Anatomy and Neuroscience - Theses

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    Investigating the human brain transcriptome and epitranscriptome: insights from long-read sequencing and transcriptional risk profiling
    Gleeson, Josie ( 2024-02)
    Complex regulation of gene expression is fundamental for the development and function of the human brain. The brain exhibits the highest levels of splicing activity and the RNA modification N6-methyladenosine (m6A) in human tissues. The expression of alternatively spliced RNA isoforms is critical for cellular specificity and development and the dysregulation of isoform expression and m6A modification processes is implicated in various neuropsychiatric and neurodegenerative disorders. Therefore, it is essential to profile the expression and modification landscape at the isoform level to elucidate the underlying pathology of complex neurological disorders. However, the short-read sequencing methods to study expression changes and m6A modifications only provide gene-level resolution. Long- read direct RNA sequencing (DRS) with Oxford Nanopore Technologies is a new method in the transcriptomics field that may address many of the remaining questions about the roles of RNA isoforms and gene regulation. We applied DRS in multiple studies with a focus on isoform quantification and isoform-level profiling of m6A modifications. DRS does not require any amplification or fragmentation steps and quantifies genes and isoforms while also characterising RNA modifications and polyA tail lengths of each isoform. However, DRS is an emerging technology and many previous transcriptomics methods have not yet been tested on DRS data. We performed DRS of human neuroblastoma cell lines and established the ability of DRS to detect differential isoform expression between synthetic RNA controls and human RNA populations. We developed and benchmarked NanoCount, a novel isoform quantification tool specifically designed for DRS data that we demonstrate outperforms other tools. DRS was subsequently applied to post-mortem human brain samples from three distinct regions: prefrontal cortex, caudate nucleus, and cerebellum. We found >15k differentially expressed isoforms between the brain regions, and our results revealed both isoform- and brain-region- specific patterning of m6A modifications and polyA tail lengths. The prefrontal cortex exhibited a distinctive profile of specifically modified isoforms enriched in excitatory neuron cell types and had the highest proportion of previously unannotated m6A sites. A population of isoforms were hypermodified with m6A and were associated with excitatory neuron cell types in all three brain regions. We also discovered >2k differentially modified m6A sites and 566 isoforms with differential polyA lengths between brain regions. Our studies utilising DRS demonstrate its applicability for investigating multiple features of RNA isoforms in the brain and provide new insights into brain region specificity and functioning with implications for neurological development and disease.
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    Optimizing Stem Cell Models of the Human Gut-immune Barriers
    Piryaei, Masoumeh ( 2024-01)
    Summary The adult human gut has an area of about 200 to 400 square meters. This massive surface is covered by a layer of epithelial cells that creates a physical barrier to the environment, whilst absorbing nutrients from food. Appropriate regulation of host immune responses to luminal contents helps maintain both tissue and microbial homeostasis in the gut tract. Impaired inflammatory regulation of the immune-epithelial interface is associated with chronic diseases such as inflammatory bowel disease (IBD). Together with the gut epithelium, intestinal macrophages produce immune regulators that control recruitment and activation of adaptive immune processes, including regulatory T cells in the intestine. We know that macrophages are recruited to the developing gut from the earliest stages of organogenesis, however, the role of these cells in healthy gut development has not been widely studied. Most of our knowledge about gut macrophages comes from studying animal models, especially rodents. This project forms part of a larger program of research aimed at understanding the role of macrophages at epithelial barriers associated with skin, gut, and mucosal surfaces. We will use induced pluripotent stem cells to derive macrophages and test optimal co-culture conditions that mimic the immune-epithelial niche. We hypothesize that introduction of macrophages to a gut epithelial model will improve the development and integrity of the in vitro gut epithelium. The hypothesis will be tested using established models of gut epithelium and introducing iPSC-macrophages to assess epithelial barrier integrity, inflammatory reactivity to injury, and immune tolerance of commensal gut bacteria.
