No description available.

No description available.

The hepatitis B virus (HBV), chronically infecting ~360 million people worldwide, accounts for over half of the cases of hepatocellular carcinoma (HCC) and contributes to over 650,000 deaths per year making HCC is the fifth most common and second most deadly form of cancer.1-3 Many genes deregulated by the hepatitis B x-antigen (HBx), the oncogenic protein encoded by HBV, are known drivers of HCC.4,5 The timing of these alterations in the initiation and progression of disease, however, are poorly understood and the treatment options for HCC are extremely limited. Here, RNA-Seq expression data originally from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) project were used to compare truly healthy liver tissues, a base-line level often excluded from cancer studies, to both non-tumor and tumor tissues of patients with HBV associated HCC. This illuminated inflammation and immune response process as dysregulated prior to tumor formation followed by disruption of cell cycle and cell survival processes once tumors have arisen. Connecting these processes are UBD, BCL6, METTL24, CHRNA4, and NFKBIZ which putatively serve as crucial early drivers in the progression from HBV infection to the development of HCC and affect the serum level of downstream targets which could serve as biomarkers for earlier disease detection. Differential methylation analysis was also carried out on samples directly from TCGA and the Gene Expression Omnibus (GEO) to determine if the differentially expressed genes were potentially deregulated due to reversible epigenetic alterations. Enriched pathways for differential methylation in non-tumor samples included the immune system and the cell cycle but none of the genes of interest from differential expression analysis were differentially methylated until stage 1 indicating that methylation is involved in the progression of disease and not initiation. / Biology

Two types of estrogen binding sites were found in the rainbow trout liver cytosol, using the equilibrium binding assay. The higher affinity, lower capacity type I site had a Kd range of 0.53-5.9 nM and a concentration range of 14-95 pmoles/g protein. The lower affinity, higher capacity type II sites had a Kd range of 65-265 nM and a concentration range of 20-180 pmoles/g protein. These estrogen binding sites are both bound by ^3H-moxestrol. The estrogen binding sites in the serum were also examined and two components were found with the higher affinity component having a Kd of 2.63 nM and a concentration of 25 pmoles/g protein while the lower affinity component had a Kd of 79 nM and a concentration of 200 pmoles/g protein. However, these estrogen binding components are unable to bind the synthetic estrogen, DES, and therefore cannot account for either of the binding components found in the liver cytosol which bind DES as readily as 17β-estradiol. The cytosol estrogen binding sites bind non-estrogens in addition to estrogens. Sucrose gradient centrifugation of the cytosol yielded two estrogen binding peaks, one at 4.4S, the other at 3.7S. The heavier peak contained binding sites able to bind progesterone as well. Gel filtration of the cytosol also resulted in two peaks, one at 43,000 daltons, the other at 33,000 daltons. Again the heavier peak could be partially competed out by progesterone. The half life of these binding components was 60 minutes at 37°C, while no decrease in binding was observed after 4 hours at either 0 or 12 0 c. After 17β-estradiol treatment in vivo type I sites (relative to type II sites) were 74% depleted after 8.5 hours and 40% depleted after 24 hours. Finally, o,p'-DDT and p,p'-DDT, components of technical grade preparations of the insecticide DDT were both able to compete for estrogen binding sites in the rainbow trout liver cytosol and therefore may be able to affect the expression of estrogen inducible genes. / Thesis / Master of Science (MSc)

In vitro liver models that closely mimic the in vivo microenvironment are central for understanding hepatic functions and intercellular communication processes. Bottom-up shotgun proteomic analysis of the hepatic cells can lend insight into such processes. This technique employs liquid chromatography-tandem mass spectrometry (LC-MS/MS) for relative quantification of protein abundances by measuring intensities of their corresponding peptides. Organotypic 3D liver models have been developed in our laboratory that consist of hepatocytes and liver sinusoidal endothelial cells (LSECs) separated by a polyelectrolyte multilayer (PEM), which serves as a mimic for the Space of Disse. Each component within these models is easily separable allowing for systematic evaluation of the cells and PEMs. In this study, proteomes of hepatocytes from PEM containing models, cultured with and without LSECs, were compared to those from monolayers. Changes in core metabolism were evaluated among all culture conditions. Overall, all cultures were ketogenic and performed gluconeogenesis. The presence of the PEM led to increases in proteins associated with mitochondrial-based β-oxidation and peroxisomal proteins. The PEMs also limited production of structural proteins, which are linked to dedifferentiation of hepatocytes, suggesting that cell-ECM interactions are essential for maintenance of their liver-like state. The presence of LSECs increased levels of carboxylesterases and other phase I and phase II detoxification enzymes suggesting that intercellular signaling mediates enzyme abundance. Taken together, these results suggest that the cell-cell (from the LSECs) and cell-ECM (from the PEMs) interactions exert different, yet crucial effects, and both are required for the preservation of metabolic liver functions and differentiated phenotypes. Changes in the PEMs as a result of cell culture were also evaluated but exhibited minimal differences at this time point. Proteomes of LSECs monolayers were also characterized. Enzymes related to the metabolism of amino acids, lipids, oxidative phosphorylation and phase I and phase II detoxification processes were all identified in LSECs monolayers highlighting their role in these processes. Characterization of 3DHL LSECs was not possible due to ion suppression resulting from the presence of excess contaminant proteins. Nonetheless, this study provides a foundation in which LSECs from 3D liver models can be compared against in future studies. / Ph. D.

Liver progenitor cells (LPCs) play a major role in the regeneration process following chronic liver damage. LPCs can differentiate into hepatocytes and cholangiocytes and thus are capable of replenishing the damaged liver. Due to their plasticity and robust nature in culture systems, they are promising candidates for use in cell therapy. However, to be able to use LPCs as tissue regenerating stem cell-like cells in the clinic, we need to fully understand how they are controlled. Although a strong association between LPCs and inflammation has been shown in many chronic liver diseases, the role of the immune system in LPC-mediated hepatic regeneration is poorly understood. We hypothesise that specific immune cells and mediators are needed to induce the LPC compartment, and that these are common to the LPC response in different injury settings. Therefore, the present study focused on the characterisation of the inflammatory environment in the LPC response, which generates this niche. The aims of this study were (i) to identify the immune cells that are important for the LPC response, (ii) to define the cytokine profile and (iii) to determine the role of the cytokine producing cells during liver regeneration. To study hepatic inflammation following liver injury, a diet-induced model of liver injury (choline-deficient, ethionine-supplemented diet, CDE diet) was compared to two transgenic mouse models of immune-mediated hepatitis (Met-Kb, 178.3). Although all three models are characterised by hepatitis, histological analysis revealed that LPCs were only detectable in the CDE and Met-Kb livers. In the 178.3 model, livers regenerated from proliferating hepatocytes. An LPC response could not be induced in these mice even when liver damage was made more severe. In the other two models, LPC numbers increased over time showing the highest numbers one week after the peak of liver injury. LPCs were often found in close proximity to inflammatory cells, in particular macrophages.

No description available.

published_or_final_version / Pharmacology / Doctoral / Doctor of Philosophy

Li Ka Shing Prizes for best PhD theses in the Faculties of Dentistry, Engineering, Medicine and Science, 2006-2007 / published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

No description available.