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(2020)ThesisDNA cytosine methylation is an important epigenetic modification that plays a key role in gene expression. DNA methylation has been shown to be involved in numerous processes, including X-chromosome inactivation in mammals, retrotransposon silencing, genomic imprinting, carcinogenesis and the regulation of tissue specific gene expression during development. Gene expression is tightly regulated via DNA methylation (5mC) and the aberrant expression of meiotic genes in mitotic cells via CpG promoter hypomethylation has been proposed to cause cancer. Cancer/Testis Antigens (CTAs) are a group of genes that encode tumour specific antigens and are expressed in the testis, certain cancers but not in normal post-natal somatic tissues. CpG island methylation and histone modifications appear to play a role in the epigenetic regulation of CTA expression, however, very little is known about their functions in vivo. A widely studied but poorly understood question to date is the mechanisms behind aberrant CTA reactivation in cancer. Given that 5mC mediated gene repression has been found to exist in vertebrate genomes and CTAs have also been identified to be a subset of highly evolutionarily conserved genes, it is critical to understand the role of 5mC mediated CTA silencing in vertebrates. By gaining a deeper understanding into the mechanisms behind this highly conserved pattern of gene repression on a specific subset of genes, we would be able to identify methods to prevent aberrant gene expression. In this study, I analysed publicly available whole genome bisulfite sequencing (WGBS), RNA-seq and chromatin immuno-precipitation followed by massively parallel sequencing (ChIP-seq) data of developing embryonic and adult somatic tissue of 3 vertebrate species to elucidate the evolutionary epigenetic regulation of CTAs in vertebrate genomes. Integrative WGBS, RNA-seq and ChIP-seq analysis revealed that CTAs are evolutionarily conserved in zebrafish, mice and humans and mechanisms of their epigenetic regulation are also conserved. I observed that histone modifications could potentially serve as an indicator of the methylation status of CTA gene promoters and that the expression of CTAs was inversely related to gene promoter 5mC levels. I demonstrate that CTAs when over-expressed cause embryonic lethality in zebrafish and the same genes are aberrantly hypomethylated at their CpG islands in a subset of human cancers. Overall, my work shows that CTAs are epigenetically regulated in an evolutionarily conserved manner and possibly via a conserved transcription factor, ETS1, that is expressed both in embryonic and cancerous tissue.
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(2022)Thesis3D computer generated biomedical animations can help audiences understand and contextualise scientific information that can be challenging to communicate due to resolution and complexity. Biomedical animators bring together multiple sources of authentic scientific data, to translate abstract information into a visual form through storytelling and visualisation. The field of biomedical animation has emerged from a long history of science visualisation and science-art endeavours, and despite there being rich discourse in the fields of data visualisation and science communication, the academic literature in the field of biomedical animation is limited, and focussed on the technical methods for visualisation, or the role these animations play in scientific research, rather than the processes through which they are created. However, as the field matures, there is a need for a deeper understanding of the creative process, and the field is now poised to expose and characterise these aspects, particularly from the perspective of the practitioner. This practice-based research project aims to expose and characterise both the visible and invisible factors that influence my personal process of creating a biomedical animation, and the tacit dimensions that influence orchestrated design choices. This research project employs a multi-method and reflective practice approach with disciplined capture and documentation of critical moments of self-reflection, that ultimately comprise the data for analysis. Thematic analysis was then used to analyse the data, and to identify themes that could contribute to frameworks that represent my personal process(es) in creating 3D biomedical animations. This has allowed me to identify and contextualise my creative process both in terms of my personal and professional position as well as within the field more broadly. I am now able to better advocate for the intangible and often undervalued aspects of my creative practice, and can articulate how a hierarchical decision matrix that considers multiple inputs contributes to my creative process. These insights will also be relevant to others in the field of biomedical animation and in the field of design more broadly, who may gain a deeper insight into their own processes of working and ways of exploring creative practice.