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    Using induced pluripotent stem cell-derived retinal pigment epithelium cells to characterise phenotypic differences associated with reticular pseudodrusen in age-related macular degeneration
    Hall, Jenna ( 2024-02)
    Age-Related Macular Degeneration (AMD) is one of the leading causes of severe vision loss in individuals aged over fifty in Western populations. A hallmark of AMD pathogenesis is the accumulation of lipid and protein deposits, termed drusen, in the macula. Past studies have established that retinal pigment epithelium (RPE) dysfunction alone initiates drusen-like deposit formation, with disease lines exhibiting greater volume and number with respect to deposit formation in vitro. Despite this, automated quantification methods for these deposits were lacking in the literature. While conventional drusen on the basal side of the RPE is a hallmark of AMD pathogenesis, the recognition of reticular pseudodrusen (RPD) on the apical side indicates a distinct AMD phenotype. This thesis focuses on the modelling of RPD using human induced pluripotent stem cells (hiPSCs) as part of this extensive investigation into molecular distinctions between conventional AMD and RPD. Initial identity confirmation of hiPSC-derived RPE cells involved rigorous characterisation and functional assays to establish baseline phenotypic presentations. Subsequent investigations delved into pathological AMD signatures, initially confirming the appearance of hallmark drusen deposits in culture. Recognising the time-intensive and manual nature of existing quantification methods, significant efforts were made to develop and publish two semi-automated quantification tools, fostering standardised approaches in the scientific community. These tools facilitated stressor experiments with N-Retinylidene-N-Retinylethanolamine (A2E), revealing its potential exacerbation of AMD phenotypes in vitro. A large-scale proteomics study aimed to identify differentially expressed proteins between AMD and RPD samples uncovered distinctions in extracellular matrix proteins, the spliceosome complex, and cellular homeostasis, including mitochondrial health. Validation of mitochondrial hits involved assays comparing baseline oxidative states. Utilising the developed pipelines for quantifying drusen-like deposits at baseline and post-stressor treatment, and investigating baseline oxidative stress in RPE cultures derived from patient-specific iPSCs revealed differences between AMD cohorts with and without RPD. This study underscores the utility of patient-specific iPSCs for in vitro modelling of AMD pathogenesis, elucidating variations in phenotypic presentations within stratified AMD disease cohorts. The data suggests discernible differences in disease profiles concerning mitochondrial dysfunction and drusen accumulation between AMD samples with or without RPD, emphasising the potential of iPSCs in unravelling the complexities of AMD.
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    Investigating gene and isoform expression quantification in human brain using nanopore single-cell long-read technologies
    Prawer, Yair David Joseph ( 2023-09)
    Alternative splicing (AS) is a fundamental mechanism that enables a single gene to generate multiple RNA products, referred to as isoforms. AS is a widespread phenomenon and is most abundant in the human brain. Nearly all multi-exon genes undergo AS giving rise to numerous isoforms that can vary in their form, function and expression profiles. The significance of RNA isoforms in the human brain is underscored by their influence over cell states and function and their regulatory role in developmental processes and disease. Despite the importance of isoforms, our comprehension of their intricate expression profiles, and their impact on development and disease has long been constrained by the limitations of short-read sequencing. However, with the advent of long-read sequencing technologies, such as Nanopore sequencing, a new frontier has emerged for studying RNA isoforms. This technology offers a more detailed examination of the isoform landscape and provides an opportunity to explore isoform structures, expression dynamics, and their role in shaping brain development and disease. Recent advances have coupled long-read sequencing with single-cell sequencing, providing an unprecedent opportunity to explore isoforms in individual cell types and along developmental trajectories. This combination of technologies provides an avenue to explore isoforms and their related role in brain development and disease at single-cell resolution. However, as is the case for any new and emerging technologies, these methods encounter several challenges often the result of error prone long reads. Using highly accurate matched short reads is one approach that has been used to combat these limitations, but this adds considerable complexity and expense in both generating and processing the data, impacting the accessibility of single-cell long-read sequencing. In this thesis I aim to combat single-cell long-read sequencing limitations and make these technologies more usable and accessible. To achieve this, I firstly co-developed and benchmarked “BLAZE”, a tool for accurate identification of single-cell barcodes without the need for matched short-read sequencing. Building on this foundation I implemented a new and novel long-read only method for single-cell sequencing and show that this method is capable of exploring the complex developmental process of neurogenesis at single-cell resolution, including identifying cell types, plotting developmental trajectories and identifying novel isoforms implicated in human brain development. Finally, I explore factors that may limit the usability and effectiveness on Nanopore single-cell tools including the impacts of RNA degradation and showcase methods to mitigate these effects. Taken together, these advancements improve the utility of Nanopore sequencing and enable a comprehensive investigation of isoforms involved in brain development and disease.
