Anatomy and Neuroscience - Theses
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ItemEarly neuronal and glial cell changes in diabetic retinopathy(University of Melbourne, 2010)
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ItemModulation of the intermediate-conductance calcium-activated potassium (IKca) channel by protein kinase a (PKA)(University of Melbourne, 2010)
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ItemNeural plasticity and gene-environment interactions in the PLC-?1 knockout mouse(University of Melbourne, 2007)
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ItemAncient DNA investigations at Samtavro, Republic of Georgia( 2016)The Samtavro cemetery, used from the Late Bronze Age through to the Early Middle Ages, is the largest known ancient burial ground in the Republic of Georgia and the Caucasus. Centrally located at the crossroads of major continents and trading networks, archaeological investigations have revealed a diverse assemblage of artefacts and cultural practices, including Greek, Roman, Persian, and Hunnic influences. In order to gain insight into the origin, affinity and legacy of the Late Antique (~300-500AD) Samtavro population, we sequenced whole mitochondrial genomes (mitogenomes) from 17 individuals buried in 14 tombs. In order to test whether tombs were being used to bury family members together, we sampled more than one individual from three different tombs. We find high mitochondrial DNA (mtDNA) diversity with all individuals from different tombs (n=14) carrying unique mtDNA haplotypes. Identical mtDNA haplotypes were found in two of the three tombs from which two individuals were sampled, probably reflecting the co-burial of kin. Fifteen individuals were included in a principal components analysis (PCA) of haplogroup composition and fourteen individuals in a multidimensional scaling plot (MDS) of Slatkin’s Fst values. These analyses show a strong west Eurasian influence with close affinity to populations from the Caucasus, Middle East and Europe. All (n=17) mitochondrial lineages belong to West Eurasian haplogroups suggesting minimal, if any, impact of ancient nomads of the Eurasian steppe. Fourteen individuals were included in a shared haplotype analysis which revealed that more than half (8/14=57%) of the hypervariable I (HVSI) haplotypes from Samtavro were previously reported in modern day Georgians. Similar haplogroup composition, low Fst values and a high haplotype sharing between ancient Samtavro and modern day Georgians suggests an autochthonous origin of the Samtavro population followed by a high degree of population continuity in the region over the last 1,500 years. However, the mtDNA represents just a single lineage precluding a fuller understanding without the co-analysis of nuclear and Y chromosomal genetic diversity.
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ItemThe role of Frizzled-7 in the normal intestinal epithelium and colorectal cancer( 2016)The Wnt signalling pathway is pivotal for many biological processes, it controls cell fate during embryonic development, is a key regulator of homeostasis in adult self-renewing tissues, is necessary for regeneration and deregulation of the pathway leads to malignant transformation. The intestinal epithelium, which is composed of a single layer of intestinal epithelial cells that line the gastrointestinal tract, is the best-understood example for the roles of Wnt signalling during homeostasis and tumourigenesis. The Wnt signalling pathway was originally linked to the genesis of colorectal cancer (CRC) through linking the genes that are mutated in human CRC to the regulation of Wnt pathway mediated transcription. Through a great deal of attention directed at understanding the link between mutated intracellular components of the pathway and the initiation of CRC, it was shown to have a fundamental role in the regulation of intestinal stem cells and in malignancies of the intestinal epithelium as well as play an important role in intestinal homeostasis. Although our knowledge about the Wnt signalling pathway has increased immensely since its discovery, there are still numerous questions regarding Wnt signalling to explore. Thus, this thesis attempts to expand our knowledge of its role in the intestine and CRC, more specifically the role of the transmembrane receptors responsible for transducing Wnt signals, the