This thesis consists of a brief description on cancer, carcinogenesis, the changes in the type and level of dietary fat available in our diets over time and association with the development of certain diseases. The main focus of this research was on omega 6 and omega 3 essential fatty acids (EFA) and their interaction with regards to carcinogenesis.

By using the acridine orange (AO) staining method to examine the release of contents from lysosomes, it was found that PD released AO into the cytosol in both cell lines. However, the releasing pattern of HepG2 and RHepG2 was quite different. Upon PD treatment, the release of AO in HepG2 cells was graduate and slow while that in RHepG2 was sudden and sharp. / Cancer is one of the leading causes of death in the world. During cancer treatment, development of multidrug resistance (MDR) is always the major cause of failures of chemotherapy in human cancers. In our project, hepatocarcinoma HepG2 and its drug-resistant derivatives RHepG2 with MDR towards doxorubicin (Dox), fenretinide and Taxol were used to examine the differences in their response towards various anti-cancer agents. / From the AO staining, most of the lysosomes were found in the cytosol near the nucleus. However, some lysosomes were found inside the nucleus occasionally. When we double stained the HepG2 cells with DiOC6(3), it was found that the lysosomes were actually located inside the nuclear tubules. However, no such lysosome migration was observed after treating the HepG2 cells with PD. Thus, lysosomes inside the nuclear tubules might not be involved in the PD-induced lysosomal pathway. The mechanism that leads to the migration of lysosomes into the nuclear tubules is still unclear. / From the Western blot analysis, cathepsin D (Cat D) and cathepsin L (Cat L) were both released from the lysosomes after treating the two cell lines with PD. Also, it seemed likely that Cat L was released earlier than that of cyt c. This implies that lysosomal permeabilization is an early event in apoptosis. With the use of siRNA technology, it was found that RHepG2 with the knockdown of Cat D and Cat L were more tolerant and vulnerable towards PD, respectively. These suggest that Cat D and Cat L might act oppositely in the apoptotic pathway. Furthermore, the addition of Cat D inhibitor, pepstatin A, blocked the PD-mediated cell death in RHepG2 cells further confirms that Cat D is a pro-apoptotic protein that is involved in the apoptotic pathway. / In conclusion, PD was a potent anti-cancer agent that could reverse the MDR properties of RHepG2 and kill more RHepG2 cells through lysosomal and mitochondrial apoptotic pathway. / Next, we investigated the underlying killing mechanism and found out that PD switched on both the mitochondrial and lysosomal apoptotic pathway in both cell lines. Our results indicate that PD was able to depolarize mitochondrial membrane potential and release apoptosis inducing factor (AIF) and cytochrome c (cyt c) from the mitochondria to cytosol. Also, PD was able to act on isolated mitochondria directly, causing a stronger mitochondrial membrane permeabilization and more AIF release from the RHepG2 than that of the parental cells. / Polyphyllin D (PD) is a saponin found in a tradition Chinese herb, Paris polyphylla, which has been used to treat liver cancers in China for many years. Interestingly, from the MTT assays, we found out that RHepG2 (IC50: 2.0 muM) was more sensitive towards PD when compared to that of its parental cells (IC50: 3.9 muM). To keep the MDR properties, RHepG2 cells were routinely cultured with 1.2 muM of Dox. When we cultured RHepG2 in the absence of Dox but with 1.2 muM of PD for 28 days, the Pgp expression could not be maintained. However, such high expression level of Pgp was maintained when RHepG2 cells were treated with vincristine (1.2 muM) in the absence of Dox. This indicates that vincristine was a substrate of Pgp to keep the Pgp expression in RHepG2 cells while PD was not. / When incubated with different concentrations of Dox, RHepG2 accumulated less Dox than that of its parental HepG2 cells. When probed by the antibody against P-glycoprotein (Pgp), RHepG2 showed a strong Pgp expression. With the addition of Pgp modulator, verapamil, RHepG2 accumulated more Dox. All these findings indicate that Pgp is a mediator giving rise the MDR in RHepG2 cells. However, RHepG2 had a higher resistance to Dox than its parental line even co-cultured with verapamil. RHepG2 remained viable at the intracellular Dox concentration that was toxic to HepG2 cells. These observations suggest that the MDR properties of RHepG2 involved multiple mechanisms in addition to the effect of Pgp. / Lee, Kit Ying Rebecca. / "August 2007." / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4735. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 241-253). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Fu Zhenming. / Thesis submitted in: December 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 138-162). / Abstracts in English and Chinese. / List of abbreviations --- p.i / Abstract (in English) --- p.ii / 摘要（中文） --- p.iii / Acknowledgement --- p.iv / Chapter Chapter 1. --- Introduction and Objectives of Study --- p.1 / Chapter 1.1 --- Hepatocellular carcinoma in general --- p.2 / Chapter 1.1.1 --- "Epidemiology, risk factors" --- p.2 / Chapter 1.1.2 --- Pathology and staging --- p.4 / Chapter 1.1.3 --- Treatment --- p.6 / Chapter 1.1.4 --- Improvement of early detection and treatment of HCC --- p.7 / Chapter 1.2 --- General aspects of mitochondria and mitochondrial DNA (mtDNA) --- p.10 / Chapter 1.2.1 --- Structure and dynamics of mitochondria --- p.10 / Chapter 1.2.1.1 --- General introduction of mitochondria --- p.10 / Chapter 1.2.1.2 --- Respiration chain of mitochondria --- p.11 / Chapter 1.2.2 --- The mitochondrial genome --- p.14 / Chapter 1.2.2.1 --- Strucure --- p.14 / Chapter 1.2.2.2 --- Genes for structure proteins --- p.16 / Chapter 1.2.2.3 --- Genes for translation --- p.17 / Chapter 1.2.2.4 --- Imported proteins and RNAs --- p.17 / Chapter 1.2.3 --- Mitochondrial DNA maintenance --- p.19 / Chapter 1.2.4 --- Mitochondrial DNA replication --- p.25 / Chapter 1.2.5 --- Mitochondrial DNA transcription --- p.30 / Chapter 1.2.6 --- Mitochondrial DNA translation --- p.32 / Chapter 1.3 --- MtDNA diseases --- p.35 / Chapter 1.4 --- MtDNA mutation and HCC --- p.35 / Chapter 1.5 --- Aims of the study --- p.39 / Chapter Chapter 2. --- Materials and Methods --- p.41 / Chapter 2.1 --- Materials --- p.42 / Chapter 2.1.1 --- Chemicals --- p.42 / Chapter 2.1.2 --- Primers --- p.42 / Chapter 2.1.3 --- Enzymes --- p.45 / Chapter 2.1.4 --- Cell line --- p.45 / Chapter 2.1.5 --- Collection of specimens --- p.46 / Chapter 2.2 --- Methodology --- p.47 / Chapter 2.2.1 --- "DNA extraction from hcc tissues, cell line Hep3B and PBMCs" --- p.47 / Chapter 2.2.1.1 --- DNA extraction from HCC tissues --- p.47 / Chapter 2.2.1.2 --- DNA extraction from cell line Hep3B --- p.49 / Chapter 2.2.1.3 --- DNA extraction from and PBMCs --- p.50 / Chapter 2.2.1.3.1 --- Preparation of PBMCs --- p.50 / Chapter 2.2.1.3.2 --- DNA extraction from and PBMCs --- p.51 / Chapter 2.2.2 --- Detection of mt whole genome mutation by direct sequencing --- p.51 / Chapter 2.2.2.1 --- Design of mtDNA primers --- p.51 / Chapter 2.2.2.2 --- PCR amplification of the whole mt genome --- p.51 / Chapter 2.2.2.3 --- Direct sequencing of the whole mt genome --- p.52 / Chapter 2.2.2.3.1 --- Primer used in sequencing --- p.52 / Chapter 2.2.2.3.2 --- Purification of the PCR products of the whole mt genome --- p.53 / Chapter 2.2.2.3.3 --- Dye terminator cycle sequencing reaction --- p.53 / Chapter 2.2.2.3.4 --- Purification of extension products --- p.54 / Chapter 2.2.3 --- Detection of mtDNA control region mutation --- p.55 / Chapter 2.2.3.1 --- PCR amplification of D310 in the mtDNA control region --- p.55 / Chapter 2.2.3.2 --- Screening of D310 mutation by PFLDA --- p.55 / Chapter 2.2.3.2.1 --- Making 8% denatured gel mixture --- p.55 / Chapter 2.2.3.2.2 --- Setting up and Pouring the denatured gel --- p.56 / Chapter 2.2.3.2.4 --- Preparing and Loading the PCR products --- p.57 / Chapter 2.2.3.2.5 --- Electrophoresis --- p.57 / Chapter 2.2.3.2.6 --- "Gel fixing, silver staining and color development " --- p.58 / Chapter 2.2.3.3 --- Direct sequencing of D310 in the mtDNA control region --- p.59 / Chapter 2.2.4 --- Detection of mt DNA coding region mutation --- p.60 / Chapter 2.2.4.1 --- PCR amplification of the 5 respiratory chain subunit genes --- p.60 / Chapter 2.2.4.2 --- Restriction enzyme digestion of 5 genes in mtDNA coding region --- p.60 / Chapter 2.2.4.3 --- Screening of mtDNA coding region mutation by SSCP --- p.61 / Chapter 2.2.4.3.1 --- Making 6% 49:1 acrylamide/Bis SSCP gel mixture --- p.61 / Chapter 2.2.4.3.2 --- "Setting up the SSCP gel, loading sample, fixing, staining and developing of the gel " --- p.62 / Chapter 2.2.4.4 --- Sequencing conformation of the mtDNA coding region mutation --- p.62 / Chapter 2.2.5 --- Statistics --- p.63 / Chapter 2.2.5.1 --- The chi-square test --- p.63 / Chapter 2.2.5.2 --- The Friedman test --- p.63 / Chapter 2.2.5.3 --- Wilcoxon signed ranks test --- p.63 / Chapter Chapter 3. --- Results --- p.64 / Chapter 3.1 --- Detection mt DNA whole genome mutation --- p.65 / Chapter 3.1.1 --- Identification of mtDNA