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    Novel therapies for nonalcoholic fatty liver disease
    Devereux, Camille Jane Bradley ( 2023-08)
    Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver disease worldwide. The hallmark of NAFLD is hepatic steatosis, which occurs when the delivery, storage, and de novo synthesis of lipids outweighs lipid catabolism through oxidation or secretion. Despite the common progression of NAFLD to more severe forms of liver disease, and the contribution of NAFLD to systemic metabolic dysfunction, insulin resistance and type 2 diabetes (T2D), there are currently no approved drugs for the treatment of NAFLD. The successful development of pharmacotherapies is likely to require innovative combination therapies targeting multiple pathways, or therapies targeting novel pathways that are dysregulated in the pathogenesis of NAFLD. The aim of this thesis was to gain a more comprehensive understanding of the molecular mechanisms underpinning dysregulated lipid metabolism and to test novel therapies for the treatment of NAFLD. The first study within this thesis investigated a novel combination therapy for the treatment of NAFLD. Increased fatty acid uptake and de novo lipogenesis (DNL) are hallmarks of NAFLD. In this regard, we investigated dual targeting of the rate limiting enzyme of DNL, Acetyl-CoA carboxylase (1), with inhibition of the fatty acid transporter fatty acid translocase (FAT/CD36). While we showed that ACC inhibition was effective for reducing hepatic steatosis in mice with NAFLD, CD36 inhibition unexpectedly exacerbated steatosis. Further, in mice lacking hepatic CD36, ACC inhibition was not as effective at lowering liver triglyceride content. These changes were associated with an upregulation of genes and proteins of DNL, including ACC, and decreased liver triglyceride secretion ex vivo. Taken together, the results from this study indicate that dual ACC and CD36 inhibition is not an effective treatment for NAFLD. The second study within this thesis investigated novel regulators of fatty acid oxidation in vivo. Mitochondrial fatty acid oxidation requires the effective transfer of fatty acids between lipid droplets and mitochondria, yet the mechanisms which allow for this transfer are not well characterised. Recent research from our lab showed that the proteins extended synaptotagmin 1 and -2 (E-Syt1, E-Syt2) transfer fatty acids at lipid droplet and mitochondria contact sites and regulate fatty acid oxidation in cells. As such, the second aim of this thesis was to investigate the role of these proteins in lipid metabolism in vivo. We showed that short term inhibition of E-Syt1 and E-Syt2 in mice reduced hepatocyte fatty acid oxidation, and that short- term overexpression of E-Syt1 and E-Syt2 reduced hepatic triglyceride content in mice. In Drosophila, we showed that inhibition of the E-Syt2 remodelled lipid droplet storage and reduced survival in response to starvation, a setting where there is an increased reliance on fatty acid oxidation for energy production. Given these findings, we investigated the effects of long-term E-Syt1 and E-Syt2 inhibition and overexpression on hepatic lipid metabolism in mice. In mice lacking both hepatic E-Syt1 and E-Syt2, although not in singular knockout mice, hepatic steatosis was significantly increased. This was associated with reduced hepatic triglyceride secretion. In mice with long- term hepatic overexpression of E-Syt1 and E-Syt2, there was no effect on liver lipid storage or lipid metabolism. The work in this thesis extends our understanding of the molecular pathways involved in dysregulated lipid metabolism in NAFLD, and the interrelationship between these pathways. These studies support the notion that combination therapies will be required to effectively treat NAFLD in the future.
