Anatomy and Neuroscience - Theses
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ItemCellular and subcellular co-storage of gastrointestinal hormones( 2018)Gastrointestinal hormones regulate a diverse range of physiological processes including digestion, metabolism, and food intake, as well as maintaining mucosal integrity and mounting defensive mechanism in response to pathogens or toxins. Disruptions of these regulatory processes are associated with disorders that include diabetes, obesity, irritable bowel syndrome, and coeliac disease. Despite this clinical significance, many aspects of the cells that contain gut hormones, enteroendocrine cells (EEC), remain poorly characterised. Historically, EEC were categorised based on their hormone content, with the assumption that each cell contained a single hormone. However, hidden in some early studies were examples that the simple ‘one cell‐one hormone’ classification system was not correct. A major focus of this thesis is the characterising of coexpression patterns of hormones in mouse and human intestine. Remarkably, examples of overlap could be found for all hormones investigated, demonstrating a need for a revised classification system for EEC. Moreover, species differences in EEC expression patterns occur, highlighting a further level of complexity in the field of research. The coexpression of EEC hormones raises new questions about how these hormones are stored at a subcellular level. Super‐resolution microscopy allowed the visualisation and quantification of vesicular stores in EEC. Overlapping and non‐overlapping vesicular stores were observed. Furthermore, the relative abundance of hormone expression varied considerably between cells and, consequentially, small amounts of hormones sometimes could not be detected with low‐resolution methods. In addition, a monoclonal antioxyntomodulin antibody was characterised, expanding the examination of hormone colocalisation patterns to the investigation of proglucagon‐derived peptides. Several hormones, including 5‐HT, influence electrolyte transport, which can be measured as the shortcircuit current (Isc) in an Ussing chamber. The effects of 5‐HT and secretin, which are frequently colocalised, on electrolyte transport across the intestinal epithelium were examined. Both hormones stimulated a secretory response in all regions of the mouse intestine, including the colon where secretin was not thought to be expressed. However, secretin gene transcripts were identified and secretin immunoreactivity was localised to EEC in the mouse colon. The functional effects of TRPA1, which is an ion channel that has previously been localised to EEC containing both 5‐HT and CCK, was also examined using an Ussing chamber. While TRPA1 agonists stimulated a secretory response, this was not mediated by 5‐HT. This research has advanced knowledge of the colocalisation patterns of gastrointestinal hormones at a cell and subcellular level and explored some aspects of the functions of costored hormones in relation to water and electrolyte movement across the gut lining.
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ItemDetermining the role of the paratrigeminal nucleus (Pa5) in airway defence( 2018)Respiratory sensations conveyed by airway sensory nerve fibres are poorly understood, yet they contribute significantly to morbidity in pulmonary disease. Our viral tracing studies identified a previously unknown airway sensory circuit in the brain projecting through an obscure medullary site known as the paratrigeminal nucleus (Pa5). The Pa5 is located in the dorsal lateral medulla and is commonly defined as a collection of interstitial cells in the dorsal tip of the spinal trigeminal tract. The Pa5 has been previously implicated in baroreceptor function and somatosensory processing. Astonishingly, very little investigations have been made to understand its role in airway afferent processing. We therefore investigated the anatomical connectivity of the Pa5, its involvement in initiating and controlling respiratory reflexes as well as in more complex respiratory behaviours including evoked cough. Conventional neuroanatomical tracing was conducted to determine the input and output connectivity of the Pa5. Firstly, retrograde tracing confirmed, in the guinea pig, that vagal afferents from the jugular versus the nodose ganglia project specifically into the brainstem primary