Anatomy and Neuroscience - Theses

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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.