by Ng Chor Yin Maggie. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (p. 358-403). / 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.

Introdução: O carcinoma hepatocelular (CHC) é o câncer primário de fígado mais comum. A cirrose hepática é o principal fator de risco para esse tumor. O rastreamento para o CHC em pacientes com cirrose tem sido recomendado há anos. Acredita-se que a detecção e o tratamento precoce do CHC melhorem a sobrevida dos pacientes. O objetivo deste estudo foi analisar a sobrevida dos pacientes cirróticos com CHC pequeno e identificar fatores preditivos de sobrevida no Brasil. Casuística e Métodos: Entre janeiro de 1998 e dezembro de 2003, 74 pacientes cirróticos com CHC foram avaliados. Eles preenchiam os seguintes critérios: CHC com até três nódulos e no máximo 30 mm de diâmetro cada. Os fatores preditores de sobrevida foram identificados através do método de Kaplan-Meier e o modelo de Cox. Resultados: A média de idade foi de 58 anos (32-77); 71% dos pacientes eram do sexo masculino; 64% tinham hepatite C; 60% eram Child-Pugh A, o valor mediano da pontuação de MELD foi de 11; 79% tinham hipertensão portal. No momento do diagnóstico, 71% tinham uma única lesão; o tamanho do principal tumor era menor que 20 mm em 47%; o valor médio de AFP foi de 131 ng/ml. Três pacientes tinham trombose de veia porta, sugestiva de invasão vascular. Cinqüenta pacientes (67,5%) foram incluídos na lista de transplante hepático, que foi realizado só em quatro pacientes. A ressecção cirúrgica do tumor foi possível em quatro pacientes. Quarenta e oito (64,8%) pacientes receberam tratamento ablativo percutâneo (ablação por radiofreqüência ou injeção percutânea de etanol). Nove pacientes não receberam nenhum tratamento específico para o tumor. A taxa de sobrevida geral foi de 80%; 62%; 41% e 17% em 12, 24, 36 e sessenta meses, respectivamente. O tempo médio de seguimento após o diagnóstico do CHC foi de 23 meses (mediana de 22 meses, variando de um a 86 meses) para todo o grupo. Durante o seguimento, ocorreram 39 óbitos ocorreram relacionados com insuficiência hepática ou progressão do CHC. A análise univariada dos 74 pacientes mostrou que escore MELD maior que 11 (p = 0,016), classificação de Child-Pugh B e C (p = 0,007), AFP > 100 ng/ml (p = 0,006), mais de uma lesão (p = 0,041), diâmetro do tumor > 20 mm (p = 0,009) e presença de invasão vascular (p < 0,0001) foram preditores independentes de sobrevida. A análise de regressão de Cox identificou invasão vascular (RR = 14,60 - IC 95% = 3,3 - 64,56 - p < 0,001) e tamanho do tumor > 20mm (RR = 2,14 - IC 95% = 1,07 - 4,2 - p = 0,030) como preditores independentes de pior sobrevida. O tratamento do CHC esteve relacionado com melhor sobrevida. Conclusão: A identificação de CHC pequeno com até 20 mm de diâmetro está relacionada com melhores taxas de sobrevida. Por outro lado, a presença de invasão vascular, apesar do tamanho pequeno das lesões, é um fator associado a péssimo prognóstico. / Introduction: Hepatocellular carcinoma (HCC) is the most common primary liver cancer. Liver cirrhosis is the major risk factor for this tumor. Screening for HCC in patients with cirrhosis has been recommended, in the belief that detection and treatment of early HCC improves patient survival. The aims of this study were to analyze the overall survival of small HCC in cirrhotic patients and identify independent predictors of survival, in Brazil. Methods: Between January 1998 and December 2003, seventy-four cirrhotic patients with hepatocellular carcinoma were evaluated satisfying the following criteria: HCC of 30 mm or smaller and a maximum of three lesions. Predictors of survival were identified using the Kaplan-Meier and the Cox model. Results: Mean age was 58 years-old (32-77), 71% of patients was male, 64% had hepatitis C, 60% were Child-Pugh A, mean MELD score was 11 and 79% had portal hypertension. At the time of diagnosis, 71% had one tumor, the size of the main tumor was smaller than 20 mm in 47%, mean AFP level was 131 ng/ml. Three patients had portal vein thrombosis, suggesting vascular invasion. Fifty patients (67.5%) were included in the liver transplant list, but it was performed in only four patients. Tumor resection was possible in four patients. Forty-eight (64.8%) patients received percutaneous treatment (radiofrequency ablation or percutaneous ethanol injection). Nine patients did not receive any cancer treatment. The overall survival rates were 80%, 62%, 41% and 17% at 12, 24, 36 and 60 months, respectively. The mean length of follow-up after HCC diagnosis was 23 months (median 22 months, range 1-86 months) for the entire group. During follow-up a total of 39 deaths related to liver failure or HCC progression occurred. Univariate analysis of the 74 patients showed that MELD score greater than 11 (p = 0.016), Child-Pugh classification (p = 0.007), AFP > 100 ng/ml (p = 0.006), more than one lesion (p = 0.041), tumor diameter > 20 mm (p = 0.009) and presence of vascular invasion (p < 0.0001) were significant predictors of survival. Cox regression analysis identified vascular invasion (RR = 14.60 - IC 95% = 3.3 - 64.56 - p < 0.001) and tumor size > 20mm (RR = 2.14 - IC 95% = 1.07 - 4.2 - p = 0.030) as independent predictors of decreased survival. Treatment of HCC was related to increased overall survival. Conclusion: Identification of small tumors of up to 20 mm diameter is related to increase survival. Nevertheless, vascular invasion, in spite of the small diameter of the lesions, is a factor associated with dismal prognosis.

by Ho Wai-chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references. / Abstract also in Chinese. / Title / Dedication / Abstract --- p.i / Table of Contents --- p.iv / Glossary of abbreviation used in the thesis --- p.vi / List of figures --- p.viii / List of tables --- p.xvii / Acknowledgement --- p.xix / Statement of Originality --- p.xx / Chapter Chapter 1 ...... --- Introduction --- p.14 / Chapter Chapter 2...... --- Basic Principle / Chapter 2.1 --- The liver - a vital organ --- p.2-1 / Chapter 2.2 --- Blood supply to the liver --- p.2-7 / Chapter 2.3 --- Normal arterial anatomy of the coeliac axis --- p.2-11 / Chapter 2.4 --- Common anomalies of the coeliac axis --- p.2-17 / Chapter 2.5 --- Previous classification of coeliac anomaies --- p.2-24 / Chapter 2.6 --- Knowledge of arterial anomaly in relation to surgery --- p.2-31 / Chapter 2.7 --- Trans-catheter treatment of hepatocellular carcinoma --- p.2-33 / Chapter 2.8 --- Prevalence of hepatocellular carcinoma in H.K