Anatomy and Neuroscience - Theses

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    The role of microglia in regulation of vasculature and blood flow in the healthy and diabetic retina
    Dixon, Michael Alexander ( 2022)
    Diabetic retinopathy is a common vascular complication of diabetes and a leading cause of blindness in those of working age. Prior to overt vascular pathology, the retina displays subtle changes to neurons, glia, and blood vessels that are likely important for disease progression. However, current treatments for diabetic retinopathy are only effective at targeting late-stage pathology. Treatments that target the early cellular changes in the diabetic retina have the potential to halt disease progression before vision is threatened. One of the earliest changes observed in the diabetic retina is a reduction in blood flow. This early vascular dysfunction has been observed in the absence of any other signs of retinopathy, suggesting it may be a key early driver of disease and a promising target for intervention. It has been suggested that the underlying cause of reduced blood flow is dysfunction of the mechanisms that regulate blood flow in the retina. However, our current understanding of these mechanisms is largely incomplete. The central aim of this thesis was, therefore, to explore how blood flow is regulated in the normal retina, and to determine how this function is altered in the diabetic retina. Recent work from our group and others have identified that microglia, the resident immune cells of the central nervous system, may play a role in regulation of blood flow. Based on this emerging evidence, our hypothesis was that microglia regulate vascular function in the retina, and that hyperglycaemia leads to changes in microglia that impair this function and result in reduced blood flow. To explore this hypothesis, we first performed RNA sequencing of retinal microglia isolated from mice lacking Cx3cr1, a chemokine receptor specific to microglia and an important regulator of many microglial functions. This revealed a role for Cx3cr1 in several possible functions related to vasculature, including vascular development, microglial-vascular adhesion, and vascular tone, which were further assessed with in vitro and in vivo imaging techniques. Imaging data revealed the Cx3cr1null retina showed increased vascular density, reduced microglial-vascular contact, and most interestingly, dilation of capillaries. This loss of vascular tone may have been due to reduced expression of angiotensin converting enzyme, a component of the renin angiotensin system (RAS), which promotes vasoconstriction. The ability of microglia to dynamically alter blood vessel diameter and hence control blood flow was then assessed by live cell imaging of the ex vivo retina. We observed frequent spontaneous calcium transients in microglia which appeared to induce vasoconstriction, which may have been mediated by purinergic signalling. Microglia also evoked vasoconstriction via a calcium-independent mechanisms, which was promoted by addition of fractalkine, the ligand for Cx3cr1. Transcriptomic data suggested FKN-Cx3cr1 signalling may promote vasoconstriction via modulation of the RAS. This was confirmed by inhibition of the RAS in the ex vivo retina, which abolished FKN-evoked vasoconstriction. As earlier work from our group has shown FKN-Cx3cr1 signalling and the microglial RAS are upregulated in the diabetic retina concurrent with reduced blood flow, we postulated that this vascular dysfunction may be caused by aberrant microglia-mediated vasoregulation. To test this, we trialled pharmacological blockade of the RAS in an animal model of type 1 diabetes. Without treatment, diabetic animals exhibited constriction of retinal capillaries, reduced blood flow, and dysfunction of inner retinal neurons. Microglia did not display classical signs of activation but did show increased accumulation on capillaries. RAS blockade successfully restored capillary diameter in the diabetic retina, but surprisingly failed to improve blood flow or neuronal function. Finally, while RAS blockade did not affect the number of microglia accumulating on capillaries, it did increase the extent to which individual microglia contacted vasculature, further alluding to the importance of the microglial RAS in regulation of retinal vascular function. In summary, our findings indicate that microglia and Cx3cr1 are important for vascular function in the retina, in particular for vascular development and maintenance of capillary tone. We also established that microglia can dynamically alter blood vessel diameter in multiple ways, suggesting these cells may be important for regulating retinal blood flow. Finally, restoration of capillary diameter by RAS blockade in the diabetic retina supports the theory that aberrant microglia-mediated vasoregulation contributes to early vascular dysfunction in diabetic retinopathy. These findings may form the basis for new treatments that can prevent vascular dysfunction in diabetic retinopathy and other CNS diseases.
