Biochemistry and Pharmacology - Theses
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ItemTranscriptional regulation and functional differences driven by STAT3 isoforms, STAT3α and STAT3β( 2018)The Signal transducer and activator of transcription 3 (STAT3) protein, a member of the STAT family of transcription factors, plays important roles in the regulation of critical biological processes. STAT3 is best known for its role in the JAK-STAT pathway, mediating the transcriptional regulation of target gene expression following its activation by extracellular signal, and continues to be the subject of intense research efforts due to its essential roles in crucial physiological processes, such as embryonic development and inflammation, as well as its involvement in numerous types of cancer. While the genome-wide DNA-binding and transcriptional regulation activities of the full-length STAT3α have been widely studied, those of a naturally occurring shorter STAT3β spliceform are less understood. Despite having identical DNA-binding domains, the STAT3 spliceforms display significant differences in their transcriptional regulation activities. Furthermore, STAT3α and STAT3β can drive opposing oncogenic and tumour suppression outcomes, respectively. Thus, the major focus of this study is to explore STAT3α- and STAT3β-specific genome-wide DNA-binding, transcriptional regulation of non-coding micro-RNAs (miRNAs), and functional outcomes in cancer cell biology. By employing ChIP-seq and miRNA expression profiling assays, this study presents results revealing that STAT3α and STAT3β display clear differences in genome-wide DNA-binding and regulation of miRNA expression. Bioinformatic analyses of transcription factor binding sites also uncovered the possible roles of co-transcription factors in the distinct STAT3 spliceform-specific transcriptional regulation activities. In addition, a Morpholino-directed splicing modulation approach to drive STAT3 knockdown and STAT3α-to-β splicing switch in cancer cells showed that the STAT3 spliceforms can differentially alter the tumourigenic properties of cancer cells, with STAT3β expression being associated with tumour suppression outcomes by driving the suppression of cell proliferation, survival and migration. Taken together, this study highlights the distinct properties of the STAT3 spliceforms in genome-wide DNA-binding which possibly underlie their different gene transcriptional regulation activities in both protein-coding and non-coding genes, and further provides evidence that STAT3β can drive distinct tumour suppression outcomes.
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ItemPopulation structure and carriage-infection dynamics of Klebsiella pneumoniae( 2018)Klebsiella pneumoniae is an opportunistic pathogen and global cause of hospital-associated (HA) infections. K. pneumoniae is also part of the healthy human microbiome, providing a potential reservoir for infection. However, the frequency of colonisation and its contribution to infections are not well characterised. A prospective, hospital-wide surveillance of all infections attributed to K. pneumoniae – across the Alfred Health network, Melbourne, Australia – was conducted over a one-year period in 2013. Concurrently, patients in the intensive care unit (Alfred Hospital; AH) and geriatric care wards (Caulfield Hospital; CH) were screened for asymptomatic colonisation. Isolates were characterised using whole genome sequencing and antimicrobial susceptibility profiling. This study aimed to address several objectives: i) To investigate the frequency of colonisation in intensive care unit (ICU) patients and whether colonising strains are a source of infection; ii) To investigate the frequency and source of antimicrobial-resistant colonisation or infection in geriatric patients; iii) To characterise the population structure and genetic diversity of K. pneumoniae causing infections in hospital patients. This study estimated community-associated (CA) asymptomatic gastrointestinal carriage among ICU patients was at 6%, rising significantly to 19% among HA individuals, the latter including all identified multi-drug resistant carriage isolates. Many patients had their own unique colonising and infecting strains, often matching within a patient, though there were instances suggestive of transmission. The combination of genomic and epidemiological data supported five clusters of recent patient-to-patient transmission, frequently involving carriage. Among the CH geriatric patients screened, GI carriage rates were 10.8% and 1.7% of patients had extended-spectrum beta-lactamase (ESBL) producing carriage strains. There were three variably MDR lineages observed among multiple patients, and though there was no direct transmission within the CH, there was evidence supporting transmission at the AH prior to CH admission. The major MDR plasmids were identified; all had regions of clustered AMR genes on transposons and/or integrons. Two of the lineages shared variants of the same plasmid indicating transmission of AMR mobile elements between strains. Across all Klebsiella infections extensive diversity was observed including multiple species, lineages, capsule types, and O-antigen types. Most patients had their own unique carriage and infection strains but there were a small number of transmission chains detected. In most transmission cases, the lineages responsible were ESBL, MDR, or carbapenemase producers. Therefore, patients with antimicrobial-resistant strains pose a greater threat to those around them, as these strains were most strongly associated with transmission. This study showed that the majority of hospital Klebsiella infections arise from the host microbiome, though there is also a smaller burden of infection due to transmission of typically antimicrobial-resistant strains. This has important implications for infection prevention and control: people who are not colonised with Klebsiella have low risk of subsequent infection, except for when infections arise from acquisition of other patients’ antimicrobial-resistant strains. This work revealed that screening for colonisation can elucidate; i) which patients are at risk from infection through self-contamination, and ii) the AMR status of patients’ strains and therefore which individuals pose a risk to other patients through potential transmission.