Anatomy and Neuroscience - Theses
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ItemCharacterisation of innate immune gene activities in rested and stimulated states( 2017)Monocytes and macrophages are among the first line of response to an infectious agent. The quality of this response will influence the type of T-lymphocyte recruited and may determine the outcome of an infection. While much is known about the repertoire of genes expressed by monocytes and macrophages in response to an acute challenge, little is known about the potential to diversify these responses through the generation of transcript isoforms. The role of transcriptional complexity in determining immune responses to stimulation has not been previously addressed in primary human monocytes. Transcriptional complexity is a measure of the abundance and diversity of transcript isoforms expressed in a genome. It has previously been demonstrated on a few genes in human myeloid cells that transcript isoforms can be expressed in a stimulus-specific manner and play important roles in immune responses. Although the expression of different transcript isoforms has been previously evaluated through the study of alternate splicing, the contribution of alternate transcription start sites has not been systematically addressed. We used CAGE-seq and ATAC-seq to characterise the extent of altering transcription start site usage following exposing monocytes to different stimuli. The comparison of the differences in alternate transcription start site usage between monocytes and macrophages revealed that these two cell types have a similar repertoire of transcription start sites. However, the transcriptional complexity focuses were different in these two cell types. Monocytes displayed dynamic changes in the alternate transcription start site usage in response to distinct stimuli, suggesting its plastic roles in immune responses. Although the mechanisms underlying the transcription start site selection have not been determined in this study, we proposed a model that differential recruitment of transcription factors to distinct promoters was predicted to initiate transcription from different transcription start sites. We have identified the potential transcription factors that were used to select transcription start sites in different conditions. The better understanding of the transcriptional complexity in primary human monocytes will assist gaining better insight into the involvement of monocytes in immune responses.
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ItemImportin α1 plays an essential role in germline stem cell maintenance in the Drosophila testes( 2017)The Drosophila testis maintains two stem cell populations at the apical tip, the germline stem cells and the cyst progenitor cells. 10-12 germline stem cells are arranged around the hub cells, a group of somatic cells responsible for maintaining the stem cell niche via expression of short range factors which promote self renewal and allow asymmetric cell division. A balance must be maintained between self renewal and differentiation to ensure the maintenance of the stem cells and production of mature spermatozoa. Spermatogenesis is a complex process requiring precise regulation to progress through the numerous stages required to produce viable spermatozoa. Importin α proteins act as adaptors between Importin β and target proteins to permit import of these target proteins. Metazoan genomes contain multiple Importin α proteins which have arisen due to multiple gene duplications and the different importin α genes show unique expression patterns during spermatogenesis in both Drosophila and mouse testes. It has been hypothesised that regulating importin α expression can drive the progression through the different stages of spermatogenesis through the targeted nuclear transport of different subsets of proteins into the nucleus. This project aims to investigate the role Importin α1 plays during spermatogenesis. It has been previously demonstrated that importin α1 null males are viable but sterile, the cause of this sterility remains unknown. To further investigate the role of Importin α1 during spermatogenesis I performed in-depth characterization of importin α1 null testes. I generated and characterised an importin α1 dominant negative transgene and used this transgene to screen for nuclear proteins that have their transport regulated by Importin α1. In this project I have shown that importin α1 is essential for the maintenance of germline stem cells in Drosophila testes and identified a potential target protein regulated by Importin α1 in spermatogonia.