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    Distinguishing features and regulatory roles of 5-HT containing enteroendocrine cells
    Koo, Ada ( 2022)
    Enteroendocrine cells (EEC) have important roles in communicating the state of the gastrointestinal (GI) tract to the rest of the body, and in signalling within the GI tract. They signal in response to nutrients and metabolites in the GI tract, potentially toxic compounds and mechanical forces. The most numerous EEC signal through 5-hydroxytryptamine (5-HT; also known as serotonin). These were also the first EEC to be identified, which was through their reactions with chrome salts, from which they gained the name enterochromaffin (EC) cells. It was at first thought that all 5-HT producing EEC were much the same. In the recent years, it has become apparent that there are functionally diverse subtypes of 5-HT containing EEC. EEC make up only approximately 1% of the intestinal epithelial cells but they are responsible for producing more than 20 peptide hormones. Of the 1% EEC population, EC cells are the most abundant cell type. I have shown that 5-HT containing EEC co-express a variety of gut hormones (Chapter 2), and their co-expression patterns are defining features that characterise them into various subpopulations based on the hormones they produce. Furthermore, EEC subpopulations express a collection of receptors that respond to different stimuli which impact their physiological effects, thus adding another layer of complexity when classifying the functional subtypes of EEC. 5-HT cells are generally depicted to be open flask-shape cells in the literature. However, one study showed an intriguing characteristic of long basal processes exhibited by some EC cells, though no study had characterised the distinct morphology of 5-HT cells in detail. Therefore, I have undertaken extensive investigations to document the morphological characteristics of 5-HT cells from the mouse stomach to rectum (Chapter 3 and Appendix A). Approximately 50% of 5-HT cells in the mouse distal colon had long basal processes, and this morphology was also observed in the gastric antrum and the rectum. These processes can reach 100 micron in length, and the abundance of this structure must serve some functional roles in the intestinal mucosa. I speculate on these in Chapters 3 and 7, and in Appendix A. An unanswered question arising from the complexity of hormone co-expression is whether co-expressed hormones could be differentially released. To address this, I examined the subcellular distribution of 5-HT and tachykinin (TK) storing secretory vesicles within the same EEC (Chapter 4). 5-HT and TK are stored in separate vesicles, and the two pools of vesicles were preferentially translocated when stimulated with glucose. In addition, duodenal 5-HT/TK cells responded differently than colonic 5-HT/TK cells under the same stimulated condition, suggesting a regional difference of EEC subpopulations. Insulin-like peptide 5 (INSL5) is co-expressed with GLP-1 and PYY in colonic L cells, and I discovered that some 5-HT cells had an intertwining relationship with L cells in the mouse large intestine (Chapter 3). In Chapter 5, I describe the development of an LC/MS assay for an INSL5 analogue that I used to investigate the role of INSL5 (Appendix B). I also discovered that RXFP4, a natural receptor for INSL5, is extensively expressed by colonic 5-HT cells and by some sensory nerve fibres in the mucosa, submucosa, and the muscle layers of the large intestine (Chapter 6). Hence, INSL5 could have an effect on both RXFP4 expressing nerve terminals and on neighbouring 5-HT cells. The regulatory role of 5-HT in the control of colorectal propulsion was demonstrated to be through an INSL5/RXFP4/5-HT/5-HT3R neuro-endocrine circuit. Collectively, my studies presented in this thesis have systematically defined the distinguishing features of 5-HT containing EEC throughout the GI tract and the regulatory role of 5-HT in colonic motility.