Frizzled receptors, particularly Frizzled-7, one of ten seven-transmembrane receptors of the Frizzled family. One of the aims was to determine which Wnt(s) interact with Frizzled-7 (Fzd7), as a role for Fzd7 in CRC morphogenesis was previously shown. Thus in Chapter 2 I demonstrate that Wnt secretion is necessary for the phenotype transitions that are associated with the dissemination of the cells from the primary tumour site and that Wnt2b and Wnt3a can cooperate with Fzd7 in this process. Furthermore, I confirm the interaction between Fzd7 and Wnt3 and demonstrate an interaction between Fzd7 and Wnt2b via co-immunoprecipitation. In chapter 3, the aim was to investigate the role of Fzd7 in the homeostasis of the intestinal epithelium. I demonstrate that conditional deletion of Fzd7 in vitro in 3D “mini-gut” organoid cultures in LGR5+ CBC stem cells is detrimental to the organoids, which verified our previous in vivo findings and suggests that Fzd7 is required for intestinal homeostasis and has a critical role in transmitting essential Wnt signals in stem cells of the intestinal epithelium. In CRC over-expression of Fzds, specifically Fzd7, has been well documented as well as its involvement in the survival, invasion and metastatic capabilities of CRC cell lines. So, in Chapter 4 I explore the potential therapeutic benefit of Fzd7 inhibition in 3d adenoma organoid cultures, by employing antibodies that target Fzd7. Blocking the Fzd7 receptors is sufficient to reduce tumour growth and limit proliferation. Furthermore, conditional deletion of Fzd7 in vitro in the cancer stem cells reveals a strong drive to restore Fzd7+ cells. Collectively, the findings from this thesis demonstrate a role for Frizzled-7 in maintaining intestinal stem cells in homeostasis and potentially in cancer. Moreover a role for the upstream components of the Wnt signalling pathway, i.e. Wnts and Fzd7, in cancer was shown.
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ItemSnail - an important regulator of Drosophila GSC homeostasis( 2016)The Drosophila testis is a long coiled tube-like structure with male germline stem cells (GSCs) residing in the apical tip. 8-10 GSCs are arranged in a rosette shape around a group of somatic cells termed the hub, which secretes factors that regulate stem cell self-renewal. Due to asymmetrical division of GSCs, one daughter cell remains in direct contact with the hub and therefore maintains stem cell identity, while the other is displaced from the hub and commences differentiation to become a gonialblast. The gonialblast undergoes four rounds of mitotic divisions with incomplete cytokinesis, resulting in a cyst of sixteen interconnected spermatogonia which then progress through pre-meiotic S-phase and differentiate to become spermatocytes. There is a delicate balance between self-renewal of GSCs and differentiation of germ cells, which is required for tissue maintenance and regeneration. Snail proteins are zinc-finger DNA-binding proteins that act as transcriptional repressors of target genes via the binding to specific sequences termed ‘E-boxes’ in the promoter sequence. Drosophila has three Snail members which include: Snail, Escargot and Worniu, while the mammalian Snail family is comprised of Snai1/Snail, Snai2/Slug and Snai3/smuc. Snail proteins play a crucial role in initiating epithelial to mesenchymal transitions (EMT), both in normal physiology and disease. This project reveals an important role for the transcription factor Snail in the regulation of GSC maintenance. I have utilised a dominant negative transgene of escargot (esgDN) in order to block the function of all three Snail family members, which resulted in a severe GSC loss phenotype. Only the expression of a WT snail transgene was able to partially rescue the phenotype. Despite its high expression in somatic cells and spermatogonia, loss of Escargot function in GSCs has no effect. Conversely, I showed that snail is expressed in very low levels in the germline but snail mutant GSCs are lost from the niche. My results demonstrate that the loss of GSCs is not due to cell death or misregulated differentiation, but due to a disruption in adhesion of GSCs to the hub, which ultimately results in loss of GSC identity. Snail proteins were previously shown to have an important role in mammalian gonads, thus suggesting a conserved role for Snail in regulating spermatogenesis.