afferent termination sites. That is the nodose vagal ganglia project almost exclusively to the nucleus of the solitary tract (nTS), while the jugular vagal ganglia project predominately to the Pa5. Furthermore, anterograde tracing was employed from the guinea pig Pa5, showing that this nucleus has extensive projections throughout the cardiorespiratory column, including the nTS, reticular nuclei, pre-bötzinger nucleus and in particular the parabrachial and Kölliker-Fuse nuclei in the pons. We have shown functional significance of these projections by implicating the Pa5 in the initiation and control of laryngeal-evoked respiratory reflexes. Initially we found that laryngeal-evoked respiratory slowing was significantly attenuated by blocking the Pa5 using the GABAA agonist muscimol (stimulus evoked change in breaths/min pre-muscimol was 40.9±3.5 breaths/min pre-muscimol vs. 21.4±2.9 post-muscimol, p=0.001), while this pharmacological modification in the nTS had no effect on the laryngeal-evoked respiratory slowing. Importantly, this novel finding showed that the Pa5 has a role in respiratory reflexes that is independent of the nTS. Furthermore, injection of glutamate receptor antagonists into the Pa5 were able to prevent respiratory slowing evoked by laryngeal stimulation (change in respiratory rate at the maximum response 40.3±4.1 breaths/min vs. 17.3±3.1 breaths/min; p=0.0002), while peptide receptor antagonists resulted in significantly smaller effects. Intriguingly, while injection of glutamate into the Pa5 resulted in respiratory slowing and in some cases apnoea, mimicking the laryngeal-evoked reflex, the direct release of neuropeptides by capsaicin microinjection into the Pa5 resulted in the paradoxical increase in respiratory rate. This unique response could be blocked by Substance P receptor antagonists but not by glutamate receptor antagonists, suggestive of two types of Pa5 postsynaptic neurons. Additional anatomical characterisation of the Pa5 revealed the existence of two phenotypically distinct subtypes of Pa5 neurons. That is, one expressing the neurokinin 1 receptor and the other expressing calbindin. Furthermore, dual retrograde tracing from nuclei important for respiratory rhythm and modulation and were shown to receive direct projections from the Pa5 (i.e. the ventrolateral medulla and pontine nuclei) revealed that less than 7% of Pa5 neurons were dual labelled and therefore revealed that Pa5 neurons project differentially throughout the cardiorespiratory column. Given the complex processing capability of the Pa5, it seemed plausible that this nociceptive region may be important in complex respiratory behaviours, such as evoked cough and the perception of airway irritations. We developed an assay in which we can study the conscious perception of an airway irritation using behaviours (enhanced grooming, moving and chewing), in addition to the standard assessment of evoked cough. Conscious unrestrained guinea pigs were aerosolised bradykinin (a jugular and nodose C-fibre stimulant) and adenosine 5’-triphosphate (ATP, a nodose selective stimulant). While bradykinin evoked both dose dependent increases in cough (8.9±2.7 at 1mg/ml and 25.7±3.7 at 3mg/ml) and behaviours (20.7±45.8 behaviour duration during saline vs., 350.4±46.7 total behaviour duration, p=0.04), ATP only evoked responses at the highest dose (22.7±5.6 coughs and 81.3 behaviour duration during saline vs. 404±109.5 total behaviour duration). These distinct cough and behavioural profiles are suggestive that jugular and nodose airway afferents differentially process respiratory sensations. This was further supported by showing that targeted toxin lesions (substance P-Saporin, 10ng/100nl) of the Pa5 resulted in decreased bradykinin evoked cough compared to controsl (i.e. 7.2±2.8 coughs control vs. 0±0 coughs lesion at 1mg/ml and 19.2±4.1 coughs control vs. 10.4±3 coughs lesion at 3mg/ml, p=0.02 and p=0.009 respectively). Alternately, lesioning NK1 receptor expressing neurons in the Pa5 had no effect on ATP evoked cough, in turn implicating NK1 receptor expressing Pa5 neurons specifically process jugular C-fibre evoked cough. Together these data are the first to reveal a complexity of airway afferent processing in the Pa5. Better understanding this putative airway somatosensory system may help identify therapeutic targets to alleviate respiratory discomfort in disease.