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    Post-translational control of cell fate decisions: The role of NEDD4 in gonad development
    Windley, Simon Peter ( 2022)
    Gonadogenesis is the process wherein two morphologically distinct organs, the testis and the ovary, arise from a common precursor. In mammals, testicular fate is determined by the expression of Sry. SRY subsequently upregulates the related family member Sox9 which is responsible for initiating testis differentiation while repressing factors critical to ovarian development such as FOXL2 and beta-catenin. The neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4) is a ubiquitin protein ligase that has been implicated in a wide array of cellular processes and the development of many organ systems. In the first part of this thesis (Chapter 3) I characterised the phenotype of XY Nedd4 deficient mice, revealing that, in the absence of NEDD4, XY fetuses exhibit complete male-to-female gonadal sex reversal owing to a reduction in gonadal-precursor cell proliferation, an early failure to initiate the testis-determining program and ectopic expression of, the ovarian transcription factor, FOXL2. This sex reversal extended to germ cells with ectopic expression of SYCP3 in XY Nedd4-/- germ cells and significantly higher Sycp3 transcripts in XY and XX Nedd4 deficient mice when compared to both XY and XX controls. Utilising conditional loss of function approaches, I then explored the cell autonomous roles of NEDD4 in the developing testis (Chapter 4) and ovary (Chapter 5). In doing so I found that, while conditional ablation of Nedd4 was unable to recapitulate the sex reversal observed in XY Nedd4-deficient mice, Nr5a1-Cre;Nedd4flox/flox testes were significantly smaller than littermate controls at all postnatal stages analysed had reduced steroidogenic capacity, owing to a reduction in Lhr and Cyp17a1, resulting in a reduction in seminal vesicle weight and anogenital distance. NEDD4 was found to be largely dispensable for ovarian development, however, although Dazl was increased upon ablation of Nedd4 in XX gonadal somatic cells. In contrast, a drastic phenotype was observed in the adrenal glands of XX Nr5a1-Cre;Nedd4flox/flox mice, which displayed adrenal dysgenesis, resulting in a reduction in adrenocortical cells. In summary, the work within this thesis establishes an essential role for NEDD4 in mammalian sex determination and gonad development and describes hitherto uncharacterised roles for this ubiquitin ligase in murine development.
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    Complex spatial 3D anatomy of the tarsal tunnel and the plantar compartments of the foot
    Bruechert, Georga Kate ( 2021)
    Anatomical understanding of the foot, to a level appropriate to inform precise clinical management, is lacking. Whilst much of the foot is in need of a clear evidence-base, the tarsal tunnel and plantar compartmentalisation of the foot are areas of particular concern for two reasons. First, the anatomy of these areas is readily visualised in a lab setting, so should be well- investigated, yet it is not. Secondly, conditions that affect these areas, such as tarsal tunnel syndrome and plantar compartment syndromes, are common and can be debilitating or even life-threatening. The clinical identification and management of these disorders is varied, has high rates of failure and high rates of patient dissatisfaction from procedures that the clinical team deem successful. Something is amiss, and the confusion and inconsistency in the reviewed literature suggests that imprecise anatomical knowledge may be a prominent contributing factor. The studies presented in this thesis aimed to explore, in sequence, the tarsal tunnel, followed by the medial and lateral columns of the foot. This sequence follows the structures coursing into the plantar aspect of the foot via the tarsal tunnel. These studies aimed to present a detailed anatomical account of these areas by using clear protocols, which involved a variety of dissection, 3D modelling, sectional anatomy and medical imaging techniques. Dissection involved following individual fascicles of soft tissue structures, identifying their orientation and attachments. Each structure was modelled during dissection, as to revaluate the spatial relationships of structures in 3D space post-dissection. Sectional anatomy and medical imaging were used to correlate the dissection findings for what is more readily applied in a clinical setting. The tarsal tunnel roof was described variably in the reviewed literature. The current data demonstrated that the roof is not formed by a singular structure, but through the combination of the passive flexor retinaculum and the dynamic abductor hallucis muscle. The boundaries of the tunnel were defined by the roof, making the description of these tissues of greater importance clinically. The tunnel was also subdivided by fibrous tissues, suggesting that the neurovasculature could be compressed by various surrounding structures. This contrasts much of the reviewed literature and provides anatomical evidence for some failures of surgical release. These data may inform tarsal tunnel surgical care and could increase the effectiveness of these procedures. The plantar aspect of foot was described in the reviewed literature as having anything from zero to ten compartments. Differences in terminology, investigative medium and methodology may have contributed to the variability. The data presented here clearly and consistently demonstrated four distinct compartments of the mid- to hind-foot region: medial, lateral and two central plantar compartments. They had well-defined anatomical boundaries formed by fibrous and muscular intermuscular septae. The relationship of the bounding structures to the muscular and tendinous structures, and the course of the plantar neurovascular bundles was demonstrated digitally in three dimensions. These models may help inform the diagnosis of causative relations for nerve impaction or vascular blockage which lead to compartment syndromes, or simply increase the likelihood of rapid decline in health, such as with foot ulceration in a patient with advanced diabetes. Lower limb amputation and death are common acute sequelae of inadequate treatment of patients in such situations, so improved anatomical understanding may hasten the diagnosis. The studies presented in the form of this thesis provide a technical foundation for future anatomical studies in the foot and other regions of the body. These data provide accurate and reproducible accounts of the complex anatomy of the tarsal tunnel and plantar foot compartments and may have a positive impact on the future care of common foot disorders.
