Anatomy and Neuroscience - Theses

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    Modelling sensory neuron plasticity in pulmonary disease with novel co-culture approaches
    Keller, Jennifer Anne ( 2022)
    The mucosa of the respiratory tree is densely innervated by sensory nerve fibres that monitor the local environment and contribute to pulmonary function and defence. Hypersensitivity of this sensory circuitry accompanies mucosal dysfunction and excessive cough in a variety of pulmonary diseases, thus contributes significantly to patient morbidity. The local mucosal mechanisms driving hypersensitivity and the accompanying phenotypic effects in sensory neurons are not well described. One hypothesis is that the epithelium-nerve functional unit is altered, perhaps mediated by disease-induced changes in the epithelium. Indeed, there exists a large body of literature demonstrating injury and reprogramming of the epithelium in many pulmonary diseases. It is proposed that this dysfunctional epithelium changes the phenotypic and/or functional properties of innervating neurons. This has not been directly confirmed, however, as it is difficult to study nerve-epithelium interactions in vivo and there are currently limited in vitro models available to directly investigate disease-specific changes to sensory neurons and their interaction with epithelial cells. In this methodological-driven project, I set out to develop novel in vitro preparations consisting of stem cell-derived or primary vagal sensory neurons and human airway epithelial cells in order to further understanding of the epithelium-nerve functional unit and facilitate the future identification of novel targets for alleviating the sensory-associated symptoms of lung disease. In the first model, human epithelial cells and murine vagal sensory neurons were utilised. The epithelial cell line BCi-NS1.1 was grown at air-liquid interface (ALI) to generate tissue that expressed important proteins and genes that define epithelial cell subtypes; secretory cells (MUC5A and MUC5B), basal cells (TUBA1A, TP63) and ciliated cells (TUBB4A, FOXJ1). Vagal ganglia were dissected from wildtype C57BL/6 mice, enzymatically dissociated and cultured, with neurons identified and characterised via their expression of pan-neuronal markers (TUBB3, ACTB) and specialised markers (P2X2, PPT-A, TRPV1 and TRPA1). Media conditions were optimised to allow ALIs to be indirectly co-cultured with these sensory neurons (shared media but no direct cellular contact). Co-cultured neurons possessed significantly longer neurites than those grown without epithelial cells (neurons only = 640 + 226.6 cm; co-culture = 916 + 350.6 cm; P<0.05) but had molecular expression profiles different to acutely isolated cells, as percentage of neurons expressing PPT-A, P2X2, TRPV1 and TRPA1 was reduced. Altered growth of neurons in co-culture conditions was indicative of the existence of epithelial paracrine mediators in the co-culture system that evoke minimal phenotypic changes in neurons. The mechanism of communication between epithelial cells and neurons was not completely dependent on growth factors since blockade of Trk tyrosine kinases did not completely attenuate the increased growth in co-culture conditions. The indirect (paracrine) co-culture model has also enabled preliminary investigation into interaction of the epithelial cells and neurons under challenge conditions, specifically looking at the viral analogue poly(I:C). This challenge was chosen because respiratory viral infections have been linked to the development and exacerbation of respiratory symptoms in a range of pulmonary diseases, and evidence has accumulated suggesting that perturbed antiviral host defence processes may contribute to disease susceptibility. Treating the ALI epithelial tissue with 12 ug/ml of poly(I:C) significantly reduced the growth of co-cultured neurons (co-culture sham (PBS in apical compartment) = 764 + 22.34 cm; co-culture PIC = 468 + 11.79 cm; P<0.05), consistent with the challenge evoking damage to the neurons. Treating neurons with the selective TRPA1 antagonist HC-030031 prevented this epithelial poly(I:C) treatment-mediated reduction in neurite growth (co-culture PIC = 468 + 11.79 cm; co-culture PIC+HC = 938 + 11.50 cm; P<0.05). The second model under development utilised human cells only. Unlike epithelial cells, human sensory neurons could not be harvested for cell culture but were derived from human embryonic stem cells (hESCs) differentiated into sensory neurons by optimising a protocol using small inhibitors and growth factors. Neural crest induction was assessed using SOX10 expression. Young protocol cultures stained positive for SOX10 at day 5 (60% of all cells), day 6 (71%), day 7 (94%), day 8 (83%), day 9 (75%) and day 10 (73%) of differenation. To determine whether the derived cells resembled sensory neurons, morphological and functional endpoints were assessed. Electrical recordings from single cells demonstrated functional properties that are consistent with immature sensory neurons (membrane potential -44.67 + 10mV, action potential threshold -36.02 + 4.88mV). Calcium recordings demonstrated transient responses to TRPV1 agonist capsaicin (21%). In addition to staining positive for TRPV1 (24%), hESC-derived cultures stained positive other important proteins and genes that define sensory neuron subtypes; MAP2 (34%), CALB (4%), PGP9.5 (46%), PRPH (32%), P2X3 (40%), P2X2 (8%), SP (0%), NKR1 (34%), CGRP (24%) and VGLUTs (23%). RNA sequencing confirmed the presence of Tac1, Ntrk2, Prph, Trpv1, Scn9a, Piezo2, and Slc17a7 in Young cultures.
