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(2023) Ireland, JakeThesisPluripotent stem cell-derived cardiomyocytes (hPSC–CM) have great importance for predicting safety parameters for pharmaceutical compounds and models of healthy versus disease states of the human heart. In recent years, there has been an insistence that all new pharmaceutical products are tested on in vitro models for potential proarrhythmic effects and the increased demand for improved biomimetic hPSC-CM in pharmaceutical safety assays such as the Comprehensive in vitro Proarrhythmic Assay (CiPA). In addition, hPSC-CM are being utilised in cell therapies to treat and reverse the effects of ischaemic heart disease, offering potential cures for cardiovascular diseases instead of treatments for delaying progressive heart failure. In the first part of this thesis, I will examine how purified extracellular matrix proteins (ECMPs) can influence pluripotent stem cell (PSC) behaviour and how we may use this to precondition cardiac progenitor lineage specifications. I use array-based techniques to investigate how protein combinations affect proliferation, pluripotency, germ layer, and cardiac progenitors. This method allows us to visualise how individual proteins can affect cells' behaviour in a larger array whilst highlighting how specific combinations can precondition pluripotent cells towards a cardiomyocyte lineage. This combinatorial approach led to the identification of several unique matrices that promote differentiation, which will aid efforts at producing therapeutically useful cell types with greater efficiency. In the second part of this thesis, I demonstrate a novel bioreactor that attenuates a magnetic field to dynamically modulate the stiffness of magnetoactive hydrogel to look at how biomimetic dynamic stiffening of a substrate can influence cardiomyocyte lineage specification. We investigate how biomimetic in vivo mechanics may influence cell fate by following the expression profiles of cells in different dynamic environments. Non-invasive electromagnetic signals affect substrate stiffness when combined with magnetic particles and magnetic fibres and how this can help direct cell orientation and accompanying lineage specification Finally, I investigate how variability in cell phenotypes and expression patterns are influenced by biomimetic cues and how these variabilities could be utilised in future safety assessment protocols and cell therapy treatments for cardiovascular disease.
(2020) Khan, MahjabeenThesisPseudomonas aeruginosa causes both contact lens and non-contact lens-related keratitis (corneal infection). This opportunistic bacterium naturally has the ability to resist the mechanism of action of many antibiotics which are used for treatment. P. aeruginosa resistance patterns and the mechanism of resistance in isolates from keratitis are not well understood. This thesis described the phenotypic and genotypic patterns of antimicrobial resistance and compared these between ocular isolates of P. aeruginosa from Australia (contact lens) and India (non-contact lens). Changes in the antimicrobial susceptibility between isolates over time were also analysed. Susceptibility to antibiotics, multipurpose disinfecting solutions and disinfectants was analysed for twenty-seven Australian isolates from contact lens-related keratitis and forty non-contact lens-related isolated from India. The whole genomes of fourteen Australian (historical and recent) and twelve Indian isolates were sequenced using Illumina® MiSeq®. Computational analysis of the genomes was performed to analyse their core and pan genomes and these were examined for the presence of acquired resistance genes, virulence genes, gene mutations, and these compared to their phenotypic resistance to antibiotics. Indian isolates possessed large pan genomes with more acquired resistance (30) genes and larger numbers of genetic variations. The Indian isolates contained clones of three sequence types ST308, ST316 and ST491, whereas Australian isolates contained only one sequence type ST233. Isolates with larger gene variations had mutations in the DNA mismatch repair system. Most multi-drug resistant Indian (non-contact lens) isolates were exoU +. Indian isolates had large accessory genes compared to Australian isolates and this increased the pan genome size of the Indian isolates. The number of core genome mutations were larger in the Indian isolates a median of 50006 (IQR=26967-50600) compared to Australian isolates a median of 26317 (IQR=25681-33780). There were differences between isolates from Australia and India with respect to their antibiotic resistance and associated genes. Indian strains had more genetic diversity and were multi-drug resistant. However, there was no evidence of substantial genetic or phenotypic changes within isolates from their respective countries.