Anatomy and Neuroscience - Theses

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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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    Ribosomal protein depletion in the Drosophila haematopoietic compartment alters cell fate determination
    Chahal, Arjun Singh ( 2020)
    Ribosomes are essential components of the translational machinery, required for cells to effectively double their protein content in order to undergo cell division. Despite the obvious need for ribosomes, due to the high energetic cost, ribosome biogenesis is tightly regulated by numerous pathways in response to cellular and extracellular signalling cues. Predictably, dysregulation of ribosome levels strongly correlates with disease. Increased ribosome biogenesis is observed in many cancers and decreased ribosome biogenesis underlies a class of developmental disorders collectively termed ribosomopathies. Somewhat surprisingly, given the ubiquitous need for ribosomes, loss of ribosomal proteins has also been associated with tissue overgrowth in both human disease and model organisms, including zebrafish and Drosophila. In the latter, reported mechanisms for overgrowth due to global reduction of ribosomal proteins comprise extrinsic effects, whereby a smaller hormone-secreting gland delays animals in their growth phase to result in larger wings and eyes. Many ribosomopathies are associated with lineage-specific defects, with ribosomal protein loss linked with haematopoietic compartment pathologies, particularly lineage depletion and anaemia. Although lineage depletion phenotypes are well established to arise from apoptosis induced by nucleolar stress, there is also an increased incidence of cancer associated with these ribosomopathies for which mechanisms remain unclear. This thesis has taken advantage of the powerful genetic tools available in Drosophila to achieve tissue-specific depletion, in the larval hematopoietic compartment (the lymph gland), of the two ribosomal proteins most commonly lost in the ribosomopathy Diamond Blackfan Anaemia (DBA), S19a (RpS19a) or S24 (RpS24). In contrast to the haematopoietic lineage depletion observed in human disease, we report cell-intrinsic overproliferation and tissue overgrowth following depletion of either RP. However, despite gross phenotypic similarities, depletion of RpS19a and RpS24 resulted in distinct cell death and differentiation defects. Moreover, while RpS24 resulted in the expected decrease in mature ribosomes, RpS19a depletion resulted in an inexplicable increase in ribosomes. Transcriptome and proteome analyses of RP-depleted lymph glands revealed upregulation of metabolic and signalling pathways; however, factors involved in transcription and translation were disproportionately increased at the protein level, consistent with altered translation. Although certain proteins were elevated after knockdown of either RP, some were specifically increased by RpS19a or RpS24 depletion. Of the 8 candidates tested for capacity of co-depletion to suppress overgrowth defects only 5 (STAT, TOP1, Osa, TCTP and Cdk12) suppressed the RpS24 lymph gland phenotype, suggesting the requirement for increased abundance of these factors in overgrowth. In the case of RpS19a, although depletion of dHEATR1 and TCTP supressed overgrowth, only depletion of Osa, a key component of the SWI/SNF chromatin remodelling complex, suppressed overgrowth through a restoration of progenitors. The increased Osa observed in RpS19a and RpS24 knockdown is, therefore, required for progenitor depletion and lymph gland overgrowth. Further studies are required to determine whether differential translation of Osa, and the other candidates, contributes to lymph gland overgrowth due to depletion of RpS19a or RpS24.
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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.