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ItemBone morphogenetic protein-4 signalling in glial cells of the central nervous system( 2017)Multiple sclerosis (MS) affects over 20,000 Australians and over 2-3 million people globally. MS is a demyelinating disease of the central nervous system (CNS) in which the insulating myelin sheath of the neuron is degraded. This leads to severe impairment of neuronal signal transmission throughout the CNS. Oligodendrocytes are specialised cells of the CNS that form the myelin sheath. Oligodendrocyte progenitor cells (OPCs) are present in the adult brain and respond to demyelinating injury, but these cells often fail to differentiate from precursor cells into oligodendrocytes to replace damaged myelin in MS lesions. One group of factors linked to this ‘differentiation block’ is the bone morphogenetic protein (BMP) family. This thesis aims to clarify the role of BMP signaling in two major classes of CNS cells: oligodendrocytes, which are formed by OPCs, and astrocytes, which play a role in regulating oligodendrocyte differentiation during CNS injury. Firstly, to understand the influence exerted by BMP4 on oligodendrocytes during remyelination, BMP4 signalling was disrupted by infusing LDN-193189, a pharmacological inhibitor of BMP4 receptors BMPRIA and BMPRIB, following cuprizone-induced demyelination in mice. This resulted in a significantly higher number of mature oligodendrocytes present in the murine corpus callosum after one week of recovery from cuprizone treatment. Furthermore, this increase in oligodendrocyte number was coupled with a significant increase in the degree of remyelinated myelin sheaths in the murine corpus callosum. In vitro analysis demonstrated that LDN-193189 has a direct positive influence on OPC differentiation into mature oligodendrocytes, and reduces the astrogliogenic effect of BMP4. Inhibiting BMP4 signalling in OPCs in vitro also promoted myelination in a dorsal root ganglion co-culture experiment. Analysis of gene transcription in OPCs treated with BMP4 and LDN-193189 suggested that the positive effect of pharmacologically inhibiting BMP4 on oligodendrocyte differentiation and myelination was mediated by downregulation of a DNA binding protein, ID4. This protein has been previously shown to inhibit oligodendrocyte differentiation in response to BMP4 signalling activity. To further understand the signalling mechanisms by which BMP4 elicits its inhibitory effect on oligodendrocyte differentiation, a transgenic mouse with an inducible conditional deletion of Bmpr1a was used to disrupt BMP4 signalling through BMPRIA. Cultures of OPCs with a BMPRIA deletion recapitulated most of the positive effects observed on oligodendrocyte differentiation and myelination as seen in OPCs treated with LDN-193189. This suggested that BMPRIA may exert a critical influence in transmitting the inhibitory BMP4 signal in OPCs compared to BMPRIB. Secondly, the influence exerted by BMP4 signalling on astrocytes was assessed using in vitro and in vivo techniques. Mature astrocyte cultures responded to BMP4 by increasing their proliferation and transcription of Gfap, a key intermediate filament protein that is upregulated in astrocytes in response to CNS injury. Furthermore, BMP4 stimulates the secretion of factors that, when applied to OPC cultures, inhibit their differentiation. This effect is normalised when astrocytes are treated with LDN-193189, suggesting the influence of BMP4 signalling on astrocytic secreted factors is mediated through BMP4 Type I receptors. To understand the function of BMP4 signalling in astrocytes during demyelination, mice with an inducible, astrocyte-specific deletion of Bmpr1a underwent cuprizone administration to cause demyelination in the murine corpus callosum. However, mice with a Bmpr1a deletion in astrocytes did not show any improvement in remyelination, or a reduction in the number of GFAP+ astrocytes in the corpus callosum compared to vehicle-treated mice. This suggests that the influence exerted by BMP4 on astrocytes during demyelination is negligible, or alternatively, that the function of BMPRIA is compensated by BMPRIB in astrocytes in vivo. This thesis has further clarified the inhibitory influence exerted by BMP4 on oligodendrocyte differentiation in the CNS. Inhibiting BMP4 signalling through its Type I receptors BMPRIA and BMPRIB following demyelination significantly enhances subsequent remyelination in the murine corpus callosum. Additionally, this thesis has identified novel astrocytic responses to BMP4 signalling in vitro, including a potential influence on secreted factors that inhibit OPC differentiation. However, further investigation is required to fully elucidate this relationship. In a broader context, it is hoped that the research reported in this project may contribute to improved clinical options through identification of treatments for improving remyelination in demyelinating disease.
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ItemInvestigating biomarkers of traumatic brain injury with preclinical models( 2017)Traumatic brain injury (TBI) results in a series of complex pathophysiological processes caused by external forces to the brain. The initial insult falls on a continuous spectrum from mild to severe and occurs in a heterogeneous patient population resulting in tremendous variability in the progression of secondary injury mechanisms. Currently, there are no biomarkers that can reliably, and objectively, assess these changes across the full spectrum of injury severity and time post-injury. This may be due, in part, to the inherent difficulty in studying TBI in the medical setting. As such, animal models have been developed as a practical platform for initial investigations into TBI and can provide a greater understanding of injury prognosis, leading to improved clinical care. These models are used here to assess the potential of neuroimaging, blood, and behavioural biomarkers to provide reliable and objective insight into the pathophysiological changes that occur following TBI. Significant contributions include evidence supporting a relationship between TBI and the later development of a motor neuron disease-like process, a comparison of diffusion-weighted imaging biomarkers in TBI, the identification of pathophysiological changes beyond the resolution of neurocognitive deficits that typically define recovery after mild TBI, and evidence that sex is an important consideration in adolescent repeated mild TBI.
