Wong Chun Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 149-160). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Abbreviations --- p.v / Acknowledgement --- p.x / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- General review of angiogenesis --- p.1 / Chapter 1.1.1 --- Angiogenesis in development and growth --- p.1 / Chapter 1.1.2 --- The process of angiogenesis --- p.2 / Chapter 1.1.3 --- Types of factors controlling angiogenesis --- p.3 / Chapter 1.2 --- Roles of VEGF and its receptors in the regulation of angiogenesis --- p.6 / Chapter 1.2.1 --- VEGF --- p.6 / Chapter 1.2.2 --- VEGF receptors --- p.8 / Chapter 1.2.3 --- Regulation of VEGF expression by hypoxia and nitric oxide… --- p.10 / Chapter 1.2.4 --- Signal transduction mechanisms of VEGFR-1 and VEGFR-2 --- p.12 / Chapter 1.2.5 --- Anti-apoptotic effect ofVEGF on endothelial cells as a result of signal transduction of VEGFR-2 --- p.14 / Chapter 1.3 --- Angiopoietins --- p.15 / Chapter 1.3.1 --- Angiopoietin 1 (Ang-1) --- p.16 / Chapter 1.3.2 --- Angiopoietin 2 (Ang-2) --- p.19 / Chapter 1.3.3 --- Angiopoietins 3 and 4 (Ang-3 and Ang-4) --- p.24 / Chapter 1.4 --- "Interaction among VEGF, angiopoietin and Tie in the maintenance of vasculature" --- p.25 / Chapter 1.5 --- Tyrosine kinase with immunoglobulin and EGF factor homology domains - Tie 1 and Tie 2 --- p.28 / Chapter 1.6 --- Angiopoietin expression in female reproductive tissues (ovary) --- p.33 / Chapter 1.7 --- Testicular angiogenesis --- p.37 / Chapter 1.8 --- Aims of the present study --- p.38 / Chapter Chapter 2 --- Materials and methods / Chapter 2.1 --- Preparation of primary cells from rat testes --- p.40 / Chapter 2.1.1 --- Sertoli cell preparation --- p.40 / Chapter 2.1.2 --- Germ cell preparation --- p.41 / Chapter 2.1.3 --- Interstitial cell and Leydig cell preparation --- p.43 / Chapter 2.2 --- Cell cultures --- p.45 / Chapter 2.2.1 --- Reagents and cell lines --- p.45 / Chapter 2.2.2 --- Cell lines of mouse TM3 Leydig cells and TM4 Sertoli cells --- p.45 / Chapter 2.2.3 --- Mouse MLTC-1 Leydig tumour cells --- p.46 / Chapter 2.2.4 --- Rat R2C Leydig tumour cells --- p.46 / Chapter 2.2.5 --- Rat LC540 Leydig tumour cells --- p.47 / Chapter 2.2.6 --- "Rat C6 glioma cells.............," --- p.47 / Chapter 2.3 --- "Analyses of Angiopoietin 1, Angiopoietin 2, Angiopoietin3, Tie 1 receptor, and Tie 2 receptor mRNA in testicular cell lines and testicular tissues" --- p.48 / Chapter 2.3.1 --- Extraction of total RNA from testicular cell lines and testicular tissues --- p.48 / Chapter 2.3.2 --- Quantitation of total RNA --- p.50 / Chapter 2.3.3 --- First strand cDNA synthesis by reverse transcription (RT) --- p.51 / Chapter 2.3.4 --- Normalization of the amounts of cDNA usedin polymerase chain reaction (PCR) --- p.52 / Chapter 2.3.5 --- Polymerase chain reaction (PCR) --- p.53 / Chapter 2.3.6 --- Purification of PCR products --- p.65 / Chapter 2.3.7 --- Confirmation of PCR product authenticity by automated DNA sequencing --- p.66 / Chapter 2.4 --- Western blot analysis --- p.68 / Chapter 2.4.1 --- Preparation of cell lysates from primary testicular cells and testicular cell lines --- p.68 / Chapter 2.4.2 --- Preparation of mouse testicular tissue and adult rat testicular tissue lysates --- p.68 / Chapter 2.4.3 --- Determination of protein concentration --- p.69 / Chapter 2.4.4 --- Reagents for Western blot analysis --- p.70 / Chapter 2.4.5 --- Preparation of protein samples and markers for Western blot analysis --- p.71 / Chapter 2.4.6 --- Sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.72 / Chapter 2.4.7 --- Transfer of proteins to membrane --- p.74 / Chapter 2.4.8 --- Blocking of the membrane --- p.74 / Chapter 2.4.9 --- Immunoblotting --- p.75 / Chapter 2.5 --- "Immunohistochemical staining for Ang-1, Ang-2，Ang-3, Tie 1 and Tie 2 in rat testes" --- p.78 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Expression of Ang-1 and Ang-1 alternatively spliced transcripts in the testis and other testicular cell types --- p.81 / Chapter 3.1.1 --- Detection of Ang-1 expression in the testis and and testicular cell types by nested PCR --- p.81 / Chapter 3.1.2 --- Detection of Ang-1 expression in testicular cell lines by nested PCR --- p.82 / Chapter 3.1.3 --- Sequence analysis of Ang-1 transcript amplified from adult rat testis --- p.84 / Chapter 3.1.4 --- Detection of alternatively spliced species of Ang-1 mRNA in the testis and other testicular cell lines --- p.87 / Chapter 3.2 --- Expression of Ang-2 and Ang-2 isoforms in the testis and