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ItemThe transcription factor grainy head regulates intestinal stem cells( 2018)The constant renewal of the intestinal epithelium is achieved through the activity of a population of intestinal stem cells (ISC). These cells maintain tissue homeostasis by ensuring balance between cell division and the production of the differentiated cell types of the intestine. This process is under strict molecular control with ISC maintenance and function controlled by the expression of a particular subset of genes and their production of multiple protein isoforms. However, the mechanism by which different protein isoforms are able to regulate intestinal regeneration remains poorly understood. Using the adult intestine of Drosophila melanogaster, we identified grainy head (grh) as a candidate gene that is able to influence regeneration using different protein isoforms. The Drosophila genome contains a single grh gene which is alternatively spliced to produce 8 mRNA transcripts and two major isoform groups, the N-isoforms and the neural specific, O-isoforms. In this study, I show using droplet digital PCR that transcripts for both isoform groups including the neural specific isoforms are expressed in the midgut, albeit at low cellular levels. Moreover, cell lineage tracing studies using grh null mutants result in a reduction of progeny arising from a single ISC. Interestingly, cell lineage tracing using an O-specific mutant resulted in a more severe reduction in progeny number compared to grh null mutants. Additionally, lineage traced ISCs mutant for the O-isoforms were lost over time. In the opposite experiment, an O-isoform, GRH.O’ was ectopically expressed in ISCs and its immediate daughter, the enteroblast (EB). Over expression solely in ISCs did not lead to a change in ISC number while over expression in EBs led to a block in differentiation and an increase in EB number. Conversely, ectopic expression of two GRH N-isoforms, GRH.N and GRH.N’ led to a loss of ISC and EBs through forced differentiation. I therefore hypothesize that GRH O-isoforms are required for ISC maintenance while the N-isoforms are required for differentiation in the Drosophila midgut. In the final part of my study, I conducted expression analysis for GRHL-2 and GRHL-3, the mammalian orthologues of GRH in the mouse small intestine. Expression studies using qPCR and immunohistochemistry reveal that both GRHL-2 and GRHL-3 are expressed in intestinal crypt cells suggesting that the function of GRH is conserved the mammalian small intestine.
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ItemThe neurovascular unit in early diabetic retinopathy: cellular changes and an emerging role for microglia( 2018)In the developed world the leading cause of blindness in working age adults is diabetic retinopathy (DR). The risk of vision loss due to diabetes increases with disease duration, and most frequently affects those who have lived with diabetes for one to two decades. For individuals with diabetes, there is a need to develop therapeutic interventions that are capable of halting or delaying degeneration of the retina as early as possible to preserve vision throughout the life of the patient. DR ultimately leads to vision loss via the death of retinal neurons, which occurs after severe dysregulation of the retinal vasculature. Retinal oedema and/or the proliferation of blood vessels both signal the advancement to a high-risk stage of vision-threatening DR, and our understanding and management of these late stages is comprehensive. However, the initial cellular changes that occur soon after the onset of diabetes and contribute to this end-stage vascular pathology are not completely understood. These include changes to the neurovascular unit: retinal blood flow, neuronal function, and glial / microglial cell reactivity. It is these early signs that are investigated in this thesis, for it is these changes at this early timepoint that hold the most promise for intervention to prevent onset of clinical vasculopathy. Using an animal model of diabetic retinopathy, the relative contribution of neurons, blood vessels, and glia/microglia to early DR pathology was assessed at two early time-points after diabetes onset. In vivo measurement of retinal blood flow, blood vessel structure, and neuronal function was undertaken; in conjunction with histological analysis of pericytes, Müller glia, astrocytes, and microglia, as well as the interactions of these cells with the vasculature. Additionally, microglia were investigated for their potential to assist in vaso-regulation using ex vivo live cell imaging and single cell population RNA-Seq. Functional measurements revealed that retinal blood flow and retinal ganglion cell function were both reduced after 4 weeks of hyperglycaemia. In depth analysis of retinal vessel structure showed capillaries to be constricted at this time-point and were also unresponsive to hyperoxic challenge. Neuronal function was further degraded by 12 weeks of diabetes, although there were no signs of cell death. Glial cells showed no difference in structure, or signs of gliosis after 4 weeks of diabetes, and did not alter contact with the vasculature. Microglia were found to be in a resting phenotype and increased their contact with retinal capillaries after 4 weeks of diabetes. The microglia specific fractalkine signalling axis was shown to initiate vasoconstriction on retinal blood vessels, a response which was absent in the diabetic retina. Vaso-active genes were identified in microglial populations, several of which were upregulated in the diabetic retinae. In summary there are two main findings to report. Firstly, this project expands our understanding of the chronopathology of cell-specific dysfunction in the early stages of DR, and indicates that capillary constriction may be a new biomarker for early stage disease. Secondly, the discovery that microglia contribute to blood flow maintenance opens new avenues for research into vascular diseases, including DR, which may unlock novel therapeutic targets.
