Pharmacology and Therapeutics - Theses
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ItemThe impact of fetal and neonatal nutrition on adult obesity and cardiovascular regulation in animal models(University of Melbourne, 2009)
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ItemRoles and regulation of glutamate transporters in neurodegenerative diseases(University of Melbourne, 2008)
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ItemCharacterisation of a novel knockin mouse model of childhood absence epilepsy(University of Melbourne, 2007)
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ItemNeuropeptide Y and feeding regulation in mice(University of Melbourne, 2006)
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ItemDevelopmental differences in the regulation and functional capacity of ABC efflux transporters at brain barrier interfaces( 2019)The safety and potential risks of many medications used in treating pregnant women and newborn children are not well defined. This is, in part, due to limited knowledge of how readily drugs transfer into integral organs such as the brain early in development. The present Thesis investigated the expression, regulation and function of ATP-binding cassette (ABC) efflux transporters at brain, cerebrospinal fluid (CSF) and placental interfaces of the Sprague Dawley rat, highlighting age-dependent differences. The expression of eight main ABC efflux transporters (abcb1a/b, abcg2, abcc1-5) was described in the rat brain, choroid plexus and placenta, revealing distinct developmental profiles. Immunostaining of the interface between the ventricular CSF and the brain identified PGP and BCRP transporters at this interface in the newborn rat but not in the adult. Drugs (rhodamine-123, digoxin, cimetidine, paracetamol) entered the developing brain more than the adult brain. Chronic drug exposure (diallyl sulfide, digoxin, paracetamol) caused an up-regulation of efflux transporter expression and functional capacity at brain barriers in adult but not fetal (E19) or newborn (P4) rats. Fetal rats exposed chronically to paracetamol via the dam had increased transfer into the brain compared to acute treatment, with this result being dose-dependent. Transcriptomic analysis revealed that chronic paracetamol exposure caused a large inflammatory profile in the placenta, indicating potential toxicity of paracetamol use in pregnancy over extended periods. The results in this Thesis suggest that drug transport into the rat brain is higher during development for acute and chronic conditions and indicate aspects of a treatment regime that may need to be considered differently for the safe prescription of medications to patients of different ages.
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ItemPhenotypic and transcriptomic alterations associated with enhanced metastatic potential in breast cancer( 2018)Despite current advances in therapies and the gradual decline in breast cancer-related mortality, metastasis is the major determinant of breast cancer survival. The treatment regimens for breast cancer metastasis are complicated by the unpredictability of metastasis development, as well as the inter- and intra-tumour heterogeneity observed in breast cancer patients. Therefore, the diagnosis and management of metastatic disease remains a major therapeutic challenge for breast cancer treatment. To overcome these obstacles, full understanding of the molecular mechanism that governs the process of metastasis is urgently needed. Annexin A1 is a protein well known for its anti-inflammatory biology and also demonstrated convincing, though controversial influences on breast cancer progression. Accumulating evidence suggest that the influence of annexin A1 may only manifest in the most aggressive breast tumour and investigation of this relationship needs to be conducted in models that reflect this specificity A powerful model developed to effectively study the complexities associated with breast cancer metastasis are breast cancer variant cell lines derived from the same parental line but displaying differing in metastatic capabilities. The MDA-MB-231HM.LNm5 is a one of such novel cell lines derived by in vivo passaging of the TN human breast adenocarcinoma MDA-MB-231 line and demonstrated robust metastatic propensity. Using this cellular model of metastasis, the body of work presented in this thesis attempted to investigate the mechanisms underlying the acquisition of metastatic phenotype in both a hypothesis and a non-hypothesis-driven manner. Part one of this thesis aimed to describe some of the phenotypic, molecular, and transcriptomic changes associated with enhanced metastatic potential found in the MDA-MB-231HM.LNm5 line, including changes in energy metabolism, proliferation, and growth-related processes. In the second part of this thesis, the endogenous expression of annexin A1 in the MDA-MB-231HM.LNm5 cells line and the parental line was silenced to explore the influence of annexin A1 on characteristics associated with elevated metastatic potential. Finally, by manipulating tumour and host ANXA1 levels, as well the host immune system, we attempted to unravel the involvement of ANXA1 in tumour initiation, growth and metastasis in vivo. Accompanying the aggressive in vivo metastasis phenotype, the MDA-MB-231HM.LNm5 metastatic daughter line exhibits heightened energy metabolism and chemo-resistance but reduced in vitro proliferative propensity. This ‘go