various testicular cell types --- p.94 / Chapter 3.2.1 --- Detection of Ang-2 expression in the testis and testicular cell types by nested PCR --- p.94 / Chapter 3.2.2 --- Detection of Ang-2 expression in testicular cell lines by nested PCR --- p.96 / Chapter 3.2.3 --- Sequence analysis of Ang-2 transcript amplified from adult rat testis --- p.98 / Chapter 3.2.4 --- Detection of the expression of Ang-2 isoforms in adult rat testis --- p.99 / Chapter 3.3 --- Expression of Ang-3 in the testis and testicular cell types --- p.103 / Chapter 3.3.1 --- Detection of Ang-3 expression in the testis and primary testicular cells by RT-PCR --- p.103 / Chapter 3.3.2 --- Detection of Ang-3 expression in testicular cell lines by RT-PCR --- p.105 / Chapter 3.3.3 --- Sequence analysis of Ang-3 transcripts amplified from TM4 mouse Sertoli cells and adult rat testis --- p.105 / Chapter 3.4 --- Expression of Tie 1 and Tie 2 in the testis and testicular blood vessel --- p.110 / Chapter 3.4.1 --- Detection of Tie 1 and Tie 2 expression in the testis and rat testicular blood vessel by RT-PCR --- p.110 / Chapter 3.4.2 --- Sequence analysis of Tie 1 transcripts amplified from adult rat testis and rat testicular blood vessel --- p.113 / Chapter 3.4.3 --- Sequence analysis of Tie 2 transcript amplified from rat testicular blood vessel --- p.113 / Chapter 3.5 --- "Western blot analysis of Ang-1 and Ang-2 expression in testicular tissues, primary testicular cells and cell lines" --- p.116 / Chapter 3.6 --- "Localization of Ang-1，Ang-2, Ang-4, Tie 1 and Tie2 proteins in adult rat testis by immunohistochemistry" --- p.122 / Chapter 3.7 --- "Comparison of angiopoietin expression patterns in testis using RT-PCR, Western immunoblotting and immunohistochemistry" --- p.128 / Chapter 3.8 --- Comparison of Tie 1 and Tie 2 expression patterns in testis using RT-PCR and immunohistochemistry --- p.128 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- "Expression of Ang-1 mRNA and protein in adult rat testis, mouse testis, rat testicular blood vessel, primary testicular cells and testicular cell lines" --- p.131 / Chapter 4.2 --- "Expression of Ang-2 mRNA and protein in adult rat testis, mouse testis, rat testicular blood vessel, primary testicular cells and testicular cell lines" --- p.136 / Chapter 4.3 --- "Expression of Ang-3 mRNA and protein in adult rat testis, mouse testis, rat testicular blood vessel, primary testicular cells and testicular cell lines" --- p.141 / Chapter 4.4 --- Expression of Tie 1 and Tie 2 mRNAs and proteinsin adult rat testis and rat testicular blood vessel --- p.143 / Chapter 4.5 --- Conclusion --- p.145 / Chapter 4.6 --- Future work --- p.146 / Chapter Chapter 5 --- References --- p.149

Single nucleotide polymorphisms present in the second intron of the fibroblast growth factor receptor 2 (FGFR2) gene have been linked with increased risk of breast cancer in several genome wide association studies. The potential effect of those SNPs appeared to be mediated through the differential binding of cis-regulatory elements, such as transcription factors, since all the SNPs in linkage disequilibrium were located in a regulatory DNA region. Preliminary studies have shown that a Runx2 binding site is functional only in the minor, disease associated allele of rs2981578, resulting in increased expression of FGFR2 in cancers from patients homozygous for that allele. Moreover, the increased risk conferred by the minor FGFR2 allele is associated most strongly in oestrogen receptor alpha positive (ERα) breast tumours, suggesting a potential interaction between ERα and FGFR signalling. Here, we have developed a human cell line model system to study the effect of those SNPs on cell behaviour. In an ERα positive breast cancer cell line, rs2981578 was edited using Zinc Finger Nucleases. Unexpectedly, the acquisition of the single risk allele in MCF7 cells failed to affect proliferation or cell cycle progression. Binding of Runx2 to the risk allele was not observed. However FOXA1 binding, an important ERα partner, appeared decreased at the rs2981578 locus in the risk allele cells. Additionally, differences in allele specific expression (ASE) of FGFR2 were not observed in a panel of 72 ERα positive breast cancer samples. Thus, the apparent increased risk of developing ERα positive breast cancer is not caused by rs2981578 alone. Rather, the observed increased risk of developing breast cancer might be the result of a coordinated effect of multiple SNPs forming a risk haplotype in the second intron of FGFR2.