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ItemStudies on mechanisms of cell death in retinal degeneration( 2018)Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinopathies affecting approximately 1.5 million people worldwide. RP is associated with genetic mutations leading to progressive deterioration of the photoreceptors followed by degeneration of the retinal pigment epithelium (RPE). The molecular mechanisms by which these mutations drive photoreceptors to death are not well understood. Accordingly, effective therapeutic strategies to stop the progression of RP and/or restore vision have yet to be established. The current study examines mechanisms of cell death in two models of RP that affect genes in visual cycle: Pro23His (P23H) rat and rd1 mice. The P23H transgenic albino rat strains are widely used models for the most common mutation of the rhodopsin (RHO) gene associated with autosomal dominant form of RP. However, detailed phenotypic characterization of the most commonly used strain (line 3, P23H-3) is lacking. Before investigating what underlying cell death signaling pathways contribute to photoreceptors death in this animal model, P23H-related functional and structural changes were evaluated using electroretinography (ERG) and in vivo imaging. In the control Sprague-Dawley (SD, control) rats, photoreceptoral and post-photoreceptoral ERG responses enhanced from postnatal day (P)18 to 1 month, when they reached adult levels. By contrast, the function of the rod and cone pathways in the P23H-3 rat showed progressive decline from 1 month of age, which correlated with loss of photoreceptors in the outer nuclear layer (ONL) from 2 months onwards. Quantitative analysis of dying P23H-3 photoreceptors in the ONL, using TUNEL assay, revealed a biphasic pattern of the cell death, with a rapid initial phase, peaking at P18, followed by a slower progressive phase. However, activation of caspase-3 and nuclear translocation of apoptosis inducing factor (AIF) were detected rarely in the nuclei of the ONL at P18, suggesting that the classical intrinsic apoptotic pathway may not be the major contributor. Consistent with this, analysis of mitochondrial membrane potential (Δψm) using flow cytometric analysis of JC-1 fluorescent assay showed similar staining patterns for both P23H-3 and age-matched SD control cells, with no significant differences between the ratio of red/green fluorescence, that is no difference in Δψm. To further investigate other potential cell death pathways involved in the degeneration of photoreceptors in P23H-3 rats, gene expression profiling was performed using a rat cell death qPCR array on RNA isolated from P14 and P18 P23H-3 and age-matched SD rat retinae. The results revealed significant dysregulation in the expression levels of several genes associated with autophagy and necrosis. The rd1 (Pde6brd1/rd1) mouse is a well-characterised animal model of human autosomal recessive RP bearing a gene mutation in the β-subunit of phosphodiesterase 6 (Pde6b). As previous studies have debated the importance of apoptotic pathways in this model, the roles of two central molecules (BAX and BAK) was investigated in mitochondrial-mediated apoptosis during normal maturation of the retina and during rd1-associated photoreceptor degeneration. C567Bl6 mice lacking Bak (Bak-/- and Bak-/- Bax+/-) exhibited normal architecture and lamination of retina, with no significant differences in thickness of retinal layers compared to wild-type (WT) at P14. Analysis of Bak and Bax expression by droplet digital PCR (ddPCR) in rd1 mice at P14 and P18 revealed elevated expression of Bak, but not Bax, compared to the WT controls. Genetic removal of Bak in rd1 mice, with or without loss of one Bax allele, resulted in a significant and transient increase in ONL thickness compared to mice rd1 at P14, that was not maintained at P18. However, in double-knockout mice (rd1/Bak-/- Bax-/-) the ONL thickness was similar to the WT, despite the presence of the rd1 mutation. Moreover, a substantial thickening was detected in inner retinal layers including inner nuclear layer (INL), inner plexiform layer (IPL) and ganglion cell layer (GCL) when compared to age-matched rd1 and WT controls. The data indicate that in the rd1 mouse model, BAK and BAX function redundantly in intrinsic cell death pathways, which are a major contributor to photoreceptor death over and above normal developmental cell death in the maturing retina. To investigate the potential to pharmacologically target intrinsic cell death mechanisms, the efficacy of a BAK antagonist was examined in vivo. Intravitreal injection of a specific BAK inhibitor (WEHI-1250993) into rd1/Bax-/- mice at P16 significantly reduced photoreceptor death at P18 when compared to vehicle controls. While these results provide proof of principle that intravitreal administration of antiapoptotic agents may be effective for ameliorating photoreceptor death, further studies are needed to develop BAX antagonists and determine the long-term effectiveness of such therapeutic approaches. In conclusion, P23H mutation in RHO induced a progressive deterioration of retinal function and structure in transgenic rats. Multiple mechanisms of cell death possibly form a complicated network of molecular events to mediate degeneration of photoreceptors in P23H-3 animal model. However, the mitochondrial-mediated apoptosis is central to both naturally occurring developmental as well as rd1-associated cell death in mouse retina. Targeting these cell death pathways may slow the progression of photoreceptor death in individuals with RP.
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ItemSuppressors of oncogenic Cbl in the Drosophila eye.( 2018)Cbl is an E3 ligase, and downregulates several cellular signalling pathways, in this role by targeting receptor tyrosine kinases for endocytosis. Mammalian Cbl was first identified as the full-length isoform of v-Cbl, a C-terminal truncated dominant negative oncogene that permits binding of v-Cbl to Cbl targets but does not facilitate their ubiquitination. This results in constitutive activation of the receptor tyrosine kinase. In this thesis, I used a Drosophila analogue of v-Cbl, named Dv-cbl. GMR>Dv-cbl had been used prior to the commencement of this study to screen for modifiers of its rough and overgrown eye phenotype using the Gene search system, a transposon-based inducible expression system. In this study, a subset of the suppressors of the GMR>Dvcbl phenotype from that screen, and two other representative lines were further investigated. The published interactions and functions of the genes implicated by the GS lines are discussed and a method of suppression of the GMR>Dv-cbl phenotype by each line is suggested. In the published work presented in this thesis, the Akap200 expressing lines EP2254 and GS2208 were further studied. Expression of Akap200 in EP2254 was confirmed via mRNA in situ hybridisation, and its ability to also suppress the Ras85DV12 phenotype was confirmed. The ability of EP2254 to suppress GMR-Dv-cbl and sev-Ras85DV12 coexpression was confirmed. When GMR-Dv-cbl and sev-Ras85DV12 are coexpressed, a phenotype that is greater than the cumulative phenotype of each would suggest arises. In fact, GMR-Dvcbl (where Dv-cbl was directly driven from the GMR promoter) was used instead of GMR-Gal4, UAS-Dv-cbl (GMR>Dv-cbl) as the coexpression of GMR>Dv-cbl and sev- Ras85DV12 results in lethality, and coexpression of GMR-Dv-cbl and sev-Ras85DV12 did not. Alone, each has a mildly rough eye. I showed that EP2254 was able to suppress this phenotype and that this suppression was partially independent of apoptosis. The endogenous function of Akap200 in the Drosophila eye was then investigated. An mRNA in situ hybridisation experiment showed that endogenous Akap200 is present in the eye disc, and a series of immunohistochemical stains showed that Akap200 was expressed in a subset of photoreceptor cells. Knockdown of Akap200 using RNAi lines showed that endogenous Akap200 was having a modifying effect on the GMR>Dv-cbl phenotype, as knockdown of Akap200 enhances the GMR>Dv-cbl phenotype. A recent study suggests that Notch is protected from internalisation by Cbl by Akap200, which is consistent with the results in this thesis.