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ItemTropomyosin related kinase B (TrkB) regulates neurite outgrowth via a novel interaction with suppressor of cytokine signalling 2 (SOCS2)( 2017)Suppressor of cytokine signalling 2 (SOCS2) negatively regulates cytokine signalling but it also has a positive effect on neurite outgrowth of cortical and dorsal root ganglion neurons. It has a high expression profile in the central nervous system in early development stages; hence any changes in the expression of SOCS2 results could result in alternations in the structure of neurites and dendritic morphology. SOCS2 has the highest expression in the hippocampus compared to other members of the SOCS family and this study aims to reveal the role of SOCS2 in the growth of hippocampal neurites in the presence of BDNF. Hippocampal neurons derived from neonatal mice are a great model of BDNF dependent study mediated by neurotrophin receptor BDNF. Neurons were derived from mice with overexpression or knock out of SOCS2 and the length of neurites were analysed in response to BDNF. While normal levels of SOCS2 did not result in any change in the length of neurites, neurons with overexpression of SOCS2 had increased length in response to BDNF. Knockout of SOCS2 resulted in shorter neurites although with BDNF treatment the length of neurites reached normal levels. The co-regulatory role of SOCS2 and BDNF in neurite outgrowth of hippocampal neurons in-vitro draws a possible link between SOCS2 and BDNF receptor, TrkB. The interaction of SOCS2 and TrkB was shown and studies in detail. Truncated isoforms and deleted mutants of TrkB and SOCS2 were employed to reveal the regions involved in the interaction. This study showed the complex interaction of SOCS2 and TrkB happens at two points. The first point is the protein-protein link between the kinase domain of TrkB and the SH2 region of SOCS2. The second point of interaction is through the juxtamembrane region of TrkB. Domains of SOCS2 that are involved in interacting with TrkB were assessed in their ability to regulate neurite outgrowth. Rat hippocampal neurons transfected with deleted mutants of SOCS2 shows that deletion in the BOX domain increases neurite length compared to SOCS2 full length indicating the importance of the interaction of the SH2 and N terminal domain in neurite growth. Intracellular events and downstream pathways stimulated by BDNF and mediated through TrkB play an important role in the outcome of TrkB signalling. SOCS2 through interacting with TrkB can influence such events. SOCS2 increases both phosphorylation and ubiquitination of TrkB, ubiquitination of TrkB being the result of the association of the juxtamembrane region of TrkB and the BOX domain. Bimolecular florescence complementation assay was employed to study the effect of SOCS2 on TrkB trafficking and it was determined that SOCS2 increases TrkB receptor turnover in the cell with an increase in trafficking of the receptor to late and recycling endosome. In summary, this study presents evidence of a novel role of SOCS2 in regulating TrkB neurotrophin signalling. The molecular and biochemical aspects of this regulation was analysed in detail in-vitro. These finding has potential implications in developing new therapeutic strategies in neurodegenerative diseases where BDNF signalling is involved.