O objetivo deste trabalho foi avaliar, in vitro, os efeitos de diferentes concentrações do fator de crescimento transformador beta 1 (TGF-β1) em células-tronco derivadas da polpa de dentes decíduos esfoliados humanos (SHED), com relação à viabilidade, proliferação, migração e diferenciação celular. As SHED foram mantidas em meio de cultura MEMα + soro fetal bovino (FBS) 10% + penicilina e estreptomicina 1% e tratadas com TGF-β1 na concentração de 1,0; 5,0 e 10,0 ng/mL. Após 1, 3, 5 e 7 dias, foram avaliadas a viabilidade celular pelo método MTT e a proliferação pelo método SRB. Após 24 h de tratamento com TGF-β1, foi realizado um ensaio de migração celular por meio de insertos com poros de 8 μm. Para a avaliação da diferenciação celular de SHED em odontoblastos foram analisados por meio da RT-PCR os marcadores DSPP e DMP-1, após tratamento com TGF-β1 nas diferentes concentrações por 14 dias. Os resultados foram submetidos à ANOVA seguido do teste de Tukey. Em relação à viabilidade celular, as diferentes concentrações de TGF-β1 não tiveram efeito citotóxico sobre SHED. As células tratadas com diferentes concentrações de TGF-β1 apresentaram maiores taxas de proliferação que as do controle negativo (MEMα + 10% de FBS) a partir do 3o dia (p=0,000). Observou-se maiores taxas de migração em direção aos meios contendo TGF-β1, mas sem diferença estatisticamente significativa entre as diferentes concentrações utilizadas, entretanto, houve diferença estatisticamente significativa entre as diferentes concentrações de TGF-β1 com o controle positivo (p=0,000), controle negativo (p=0,000) e entre o controle positivo e negativo (p=0,002). A expressão de DMP-1 foi observada de forma crescente nas doses de 1,0 e 5,0 ng/mL de TGF-β1 ao longo do período (1, 7 e 14 dias) e na dose de 10,0 ng/mL a marcação foi mais intensa desde o primeiro dia do estímulo. Em relação à expressão de DSPP, o grupo tratado com 10,0 ng/mL apresentou marcação após 14 dias de tratamento. Sendo assim, este estudo permite concluir que as diferentes concentrações de TGF-β1 estimularam a proliferação e migração celular, sem efeito citotóxico sobre as células ao longo do período do estudo. Em relação à diferenciação celular a concentração 10,0 ng/mL de TGF-β1 estimulou à expressão de DMP-1 e DSPP. / The aim of this study was to evaluate, in vitro, the effect of transforming growth factor beta 1 (TGF-β1) in stem cells derived from the pulp of human exfoliated deciduous teeth (SHED) regarding to cell viability, proliferation, migration and differentiation. SHED were maintained in MEMα culture medium + 10% fetal bovine serum (FBS) + 1% penicillin and streptomycin, and treated with TGF-β1 at the following concentrations of 1.0; 5.0 and 10.0 ng/mL. After 1, 3, 5 and 7 days, cell viability was assessed by MTT assay and proliferation by the SRB method. After 24h of TGF-β1 treatment, cell migration assay was carried out using inserts of 8 μm pore size. To evaluate SHED differentiation into odontoblasts, DMP-1 and DSPP markers were analyzed by RT-PCR, after treatment at different concentrations of TGF-β1 for 14 days. The results were submitted by ANOVA and Tukey test. With respect to cell viability, the different TGF-β1 concentrations did not have cytotoxic effect on SHED. The cells treated by different TGF-β1 concentrations showed higher proliferation rates than those of the negative control (MEMα + 10% FBS) after the third day (p = 0.000). Higher rates of migration towards the media containing TGF-β1 were observed, but there were no statistically significant differences among the concentrations. All different TGF-β1 concentrations showed statistically significant differences with the positive control (p=0.000) and negative control (p=0.000). Statistically significant differences were observed between positive and negative control (p=0.002). DMP-1 expression was observed incrementally at TGF-β1 concentrations of 1.0 and 5.0 ng/mL at 1, 7, and 14 days and the concentration of 10.0 ng/mL was more intense from day one of the stimulus. DSPP expression was more intense after 14 days of treatment with the concentration of 10.0 ng/mL. Thus, this study concluded that different TGF-β1 concentrations stimulated cell proliferation and migration, without cytotoxic effect on the cells throughout the study period. From the perspective of cell differentiation, TGF-β1 concentration of 10.0 ng/mL was capable of stimulating DMP-1 and DSPP expression.

A formação de novos vasos sanguíneos é fundamental para o crescimento tumoral e a disseminação metastática, sendo o fator de crescimento endotelial vascular (VEGF) uma das chaves reguladoras deste processo. O objetivo do presente estudo foi avaliar a expressão imunohistoquímica de VEGF nos hemangiossarcomas e hemangiomas esplênicos, e rever a prevalência das demais afecções esplênicas através da análise retrospectiva do diagnóstico histopatológico de cães submetidos à esplenectomia. Os resultados foram confrontados com os exames laboratoriais, as manifestações clínicas, a presença de arritmias cardíacas e de hemoperitôneo. Participaram do estudo retrospectivo 109 cães atendidos no Serviço de Cirurgia de Pequenos Animais do Hospital Veterinário da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, entre os anos de 2002 e 2009. A média de idade foi de 10 anos (± 3), não foi observado predileção sexual. Cães sem raça definida foram os mais acometidos, com peso médio de 22 kg (± 13). Cinqüenta e dois por cento (57/109) dos animais foram esplenectomizados devido a afecções não neoplásicas, enquanto que 48% (52/109), por neoplasias esplênicas. Dentre estes, o diagnóstico mais freqüente foi o hemangiossarcoma, acometendo 28 (54%) animais. Os sintomas mais freqüentes foram disorexia, apatia e emese. Cães com neoplasias malignas apresentaram níveis de hematócrito e hemácias significativamente menores que os acometidos por massas benignas. Do mesmo modo, a presença de hemoperitôneo, secundário à ruptura esplênica, correlacionou-se significativamente com a presença de neoplasia maligna. Arritmias cardíacas não foram fatores preditivos para a diferenciação da esplenomegalia. A avaliação imunohistoquímica da expressão tecidual de VEGF foi realizada em 23 hemangiossarcomas e 7 hemangiomas, revelando-se significativamente maior nas neoplasias malignas. Tal resultado sugere que a expressão deste fator pode estar relacionada à proliferação maligna observada nos hemangiossarcomas. / New blood vessel formation is a fundamental event in the process of tumor growth and metastatic dissemination, being the vascular endothelial growth factor (VEGF) one of the key regulators of this process. The aim of this study was evaluate the VEGF immunohistochemical expression in splenic hemangiosarcomas and hemangiomas, and review the prevalence of canine splenic disorders through retrospective analysis of histological diagnosis after splenectomy. The results were confronted with laboratory findings, clinical signs and presence of cardiac arrhythmia and hemoperitoneum. A hundred nine dogs were included in the retrospective study at Veterinary Hospital of School of Veterinary Medicine, University of Sao Paulo, between 2002 and 2009. The average age was 10 year ± 3, without sexual predilection. Mix breeds were the most frequent, and average weigh was 22kg ± 13. Overall, 52% (57/109) of dogs were splenectomized for nonneoplastic disease, although 48% (52/109) were splenectomized for neoplasia. Among these dogs the most common diagnosis was hemangiossarcoma (28 dogs, 54%). Frequently clinical signs included anorexia, lethargy and vomiting. Dogs with malignant neoplasia had significantly lower red blood cells counts and packed cell volume compared with values for dogs with benign masses. Similarly, hemoperitoneum secondary to splenic rupture had a significant correlation with malignant tumor. Cardiac arrhythmia was not useful in differentiating dogs with splenomegaly. Expression of vascular endothelial growth factor was made by immunohistochemical analyses in 23 hemangiosarcomas and 7 hemangiomas being significantly higher in malignant tumor. These data suggest that VEGF expression may contribute to malignant proliferation of hemangiossarcoma.

