Anatomy and Neuroscience - Theses

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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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    Characterisation of a novel liver progenitor cell marker in biliary atresia
    Leal, Marcelo Cerf ( 2022-06)
    The characterisation of liver progenitor cells (LPC) has proven to be difficult, likely due to plasticity in the regenerative/reparative processes of the liver based on the type and extent of injury. The recent development of an antibody to the GCTM-5 epitope has identified a putative liver progenitor cell population which was not previously described in the paediatric population. GCTM-5 was previously identified to strongly mark cells in the foetal ductal plate, early in the developmental pathway of both hepatocytes and cholangiocytes. This study describes the distribution of this antibody in patients with biliary atresia at time of paediatric liver transplant, and compares it to other known markers for cells with progenitor-like properties. We conducted a chart review of all patients with biliary atresia undergoing a liver transplant between 1995-2015 at the Royal Children’s Hospital, Melbourne. Multiple samples that had been stored from the explanted liver of patients with biliary atresia were stained for ENPRO-1, a second-generation antibody with greater affinity for the same epitope stained by GCTM-5. We found that the ENPRO-1 antibody most strongly marked ductular reaction cells in this human population, which is a histological description known to contain the LPC niche. It also identified undifferentiated EpCAM positive cells, most SOX9-positive cells, all NCAM-positive cells and some Lgr5-positive cells. As such, ENPRO-1 was found to mark a novel subset of liver cells of previously identified cells with progenitor properties. This is critical to validate future studies for a serum biomarker for activation of this LPC niche, as the epitope marked by the ENPRO-1 antibody is known to be secreted into blood. Unexpectedly, we found CD133/PROM1 to strongly mark almost all parenchymal hepatocytes in regenerative nodules, which has not previously been described, and may be unique to patients with end-stage liver failure due to biliary atresia.
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    Commitment to Serotonergic Signalling: Evolution and Distribution of the Serotonin Receptors
    Fernando, Sabrina ( 2022)
    Serotonin is a subclass of neurotransmitters and a relatively simple metabolite. The serotonin system underpins a multitude of biological functions by coupling with diverse serotonergic signalling. The effectors of serotonergic signalling are the serotonin receptors: a family of GPCRs (G protein-coupled receptors). These receptors are associated with diverse biological functions and are the primary sites of action for many psychotherapeutic drugs. We describe what commitment to serotonergic signalling entails by quantifying the diversity of this family in terms of receptor sequences, and expression patterns in the brain and systemic tissues. Sampling receptor sequences from the HTR (serotonin receptors) family, we constructed an updated phylogeny. We interpolated Bayesian priors to include key evolutionary events and to date the evolution of the family. Using RNA sequencing data, we provide the first systematic evaluation of the HTR distribution in the human body and organised the family based on their shared expression patterns. The phylogeny recapitulated early radiation of HTRs predating vertebrate evolution and demonstrated the three present clades of Galphas-coupled HTRs lack a singular ancestral node. The RNA-sequence analysis identified the systemic as well as brain-specific receptors, and reproducibly detected ten family members in the brain, which could be sub-classified by their co-expression patterns in cortical and subcortical regions. Together, the phylogenetic tree and the transcriptome map underscore the diversity of the HTR family, with multiple members evolved to activate all types of Galpha pathways specifically in the brain as well as systemically. Thus, through applying ‘omics data, the thesis outputs present the first systematic description of the family. The findings of this thesis reinforce the fact that multiple serotonin receptors are not evolutionary redundancies, but rather each receptor corresponds to specialised tissue distribution. An accurate understanding of the commonalities and contrasts among the subtypes would aid the development of subtype-specific drug targets.
