhibition appeared to be a later event as compared with VEGFR-2. Tie2 overexpression in HUVEC could attenuate the inhibitory effect of triptolide on HUVEC proliferation. Tie2 knockdown mimicked the inhibition activity of triptolide in tube formation assay. These phenomemon revealed that Tie2 signaling pathway plays a crucial role in triptolide-mediated angiogenesis inhibition. In in vivo Matrigel Plug assay, triptolide showed inhibition effect at as low as 100 nM. / T. wilfordii is an immune-suppressive, anti-inflammatory herb used in China for centuries. Through bioassay-guided purification, three antiangiogenic terpenoids were isolated from the ethyl acetate fraction, namely, celastrol, cangoronine and triptolide. Among them, triptolide manifested the most potent antiangiogenic activities against vessel formation. As low as 0.31microM, triptolide inhibited 20% of vessel formation, and the inhibition reached a plateau of 50% at 1.2 microM. Celatrol reduced vessel formation by more than 30% at 0.62microM, but killed 50% of the embryos at higher concentrations. Cangoronine was much weaker, inhibiting vessel formation by 20% at 2.5microM. These three components all showed stronger antiangiogenic activities than 2-methoxyestradiol, a putative compound currently under clinical trials as an antiangiogenic agent for cancer treatment, as the latter inhibited angiogenesis in zebrafish embryos by 34% at 10microM. The loss of vessel formation in the embryos treated with triptolide was further confirmed using endogenous alkaline phosphatase staining. Semi-quantitative RT-PCR analysis revealed that triptolide dose- and time-dependently reduced the mRNA expression of angiopoietin (angpt2) and tie2 in zebrafish, indicating the involvement of angpt2/tie2 signaling pathway in the antiangiogenic action of triptolide. / This research revealed that zebrafish model is a promising antiangiogenic model for both the screening of antiangiogenic agents from Chinese herbal medicine and the subsequent discovery for the drug targets. Triptolide, an anti-inflammatory component from T. wilfordii, is a potent angiogenic inhibitor through targeting VEGFR-2 and Tie2 pathways in mammal models whereas targeting ang2-tie2 pathway in zebrafish model. The anti-tumor action of triptolide was demonstrated to be partly through inhibition of tumor angiogenesis. Moreover, the potent antiangiogenic action exerted by triptolide at nanomolar dosage level gives proof that it is a promising lead compound for the development of antiangiogenic drug for cancer treatment. (Abstract shortened by UMI.) / He, Mingfang. / Adviser: Paul Pui-Hey Bot. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0247. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 84-106). / 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. / Abstracts in English and Chinese.

*Please refer to dissertation for diagrams. / In this thesis, a review concerning enantiospecific syntheses and structure activity relationship of N-linked carbadisaccharides from 1994 to 2005 is presented. / Valienamine was isolated from microbial degradation products of validoxylamine A with Pseudomonas denitrificans. It showed alpha-glycosidase inhibitory activity and antibiotic activity. Isopropylidene protected allylic chlorides, 2-epi-valienamine and 4-amino-6-deoxy-alpha- D-pseudomannopyranose were synthesized from (-)-quinic acid. The acetonide blocking groups were shown to be the best hydroxyl protecting groups for coupling precursors, compatible with palladium-catalyzed coupling reaction which afforded high yields of the 1,1'- and 1,4'-N-linked carbadisaccharides 83-88 and 89-91, respectively, with a minimum amount of an elimination diene side-product. 2-epi-Valienamines 92 and N-alkyl 2-epi-valienamine 93 and 94 were also prepared. The key palladium-catalyzed coupling reaction was shown to be a regio- and stereospecific reaction. Acid hydrolysis was used to obtain free pseudoaminosugars as glycosidase inhibitors. The inhibitory activities of these pseudoaminosugars were evaluated by the Biochemistry Department of the Chinese University of Hong Kong.* / Kwong Suk Kwan. / "January 2006." / Adviser: Tony K. M. Shing. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6409. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 146-154). / 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.