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ItemImaging and neuropsychological correlates of neurodegeneration and cerebrovascular disease in elderly Australians: vascular risk factors, vascular pathology and β-amyloid positron emission tomography (PET)( 2016)Vascular risk factors increase risk for dementia, however whether they contribute to the dementia phenotype through the pathology of Alzheimer’s disease (AD) or cerebrovascular disease (CVD) is unclear. This thesis utilizes multimodal neuroimaging with 3-Tesla Magnetic Resonance Imaging, 11C-PiB and 18F-NAV4694 Positron Emission Tomography (β-amyloid PET), and Carotid Artery Ultrasound (Intima-Media Thickness) in a cohort of older Australians to characterize associations between vascular risk, vascular pathology and AD-biomarkers, and their relative contributions to cognitive change over time. The broad aims of the thesis are to determine the associations between vascular risk factors and vascular pathology with molecular and structural neuroimaging markers of Alzheimer’s disease in a cohort of pre-symptomatic older Australians, and to identify potentially-modifiable factors associated with increased AD-pathology or cognitive loss which could represent potential targets for intervention studies in the future. This thesis comprises three areas of study. In the first (Chapter 6 and 7), Susceptibility- Weighted Magnetic Resonance Imaging (SWI) was used to assess prevalence and incidence of asymptomatic cerebral microhaemorrhage, and their relationship with Aβ- burden, other imaging biomarkers, and cognitive changes. In older cognitively-normal controls, lobar microhaemorrhage were common (19.1%) and associated with presence of Aβ (PiB+ status) and age, but not vascular risk factors. In this cohort, microbleeds were not independently associated with poorer cognition at baseline, nor incident cognitive decline, after adjustment for covariates including baseline Aβ-burden. Longitudinally, incident microbleeds were associated with Aβ burden, and markers of vascular disease (baseline microbleeds, white matter hyperintensity severity and lacunar infarction). The second area (Chapters 8 and 9) examined the relationship between vascular risk factors and vascular disease (cerebrovascular disease, microhaemorrhage and CIMT) with in vivo neuroimaging biomarkers of AD (Aβ-burden and brain atrophy), both cross-sectionally, and over six years’ follow-up. Burden of vascular risk was associated with the presence of Aβ, and vascular risk burden and genetic risk (Apolipoprotein [APOE] ε4 status) interacted significantly to influence Aβ most strongly in APOE ε4 carriers. Important independent relationships with Aβ also emerged for hypertension, hypercholesterolaemia, insulin resistance, and body mass index, some of which were also influenced by genetic risk (APOE ε4 status). An interaction between APOE ε4 and CVD was associated with greater Aβ, such that APOE ε4 carriers with CVD had greater Aβ burden. However, as this finding was negated by controlling for presence of microbleeds, the finding may be explained by presence of cerebral amyloid angiopathy in these participants. Carotid artery disease (high CIMT) was not associated with higher Aβ overall or in APOE ε4 carriers, however high CIMT was associated with greater Aβ accumulation over time in APOE ε4 non-carriers. The final area of study (Chapter 10) sought to determine the relative contributions between (MRI-defined) cerebrovascular disease and Aβ (PET imaging) biomarkers to change in cognitive performance and incident dementia in preclinical and prodromal AD. In cognitively-normal controls, presence of Aβ and CVD were each independently associated with longitudinal performance in memory, visuospatial function, attention and processing speed, however an interaction between Aβ and CVD was seen for change in executive function. Second, in a non-demented cohort of controls and MCI participants, risk of incident cognitive decline and dementia were most strongly influenced by presence of Aβ, rather than CVD, with an additive, rather than synergistic association seen between the two pathologies. These chapters describe the relationships between markers of increased vascular risk, evidence of peripheral and cerebral vascular pathology, Alzheimer’s disease biomarkers, and cognitive changes. Taken together, the findings suggest that several risk factors for vascular disease may also be associated with Alzheimer’s disease pathology, and that genetic risk (APOE ε4 status), and timing of assessment may influence observed associations. Interventions to mitigate vascular risk are likely to prove beneficial to reduce the imposte of dementia on future generations and intervention trials targeting modifiable lifestyle factors will be vital in this regard. The thesis also confirms the benefit of multimodal neuroimaging in combination with longitudinal clinical assessment in providing new insights into the natural history of dementia due to Alzheimer’s disease, particularly its presymptomatic stages.