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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 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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    The effect of genetic susceptibility and immunosuppressant treatment on mononuclear cell phenotype in multiple sclerosis
    Ayoub, Sinan ( 2019)
    Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). The immune system plays a significant role in the pathophysiology of the disease and research into the mechanisms that may be potentially contributing to the disease is essential to help advance knowledge in the field and ultimately seek better outcomes for MS patients. A key set of cells that are believed to play a role in MS immunopathology are mononuclear cells which consist of B-cells, T-cells, monocytes and their derivatives. The aims of this thesis was to assess the effects of 1) genetic susceptibility and 2) immunosuppressant treatment, on mononuclear cell phenotype in MS. The first aim involved interrogating a key co-stimulatory molecule, cluster of differentiation 40 (CD40), expressed on mononuclear cells which is known to play a fundamental role in the regulation of the humoral immune response. CD40 is a risk gene for the development of MS and I sought to assess the effects of the single nucleotide polymorphism (SNP) rs1883832 on the phenotype of CD40-expressing mononuclear cells. Utilising a range of bimolecular methods and samples taken from MS individuals and healthy matched-controls (HC), I showed that SNP rs1883832, particularly the ‘risk’ genotype (homozygous TT) was correlated with reduced soluble CD40 (sCD40) levels in HC, but not MS patients. I did not observe any phenotype-dependent differences in sCD40 levels between MS (in quiescent state) and during active demyelination compared to HC. Upon assessment of key cytokines produced by B-cell and monocytes, I observed elevated gene expression of the pro-inflammatory cytokine interleukin-1 beta (IL-1β) in the MS population linked to the CD40 risk TT-genotype. In the second component, I showed that the immunosuppressant drug Cladribine, is highly toxic to mononuclear cells, and that it down-regulates the expression of the key phagocytic marker MERTK at the protein level (preliminary data, study is ongoing). In summary, the data presented in this thesis suggests that the CD40 risk gene and the immunosuppressant Cladribine play roles in altering the phenotype in mononuclear cells. The understanding from this body of work provides some insights into how genetic susceptibility can drive changes in mononuclear cell phenotype which may impact on the modulation of co-stimulatory mechanisms in humoral immunity. Moreover, the findings from the Cladribine study may assist the field in the discovery of alternative mechanisms of the drug on mononuclear cells and therefore inform a refined approach to treatment of MS.
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    Suppressors of oncogenic Cbl in the Drosophila eye.
    Sannang, Rowena Tenri ( 2018)
    Cbl is an E3 ligase, and downregulates several cellular signalling pathways, in this role by targeting receptor tyrosine kinases for endocytosis. Mammalian Cbl was first identified as the full-length isoform of v-Cbl, a C-terminal truncated dominant negative oncogene that permits binding of v-Cbl to Cbl targets but does not facilitate their ubiquitination. This results in constitutive activation of the receptor tyrosine kinase. In this thesis, I used a Drosophila analogue of v-Cbl, named Dv-cbl. GMR>Dv-cbl had been used prior to the commencement of this study to screen for modifiers of its rough and overgrown eye phenotype using the Gene search system, a transposon-based inducible expression system. In this study, a subset of the suppressors of the GMR>Dvcbl phenotype from that screen, and two other representative lines were further investigated. The published interactions and functions of the genes implicated by the GS lines are discussed and a method of suppression of the GMR>Dv-cbl phenotype by each line is suggested. In the published work presented in this thesis, the Akap200 expressing lines EP2254 and GS2208 were further studied. Expression of Akap200 in EP2254 was confirmed via mRNA in situ hybridisation, and its ability to also suppress the Ras85DV12 phenotype was confirmed. The ability of EP2254 to suppress GMR-Dv-cbl and sev-Ras85DV12 coexpression was confirmed. When GMR-Dv-cbl and sev-Ras85DV12 are coexpressed, a phenotype that is greater than the cumulative phenotype of each would suggest arises. In fact, GMR-Dvcbl (where Dv-cbl was directly driven from the GMR promoter) was used instead of GMR-Gal4, UAS-Dv-cbl (GMR>Dv-cbl) as the coexpression of GMR>Dv-cbl and sev- Ras85DV12 results in lethality, and coexpression of GMR-Dv-cbl and sev-Ras85DV12 did not. Alone, each has a mildly rough eye. I showed that EP2254 was able to suppress this phenotype and that this suppression was partially independent of apoptosis. The endogenous function of Akap200 in the Drosophila eye was then investigated. An mRNA in situ hybridisation experiment showed that endogenous Akap200 is present in the eye disc, and a series of immunohistochemical stains showed that Akap200 was expressed in a subset of photoreceptor cells. Knockdown of Akap200 using RNAi lines showed that endogenous Akap200 was having a modifying effect on the GMR>Dv-cbl phenotype, as knockdown of Akap200 enhances the GMR>Dv-cbl phenotype. A recent study suggests that Notch is protected from internalisation by Cbl by Akap200, which is consistent with the results in this thesis.