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    Identification of novel biomarkers and treatments for non-alcoholic fatty liver disease and associated co-morbidities
    De Nardo, William ( 2022)
    Non-alcoholic fatty liver disease (NAFLD) is characterised by impaired lipid metabolism, hepatic fibrosis and linked to systemic insulin resistance, which is partly mediated by remodelling of liver-secreted proteins or “hepatokines.” There are no effective screening tools or approved pharmacotherapies for NAFLD and NAFLD-associated fibrosis. Proteomic studies estimate that ~25% of circulating proteins are liver-derived, suggesting that hepatokines could be biomarkers and, due to well described impacts on energy homeostasis, effective treatments for NAFLD and NAFLD-associated fibrosis. This thesis aimed to discover novel biomarkers and treatments for NAFLD, non-alcoholic steatohepatitis (NASH), and NAFLD with significant fibrosis (NAFLD F2-3). For biomarker discovery, bariatric patients were prospectively recruited, plasma and a liver wedge biopsy were procured, livers were precision-cut to assess hepatokine secretion and were grouped based on histological confirmation of NAFLD, NASH, and NAFLD F2-3. Chapter 3 identified 3333 liver-secreted proteins, of which, 107 and 5 hepatokines were remodelled with NASH and NAFLD F2-3, respectively. EMILIN1 secretion was increased in NASH, NAFLD F2-3, and in the plasma of NASH patients. However, plasma EMILIN1 poorly stratified patients NASH. In Chapter 4, we speculated that hepatokine secretion may not correspond to changes in plasma levels. Notably, highly abundant plasma proteins corresponded to ~62% of the proteins detected in the human liver-secreted proteome. Plasma proteomics showed few changes in patients with NASH and NAFLD F2-3. Proteins APOF and FCN3 stratified patients with NASH and IGKV2-28 and APCS outcompeted existing non-invasive scores to stratify patients with NAFLD F2-3. Together, this provides a novel resource characterising human liver-secreted and highly abundant plasma proteins and identified IGKV2-28, APCS, APOF, and FCN3 as biomarkers for NAFLD-associated fibrosis and NASH. In Chapters 5 and 6, we exploited hepatokine remodelling to uncover novel treatments for NAFLD and hepatic fibrosis. In Chapter 3, we detected EMILIN1, which was previously reported to reduce TGF signalling. Hepatocyte-specific overexpression of EMILIN1 with severe NASH and advanced fibrosis reduced TGF signalling and hepatic fibrosis, while overexpression of EMILIN1 mice in a mouse model of mild NASH did not impact fibrosis or metabolic co-morbidities, suggesting that EMILIN1 could be a therapeutic target for advanced, but not mild, fibrosis. Physical activity can resolve NAFLD and improve its metabolic co-morbidities, however, the effects of exercise-training on hepatokine secretion and the metabolic impact of exercise-regulated hepatokines in NAFLD remain unresolved. Chapter 6 assessed whether exercise-training could remodel hepatokine secretion to mediate improvements in NAFLD and associated co-morbidities. Mass spectrometry proteomics analysis detected 2657 intracellular and 1593 secreted proteins from isolated mouse hepatocytes. Exercise-training remodelled the hepatocyte proteome, with differences in 137 intracellular and 35 secreted proteins. Hepatocyte-secreted factors from exercise-trained mice improved insulin action in skeletal muscle and increased hepatic fatty acid oxidation. Hepatocyte-specific overexpression of SDC4 reduced hepatic steatosis, which was associated with reduced fatty acid uptake, and blunted pro-inflammatory and pro-fibrotic gene expression. Treating hepatocytes with recombinant SDC4 recapitulated these effects. Together, the work in this thesis addresses an unmet clinical need to discover novel biomarkers and therapeutics to treat NAFLD and co-morbidities.
