Medicine & Health

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  • (2020) Khan, Mahjabeen
    Thesis
    Pseudomonas 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.

  • (2020) Rowlands, Benjamin
    Thesis
    Sirtuins (SIRTs) comprise a family of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases, capable of affecting health-span and DNA expression. In cell-culture and peripheral-tissue models, researchers have identified that SIRT1 and SIRT2 are also capable of changing enzymatic activity in glycolysis and Krebs cycle. In brain, the impact of SIRT1 and SIRT2 deacetylase activity on metabolism is poorly understood. The aim of this project was to determine if metabolic pathways in brain could be regulated by SIRT1 and SIRT2-mediated deacetylation in mammalian systems. An established ex vivo reductionist model of brain metabolism was used to test the hypothesis that direct inhibition, activation or ablation of SIRT1 or SIRT2 deacetylase activity would result in significant changes in brain metabolism. Alterations in brain metabolism were assessed by examining changes in 13C-enriched substrates, and metabolite pools with 13C and 1H nuclear magnetic resonance spectroscopy. Chapter three provides evidence that approximately 30% of the GABA synthesized from [1,2-13C]acetate was made directly in neurons. Activation of neuronal specific SIRT1 caused an increase in the incorporation of [1,2-13C]acetate into brain, while activation of astrocytes with potassium depolarization caused a decrease in [1,2-13C]acetate incorporation. These results indicate that acetate is not a reliable marker, nor exclusively metabolised in astrocytes. Further, brain metabolism of acetate is modulated through enzyme acetylation regulated by SIRT1 deacetylase activity. Results in chapter four posit that activation of SIRT1 with SRT 1720 directly stimulated incorporation of 13C into Krebs cycle intermediates and reduced incorporation into lactate. Several off-target effects were observed for SIRT1 activator resveratrol and SIRT1 inhibitory EX-527 that questions their suitability for study of SIRT1 activity. Chapter five concludes that inhibition of SIRT deacetylase activity by AGK2 produced an effect consistent with glutamatergic AMPA receptor activation, in keeping with known SIRT2 targets. Potent SIRT2 inhibitor C64 increased 13C label incorporation into GABA from [1-13C]D-glucose in guinea pigs, and glutamine from [1,2-13C]acetate in WT mice, an effect that was also observed in SIRT2 KO mice. These results indicate that SIRT2 deacetylase activity may impact neurotransmitter systems. This thesis supports the theory that SIRT1 and SIRT2 deacetylase activity can influence brain metabolism in mammalian systems.