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    The effect of the ablation of the SEZ6 protein family on microglial structure and function in the central nervous system
    Schomann, Anja ( 2023-09)
    Microglia are the resident immune cells of the brain and are responsible for mediating the central nervous system (CNS) response to infection, injury and disease. More recently, roles for microglia in the developing brain have become evident, including maintaining tissue homeostasis, vascular control, and synaptic pruning, a normal developmental process during which synaptic connections are refined. Microglia have been shown to remove weaker synaptic connections through the involvement of the complement system, particularly the complement proteins C3 and C1q which label the synapse for microglial mediated removal. Reflecting their important role in this process, microglia are the only cells of the CNS that express complement receptor (CR) 3 and lack of C1q, C3 or CR3 results in reduced synaptic pruning during development. The Seizure-related protein 6 (SEZ6) family contain similar structural domains to those of complement regulatory proteins, namely Complement Control Protein (CCP) and CUB domains and SEZ6 proteins were recently shown to protect expressing cells against complement deposition. In genetic knockout models [SEZ6 knockout (KO) or SEZ6 triple knockout (TKO) mice lacking all three SEZ6 family members], roles for SEZ6 proteins in the development and maintenance of excitatory synapses have been identified. Preliminary data from our laboratory indicate that lack of SEZ6 proteins leads to reductions in microglial biomarker proteins in synaptosome preparations from mouse brain, suggesting that SEZ6 proteins are involved in regulating microglial-mediated synapse remodelling. This thesis will explore the role of SEZ6 proteins in regulating microglial structure and function in the brain. The aims of this work were to validate the preliminary synaptosome data and determine whether loss of SEZ6 proteins results in altered microglial expression of key proteins, including the cluster of differentiation (CD) 11B protein which, together with CD18, comprises CR3. Western analysis of CD11B protein in synaptosome preparations isolated from SEZ6 TKO showed reduced CD11B levels compared to control mice, supporting our preliminary data. Interestingly, this reduction was observed in female SEZ6 TKO mice only, with no change in synaptosome CD11B levels observed in males. In order to determine whether the decrease in synaptosome CD11B was due to a decrease in total expression in microglia, CD11B protein expression was also quantified in fluorescence-activated cell sorting (FACS)-isolated microglia. No change in total CD11B protein expression was found in either male or female SEZ6 TKO mice. However, when surface expression of CD11B and F11R were quantified via flow cytometry, a reduction in the surface expression of CD11B was found in both sexes and surface F11R expression was reduced in female SEZ6 TKO mice. Work in this chapter showed that loss of SEZ6 reduced the presence of the complement receptor CR3 subunit CD11B in synaptosomes of female mice and this may be due to altered surface expression of this receptor on microglia, supporting a role for SEZ6 protein in modulating the interaction between microglia and synapses. To further investigate the interaction of microglia and synapses in the absence of SEZ6 proteins, morphology of microglia was analysed as morphological changes in microglia are indicative of an altered state and associated with the microglial response to changes in their environment. In addition, microglia density was analysed as it was hypothesized that a lower density of microglia might lead to synapses being less frequently contacted. Quantification of the microglial density and morphology revealed strong regional differences, particularly in the cerebellum compared to the rest of the brain. While the density of microglia remained unchanged in the absence of SEZ6 proteins, microglia were found to be hyper-ramified in the hippocampus and striatum of SEZ6 TKO mice. This finding further supports an altered state of microglia and changed interaction with synapses. Lastly, the effect of the lack of SEZ6 proteins on synaptic pruning was analysed to investigate whether lack of SEZ6 proteins affects microglial function. Anterograde tracers [cholera toxin subunit beta (CTB) linked to different fluorophores] were injected separately into the left and right eye of P9 mice and the overlap of retinal ganglion cell (RGC) terminal fields was analysed in the P11 dorsal lateral geniculate nucleus (dLGN). No significant difference in the RGC terminal overlap was observed between SEZ6 TKO and control mice but pruning effects could not be excluded due to the incomplete labelling of the dLGN, even after troubleshooting this challenging intravitreal injection procedure. Together, the reduction of CR3 subunits in synaptosomes and on the surface of cortical microglia as well as the hyper-ramification of microglial processes in the striatum and hippocampus indicates that microglia have an altered interaction with synapses in the absence of SEZ6 proteins. While a direct functional effect on developmental synaptic pruning in SEZ6 TKO mice could not be shown, these results indicate novel roles for SEZ6 proteins as regulators of microglial function.
