Yip, Wing Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 100-119). / Abstract also in Chinese. / Abstract (English version) --- p.i / Abstract (Chinese version) --- p.iii / Acknowledgments --- p.v / Table of Contents --- p.vii / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xiii / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Hepatocellular Carcinoma --- p.1 / Chapter 1.1.1 --- Epidermiology --- p.1 / Chapter 1.1.2 --- Etiology --- p.1 / Chapter 1.2 --- Hepatitis B Virus --- p.3 / Chapter 1.2.1 --- The Epidermiology of Hepatitis B Virus Infection --- p.3 / Chapter 1.2.2 --- The Morphology and Genome of Hepatitis B Virus --- p.4 / Chapter 1.2.3 --- HBV Genotypes and Their Significance --- p.8 / Chapter 1.3 --- Hepatitis B Virus X Protein --- p.9 / Chapter 1.3.1 --- HBx Alters Various Signal Transduction Pathways --- p.10 / Chapter 1.3.2 --- HBx Interacts with Various Transcription Factors and Co-activators --- p.12 / Chapter 1.3.3 --- HBx Induces Epigenetic Alterations --- p.14 / Chapter 1.3.4 --- Identification of COOH-terminal Truncated HBx in Liver Tumors --- p.15 / Chapter 1.4 --- MicroRNAs --- p.17 / Chapter 1.4.1 --- Transcriptional Regulation and Biogenesis of MicroRNAs --- p.18 / Chapter 1.4.2 --- MicroRNAs and Cancer --- p.21 / Chapter 1.4.3 --- MicroRNAs and HCC --- p.25 / Chapter 1.5 --- Hypothesis and Aims of the Study --- p.29 / Chapter CHAPTER 2 --- MATERIALS and METHODS --- p.30 / Chapter 2.1 --- Patients --- p.30 / Chapter 2.2 --- Cell Lines --- p.30 / Chapter 2.3 --- Cloning of Various HBx Constructs --- p.32 / Chapter 2.3.1 --- PCR Amplification of HBx Fragments --- p.32 / Chapter 2.3.2 --- Cloning of HBx Fragments into TA-vectos --- p.33 / Chapter 2.3.3 --- Heat Shock Transformation --- p.33 / Chapter 2.3.4 --- Sub-cloning of HBx Fragments into Lentiviral Vectors --- p.34 / Chapter 2.4 --- Generation of Lentivirus --- p.37 / Chapter 2.4.1 --- Lentivirus Infection --- p.37 / Chapter 2.5 --- RNA Extraction --- p.38 / Chapter 2.6 --- Western Blot Analysis --- p.39 / Chapter 2.7 --- MiRNA Microarray --- p.40 / Chapter 2.7.1 --- Cyanine3-pCp Labeling of RNA Samples --- p.40 / Chapter 2.7.2 --- Sample Hybridization --- p.41 / Chapter 2.7.3 --- Microarray Wash --- p.41 / Chapter 2.7.4 --- Array Slide Scanning and Processing --- p.41 / Chapter 2.8 --- Detection of HBx Gene Deletion by PCR --- p.43 / Chapter 2.9 --- Immunohistochemistry --- p.44 / Chapter 2.10 --- Quantitative Real-time PCR --- p.45 / Chapter 2.11 --- Proliferation Assay --- p.47 / Chapter 2.12 --- Cell Cycle Analysis --- p.48 / Chapter 2.13 --- Annexin V Apoptosis Assay --- p.49 / Chapter 2.14 --- Colony Formation Assay --- p.50 / Chapter 2.15 --- Statistical Analysis --- p.51 / Chapter CHAPTER 3 --- RESULTS --- p.52 / Chapter 3.1 --- Detection of Full-length and COOH-terminal Truncated HBx in HCC Tissues --- p.52 / Chapter 3.2 --- Confirmation of HBx Expression in HCC Tissues --- p.55 / Chapter 3.3 --- Comparison of HBx from Different HBV Genotypes for Study --- p.61 / Chapter 3.4 --- Functional Characterization of COOH-tterminal Truncated HBx --- p.64 / Chapter 3.4.1 --- Selection of COOH-terminal Truncated HBx --- p.64 / Chapter 3.4.2 --- Generation of Various HBx-expressing Hepatocyte Cell Lines --- p.66 / Chapter 3.4.3 --- Effect of Full-length and COOH-terminal Truncated HBx on Cell Proliferation --- p.69 / Chapter 3.4.4 --- Effect of Full-length and COOH-terminal Truncated HBx Cell Cycle --- p.34 / Chapter 3.4.5 --- Effect of Full-length and COOH-terminal Truncated HBx on Apoptosis --- p.45 / Chapter 3.5 --- MicroRNA Profiling of Various HBx-expressing Hepatocyte Cell Lines --- p.76 / Chapter 3.5.1 --- Identification of Deregulated MicroRNAs by Microarray --- p.76 / Chapter 3.5.2 --- Validation of Deregulated MicroRNAs by Real-time PCR Analysis --- p.80 / Chapter 3.5.3 --- Confirmation of Deregulated MiRNAs in HCC and Adjacent Non-tumor Tissues --- p.84 / Chapter 3.5.4 --- Potential Downstream Targets of the HBx-deregulated MiRNAs --- p.87 / Chapter CHAPTER 4 --- DISCUSSION --- p.91 / Chapter 4.1 --- The Impact of COOH-terminal Truncated HBx in HCC --- p.91 / Chapter 4.2 --- The Biological Significance of COOH-terminal Truncated HBx Induced MiRNAs --- p.94 / Chapter 4.3 --- Limitations of the Present Study --- p.97 / Chapter 4.4 --- Future Studies --- p.98 / Chapter CHAPTER 5 --- CONCLUSION --- p.99 / REFERENCES --- p.100

Leung, Chung Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 100-113). / Abstracts in English and Chinese. / Abstract --- p.I / 摘要 --- p.III / Acknowledgements --- p.V / List of figures --- p.VI / List of tables --- p.VIII / Abbreviations --- p.IX / Table of Contents --- p.XII / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Epidemiology of hepatocellular carcinoma (HCC) --- p.1 / Chapter 1.2 --- Etiology of heptocellular carcinoma (HCC) --- p.1 / Chapter 1.3 --- HBV genome structure --- p.2 / Chapter 1.4 --- HBV pathogenesis --- p.2 / Chapter 1.5 --- Hepatitis B virus X protein (HBx) --- p.3 / Chapter 1.6 --- Oxidative stress and antioxidant --- p.5 / Chapter 1.6.1 --- Glutathione (GSH) --- p.5 / Chapter 1.6.2 --- Superoxide dismutase (SOD) --- p.7 / Chapter 1.6.3 --- Oxidative stress in HBV-related liver disease and HCC --- p.8 / Chapter 1.7 --- Apoptosis and necrosis --- p.9 / Chapter 1.7.1 --- Apoptotic pathways --- p.9 / Chapter 1.8 --- Role of HBx in apoptosis --- p.10 / Chapter 1.9 --- Transcriptional activity by HBx --- p.12 / Chapter 1.10 --- Chemotherapy drug resistance --- p.13 / Chapter 1.11 --- Objectives of study --- p.14 / Chapter Chapter 2: --- Methods and materials / Chapter 2.1 --- Construction of plasmid --- p.23 / Chapter 2.1.1 --- PCR amplification of wild-type and mutant HBx --- p.23 / Chapter 2.1.2 --- Agarose gel extraction --- p.25 / Chapter 2.1.3 --- Restriction enzyme digestion --- p.26 / Chapter 2.1.4 --- Ligation of vectors and gene of interest --- p.26 / Chapter 2.1.5 --- Preparation of competent cells for transformation --- p.27 / Chapter 2.1.6 --- Transformation of plasmid in competent cells --- p.27 / Chapter 2.1.7 --- Plasmid extraction by mini-prep --- p.28 / Chapter 2.1.8 --- DNA sequencing of the inserted genes --- p.29 / Chapter 2.2 --- Transfection --- p.30 / Chapter 2.2.1 --- Cell line --- p.30 / Chapter 2.2.2 --- Lipofectamine transfection --- p.31 / Chapter 2.2.3 --- Construction of stably-transfected cell lines --- p.31 / Chapter 2.3 --- Detection of expression of transfected gene in mRNA level by RT-PCR --- p.32 / Chapter 2.3.1 --- RNA extraction --- p.32 / Chapter 2.3.2 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.33 / Chapter 2.3.3 --- Agarose gel electrophoresis --- p.36 / Chapter 2.4 --- Detection of expression of transfected gene in protein level by Western blot --- p.36 / Chapter 2.4.1 --- Sample preparation --- p.36 / Chapter 2.4.2 --- Measurement of protein concentration --- p.36 / Chapter 2.4.3 --- Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) --- p.37 / Chapter 2.4.4 --- Transfer of proteins to nitrocellulose membrane --- p.38 / Chapter 2.4.5 --- Immunoblotting of protein --- p.38 / Chapter 2.5 --- Measurement of reduced glutathione (GSH) concentration in cell lines --- p.39 / Chapter 2.5.1 --- Sample preparation --- p.39 / Chapter 2.5.2 --- Measurement of GSH concentration --- p.39 / Chapter 2.6 --- Superoxide dismutase (SOD) activity in cell lines --- p.40 / Chapter 2.6.1 --- Sample preparation --- p.40 / Chapter 2.6.2 --- Measurement of total SOD activity --- p.41 / Chapter 2.6.3 --- Measurement of Cu/ZnSOD and MnSOD by Western blot --- p.42 / Chapter 2.7 --- Cell proliferation assay --- p.43 / Chapter 2.7.1 --- Drugs and concentration --- p.43 / Chapter 2.7.2 --- "MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assay" --- p.43 / Chapter 2.7.3 --- Cell proliferation and cytotoxicity of the drugs --- p.44 / Chapter 2.8 --- Detection of apoptosis by flow-cytometry --- p.44 / Chapter 2.8.1 --- Cell culture --- p.44 / Chapter 2.8.2 --- Cell fixation --- p.45 / Chapter 2.8.3 --- Cell staining --- p.45 / Chapter 2.8.4 --- Flow cytometry analysis --- p.46 / Chapter 2.9 --- Detection of protein involved in apoptotic pathway --- p.46 / Chapter 2.9.1 --- Antibodies --- p.46 / Chapter 2.9.2 --- Sample Preparation --- p.47 / Chapter 2.9.3 --- Measurement of protein concentration --- p.48 / Chapter 2.9.4 --- Western blotting --- p.49 / Chapter Chapter 3: --- Establishment of HBx transfected stable cell lines / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Results --- p.56 / Chapter 3.2.1 --- Plasmid construction --- p.56 / Chapter 3.2.2 --- Stable transfection of cell lines --- p.57 / Chapter 3.2.3 --- Morphology of wild type and mutant HBx-transfected cell lines --- p.58 / Chapter 3.3 --- Discussion --- p.58 / Chapter Chapter 4: --- Antioxidant level in HBx transfected cell lines / Chapter 4.1 --- Introduction --- p.68 / Chapter 4.2 --- Results --- p.70 / Chapter 4.2.1 --- Glutathione (GSH) concentration in different cell lines --- p.70 / Chapter 4.2.2 --- Superoxide dismutase (SOD) activity in different cell lines --- p.71 / Chapter 4.2.2.1 --- Total SOD activity --- p.71 / Chapter 4.2.2.2 --- Cu/ZnSOD --- p.71 / Chapter 4.2.2.3 --- MnSOD --- p.72 / Chapter 4.3 --- Discussion --- p.72 / Chapter Chapter 5: --- Involvement of HBx in apoptotic pathway / Chapter 5.1 --- Introduction --- p.81 / Chapter 5.2 --- Results --- p.82 / Chapter 5.2.1 --- Cell proliferation of HBx transfected cells --- p.82 / Chapter 5.2.2 --- Apoptosis of HBx transfected cells --- p.83 / Chapter 5.2.3 --- Cytotoxicity of fluorouracil (5FU) and doxorubicin (DOX) in HBx transfected cells --- p.84 / Chapter 5.2.4 --- Detection of anti-apoptotic proteins cIAP2 and Bcl-2 in HBx-transient and stably transfected cells --- p.84 / Chapter 5.3 --- Discussion --- p.85 / Chapter Chapter 6: --- Concluding remarks and general discussion / Chapter 6.1 --- General discussion --- p.93 / Chapter 6.2 --- Future work --- p.97 / Chapter 6.3 --- Summary --- p.99 / References --- p.100 / Appendix 1 --- p.114

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Feb. 3, 2010). Includes bibliographical references.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2010. / Härtill 4 uppsatser.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Not embargoed. Vita. Bibliography: 155-182.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 82-96).