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    Iron homeostasis in models of skeletal muscle atrophy
    Alves, Francesca Maria ( 2022)
    Skeletal muscle is one of the most active, adaptive, and resilient tissues in the human body, with an innate capacity to regenerate after injury. Dysregulation of protein synthesis and protein breakdown can lead to skeletal muscle wasting which is associated with a wide range of conditions, including genetic mutations (e.g., Duchenne muscular dystrophy), age-related wasting (sarcopenia) and different forms of injury (including ischemic reperfusion damage). Despite differences in their causality, all of these chronic and acute illnesses/injuries are linked by mechanisms that include abnormal ROS generation and inflammation. Iron, one of the most abundant trace metals, plays a crucial role in oxidative metabolism but it can also generate ROS. Due to the high energetic demand, skeletal muscle contains a significant iron load, but few studies have investigated the detrimental consequences of excess iron. To this end, my thesis research investigated the contribution of iron dysregulation to muscle atrophy. We discovered and characterised skeletal muscle iron overload using novel laser ablation-inductively coupled-mass spectrometry (LA-ICP-MS) technology in multiple models of muscle atrophy, including genetic murine models of Duchenne muscular dystrophy (mdx and dko), aged mice relative to adult controls, and in injured muscles using a model of ischemia-reperfusion injury. We subsequently investigated the therapeutic potential of reducing iron levels via iron chelation (deferiprone; DFP) and by overexpressing myoglobin, the muscle specific iron binding protein. In Chapter 3, we showed that DFP treatment (4 weeks; 100 mg/kg/day) could improve aspects of the dystrophic pathology: fibrosis and ROS generation (DHE) were reduced in diaphragm muscles of DFP treated mdx mice. However, the reduction in iron decreased the abundance of haemoproteins (myoglobin and cytochrome c) and compromised mitochondrial function (reduced citrate synthase activity). To overcome this paradox, in Chapter 4 we demonstrated that overexpression of myoglobin in dko mice maintained haemoprotein expression while decreasing fibrosis. In Chapter 5 we review the literature regarding iron chemistry in skeletal muscle and discuss the emerging field of iron homeostasis in sarcopenia. In Chapter 6 we identified an exacerbated iron overload in an aged mouse model of haemochromatosis (a common genetic disorder). The increased skeletal muscle iron was associated with decreased haemoproteins, and proteins involved in oxidative metabolism. In Chapter 7 we found chronic (12-week) DFP treatment in aged mice reduced iron and ferritin in the liver but not in skeletal muscle. We found that iron overload was associated with increased lipid peroxidation (4HNE) and ischemia-reperfusion injury exacerbated the accumulation of iron, ferritin and lipid peroxidation in muscles of aged mice compared to adult controls. However, an attempt to reduce iron pre- or post-injury in aged mice exacerbated the already impaired regeneration. The lack of efficacy of DFP prompted a shift from iron to ferritin. In Chapter 8 we conducted preliminary experiments regarding the administration of ferritin to skeletal muscle and found it impeded muscle regeneration (after ischemia-reperfusion injury), which was associated with an exacerbated inflammatory response and dysregulation of haem metabolism. In conclusion, my thesis research characterised an underlying iron dyshomeostasis (increase in iron and ferritin) in multiple models of muscle atrophy associated with chronic inflammation and elevated ROS (including DMD, sarcopenia and IRI). We identified that iron chelation/reduction in skeletal muscle is largely ineffective and propose that future studies should alter iron distribution via modulating haem synthesis, increasing myoglobin and/or ferritin breakdown/clearance.