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    Benchmarking and developing human pluripotent stem cell models of macrophage biology
    Rajab, Nadia ( 2021)
    Macrophages are innate immune cells that are found resident in every tissue, and are not only important for host defense, but are also involved in tissue homeostasis, injury, and disease. The advent of human pluripotent stem cells (hPSCs) provides new opportunities to model human macrophages. However, a deeper appreciation of the functional diversity of macrophages, including contextual responsiveness, and innate memory is currently missing from stem cell differentiation studies. Immune responsiveness to pathogenic challenge is known to be impacted by a macrophage's history of prior exposure, but this has not been evaluated in the context of hPSC-derived myeloid cells.    This thesis describes the development of a transcriptomic atlas, the Myeloid Atlas, to assess impact of macrophage ontogeny, experimental treatment, and tissue residency on molecular phenotype. In doing so, we have revealed gaps in hPSC-macrophage models, revealing several differences between hPSC-macrophages, peripheral blood monocyte-derived macrophages and primary tissue resident cells. These differences include poor maturation in hPSC-macrophages in the absence of priming signals such as IFN, or repeated exposure to LPS. We demonstrate the requirement for priming in hPSC-macrophages and discover the importance of re-stimulation events in shaping macrophage activation. We further assess phenotypic heterogeneity in both peripheral blood monocyte-derived macrophages and hPSC-macrophages using single-cell RNA sequencing. We demonstrate synchronized population responses to LPS activation and further provide evidence for priming in shaping macrophage responsiveness. Overall, these findings highlight that macrophages are shaped by prior activating, or priming, signals which can be recapitulated in the laboratory using re-stimulation events that shape population phenotypes. The outcomes of this work are expected to improve routine macrophage derivation from hPSC sources, as exploitation of the requirement for in vitro macrophage priming provides future opportunities to shape the quality of acute or long-term macrophage responsiveness for diverse applications.
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    Nanosecond Laser Therapy for the Management of Age-related Macular Degeneration
    Findlay, Quan ( 2021)
    Age-related macular degeneration (AMD) is a chronic, progressive disease affecting the central retina and remains the leading cause of irreversible vision loss in developed nations. AMD is a multifactorial disease encompassing a complex interplay between ageing, environmental and genetic risk factors which ultimately lead to chronic inflammation and death of photoreceptors. While the pathophysiology of AMD is still not fully understood, recent advances have highlighted the involvement of the innate immune system and RPE dysfunction to be fundamental to the development and progression of this debilitating condition. As such, therapies that can influence these two systems have the potential to slow progression of disease. Despite advances in the treatment of neovascular AMD, there remains no effective treatment for non-neovascular AMD and importantly, interventions that address the progression from early to sight-threatening late AMD remain limited. The progression to late stages is usually slow, providing a large window of opportunity to intervene. Novel laser treatments, specifically short-duration pulsed lasers such as the subthreshold nanosecond laser (SNL), offer a potential therapeutic option. The fundamental aims of this study were to investigate SNL-induced cellular response in the RPE that may have potential benefits for addressing pathologic changes that occur in the RPE/BM interface in AMD, and to determine whether changes to the RPE are associated alterations to systemic innate immunity. SNL treatment, delivered in a single session, to the posterior eye in wildtype mice was confirmed using immunohistochemistry to selectively target the RPE without causing damage to the overlying retina. The laser-ablated RPE monolayer was healed within 3 days post-treatment with evidence indicative of newly proliferated daughter cells. Importantly, this proliferative response was also observed in the non-laser treated fellow eye. RNA sequencing and pathway analysis of laser-treated RPE identified biologically-relevant pathways including the promotion of cell survival and cell proliferation, and the inhibition of inflammation and apoptosis. Together, the fellow eye effect and RNA sequencing results suggest that a SNL-induced systemic change involving innate immunity may likely be involved. Systemic change in leukocyte phagocytosis function following SNL treatment was