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    Exploring concepts of cardiomyocyte 'stiffness' in cardiac diastolic pathology
    Janssens, Johannes Vasilios ( 2023-12)
    Background Diastolic dysfunction is a common and early feature of cardiometabolic diseases. In patients with cardiometabolic disease it is a predictor of clinical outcomes. It typically progresses silently over a period of years in the worst case scenario resulting in heart failure. Despite this, there are no in vitro diagnostics or treatments that directly target diastolic dysfunction in this population. This may reflect absence of understanding about the combination of cellular and molecular characteristics that manifest diastolic dysfunction. At the intact heart level, diastolic dysfunction can be characterized as an impaired ability to relax and increased left ventricular stiffness. Consistent functional and molecular features of cardiomyocytes which may contribute to diastolic dysfunction in cardiometabolic disease settings remain unresolved. At the cardiomyocyte level, emerging evidence suggests that intrinsic stiffness of cardiomyocytes may be an important contributor to diastolic dysfunction while at the molecular level a sugar replete heart is a characteristic feature in cardiometabolic disease. The ability for excess sugar availability to predispose to non-enzymatic and irreversible modification (glycation) of the structure and function of the myofilament proteins responsible for cardiomyocyte stiffness and relaxation remains unexplored. Importantly, these cellular and molecular characteristics may form the basis of novel diagnostic or treatment strategies for early detection of diastolic dysfunction. The aim of this Thesis was to explore and define concepts and characteristics of cardiomyocyte stiffness and the molecular features thereof, especially as it relates to cardiometabolic disease. Research questions Question 1: Does intrinsic cardiomyocyte stiffness measured in vitro contribute to in vivo diastolic dysfunction in diet-induced cardiometabolic disease? (Chapter 2) Question 2: Are human purified troponin complex and troponins enriched from rodent myocardium modified by advanced glycation end products endogenously and are these modification sites in domains of functional relevance as described in the literature? (Chapter 3) Question 3: Can a liquid chromatography coupled MS/MS approach be developed to quantify the extent of amino acid site specific carboxymethyllysine modification on cardiac troponin I. (Chapter 4) Methods Cardiometabolic disease was induced in rodents via dietary (High fat/sugar; prediabetes or Type 2 diabetes) or pharmacological (streptozotocin; Type 1 diabetes) interventions. Echocardiography was used to assess in vivo heart function in cardiometabolic disease rodent models. Cardiomyocytes were isolated from high fat/sugar fed mice and respective controls by collagenase dissociation. Glass fibers were attached (MyoTak) at cardiomyocyte longitudinal ends, and paced cardiomyocytes (2, 4Hz, 2.0mM Calcium, 37C) were subjected to progressive stretch protocol. Force development, sarcomere length, and intracellular calcium transients (Fura2AM, 5uM) were simultaneously measured (Myostretcher, IonOptix). Liquid chromatography coupled MS/MS was used to map the occurrence of Advanced Glycation End product modifications (CML, carboxymethyllysine; CEL, carboxyethyllysine; MGH1, Methylglyoxal derived hydroimidazolone) across the human troponin complex. Western blot was used to quantify total CML amounts on myocardial proteins of interest in Type 2 diabetic human and Type 1 diabetic rat myocardial samples. A novel immuno enrichment, on-bead AspN digestion, and inclusion list assisted LC MS/MS acquisition approach was developed to quantify site specific abundance of CML modifications on myocardial proteins of interest. Results Answer 1: Intrinsic cardiomyocyte stiffness is a contributing characteristic of diastolic dysfunction in diet induced cardiometabolic disease. This shows that load and stretch are important factors to consider in assessing diastolic function of cardiomyocytes. (Chapter 2) Answer 2: The human troponin complex is modified by advanced glycation end-products at 18 lysine and arginine sites. Additionally, the advanced glycation end product carboxymethyllysine was detected at 3 sites in streptozotocin-induced Type 1 Diabetic rats but not control counterparts. These modifications were detected in regions of protein protein interaction, adjacent to phosphorylation sites and in key regions of conformational change. (Chapter 3) Answer 3: A new approach for quantifying carboxymethyllysine modified protein was demonstrated. The approach employed a combination CML-modified reference library paired with specific protein immuno enrichment and yielded a set of 4 criteria to use to validate peak selection for quantification. (Chapter 4) Conclusion This Thesis reveals novel pathologic features of cardiomyocyte function in cardiometabolic disease and links them with in vivo diastolic dysfunction. New molecular mechanisms relating to disease specific post translational modification which may underly cardiomyocyte diastolic dysfunction at the contractile myofilament were discovered. Finally, novel quantitative methodology was developed to advance the translational application of these myofilament modifications as potential diagnostic markers for the early detection of diastolic dysfunction in cardiometabolic disease. Specific information pertaining to Chapter 4 of the Thesis has been omitted from this publicly published abstract for the purpose of intellectual property protection.
