Wu, Xinggang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 124-161). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

To, Ka Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (p. 115-120). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / 中文摘要 --- p.vi / List of abbreviations --- p.ix / List of tables --- p.xii / List of figures --- p.xiii / Contents --- p.xvi / Chapter Chapter One: --- General introduction --- p.1 / Chapter 1.1 --- Background of Hepatocellular carcinoma (HCC) --- p.2 / Chapter 1.2.1 --- ZBP-89 structure and its expression in cancers --- p.3 / Chapter 1.2.2 --- Transcriptional regulation of ZBP-89 --- p.5 / Chapter 1.3.1 --- Apoptosis and necrosis --- p.6 / Chapter 1.3.2 --- Mechanisms of Apoptosis --- p.7 / Chapter 1.4 --- Bcl-2 family --- p.11 / Chapter 1.5 --- Regulation of p53 cancer cells --- p.12 / Chapter 1.6 --- The aim of the study --- p.13 / Chapter Chapter Two: --- Up-regulation of Bak expression by Ad-ZBP-89 induce apoptosis in human liver cancer cells --- p.15 / Chapter 2.1. --- Introduction --- p.16 / Chapter 2.2. --- Materials and methods --- p.18 / Chapter 2.2.1. --- Cell culture --- p.18 / Chapter 2.2.2. --- RT-PCR --- p.19 / Chapter 2.2.3. --- Western blotting --- p.21 / Chapter 2.2.4. --- Adenovirus infection and Cell viability assay --- p.24 / Chapter 2.2.5. --- Detection of apoptosis --- p.26 / Chapter 2.2.6. --- RNA interference --- p.27 / Chapter 2.2.7. --- Statistical analysis --- p.29 / Chapter 2.3. --- Results --- p.30 / Chapter 2.3.1. --- Endogenous expression of ZBP-89 and Bak of human liver cancer cells --- p.30 / Chapter 2.3.2. --- Effects of Ad-ZBP-89 on proliferation in HCC cell lines --- p.31 / Chapter 2.3.3. --- Effects of Ad-ZBP-89 on the expression of Bcl-2 family members --- p.34 / Chapter 2.3.4. --- ZBP-89 induced Bak expression and release of cytochrome c --- p.39 / Chapter 2.3.5. --- Effects of Ad-ZBP-89 on apoptosis rate in HCC cell lines --- p.41 / Chapter 2.3.6. --- Effects of ZBP-89 siRNA on expression of Bcl-2 family members and proliferation in HCC cell lines --- p.43 / Chapter 2.3.7. --- Effects of Bak siRNA and its combined effect with Ad-ZBP-89 on the expression of Bak and reduced apoptosis in HCC cell lines --- p.50 / Chapter 2.4. --- Discussion --- p.55 / Chapter Chapter Three: --- Identification of ZBP-89 protein as an apoptosis activator for a pro-apoptotic Bak gene promoter --- p.60 / Chapter 3.1. --- Introduction --- p.61 / Chapter 3.2. --- Materials and methods --- p.64 / Chapter 3.2.1. --- Cell lines and tissues --- p.64 / Chapter 3.2.2. --- Transient transfection and Luciferase activity assay --- p.64 / Chapter 3.2.3. --- pGL3-Bak-promoter vector construction --- p.67 / Chapter 3.2.4. --- "Preparation of mitochondrial, cytosolic and nuclear fractions" --- p.74 / Chapter 3.2.5. --- Electrophoretic mobility shift assay --- p.75 / Chapter 3.2.6. --- Overexpression of Bak --- p.76 / Chapter 3.2.7. --- RT-PCR and Western blot analysis on HCC tissues samples --- p.80 / Chapter 3.2.8. --- Statistical Analysis --- p.80 / Chapter 3.3. --- Results --- p.81 / Chapter 3.3.1. --- ZBP-89 activates Bak-luciferase promoter genes in HCC cells --- p.81 / Chapter 3.3.2. --- ZBP-89 activates shortened Bak-luc-promoter in PLC/PRF/5 and SK-Hep-1 cells --- p.82 / Chapter 3.3.3. --- ZBP-89 is a potential binding protein to the Bak promoter gene region -457/-407 --- p.85 / Chapter 3.3.4. --- The combined effects of Bak overexpression and Ad-ZBP-89 induce apoptosis in HCC cells --- p.89 / Chapter 3.3.5. --- The combined effects on Bak protein expression --- p.94 / Chapter 3.3.6. --- Bak expression in HCC tissues --- p.98 / Chapter 3.4. --- Discussion --- p.99 / Chapter Chapter Four: --- Conclusion and Future Perspectives --- p.104 / Chapter 4.1. --- Conclusion --- p.104 / Chapter 4.2. --- Future Perspectives --- p.112 / Reference --- p.113

