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Chronic liver diseases (including liver cancer) are a common cause of morbidity and mortality worldwide, accounting for almost 2 million deaths per year, with non-alcoholic fatty liver disease (NAFLD) becoming a growing burden on the healthcare systems in westernised countries. Liver fibrosis, driven by persistent, low-grade inflammation, is a common feature of chronic liver diseases, characterised by the unregulated wound healing processes that result in excessive extracellular matrix formation. Irrespective of aetiology, liver fibrosis and its most severe form cirrhosis, contributes to declining liver function and predisposes to hepatocellular carcinoma. The mechanisms by which liver injury progresses to more advanced stages are still being elucidated, resulting in the slow development of effective therapeutics for fibrosis prevention or reduction, where no effective therapeutic modalities to effectively treat liver fibrosis currently exist. Therefore, there is an unmet need for identification of new therapeutic targets to prevent disease progression, requiring suitable modelling systems to understand the pathogenesis of fibrosis and for efficient drug development. Mice have been widely utilised as preclinical models to understand the molecular mechanisms driving liver disease progression in an in vivo setting. Significant variations in the methodologies have contributed to a failure to produce standardised models with known and predictable fibrotic outcomes, resulting in discrepancies in the literature. Differing techniques utilised to measure liver injury and fibrosis also influence the study outcomes. This thesis aims to compare and contrast commonly used fibrosis models (Carbon tetrachloride, thioacetamide and atherogenic diet) while controlling for mouse strain, sex and age to assess their congruence with expected fibrosis or cirrhotic outcomes observed in the human condition. Thioacetamide (TAA) administration in the drinking water method produced the most advanced cirrhotic phenotype though requires a long period for induction, whereas carbon tetrachloride (CCl4) model is more suitable as an early inflammatory model with moderate fibrosis to early-stage cirrhosis. Thioacetamide administration by intraperitoneal injection with increasing dosage over 6 weeks presented a rapid fibrosis model, intermediate between the oral TAA and CCl4 in terms of liver injury and fibrotic outcome. Atherogenic diet for 12 weeks resulted in simple steatosis without fibrotic scar. No single model was able to recapitulate human liver disease progression. The injury phenotype in these ‘standardised’ models is useful to study the development of progressive liver injury stage in a reproducible manner, enabling specific experimental questions to be addressed based on expected liver injury and fibrotic outcomes. In the search for new therapeutics for liver fibrosis, repositioning of approved drugs is advantageous in the drug discovery process by identification of targets common to liver injury pathways. Oxidative stress has been implicated as driver of hepatocyte injury and in the pathogenesis of liver fibrosis progression to cirrhosis. The effects of a new pro-drug NP202, with proven efficacy in prevention of myocardial fibrosis in other pre-clinical models of heart failure and injury via antioxidant pathways, has not been previously investigated in the liver disease context, with potential use as an anti-fibrotic therapeutic. Mouse models of fibrosis and NAFLD/NASH development with the addition of NP202 were used to determine the efficacy of this pro-drug in the prevention of TAA, western diet or atherogenic diet-induced liver injury during disease progression and resolution of fibrotic scar after injury cessation. NP202 treatment did not prevent fibrosis development in TAA-injured livers, and had little effect on liver injury, steatosis or fibrosis development in the dietary models of NAFLD/NASH. After injury cessation, continued administration of the pro-drug failed to improved resolution of liver injury, steatosis and fibrotic scar. Unexpectedly, some mice administered concurrent TAA and NP202 treatment developed primary liver tumours. As inflammation is a major driver of fibrotic injury, a tumour necrosis factor (TNF) knockout mouse was utilised to model inhibition of a major proinflammatory mediator, as a potential therapeutic strategy to prevent fibrogenesis in hepatotoxin or diet-induced liver injury. During early phase injury, both inflammation and fibrosis were impaired with hepatotoxin induction, resulting in significantly lower level of hepatic injury. While inhibition of TNF did not inhibit the fibrogenic processes, the rate of inflammatory injury and fibrotic scar deposition was slowed in oral TAA administered mice compared to control mice. Further, Loxl2-mediated collagen crosslinking was impaired in TNF-deficient mice undergoing TAA or CCl4 induced liver injury, with resolution of fibrotic scar also impaired in these mice. In contrast, atherogenic diet caused significant hepatocyte ballooning and steatotic injury in TNF-deficient animals, suggestive that the role of TNF is dynamic and heavily dependent on the type of hepatotoxicant. Taken together, anti-TNF therapies have the potential to reduce the inflammatory injury driving early fibrogenesis, though alone cannot prevent fibrotic scar formation. Furthermore, TNF is required of efficient fibrotic scar dissolution after cessation of injury. Overall, while there is no single liver injury mouse model that can effectively recapitulate the natural liver disease progression observed in humans, mouse models continue to serve as an essential and irreplaceable pre-clinical model for understanding of pathological processes. The utility of well characterised liver fibrosis models allows the study of inhibition of major drivers of liver injury such as TNF and oxidative stress in a controlled manner, which is particularly important when testing novel therapeutics for safety and efficacy purposes. The expected injury phenotypes and examples of their use in practice, as described in this thesis can assist in the identification of the most suitable liver injury model for pre-clinical assessment of future anti-fibrotic therapeutics.