Lai, Ching Janice. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 148-168). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese --- p.iii / Acknowledgements --- p.v / Table of Content --- p.vi / List of Tables --- p.x / List of Figures --- p.xi / Abbreviations --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Nervous System and the Blood-Brain-Barrier --- p.3 / Chapter 1.3 --- Glial cells --- p.3 / Chapter 1.4 --- Studying Astrocyte Responses As a New Direction in Neuroscience --- p.4 / Chapter 1.5 --- The Roles of Astrocyte in the CNS --- p.5 / Chapter 1.5.1 --- Energy-Dependent Communication Between Neurons and Astrocytes --- p.7 / Chapter 1.5.2 --- Strategies for Metabolic Exchange Between Astrocytes and Neurons --- p.8 / Chapter 1.5.2.1 --- Provision of Energy Metabolites to Neurons by Astrocytes --- p.9 / Chapter 1.5.2.2 --- Glucose Transporters in the CNS --- p.10 / Chapter 1.5.2.3 --- The Lactate Shuttle Hypothesis --- p.12 / Chapter 1.5.2.4 --- The Regulation of Glucose Uptake at the Blood-Brain-Barrier (BBB) by the Activity of Neurons --- p.14 / Chapter 1.5.3 --- Alternation of Energy Metabolism in Neuropathy --- p.15 / Chapter 1.5.3.1 --- Ketone Body Shuttle Hypothesis --- p.15 / Chapter 1.5.3.2 --- The Utilization of Free Fatty Acids by the Brain --- p.17 / Chapter 1.5.4 --- The Provision of Neurotrophic Factors to Neurons by Astrocytes --- p.17 / Chapter 1.5.4.1 --- Neurotrophins --- p.18 / Chapter 1.5.4.1.1 --- Relationship Between Neurotrophins and Glucose --- p.20 / Chapter 1.5.4.2 --- S100B --- p.21 / Chapter 1.5.5 --- Astrocytic Cholesterol in Astrocytes as a Neurotrophic Factor --- p.22 / Chapter 1.6 --- Neuroprotective Effect of Glucose vi - --- p.23 / Chapter 1.7 --- Diseases Associated with Decreased Glucose Transport at the BBB --- p.24 / Chapter 1.7.1 --- Glucose Transporter Type 1 Deficiency Syndrome (GlutlDS) --- p.24 / Chapter 1.7.2 --- Hypoglycemia with Insulin Therapy for Diabetes Patients --- p.24 / Chapter 1.8 --- Aims and Hypothesis of Study --- p.26 / Chapter Chapter 2. --- 2 Materials and Methods --- p.27 / Chapter 2.1 --- Materials --- p.27 / Chapter 2.1.1 --- Cell Culture --- p.27 / Chapter 2.1.1.1 --- Cells --- p.27 / Chapter 2.1.1.1.1 --- C6 cells --- p.27 / Chapter 2.1.1.1.2 --- Primary Astrocytes --- p.27 / Chapter 2.1.1.2 --- Cell Culture Reagent --- p.27 / Chapter 2.1.2 --- Study of Growth Properties --- p.31 / Chapter 2.1.2.1 --- Equipment for Growth Curve Construction --- p.31 / Chapter 2.1.2.2 --- Reagents for Flow Cytometry --- p.32 / Chapter 2.1.2.3 --- Reagents for 3H-thymidine Incorporation Assay --- p.32 / Chapter 2.1.3 --- Study of Neurotrophic Properties --- p.33 / Chapter 2.1.3.1 --- Determination of Neurotrophic Factor Productions --- p.33 / Chapter 2.1.3.1.1 --- Reagents and Buffers for Northern Blot Analysis --- p.33 / Chapter 2.1.3.2 --- Reagents and Buffers for Western Blot Analysis --- p.43 / Chapter 2.1.3.2.1 --- Protein Assay --- p.43 / Chapter 2.1.3.2.2 --- Reagents for SDS Polyacrylamide Electrophoresis of Proteins --- p.44 / Chapter 2.1.3.2.3 --- Reagents for the Transfer of Protein to Membrane and Signal Detection --- p.47 / Chapter 2.1.4 --- Study of Lipid in Glial cells --- p.50 / Chapter 2.1.4.1 --- Determination of Genes Expression in Lipid Metabolism --- p.50 / Chapter 2.1.4.2 --- Reagents for Determination of Cholesterol and Fatty Acid Levels by Gas Chromatography --- p.50 / Chapter 2.2 --- Methods --- p.54 / Chapter 2.2.1 --- Cell