Medicine & Health

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  • (2021) Collins, Scott
    Thesis
    Chronic liver diseases including cirrhosis and primary liver cancer are a significant health burden worldwide. Liver cirrhosis is end stage liver injury resulting in a progressive fibrosis phenotype, in which the hepatic architecture is distorted. The most common cause of cirrhosis is chronic liver injury caused by hepatitis B, alcohol related liver disease, hepatitis C or non-alcoholic steatohepatitis. Primary liver cancer is a leading cause of cancer mortality globally and is commonly observed as a progression of liver cirrhosis. Liver injury usually occurs because of immune-mediated or direct injury to the hepatocytes and involves multiple cellular subsets; including hepatic stellate cells, liver adipocytes, liver resident macrophages kupffer cells, endothelial cells and infiltrating immune cells. Injury to these cells result in the release of reactive oxygen species, proinflammatory signals, proliferation-associated cytokines, and the activation of repair pathways. A chronic activation of these signals can result in dysregulation of the normal repair response and generation of a pathogenic fibrotic response. A broadly canonical response, chronic inflammation drives fibrosis and cirrhosis irrespective of liver injury aetiology. The burden of liver disease provides the impetus to pursue the use of representative in vitro models of liver function and responses to injury. Improved 2D and 3D in vitro disease models would enhance our understanding of the causes of liver injury and the development of cirrhosis and primary liver cancer while increasing the efficacy of preclinical drug discovery. Current 2D in vitro assays based on cell lines such as HepG2 that have reduced metabolic capacities compared to primary hepatocytes ex vivo, and the use of primary human hepatocytes suffers from high donor-to-donor variation and only retain in vivo characteristics for a short time ex vivo. The shortcomings of 2D cell culture models have driven the development of 3D cell culture techniques. The advantages of 3D models include replicating the complex attributes of the liver beyond liver specific metabolism, such as increased cell density, organisation, and cell-cell signalling, O2 zonation, as well as the anatomy of the liver lobule and the circulatory system. After a comprehensive review of all the current in vitro models of the liver we hypothesised that a liver organoid cell culture model co-cultured with myofibroblast like hepatic stellate cells can model liver injury. An organoid cell culture is defined as a collection of cells culturing several cell types that develop from stem cells or organ progenitors and self-organise through cell sorting and spatially restricted lineage commitment, similar to organogenesis in vivo. Liver organoids have demonstrated many advantages over conventional in vitro models such as long-term genetic stability, 2D in vivo-like organisation, and maintaining the necessary cellular cross talk and behavioural characteristics of their primary corresponding cells. The focus of this thesis is the application of 3D liver organoids to model and analyse the molecular and cellular effects of liver injury. We established a 3D liver organoid cell culture model from primary mouse tissue and characterised the capacity of these organoids to model liver characteristics in vitro and used this model to define the interactions between organoid hepatocytes and hepatic stellate cells in a co-culture trans-well system. The impact of inflammatory cytokines tumour necrosis factor-α and transformation growth factor-β on this model, as well as other variables such as hypoxia and the anti-fibrosis drug Halofuginone were assessed. Hepatic stellate cell dependent decreases in organoid viability and organoid dependent increases in hepatic stellate cell viability were observed, as well as Halofuginone dependent decreases in hepatic stellate cell viability were also observed. Markers characteristic of liver injury and fibrosis, such as Actn1 and Lamb3 were upregulated in hepatic stellate cells, although collagen expression was downregulated in these cells. Transcriptional profiling revealed a tumour necrosis factor-α mediated apoptotic response in organoids and an inflammatory response in both the organoids and hepatic stellate cells. We concluded that while liver organoids and hepatic stellate cells responded to experimental variables, there were limitations when it came to the cross talk between the cultures in the trans-well system. While apoptotic bodies from the organoids may have stimulated proliferation of hepatic stellate cells, many key genes responsible for liver injury were either not upregulated or were downregulated in co-culture. Electron microscopy analysis of liver organoids showed important ultrastructural changes compared to a whole liver section. Our findings of secreted exosomes, microvilli within the lumen of the organoids, and many ultrastructural features found within liver cells in vivo confirm that our 3D liver organoids closely resemble the liver. We also demonstrated how the use of high-resolution field emission scanning electron microscopy with automated scan resolution can generate a high-resolution ultrastructure map of the whole organoid. This method can also be combined with correlative light electron microscopy for immunofluorescent labelling of proteins of interest using quantum dot nanoparticles. Overall, our 3D organoid model of liver injury had encouraging results and furthering our understanding of pathogenesis of liver fibrogenesis in vitro and the study of novel anti-fibrotic therapeutic agents.

  • (2021) Ng, Olivia
    Thesis
    The arteriovenous fistula (AVF) is a surgically-made vascular structure connecting an artery to a vein. It is the optimal form of vascular access for haemodialysis-dependent end-stage renal disease patients. However, AVF are prone to access dysfunction through the formation of stenoses, which compromise the structure’s utility. To date, a plethora of clinical models are used to predict AVF formation failure based on patient factors and other models predicting late AVF failure by assessing haemodynamics and quantifying disturbed flow behaviours and wall shear stress metrics with stenosis formation. That said, inconsistencies were identified in the correlation between these metrics and diseased AVFs. This thesis aims to assess the suitability of another haemodynamic-related metric, resistance, derived from pressure drop and flow rates through patient-specific CFD modelling, for diagnosing and predicting AVF failure. A three-dimensional ultrasound scanning system was used to obtain patient-specific geometry and flow profiles, used for CFD models which were then analysed, with resistance calculated for each patient. The significance of patient-specific CFD modelling was demonstrated in its usefulness to generate a patient-targeted indicator of diseased AVF. To study the effectiveness of resistance as a metric, the relationship between CFD-derived resistance and the potential for AVF failure was evaluated, starting with classification of resistance results among patients who had undergone treatment for stenosis. An exploratory study into the suitability of CFD-derived resistance and its association with patients’ AVF conditions was further conducted by classifying data from a larger patient dataset and fitting the classified data to a multilevel regression model. CFD-derived resistance was found to be higher at the proximal vein of problematic AVF, however this figure was 76% lower among patients who had undergone stenosis treatment. Meanwhile, no correlation was found between resistance at the proximal artery and patency status. An area under curve of 92.1% was found from the receiver operating characteristic analysis, noting an outstanding discrimination of the classification. CFD-derived resistance appears to be a promising metric in the assessment of a suitable diagnostic marker for AVF failure. This research concludes with aspirations for clinical implementation of a related system, alongside routine surveillance of AVF.