or grow’ dichotomy, underlined by an increase in the quiescent cell population and dampened Ca2+ signalling, can be restored by the knock-down of ANXA1. In comparison, phenotypes of the non-metastatic parental MDA-MB-231 cells are unaltered by ANXA1 KD. In vivo studies showed that the expression of annexin A1 is required for growth and progression of breast cancer in specific metastatic mouse models. Overall, the studies presented in this thesis deepens our knowledge of the complex biological processes underlying the acquisition of metastatic propensity in breast cancer and reported evidence that suggested annexin A1 to be an important contributor of metastatic alterations. Further targeted investigation could facilitate the development of new strategies for therapeutic interventions and clinical management of patients with metastatic breast cancer
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ItemDevelopment and application of advanced bioimaging techniques to investigate stress pathways and drug action in neurodegeneration( 2019)Neurodegenerative diseases such as Frontotemporal Dementia (FTD), and Amyotrophic Lateral Sclerosis (ALS), also known as Motor Neuron Disease (MND), have limited therapeutics available, placing a high demand on healthcare systems and overall economic and social burden. Recently, it has been found that pathological mutations in RNA binding proteins such as TDP-43 (Tar DNA-binding Protein, 43kDa), FUS (Fused in Sarcoma), and hnRNP (heterogenous nuclear RiboNucleoProteins) can be causative of ALS or FTD. Cell stress has been implicated in ALS and FTD, particularly oxidative stress, mitochondrial dysfunction, ATP depletion and metal dyshomeostasis. In cell culture models, it has been shown that TDP-43 and hnRNP proteins can incorporate into stress granules in conditions of cell stress, including during oxidative stress, metal dyshomeostasis or conditions of ATP depletion, however this is not well defined. Furthermore, there is limited evidence of FUS incorporating within stress granules. The study described here has enhanced the understanding of RNA binding proteins in vitro. Utilising neurodegeneration-associated cell stress pathways in conjunction with advanced bioimaging techniques, we show that both non-mutated FUS and non-mutated TDP-43 can occur within the same stress granule. Furthermore, we show different populations of TDP-43 and FUS within stress granules from different stress types. This study also enhanced our understanding of the sub-cellular distribution of a neuroprotective metal-complex therapeutic, utilising fluorescence lifetime imaging on live cells; this therapeutic has previously been shown to be neuroprotective against TDP-43 aggregation. To extend the study, a novel metal complex was utilised in attempt to enhance the field of techniques available to study RNA binding proteins in vitro. Overall, this thesis has advanced our understanding of disease pathways, therapeutic action, and new investigative tools for neurodegeneration.
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ItemInvestigating Aβ toxicity and binding to neurons from differentiated human stem cells( 2019)Alzheimer’s disease (AD) is a neurodegenerative disease that is pathologically characterized by abnormal deposition of extracellular amyloid plaques and intraneuronal neurofibrillary tangles. The deposition of these aggregated proteins causes progressive brain atrophy resulting from gradual synaptic loss and neuronal cell death. Although the aetiology of AD remains elusive, studies have shown that amyloid beta (Aβ) peptide, a cleavage product from amyloid precursor protein (APP), is a key protein causing AD pathogenesis. Recent studies have identified how the presence of soluble low molecular weight Aβ oligomers in the brain correlate best with synaptic loss, and they are a better predictor of disease progression compared to the presence of amyloid plaques or neurofibrillary tangles. In the AD brain, neuronal subpopulations appear to exhibit different levels of vulnerability to Aβ, particularly the basal forebrain cholinergic and hippocampal glutamatergic neurons, while GABAergic neurons appear to remain unaffected till later disease stages. Current treatments based on knowledge gathered from mouse models targeting the cholinergic and glutamatergic systems only alleviate symptoms and are ineffective in halting disease progression. Therefore, we hypothesize that Aβ exerts its neurotoxic effect by binding to a subpopulation of mature neurons. To address this, human embryonic stem cells (hESCs) were differentiated into mature glutamatergic and GABAergic neurons and cultured up to 12 weeks. These cultures were treated fortnightly with soluble synthetic Aβ peptide for 96 hrs. We found that Aβ bound to neurites in culture, altered gene expression and neurotoxicity was more pronounced in 6-week old glutamatergic than GABAergic cultures. Further investigations in determining the specific toxic species of Aβ oligomers revealed that 12 week old cultures were more susceptible to Aβ oligomer induced toxicity, with Aβ dimers being most toxic to glutamatergic neurons while GABAergic neurons were most susceptible to Aβ tetramers. In summary, we successfully established a simple hESC based model to study Aβ toxicity. Our findings also highlight the importance of using relevant human cell-based models to study AD pathogenesis as well as identify potential AD modifying therapeutic strategies.