whole genome by direct sequencing --- p.65 / Chapter 3.2 --- Detection mt DNA D-loop mutation --- p.76 / Chapter 3.2.1 --- Screening of C-tract alteration in HCC tissus by PCR fragments length detection assay (PFLDA) --- p.76 / Chapter 3.2.2 --- Screening of coding region alteration in HCC tissues by SSCP --- p.77 / Chapter 3.2.2.1 --- Identification of C-tract alterations in HCC and non-tumorous tissues by direct sequencing --- p.77 / Chapter 3.2.3 --- Identification of C-tract alterations by direct sequencing --- p.82 / Chapter 3.2.3.1 --- Identification of C-tract alterations in HCC tissues by direct sequencing --- p.82 / Chapter 3.2.3.2 --- Identification of C-tract alteration in PBMC of normal subjects by direct sequencing --- p.82 / Chapter 3.2.3.3 --- Identification of C-tract alteration in PBMC of HCC patients by direct sequencing --- p.82 / Chapter 3.2.4 --- Statistics of the analysis of C-tract alterations --- p.82 / Chapter 3.3 --- Detection mt DNA mutation in the coding region --- p.87 / Chapter Chapter 4. --- Discussion --- p.98 / Chapter 4.1 --- Detection mtDNA whole genome mutation --- p.99 / Chapter 4.2 --- Detection mtDNA D-loop mutation --- p.107 / Chapter 4.3 --- Detection mtDNA mutation in the coding region --- p.119 / Chapter 4.4 --- Possible mechanisms of mtDNA mutation in HCC carcinogenesis --- p.125 / Chapter 4.5 --- Proposals for prospective studies --- p.126 / Chapter 4.5.1 --- Function of C7 in D310 --- p.128 / Chapter 4.5.2 --- Function changes of mtDNA coding region mutation --- p.130 / Chapter 4.5.3 --- Detection of D310 C-tract mutation in patients' plasma --- p.131 / Chapter 4.5.4 --- Relationship between nMSl and mtMSI --- p.132 / Chapter 4.6 --- Summary --- p.134 / References --- p.137

肝癌是造成癌症相關死亡的主要原因之一。而常規化療受耐藥性的發展和各種副作用的限制。由於無毒性和鲜明的生物药物能力，從蘑菇提取的代謝物在癌症治療中獲得更多的注意和关注。我們以前的研究已經證明來自平菇香菇多醣蛋白複合物的抗癌作用。本研究的目的是探討一種含有多醣蛋白複合物的秀珍菇（PP）熱水提取物在肝癌細胞中抗癌活性的分子機制。 / 我們的研究結果表明，用PP处理过的肝癌細胞，不僅顯著的显示出降低的體外腫瘤細胞的增殖和侵襲，也增強化療藥物順鉑的藥物敏感性。無論是口服和腹腔注射都顯著抑制移植免疫BALB / c裸小鼠的腫瘤生長。同时，PP也能在體外和體內实验顯著抑制PI3K/Akt信號通路在肝癌細胞。有趣的是，当过表达AKT时，Myr-AKT，PP的這種抑制癌细胞生长的效果有减弱的趋势，同时也反映在PP对癌细胞侵襲抑制的作用上。印跡和酶聯免疫吸附試驗結果表明，在PP处理过的肝癌細胞中，血管內皮生長因子（VEGF）的表達和分泌減少了。此外， rhVEGF的加入减弱了 PP对PI3K/Akt通路和肝癌细胞表型的抑製作用。 / 我們的研究結果表明，PP能在體外和體內试验中抑制肝癌細胞增殖，侵襲和耐藥性，通过抑制分泌血管內皮生長因子誘導PI3K/Akt的信號通路。這項研究表明了PP的潛在治療肝癌的治療意義。 / Liver cancer or hepatocellular carcinoma is one of the leading causes of cancer-related deaths. Conventional chemotherapies are limited by the development of drug resistance and various side effects. Because of its non-toxicity and potent biopharmacological activity, metabolites derived from mushrooms have received more attention in cancer therapy. Our previous studies have demonstrated the anti-cancer effects of polysaccharide-protein complexes derived from the Pleurotus mushrooms. The aim of this study was to investigate the underlying molecular mechanism of the anti-cancer activity of a hot water extract containing a polysaccharide-protein complex isolated from Pleurotus pulmonarius (PP) in liver cancer cells. / Our results indicated that exposure of liver cancer cells to PP not only significantly reduced the in vitro cancer cell proliferation and invasion but also enhanced the drug-sensitivity to the chemotherapeutic drug Cisplatin. Both oral administration and intraperitoneal injection of PP significantly inhibited the tumor growth in xenograft BALB/c nude mice. PP triggered a marked suppression of the PI3K/AKT signaling pathway in liver cancer cells in vitro and in vivo, and overexpression of the constitutively active form of AKT, Myr-AKT, abrogated this effect and the inhibited proliferation and invasion by PP. Both western blot and ELISA results showed that PP-treated liver cancer cells had reduced expression and secretion of vascular endothelial growth factor (VEGF). Addition of recombinant human VEGF attenuated the inhibitory effects of PP on PI3K/AKT pathway and the cancer phenotypes. / Our results demonstrated that PP suppressed the proliferation, invasion, and drug-resistance of liver cancer cells in vitro and in vivo, mediated by the inhibition of autocrine VEGF-induced PI3K/AKT signaling pathway. All these results suggest the potential therapeutic implication of PP in the treatment of human liver cancer. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Xu, Wenwen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 83-99). / Abstracts also in Chinese. / Thesis Committee --- p.i / English Abstract --- p.ii / Chinese Abstract --- p.iv / Acknowledgements --- p.v / List of Tables --- p.vi / List of Figures --- p.vii / Abbreviations --- p.x / Content page --- p.xiv / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Mushroom as functional foods --- p.1 / Chapter 1.1.1 --- Introduction of functional food --- p.1 / Chapter 1.1.2 --- Functional food and cancer --- p.1 / Chapter 1.1.3 --- Edible Mushroom as functional food --- p.4 / Chapter 1.1.4 --- Pleurotus pulmonarius and its function --- p.7 / Chapter 1.2 --- Hepatocellular carcinoma --- p.9 / Chapter 1.2.1 --- Liver and hepatocellular carcinoma --- p.9 / Chapter 1.2.2 --- Carcinogenesis of liver cancer --- p.12 / Chapter 1.2.2.1 --- Hallmarks of cancer --- p.12 / Chapter 1.2.2.2 --- Cell cycle --- p.13 / Chapter 1.2.2.3 --- Apoptosis --- p.15 / Chapter 1.2.2.4 --- Angiogenesis --- p.17 / Chapter 1.2.2.5 --- Invasion and metastasis --- p.19 / Chapter 1.2.2.6 --- Drug resistance --- p.21 / Chapter 1.2.3 --- The role of PI3K/AKT pathway --- p.23 / Chapter 1.2.4 --- The role of growth factor Vascular endothelial growth factor (VEGF) in HCC --- p.25 / Chapter 1.3 --- Research objectives --- p.27 / Chapter 1.3.1 --- Hypothesis and objectives --- p.27 / Chapter 1.3.2 --- Experimental design --- p.28 / Chapter Chaper 2 --- Materials and Methods --- p.29 / Chapter 2.1 --- Materials --- p.29 / Chapter 2.1.1 --- Mushroom Pleurotus pulmonarius --- p.29 / Chapter 2.1.2 --- Drugs and cell lines --- p.29 / Chapter 2.1.3 --- Antibodies list --- p.30 / Chapter 2.1.4 --- Animal models --- p.32 / Chapter 2.2 --- Sample preparation and structure investigation --- p.32 / Chapter 2.2.1 --- Polysaccharide extraction from mushroom --- p.32 / Chapter 2.2.2 --- Endotoxin test --- p.32 / Chapter 2.2.3 --- Determination of monosaccharide profile by gas chromatography and mass spectrometry (GC/MS) --- p.33 / Chapter 2.2.3.1 --- Sample preparation for gas chromatography analysis --- p.33 / Chapter 2.2.3.1.1 --- Acid depolymerisation --- p.33 / Chapter 2.2.3.1.2 --- Neutral sugar derivatization --- p.33 / Chapter 2.2.3.1.3 --- External monosaccharide standard preparation --- p.34 / Chapter 2.2.3.2 --- Gas chromatography-mass spectrometry (GC/MS) --- p.34 / Chapter 2.2.4 --- Determination of total sugar by phenol-sulfuric acid method (Dubois, 1956) --- p.36 / Chapter 2.2.5 --- Determination of protein content by Lowry-Folin method (Lowry et al.,1951) --- p.37 / Chapter 2.3 --- Biological assays --- p.38 / Chapter 2.3.1 --- In vitro assays --- p.38 / Chapter 2.3.1.1 --- MTT assay --- p.38 / Chapter 2.3.1.2 --- Colony formation assay --- p.38 / Chapter 2.3.1.3 --- Plasmid transfection --- p.39 / Chapter 2.3.1.4 --- In vitro cell invasion assay --- p.39 / Chapter 2.3.1.5 --- Cell cycle analysis --- p.39 / Chapter 2.3.1.6 --- Western blot analysis --- p.40 / Chapter 2.3.1.7 --- VEGF ELISA Kit --- p.42 / Chapter 2.3.2 --- In vivo assays --- p.43 / Chapter 2.3.2.1 --- Tumor xenograft nude mouse model --- p.43 / Chapter 2.3.2.2 --- Immunohistochemistry --- p.45 / Chapter 2.3.2.3 --- H&Estaining --- p.45 / Chapter 2.3.3 --- Statistical analysis --- p.45 / Chapter Chaper 3 --- Results and discussion --- p.46 / Chapter 3.1 --- The yield and chemical characteristic of PP --- p.46 / Chapter 3.1.1 --- The yield of PP from mushroom Pleurotus pulmonarius --- p.46 / Chapter 3.1.2 --- Total carbohydrate and protein content --- p.47 / Chapter 3.1.3 --- Monosaccharide composition by GC-MS --- p.48 / Chapter 3.2 --- Toxicity of the PP water by Limulus amebocyte lysate (LAL) test --- p.48 / Chapter 3.2.1 --- Limulus amebocyte lysate (LAL) test --- p.48 / Chapter 3.3 --- Effects of PP on the proliferation of liver cancer cell lines --- p.50 / Chapter 3.3.1 --- MTT assay --- p.50 / Chapter 3.3.2 --- Colony-formation assay --- p.51 / Chapter 3.3.3 --- Cytotoxic effects of PP against normal liver cell --- p.52 / Chapter 3.3.4 --- The anti-proliferative effect of PP on other cancer types --- p.53 / Chapter 3.3.5 --- Cell cycle analysis by flow cytometry of PP treated liver cancer cells --- p.54 / Chapter 3.3.6 --- Protein expression by western blot analysis of P treated liver cancer cells --- p.56 / Chapter 3.4 --- Anti-cancer effect of PP on liver cancer cells through inactivation of PI3K/AKT signaling pathway --- p.57 / Chapter 3.4.1 --- Effect of PP on inactivation of PI3K/AKT pathway --- p.57 / Chapter 3.4.2 --- The abrogated inhibitory effect of PP on Huh7 with overexpression of AKT. --- p.59 / Chapter 3.4.3 --- The abrogated inhibitory effect of PP on PI3K/AKT signal pathway with overexpression of the constitutively active form of AKT, Myr-AKT --- p.60 / Chapter 3.5 --- Inhibition of VEGF expression and secretion by PP --- p.62 / Chapter 3.5.1 --- ELISA result of PP on VEGF secretion --- p.62 / Chapter 3.5.2 --- The attenuated inhibitory effect of PP on cell proliferation with addition of rhVEGF --- p.63 / Chapter 3.5.3 --- The attenuated