examined for 12-month old wildtype and in the mouse model of AMD (P2X7-null mice). Real-time flow cytometry showed a laser-induced reduction in total phagocytosis capacity of yellow-green fluorescent beads 3 months following SNL treatment. Subsequent ultrastructural studies in the P2X7-null mice showed a significant thinning of the pathologically thickened Bruch’s Membrane (a hallmark feature in AMD). The role of SNL in altering the systemic response, including phagocytosis and cytokine changes, was investigated in a cohort of patients with intermediate AMD. Similar to the rodent results, a substantial reduction in peripheral monocyte phagocytosis was also observed for all subsets of monocyte populations occurring from 3 months following SNL treatment. This reduction was sustained for at least 12 months post-treatment without any significant recovery to pre-laser levels. Cytometric bead array demonstrated significant changes to cytokine-profile with an acute elevation of 9 to 13 cytokines involved in inflammation (of a total of 13 cytokines) at the 2 weeks to 3 month post-treatment time points using 200 SNL spots. At the 6 month time point, a significant increase in interleukin-6 remains in the laser-treated participants compared to pre-laser levels. In summary, these findings indicate that SNL can induce a rejuvenating response by promoting RPE proliferation in both the treated eye and the non-treated eye while also reducing the thickness of BM in the eye that received SNL treatment. This study also provides evidence that SNL therapy has the potential to modify systemic immune responses, including phagocytosis function and cytokine-profile. Taken together, our results indicate a potential role for SNL treatment of AMD to slow progression of disease.
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    Investigating regulation of vagally-evoked respiratory responses by the parabrachial complex in the mouse
    Behrens, Robert ( 2022)
    The presence of respiratory irritants or infection can promote aberrant activity of central nervous system (CNS) neural circuits, which conduct interoceptive and homeostatic activity. Dysfunctional action in these CNS circuits contribute to morbidity in disease and decrease in quality of life. Incomplete understanding of CNS respiratory sensory relay circuits likely precludes effective medical treatment in instances of aberrant neural activity. Thus far, our understanding of respiratory relay aspects of the parabrachial complex (PBC) is unassessed. The PBC has been known to relay pain, thermoregulatory, gustatory, fluid and salt intake, and other sensory inputs to forebrain regions. Additionally, the PBC is known to receive respiratory-relevant sensory information to its pontine respiratory group (PRG) division, which in turn regulates respiratory pattern. However, the potential of the PBC in relaying vagally-derived respiratory-relevant information to forebrain regions has not been assessed. This thesis investigated physiological, neurochemical, and neuroanatomical components of the PBC in mice to assess its role in relaying vagal sensory information. To achieve this, a novel methodological approach was developed to facilitate physiological investigation of changes in respiratory rate, in response to electrical stimulation of the vagus nerve (eVNS), combined with muscimol inhibition of select PBC areas. Investigation identified region-dependent modulation of eVNS-induced activity by the PBC, with distinct roles for the medial parabrachial (MPB) and lateral parabrachial (LPB). Muscimol microinjection into the caudal LPB resulted in the complete abolishment of eVNS-induced apnoea. Inhibition of the caudal or intermediate MPB potentiated eVNS-induced tachypnoea, with inhibition of intermediate MPB regions additionally increasing the eVNS frequency required to induce apnoea. Given these physiological findings, the neurochemical phenotypes and neuroanatomical connectivity of the PBC were next investigated with immunohistochemistry and viral tracing, with an emphasis on description of the caudal PBC, a less understood subregion. The immunohistochemical findings obtained here suggest that the caudal LPB appears to be a unique entity, distinct from the intermediate and rostral LPB, due to the absence of CGRP or ChAT somal labelling in the caudal LPB which was present in the intermediate and rostral LPB. Additionally, calbindin immunolabelling identified a previously undescribed ‘axonal bundle’ principally present in caudal PBC regions. Viral tracing used here did not allow a discrete investigation of the caudal PBC and its connectivity in isolation, yet replicated projection patterns of the PBC that had previously been comprehensively assessed only in rats. Overall, this thesis has identified the caudal PBC to be a novel region of vagal sensory relay, deserving of future investigation.