Chinese --- p.2-42 / Chapter 2.9 --- Management of hepatocellular carcinoma in Hong Kong --- p.2-43 / Chapter Chapter 3...... --- Definitions --- p.3-1 / Chapter Chapter 4...... --- Objectives of the study --- p.4-1 / Chapter Chapter 5...... --- "Materials, methods and subjects" / Chapter 5.1 --- Materials --- p.5-1 / Chapter 5.2 --- Methods --- p.5-3 / Chapter 5.3 --- Subjects --- p.5-10 / Chapter Chapter 6...... --- Results / Chapter 6.1 --- Coeliac axis --- p.6-5 / Chapter 6.2 --- Common hepatic artery --- p.6-9 / Chapter 6.3 --- Proper hepatic artery --- p.6-11 / Chapter 6.4 --- Right hepatic artery --- p.6-12 / Chapter 6.5 --- Middle hepatic artery --- p.6-20 / Chapter 6.6 --- Left hepatic artery --- p.6-28 / Chapter 6.7 --- Gastroduodenal artery --- p.6-33 / Chapter 6.8 --- Right gastric artery --- p.6-37 / Chapter 6.9 --- Left gastric artery --- p.5-45 / Chapter 6.10 --- Splenic artery --- p.6-49 / Chapter 6.11 --- Summary of results --- p.6-51 / Chapter Chapter 7...... --- Discussion / Chapter 7.1 --- Introduction --- p.74 / Chapter 7.2 --- Selective Internal Radiation --- p.7-3 / Chapter 7.3 --- Coeliac axis --- p.7-10 / Chapter 7.4 --- Common hepatic & proper hepatic artery --- p.7-7 / Chapter 7.5 --- Right hepatic artery --- p.7-14 / Chapter 7.6 --- Middle hepatic artery --- p.7-18 / Chapter 7.7 --- Left hepatic artery --- p.7-25 / Chapter 7.8 --- Gastroduodenal artery --- p.7-30 / Chapter 7.9 --- Right gastric artery --- p.7-35 / Chapter 7.10 --- Left gastric artery --- p.7-43 / Chapter 7.11 --- Splenic artery --- p.7-45 / Chapter 7.12 --- Comparison with the golden classics --- p.7-47 / Chapter 7.13 --- Comparison of subjects with HCC & without HCC --- p.7-50 / Chapter 7.14 --- Comparison of the male group and the female group --- p.7-51 / Chapter Chapter 8...... --- Conclusions / References --- p.B-1 / Bibliography --- p.B-1 / Appendix I Schematic diagram of histological anatomy of the liver --- p.A-l / Appendix II Embryology --- p.A-2 / "Appendix III Percentages of occurrence of the different types of coeliac axis, by Michels' study" --- p.A-3 / "Appendix IV Percentages of occurrence of the different types of the hepatic arterial blood supply, by Michels' study" --- p.A-4 / Appendix V No. of deaths from malignant liver cancer in Hong Kong froml984 to1993 --- p.A-5 / Appendix VI Flow chart for HCC management in PWH of Hong Kong --- p.A-6 / Appendix VII Comparison with Michels' study --- p.A-7 / Appendix VIII Comparison of the group with HCC and the group without HCC --- p.A-8 / Appendix IX Comparison of the male and female group --- p.A-9

Chan Ming Lok. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 93-108). / Abstracts in English and Chinese. / Ackowledgments --- p.i / Abstract in English --- p.ii / Abstract in Chinese --- p.iii / List of Abbreviations --- p.iv / List of Tables --- p.v / List of Figures --- p.vi / Chapter Chapter 1 --- Introduction and Objectives / Chapter 1.1 --- Hepatocellular Carcinoma --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Geographical Distribution --- p.1 / Chapter 1.1.3 --- Sex and Age --- p.1 / Chapter 1.1.4 --- Etiology --- p.2 / Chapter 1.1.5 --- Molecular Basis of HCC --- p.3 / Chapter 1.1.6 --- Situation in China and Hong Kong --- p.4 / Chapter 1.2 --- The Hepatitis B Virus --- p.5 / Chapter 1.2.1 --- Morphology --- p.5 / Chapter 1.2.2 --- Structure of the HBV Genome --- p.6 / Chapter 1.2.3 --- Functional Domains of the HBV Genome --- p.9 / Chapter 1.2.4 --- Pathogenesis of HBV Infection --- p.11 / Chapter 1.3 --- HBx --- p.12 / Chapter 1.3.1 --- The HBV x Gene --- p.12 / Chapter 1.3.2 --- The HBX Protein --- p.13 / Chapter 1.3.3 --- "Preferential HBX Expression in Sera, Hepatitis, Cirrhosis and HCC" --- p.13 / Chapter 1.3.4 --- Cellular Localization of HBX --- p.14 / Chapter 1.3.5 --- Animal Studies --- p.15 / Chapter 1.3.6 --- Functional Studies on HBX --- p.15 / Chapter 1.3.7 --- Variations in the HBx Gene --- p.21 / Chapter 1.4 --- Objectives of this Study --- p.24 / Chapter Chapter 2 --- Methods and Materials Methods / Chapter 2.1 --- Paraffin Embedding of Patient Tissue Samples --- p.26 / Chapter 2.1.1 --- Tissue Processing --- p.26 / Chapter 2.1.2 --- Paraffin Embedding of Tissue Samples --- p.26 / Chapter 2.2 --- Sectioning of Paraffin Embedded Tissue Sections --- p.26 / Chapter 2.3 --- Immunohistochemical Staining of Paraffin Embedded Tissue Sections --- p.26 / Chapter 2.3.1 --- Dewaxing of Paraffin-Embedded Tissue Sections --- p.26 / Chapter 2.3.2 --- Rehydration of Tissue Sections --- p.27 / Chapter 2.3.3 --- Antigen Retrieval --- p.27 / Chapter 2.3.4 --- Quenching of Endogenous Hydrogen Peroxidase --- p.27 / Chapter 2.3.5 --- Blocking of Endogenous Biotin and Non-Specific Protein Binding --- p.27 / Chapter 2.3.6 --- Antibody Incubation and Color Development --- p.27 / Chapter 2.3.7 --- Counterstaining and Coverslip Mounting --- p.28 / Chapter 2.3.8 --- Interpretation of Immunostaining Results --- p.28 / Chapter 2.4 --- DNA Extraction from HCC Tissues --- p.28 / Chapter 2.4.1 --- Sectioning of Frozen HCC Specimens --- p.28 / Chapter 2.4.2 --- Proteinase K Digestion and Phenol Chloroform Extraction --- p.29 / Chapter 2.4.3 --- Ethanol Precipitation and Re-suspension in Tris-EDTA (TE) Buffer --- p.29 / Chapter 2.5 --- Quantitation and Purity Check of Extracted DNA --- p.29 / Chapter 2.6 --- Quality Check for Extracted Genomic DNA --- p.30 / Chapter 2.6.1 --- Agarose Gel Electrophoresis --- p.30 / Chapter 2.6.2 --- Polymerase Chain Reaction (PCR) of the β-globin Gene --- p.30 / Chapter 2.6.3 --- Analysis of PCR Fragments by Agarose Gel Electrophoresis --- p.30 / Chapter 2.7 --- Polymerase Chain Reaction Amplification of HBs and HBx Genes of the Hepatitis B Virus --- p.31 / Chapter 2.8 --- Southern Blot of HBx PCR Fragments --- p.31 / Chapter 2.8.1 --- Immobilization of DNA onto a Positively Charged Nylon Membrane and Pre-hybridization --- p.31 / Chapter 2.8.2 --- Radio-labeling of an HBV Probe --- p.32 / Chapter 2.8.3 --- Hybridization of a 32P-labeled HBV Probe and Film Exposure --- p.32 / Chapter 2.9 --- Cloning of PCR Fragments into pGEM®-T Vector for Sequencing --- p.33 / Chapter 2.9.1 --- Gel Extraction and Purification --- p.33 / Chapter 2.9.2 --- Ligation --- p.33 / Chapter 2.10 --- Transformation of Competent DH5a cells --- p.34 / Chapter 2.10.1 --- Preparation of Competent DH5α Using Calcium Chloride --- p.34 / Chapter 2.10.2 --- Heat Shock of Competent DH5α Cells --- p.34 / Chapter 2.10.3 --- Plating of Transformed Cells onto LB Agar Plates --- p.34 / Chapter 2.10.4 --- Screening of