inhibitory effect of PP on PI3K/AKT signal pathway with addition of rhVEGF --- p.64 / Chapter 3.6 --- Effect of PP on enhancing the chemosensitivity of liver cancer cells to Cisplatin --- p.66 / Chapter 3.6.1 --- Synergistic effect of PP with cisplatin (DDP) in liver cancer cells --- p.66 / Chapter 3.6.2 --- The abrogated drug-resistant effect by PP by overexpression of the constitutively active form of AKT, Myr-AKT --- p.67 / Chapter 3.6.3 --- The abrogated drug-resistant effect of PP with addition of rhVEGF --- p.68 / Chapter 3.7 --- The anti-invasive potential of PP on liver cancer cells. --- p.69 / Chapter 3.7.1 --- Boyden chamber assay --- p.69 / Chapter 3.7.2 --- The attenuated anti-invasive effect of PP on liver cancer cells with overexpression of constitutively activated AKT --- p.71 / Chapter 3.7.3 --- The attenuated anti-invasive effect of PP on liver cancer cells with addition of rhVEGF --- p.72 / Chapter 3.8 --- The anti-tumor effect of PP in vivo --- p.73 / Chapter 3.8.1 --- The anti-tumor effect of PP by using tumor xenograft model --- p.73 / Chapter 3.8.2 --- Body weight of nude mice treated with PP --- p.75 / Chapter 3.8.3 --- Harmful effect of PP on nude mice --- p.76 / Chapter 3.8.4 --- Immunohistochemist analysis of mice tumor xenograft treated with PP --- p.77 / Chapter 3.8.5 --- Western blot anaylysis using the tumor tissues harvested from mice xenograftes treated with PP --- p.78 / Chapter Chapter 4 --- Conclusion and future Plan --- p.81 / Reference --- p.83 / Related Publication List --- p.100

BTBD7 is a newly identified candidate gene for HCC using a high-throughput cDNA/EST microassay. This gene encodes for a protein of 410 amino acid residues. This protein was previously named as the function unknown protein 1 (FUP1) because the biological function of this protein was unknown at that time. Bioinformatics analysis revealed that this protein contains two bric-a-brac, tramtrack, broad-complex (BTB) domains located at amino acid positions 143 to 230 and 274 to 342. In order to reflect its structure and functions, and to be consistent with the GeneBank database (Accession No. NM_018167), we rename it as BTBD7 (BTB domain containing 7). / In conclusion, our study demonstrated that BTBD7 is a novel oncogene, which is associated with hepatocellular carcinoma and is essential for the inhibition of cell growth and tumorigenesis. To our knowledge, BTBD7 is the first identified regulator of p16INK4A through inhibiting the promoter activity of p16INK4A. BTBD7 may thus serve as a new tumor marker or as a potential target of treating hepatocellular carcinoma. / In previous studies, the expression of BTBD7 was shown to be tissue-specific as demonstrated by Northern blot. Furthermore, we collected 18-paired HCC samples to further reveal the correlation of BTBD7 gene expression profiles with tumorigenesis. Our data showed that BTBD7 was significantly elevated in 44.4% of the HCC samples. Compared with immortalized hepatocyte cell lines MIHA or LO2, both mRNA level and protein level of BTBD7 were also elevated in the hepatoma cell lines HepG2, BEL7404, Hep3B and Huh7. This gave a due that the expression of BTBD7 may be correlated with carcinogenesis of liver cells. / In the present study, the function of BTBD7 was investigated. We used RNAi approach to silence BTBD7. Compared with the control, siBTBD7 induced cell cycle arrest at G1 phase and later caused obvious cell death. The cell death was further demonstrated to be apoptosis through activation of caspase 3. Furthermore, we carried out candidate gene search using knockdown of BTBD7. The mRNA level of tumor suppresser p16INK4A was upregulated and hTERT was downregulated in BTBD7 knocked down cells. The other key genes involved in cell growth, cell cycle control, cell death and survival (c-myc, c-fos, c-jun, p21CIP1, p27KIP1, p53, Survivin, E2F, NF-kappaB, Bax, p14ARF, p16INK4A and hTERT) did not respond to the reduced BTBD7 levels. On the other hand, double knockdown of p16INK4A and BTBD7 markedly reduced the effects of cell cycle arrest and the death ratio caused by dysfunction of BTBD7 or overexpression of p16INK4A, suggesting that p16 INK4A is a downstream target of BTBD7. We further adopted a dominant negative approach to confirm these results. / Liu, Zheng. / Advisers: C. H. K. Cheng; Mingliang He. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3449. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 120-161). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