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    The role of neurotrophic factors in osteoarthritis pain
    Nazemian, Vida ( 2023-10)
    Introduction: Osteoarthritis (OA) is a progressive disease of synovial joints and subchondral bone characterized by swelling, stiffness and pain. Brain-derived neurotrophic factor (BDNF) and artemin (ARTN) are neurotrophic factors that are important regulators of pain, and have recently been implicated in the pathogenesis of OA pain. This study aimed to explore roles for BDNF and ARTN in OA pain, by investigating whether the expression of BDNF and ARTN, and their receptors (TrkB and GFRa3), is altered in different tissues at different stages of OA, and whether blocking their signalling during late-stage OA can alleviate pain. Methods: The monoiodoacetate (MIA)-induced OA of the rat knee joint was used to explore roles for BDNF and ARTN signalling in OA pain. Pain behaviour was assessed using the dynamic weight-bearing apparatus to assay OA-induced changes in hindlimb weight bearing behaviour, at different stages of disease (early vs late). Histopathological alterations in the knee joint and surrounding bones were assayed using Haematoxylin and Eosin staining and scored using a modified OARSI scale. Changes in expression of BDNF/TrkB and ARTN/GFRa3 were explored using Western blot analysis of lysates from different tissues (joint, bone, and DRG), and at different timepoints of the disease (early vs late). The dynamic weight-bearing assay was used to determine if inhibiting BDNF signalling (with a peptide mimetic TrkB inhibitor) or ARTN signalling (with a sequestering antibody) could relieve pain at late-stage disease. Results: The results of this thesis highlight differential histopathological changes occurring in the early and late stages of OA, with joint involvement being prominent in early OA, and bone and cartilage involvement in late OA. BDNF expression was increased in the joint in early OA and in the bone in late OA. ARTN expression was also increased in the joint in both early and late OA and in the bone in late OA. Attempts to alleviate pain in MIA-injected animals by targeting the BDNF/TrkB and ARTN/GFRa3 signalling pathways did not yield pain relief outcomes with the therapeutic approach chosen in this study. Conclusion: Our findings suggest that altered pain behaviour in early MIA-induced OA is associated with changes in the joint not surrounding bones, while altered pain behaviour in late MIA-induced OA are attributable to the surrounding bones. Furthermore, BDNF and ARTN may contribute differentially to pain in early and late stages of MIA-induced OA through actions in joint versus bone. These findings support further investigations into the role of BDNF and ARTN signalling in OA pain and the development of novel targeted therapeutic approaches for managing OA pain.
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    Targeting lipid metabolism for prostate cancer therapy.
    Fidelito, Gio ( 2023-10)
    Deregulating cellular metabolism as a hallmark of cancer involves adaptation in nutrient acquisition, preferential utilisation of substrates, and transcriptional changes that alter intracellular metabolic signalling pathways. Understanding aspects of metabolism that are essential for cancer cells to sustain their uncontrolled proliferation can help to identify metabolic vulnerabilities, which can be therapeutically harnessed to limit cancer growth. Although prior studies in cells and mice have demonstrated the importance of oxidative metabolism and lipogenesis in prostate cancer growth and progression, however, the metabolic landscape of human prostate cancer remains unclear. Accordingly, this thesis aimed to elucidate the metabolic landscape of human prostate cancer and to identify targetable metabolic dependency for the development of novel therapeutic strategies for patients with prostate cancer. To define the metabolic landscape of human prostate cancer, we assessed substrate metabolism using radiolabelled (14C) and stable (13C) isotope tracing in precision-cut slices of patient-derived xenografts (PDXs). Glucose, glutamine, and fatty acid oxidation was variably upregulated in malignant compared to benign PDXs. De novo lipogenesis (DNL) and storage of free fatty acids into phospholipids and triacylglycerols were increased in malignant PDXs. There was no difference in substrate utilisation between localised and metastatic PDXs and hierarchical clustering revealed marked metabolic heterogeneity across all PDXs. Mechanistically, glucose utilisation was mediated by acetyl-CoA production rather than carboxylation of pyruvate, while glutamine entered the TCA cycle through transaminase reactions before being utilised via oxidative or reductive pathways. Blocking fatty acid uptake or oxidation with pharmacologic inhibitors was sufficient to reduce cell viability in PDX-derived organoids, whereas blockade of DNL, or glucose or glutamine oxidation induced variable and limited therapeutic efficacy. These findings demonstrate that human prostate cancer, irrespective of disease stage, can effectively utilise all metabolic substrates, albeit with marked heterogeneity across tumours. We also