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ItemTau and beta-amyloid deposition, structural integrity, and cognitive function following traumatic brain injury in Australian war veterans( 2018)Background: Traumatic brain injury (TBI), has been diagnosed in over 355,000 US military service personnel since 2000. Epidemiological research indicates that veterans with TBI are two-to-four times more likely to develop dementia than controls; however, mechanisms contributing to this relationship are poorly understood. The aim of this study was to investigate if Vietnam war veterans with a TBI show evidence of Alzheimer’s disease (AD) pathological markers, as assessed by A-amyloid, tau and glucose metabolism using PET, as well as structural MRI, including diffusion tensor imaging, and neuropsychological testing. Methods: Sixty-nine male veterans - 40 with TBI (aged 68.0±2.5 years) and 29 controls (aged 70.1±5.3 years) - underwent A-amyloid (18F-Florbetaben), tau (18F-AV1451) and 18F-FDG PET, MRI, psychiatric and neuropsychological assessment. The TBI cohort included 15 participants with mild, 16 with moderate, and 9 with severe injury. Fractional Anisotropy (FA) was employed as a measure of white matter tissue integrity. PET Standardized Uptake Value Ratios (SUVR) were calculated using the cerebellar cortex as reference region. Analyses were adjusted for IQ, age, ApoE status and psychiatric comorbidities. Results: Veterans with moderate-to-severe TBI performed significantly worse than controls on composite measures of memory and learning (M = -0.55 0.69, t(67) = 2.86, p=0.006, d=0.70) and attention and processing speed (M = -0.71 1.08, t(52) = 2.53, p=0.014, d=0.69). The moderate-to-severe TBI group had significantly lower FA than controls in the genu (F(3,36)=8.81, p<0.05, partial 2 = 0.17), and body (F(3,36)=4.39, p <0.05, partial 2=0.14) of the corpus callosum, as well as in global white matter (F(3,36)=5.35, p <0.05, partial 2=0.13). There were no significant differences in 18F-Florbetaben or 18F-AV1451 uptake amongst the groups, however the moderate-to-severe TBI group had significantly lower 18F-FDG retention than controls in the mesial temporal region (F(8,44) = 2.21, p <0.05, partial 2 = 0.13). No differences were found between the mTBI group and controls on any of the outcome measures. Conclusions: These findings indicate that moderate-to-severe TBI, but not mTBI, is associated with later-life cognitive deficits, and diminished global white matter integrity, specifically in the corpus callosum. However, these deficits are not associated with AD pathology. These results are consistent with current evidence of white matter axonal damage as the primary source of cognitive impairment in TBI, and are not reflective of a neurodegenerative process.