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(2020)ThesisWhole genome sequencing (WGS) is a powerful tool for diagnosis of Mendelian disorders. This study is aimed at evaluating the utility of WGS for molecular diagnosis of a multiethnic Intellectual Disability(ID) cohort. Individuals were recruited through the Clinical Genetics department of a tertiary hospital in New South Wales, Australia, over three years. All patients had varying degrees of syndromic or non-syndromic ID; some had neurological syndromes. WGS was undertaken using singleton, duo or trio approach after assessment of clinical features, family history and screening genetic investigations. Next Generation Sequencing (NGS) technology was utilised for sequencing and analysing genomic data at Genome.One, a NATA accredited WGS laboratory in Australia. Analysis included sequence variation and copy number limited to exonic and flanking splice site regions of known Mendelian disease-causing genes. Families where no diagnosis was made on initial analysis, were reanalysed two years later. A total of 46 probands from 43 families underwent WGS. There were 8/43 (18%) consanguineous families. A final diagnosis was made in 22/43 (51%) families. A variant providing a partial explanation of the phenotype was found in 3/43(6.9%) families. Actionable incidental findings were reported in 3/43 families. No copy number variants were identified. The RAS-MAPK pathway and microtubule related proteins emerged as predominant causative pathways within this phenotypically diverse cohort. Reanalysis revealed candidate variants in 6/14 families reanalysed representing a potential increased yield of 14%. In summary, application of WGS for investigation of an unselected ID cohort demonstrated a significant diagnostic yield. A clinical and genomic data review two years after the initial analysis was an achievable and worthwhile exercise, increasing the diagnostic yield to 65%.
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(2021)ThesisBackground: Methicillin sensitive staphylococcus aureus bacteraemia (SAB) is a common cause of bacteraemia with substantial associated mortality. Flucloxacillin is one of the first line antibiotics used in the treatment of this condition. The relationship between serum flucloxacillin concentrations below the minimum inhibitory concentration (MIC) of the pathogen and clearance of bacteraemia based on blood cultures remains uncertain. In addition, flucloxacillin is renally cleared by active tubular secretion, however the current Australian dosing guidelines do not recommend dose adjustment until the eGFR is <10 ml/min/1.73m2. We investigated the impact of estimated glomerular filtration rate (eGFR) on the serum flucloxacillin concentration. Methods: Two retrospective observational studies are presented. In the first study, 138 episodes of SAB between January 2015 and December 2018 were analysed. Inclusion criteria were age > 16 years and a positive blood culture for methicillin sensitive staphylococcus aureus. Of these, there were 30 and 41 episodes in which appropriately timed trough and mid-dosage estimated free flucloxacillin concentrations [FLX]efree, respectively, were obtained within 7 days of the initial MSSA blood culture. Differences between the persistent (blood cultures positive >72 hours) and non-persistent (<72 hours) bacteraemia groups in age, sex, focus of infection and risk factors for complicated clinical course were determined. In the second study, we analysed 396 serum flucloxacillin concentrations [FLX]total, restricted to the first per hospital admission or outpatient encounter and those simultaneously co-measured with creatinine. Inclusion criteria were age > 16 years and a serum flucloxacillin concentration measured at the study institution between May 2015 and June 2020. Free flucloxacillin concentrations [FLX]efree were estimated from total concentrations using a Michaelis-Menten model for serum albumin binding. We analysed the proportion of episodes with [FLX]efree below the upper limit oxacillin MIC for methicillin-susceptible Staphylococcus aureus (1mg/L) in this cohort, and [FLX]total>125.1mg/L - a previously identified neurotoxicity threshold, respectively, by GFR quintile. Results: In the first study, the persistent and non-persistent bacteraemia groups were found to be similar in age, sex and types of infection. Sub-MIC trough concentrations were identified in 3/17 cases of persistent bacteraemia, as compared with 0/13 cases of non-persistent bacteraemia (OR 0.0; 95% CI 0.0 to 1.4; p=0.24). Sub-MIC mid-dosage concentrations were identified in 3/22 cases of persistent bacteraemia, as compared with 0/19 cases of non-persistent bacteraemia (OR 0.0; 95% CI 0.0 to 1.28; p=0.24). In the second study, of the 396 [FLX]total samples, 242 [64.7%]) were from hospital ward inpatients, 70 (18.7%) samples were obtained from patients in ICU or HDU care, while 62 (16.6%) were from outpatients. In a multivariable regression analysis, [FLXfree] negatively correlated with eGFR. Flucloxacillin concentrations stratified by GFR quintiles were determined. The proportion of episodes with [FLXfree]<1 mg/L was significantly lower in the lower GFR quintiles when compared with the higher GFR quintile (p<0.001 for trend). Conversely, [FLX] above a previously identified neurotoxicity threshold was found to be significantly more common in lower GFR quintiles as compared with higher GFR quintiles (p<0.001 for trend). Conclusions: Sub-MIC trough or mid-dosage estimated free flucloxacillin concentrations were not associated with a lower rate of persistent bacteraemia, however the wide confidence intervals include a clinically important effect, and larger studies are warranted. Flucloxacillin concentrations were significantly negatively correlated with eGFR. Potentially subtherapeutic concentrations were more common in patients in higher eGFR quintiles, while potentially neurotoxic concentrations were more common in patients in lower eGFR quintiles but above the current threshold for dose adjustment.