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ItemInvestigating the role of Amyloid Precursor-Like Protein 2 in Motor Neurone Disease( 2019)Motor neurone disease (MND) is a fatal human neurodegenerative disorder. The most common form of MND is amyotrophic lateral sclerosis (ALS). MND is characterised by the progressive destruction of motor neurons in the central nervous system which causes muscle weakness, muscle atrophy, paralysis and ultimately death. The sporadic forms of the disease account for the majority of patients, and 5-10% of MND cases are inherited (familial MND) (Marin et al., 2017). Both sporadic and familial MND share similar clinical and pathological features, suggesting common molecular mechanisms of degeneration. Among the familial MND patients approximately 20% possess a mutation in the SOD1 gene encoding for the enzyme Cu/Zn superoxide dismutase (Rosen et al., 1993). There are more than 170 different SOD1 gene mutations described, and the majority are missense substitutions resulting in a toxic gain of enzyme function (http://alsod.iop.kcl.ac.uk/). Transgenic mouse models over-expressing mutant forms of the human SOD1 gene replicate key pathological symptoms seen in MND patients and are widely used to study MND. Despite progress in deciphering the molecular mechanisms of this disease, the cause and modulation of MND remains unclear. The Amyloid Precursor Protein (APP), is well-known for its association with Alzheimer's Disease, and it has been shown to be a modulator of MND. APP protein expression levels were increased in the spinal cords from MND patients as well as in SOD1 transgenic mice at symptomatic stage of the disease (Koistinen et al., 2006; Rabinovich-Toidman et al., 2015). The resultant SOD1-G93A:APP-/- mice from the cross breeding between APP homozygous deletion and SOD1-G93A transgenic mice (overexpress human SOD1 gene with G93A familial mutation) showed significant decrease in MND pathogenesis and reduced disease progression (Bryson et al., 2012). The SOD1-G93A:APP-/- mice also displayed significantly ameliorated muscle contractility, improved neuromuscular junction innervation and decreased motor neuron loss. Taken together these findings suggest an important role for APP in MND pathophysiology. APP is part of a gene family that includes the amyloid precursor-like protein 1 (APLP1) and amyloid precursor-like protein 2 (APLP2) genes. To understand if other APP-family members modulated MND we investigated the role of APLP2 in the SOD1-G37R transgenic mouse model. We found a significant sex-dependent increase in the expression of APLP2 protein in the spinal cord of the SOD1-G37R mice. To test if APLP2 gene expression can modulate disease outcomes in MND we crossed the SOD1-G37R and APLP2 knockout (KO) mice to generate the SOD1:APLP2+/- and SOD1:APLP2-/- lines. We found the lack of APLP2 expression improved motor performance and extend survival in a sex-dependent manner. The molecular basis for APLP2’s actions identified effects on muscle physiology and synaptic function at the neuromuscular junction. Taken together, our novel results demonstrate there are sex-dependent differences in the SOD1 mouse model, and this is affected by APLP2 expression. These data extend the modulatory role by the amyloid precursor protein family in MND, and identify the APP-family as an important target for further investigation into the cause and regulation of MND.
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ItemMolecular basis for amyloid precursor protein mediated neuroprotection in traumatic brain injury( 2019)Amyloid precursor protein (APP) is neuroprotective in traumatic brain injury (TBI). Treatment with soluble amyloid precursor protein (sAPP) can rescue motor and cognitive deficits following TBI in mouse and rat models (Corrigan et al., 2012c). The neuroprotective active site in sAPP is located in residues 96 to 110 (APP96-110) (Corrigan et al., 2014). We hypothesize that APP96-110 interacts with a specific molecule(s) to trigger its neuroprotective response in TBI. To identify protein(s) interacting with the APP96-110 peptide, a biotin-streptavidin affinity capture method combined with mass spectrometry was utilised. Among the proteins identified, the Amyloid Precursor-like Protein 2 (APLP2) was found to be a robust interacting target for APP96-110. Previous reports showed APLP2 binds to a region in APP which includes 96-110 (Soba et al., 2005). To test the role of APLP2 in TBI, APLP2 wildtype (APLP2+/+) and APLP2 knockout (APLP2-/-) mice, from both sexes, were subjected to mild controlled cortical impact injury. Brains were collected following 7 days of surgery and histopathological assessment was done looking at primary and secondary effects of injury. These include tissue morphology, neuronal loss, axonal injury, tau pathology, astrogliosis and microgliosis. Motor function was assessed by DigiGait over 7 days post-surgery. Initial gait analysis showed the craniotomy procedure itself induced gait disturbances, but to a lesser extent and that injury worsened gait performance in both genotypes. Sex differences were observed in brain injury, with males more susceptible at acute phase of injury with increased motor deficits and astrogliosis in both genotypes. There was greater axonal damage and tau pathology detected in males than females expressing endogenous APLP2. This study highlights the importance of considering craniotomy controls and female and male mice in TBI study. Sex-specific comparisons made between APLP2+/+ and APLP2-/- mice following injury showed the lack of APLP2 in males leads to decreased motor deficits, axonal damage and tau pathology compared to males expressing endogenous APLP2. In the case of females, APLP2-/- mice were less susceptibility to brain injury compared to APLP2+/+ females. This suggests APLP2 expression may be modulated by sex hormones. Using an in silico approach, progesterone and estrogen transcription binding motifs were identified in the mouse and human APLP2 promoter sequence.