confirm that fatty acid uptake and oxidation are targetable metabolic dependencies in human prostate cancer. Considering the requirement of fatty acids in most tissues, targeting these processes may not be achievable. Therefore, we posited that identification of essential proteins of lipid metabolism, which are required for prostate cancer survival, would guide the development of novel strategies for treating prostate cancer. In Chapter 3, we developed a bespoke human lipid metabolism knockout library, performed CRISPR-Cas9-based functional genomics screening, and identified 63 commonly shared essential genes between LNCaP, C4-2B, and MR-49F. Having identified multiple genes within the mevalonate-dolichol-N-glycosylation biosynthesis to be essential, we subsequently validated the requirement of this pathway in Chapter 4. Deletion of genes encoding enzymes within this pathway resulted in reduced cell proliferation and anchorage-independent growth. This was partly mediated by G1 cell cycle arrest, induction of cell death and senescence. Further in vivo validation of genes within the dolichol-N-glycosylation biosynthesis (NUS1, DOLK, DPAGT1) in xenograft model demonstrated the requirement of NUS1 for tumour growth. Collectively, the work in this thesis highlights the heterogeneity and complexity of metabolic profiles in human prostate cancer. Furthermore, we have identified putative essential genes of lipid metabolism which may provide novel strategies for targeting lipid metabolism for patients with prostate cancer.
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    Integrated Control of Gastric Function - Contributing to the Development of The Virtual Stomach
    Di Natale, Madeleine Rose ( 2023-12)
    The stomach is the first reservoir of the gastrointestinal tract, it has been referred to as the portal to the digestive system as it is the centre which assesses and processes content before it is passed to the small intestine where the great majority of nutrient absorption occurs. Functional gastric disorders which chronically impact the motility patterns cause symptoms such as severe abdominal pain, early satiety, nausea and vomiting which can impact one’s quality of life. Current treatments for these conditions are largely ineffective and given the chronic nature of these disorders many pharmacological treatments become less effective over time. Approximately 10% of the world’s population are affected by functional gastric disorders. These disorders can be caused by changes in the extrinsic or intrinsic neuronal circuitry. Therefore, neuromodulation therapies have been seen as a promising alternative therapy. However, there are still gaps in our knowledge of the stomach anatomy and enteric integrated control which is required to effectively treat these conditions. This thesis aims to detail the anatomical structures of the stomach and map the intrinsic neuronal inputs of the stomach muscle which will fill these knowledge gaps and further our understanding of the integrated control of gastric function at the autonomic neuromuscular junction. Anatomical investigations indicate that the organisation and directions of the muscle layers in the rat are quite similar to the arrangements seen in the human stomach; however, there are some intricate features that are different to standardised textbooks. This includes the hair-pin turn of the longitudinal muscle bundles on the proximal stomach ventral and dorsal surfaces as well as the identification of the esophago-pyloric ligaments which had not been correctly identified in the literature prior to my publication (Chapter 2). The alignment between anatomical structure and function across species has also been discussed and a standardised nomenclature has been proposed in Chapter 6. The morphologies, distribution patterns and targets of the neurons were investigated and demonstrate different ganglia patterns from proximal to distal stomach and differences at the lesser and greater curvatures. These innervation differences turned out to correspond to functional innervation patterns (Chapter 3). We have demonstrated that there are multiple neural inputs to any single smooth muscle cell as increasing the stimulus voltage recruits additional inputs, recruiting axons with different thresholds which increases the junction potential amplitude that is recorded. The influence of inhibitory neurons declined with increased distance from the stimulation site. Differences in neuromuscular transmission across anatomical regions of the stomach and the influence of interstitial cells within the antrum were investigated in Chapter 5. The inhibitory neurons produced a hyperpolarising event across all three anatomical regions of the stomach. The excitatory neurons produced depolarising events in the fundus, intermittent and smaller depolarisation in the corpus, whilst depolarisation events were not recorded in the antrum. The data generated in this thesis are part of a larger collaborative initiative and will contribute to the development of a computer simulation model of the stomach which aims to realistically replicate the mechanisms and motility patterns of the stomach: This initiative is entitled The Virtual Stomach.