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ItemAn investigation of intentionally modified crania in Georgia and Europe in the Migration Period (4th – 7th c AD)( 2018)This aim of this research is to examine the custom of intentional cranial modification in the Mtskheta region of Georgia during the Migration Period (4th ‒ 7th c AD) by comparing it with that in Europe, particularly investigating the role of the Huns in intensifying the practice during this time. Thirty-two intentionally modified adult crania from Georgia were compared with thirty-one from Hungary, which included eleven juveniles, thirteen adult crania from Germany, two from the Czech Republic, one from Austria and one from Crimea. The control group comprised nineteen non-modified adult crania from worldwide collections in the department of Anatomy at the University of Melbourne. Geometric morphometric techniques were used to provide a quantitative comparison of the modified cranial shape. There were four main areas of study commencing in Georgia, moving to Hungary and then to other European sites. 1. The analysis of modified crania in Mtskheta, Georgia revealed a proliferation of the practice corresponding to the European Migration Period. The predominance of females and absence of juveniles indicated that cranial modification was not an indigenous practice but occurred due to migration. 2. To investigate the presence or influence of the Huns in the practice of cranial modification in Georgia, eigenshape analysis was used to compare modified cranial shape outlines in Georgia with Hungary, the centre of Hunnic administration and cultural influence, with modern non-modified crania for comparison. Firstly, this method could distinguish between modified and non-modified crania and secondly found that the shape of the Georgian modified crania differed significantly from those in Hungary. The Georgian crania had considerable variation in shape whereas those from Hungary revealed great homogeneity. These findings suggested that Huns were not directly involved in the practice of cranial modification in Georgia. 3. Juvenile modified crania were common in Hungary, which confirmed that the practice was indigenous in this region and provided the impetus for a three-dimensional analysis to compare juvenile and adult modified cranial surface in Georgia and Hungary. This demonstrated that the Hungarian crania had been modified by two bindings distinguishing them from the Georgian crania which were modified with single or double bindings and thus showed greater variation. This confirmed the hypothesis that the Huns used this specific technique of cranial modification to differentiate individuals within their area of dominance. 4. Three-dimensional surface analysis of modified crania was extended to Crimea, Czech Republic, Austria and Germany. Principal component and discriminant function analyses indicated that the modified crania from these sites did not strictly conform to the Hungarian double binding form, but showed increased variation as in Georgia, indicating that there were multiple conflicting social influences in pursuing the practice of intentional cranial modification among nomadic groups in the Migration Period of Europe. Quantitative shape analysis of three dimensional images revealed regional differences in cranial morphology and demonstrated the importance of the practice in promoting social identity. This provided an understanding of the cultural interactions taking place during this period of intense social change in Europe.
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ItemTackling bone pain at the source: identifying and exploring new therapeutic targets( 2018)Pain associated with bone pathology puts a significant burden (both in terms of quality of life and cost) on individuals, society, and the health care system in Australia and worldwide. Current strategies for management are either ineffective or have serious side effect profiles that prohibit use for long-term treatment of bone pain. A major impediment to the development of more targeted strategies is the lack of detailed knowledge of the mechanisms that drive bone pain. To address this, I have developed an exciting, novel in vivo bone-nerve preparation that has been extensively exploited in this thesis to monitor the electrophysiological activity of single sensory neurons that innervate the marrow cavity of the tibial bone of the rat. I provide the first evidence that single sensory neurons that innervate the bone marrow respond to high threshold mechanical stimulation, have response properties consistent with a role in nociception and provide information about different features of an intra-osseous pressure stimulus. These findings show how some bone marrow afferent neurons signal pain in bone diseases and pathologies that involve a mechanical disturbance and/or increased intra-osseous pressure. I have also used this novel in vivo bone-nerve preparation to report roles for a number of growth factors in the pathogenesis of bone pain. In particular, I demonstrate that nerve growth factor (NGF) injected directly into the tibia rapidly activates and sensitizes bone marrow afferent neurons. NGF-induced changes in the activity and sensitivity of bone marrow afferent neurons are dependent on signalling through the TrkA receptor, but are not affected by mast cell stabilization. This provides the conceptual framework for understanding NGF sequestration therapy used to treat inflammatory bone pain. I also report that, in addition to NGF, the Glial cell line-derived neurotrophic factor family ligands (GFLs) (artemin, GDNF, and neurturin), also rapidly activate and sensitize bone afferent neurons, albeit with different time courses. This is exciting because it suggests that some of these GFL signalling pathways could be targeted for the treatment of bone pain. I have further developed and report a new model of carrageenan-induced inflammation of bone to provide evidence that sequestration of artemin reduces inflammation-induced activation and sensitization of bone marrow nociceptors. This provides proof-of-principle that sequestering artemin could be beneficial for the treatment of inflammatory bone pain. Together these results contribute significantly to our understanding of the mechanisms that drive bone pain and have opened the door to opportunities to explore the role of a number of signalling molecules that could be targeted for therapeutic benefit to reduce the significant burden of bone pain.
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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.