Transformants for Inserts --- p.35 / Chapter 2.11 --- Miniprep of Plasmid DNA --- p.35 / Chapter 2.11.1 --- Inoculation of Bacterial Clones --- p.35 / Chapter 2.11.2 --- DNA Extraction by Alkaline Lysis and Phenol/Chloroform --- p.35 / Chapter 2.11.3 --- Ethanol Precipitation and Re-suspension in TE Buffer --- p.35 / Chapter 2.11.4 --- Confirmation of Positive Clones --- p.36 / Chapter 2.12 --- Sequencing of pGEM®-T Cloned HBx PCR Fragments --- p.36 / Chapter 2.13 --- Construction of the HBx-GFP Plasmid --- p.36 / Chapter 2.13.1 --- PCR Amplification of HBx Gene Inserts --- p.36 / Chapter 2.13.2 --- Confirmation of HBx Insert Sequence by DNA Sequencing --- p.37 / Chapter 2.13.3 --- Restriction Digest of HBx-pGEM®-T Plasmids to Obtain HBx Inserts --- p.37 / Chapter 2.13.4 --- Restriction Digest of pEGFP-Nl Cloning Vector for Cloning --- p.37 / Chapter 2.13.5 --- Ligation of HBx Inserts into the pEGFP Cloning Vector --- p.37 / Chapter 2.14 --- Large Scale Plasmid DNA Preparation --- p.38 / Chapter 2.15 --- Cell Culture --- p.39 / Chapter 2.16 --- Transfection using LipofectAminéёØ --- p.39 / Chapter 2.16.1 --- Seeding of Cells for Coverslip Growth --- p.39 / Chapter 2.16.2 --- Transfection using LipofecAminéёØ --- p.39 / Chapter 2.17 --- Cell Fixation and DAPI Staining Materials --- p.40 / Chapter 2.18 --- Chemicals --- p.41 / Chapter 2.19 --- Antibodies --- p.41 / Chapter 2.20 --- "Formalin-fixed, Paraffin Embedded Tissues of HCC Tissues from Xiamen" --- p.41 / Chapter 2.21 --- Frozen Liver Tissues --- p.41 / Chapter 2.22 --- PCR Reagents --- p.43 / Chapter 2.23 --- Primers --- p.43 / Chapter 2.24 --- Plasmid --- p.43 / Chapter 2.25 --- Enzymes --- p.43 / Chapter 2.26 --- Ligation Reagents --- p.43 / Chapter 2.27 --- Cloning Vectors --- p.45 / Chapter 2.28 --- Competent Cell --- p.45 / Chapter 2.29 --- Hela and HepG2 Cell Line --- p.45 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Hepatitis B Virus Status of HCC Patients from Hong Kong and Xiamen --- p.46 / Chapter 3.2 --- Immunohistochemical Studies of the HBx Protein in Hong Kong and Xiamen HCC --- p.46 / Chapter 3.2.1 --- Cross Reaction of Anti-99 with Cytokeratin 18 (CK18) --- p.46 / Chapter 3.2.2 --- HBx Expression in HCC Patient Tissue Samples from Hong Kong --- p.50 / Chapter 3.2.3 --- HBxAg Staining in HCC Tissue Samples from Xiamen --- p.50 / Chapter 3.3 --- Agarose Gel Electrophoresis of DNA Extracted from Frozen Liver Tissues --- p.50 / Chapter 3.4 --- PCR Amplification of the β-globin Gene --- p.55 / Chapter 3.5 --- PCR Amplification of the HBs Gene from Liver Samples of HCC Patients from Hong Kong --- p.55 / Chapter 3.6 --- PCR Amplification of the HBx Gene from Liver Samples of HCC Patients from Hong Kong --- p.55 / Chapter 3.7 --- Amplification of the HBx Gene from Serum Samples of Chronic Hepatitis B Virus from Hong Kong Using Nested PCR --- p.61 / Chapter 3.8 --- Southern Blot of HBx PCR Fragments --- p.61 / Chapter 3.9 --- Cloning and Sequencing of the HBx Gene in HCC and Chronic Hepatitis B Patient Samples from Hong Kong --- p.61 / Chapter 3.10 --- Expression Pattern of Wild-type HBx-GFP Fusion Protein in Transiently Transfected HeLa and HepG2 Cells --- p.73 / Chapter 3.11 --- Expression Patterns of HBx-GFP with and without Mutations at Codons 130 and 131 in HeLa and HepG2 Cell Line --- p.78 / Chapter 3.12 --- Growth Kinetics of HeLa Cells Transfected with GFP and Wild-type HBx-GFP with and without Mutations in Codons 130 and131 --- p.81 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- HBxAg Expression in Tumorous and Surrounding Non-tumorous Tissues --- p.83 / Chapter 4.2 --- "Detection of the HBx Gene in Sera, Non-tumorous and Tumorous Tissues" --- p.84 / Chapter 4.3 --- HBx Gene Mutations in Chronic Hepatitis and HCC --- p.85 / Chapter 4.3.1 --- Codon 127 (HBV nt 1752-1754) --- p.85 / Chapter 4.3.2 --- Codons 130 and 131 (HBV nt 1761-1766) --- p.86 / Chapter 4.3.3 --- Lack of Correlation between HBx Gene Mutations and Lack of HBxAg Expression --- p.87 / Chapter 4.4 --- Cellular Localization of HBxAg in Transiently Transfected Cells Lines --- p.88 / Chapter 4.5 --- Functional Difference Between Wild-type and Mutant HBX Protein --- p.89 / Chapter Chapter 5 --- Conclusions and Directions for Further Studies / Chapter 5.1 --- Conclusions --- p.91 / Chapter 5.2 --- Directions for Further Studies --- p.92 / References --- p.93 / Appendix / Chapter A1 --- Recipes of Reagents Used in this Study --- p.109 / Chapter A2 --- Schematic Setup of Downward Capillary Transfer of DNA --- p.112 / Chapter A3 --- Circle Map of the pGEM®-T Cloning Vector and Construct of the HBx-pGEM®-T Plasmid --- p.113 / Chapter A4 --- Circle Map of the pEGFP-Nl Cloning Vector and Construct of the HBx-GFP Plasmid --- p.114

Siu Pak-yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 158-174). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Contents --- p.vi / List of Figures and Tables --- p.xiii / List of Abbreviations --- p.xviii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Characteristics of Arsenic Compound --- p.1 / Chapter 1.1.1 --- Arsenic Compounds are Used as Poison --- p.1 / Chapter 1.1.2 --- Arsenic Compounds are Used as Medicine --- p.2 / Chapter 1.2 --- Arsenic Trioxide is a Traditional Chinese Medicine --- p.3 / Chapter 1.3 --- Properties of Arsenic Trioxide --- p.5 / Chapter 1.4 --- Use of Arsenic Trioxide in Cancer Treatment --- p.7 / Chapter 1.4.1 --- Arsenic Trioxide as a Therapeutic Agent in the Treatment of Acute Promyelocytic Leukemia --- p.7 / Chapter 1.4.1.1 --- Characteristics of Acute Promyelocytic Leukemia --- p.7 / Chapter 1.4.1.2 --- Treatment of Acute Promyelocytic Leukemia with All-Trans Retinoic Acid --- p.10 / Chapter 1.4.1.3 --- Treatment of Acute Promyelocytic Leukemia with Arsenic Trioxide --- p.11 / Chapter 1.4.1.4 --- Action Mechanism of Arsenic Trioxide --- p.13 / Chapter 1.4.2 --- Arsenic Trioxide as a Therapeutic Agent in the Treatment of Non-APL Leukemia --- p.15 / Chapter 1.4.3 --- Arsenic Trioxide as a Therapeutic Agent in the Treatment of Solid Tumors --- p.16 / Chapter 1.5 --- Human Hepatocellular Carcinoma --- p.16 / Chapter 1.5.1 --- The Incidence of Liver Cancer --- p.16 / Chapter 1.5.2 --- Classification of Liver Cancer --- p.17 / Chapter 1.6 --- Aim of the Project --- p.17 / Chapter 1.6.1 --- In Vitro Study of the Effect of Arsenic Trioxide on HepG2 Cells --- p.19 / Chapter 1.6.2 --- In Vivo Study of the Effect of Arsenic Trioxide by Tumor-Bearing Nude Mice Model --- p.20 / Chapter 1.6.3 --- "In Vitro Study of the Effect of Arsenic Trioxide on