culture --- p.54 / Chapter 2.2.1.1 --- Maintenance of C6 cells --- p.54 / Chapter 2.2.1.2 --- Primary Culture of Rat Astrocytes --- p.54 / Chapter 2.2.2 --- Study of Growth Properties of Glial Cells vii - --- p.56 / Chapter 2.2.2.1 --- Construction of cell growth curve --- p.56 / Chapter 2.2.2.2 --- Flow Cytometric Analysis of Cell Cycle Profile --- p.56 / Chapter 2.2.2.3 --- Measurement of DNA Synthesis --- p.57 / Chapter 2.2.3 --- Study of Neurotrophic Properties --- p.58 / Chapter 2.2.3.1 --- Determination of Neurotrophic Facotor Production --- p.58 / Chapter 2.2.3.1.1 --- Northern Blot Analysis --- p.58 / Chapter 2.2.3.1.2 --- Western Blot Analysis --- p.67 / Chapter 2.2.3.2 --- Determination of Gene Expression in Lipid Metabolism --- p.72 / Chapter 2.2.3.2.1 --- Northern Blot Analysis --- p.72 / Chapter 2.2.3.2.2 --- RT-PCR --- p.72 / Chapter 2.2.3.3 --- Study of Lipid Profiles in Glial Cells --- p.73 / Chapter 2.2.3.3.1 --- Sample preparation --- p.73 / Chapter 2.2.3.3.2 --- Total Cholesterol Determination --- p.73 / Chapter 2.2.3.3.3 --- Total Fatty Acid Determination --- p.75 / Chapter 2.2.3.3.4 --- Quantification of Proteins --- p.76 / Chapter 2.2.4 --- Statistical Analysis --- p.77 / Chapter Chapter 3 --- Results --- p.78 / Chapter 3.1 --- The effects of glucose deficiency on cell proliferation --- p.78 / Chapter 3.1.1 --- Direct Cell Count Assay --- p.78 / Chapter 3.1.2 --- Flow Cytometry Assay --- p.83 / Chapter 3.1.3 --- 3H-Thymidine Uptake Assay --- p.85 / Chapter 3.2 --- The Effects of Glucose Deficiency on Neurotrophic Properties of Glial Cells --- p.87 / Chapter 3.2.1 --- The Effects of Glucose Deficiency on mRNA and Protein Expressions of Neurotrophins --- p.88 / Chapter 3.2.1.1 --- Northern Blot Assays --- p.88 / Chapter 3.2.1.2 --- Western Blot Assays --- p.93 / Chapter 3.2.2 --- The Effects of Glucose Deficiency on Lipid Homeostasis --- p.96 / Chapter 3.2.2.1 --- Northern Blot Assays --- p.96 / Chapter 3.2.2.2 --- Gas Chromatography Assays --- p.101 / Chapter 3.2.2.2.1 --- Cholesterol Analyses --- p.102 / Chapter 3.2.2.2.2 --- Fatty Acid Analyses --- p.105 / Chapter Chapter 4 --- Discussion --- p.115 / Chapter 4.1 --- The in vitro Model of Hypoglycorrhachia --- p.115 / Chapter 4.2 --- Decreased Glucose Level Triggers Changes of Gial Cells Proliferation --- p.117 / Chapter 4.3 --- Expression of Neurotrophic Factor under Glucose Deficiency viii - --- p.120 / Chapter 4.3.1 --- Alteration of the Expression of Neurotrophins --- p.120 / Chapter 4.3.1.1 --- NGF --- p.122 / Chapter 4.3.1.2 --- BDNF --- p.123 / Chapter 4.3.1.3 --- NT-3 --- p.126 / Chapter 4.3.1.4 --- NT-4/5 --- p.128 / Chapter 4.3.2 --- Alteration of the mRNA Expression of Calcium Binding ProteinS100B --- p.128 / Chapter 4.4 --- Alteration of Lipid Metabolism in Decreased Glucose Supply --- p.130 / Chapter 4.4.1 --- Up-regulation of ApoE mRNA Expression in Glucose Deficiency --- p.133 / Chapter 4.4.2 --- Cholesterol Homeostasis in Glial Cells --- p.133 / Chapter 4.4.3 --- Fatty Acids Homeostasis in Glial Cells --- p.135 / Chapter 4.4.4 --- Decreased Ketone Bodies synthesis in Glucose Deficiency --- p.143 / Chapter 4.5 --- Limitations of the Current Study --- p.144 / Chapter 4.6 --- Future Directions --- p.145 / Chapter Chapter 5 --- Conclusion --- p.147 / References --- p.148 / Appendix --- p.169

Tse Chui-ling. / "July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 172-185). / 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.

