http://hdl.handle.net/11343/159 Mon, 25 Mar 2019 13:12:24 GMT 2019-03-25T13:12:24Z http://hdl.handle.net/11343/221683 Investigating the molecular basis of Parkinson's disease Gao, Yujing Loss of function mutations in Ras analog in brain 39B (RAB39B) can cause Parkinson’s disease (PD), a common and incurable neurodegenerative disorder. RAB39B is a small GTPase protein that functions to regulate cellular trafficking events, and is important for maintaining the intracellular homeostasis of their target proteins. Currently, the normal physiological function(s) of RAB39B are unclear, and the link between its function(s) and the development of PD are yet to be elucidated. The pathological features of RAB39B-mediated PD are consistent with the classical presentation of PD, and include dopaminergic neuron loss and the presence of alpha-synuclein (αSN) positive Lewy bodies in the substantia nigra. Further, there are now several reports demonstrating that loss of RAB39B in vitro can lead to dysregulated αSN homeostasis. Therefore, the overall hypothesis of this study is that the pathological mechanisms underlying RAB39B-mediated PD are associated with impaired protein trafficking and perturbed αSN homeostasis. The primary aim of this study is to test this hypothesis, and more generally, to investigate the function(s) of RAB39B using novel cell and animal models. The first part of this study focused on determining the distribution of RAB39B in mouse and human brain, and identifying the protein interactor(s) of RAB39B. The spatiotemporal localisation of RAB39B was systematically assessed in postnatal mouse brain tissue. The distribution of RAB39B in brain tissue from healthy controls, and individuals with idiopathic PD, were also assessed. Overall, RAB39B was found to be localised to the cortex and hippocampus, brain regions involved with cognitive function, and the substantia nigra pars compacta, the brain region most affected in PD. Further, RAB39B was dysregulated in the cortex and substantia nigra of individuals with idiopathic PD, implicating for the first time a role for RAB39B in the pathological mechanisms underlying idiopathic PD. A number of putative RAB39B interactors were identified in this study, suggesting multiple possible neuronal function(s) for RAB39B. In the second part of this study, the pathological mechanisms underlying RAB39B-mediated PD were investigated using stem cell derived neuron models. Stem cell lines with RAB39B deletion were derived from patient fibroblasts, and also independently generated using CRISPR/Cas9 in a parental embryonic stem cell line. Neuron cultures differentiated from RAB39B deletion stem cell lines show increased SNCA expression and αSN levels. These results implicated RAB39B in the regulation of SNCA expression, and suggested that the pathological mechanisms underlying PD mediated by RAB39B mutations, and increased SNCA dosage, are potentially converging. In the third part of this study, the pathological mechanisms underlying RAB39B-mediated PD were investigated using mouse models. Mice with Rab39b deletion were generated using CRISPR/Cas9. Behavioural assessments of mice with Rab39b deletion demonstrated altered motor activity compared to wildtype littermates. Global proteomic analysis of the substantia nigra of Rab39b knockout mice demonstrated altered proteomic profiles compared to wildtype littermates. Specifically, aged Rab39b knockout mice showed significant reductions in enzymes involved in dopamine metabolism and an increase in αSN. These results implicated RAB39B in the regulation of dopamine and αSN homeostasis, and suggested that the pathological mechanisms underlying RAB39B-mediated PD involve dysregulated dopamine and αSN. In conclusion, this study provided significant insights into the potential function(s) of RAB39B, and its role PD pathogenesis. Overall, the results supported the hypothesis that RAB39B participates in the regulation of αSN homeostasis. In addition, a range of cell and animal models for RAB39B-mediated PD were generated in this study, and are valuable tools for future studies. More broadly, knowledge gained from this project can improve understanding of idiopathic PD, due to shared disease features and thus pathological mechanisms of genetic and idiopathic PD. Ultimately, improved understanding of disease mechanisms can aid the development of novel therapeutics, which could potentially modify the development and progression of PD and related disorders characterised by Lewy pathology. © 2018 Dr Yujing Gao Mon, 01 Jan 2018 00:00:00 GMT http://hdl.handle.net/11343/221683 2018-01-01T00:00:00Z http://hdl.handle.net/11343/221682 Understanding the role of ATP13A2 in endo-lysosomal pathways Chiam, Mathew Parkinson’s disease (PD) is a common neurodegenerative disease affecting 1 to 2% people aged over 65. Cytoplasmic inclusions are pathognomonic of PD and point to dysregulation of protein clearance as a central part of the pathology. Two forms of familial PD result from mutations in the genes for the ATP13A2 and α-synuclein proteins. One of these proteins (α-synuclein) forms the characteristic inclusions of PD and is thus likely to be affected by dysregulated protein clearance, whereas the other is part of the endo-lysosomal system (ELS), which is a central component of protein clearance. The predictions on which these studies were based on were that removal of ATP13A2 would have i) a deleterious effect on ELS function; ii) induce aspects of neurodegeneration; iii) dopaminergic systems would be most susceptible; iv) the presence of high levels of α-synuclein would exacerbate these changes. None of these proved correct in these mice and instead it appears that removal of ATP13A2 results in i) improved capacity of ELS function; ii) there is no loss of neurons but there is neuro gliosis; iii) there is plasticity in the dopaminergic systems that suggest a response to altered neurotransmission; iv) the presence of high levels of α-synuclein did not exacerbate any aspect of the pathology and the absence of ATP13A2 in the presence of increased α-synuclein expression resulted in improved clearance of α-synuclein. These unexpected findings open up several new avenues for investigation of the normal physiological role of these proteins in the ELS