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    Tracing Diet and Mobility of Past Human Populations in Greater Mtskheta, Georgia
    Langowski, Natalie Ann ( 2021)
    The region of Greater Mtskheta (Republic of Georgia) lies in the Southern Caucasus and presents a near continuous record of human occupation throughout the Late Bronze-Early Iron Age (LBA-EIA, 1500-500 BC), Hellenistic Period (400-1 BC) and Roman-Late Antique Period (RLA; AD 1-700). Greater Mtskheta became increasingly urbanised, densely populated and hosted an increasingly complex society during these time periods. Contemporary written sources provide little insight into the lifestyles and social organisation maintained by Mtskheta’s inhabitants; researchers rely heavily on the trace remains of Mtskheta settlements and cemeteries to reconstruct how the inhabitants lived and what resources they consumed. Archaeological investigations reveal that by the 1st century AD, the resident society was multi-cultural, socially stratified and maintained far-reaching trade networks with the Greeks, Romans, Parthians, and Sassanid Persians. Following the 4th century AD a series of cultural changes emerge in Greater Mtskheta cemeteries including a shift in burial customs, the appearance of people with intentionally modified crania and new ‘Eurasian’ styles of grave goods. These changes suggest a novel cultural influence arrived in Mtskheta at this time, which has been tentatively attributed to contact with Eurasian nomadic-pastoralists from the steppe of southern Russia. Stable Isotope analysis of archaeological human and faunal remains can provide insight into the diet composition and mobility of people from ancient times. This research examines carbon, nitrogen and strontium isotope ratios (d13C, d15N, and 87Sr/86Sr) of humans excavated from Greater Mtskheta cemeteries remains dating between 1500 BC-AD 700. Diet was compared between time periods, sites, demographic groups (age-at-death, sex) and cultural groups (burial types, modified/unmodified skulls) to illustrate how dietary access or preferences differed over time and between these groups. Further, analyses were used to examine the role migration played in the onset of cultural changes after the 4th century. d13C and d15N results show Greater Mtskheta residents consumed a mixed C3 and C4 diet in the LBA-EIA, which is consistent with a trend in similar studies of human populations in Inner Asia and the South Caucasus during this time. The human diet in Mtskheta transitioned to a C3-dominated diet by the RLA Period, a trend which was also displayed by 87Sr/86Sr populations in the Kislovodsk basin (North Caucasus). LBA-EIA diets differed between men and women, possibly indicating greater animal product consumption among males of this period; while diets became isotopically homogeneous between sites and demographic groups in the RLA period, at a time when social stratification and complexity in Mtskheta was at its peak. The 87Sr/86Sr results demonstrate some individuals with intentionally modified skulls immigrated to Mtskheta after the 4th century, and these individuals may have originated from the vicinity of the North Caucasus or Alazani Valley. This study demonstrates the Greater Mtskheta human diet changed significantly between the LBA-EIA and the RLA with increasing social complexity, and indicates the Greater Mtskheta inhabitants maintained cultural connections with the surrounding regions throughout these time periods, apparently mirroring regional isotopic trends in diet, and evidently accommodating migrants after the 4th century AD.
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    Investigating oligodendrocyte population expansion in the central nervous system
    Craig, Georgina Alice ( 2022)
    The myelin sheath is an essential component of central nervous system (CNS) health, without which neuronal function is compromised. Oligodendrocytes are the myelinating cells of the CNS and are produced throughout life through the division and differentiation of oligodendrocyte precursor cells, or OPCs. Interestingly, the processes driving oligodendrocyte production appear distinct between humans and mice. Human OPCs divide quickly and stop dividing with age. There is little oligodendrocyte addition to human white matter tracts in adulthood, and few pre-existing oligodendrocytes are replaced by new cells. By contrast, murine OPCs divide slowly, continue generating new oligodendrocytes in adulthood, and evidence suggests oligodendrocyte replacement is high in adult murine white matter. For these reasons, the relevance of using mice to study human conditions in which oligodendrocytes and myelin are compromised–such as multiple sclerosis (MS)–has recently been questioned. This presents a fundamental hurdle to performing translatable basic research into demyelinating diseases like MS. Oligodendrocyte addition is also required to facilitate new learning, in a phenomenon known as ‘adaptive myelination’. Therefore, distinct oligodendrocyte production dynamics in the human versus the mouse challenges whether studies of murine learning are of clinical relevance to the human. This has severe implications for pre-clinical animal studies which attempt to mitigate the effects of cognitive decline. In this thesis, I use novel techniques to show that both human and mouse OPCs divide quickly and stop dividing with age. I show humans and mice have similar profiles of oligodendrocyte integration over life, and that oligodendrocyte replacement is low in mice as it is in humans. I mount the argument that previous distinctions between human and mice are primarily driven by a tendency to use relatively young mice to assess ‘adult’ white matter change, in conjunction with a tendency to use density-based metrics, rather than measuring total numbers of oligodendroglia. Finally, I investigate whether oligodendrocyte production or survival alters in two established murine models of adaptive myelination: social isolation and environmental enrichment. I find that juvenile social isolation does not alter the course of oligodendrocyte production nor survival in the prefrontal cortex, but that juvenile environmental enrichment transiently increases oligodendrocyte survival. Consistent with recent studies, this suggests that environmental interventions have an exciting potential to promote more efficient modes of oligodendrocyte addition in the CNS. Importantly, this thesis realigns mouse and human oligodendrocyte growth systems to provide credence to the translatability of mouse studies. While the number of oligodendrocytes may be plastic in juvenile development, there is little ability for extensive cellular remodelling in adulthood. This brings forth important questions such as: are there limits to adaptive myelination with ageing? Are there limits to myelin repair? As the human population experiences an increase in the average lifespan, investigating such questions will become key to our ability to therapeutically target white matter repair in neurodegenerative conditions, or preserve cognitive function in age-related cognitive decline.
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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.