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ItemRelationship between structure, molecular phenotype and function of corneal sensory neurons and their nerve terminal endings( 2017)The ability to sense and react to our environment is governed by activity in peripheral sensory neurons, which are widely distributed in the different body tissues. Embedded in their membrane there are specific types of ion channels and receptors that are responsible for the transduction of the different stimulus modalities. This thesis has used immunohistochemistry, in situ hybridization and electrophysiology to explore the structure and molecular phenotype of the different functional sub-types of sensory neurons innervating the cornea, and their nerve terminal endings in guinea pigs and mice. In addition, the thesis has investigated the effects of metabolic stress induced by a high fat diet and diabetes on the nerves fibres of sensory neurons in the corneal epithelium of mice. Previous studies have shown that corneal afferent neurons have heterogeneous molecular identities(Bron et al., 2014; Ivanusic et al., 2013). The results presented in Chapter 2 confirmed this heterogeneity and found that the majority of corneal afferent neurons could be subdivided into the 3 functionally defined corneal receptors on the basis of their selective expression of sensory transducer molecules. Transient receptor potential cation channel subfamily V member 1 (TRPV1) was expressed in polymodal nociceptors, transient receptor potential cation channel subfamily M member 8 (TRPM8) was expressed in cold thermoreceptors and Piezo2 was expressed in mechano-nociceptors. Furthermore, the TRPV1-IR corneal afferent neurons were further subdivided into three sub-populations on the basis of their molecular phenotype. These findings led to the hypothesis that the neurochemically defined sub-populations of TRPV1-IR corneal afferent neurons form morphologically distinct nerve terminal endings that terminate at different locations within the corneal epithelium. The results in Chapter 3 confirmed this hypothesis, showing that CGRP expressing TRPV1-IR nerve terminals in the guinea pig cornea had simple endings that terminated in the basal or wing cell layers, whereas the TRPV1-IR nerve terminals that did not express CGRP had ramifying endings that terminated in squamous cell layer. A previous study had demonstrated that the TRPM8-IR nerve terminals of putative cold thermoreceptors form complex endings that terminate in the wing and squamous cell layers(Ivanusic et al., 2013). The results presented in Chapter 4 confirmed that functionally identified polymodal nociceptors and cold thermoreceptors in the corneal epithelium have distinct nerve terminal morphologies and neurochemical phenotypes. The experiments in Chapter 5 investigated the effects of high fat-diet and Type 2 diabetes on the density of TRPV1-IR and TRPM8-IR nerve fibres in the corneal epithelium. It was demonstrated that TRPV1-IR and TRPM8-IR fibres in the corneal epithelium were affected differently by the metabolic stress associated with these pathological conditions. The findings of this thesis define the different sub-populations of corneal afferent neurons and show morphological and/or neurochemical specialisation of the TRPV1 expressing corneal nerve terminals in normal cornea. Furthermore, the findings demonstrate directly for the first time that pathology induced by metabolic stress can differentially affect some of these sub-populations of corneal sensory neurons.
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ItemA characterisation of neurons involved in learning and memory( 2017)Studies into the mechanisms of memory have long searched for a concept known as the engram, defined as the change that occurs in a neuronal circuit to underlie the encoding of memory. However, a complete description of the specific neuronal changes that occur following the encoding of any type of memory has not yet been published. This thesis used context- and auditory-fear conditioning as a model of learning, and a transgenic approach of identifying functionally activated neurons, to identify neurons throughout the brain that were specifically activated by fear learning. Discrete populations of neurons were identified within the amygdala, hypothalamus and septum, and neuronal activation was shown to occur specifically following the acquisition of auditory fear memories, with minimal activation in these regions due to the non-learning aspects of the conditioning process, such as tone exposure, shock exposure, or expression of fearful behaviour. These different populations of neurons may form nodes in the circuit for fear conditioning. One population of activated neurons was identified within the lateral amygdala, a key region involved in fear conditioning. Activated neurons in this region also expressed the phosphor-activated form of ERK1/2 kinase. As fear-learning has previously been shown to require phosphor-activation of ERK1/2 in the lateral amygdala, this suggests that the identified population of neurons in the lateral amygdala is not only activated by, but directly involved in fear learning. The electrophysiological properties of neurons within this region were then investigated via random patch-clamp recording. Neurons from fear-conditioned mice show modified action potential dynamics when compared to neurons from home cage mice, and a subset of neurons recorded from fear conditioned mice display modified synaptic activity. Finally, the activated neurons within the lateral amygdala were specifically targeted for patch-clamp experiments via the development of a novel method for visualising activated neurons in live brain slices. The population of activated neurons within the lateral amygdala displayed significant differences in synaptic activity when compared with neighbouring, nonactivated neurons. Given that these changes occur in neurons shown to be directly involved in fear learning, they may underlie the encoding of auditory fear memory in vertebrates, thus bringing a description of the engram one step closer.