"Epigenetic" refers to a heritable change in the gene expression pattern that is not mediated by any alterations in the primary nucleotide sequence of a gene in the genome. This change involves methylation of DNA in the gene promoter regions, modification of histone residues and chromatin remodeling. Among them, methylation of DNA promoter region is an essential step in epigenetic gene silencing and is known to be closely related to carcinogenesis and cancer progression. / Our preliminary study on effect of treatments of some potential anti-cancer drug candidates, namely Pheophorbide A (Pa), Pa combining with photodynamic therapy, Polyphyllin D (designated as HK-18), and its derivative designated as HK-27 on human breast cancer cell lines MCF-7 and MDA-MB-231 showed that the promoter methylation of CDH1 was decreased in response to treatments of Pa, HK-18, and HK-27 in MDA-MB-231 cells. / The aim of this study was to explore whether any methylation of DNA promoters mechanism is involved in drug resistance of a doxorubicin-induced human multidrug resistant hepatocellular carcinoma sub-linage R-HepG2 which was established from the doxorubicin sensitive HepG2 cell line in our laboratory. In this project, it was observed that the DNA promoter methylations of ESR1, Rassf2A, CDH1 and MDR1 in R-HepG2 were higher than those in HepG2 cells respectively by methylation specific polymerase chain reaction method. Bisulfite sequencing showed that the total 32 CpGs of CDH1 promoter region in R-HepG2 cells were hypermethylated while they were hypomethylated in HepG2 cells. CDH1 is the encoding gene of E-cadherin. The promoter hypermethylation induced CDH1 silencing in R-HepG2 cells was confirmed by reverse transcription polymerase chain reaction and Western blotting that CDH1 transcription and E-cadherin expression were maintained in HepG2 cells but both were lost in R-HepG2 cells. RT-PCR of 10 multidrug resistant related genes revealed that transcription of MDR1 was obviously increased in R-HepG2 cells, transcription of MRP1 and MRP5 were slightly increased in R-HepG2 cells, transcription of MRP6 and BCRP were slightly decreased in R-HepG2 cells comparing to those in the parental HepG2 cells. This result suggests that up-regulation of P-glycoprotein expression which is the protein product of MDR1 may be one of the major causes of multidrug resistance in R-HepG2 cells. Transient transfection of CDH1 cDNA increased the CDH1 transcription and E-cadherin expression in R-HepG2 cells. I also found that the CDH1 transfected R-HepG2-CDH1 cells showed increased amount of doxorubicin uptake, increased apoptotic population of cells exposed to doxorubicin, suppressed cell migration, and decreased P-glycoprotein expression comparing to those in R-HepG2 cells. It was also found that the transcription levels of SNAI2, TWIST1, ASNA1 and FYN were obviously higher in R-HepG2 cells than those in HepG2 cells. The transcription of FYN and TWIST1 were obviously decreased in CDH1 cDNA transfected R-HepG2-CDH1 cells which displayed a negative correlation with the transcription level of CDH1 and these results imply a suppressive role of CDH1 in regulating these genes which were involved in cancer metastasis and multidrug resistance. / Jiang, Lei. / Adviser: Kwok-Pui, Fang. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 144-171). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

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

Li, Sai Kam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 162-179). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese (摘要） --- p.ii / Acknowledgements --- p.iii / Table of Content --- p.iv / Abbreviations --- p.xi / List of Figures --- p.xiv / List of Tables --- p.xvii / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Hepatitis B Virus / Chapter 1.1.1 --- General information --- p.1 / Chapter 1.1.2 --- Classification --- p.2 / Chapter 1.1.3 --- Virus life cycle and genome --- p.3 / Chapter 1.1.4 --- Hepatitis B virus X protein (HBx) --- p.7 / Chapter 1.2 --- Enigmatic functions of HB --- p.x / Chapter 1.2.1 --- HBx as a transactivator --- p.10 / Chapter 1.2.2 --- HBx as a cell cycle regulator --- p.12 / Chapter 1.2.3 --- HBx as an apoptosis modulator --- p.13 / Chapter 1.3 --- Etiology of HBV-mediated hepatocarcinogenesis --- p.14 / Chapter 1.4 --- Clinical mutants of HBV --- p.16 / Chapter 1.5 --- Hypothesis and aims of the research --- p.16 / Chapter 1.6 --- Basis of Tet-On system --- p.18 / Chapter CHPATER 2 --- EXPERIMENT MATERIALS / Chapter 2.1 --- Cell culture / Chapter 2.1.1 --- Cell-lines --- p.21 / Chapter 2.1.2 --- Culture medium --- p.22 / Chapter 2.1.3 --- Culture medium supplements --- p.23 / Chapter 2.2 --- Reagents for subcloning / Chapter 2.2.1 --- Reagents for polymerase chain reaction (PCR) --- p.24 / Chapter 2.2.2 --- Reagents for restriction enzyme digestion --- p.24 / Chapter 2.2.3 --- Reagents for ligation --- p.25 / Chapter 2.2.4 --- Reagents for electrophoresis --- p.25 / Chapter 2.2.5 --- Reagents for E. coli DH5a preparation --- p.25 / Chapter 2.2.6 --- Materials for bacterial culture work --- p.27 / Chapter 2.3 --- Reagents for subcellular localization study / Chapter 2.3.1 --- Reagents for cell staining --- p.28 / Chapter 2.3.2 --- Reagents for mounting slides --- p.29 / Chapter 2.3.3 --- Materials for site-directed mutagenesis --- p.29 / Chapter 2.4 --- Reagents for cell cycle analysis and cellular proliferation / Chapter 2.4.1 --- Reagents for cell cycle analysis --- p.29 / Chapter 2.4.2 --- Reagents for cellular proliferation study --- p.30 / Chapter 2.5 --- Reagents for protein expression study / Chapter 2.5.1 --- Cell lysis buffer --- p.30 / Chapter 2.5.2 --- Reagents for SDS-PAGE --- p.30 / Chapter 2.5.3 --- Reagents for Western blot --- p.33 / Chapter 2.5.4 --- Antibodies --- p.34 / Chapter 2.6 --- Reagents for gene expression study / Chapter 2.6.1 --- Reagents for RNA extraction --- p.36 / Chapter 2.6.2 --- Reagents for first strand cDNA synthesis --- p.37 / Chapter 2.6.3 --- Reagents for real-time PCR --- p.37 / Chapter 2.7 --- Reagents for establishment of Tet-On inducible stable cell-lines / Chapter 2.7.1 --- Reagents for MTT assay --- p.38 / Chapter 2.7.2 --- Reagents for selection of stable clones --- p.38 / Chapter 2.8 --- Vectors used in the project / Chapter 2.8.1 --- Vectors for subcellular localization study --- p.39 / Chapter 2.8.2 --- Vectors for establishment of Tet-on inducible cell-lines --- p.39 / Chapter 2.9 --- Primers used in the project / Chapter 2.9.1 --- Primers used for subcloning --- p.42 / Chapter 2.9.2 --- Primers used for site-directed mutagenesis --- p.43 / Chapter 2.9.3 --- Primers used in real-time chain polymerase reaction --- p.43 / Chapter CHAPTER 3 --- RESEARCH METHODS / Chapter 3.1 --- Subcloning of HBx and mutant genes into a green fluorescence protein (GFP) expression vector / Chapter 3.1.1 --- Amplification of HBxWt，HBxΔC44 and HBxAN60 genes --- p.45 / Chapter 3.1.2 --- Purification of PCR products --- p.46 / Chapter 3.1.3 --- Restriction enzyme digestion --- p.47 / Chapter 3.1.4 --- Ligation of gene products with pEGFP-C 1 vector --- p.47 / Chapter 3.1.5 --- Preparation of chemically competent bacterial cells E. coli strain DH5α --- p.47 / Chapter 3.1.6 --- Transformation of the ligation product into competent cells --- p.48 / Chapter 3.1.7 --- PCR confirmation of successful ligation --- p.48 / Chapter 3.1.8 --- Small scale preparation of bacterial plasmid DNA --- p.49 / Chapter 3.1.9 --- DNA sequencing of the cloned plasmid DNA --- p.50 / Chapter 3.1.10 --- Large scale preparation of target recombinant plasmid DNA --- p.50 / Chapter 3.2 --- Subcellular localization pattern study / Chapter 3.2.1 --- Cell transfection --- p.51 / Chapter 3.2.2 --- Mitochondria and nucleus staining --- p.52 / Chapter 3.2.3 --- Epi-fluorescence microscopy --- p.53 / Chapter 3.2.4 --- Analysis of fluorescence images --- p.53 / Chapter 3.2.5 --- In vitro site-directed mutagenesis --- p.53 / Chapter 3.3 --- Cell cycle phase analysis with flow cytometry / Chapter 3.3.1 --- Cell transfection --- p.55 / Chapter 3.3.2 --- Cell staining --- p.55 / Chapter 3.3.3 --- Flow cytometry --- p.55 / Chapter 3.4 --- Cellular proliferation quantification by BrdU proliferation assay / Chapter 3.4.1 --- Cell transfection --- p.57 / Chapter 3.4.2 --- BrdU ELISA measurement --- p.57 / Chapter 3.5 --- Protein expression / Chapter 3.5.1 --- Cell lysate collection --- p.58 / Chapter 3.5.2 --- Quantification of protein samples --- p.59 / Chapter 3.5.3 --- SDS-PAGE --- p.59 / Chapter 3.5.4 --- Western blot --- p.60 / Chapter 3.5.5 --- Western blot luminal detection --- p.60 / Chapter 3.6 --- Gene expression / Chapter 3.6.1 --- Primer design --- p.61 / Chapter 3.6.2 --- Cell transfection --- p.61 / Chapter 3.6.3 --- RNA extraction --- p.61 / Chapter 3.6.4 --- Reverse transcription for first strand complementary DNA (cDNA) --- p.63 / Chapter 3.6.5 --- Quantitative real-time PCR --- p.63 / Chapter 3.7 --- Establishment of Tet-On inducible stable cell-lines / Chapter 3.7.1 --- Subcloning of HBx gene into pTRE2 vector --- p.64 / Chapter 3.7.2 --- Construction of WRL68/Tet-On stable cell-lines --- p.64 / Chapter 3.7.3 --- Construction of WRL68/Tet-On HBx and mutants expression cell-lines --- p.68 / Chapter 3.7.4 --- Characterization of Tet-On gene expression monoclones --- p.69 / Chapter 3.8 --- Statistical analyses --- p.70 / Chapter CHPATER 4 --- STUDY ON MITOCHONDRIA TARGETING / Chapter 4.1 --- Establishment of pEGFP-Cl-HBx and mutants constructs --- p.71 / Chapter 