Cholestasis is an important liver pathology. During cholestasis bile acids accumulate in the bile canaliculus affecting hepatocyte viability. The actions of bile acids require changes in the release of Ca2+ from intracellular stores and in Ca2+ entry. The target(s) of the Ca2+ entry pathway affected by bile acids is, however, not known. The overall objective of the work described in this thesis was to elucidate the target(s) and mechanism(s) of bile acids-induced modulation of hepatocytes calcium homeostasis. First, it was shown that a 12 h pre-incubation with cholestatic bile acids (to mimic cholestasis conditions) induced the inhibition of Ca2+ entry through store-operated Ca2+ channels (SOCs), while the addition of choleretic bile acids to the incubation medium caused the reversible activation of Ca2+ entry through SOCs. Moreover, it was shown that incubation of liver cells with choleretic bile acids counteracts the inhibition of Ca2+ entry caused by pre-incubation with cholestatic bile acids. Thus, it was concluded that SOCs are the target of bile acids action in liver cells. Surprisingly, despite the effect of choleretic bile acids in activating SOCs, the Ca2+ dye fura-2 failed to detect choleretic bile acid-induced Ca2+ release from intracellular stores in the absence of extracellular Ca2+. However, under the same conditions, when the sub-plasma membrane Ca2+ levels were measured using FFP-18 Ca2+ dye, choleretic bile acid induced a transient increase in FFP-18 fluorescence. This evidence suggested that choleretic bile acids-induced activation of Ca2+ entry through SOCs, involving the release of Ca2+ from a region of the endoplasmic reticulum (ER) located in the vicinity of the plasma membrane.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 283-321).

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 192-197).

published_or_final_version / Surgery / Master / Master of Philosophy

This project studied the biodegradation of a plasticizer, di-(2-ethylhexyl) adipate (DEHA), by two mammalian cell lines, HepG2 and WIF-B, in vitro . An MTT assay showed that DEHA had a toxic effect on both cell lines. Despite this, both hepatocyte cell lines successfully degraded the plasticizer. Metabolites were identified and quantified by gas chromatography. HepG2 cells showed minimal alcohol dehydrogense activity and this resulted in the accumulation of 2-ethylhexanol. WIF-B cells were able to breakdown the alcohol and produced 2-ethylhexanoic acid. It is important to note that an enzyme was essential for this step in the degradation of the plasticizer, as this proves that it was biodegradation and not physical degradation. By comparing the metabolites formed and the order of their appearance, the degradation pathway in these mammalian cells was found to be similar to the established degradation pathways for bacteria, fungi and yeast.