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    The Clinical Anatomy of the Hallucal Sesamoids and their Attachments
    Thorpe Lowis, Casper ( 2021)
    The hallucal sesamoid bones are essential, but poorly understood, features of the first, or great, toe complex. With the associated structures of the foot, collectively termed the first ray, this complex is essential for optimal foot function. Disorders of this region present symptomatically in a broad range from intermittent discomfort through to a complete inability to walk or weight-bear. Pain in this region may also be enough to interrupt sleep and a range of daily tasks. Given the perceived importance of the hallucal sesamoids to this complex, it is perhaps surprising to discover the anatomy of the hallucal sesamoids, and particularly their interaction with neighbouring tissues, is poorly understood. One major consequence of this is the lack of a clear, consistent anatomical foundation for the diagnosis, treatment (including surgically) and management of these disorders. This thesis will document a series of studies which aim to collectively enhance the anatomical understanding of the hallucal sesamoids and their relations. After exploring the historical context of the hallucal sesamoids, reviewing the current anatomical and clinical literature, and establishing some key concepts about the presence, absence and variability of these bones, a series of detailed studies will be presented. Using a multi-modal approach that includes dissection, 3D modelling, medical imaging and sectional anatomy (macro and micro), the key tissues thought to interact with the hallucal sesamoids will be investigated: the adductor and abductor hallucis muscles, the plantar aponeurosis, the flexor hallucis brevis muscle and a range of highly specialised ligaments. The ligaments include the deep transverse metatarsal and sesamophalangeal, , amongst others, along with interactions with other tissues such as tendon tunnels, plantar plates and joint capsules. Of particular importance when considering the function of these structures is their attachment to skeletal tissues, such as the hallucal sesamoids. There is contention over what tissues are attached to each of the two hallucal sesamoids. These studies provide a detailed, quantified and uniquely visualised account of the attachments to each bone, and to neighbouring structures. This comprehensive account provides a strong basis for the development of more anatomically informed diagnostic approaches and clinical management of disorders of this area. The key findings presented in this thesis include definitive three dimensional mapping of the detailed attachments to each hallucal sesamoid. These dispel common misconceptions that the sesamoids are engulfed by a single tissue, or that they do not have substantial connections to a range of structures. These data strongly demonstrate these sesamoids have multidirectional interactions which maintain their dynamic position through the gait cycle, but are also particularly vulnerable to injury, including iatrogenic injury. In fact, poor post-operative outcomes from surgical treatments in this area may be attributable to a lack of anatomical understanding. Studies demonstrate the intimate relationships between structures often targeted for surgical release and those which must be maintained. These relations, and particularly the innovative three-dimensional visualisations developed from these data, may inform significant advances in first ray health care.