Multidrug-Resistant Human Hepatocellular Carcinoma Cell Line, R-HepG2" --- p.22 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.24 / Chapter 2.1.1 --- Cell Lines and Culture Medium --- p.24 / Chapter 2.1.1.1 --- Cell Lines --- p.24 / Chapter 2.1.1.2 --- Culture Medium --- p.25 / Chapter 2.1.2 --- Chemicals --- p.26 / Chapter 2.1.3 --- Reagents and Buffers --- p.27 / Chapter 2.1.3.1 --- Phosphate Buffered Saline (PBS) --- p.27 / Chapter 2.1.3.2 --- "3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) Solution" --- p.27 / Chapter 2.1.3.3 --- Reagents for DNA Fragmentation Assay --- p.21 / Chapter 2.1.3.3.1 --- DNA Lysis Buffer --- p.27 / Chapter 2.1.3.3.2 --- Tris-EDTA (TE) Buffer --- p.27 / Chapter 2.1.3.3.3 --- Tris-Acetate (TAE) Buffer --- p.28 / Chapter 2.1.3.3.4 --- Proteinase K and Ribonuclease A (RNase A) --- p.28 / Chapter 2.1.3.3.5 --- 6X DNA Loading Dye --- p.28 / Chapter 2.1.3.3.6 --- One Hundred Base-Pair DNA Ladder --- p.28 / Chapter 2.1.3.4 --- Reagents for Western Blot Analysis --- p.29 / Chapter 2.1.3.4.1 --- SDS Lysis Buffer --- p.29 / Chapter 2.1.3.4.2 --- 4X Lower Gel Buffer --- p.29 / Chapter 2.1.3.4.3 --- 4X Upper Gel Buffer --- p.29 / Chapter 2.1.3.4.4 --- 10X SDS Running Buffer --- p.29 / Chapter 2.1.3.4.5 --- 2X SDS Sample Loading Dye --- p.30 / Chapter 2.1.3.4.6 --- Electroblotting Buffer --- p.30 / Chapter 2.1.3.4.7 --- Tris-Buffered Saline with 01% Tween-20 (TBS-T) --- p.30 / Chapter 2.1.3.4.8 --- Lysis Buffer for Detection of the Release of Cytochrome C --- p.31 / Chapter 2.1.3.5 --- Propidium Iodide (PI) --- p.31 / Chapter 2.1.3.6 --- "5，5 ´ة,6,6´ة-tetrachloro-1,1',3,3 '-tetraethylbenzimidazolyl carbocyanine Iodide (JC-1)" --- p.31 / Chapter 2.1.3.7 --- Reagents for In Vivo Study --- p.32 / Chapter 2.1.3.7.1 --- Saline --- p.32 / Chapter 2.1.3.7.2 --- Homogenizing Buffer --- p.32 / Chapter 2.1.3.7.3 --- 10% Buffered Formalin --- p.32 / Chapter 2.1.3.7.4 --- Acid Alcohol --- p.32 / Chapter 2.1.3.7.5 --- Scott's Tap Water --- p.32 / Chapter 2.1.3.7.6 --- 0.5% Aqueous Eosin --- p.33 / Chapter 2.2 --- Methods --- p.33 / Chapter 2.2.1 --- MTT Assay --- p.33 / Chapter 2.2.2 --- Trypan Blue Exclusion Assay --- p.34 / Chapter 2.2.3 --- Analysis of Cell-Cycle Phase Distribution by Flow Cytometry with PI Staining --- p.34 / Chapter 2.2.4 --- DNA Fragmentation Assay --- p.35 / Chapter 2.2.5 --- Quantification of Apoptosis by Flow Cytometry with Annexin V-PI Staining --- p.36 / Chapter 2.2.6 --- Assessment of the Change in Mitochondrial Membrane Potential (ΔΦm) --- p.37 / Chapter 2.2.7 --- Western Analysis --- p.38 / Chapter 2.2.8 --- Glucose Uptake Assay --- p.40 / Chapter 2.2.9 --- ATP Production Assay --- p.41 / Chapter 2.2.10 --- In Vivo Study --- p.44 / Chapter 2.2.10.1 --- Animal Model --- p.44 / Chapter 2.2.10.2 --- Cell Line --- p.44 / Chapter 2.2.10.3 --- Treatment with Arsenic Trioxide --- p.44 / Chapter 2.2.10.4 --- Assessment of the Anti-Cancer Activity of Arsenic Trioxide --- p.45 / Chapter 2.2.10.5 --- Tissue Sample Preparation --- p.45 / Chapter 2.2.10.5.1 --- Preparation of Plasma --- p.45 / Chapter 2.2.10.5.2 --- Preparation of Liver Tissue Homogenate --- p.46 / Chapter 2.2.10.5.3 --- Preparation of Cytosolic Fraction --- p.46 / Chapter 2.2.10.6 --- Measurement of the Plasma Enzyme Activity --- p.46 / Chapter 2.2.10.6.1 --- "Plasma Creatine Kinase (CK) Activity, Plasma Lactate Dehydrogenase (LDH) Activity, Plasma Alanine Transaminase (ALT) Activity and Plasma Asparate Transaminase (AST) Activity" --- p.46 / Chapter 2.2.10.7 --- Preparation of Tissue for Light Microscopic Study --- p.48 / Chapter 2.2.10.8 --- Measurement of the Basal Reduced Glutathione (GSH) Level of Liver Tissue --- p.51 / Chapter 2.2.10.9 --- "Measurement of the Activity of Antioxidant Enzyme, Glutathione S-Transferase (GST) of Liver Tissue" --- p.53 / Chapter 2.3 --- Statistical Analysis --- p.54 / Chapter Chapter 3 --- "In Vitro Study of Arsenic Trioxide on Acute Promyelocytic Leukemia Cell Line, NB-4" / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Principle of Flow Cytometry with Annexin V-PI Staining --- p.56 / Chapter 3.3 --- The Effect of Arsenic Trioxide on Cell Proliferation of NB-4 Cells --- p.59 / Chapter 3.4 --- Study of the Action Mechanism of Arsenic Trioxide upon Treatment of NB-4 Cells --- p.61 / Chapter 3.5 --- Summary --- p.63 / Chapter Chapter 4 --- "In Vitro Study of Arsenic Trioxide on Human Hepatocellular Carcinoma Cell Line, HepG2" / Chapter 4.1 --- Introduction --- p.64 / Chapter 4.2 --- The Effect of Arsenic Trioxide on Cell Proliferation of HepG2 Cells by MTT Assay --- p.66 / Chapter 4.3 --- The Effect of Arsenic Trioxide on HepG2 Cells at Clinically Achievable Concentration --- p.68 / Chapter 4.3.1 --- The Cytotoxicity of Arsenic Trioxide on HepG2 Cells by Trypan Blue Exclusion Assay --- p.68 / Chapter 4.3.2 --- The Effect of Arsenic Trioxide on Cell-Cycle Phase Distribution --- p.71 / Chapter 4.3.3 --- The Underlying Mechanism of the Cytotoxic Effect of Arsenic Trioxide 一 Necrosis or Apoptosis? --- p.74 / Chapter 4.3.3.1 --- DNA Fragmentation Assay --- p.74 / Chapter 4.3.3.2 --- Flow Cytometry with Annexin V-PI Staining --- p.76 / Chapter 4.3.3.3 --- Brief Conclusion --- p.78 / Chapter 4.3.4 --- The Study of the Mechanism of Apoptotic Pathway --- p.78 / Chapter 4.3.4.1 --- Activation of Caspase-3 upon Arsenic Trioxide Treatment --- p.79 / Chapter 4.3.4.2 --- The Participation of Mitochondria in Arsenic Trioxide-Induced Apoptosis --- p.81 / Chapter 4.3.4.2.1 --- The Change in Mitochondrial Membrane Potential upon Arsenic Trioxide Treatment --- p.81 / Chapter 4.3.4.2.2 --- The Study of the Release of Cytochrome C from the Mitochondria to Cytosol upon Treatment with Arsenic Trioxide --- p.85 / Chapter 4.3.4.2.3 --- Brief Conclusion --- p.87 / Chapter 4.4 --- Arsenic Trioxide Mediated Its Effect via Other Action Mechanisms --- p.87 / Chapter 4.4.1 --- The Effect of Arsenic Trioxide on the Expression of Glucose Transporters 1 and2 --- p.88 / Chapter 4.4.2 --- The Effect of Arsenic Trioxide on Glucose Uptake --- p.91 / Chapter 4.4.3 --- The Effect of Arsenic Trioxide on ATP Production --- p.93 / Chapter 4.4.4 --- Brief Conclusion --- p.93 / Chapter 4.5 --- Summary --- p.95 / Chapter Chapter 5 --- In Vivo Study of Arsenic Trioxide on HepG2-Bearing Nude Mice / Chapter 5.1 --- Introduction --- p.96 / Chapter 5.2 --- Treatment with Arsenic Trioxide --- p.97 / Chapter 5.3 --- Assessment of the Anti-Tumor Effect of Arsenic Trioxide --- p.99 / Chapter 5.4 --- The Effect of Arsenic Trioxide toward Normal Tissues --- p.103 / Chapter 5.4.1 --- The Effect of Arsenic Trioxide on Liver --- p.104 / Chapter 5.4.1.1 --- Morphological Study --- p.104 / Chapter 5.4.1.2 --- Enzymatic Study --- p.107 / Chapter 5.4.1.3 --- Brief Conclusion --- p.107 / Chapter 5.4.2 --- The Effect of Arsenic Trioxide on Heart --- p.110 / Chapter 5.4.2.1 --- Morphological Study --- p.110 / Chapter 5.4.2.2 --- Enzymatic Study --- p.112 / Chapter 5.4.2.3 --- Brief Conclusion --- p.112 / Chapter 5.5 --- Involvement of the Glutathione Redox System --- p.115 / Chapter 5.5.1 --- Basal GSH Level --- p.115 / Chapter 5.5.2 --- The Activity of Glutathion S-Transferase --- p.117 / Chapter 5.5.3 --- Brief Conclusion --- p.117 / Chapter 5.6 --- Summary --- p.120 / Chapter Chapter 6 --- "In Vitro Study of Arsenic Trioxide on Multidrug-Resistant Human Hepatocellular Carcinoma Cell Line, R-HepG2" / Chapter 6.1 --- Introduction --- p.121 / Chapter 6.2 --- The Effect of Doxorubicin on the Parental HepG2 Cells and R-HepG2 Cells by MTT Assay --- p.123 / Chapter 6.3 --- The Effect of Arsenic Trioxide on Cell Proliferation of R-HepG2 Cells by MTT Assay --- p.126 / Chapter 6.4 --- The Effect of Arsenic Trioxide on Cell-Cycle Phase Distribution of R-HepG2 Cells --- p.129 / Chapter 6.5 --- Trioxide on R-HepG2 Cells ´ؤ Necrosis or Apoptosis? --- p.131 / Chapter 6.5.1 --- DNA Fragmentation Assay --- p.131 / Chapter 6.5.2 --- Flow Cytometry with Annexin V-PI Staining --- p.133 / Chapter 6.5.3 --- Brief Conclusion --- p.133 / Chapter 6.6 --- Examination of the Probable Involvement of Arsenic Trioxide as a Substrate of P-Glycoprotein --- p.135 / Chapter 6.7 --- Summary --- p.137 / Chapter Chapter 7 --- Discussion / Chapter 7.1 --- The Significance of the Study of Arsenic Trioxide in the Treatment of Arsenic Trioxide --- p.138 / Chapter 7.2 --- Comparison of Preparation of Drug in Present Study with Others --- p.140 / Chapter 7.3 --- Effect of Arsenic Trioxide on Human Hepatocellular Carcinoma --- p.142 / Chapter 7.4 --- Mechanism Study of Arsenic Trioxide --- p.142 / Chapter 7.5 --- Dosage of Arsenic Trioxide Used in In Vivo Study --- p.152 / Chapter 7.6 --- Cytotoxicity of Arsenic Trioxide toward Normal Tissues --- p.153 / Chapter 7.7 --- "Effect of Arsenic Trioxide on Multidrug-Resistant Human Hepatocellular Carcinoma Cell Line, R-HepG2" --- p.154 / Chapter 7.8 --- Conclusions and Future Prospect --- p.156 / Chapter Chapter 8 --- References / Chapter 8.1 --- English References --- p.158 / Chapter 8.2 --- Chinese References --- p.174 / Chapter 8.3 --- Online References --- p.174

Ong Chik Ying Rose. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 181-195). / Abstracts in English and Chinese. / Acknoledgements --- p.i / List of Abbreviations --- p.ii / Abstract --- p.iii / Abstract in Chinese --- p.v / List of Publications and Abstracts --- p.vii / List of Figures --- p.viii / List of Tables --- p.xi / Contents --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Saponins --- p.3 / Chapter 1.1.1 --- Structure of saponins --- p.3 / Chapter 1.1.2 --- Occurrence of saponins --- p.7 / Chapter 1.1.3 --- Bioactivities of saponins --- p.8 / Chapter 1.2 --- Paris Polyphylla --- p.9 / Chapter 1.2.1 --- Chonglou --- p.11 / Chapter 1.3 --- Polyphyllin D --- p.12 / Chapter 1.4 --- Apoptosis --- p.15 / Chapter 1.4.1 --- Apoptosis and necrosis --- p.16 / Chapter 1.4.2 --- Initiation phase of apoptosis --- p.16 / Chapter 1.4.2.1 --- Extrinsic apoptotic pathway --- p.18 / Chapter 1.4.2.2 --- Intrinsic apoptotic pathway --- p.19 / Chapter 1.4.3 --- Execution phase of apoptosis --- p.19 / Chapter 1.4.4 --- Termination phase of apoptosis --- p.20 / Chapter 1.5 --- Multi-drug resistance (MDR) --- p.21 / Chapter 1.5.1 --- MDR mediated by decreased drug accumulation --- p.21 / Chapter 1.5.2 --- MDR mediated by enhanced anti-oxidant enzyme activities --- p.25 / Chapter 1.5.3 --- MDR mediated by enhanced detoxification of drugs --- p.25 / Chapter 1.5.4 --- MDR mediated by enhanced DNA repair system --- p.26 / Chapter 1.5.5 --- MDR mediated by altered apoptotic pathway --- p.26 / Chapter 1.5.6 --- Current strategies for overcoming multidrug resistance in cancer --- p.27 / Chapter 1.6 --- Hepatocellular carcinoma (HCC) --- p.30 / Chapter 1.7 --- Objectives of project --- p.32 / Chapter Chapter 2 --- Materials and Methods --- p.33 / Chapter 2.1 --- Materials --- p.34 / Chapter 2.1.1 --- Culture of Cells --- p.34 / Chapter 2.1.1.1 --- Cell lines --- p.34 / Chapter 2.1.1.2 --- Preservation of Cells --- p.35 / Chapter 2.1.2 --- Culture Media --- p.36 / Chapter 2.1.2.1 --- RPMI 1640 (Phenol Red Medium) --- p.36 / Chapter 2.1.2.2 --- RPMI 1640 (Phenol Red-free Medium) --- p.36 / Chapter 2.1.3 --- Buffers and Reagents --- p.37 / Chapter 2.1.3.1 --- Buffer for Common Use --- p.37 / Chapter 2.1.3.2 --- Reagents for Annexin-V-FITC/PI Assay --- p.37 / Chapter 2.1.3.3 --- Reagents for Western Blotting Analysis --- p.37 / Chapter 2.1.4 --- Chemicals --- p.40 / Chapter 2.1.4.1 --- Fluorescence Dyes --- p.40 / Chapter 2.1.4.2 --- Antibodies --- p.41 / Chapter 2.1.4.3 --- Other Chemicals --- p.42 / Chapter 2.1.5 --- Summary of chemicals used in this study --- p.43 / Chapter 2.2 --- Methods --- p.48 / Chapter 2.2.1 --- In vitro Cell Cytotoxicity Assay --- p.48 / Chapter 2.2.1.1 --- AlamarBlue Assay --- p.48 / Chapter 2.2.2 --- Flow Cytometry --- p.50 / Chapter 2.2.2.1 --- Analysis by Flow Cytometry --- p.50 / Chapter 2.2.2.2 --- Determination of Apoptotic and Late Apoptotic/Necrotic Cells with Annexin-V-FITC/PI Cytometric Analysis --- p.50 / Chapter 2.2.2.3 --- Determination of Mitochondrial Membrane Potential in cells --- p.51 / Chapter 2.2.2.4 --- Determination of Hydrogen Peroxide (H2O2) Release in cells --- p.52 / Chapter 2.2.2.5 --- Measurement of doxorubicin accumulation in cells --- p.53 / Chapter 2.2.2.6 --- Determination of P-glycoprotein expression level in cells --- p.54 / Chapter 2.2.2.7 --- Determination of mitochondrial depolarization and swellingin isolated mitochondria --- p.54 / Chapter 2.2.3 --- Methods involved in DNA sequencing of MDRl promoter --- p.56 / Chapter 2.2.3.1 --- DNA extraction --- p.56 / Chapter 2.2.3.2 --- DNA and Gel Band Purification --- p.56 / Chapter 2.2.3.3 --- Assessment of DNA amount --- p.57 / Chapter 2.2.3.4 --- Polymerase Chain Reaction --- p.57 / Chapter 2.2.3.5 --- Agarose Gel Electrophoresis --- p.59 / Chapter 2.2.3.6 --- Preparation for DNA sequencing --- p.59 / Chapter 2.2.4 --- Western Blotting Analysis --- p.61 / Chapter 2.2.4.1 --- Preparation of Proteins from Cells --- p.61 / Chapter 2.2.4.2 --- Preparation of proteins from isolated