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ItemNew insights into molecular and cellular pathways of neurodegeneration in amyotrophic lateral sclerosis models( 2016)Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and paralysing neurological disorder usually fatal within 2-5 years from diagnosis. First described by Jean-Martin Charcot in the late 1860s, ALS still remains a terminal disease with no effective treatments or cure. Riluzole is the only clinically-approved drug for ALS that may extend survival by 2-3 months. Therefore, there is an urgent need to understand the underlying pathogenesis of ALS to better guide development of disease-modifying treatment strategies. This thesis investigates the molecular basis of three inter-related pathogenic mechanisms implicated in motor neuron vulnerability and loss in ALS: defective energy homeostasis; disruption of protein homeostasis and abnormal RNA homeostasis. Two leading mouse models of ALS were implemented in these studies; transgenic SOD1G93A and TDP-43A315T mice in which novel pharmacological and genetic interventions were evaluated for efficacy. To examine whether defective energy metabolism is causal or consequential in the pathological cascade of ALS, the role of the key metabolic and stress sensor; AMP-activated protein kinase (AMPK) was investigated for the first time in two ALS mouse models. AMPK activation in the spinal cord associated with symptom progression, but not onset, in SOD1G93A mice, implicating AMPK activity in mediating disease course. Conversely, AMPK inactivation occurred in spinal cord and brain of pre-symptomatic TDP-43A315T mice by a protein phosphatase 2A-dependent mechanism, identifying a novel regulation of AMPK activity by pathogenic TDP-43. AMPK inactivity may therefore drive disease initiation in this mouse model. Hence, mutant SOD1 and TDP-43 exert contrasting effects on regulation of AMPK activation which may reflect intrinsic differences in energy metabolism and neurodegeneration in these two ALS mouse models. Next, a novel pharmacological strategy to improve protein homeostasis and motor neuron health was developed and evaluated for ALS. The intracellular catabolic pathway, autophagy, particularly macroautophagy, was robustly induced in mutant SOD1 and TDP-43 models of ALS. To potentiate autophagy in ALS, a novel autophagy enhancer rilmenidine was used to stimulate mTOR-independent macroautophagy in mutant SOD1 cell and mouse models. Rilmenidine treatment achieved efficient macroautophagy induction in vitro and in vivo. However, the treatment worsened motor neuron degeneration and survival of male SOD1G93A mice by exacerbating accumulation of insoluble and misfolded SOD1 species and aggregates in spinal cords. Thus, macroautophagy stimulation using rilmenidine may mediate disease progression in this specific mouse model of ALS. Lastly, a new gene therapy strategy to alleviate defects in the RNA binding protein TDP-43 was investigated. Survival motor neuron (SMN) protein deficiency causes progressive motor neuron degeneration in spinal muscular atrophy (SMA) and may be linked to pathology in ALS. SMN overexpression was previously determined to be beneficial in mutant SOD1 models of ALS. To extend these studies to TDP-43 proteinopathy, upregulation and accumulation of endogenous SMN protein into stress granules within motor neurons was demonstrated for the first time in TDP-43A315T mice. The impact of forced SMN overexpression in TDP-43A315T mice was examined, revealing improved SMN nuclear targeting, motor neuron survival, neuroinflammation and metabolic deficits as shown by AMPK activation, in female mice. Furthermore, levels of androgen receptor (AR), mutations of which cause spinal bulbar muscular atrophy (SBMA), were significantly impaired in spinal cords of male TDP-43A315T mice. This provides evidence for shared biochemical pathways in ALS, SMA and SBMA, mediated by deficiency of factors such as SMN and AR which confer motor neuron vulnerability. In summary, in mutant SOD1-linked disease, persistent AMPK signalling and autophagy activation in motor neurons may be key determinants of disease progression. In mutant TDP-43-mediated ALS, AMPK inactivation and cytoplasmic accumulation of SMN in motor neurons may be early events triggering disease onset. In conclusion, this thesis provides novel insights into pathogenic mechanisms underlying disruption of energy, protein and RNA homeostasis within motor neurons and significant clues to therapeutic alleviation of these defective pathways in ALS. In addition, this thesis identifies new links between three main neurological disorders affecting the motor system of humans; ALS, SMA and SBMA, mediated by dysregulation of SMN and AR, suggesting shared pathogenic pathways. Finally, this work importantly extends the spectrum of motor neuron diseases that may benefit from SMN restoration, excitingly paving the way for future therapeutic development and testing of SMN enhancing agents for ALS.