Cette thèse décrit le développement de nouveaux systèmes catalytiques à base de fer ainsi que l'étude de leur réactivité vis-à-vis de l'éthylène. Dans un premier temps, nous nous sommes intéressés au développement de précurseurs de fer(III) associés à des ligands monoanioniques tridentes. Deux voies de synthèse ont été envisagées. La première décrit la complexation d'un ligand anionique sur le précurseur FeCl3 et la seconde passe par l’oxydation d'un complexe de fer(II) associé à un ligand neutre conduisant à une espèce binucléaire. Activés par le MAO, ces catalyseurs de fer(III) constituent les premiers complexes du genre permettant l’oligomérisation de l'éthylène. L’accent a également été porté sur la recherche de nouveaux activateurs. Des complexes d’aluminium répondant à nos attentes ont été obtenus par réaction entre un alcool et le triméthylaluminium. Selon la nature de l'alcool, la structure des activateurs peut être soit binucléaire ou trinucléaire. Enfin, des complexes de fer et de nickel associés à des ligands imino-imidazoles possédant un bras hémilabile ont été synthétisés. Une fois activés, les systèmes à base de nickel ont montré de bonnes activités en catalyse. / This thesis describes the development of new catalytic systems based upon iron complexes and their reactivity toward ethylene. First, we focused our interest on the synthesis of iron(III) precursors chelated by monoanionic ligand. Those complexes were obtained either by reaction of the monoanionic ligand with FeCl3 or through oxidation of the iron(II) complex. The second reaction led to binuclear complexes. Then, another aim of the thesis was to design new well-defined cocatalysts for the activation of iron complexes. The study of the reaction between an alcohol and the trimethylaluminum allowed us to reach this aim. Aluminum complexes adopted either a binuclear framework or a trinuclear one, depending on the nature of alcohol reagent. Besides this work, new iron(II) and nickel(II) complexes chelated by imino-imidazole ligands bearing a pendant donor function L were synthesized. All complexes have been evaluated for the oligomerization of ethylene in the presence of EtAlCl2 or MAO as cocatalyst. Only nickel complexes were active toward ethylene transformation.