Hepatocyte-directed liposomal gene delivery systems have received much attention in view of the present lack of suitable treatment alternatives for several liver-associated disorders. While targeting of liposomes to the asialoglycoprotein receptor (ASGP-R), nearly-exclusive to hepatocytes, is a well-documented means of achieving cell-specificity, several intra- and extracellular barriers reduce the efficacy of liposomal gene transfer. These include the aggregation and opsonisation of lipoplexes by serum components; and endo/lysosomal degradation of internalised DNA. This study has attempted to address the individual concerns by modifying hepatotropic liposomes with a steric stabilising, polyethylene glycol (PEG) shroud, and an endosomal escape-inducing proton sponge moiety. Novel galactosylated (SH02) and imidazolylated (SH04) cholesterol derivatives were successfully synthesised with the aim of conferring the respective functions of ASGP-R-specificity and proton sponge capability upon cationic liposome formulations. The individual derivatives afforded stable, unilamellar vesicles (< 200 nm, Z-average diameter) when incorporated at 10 % on a molar basis with the cytofectin, 3β[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) and co-lipid, dioleoylphosphatidylethanolamine (DOPE). Modification of these formulations with 1,2-distearoyl-sn-glycero-phosphoethanolamine-N-[carboxy(polyethylene glycol)2000] (DSPEPEG₂₀₀₀), at 5 mol %, gave smaller vesicles (< 110 nm, Z-average diameter) and moderately reduced the instability associated with the combination of both SH02 and SH04 in a single formulation. Individual preparations formed electrostatic complexes with pCMV-luc plasmid DNA, as demonstrated by gel retardation assays and electron microscopy. Furthermore, the liposomes afforded some protection to the DNA cargo against serum nuclease attack during a 4 hour-long exposure to foetal calf serum at 37 °C. However, the DNA-binding and protecting capabilities of the liposomes were reduced upon addition of the PEG coating. Growth inhibition assays showed that lipoplexes derived from individual formulations were well tolerated by human hepatocyte-derived, HepG2, and embryonic kidney, HEK293, cell lines. Expression of the luciferase transgene mediated by non-pegylated formulations containing SH02 was significantly higher in hepatocytes than in the ASGP-R-negative, kidney cells. Furthermore, receptor-mediated internalisation of non-pegylated, galactosylated carriers by hepatocytes was demonstrated by the gross inhibition of transfection in the presence of excess asialofetuin, a natural ligand to the ASGP-R. Liposome acid titration profiles highlighted the endosomal pH-buffering capacity afforded by SH04. However, the imidazolylated lipid enhanced the transfection activity of the non-sterically stabilised Chol-T/DOPE system, but not that of its targeted counterpart, and only with respect to HEK293 cells. Finally, pegylation reduced the transfection capability of liposomes by at least three orders of magnitude in both cell lines. The results suggest that further optimisation of liposome composition is necessary in order to achieve a liposomal system that simultaneously embodies hepatocyte-targeting, proton sponge and long-circulating properties. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2012.

Bibliography: leaves 116-138 / [14], 138 leaves, 5 leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Clinical & Experimental Pharmacology, 1988

[Truncated abstract] Hepatic progenitor cells (HPC) are becoming increasingly recognized as facultative stem cells capable of regenerating the liver during chronic liver injury and also as targets of malignant transformation. Similar markers are expressed by hepatocellular carcinoma (HCC) and HPC, and a precursor-product relationship is well established. This thesis focuses on the ways in which the HPC population can be controlled under circumstances of chronic liver injury, and in this manner, reduce the risk of progression to HCC reduced. The major aim of Chapters 3 to 5 was to elucidate the effect of interferon α (IFNα) therapy on HPC. Chronic hepatitis C affects approximately 250 million individuals world wide. Approximately 80% of infections progress to chronicity, which places the individuals at greater risk of developing HCC. The gold standard of treatment of chronic hepatitis C is a combination of pegylated IFNα and ribavirin. ...The results were surprising. While IFNγ exerted a pro-apoptotic and antiproliferative effect on HPC in vitro, administration of IFNγ to CDE-fed mice for 14 days increased fibrosis, enhanced inflammatory infiltration and exacerbated the HPC response, with concurrent hepatocyte cell death. In addition, increased morbidity and mortality were observed in the IFNγ-treated mice compared to control. IFNγ treatment was found to prime the liver for the HPC response by recruiting inflammatory cells and altering the hepatic cytokine profile, both of which may facilitate an increased HPC response. Numbers of activated HSC were also increased in the IFNγ-treated, CDE-fed mice, correlating with the increased fibrosis seen in these animals. This data contradicts the current experimental use of IFNγ for treatment of fibrosis. Based on our results, we suggest that IFNγ promotes HPC proliferation in the CDE model, by encouraging inflammatory infiltration and hepatocyte damage and this initiates pro-fibrotic events. Concurrent proliferation of HPC and activated HSC further supports the view that there is a close relationship between the two cell types, and thus, a link between the HPC response and fibrosis. In conclusion, findings documented in this thesis suggest that administration of IFNα and IFNγ can contribute to shaping the HPC response. IFNα therapy may reduce HCC risk in chronic hepatitis C patients by bringing the HPC population under control. In contrast, IFNγ treatment can exacerbate the HPC response, liver fibrosis and parenchymal damage, illustrating the need to approach this method of fibrosis treatment with caution.