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ItemMolecular mechanisms of BDNF signalling in central nervous system myelination( 2016)Myelination within the CNS allows the fast and efficient transmission of action potentials along axons. Both myelin and the oligodendrocytes that generate myelin and wrap the axons are crucial for the development and maintenance of an adaptive and healthy nervous system. Interestingly, the process of myelination is responsive to neural activity so myelination also contributes to a form of neuroplasticity called white matter plasticity. Disorders as varied as multiple sclerosis and schizophrenia result when myelination is compromised, and experiences in the world, from childhood neglect and maltreatment to learning a second language, leave their trace in the white matter of our brains. Much remains to be discovered about the factors that regulate myelination and the signalling networks these factors utilise. Our laboratory has taken an interest in a growth factor that has a recognised role in synaptic plasticity and is epigenetically regulated. This molecule is brain-derived neurotrophic factor (BDNF). Our studies reveal that BDNF exerts a pro-myelinating effect in vitro and utilises Erk1/2 to mediate this effect. Specifically, BDNF acts on oligodendroglial TrkB receptors to myelinate the axons of DRG neurons in co-culture with oligodendrocytes. Studies in vivo confirm a role for BDNF, TrkB and Erk1/2 in myelination. Previous work from our laboratory indicates that loss of TrkB from myelinating oligodendrocytes leads to a phenotype of thinner myelin in the CNS, and my results reveal that when TrkB is deleted from the time of oligodendrocyte specification, this phenotype is restricted to thinner myelin only around large diameter axons. This suggests that there compensation occurs for the loss of TrkB but the factors that contribute to this putative mechanism of compensation are not yet known. Surprisingly, despite TrkB null oligodendrocytes only yielding a relatively mild hypomyelinating phenotype in vivo, I discovered that in the context of a myelinating co-culture, TrkB null oligodendrocytes exhibit a severely reduced capacity to myelinate. This led me to pose the question: how were oligodendrocytes that had a very limited capacity to myelinate in vitro able to myelinate the majority of axons normally in vivo? The result suggested that a cell or factor that was present in vivo but absent in the myelinating co-culture system was enhancing the oligodendrocytes capacity to myelinate. Interestingly, in vitro studies demonstrate that Fyn kinase associates with and is activated by TrkB, and that Fyn translocates neuronal TrkB receptors to lipid rafts in response to BDNF treatment. Similarly to BDNF, in vivo studies on Fyn kinase reveal that it too has a role in synaptic plasticity and myelination and this potentially suggests they utilise a common signalling pathway. In this work I interrogated BDNF signalling in oligodendrocytes and identified that BDNF and Fyn kinase do share a common signalling pathway: Fyn kinase is a downstream mediator of the pro-myelinating effect of BDNF. I then hypothesised that Fyn kinase could form part of a mechanism that compensates for the loss of oligodendroglial TrkB in myelination in vivo. I demonstrated that forced over-expression of Fyn kinase by TrkB null oligodendrocytes enhanced myelination in vitro. However, I also found that in isolated, cultured TrkB null oligodendrocytes the expression of Fyn kinase protein was lower than control oligodendrocytes although proportionally more of it was active. Taken together, these results suggest that Fyn kinase could, in principle, contribute towards redundancy in myelination but requires factors or cells, absent from the in vitro myelination assay, but present in vivo that increase its activation or expression level. Further studies are required to identify these factors and to determine if this putative compensation mechanism occurs in vivo. BDNF and Fyn kinase both have roles in neural plasticity and it is consistent with a maxim of biological economy that they would have complementary roles in white matter plasticity. BDNF switches oligodendrocytes into an activity dependent mode of myelination, and the findings of this study indicate that Fyn kinase mediates BDNF driven myelination, suggesting a role for Fyn kinase in white matter plasticity as well. Due to the burgeoning burden of neurodegeneration and the recognition that white matter plasticity is affected in mental illness and by experiences of trauma, it is imperative that we interrogate the molecular mechanisms of white matter plasticity to better target treatments to alleviate the distress caused by these challenges.