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Item Investigating HDAC4 aggregation in a Drosophila model of neuronal development : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand(Massey University, 2024-06-21) Hawley, Hannah RoseHistone deacetylase four (HDAC4) is essential in neuronal development and function, and dysregulation of HDAC4 has been observed in a number of neurodevelopmental and neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases. In particular, its aberrant nuclear accumulation is a common feature among these diseases, and it has been observed that upon upregulation or accumulation in the nucleus, HDAC4 forms punctate foci in neuronal nuclei. Previous research in a Drosophila model determined that overexpression of HDAC4 disrupted both neuronal development and long-term memory, and this was largely mediated by the nuclear pool of HDAC4. Based on these data, it was hypothesised that aggregates of HDAC4 are responsible for the neurotoxicity that leads to disrupted neurodevelopment and memory. Therefore, this study aimed to determine whether the presence of HDAC4 nuclear aggregates correlated with neurodevelopmental deficits in a Drosophila model of neurodevelopment, and if so, how they mediate their toxic effects. The N-terminus of HDAC4 forms homo-oligomers in solution, and it was hypothesised that full-length HDAC4 similarly oligomerises, and that this is required for its aggregation in neuronal nuclei. Mutations predicted to prevent oligomerisation were introduced into the N- terminus of HDAC4 and were shown to significantly reduce aggregation of HDAC4 in Drosophila neurons. Furthermore, their presence also reduced the severity of HDAC4 overexpression-induced impairments in neurodevelopment. Conversely, stabilisation of oligomerisation increased aggregation and the severity of neurodevelopmental phenotypes, together indicating that aggregation positively correlates with the severity of neurodevelopmental deficits. HDAC4 aggregates have been previously shown to sequester the transcription factor MEF2, and further investigation revealed that the presence of MEF2 stabilised aggregation and increased the severity of defects in neuronal development. Importantly, targeting the interaction between HDAC4 and MEF2 reduced the severity of these defects. Other than MEF2, the composition of HDAC4 aggregates is unknown, and therefore immunoprecipitation-coupled mass spectrometry was performed on nuclear HDAC4 to identify candidate interactors of aggregates. This revealed a number of proteins with roles in neuronal development and function, as well as those involved in splicing and protein homeostasis, suggesting that aggregates may be impairing these processes to mediate toxicity. Together these data indicate that nuclear aggregation of HDAC4 impairs neurodevelopment, and may constitute a novel biomarker of disease or therapeutic target. Given the overlap in aetiology between neurodevelopmental and neurodegenerative diseases, further investigation of whether HDAC4 aggregation contributes to the severity and/or progression of neurodegenerative disorders is warranted.Item Impact of quorum sensing on cell aggregation in enteropathogenic and enterohaemorrhagic Escherichia coli : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosphy in Microbiology and Genetics at Massey University, Turitea campus, New Zealand(Massey University, 2013) Govan, Barbara Siobhansensing has been shown to regulate phenotypic traits such as motility and biofilm production in pathogenic bacteria. Escherichia coli utilise both AI-2 and N-acylhomoserine lactones (AHL) quorum sensing systems to initiate phenotypic switches, such as changes in dissemination, in response to overall microbial population density. In addition HosA, a transcriptional regulator present in pathogenic strains of E. coli, was shown to be important in bacterial dissemination during this study. Deletion of hosA from enteropathogenic E. coli strain E2348/69 resulted in a non-motile population at lower temperatures, an effect that was reversed in the presence of exogenous AHL. Furthermore, addition of the same AHLs to wild-type E2348/69 decreased population motility. Bacterial aggregation has been linked to the motility of the population. Deletion of hosA was shown to increase aggregation, corresponding to an observed decrease in motility. Furthermore, addition of AHL was shown to decrease the propensity of the HosA mutant population to aggregate. Opposing effects were observed in the non-aggregating wildtype population. A hypothetical hierarchical association between HosA and quorum sensing was modelled to explain the relationship between motility and aggregation in E2348/69. It was observed the EAF plasmid is not essential for the formation of A/E lesions on human cell-line HT-29 by E2348/69. Infection of Galleria mellonella revealed increased virulence in highly aggregative populations and further highlighted the effect of HosA and environmental conditions on the pathogenicity of E2348/69. Ruminant animals, in particular cattle, are the main reservoir of enterohaemorrhagic E. coli O157:H7 with infection remaining asymptomatic. By comparison infection in humans can result in a range of sequalae from mild to life-threatening. E. coli O157:H7 is derived from an EPEC progenitor and as expected, dissemination was affected by specific AHLs in a similar manner to E2348/69. However, no aggregation was observed in the presence or absence of signal. This suggested a difference in the genes affected by quorum sensing between enteropathogenic and enterohaemorrhagic E. coli. Development of biocontrol strategies targeting AHL-dependent quorum sensing regulated processes, such as aggregation to reduce E. coli O157:H7 contamination of meat products is possible, based on the data presented in this thesis.