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ItemThe development of sympathoadrenal progenitor cells into sympathetic neurons and adrenal chromaffin cells( 2017)Sympathetic neurons and adrenal chromaffin cells originate from a common precursor, the neural crest-derived sympathoadrenal (SA) progenitor cell. Specification of trunk neural crest cells into SA progenitor cells is under the influence of bone morphogenetic proteins (BMPs) produced from the wall of the dorsal aorta. SA progenitor cells then further differentiate into either sympathetic neurons of the sympathetic ganglia or chromaffin cells in the adrenal medulla. However, the mechanism underlying the segregation of chromaffin and neuronal cell fates remain unknown and the time course of SA segregation and regulation of cell fate is poorly understood. To discover novel regulators, and to have a broader view of the transcriptional networks involved in the segregation events, large scale gene screening is required. Isolation of sympathetic neuroblasts and adrenal chromaffin precursor cells for molecular studies, however, is challenging, as they are hard to discriminate during early development. Therefore, the aims of this study were to 1), identify effective marker(s) for early discrimination of neuroblasts and chromaffin precursor cells in order to 2), investigate their patterns of proliferation and differentiation and 3), develop an isolation method by fluorescence-activated cell sorting (FACS) for the two cell types, and finally 4), perform a comparative transcriptomic analysis by using RNA sequencing (RNA-seq) to identify candidate regulator genes that control fate determination of developing adrenal chromaffin cells and sympathetic neuroblasts. The temporal and spatial expression patterns of tyrosine hydroxylase (TH) and cocaine and amphetamine regulated transcript (CART) were first analysed by quadruple-label immunofluorescence. The proliferative behaviour of SA cells and their derivatives were also analysed. Our data showed that sympathetic neuroblasts and chromaffin precursor cells could be discriminated as early as E12.5 by differences in the intensity of TH immunostaining, expression of CART and by proliferative activity. Quantitative data showed higher TH immunostaining in chromaffin cells compared with most ganglion cells, while CART expression was widespread in embryonic sympathetic neuroblasts but absent from adrenal chromaffin cells. Thus CART expression and the levels of TH expression are identified as markers for distinguishing between sympathetic neurons and chromaffin cells as they differentiate. The possibility of isolating sympathetic neuroblasts and adrenal chromaffin precursor cells by FACS was then examined. Isolation based on the differential levels of TH expression by FACS was first investigated in transgenic animals that express fluorescent marker proteins driven from the TH promoter. In cells from transgenic mice where TH-Cre drove enhanced yellow fluorescent protein (EYFP) expression, the intensity of EYFP expression was found to separate the two cell types for isolation. A FACS-based isolation strategy developed in this study permitted routine isolation of purified sympathetic neuroblasts and adrenal chromaffin cells in E12.5 mice. The cell type-specific transcription profiles of the isolated sympathetic neuroblasts and adrenal chromaffin precursor cells during the key segregation event at E12.5 mice were examined by RNA-seq. Comparative transcriptome analysis revealed more than 4,000 differentially expressed genes between adrenal chromaffin cells and sympathetic neuroblasts. Among these, 10 genes including Elf3, Elf4, Nrk, Msx2, Dlx1, Dlx2, Dll4, Dlk1, Foxq1 and Fzd10, were identified as potential regulators based on their differential expression level and potential relevancy to development. The temporal gene expression patterns of these 10 genes during SA development in E11.5 to E14.5 mice were further examined by droplet digital PCR (ddPCR) and all of 10 genes were shown to be expressed transiently at E12.5 and E13.5, suggesting a potential role during the key stage of lineages segregation. Among these, the differential protein expression patterns of DLK1 and FOXQ1 in the adrenal medulla and sympathetic ganglia, revealed by immunohistochemistry, were also found to be highly consistent with the gene expression patterns revealed by both RNA-seq and ddPCR. For Nrk, loss-of-function analysis in E18.75 mutant mice showed a defect in adrenergic phenotype acquisition in adrenal chromaffin cells and cell proliferation, suggesting a role in adrenergic chromaffin cell fate acquisition. The RNA-seq data also raised the possibility of epigenetic regulation in SA lineage cell fate acquisition through genomic imprinting and X-chromosome inactivation.