Browsing by Subject "Drosophila melanogaster"
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(2023)Intestinal stem cells maintain the regenerative potential of intestinal epithelium, which needs to be renewed constantly. Dysregulation of intestinal stem cell proliferation is associated with aging and intestinal diseases. The midgut of Drosophila melanogaster is a valuable model for studying intestinal stem cell driven tissue regeneration. It is similar in function to the mammalian small intestine but has a simpler cellular structure. Drosophila midgut is divided into five major regions with specialized physiological functions, characteristic morphological features and distinctive gene expression profiles. The midgut consists of a monolayer of absorptive enterocytes, small secreting enteroendocrine cells, intestinal stem cells and enteroblasts. Intestinal stem cells differentiate into enterocytes through a transient enteroblast phase. 5-hydroxytryptamine has been shown to activate proliferation of intestinal epithelium in mammals, but its mechanism of action is still unknown. Also, sex-specific differences in 5-hydroxytryptamine signalling have been recorded in mammals. 5-hydroxytryptamine signalling pathway has several downstream targets that have diverse downstream effect. Thus, 5-hydroxytryptamine signalling creates a complex and versatile regulatory network. The aim of my thesis is to study the effects of 5-hydroxytryptamine signalling on intestinal stem cell proliferation and cellular turnover in Drosophila midgut in both sexes. The effects of excessive amounts of 5-hydroxytryptamine are first studied by feeding Drosophila with 5-hydroxytryptophan, a product of the rate-limiting step in the 5-hydroxytryptamine synthesis pathway. The effects of 5-hydroxytryptamine signalling are further studied by knocking down and overexpressing a component of the 5-hydroxytryptamine signalling pathway in intestinal stem cells and enteroblasts. Dissected midguts are immunofluorescently stained, imaged and analysed both visually and with bioinformatics tools. The results indicate that 5-hydroxytryptamine signalling has both regional and sex-specific functions that affect intestinal stem cell proliferation and cellular turnover in Drosophila midgut. The most dramatic effects are seen in cellular turnover, which indicates that 5-hydroxytryptamine signalling plays a role in enteroblast differentiation. Furthermore, the results suggest that bidirectional signalling between enteroblasts and dying enterocytes facilitates cellular turnover in the midgut. As 5-hydroxytryptamine signalling is indicated in inflammatory bowel diseases such as Crohn’s disease, my results might help in the development of treatments for such conditions.
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(2019)Glutamine, the conditionally essential amino acid, is a major carbon and nitrogen carrier required for a range of cell functions, such as protein synthesis and maintaining redox balance. While healthy cells adjust their activities in response to glutamine availability, tumor cells display deregulated glutamine uptake and metabolism allowing quick proliferation and survival in cellular stress conditions. Hence, further knowledge of the glutamine sensing network is of interest. Utilizing Drosophila melanogaster, the roles of formerly identified glutamine sensing regulator candidates, Forkhead box O (FoxO), Super sex combs (Sxc), Spalt major (Salm) and Spalt-related (Salr), were explored. Drosophila is an efficient model organism for analyzing gene regulatory mechanisms, with its simple genome but conserved genes and metabolic pathways. Loss-of function and gain-of-function mutants of the candidates were cultured with/without glutamine, and their physiological response and gene expression changes were analyzed. The results show the glutamine intolerant phenotype of FoxO and Sxc deficiency, not dependent on altered food intake levels of larvae. However, glutamine intolerance of Salr and Salm deficiency was not observed. Moreover, we aimed to gain further insight to the roles of FoxO and Sxc in glutamine metabolism. Since amino acid catabolism produces ammonia, and glutamine metabolism plays a vital role in ammonia detoxification, we performed a pH-based measurement of foxo and sxc mutant larvae hemolymph on food with/without glutamine. However, we could not associate FoxO or Sxc with regulation of glutamine-derived ammonia clearance. In addition, we explored FoxO downstream regulator candidates. Putative promoter areas of Paics, Uro, Sesn, salr, Prat2 and Gdh were cloned into reporter vectors and the luciferase activity was analyzed under the expression of foxo. The results indicate that FoxO is a regulator of all of the 6 genes. Next we could utilize the here constructed plasmids to see whether the FoxO-mediated regulation is affected by altered glutamine levels in cell culture.
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(2019)Epithelial cells form a barrier between the tissue and the external environment. Epithelial morphogenesis refers to the shaping of epithelial layers and is a key step in the development of organisms. The actin cytoskeleton provides the cell its form and during epithelial morphogenesis, produces force to shape the cells. To achieve this, the actin cytoskeleton is organized into protrusive and contractile networks. In a living cell, these actin networks are dynamic, as the filaments are constantly undergoing assembly and disassembly. Actin-binding proteins regulate the turnover of actin filaments, but in epithelial morphogenesis, the regulatory role of most of these proteins is still relatively unknown. In all multicellular organisms, actin disassembly is controlled by ADF/cofilin. ADF/cofilin activity is furthermore enhanced by other actin-binding proteins, one of which is cyclase-associated protein (CAP). CAP promotes actin turnover by accelerating ADF/cofilin mediated actin disassembly and in recycling actin monomers to sites of actin polymerization. Unlike ADF/cofilin that regulates actin disassembly throughout the whole cell, CAP could be subject to more specific spatial regulation, as loss of CAP leads to F-actin accumulation on the apical side of epithelial cells. However, the role of CAP in morphogenetic cell rearrangements remains poorly known. In addition, the in vivo role of the biochemical functions of CAP has not been elucidated. The aim of this master’s thesis is to describe the role of CAP in regulating the actin cytoskeleton in the follicular epithelium of the fruit fly Drosophila melanogaster. For this purpose, chimeric mutant flies with homozygous CAP loss of function mutation were generated. Subsequently, the effect of the CAP loss of function was observed in follicle cell populations undergoing morphogenetic changes. In addition, CAP loss of function was rescued with different transgenes producing mutant CAP proteins to identify the protein domains of CAP with in vivo significance. In addition, a Drosophila CAP specific antibody was purified to be used in immunostaining. The ovaries were imaged using confocal microscopy. In this thesis, it is shown that CAP loss of function caused accumulation of filamentous actin in all observed follicular cell populations. Surprisingly, the actin turnover was rescued by all of the used CAP rescue transgenes, but the mutant transgenes exhibited phenotypes resembling the CAP loss of function in other epithelial tissues. Moreover, CAP loss of function caused defects in the follicle cell movement and cell spreading. The loss of function also caused expression changes in other actin-binding proteins. The findings of these thesis support the current knowledge of CAP importance for functional actin turnover in the follicle cells, even though the protein domain necessary for in vivo function could not be deciphered. Moreover, this project provides indication that CAP has an indispensable role in dynamic morphogenetic processes in the epithelium. Together with other actin-binding proteins, CAP could regulate epithelial actin turnover in spatially directed manner, providing force for epithelial cell adhesions or protrusions.
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