生長滯後是包括炎症性腸病在內的炎症疾病引起的併發症。實驗表明，炎症使肝臟對生長激素(GH)的作用變得不敏感或引起生長激素抵抗。生長激素抵抗會引起胰島素生長因子-1 (IGF-I)的表達下降，並且會啟動一系列的代謝反應。多年來的研究證明炎症因子白介素-6 (IL-6)，腫瘤壞死因子 -α (TNF-α)和白介素-1β(IL-1β)參與肝臟生長激素抵抗的病理過程。然而這些炎症因子調控生長激素通路的具體機理尚不清楚。通過用人肝癌細胞系Huh-7和慢性炎症及急性炎症兩種老鼠模型，我們發現: 1) TNF-α和IL-1β抑制生長激素受體(GHR)的表達; 2) IL-6誘導細胞因子信號轉導抑制因子-3 (SOCS3)的高表達; 3) IL-6-SOCS3途徑對GH-IGF-I信號通路的抑制作用依賴于GHR的表達量，當TNF-α及IL-1β升高而使GHR的表達量下降後，IL-6就不再對GH-IGF-I信號通路有抑制作用。以上結果表明IL-6， TNF-α和IL-1β抑制肝臟生長激素信號通路的機制是不一樣的，這些結果或許對臨床上治療青少年中炎症引起的生長激素抵抗疾病有一定的指導意義。 / 成纖維細胞生長因子(FGF) 通過結合和啟動成纖維細胞生長因子受體(FGFR)而參與許多生理過程。FGF19屬於FGF15/19亞家族，這個亞家族還包括FGF21和FGF23。FGF19調節肝臟中膽汁酸的穩態及蛋白和糖原的合成。FGF19通過與FGFR4及共受體β-klotho結合來啟動信號通路。研究表明，TNF-α通過抑制共受體β-klotho的表達來抑制脂肪細胞中的FGF21信號通路。然而IL-6，TNF-α和IL-1β在調節肝臟FGF19信號通路中的作用尚不清楚。我們的體外細胞和體內動物實驗結果表明，IL-1β通過JNK和NF-κB通路抑制肝臟中β-klotho的表達。IL-6與TNF-α不調節Huh-7細胞中β-klotho的表達。 / 綜上所述，IL-6，TNF-α及IL-1β在肝臟生長激素及FGF19通路中起不同的調節作用。 / Growth failure is a major complication of inflammatory diseases including inflammatory bowel disease. Evidence suggests that during inflammation, the liver becomes resistant to growth hormone (GH) actions, leading to downregulation of the anabolic gene IGF-I and the activation of catabolic processes. Decades of studies demonstrated that pro-inflammatory cytokines IL-6, TNF-α and IL-1β are involved in the pathogenesis of hepatic GH resistance. However, the exact mechanisms used by these individual cytokines to regulate GH signaling are not defined. Using Huh-7 human hepatoma cells and mouse models of chronic and acute inflammation, we show that TNF-α and IL-1β but not IL-6 inhibited hepatic GH receptor (GHR) expression, and that IL-6 but not TNF-α and IL-1β stimulated expression of suppressor of cytokine signaling-3 (SOCS3). TNF-α/IL-1β and IL-6 acted primarily at GHR and SOCS3 respectively to inhibit the GH-IGF-I pathway. While TNF-α/IL-1β exerted a tonic inhibition on hepatic GH signaling, IL-6 activity is dependent on the active GH pathway. IL-6 lost its inhibition on the GH-IGF-I pathway when GHR expression was blocked as the inflammation progressed. These results reveal previously undefined distinct mechanisms used by TNF-α/IL-1β and IL-6 to inhibit the hepatic GH pathway. Our results may provide a new guidance for clinical practice in treating pediatric infammation-induced GH resistance. / Fibroblast growth factors (FGFs) play critical roles in many physiological processes by binding to and activating FGF receptor (FGFR) family. FGF19 belongs to FGF15/19 subfamily of FGFs that includes FGF15/19, FGF21 and FGF23. FGF19 has been shown to regulate bile acid homeostasis, and protein and glycogen synthesis in the liver. FGF19 binds FGFR4 and the co-receptor β-klotho to initiate signaling. Studies have shown that proinflammatory cytokines such as TNF-α can impair FGF21 signaling in adipose cells by repressing the expression of β-klotho. However, little is known about the effects of IL-6, TNF-α and IL-1β on regulating hepatic FGF19 signaling. In the present study, we found that IL-1β inhibited β-klotho expression both in vitro and in vivo, and this inhibition required JNK and NF-κB pathways. IL-6 and TNF-α did not inhibit β-klotho expression in Huh-7 cells. / Taken together, our results demonstrate that IL-6, TNF-α and IL-1β play different roles in regulating the GH and FGF-19 pathways in the liver. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhao, Yueshui. / Thesis (Ph.D.) Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 147-182). / Abstracts also in Chinese.

Siu, Yeung Tung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 65-75). / Abstracts in English and Chinese. / Title Page --- p.i / Abstract in English --- p.ii / Abstract in Chinese --- p.iii / Acknowledgement --- p.iv / Table of Contents --- p.v-viii / List of Figures --- p.ix / List of Tables --- p.x / Abbreviations --- p.xi-xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Growth factors involved in hepatocytes proliferation --- p.1-6 / Chapter 1.1.1 --- Hepatocyte growth factor (HGF) --- p.1 / Chapter 1.1.2 --- Tumor necrosis factor-a (TNF-α) and interleukin-6 (IL-6) --- p.2 / Chapter 1.1.3 --- Epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) --- p.3 / Chapter 1.1.4 --- Other comitogens --- p.4 / Chapter 1.1.5 --- Transforming growth factor-β (TGF-β) --- p.5 / Chapter 1.2 --- Bone marrow stromal cells and hepatocytes proliferation --- p.7-12 / Chapter 1.2.1 --- Role of bone marrow stromal cells in bone marrow --- p.7 / Chapter 1.2.2 --- Bone marrow as a source of hepatic oval cells --- p.8 / Chapter 1.2.3 --- Growth factors secreted by bone marrow stromal cells involved in hepatocytes proliferation --- p.9 / Chapter 1.2.4 --- Endocrine in hepatocytes proliferation --- p.12 / Chapter 1.3 --- Objective of this study --- p.13-15 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Cell cultures --- p.16 / Chapter 2.2 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.17-18 / Chapter 2.2.1 --- "Enrichment of human hepatic cell lines, Hep 3B, Hep G2, C3A, SK-Hep-1 and Chang cells