and also for investigating whether understanding the mechanisms of the improved α-synuclein clearance might reveal new therapeutic targets for protein clearance disorders. © 2018 Dr Mathew Chiam Mon, 01 Jan 2018 00:00:00 GMT http://hdl.handle.net/11343/221682 2018-01-01T00:00:00Z http://hdl.handle.net/11343/221681 Vancomycin pharmacokinetics and pharmacodynamics in young infants Gwee, Amanda Vancomycin is a glycopeptide antibiotic commonly used for the treatment of late-onset sepsis in young infants. Vancomycin dosing is challenging in young infants due to the rapid physiological changes that occur after birth which effect both vancomycin disposition and clearance. Vancomycin is routinely administered to young infants as intermittent infusions (IIV) but studies show that current recommended IIV dosing regimens in paediatric drug formularies lead to subtherapeutic vancomycin levels in the majority of young infants. Therefore, in some neonatal units, continuous infusions of vancomycin (CIV) are used routinely based on data from adult studies that suggest CIV is associated with improved attainment of target concentrations and fewer drug side effects. Therapeutic drug monitoring of vancomycin is used to determine both treatment efficacy as well as toxicity. Current recommendations for target trough concentrations of 10 to 20 mg/L are based on the pharmacodynamic target (relationship between drug concentration and drug effect) for Staphylococcus aureus. However, there are limited data on the pharmacodynamic target for other staphylococci, in particular coagulase-negative staphylococci that are also important pathogens in young infants. My PhD aims to optimise vancomycin dosing in young infants, as well as to study the pharmacodynamics of vancomycin in vitro to improve the treatment of late-onset infections in young infants. The first section of this thesis focuses on determining the optimal vancomycin dosing regimen in young infants. From my literature review on CIV dosing in young infants (see Appendix 1) the existing data suggest that CIV is well tolerated and achieves target vancomycin levels in the majority of study participants. Therefore, to evaluate CIV compared to IIV (the current standard of care), chapters 3 and 4 of my thesis comprise: (i) a protocol for a multicentre randomised controlled trial (RCT) of CIV compared to IIV in young infants aged 0 to 90 days (ii) the results of this RCT in 111 young infants in the neonatal intensive care setting. The second section of this thesis (chapter 5) describes an in vitro study designed to determine the in vitro pharmacodynamics of vancomycin for one of the most common coagulase-negative staphylococci causing late-onset sepsis in young infants, S. epidermidis. To guide recommendations for target vancomycin levels, this study determined whether vancomycin exhibits time- or concentration-dependent killing of S. epidermidis. The final section of my thesis (chapter 6) focuses on the development of a population pharmacokinetic model for vancomycin in young infants using non-linear mixed-effects modelling (NONMEM). The model is based on data from the RCT (chapter 4). Using this model, the relationship between trough concentrations and the area under the concentration-time curve over a 24-hour period (AUC24) were determined as well as whether vancomycin therapeutic drug monitoring (TDM) can be done earlier during treatment. © 2018 Dr Amanda Gwee Mon, 01 Jan 2018 00:00:00 GMT http://hdl.handle.net/11343/221681 2018-01-01T00:00:00Z http://hdl.handle.net/11343/221680 ‘Female performers on a male stage’: can social media reignite the Women’s Liberation Movement? Megarry, Jessica Lee This thesis uses radical feminist political theory to investigate whether women’s use of social media can reignite the Women’s Liberation Movement (WLM). In doing so, the thesis provides the first internal critique of contemporary radical feminist digital organising strategies. Emerging in the late 1960s across Western democracies, radical feminist communication in the WLM was characterised by face-to-face organising in small women-only groups and the development of an autonomous print culture. More recently, the time-space compression facilitated by social media has unleashed an avalanche of celebratory rhetoric championing the new opportunities now available to women to digitally connect across geographical borders and instantly raise their voices against male dominance. On this basis, some scholars have now claimed that digital space has propelled feminism into a fourth wave. This claim is curious from a radical feminist perspective, because social movement studies, feminist media studies and critical internet studies scholars have yet to critically analyse how the shift away from face-to-face communication and the adoption of male-controlled and algorithmically curated digital platforms is helping or hindering women’s ability to revive a revolutionary feminist movement. Drawing on in-depth semi-structured interviews with 26 feminist activists across the USA, UK, Australia, Canada and New Zealand, as well as archival and digital primary activist materials, this thesis is the first academic work to critically analyse how male power in digital space is shaping women’s ability to raise their consciousness, build feminist theory, and engage in politically-informed feminist praxis. I argue that the use of social media for feminist communication prevents women from reviving a strong, woman-centred liberation movement because they remain physically separated from each other whilst also placed under male surveillance. Feminist organising on social media is subject to male approval; not only does social media expand male access to both individual women and feminism's strategic directions, but it also impedes women’s ability to advance a feminist ethic of engagement based upon collectivity. Instead of providing a space conducive to the development of new forms of engagement between women outside of male control, social media forces feminists to work within a male infrastructure and pursue an ethic of engagement based upon antagonism, individualism, mistrust and celebrity worship. © 2019 Dr Jessica Lee Megarry Mon, 01 Jan 2018 00:00:00 GMT http://hdl.handle.net/11343/221680 2018-01-01T00:00:00Z