mitochondria --- p.63 / Chapter 2.2.4.3 --- Protein analysis with Western analysis --- p.63 / Chapter 2.2.5 --- Confocal laser scanning microscopy (Confocal microscopy) --- p.66 / Chapter 2.2.5.1 --- Analysis with confocal microscopy --- p.66 / Chapter 2.2.5.2 --- Determination of mitochondrial changes in cells by confocal microscopy --- p.66 / Chapter 2.2.5.3 --- Determination of lysosomal rupture in cells by confocal microscopy --- p.67 / Chapter 2.2.6 --- Mitochondrial isolation --- p.68 / Chapter Chapter 3 --- Results 一 Resistance Mechanisms in RHepG2 cells --- p.69 / Chapter 3.1 --- Resistance of RHepG2 cells towards various chemical agents --- p.70 / Chapter 3.1.1 --- RHepG2 cells are resistant to doxorubicin --- p.70 / Chapter 3.1.2 --- RHepG2 cells are resistant to taxol --- p.72 / Chapter 3.1.3 --- RHepG2 cells are resistant to valinomycin --- p.74 / Chapter 3.2 --- Resistance mechanism in RHepG2 cells --- p.76 / Chapter 3.2.1 --- Reduced doxorubicin accumulation is observed in RHepG2 cells --- p.76 / Chapter 3.2.2 --- More P-glycoproteins on the cell surface was observed in RHepG2 cells --- p.80 / Chapter 3.2.3 --- Inhibition of P-glycoprotein activity increased doxorubicin accumulation in RHepG2 cells --- p.82 / Chapter 3.2.4 --- HepG2 and RHepG2 cells contain the same P-glycoprotein promoter region --- p.86 / Chapter 3.2.5 --- RHepG2 over-expressed Bcl-2 --- p.91 / Chapter 3.2.6 --- HepG2 and RHepG2 cells had the same level of Bax protein --- p.93 / Chapter Chapter 4 --- Results - Effects of Polyphyllin D in HepG2 and RHepG2 cells --- p.95 / Chapter 4.1 --- Cytotoxicity of Polyphyllin D in HepG2 and RHepG2 cells --- p.96 / Chapter 4.1.1 --- Polyphyllin D exhibited cytotoxic effect in both HepG2 and RHepG2 cells --- p.96 / Chapter 4.2 --- Apoptotic mechanism caused by Polyphyllin D in HepG2 and RHepG2 cells --- p.93 / Chapter 4.2.1 --- Polyphyllin D caused apoptosis in HepG2 and RHepG2 cells --- p.98 / Chapter 4.2.2 --- Polyphyllin D did not activate caspase8 --- p.103 / Chapter 4.2.3 --- Polyphyllin D did not concentrate on the plasma membrane of cells --- p.106 / Chapter 4.2.4 --- Polyphyllin D did not change Bcl-2 level in HepG2 and RHepG2 cells --- p.109 / Chapter 4.2.5 --- Polyphyllin D treatment enhanced Bax protein expression in both HepG2 and RHepG2 cells --- p.111 / Chapter 4.2.6 --- Polyphyllin D caused cytochrome c and AIF release in HepG2 and RHepG2 cells --- p.113 / Chapter 4.2.7 --- Polyphyllin D induced mitochondrial membrane depolarization in HepG2 and RHepG2 cells --- p.118 / Chapter 4.2.8 --- Polyphyllin D caused mitochondrial swelling in HepG2 and clustering of mitochondriain RHepG2 cells --- p.122 / Chapter 4.2.9 --- "Polyphyllin D caused H202 release in HepG2 and RHepG2 cells, and the cytotoxic effects of Polyphyllin D could be reduced by NAC" --- p.127 / Chapter 4.2.10 --- Polyphyllin D caused permeabilization of lysosomes --- p.132 / Chapter 4.3 --- Site of action of Polyphyllin D in cells --- p.135 / Chapter 4.3.1 --- Purity of isolated mitochondria --- p.135 / Chapter 4.3.2 --- Polyphyllin D caused cytochrome c release from the HepG2 and RHepG2 isolated mitochondria --- p.137 / Chapter 4.3.3 --- Polyphyllin D induced mitochondrial depolarization in HepG2 and RHepG2 isolated mitochondria --- p.139 / Chapter 4.3.4 --- Polyphyllin D caused mitochondrial swelling in HepG2 and RHepG2 isolated mitochondria --- p.142 / Chapter 4.4 --- Resistance reversal effects of Polyphyllin D in RHepG2 cells --- p.144 / Chapter 4.4.1 --- Polyphyllin D increased doxorubicin accumulation in RHepG2 cells --- p.144 / Chapter 4.4.2 --- P-glycoprotein expression was not down-regulated after Polyphyllin D treatment --- p.146 / Chapter 4.4.3 --- Co-treatment of doxorubicin with Polyphyllin D had enhanced cytotoxic effect --- p.148 / Chapter Chapter 5 --- Discussion - Resistance mechanisms in RHepG2 cells --- p.150 / Chapter 5.1 --- Resistance of RHepG2 cells towards various chemical reagents --- p.151 / Chapter 5.2 --- Resistance mechanisms in RHepG2 cells --- p.154 / Chapter Chapter 6 --- Discussion - Effects of Polyphyllin D in HepG2and RHepG2 cells --- p.159 / Chapter 6.1 --- Cytotoxicity of Polyphyllin D in HepG2 and RHepG2 cells --- p.160 / Chapter 6.2 --- Apoptotic mechanisms caused by Polyphyllin D in HepG2 and RHepG2 cells --- p.162 / Chapter 6.3 --- Site of action of Polyphyllin D in HepG2 andRHepG2 cells --- p.172 / Chapter 6.4 --- Resistance reversal effects of Polyphyllin D in RHepG2 cells --- p.175 / Chapter Chapter 7 --- Future Perspectives --- p.177 / Chapter Chapter 8 --- Conclusion --- p.179 / References

Lau Yee Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 147-157). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Abbreviations --- p.v / List of Figures --- p.viii / List of Tables --- p.xi / Contents --- p.xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Hepatocellular carcinoma (HCC) --- p.1 / Chapter 1.1.1 --- Background of hepatocellular carcinoma (HCC) --- p.1 / Chapter 1.1.2 --- Etiology of HCC --- p.2 / Chapter 1.1.3 --- Relationship between HCC and HBV --- p.3 / Chapter 1.1.4 --- Differential gene expression under induction of HBx protein by microarray analysis --- p.5 / Chapter 1.1.5 --- Confirmation of candidate genes --- p.6 / Chapter 1.2 --- Ras-Oncogene --- p.8 / Chapter 1.2.1 --- Ras superfamily --- p.8 / Chapter 1.2.1.1 --- Rho family --- p.9 / Chapter 1.2.1.2 --- Rab family --- p.10 / Chapter 1.2.2 --- Functional mechanism of small GTPase --- p.11 / Chapter 1.2.3 --- Possible functions of Rho and Rab family members --- p.14 / Chapter 1.3 --- RhoC --- p.16 / Chapter 1.3.1 --- The genomic and protein structures of RhoC --- p.16 / Chapter 1.3.2 --- Relationship between RhoC and tumours --- p.19 / Chapter 1.4 --- Rabl4 --- p.20 / Chapter 1.4.1 --- The genomic and protein structures of Rabl4 --- p.20 / Chapter 1.4.2 --- Relationship between Rabl4 and tumours --- p.23 / Chapter 1.5 --- Aims of study --- p.23 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.25 / Chapter 2.1.1 --- Cell lines --- p.25 / Chapter 2.1.2 --- Cell culture reagents --- p.26 / Chapter 2.1.3 --- Reagents for total RNA isolation --- p.29 / Chapter 2.1.4 --- Reagents for reverse transcription polymerase chain reaction (RT-PCR) --- p.30 / Chapter 2.1.5 --- Reagents and buffers for Western blot analysis --- p.31 / Chapter 2.1.6 --- Vectors for cloning --- p.39 / Chapter 2.1.7 --- Reagents for polymerase chain reaction (PCR) --- p.39 / Chapter 2.1.8 --- Restriction digestion reagents --- p.42 / Chapter 2.1.9 --- Reagents for agarose gel electrophoresis --- p.42 / Chapter 2.1.10 --- Ligation reagents --- p.44 / Chapter 2.1.11 --- Bacterial culture medium --- p.44 / Chapter 2.1.12 --- Dyes and reagents for fluorescent microscope --- p.46 / Chapter 2.1.13 --- Reagents for flow cytometry --- p.48 / Chapter 2.1.14 --- Detection of apoptosis --- p.48 / Chapter 2.2 --- Methods --- p.50 / Chapter 2.2.1 --- Identification of gene expression of candidate genes in HCC --- p.50 / Chapter 2.2.1.1 --- cDNA preparation --- p.50 / Chapter (1) --- Cell culture of HepG2 and WRL-68 cell lines --- p.50 / Chapter (2) --- Total RNA isolation --- p.50 / Chapter (3) --- First-strand cDNA synthesis --- p.51 / Chapter 2.2.1.2 --- RT-PCR of candidate genes --- p.52 / Chapter 2.2.1.3 --- Western blotting --- p.53 / Chapter (1) --- Cell culture --- p.53 / Chapter (2) --- Protein extraction --- p.53 / Chapter (3) --- Quantification of proteins --- p.53 / Chapter (4) --- Detection of RhoC and Rabl4 protein by western blot analysis --- p.54 / Chapter (5) --- Western blotting luminol detection --- p.56 / Chapter 2.2.2 --- Cloning protocol --- p.57 / Chapter 2.2.2.1 --- Amplification of RhoC and Rabl4 genes --- p.57 / Chapter 2.2.2.2 --- Purification of PCR product --- p.58 / Chapter 2.2.2.3 --- Restriction enzymes digestion --- p.53 / Chapter 2.2.2.4 --- Insert/vector ligation --- p.59 / Chapter 2.2.2.5 --- Preparation of chemically competent bacterial cells (E. coli strain DH5a) --- p.60 / Chapter 2.2.2.6 --- Transformation of ligation product into chemically competent bacterial cells --- p.61 / Chapter 2.2.2.7 --- Small-scale preparation of bacterial plasmid DNA --- p.61 / Chapter 2.2.2.8 --- Screening for recombinant clones --- p.62 / Chapter 2.2.2.9 --- DNA sequencing of cloned plasmid DNA --- p.63 / Chapter 2.2.2.10 --- Midi-scale preparation of recombinant plasmid DNA --- p.64 / Chapter 2.2.3 --- Visualization of the subcellular localization patterns --- p.66 / Chapter 2.2.3.1 --- Cell culture of AML12 and HepG2 cell lines --- p.66 / Chapter 2.2.3.2 --- Transfection of GFP fusion constructs into cells --- p.66 / Chapter 2.2.3.3 --- DAPI staining --- p.67 / Chapter 2.2.3.4 --- ER-Tracker´ёØ Blue-White DPX staining --- p.68 / Chapter 2.2.3.5 --- Subcellular localization study using Epi-fluorescence microscopy --- p.68 / Chapter 2.2.4 --- Analysis of cell cycle --- p.69 / Chapter 2.2.4.1 --- Transfection of GFP vectors / GFP-tagged proteins into cells --- p.69 / Chapter 2.2.4.2 --- Analysis of cell cycle by flow cytometry --- p.69 / Chapter 2.2.5 --- Detection of apoptosis --- p.70 / Chapter 2.2.5.1 --- Transfection --- p.70 / Chapter 2.2.5.2 --- Detection of DNA fragmentation --- p.70 / Chapter 2.2.6 --- Reorganization of Actin cytoskeleton by RhoC --- p.71 / Chapter 2.2.6.1 --- Transfection of GFP vectors/GFP-tagged proteins into cells --- p.71 / Chapter 2.2.6.2 --- Rhodamine phalloidin (RP) staining --- p.71 / Chapter 2.2.6.3 --- Epi-fluorescence microscopy --- p.72 / Chapter 2.2.7 --- Analysis of cell invasion under induction of RhoC --- p.72 / Chapter 2.2.7.1 --- "Sub-cloning of human RhoC gene into a mammalian expression vector, pHM6" --- p.72 / Chapter 2.2.7.2 --- Transfection of pHM6-RhoC --- p.73 / Chapter 2.2.7.3 --- Cell invasion assay --- p.73 / Chapter 2.2.8 --- Analysis of downstream effectors in RhoC-mediated pathway --- p.75 / Chapter 2.2.8.1 --- RT-PCR --- p.75 / Chapter 2.2.8.2 --- Western blotting --- p.75 / Chapter 2.2.9 --- Analysis of role of Rabl4 in membrane trafficking --- p.76 / Chapter 2.2.9.1 --- Cloning and transfection --- p.76 / Chapter 2.2.9.2 --- Alexa 594 transferrin conjugate staining --- p.76 / Chapter 2.2.9.3 --- Epi-fluorescence microscopy --- p.77 / Chapter 2.2.10 --- Statistics --- p.77 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Expression of RhoC and Rabl4 in hepatoma cells --- p.78 / Chapter 3.1.1 --- RT-PCR --- p.78 / Chapter 3.1.2 --- Western blotting --- p.81 / Chapter 3.2 --- Subcellular localization of RhoC and Rab 14 --- p.85 / Chapter 3.3 --- Characterization of RhoC --- p.93 / Chapter 3.3.1 --- Cell cycle analysis --- p.93 / Chapter 3.3.2 --- Apoptosis --- p.95 / Chapter 3.3.3 --- Actin cytoskeleton reorganization --- p.97 / Chapter 3.3.4 --- Cell invasion ability --- p.99 / Chapter 3.3.5 --- Downstream effectors of RhoC in cytoskeletal reorganization --- p.102 / Chapter 3.4 --- Characterization of Rabl4 --- p.107 / Chapter 3.4.1 --- Cell cycle analysis --- p.107 / Chapter 3.4.2 --- Apoptosis --- p.109 / Chapter 3.4.3 --- Roles in intracellular transportation --- p.111 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Strong expression of RhoC and Rabl4 in hepatoma cells --- p.117 / Chapter 4.2 --- Subcellular localization of RhoC and Rabl4 --- p.119 / Chapter 4.3 --- The effects of RhoC in normal liver cells --- p.122 / Chapter 4.3.1 --- Cell cycle progression by RhoC through regulating of G1 to S phase transition --- p.122 / Chapter 4.3.2 --- RhoC shows no apoptotic effect in normal liver cell systems --- p.123 / Chapter 4.3.3 --- Formation of actin filaments and stress fibers --- p.124 / Chapter 4.3.4 --- Induction of cell invasion in RhoC-expressing cells --- p.125 / Chapter 4.3.5 --- Downstream effectors in signaling pathway of RhoC in actin filment reorganization and cell invasion --- p.126 / Chapter 4.4 --- The effects of Rabl4 in normal liver cells --- p.132 / Chapter 4.4.1 --- Cell proliferation effects of Rabl4 by increasing percentage of cells in S phase for DNA synthesis --- p.132 / Chapter 4.4.2 --- Rabl4 has no apoptotic effects --- p.133 / Chapter 4.4.3 --- Roles of Rabl4 in vesicular transport --- p.134 / Chapter 4.5 --- Conclusion --- p.138 / Chapter 4.6 --- Future prospects --- p.140 / Appendix --- p.143 / References --- p.147