Conference poster presentation at the Gastroenterological Society of Australia Symposium 2020.Accompanying abstract:Background and Aim: Three‐dimensional (3D) cultures in materials like Matrigel (Corning Life Sciences) are considered more biosimilar to the in vivo tissue than two‐dimensional (2D) culture, due to effect of cell–extracellular matrix interactions in a more physiologically relevant spatial orientation. Major disadvantages include the considerable labor, sample‐to‐sample variation in 3D cultures, and low batch‐to‐batch reproducibility, rendering them more biologically relevant models but tedious for high‐throughput culture and downstream assays. To potentially overcome these issues, we aimed to use the RASTRUM 3D bioprinter (Inventia Life Science) with tunable synthetic polyethylene glycol (PEG)‐based matrices to efficiently and reproducibly create encapsulated 3D liver models.Methods: A range of human liver cell subpopulations, PH5CH8, LX‐2, and HepG2, were assessed for cell line growth and morphological changes when manually encapsulated in functionalized PEG‐based hydrogels of stiffness similar to healthy liver. Cells were also bioprinted using the drop‐on‐demand printing technology of the RASTRUM 3D bioprinter into 96‐well culture plates for encapsulation within a 3D matrix and monitored for cell growth, morphology, and viability for up to 7 days after printing by PrestoBlue cell viability assay and in situ calcein AM/propidium iodide staining. Intracellular albumin was assessed in PH5CH8 and HepG2 cells by in situ immunofluorescent staining. In a separate experiment, 3D‐bioprinted PH5CH8 and LX‐2 were treated with thioacetamide, a hepatotoxin used for robust cirrhosis induction in animal models, for IC50 determination compared with 2D cell cultures.Results: In all cell types, the addition of RGD peptide to PEG matrices had the greatest effect on cell morphology and growth. LX‐2 cells reacted strongly to the presence of collagen type I, with spindle‐like morphology more similar to an in vivo activated state. Bioprinting of each cell type into 96‐well culture plates took about 20 min per plate and resulted in consistently formed and encapsulated spheroidal 3D cell models, with high viability at 7 days in culture (> 95%). In post‐print encapsulation with 3D culture, PH5CH8 and HepG2 spheroids were positively stained for albumin. In vitro toxicity studies showed both PH5CH8 and LX‐2 cells printed and grown in 3D in 96‐well plates were 2–4 times more resistant to thioacetamide‐induced toxicity than 2D grown cultures. Importantly, bioprinting was reproducible, with the same morphology observed across the 96‐well plate and between non‐consecutive printing events for each cell line within 10 passages.Conclusion: We show that the RASTRUM 3D bioprinter is able to efficiently and reproducibly create 3D liver cell models into 96‐well plates, with high cell viability in functionalized PEG‐based matrices. Moreover, PH5CH8 and LX‐2 spheroidal structures in these 3D cultures are more resistant to toxic insults, such as thioacetamide, than 2D culture cells and, therefore, are more similar to in vivo conditions. We conclude that RASTRUM‐printed cells in PEG‐based matrices are an alternative for 3D‐based cell culture models for use in high‐throughput assays of more biological relevance.

Sequence signals may be defined as linguistic forms that ostensively mark connections and relationships between one part of a piece of continuous writing and another. This study demonstrates that a wide range of sequence signals occur in technical writing, and hence must be taught to students who read technical English. (Author)

The thesis discusses the metaphors used in the speeches of US President Barack Obama and Chinese Premier Wen Jiabao. The framework is Cognitive Metaphor Theory, which introduces the idea that cognitive metaphors are conceptualizations or patterns of thought, not lingusitic phenomena, although these metaphors give rise to linguistic metaphors. The metaphorical mapping, the relations between conceptual metaphors and our experience, the differences between conceptual metaphors and poetic metaphors, as well as the functions of metaphors are also discussed. It is the purpose of the thesis to show the ubiquity of metaphor by analyzing the metaphors used in the West (represented by Obama) and the East (represented by Wen). A comparison of the similarities and differences of the conceptual metaphors used by the two politicians is made. The topics of speeches vary, from internal affairs (such as economy and domestic politics) to international affairs. The findings show that the conceptual metaphors TO PURSUE WELL-BEING IS A JOURNEY, PERSONIFICATION and POLITICS IS WAR are found to be the most dominant in both the English and the Chinese corpus, but some details or linguistic expressions in the same conceptual metaphors differ. Some specific American and Chinese metaphors are also highlighted, for instance, the American Dream and the Chinese flag. Metaphors are extensively used in both Obama and Wen’s speeches, and in fact among politicians as well, since metaphors not only have rhetoric functions, but also the power function of legitimization and delegitimization.

Older people who experience declining health are often faced with difficult decisions about possible residential relocation. The research aim was to determine how five distinct dimensions-functional status, features of current housing, social networks, features of retirement communities, and financial considerations-affect decisions to relocate to a retirement community. A vignette experiment with a factorial design was conducted involving both older people and adult children who were concerned with an aging parent. Use of the Internet for administration of the experiment made it possible to deliver information to research participants through video clips. Research participants were influenced by each of the dimensions; however, functional status of the vignette persons had the greatest impact, and financial considerations the least. Adult children were more likely to recommend moves than were older people. The research is suggestive of the potential for use of vignette experiments for a fuller understanding of relocation decisions.