at G0-G1 phases by serum deprivation" --- p.17 / Chapter 2.2.2 --- "Incubation of serum deprived Hep 3B, Hep G2, C3A, SK- Hep-1 and Chang cells with mitogenic stimuli" --- p.17 / Chapter 2.2.3 --- Cell cycle analysis by flow cytometry using propidium iodide staining --- p.17 / Chapter 2.3 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.18-20 / Chapter 2.3.1 --- Partially growth arrested human SK-Hep-1 cells --- p.18 / Chapter 2.3.2 --- Human bone marrow stromal cells --- p.19 / Chapter 2.3.2.1 --- Bone marrow stromal cellular extract --- p.19 / Chapter 2.3.2.2 --- Total protein assay --- p.19 / Chapter 2.3.3 --- Incubation of SK-Hep-1 cells with bone marrow stromal cellular extracts --- p.20 / Chapter 2.4 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.21-22 / Chapter 2.4.1 --- Dialysis --- p.21 / Chapter 2.4.2 --- Temperature treatment --- p.21 / Chapter 2.4.3 --- Proteolysis --- p.22 / Chapter 2.5 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.22-26 / Chapter 2.5.1 --- Incubation of SK-Hep-1 cells with bone marrow stromal cellular extract or other growth factors --- p.22 / Chapter 2.5.2 --- Metabolic labeling of SK-Hep-1 cells with [32P]orthophosphate --- p.23 / Chapter 2.5.3 --- Incubation of labeled SK-Hep-1 cells with bone marrow stromal cellular extract or other growth factors --- p.23 / Chapter 2.5.4 --- SK-Hep-1 cells lysate extraction --- p.23 / Chapter 2.5.5 --- Two-dimensional electrophoresis --- p.24 / Chapter 2.5.5.1 --- First dimension isoelectric focusing --- p.24 / Chapter 2.5.5.2 --- Second dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis --- p.25 / Chapter 2.5.6 --- Amplification of radiolabeled signal by EN3HANCE --- p.25 / Chapter 2.5.7 --- Visualization of autoradiography --- p.26 / Chapter 2.5.8 --- Visualization by silver staining --- p.26 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.27-30 / Chapter 3.1.1 --- "Enrichment of human hepatic cell lines, Hep 3B, Hep G2, C3A, SK-Hep-1 and Chang cells at G0-G1 phases by serum deprivation" --- p.27 / Chapter 3.1.2 --- DNA synthesis of hepatic cell lines in response to 10 % FBS after serum deprivation --- p.29 / Chapter 3.2 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.31-39 / Chapter 3.2.1 --- Cell cycle distribution of partially growth arrested SK-Hep-1 cells in response to mitogens --- p.31 / Chapter 3.2.2 --- Time course on DNA synthesis of partially growth arrested SK-Hep-1 cells in response to FBS and bone marrow stromal cellular extract --- p.36 / Chapter 3.2.3 --- Dose response on DNA synthesis of partially growth arrested SK-Hep-1 cells in response to bone marrow stromal cellular extracts --- p.38 / Chapter 3.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.40-44 / Chapter 3.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.45-49 / Chapter 3.4.1 --- Mitogenic response of SK-Hep-1 cells in response to bone marrow stromal cellular extract and other growth factors --- p.45 / Chapter 3.4.2 --- Early intracellular signaling of SK-Hep-1 cells in response to bone marrow stromal cellular extract and other growth factors --- p.47 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.50 / Chapter 4.2 --- "Mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.51 / Chapter 4.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.52 / Chapter 4.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.53 / Chapter 4.5 --- Possible directions for future investigation --- p.55 / Chapter 4.6 --- Conclusions --- p.56 / Chapter Chapter 5 --- Appendices / Chapter 5.1 --- Reagents and solutiuons --- p.57-64 / Chapter 5.1.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.57 / Chapter 5.1.2 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.59 / Chapter 5.1.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.60 / Chapter 5.1.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.61 / Chapter Chapter 6 --- References --- p.65-75

Tang Yin Nei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves xv-xxv). / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.i / ABBREVIATIONS --- p.ii-iii / INDEX FOR FIGURES --- p.iv-v / INDEX FOR TABLES --- p.vi / ABSTRACT (Chinese and English) --- p.vii-xi / TABLE OF CONTENTS --- p.xii-xiv / Chapter CHAPTER I 226}0ؤ --- INTRODUCTION --- p.1 / Chapter 1.1 --- Background --- p.2 / Chapter 1.2 --- Tendon / Chapter 1.2.1 --- Structure and function --- p.3 / Chapter 1.2.2 --- Tendon fibroblast --- p.6 / Chapter 1.2.3 --- Components of the extracellular matrix --- p.7 / Chapter 1.2.3.1 --- Collagen --- p.8 / Chapter 1.2.3.2 --- Proteoglycan --- p.9 / Chapter 1.2.3.3 --- Non-collagenous structural glycoprotein --- p.10 / Chapter 1.3 --- Inflammation disorders of tendon / Chapter 1.3.1 --- Inflammation --- p.11 / Chapter 1.3.2 --- Treatment --- p.12 / Chapter 1.3.2.1 --- Glucocorticoid as an anti-inflammatory agent --- p.12 / Chapter 1.3.2.2 --- Dexamethasone --- p.14 / Chapter 1.3.3 --- Clinical occurrence of tendon rupture --- p.15 / Chapter 1.3.4 --- Animal research related to