This master thesis presents the effects of fly ash on the properties of alkali activated slag concrete, commonly referred as Geopolymer concrete (GPC). Cement manufacturer are major producers of CO2 which negatively affects the environment. Due to the increased construction activities and environmental concern, it is necessary to introduce alternative and eco-friendly binders for concrete. Slag and fly ash based concrete, which is by-product from industrial waste, is probably the best replacement for OPC concrete due to less or nil environmental issue. Most of the researchers have already concluded that slag and fly ash can be used as binders in concrete by activating them with alkali activator solution (e.g. by sodium silicate or sodium carbonate). In the present work concretes were produced by varying the proportion of slag to fly ash (40:60, 50:50, 60:40 & 80:20); amount of alkali activators (5, 10 & 14) and chemical modulus of sodium silicate (Ms) (0.25, 0.5 & 1).  Setting times and compressive strength values were evaluated. Results showed that decrease in fly ash content irrespective of % of alkali activators and alkali modulus (Ms), the compressive strength was increasing and setting time was getting shorter. The produced concretes showed increasing compressive strength with increase in % of alkali activator for Ms 0.5 and 1, while for Ms=0.25 the strength was decreasing with increase in % of alkali activators. From this it can be concluded that, Ms=0.5 was the optimum point below which the reaction got slower. Based on the initial investigations, mix S8:F2-SS10(1) and S8:F2-SS10(0.5) showed most promising results in terms of fresh and hardened concrete properties and were easy to handle. Consequently, the above mentioned mixture was chosen to be studied in more detail. The experimental program for these mixes included determination of slump flow, compressive strength (7, 14, 28 days) and shrinkage (drying and autogenous). The results shows that, strength increased with time and comparatively mix with Ms=0.5 showed higher compressive strength than mix with Ms=1, due to higher alkalinity of the pore solution. Mix with Ms=1 showed higher drying shrinkage compared to mix with Ms=0.5, which was explained by higher alkalinity of the solutions (Ms=0.5) leading to rapid formation of aluminosilicate gel. Autogenous shrinkage appeared to be higher for mix with Ms=0.5. This was associated with lower modulus which leads to densification of concrete microstructure at early ages. Pore diameter decrease and the water trapped in the pores exerted increasing tensile stress resulting for higher autogenous shrinkage.

Anticoagulants are the basis for treatment and prevention of thrombotic diseases. The currently available medicines are associated with a wide range of adverse reactions that mandates developing new anticoagulants. Several lines of evidence support the superiority of factor Xa (FXa) as a promising target to develop novel anticoagulants. This work focuses on the design of direct and indirect FXa inhibitors using an interdisciplinary approach. As indirect FXa inhibitors, a focused library of tetrasulfated N–arylacyl tetrahydroisoquinoline (THIQ) nonsaccharide allosteric antithrombin activators was designed, synthesized, and biochemically evaluated to establish their structure–activity relationship (SAR). An N–arylacyl THIQ analog having carboxylate at position–3, two sulfate groups at positions–5 and –8 of THIQ moiety, butanoyl linker, and two sulfate groups at positions–2 and –5 of the phenolic monocyclic moiety was identified as the most promising nonsaccharide antithrombin activator with KD of 1322 ± 237 μM and acceleration potential of 80–fold. Its biochemical profile indicates a strong possibility that it activates antithrombin by the pre–equilibrium pathway rather than the induced–fit mechanism utilized by heparin analogs. A similar interdisciplinary approach was exploited to design direct FXa inhibitors that possess high selectivity and are potentially orally bioavailable. Structurally, the designed direct FXa inhibitors are neutral THIQ dicarboxamides. THIQ dicarboxamide is a privileged structure with a semi–rigid character, a structural feature that potentially offers high selectivity for targeting FXa over other coagulation and digestive proteases. It can also be thought of as an amino acid–like structure, which affords accessibility to a large number of compounds using well established peptide chemistry. Mechanistically, the designed inhibitors were expected to bind to FXa in the active site and function as orthosteric inhibitors. These direct FXa active site inhibitors are also likely to inhibit clot–bound enzyme. Nearly 60 THIQ dicarboxamides were synthesized and biochemically evaluated. Through detailed SAR analysis, the most potent analog was designed and found to exhibit an IC50 of 270 nM (Ki = 135 nM), an improvement of more than 207–fold over the first inhibitor synthesized in the study. The most potent inhibitor displayed at least 1887–fold selectivity for FXa over other coagulation enzymes and a selectivity index of at least 279–fold over the digestive serine proteases. This analog doubled plasma clotting times at 17–20 μM, which are comparable to those of agents being currently studied in clinical trials. Overall, allosteric and orthosteric approaches led to the design of indirect and direct small molecule inhibitors of FXa based on the THIQ scaffold. This work introduces two promising molecules, a tetrasulfated N–arylacyl THIQ analog as a heparin mimetic and a neutral THIQ dicarboxamide as a potent, selective, and potentially bioavailable peptidomimetic, for further advanced medicinal chemistry studies.