4.2 --- Transactivation C-terminus domain is essential for granular localization --- p.73 / Chapter 4.3 --- Wild-type HBx localizes in mitochondria --- p.76 / Chapter 4.4 --- C-terminal transactivation domain is sufficient for mitochondria targeting --- p.79 / Chapter 4.5 --- Mapping of the HBx region crucial for mitochondria targeting --- p.81 / Chapter 4.6 --- The 111-117 amino acids in HBx do not work as a signal peptide --- p.83 / Chapter 4.7 --- Site-directed mutagenesis identifies the key amino acid at 115 in HBx for mitochondrial targeting --- p.85 / Chapter CHAPTER 5 --- CELL PROLIFERATION AND REGULATION / Chapter 5.1 --- Alteration of S-phase distribution in cell cycle --- p.88 / Chapter 5.2 --- Analysis of DNA synthesis using BrdU proliferation ELISA --- p.92 / Chapter 5.3 --- Differential molecular regulation of cell cycle --- p.94 / Chapter 5.4 --- Regulation of the mRNA expression levels of cyclin-dependent kinases inhibitors p2raf/cipl and p27kipl --- p.98 / Chapter CHAPTER 6 --- TRANSACTIVATION AND RAS/RAF/MAPK PHOSPHORYLATION / Chapter 6.1 --- Determination of p53-dependency of p21、vaf/cipl expression --- p.101 / Chapter 6.2 --- Ras/Raf/MAPK pathway activation by HBx variants / Chapter 6.2.1 --- ERK1/2 phophorylation by HBx variants --- p.104 / Chapter 6.2.2 --- ERK inhibition blocks the regulation effect on p53Wt and p21waf/cipl --- p.107 / Chapter 6.3 --- Transactivation activity on oncogenes/ proto-oncogenes / Chapter 6.3.1 --- Effect on c-myc (NM´ؤ002467) mRNA expression --- p.109 / Chapter 6.3.2 --- Effect on RhoC (NM_017744) and Rabl4 (NM´ؤ016322) mRNA expression --- p.112 / Chapter CHAPTER 7 --- CONSTRUCTION OF TET-ON INDUCIBLE CELL-LINES / Chapter 7.1 --- Establishment of WRL/Tet-On monoclonal cell-lines Page / Chapter 7.1.1 --- Determination of geneticin selection dosage --- p.116 / Chapter 7.1.2 --- Selection of the best WRL/TOn clone using luciferase assay --- p.118 / Chapter 7.2 --- Establishment of inducible WRL/TOn/Gene monoclonal cell-lines / Chapter 7.2.1 --- Determination of hygromycin selection dosage --- p.120 / Chapter 7.2.2 --- Selection of positive WRL/TOn/Gene clones with viral genes --- p.122 / Chapter 7.3 --- Characterization of TOXDC1 cell-line / Chapter 7.3.1 --- Cell morphology --- p.125 / Chapter 7.3.2 --- Growth pattern of TOXDC1 --- p.126 / Chapter 7.3.3 --- HBxAC44 induced p21waf/cipl mRNA expression --- p.127 / Chapter 7.3.4 --- Doxycycline concentration dependent HBxAC44 expression in TOXDC1 --- p.129 / Chapter CHAPTER 8 --- DISCUSSION / Chapter 8.1 --- Selection of cell model / Chapter 8.1.1 --- Selection of cell models --- p.130 / Chapter 8.1.2 --- Selection of truncation mutant --- p.131 / Chapter 8.2 --- Differential sub-cellular localization of HBx and its variants / Chapter 8.2.1 --- Mechanisms of mitochondria targeting --- p.132 / Chapter 8.2.2 --- Mitochondria as site of HBx-induced apoptosis --- p.134 / Chapter 8.2.3 --- Stimulation of calcium release from mitochondria by wild-type HBx --- p.135 / Chapter 8.3 --- Cell cycle distribution profiling and its regulations / Chapter 8.3.1 --- Cell cycle pattern and cell proliferation --- p.136 / Chapter 8.3.2 --- Differential cell cycle molecular pathway activation --- p.138 / Chapter 8.4 --- Ras/Raf/MAPK mediated transactivation by HBxWt and its mutants / Chapter 8.4.1 --- p53-mediated p21waf/cipl expression --- p.142 / Chapter 8.4.2 --- ERK-mediated p21waf/cipl and wild-type p53 mRNA expression --- p.143 / Chapter 8.4.3 --- Regulation of oncogenes/ proto-oncogenes expression --- p.147 / Chapter 8.5 --- General discussions on differential effects of HBxWt and HBxAC44 --- p.149 / Chapter 8.6 --- Establishment of Tet-On/HBxAC44 cell-line TOXDC1 --- p.153 / Chapter 8.7 --- Conclusions --- p.154 / Chapter 8.8 --- Future Prospects / Chapter 8.8.1 --- From mitochondria targeting to calcium signaling --- p.157 / Chapter 8.8.2 --- Construction of a complete cell cycle regulation pathway --- p.158 / Chapter 8.8.3 --- Elucidation of the transcriptional transactivation regulation --- p.159 / Chapter 8.8.4 --- To make the best use of the Tet-on stable cell-line TOXDC1 --- p.159 / Chapter 8.8.5 --- Study with other carboxy-terminal truncation mutants --- p.160 / Chapter 8.8.6 --- In vivo study --- p.160 / REFERENCES --- p.162

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

This thesis consists of a brief description on cancer, carcinogenesis, the changes in the type and level of dietary fat available in our diets over time and association with the development of certain diseases. The main focus of this research was on omega 6 and omega 3 essential fatty acids (EFA) and their interaction with regards to carcinogenesis.