glucocorticoids and tendon rupture --- p.18 / Chapter 1.4 --- Platelet-derived growth factor isoform B (PDGFBB) / Chapter 1.4.1 --- Structure and function --- p.21 / Chapter 1.4.2 --- PDGFbb effects on connective tissue --- p.22 / Chapter CHAPTER II 226}0ؤ --- AIM OF THE STUDY --- p.23 / Chapter 2.1 --- Limitations of the past researches --- p.24 / Chapter 2.2 --- Hypothesis of this study --- p.25 / Chapter 2.3 --- Objectives --- p.26 / Chapter 2.4 --- Long term significance --- p.26 / Chapter CHAPTER III 226}0ؤ --- METHODOLOGY --- p.27 / Chapter 3.1 --- Chemicals and materials used / Chapter 3.1.1 --- Chemicals --- p.28 / Chapter 3.1.2 --- Materials --- p.28 / Chapter 3.2 --- Specimen collection and preparation / Chapter 3.2.1 --- Collection --- p.29 / Chapter 3.2.2 --- Preparation and isolation --- p.30 / Chapter 3.2.3 --- Cell culture --- p.31 / Chapter 3.3 --- Reagent preparation / Chapter 3.3.1 --- Charcoal-stripped serum --- p.32 / Chapter 3.3.2 --- Phenol-red free DMEM --- p.33 / Chapter 3.3.3 --- MTT --- p.33 / Chapter 3.3.4 --- Dexamethasone --- p.34 / Chapter 3.3.5 --- PDGFbb --- p.34 / Chapter 3.3.6 --- Trypan blue --- p.35 / Chapter 3.3.7 --- TCA/Tannic acid --- p.35 / Chapter 3.3.8 --- Collagenase buffer --- p.35 / Chapter 3.4 --- Morphology / Chapter 3.4.1 --- Inverted phase contrast light microscopy --- p.36 / Chapter 3.4.2 --- Scanning electron microscopy --- p.36 / Chapter 3.5 --- Biological assays / Chapter 3.5.1 --- "MTT (3-[4,5-Dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide) assay" --- p.38 / Chapter 3.5.1.1 --- Correlation between MTT assay and trypan blue dye method --- p.38 / Chapter 3.5.1.2 --- Growth kinetics for tendon fibroblasts --- p.41 / Chapter 3.5.1.3 --- Cell viability --- p.43 / Chapter 3.5.2 --- Brdu (5-bromo-2'-deoxyuridine) assay --- p.44 / Chapter 3.5.3 --- Flow cytometry --- p.45 / Chapter 3.5.4 --- Apoptosis --- p.47 / Chapter 3.5.5 --- 3H-Proline incorporation assay --- p.48 / Chapter 3.5.6 --- 35Sulfate incorporation assay --- p.51 / Chapter 3.5.7 --- Immunocytochemistry (PDGF-β receptor) --- p.54 / Chapter 3.6 --- Statistical analysis / Chapter 3.6.1 --- Dose-response curve of dexamethasone on cell viability and proliferation --- p.55 / Chapter 3.6.2 --- Comparison among various treatments of fibroblasts --- p.55 / Chapter CHAPTER I´Vؤ --- RESULTS --- p.56 / Chapter 4.1 --- In vitro effect of dexamethasone on rat tendon fibroblasts / Chapter 4.1.1 --- Viable cell number between two sexes --- p.57 / Chapter 4.2 --- In vitro effect of dexamethasone and PDGFBB on human tendon fibroblasts / Chapter 4.2.1 --- Gross morphology --- p.58 / Chapter 4.2.2 --- Cell cycle --- p.60 / Chapter 4.2.3 --- Apoptosis --- p.61 / Chapter 4.2.4 --- Viable cell number / Chapter 4.2.4.1 --- Effect of dexamethasone --- p.62 / Chapter 4.2.4.2 --- Effect of PDGFBB --- p.63 / Chapter 4.2.5 --- Cell proliferation / Chapter 4.2.5.1 --- Effect of dexamethasone --- p.65 / Chapter 4.2.5.2 --- Effect of PDGFbb --- p.67 / Chapter 4.2.6 --- Collagen synthesis --- p.68 / Chapter 4.2.7 --- Proteoglycan synthesis --- p.72 / Chapter 4.2.8 --- PDGF-rβ expression --- p.74 / Chapter CHAPTER V 226}0ؤ --- DISCUSSION --- p.75 / Chapter 5.1 --- Dexamethasone and PDGFBB induced change of cell morphology --- p.77 / Chapter 5.2 --- Dexamethasone retarded cell growth of human tendon fibroblast --- p.80 / Chapter 5.3 --- Dexamethasone inhibited collagen synthesis --- p.82 / Chapter 5.4 --- Dexamethasone inhibited proteoglycan synthesis --- p.86 / Chapter 5.5 --- PDGFbb could counteract the inhibitory effects of dexamethasone --- p.88 / Chapter 5.6 --- Expression of PDGF-(3 receptor is regulated by dexamethasone and PDGFBB --- p.90 / Chapter 5.7 --- Limitations of this study / Chapter 5.7.1 --- Not enough sample to differentiate different between two sexes --- p.92 / Chapter 5.7.2 --- Small sample size and few assays --- p.92 / Chapter 5.7.3 --- Limitations of the cell culture model --- p.93 / Chapter 5.7.4 --- Difficult to further in vivo study on human --- p.93 / Chapter 5.8 --- Contributions of this study / Chapter 5.8.1 --- Improve the limitation of the past research --- p.94 / Chapter 5.8.1.1 --- Human tendon specimen --- p.94 / Chapter 5.8.1.2 --- In vitro system --- p.94 / Chapter 5.8.2 --- Understand the effect of dexmaethasone on human tendon fibroblasts --- p.95 / Chapter 5.8.3 --- Counteract the deleterious effects of dexamethasone by PDGFBB --- p.95 / Chapter CHAPTER VÍؤ --- CONCLUSION & FUTURE STUDY --- p.96 / Chapter 6.1 --- Conclusion --- p.97 / Chapter 6.2 --- Future study --- p.98 / Chapter 6.2.1 --- Study the balance between matrix synthesis and degradation --- p.98 / Chapter 6.2.2 --- Determine collagen typing --- p.99 / Chapter 6.2.3 --- Further explore the effect of glucocorticoid in organ culture model --- p.100 / Chapter 6.2.4 --- Investigate molecular mechanism of dexamethasone and PDGFBB --- p.100 / REFERENCES --- p.xv-xxv / APPENDIX --- p.xxvi

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