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    The adult Drosophila salivary gland: developing a new epithelial research model
    Van Ree, Caitlin ( 2021)
    Arthropod-borne viruses, also known as arboviruses, are transmitted to humans through arthropod bites. Viruses such as Dengue, West Nile, and Zika are transmitted through mosquito bites and cause serious illness in humans. These viruses are injected into a human host in the saliva of a feeding mosquito, a process that hinges on the virus invading the mosquito’s salivary glands. Therefore, a deep understanding of insect salivary glands is an important step in learning how to control arboviruses. One of the world’s most popular research organisms, Drosophila melanogaster, is a relative of the mosquito and of other insect disease vectors. Drosophila salivary glands could provide an excellent model for studying the transmission of arboviruses, unfortunately extraordinarily little is known about the glands of adult Drosophila. The aim of this research project was to develop the adult Drosophila salivary glands as a research model for studying the interactions between arboviruses and insect salivary glands. Since little is known about the glands, my investigations focused on understanding the structure, function, and maintenance of the cells within the salivary gland. To understand the structure of salivary glands, I first investigated the structure of the organ, before looking closely at individual cells. I characterised the structure of the cells by investigating localisation of cell-junctions, cytoskeletal elements, and cell-polarity markers. I also observed the establishment of these morphological features throughout different stages of development. Second, by combining the structural data with investigations into intracellular signals and membrane channels, I provided a hypothesis of the functions of salivary gland cells. Then, by analysing cell division and cell-maintenance pathways in the salivary glands, I provided an insight to how the salivary gland cell population is maintained. From this project the salivary glands emerged as a multifaceted research model that could be used to investigate arboviral diseases, epithelial tissues, and amitotic division.
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    Changes in Retinal Ganglion Cells during Disease and Aging
    Wang, Yao Mei ( 2020)
    Healthy retinal ganglion cell (RGC) function is vital for vision, and diseases that cause RGC degeneration can be debilitating. RGC degeneration features prominently during age-related diseases such as glaucoma and diabetic retinopathy. This thesis investigates how RGCs change in models of these diseases and if similar changes can be observed in normal aging. Exploring and comparing functional, structural, and molecular changes that develop in rodent RGCs during pathological states and normal aging can expand our understanding of how they degenerate during disease. Glaucoma is characterised by gradual RGC degeneration and is usually accompanied by increased intraocular pressure (IOP). We first examined the effect of an acute, non-ischemic IOP insult on RGC activity in wildtype mice. In wildtype mice, OFF-RGCs alone showed reduced spontaneous and light-elicited activity after IOP elevation. Next, we explored the role of the P2X7-receptor (P2X7-R) following IOP elevation, as it has been suggested to contribute to RGC death in glaucoma. After IOP elevation in P2X7-R knockout mice, both ON- and OFF-RGCs exhibited reduced light-elicited activity. Staining for P2X7-R in Thy1-YFP-H mice showed greater expression on ON-RGC dendrites than in other RGC cell subtypes. This study demonstrated the dysfunction of OFF-RGCs after acute, non-ischemic IOP elevation was not prevented by P2X7-R ablation. P2X7-R knockout seemed to worsen the effects of IOP elevation as it also caused ON-RGC dysfunction. In early stages of diabetic retinopathy, there is increasing evidence for RGC degeneration, prior to perturbation of other retinal neurons. Examining individual RGC function, we found that after 4 weeks of STZ-induced diabetes OFF-RGCs showed an increase in spiking activity at a single light intensity (220 photoisomerisations/sec/rod) compared to control. No changes in RGC density, synaptic protein puncta counts or Muller glia gliosis were identified. Microglia, however, showed a reduction in volume. These changes early in diabetes, though subtle, suggest dysfunction of the retinal circuitry alongside the development of inflammation. Aging can exacerbate RGC degeneration and contribute to the development of diseases such as glaucoma and diabetes. To probe the effects of age on RGCs in greater detail we used a combination of functional, structural, and molecular techniques. By examining the transcriptomes of isolated RGCs from young and aged mice using RNA-sequencing, we found upregulated genes in the pathways for oxidative stress, protein degradation and synaptic function. The upregulation of these genes may be a defensive strategy against age-related stresses during normal, healthy aging. This appears to be supported by our finding that RGCs were not as susceptible to structural or functional loss with age in comparison to photoreceptors and other cell classes. Overall, we found RGCs were dysfunctional prior to death by using early-stage disease models. The results of this thesis provide evidence that age- and disease-related stressors may invoke divergent responses in RGCs, despite aging being a risk factor for retinopathies. Stressors like increased IOP and hyperglycaemia worsen RGC function in a subtle manner; yet aging itself does not seem to pose a threat to RGC survival or function as RGCs seem more robust when compared to other retinal neurons. Future explorations could consider whether an additive effect of aging and disease may cause RGC defence mechanisms to become compromised