Chau, Kin Fan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 106-119). / Abstract also in Chinese. / Abstract --- p.II / 摘要 --- p.VI / Acknowledgements --- p.IX / List of figures --- p.X / List of Abbreviations --- p.XII / Table of Contents --- p.XV / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Incidence and etiology of hepatocellular carcinoma (HCC) --- p.1 / Chapter 1.2 --- Structure of Hepatitis B Virus (HBV) --- p.2 / Chapter 1.3 --- Hepatitis B X protein (HBx) and HCC --- p.5 / Chapter 1.4 --- HBx and Apoptosis --- p.8 / Chapter 1.5 --- The role of Bcl-2 family in apoptosis and cell survival --- p.10 / Chapter 1.6 --- "Bid, the BH3-domain only protein" --- p.14 / Chapter 1.7 --- Dual Functions of Bid --- p.16 / Chapter 1.8 --- The relationship between Bid and HBx --- p.19 / Chapter 1.9 --- Hypoxia and HCC --- p.21 / Chapter 1.10 --- Hypoxia and HBx --- p.25 / Chapter 1.11 --- Hypoxia and Bid --- p.28 / Chapter 1.12 --- Aim of study --- p.29 / Chapter Chapter 2: --- Methods and materials / Chapter 2.1 --- Confirmation of the culture of the stable cell lines --- p.30 / Chapter 2.2 --- Doxorubicin treatment to the cell lines --- p.34 / Chapter 2.3 --- Culture of the cell lines under hypoxic conditions --- p.35 / Chapter 2.4 --- Protein sample preparations --- p.37 / Chapter 2.5 --- Determination of protein samples --- p.38 / Chapter 2.6 --- Sodium dodecyl sulfate 226}0ؤ polyacrylamide gel electrophoresis (SDS- PAGE) --- p.39 / Chapter 2.7 --- Transfer of protein to nitrocellulose membranes --- p.39 / Chapter 2.8 --- Western blot analysis of proteins --- p.41 / Chapter 2.8.1. --- Antibodies --- p.41 / Chapter 2.8.2. --- Determination of expression profiles of desired proteins by immunoblotting --- p.45 / Chapter 2.9 --- "Measurement of cell viability by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay" --- p.46 / Chapter 2.10 --- Determination of cell proliferation by BrdU proliferation assay --- p.47 / Chapter 2.11 --- Detection of apoptosis of the cell lines by TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling) --- p.50 / Chapter 2.12 --- Determination of the involvement of p38 MAPK in the generation of truncated Bid by p38 MAPK inhibitor SB203580 --- p.52 / Chapter Chapter 3: --- Results / Chapter 3.1 --- Confirmation of plasmids and the stable cell lines --- p.53 / Chapter 3.2 --- Morphology and the basic parameters of the cells with full-length HBx or mutant HBx --- p.53 / Chapter 3.3 --- Cell viability under doxorubicin treatment with or without hypoxia --- p.59 / Chapter 3.4 --- Determination of cell proliferation under stress --- p.70 / Chapter 3.5 --- Expression profiles of various proteins in the stable cell lines under doxorubicin treatment with or without hypoxia --- p.74 / Chapter 3.5.1. --- Verification of hypoxia --- p.74 / Chapter 3.5.2. --- Pro-apoptotic proteins --- p.74 / Chapter 3.5.3. --- Anti-apoptotic proteins --- p.74 / Chapter 3.6 --- Determination of apoptosis of various cell lines under stress --- p.82 / Chapter 3.7 --- "p38 MAPK, but not Akt, was activated by doxorubicin" --- p.87 / Chapter 3.8 --- The p38 MAPK inhibitor SB203580 could attenuate the cleavage of Bid --- p.89 / Chapter Chapter 4: --- Discussion --- p.92 / Chapter Chapter 5: --- Conclusion and future prospective --- p.103 / Chapter Chapter 6: --- References --- p.106