Leung Chin-lung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 133-164). / Abstracts in English and Chinese. / Abstract --- p.iv / 摘要 --- p.vi / List of abbreviation --- p.viii / Chapter Chapter 1 --- Hepatocellular Carcinoma --- p.1 / Chapter 1.1 --- A Health Burden --- p.1 / Chapter 1.2 --- Pathology --- p.3 / Chapter 1.3 --- Epidemiology --- p.7 / Chapter 1.3.1 --- Global HCC distribution --- p.7 / Chapter 1.3.2 --- Age and Gender --- p.10 / Chapter 1.4 --- Risk Factors of HCC --- p.12 / Chapter 1.4.1 --- Hepatitis B virus (HBV) --- p.13 / Chapter 1.4.1.1 --- Chronic HBV infection --- p.13 / Chapter 1.4.1.2 --- Role of HBV in hepatocarcinogenesis --- p.16 / Chapter 1.4.1.2 a) --- Direct Oncogenesis --- p.16 / Chapter 1.4.1.2 b) --- Indirect Oncogenesis --- p.17 / Chapter 1.4.2 --- Hepatitis C virus (HCV) --- p.23 / Chapter 1.4.2.1 --- Chronic HCV infection --- p.23 / Chapter 1.4.2.2 --- Role of HCV in hepatocarcinogenesis --- p.23 / Chapter 1.4.3 --- Chemicals as liver carcinogens --- p.27 / Chapter 1.4.3.1 --- Aflatoxin Bi (AFB1) --- p.28 / Chapter 1.4.3.2 --- Vinyl chloride --- p.29 / Chapter 1.4.3.3 --- Alcoholic beverages --- p.29 / Chapter 1.4.4 --- Inborn Errors in Metabolisms --- p.30 / Chapter 1.4.4.1 --- Hereditary tyrosinemia --- p.30 / Chapter 1.4.4.2 --- Hereditary haemochromatosis --- p.30 / Chapter 1.4.4.3 --- α1-antitrypsin deficiency --- p.31 / Chapter 1.4.5 --- Liver lesions --- p.32 / Chapter 1.5 --- Genetic alterations in HCC --- p.33 / Chapter Chapter 2 --- Rationale of the study --- p.39 / Chapter Chapter 3 --- LOH study on 8p in HCC --- p.48 / Chapter 3.1 --- Introduction --- p.48 / Chapter 3.1.1 --- "Knudson's ""two-hit"" model and LOH" --- p.48 / Chapter 3.1.2 --- Microsatellite DNA and LOH study --- p.49 / Chapter 3.2 --- Materials and Methods --- p.51 / Chapter 3.2.1 --- Patients and Specimens --- p.51 / Chapter 3.2.1.1 --- Genomic DNA extraction from liver tissues --- p.53 / Chapter 3.2.1.2 --- Genomic DNA extraction from buffy coat --- p.55 / Chapter 3.3 --- LOH study on 8p in HCC --- p.57 / Chapter 3.3.1 --- Microsatellite markers --- p.57 / Chapter 3.3.2 --- 5-end labeling --- p.60 / Chapter 3.3.3 --- Amplification of microsatellite DNA --- p.60 / Chapter 3.3.4 --- Denaturing polyacrylamide gel electrophoresis --- p.61 / Chapter 3.3.5 --- Detection of LOH --- p.62 / Chapter 3.4 --- Results --- p.63 / Chapter 3.4.1 --- LOH status of 52 HCC cases --- p.63 / Chapter 3.4.2 --- Clinicopathological correlation --- p.67 / Chapter 3.4.3 --- Delineation of common deletion region (CDR) --- p.67 / Chapter 3.4.4 --- Common deletion region of interest --- p.77 / Chapter Chapter 4 --- Study on LZTS1 --- p.83 / Chapter 4.1 --- Introduction 一 LZTS1 --- p.83 / Chapter 4.2 --- Mutation analysis of LZTS1 in HCC --- p.87 / Chapter 4.2.1 --- Materials and Methods --- p.87 / Chapter 4.2.1.1 --- Patients and HCC cell lines --- p.87 / Chapter 4.2.1.2 --- Genomic DNA extraction from HCC cell lines --- p.87 / Chapter 4.2.1.3 --- Amplification of exons of LZTS1 --- p.89 / Chapter 4.2.1.3a) --- Primer pairs --- p.89 / Chapter 4.2.1.3b) --- PCR conditions --- p.90 / Chapter 4.2.1.4 --- Purification of PCR products --- p.93 / Chapter 4.2.1.5 --- Cycle sequencing reaction --- p.94 / Chapter 4.2.1.6 --- Purification of cycle sequencing reaction product --- p.94 / Chapter 4.2.1.7 --- Sequence analysis by automated sequencer --- p.95 / Chapter 4.2.1.8 --- Search for sequence variants of LZTS1 --- p.96 / Chapter 4.2.2 --- Results --- p.97 / Chapter 4.3 --- Expression analysis of LZTS1 in HCC with preliminary results --- p.103 / Chapter 4.3.1 --- Materials and Methods --- p.103 / Chapter 4.3.1.1 --- Patients and Specimens --- p.103 / Chapter 4.3.1.2 --- Total RNA extraction --- p.103 / Chapter 4.3.1.3 --- Reverse transcription --- p.104 / Chapter 4.3.1.4 --- Semi-quantitative PCR --- p.105 / Chapter 4.3.1.4a) --- Primer pairs --- p.105 / Chapter 4.3.1.4b) --- PCR conditions --- p.106 / Chapter 4.3.2 --- Results --- p.109 / Chapter Chapter 5 --- Discussion --- p.111 / Chapter 5.1 --- LOH study on 8p in HCC --- p.111 / Chapter 5.2 --- Study on LZTS1 in HCC --- p.125 / Chapter 5.2.1 --- Mutation analysis of LZTS1 --- p.125 / Chapter 5.2.2 --- Expression analysis of LZTS1 --- p.129 / Chapter 5.3 --- Future Study --- p.132 / References --- p.133

Yip, Ka Yan. / "August 2011." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 87-113). / Abstracts in English and Chinese. / Acknowledgement --- p.III / Abstract --- p.V / 論文摘要 --- p.VI / Table of Contents --- p.VII / List of Figures --- p.IX / List of Abbreviations --- p.XI / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Hepatocellular carcinoma --- p.1 / Chapter 1.1.1 --- Overview --- p.1 / Chapter 1.1.2 --- Risk factors --- p.3 / Chapter 1.1.3 --- Treatment ofHCC --- p.12 / Chapter 1.2 --- Berberine - a compound derived from Traditional Chinese Medicine --- p.15 / Chapter 1.2.1 --- Traditional Chinese Medicine --- p.15 / Chapter 1.2.2 --- Berberine --- p.16 / Chapter 1.3 --- Cell cycle --- p.18 / Chapter 1.3.1 --- An Overview of cell cycle --- p.18 / Chapter 1.3.2 --- Cell cycle and carcinogenesis --- p.18 / Chapter 1.4 --- Molecular mechanism of apoptosis --- p.20 / Chapter 1.4.1 --- Overview of apoptosis --- p.20 / Chapter 1.4.2 --- Caspases cascade --- p.22 / Chapter 1.4.3 --- Bcl-2 family --- p.24 / Chapter 1.5 --- Apoptosis as a target of cancer therapy --- p.26 / Chapter 1.6 --- Aims of study --- p.27 / Chapter Chapter 2 --- Materials and Methods --- p.28 / Chapter 2.1 --- Cell culture and treatment --- p.28 / Chapter 2.1.1 --- Cell lines --- p.28 / Chapter 2.1.2 --- Berberine --- p.29 / Chapter 2.1.3 --- Chemicals and reagents --- p.29 / Chapter 2.1.4 --- Preparation of solutions --- p.29 / Chapter 2.1.5 --- Procedures --- p.31 / Chapter 2.2 --- Apoptosis detection by FITC Annexin V and PI co-staining --- p.33 / Chapter 2.2.1 --- Chemicals and reagents --- p.33 / Chapter 2.2.2 --- Procedures --- p.33 / Chapter 2.3 --- Gene expression in Berberine-induced apoptotic cells --- p.35 / Chapter 2.3.1 --- Chemicals and Reagents --- p.35 / Chapter 2.3.2 --- Procedures --- p.35 / Chapter 2.4 --- Protein expression in Berberine-induced apoptotic cells --- p.38 / Chapter 2.4.1 --- Chemicals and Reagents --- p.38 / Chapter 2.4.2 --- Preparation of solution --- p.39 / Chapter 2.4.3 --- Procedures --- p.41 / Chapter 2.5 --- Caspase cascade studies in berberine-induced apoptosis --- p.43 / Chapter 2.5.1 --- Chemicals and reagents --- p.43 / Chapter 2.5.2 --- Procedures --- p.43 / Chapter 2.6 --- Cell cycle study in berberine-induced apoptotic cells --- p.44 / Chapter 2.6.1 --- Chemicals and Reagents --- p.44 / Chapter 2.6.2 --- Preparation of solutions --- p.44 / Chapter 2.6.3 --- Procedures --- p.44 / Chapter Chapter 3 --- Results --- p.46 / Chapter 3.1 --- Berberine induces apoptosis in hepatocellular cells --- p.46 / Chapter 3.2 --- Gene expression in Berberine-induced apoptotic cells --- p.53 / Chapter 3.3 --- Caspase cascade studies in berberine-induced apoptosis --- p.58 / Chapter 3.4 --- Protein expression in Berberine-induced apoptotic cells --- p.62 / Chapter 3.5 --- Berberine caused G1 cell cycle arrest in HCC cell lines --- p.65 / Chapter Chapter 4 --- Discussion --- p.76 / References --- p.87

by Zhong Sheng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (p. 186-203). / 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.