Bio21 - Theses
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ItemA journey of synthetic chemistry towards immunogenic glycolipids and non-lipidic antigens( 2018)Microbes, both pathogenic and commensal, produce a wide range of glycolipids that act as unique molecular signatures. The ability of the human immune system to fight infection as well as to modulate commensal organisms are active areas of research. Microbial glycolipids are known to interact with the immune system though discrete protein families including CD1 and Mincle. The main challenge in the study of such systems is the difficulty in, and often impossibility of, obtaining pure, homogeneous material from natural sources. We synthesised four classes of molecules of both natural and unnatural origin to investigate their potential to modulate the human immune system through the CD1 and Mincle axes. Chapter 2 describes the synthesis of a range of cholesteryl α-glucosides that are found in members of the Helicobacter family, including the prominent gut bacterium Helicobacter pylori. As part of this work we investigated the effect of remote protecting groups on the sugar on the stereochemical outcome of glucosylation reactions. In chapter 3 we designed and synthesised a set of purely synthetic glycolipids drawing upon the structures of known Mincle agonists. We investigated these compounds for their ability to signal through Mincle as a prelude to the development of improved vaccine adjuvants that promote cellular and humoral immunity. Chapter 4 discloses the total synthesis of α-glucosyl and α-glucuronosyl diglycerides, found in both pathogenic and commensal organisms relevant to human health. Finally, we prepared a set of analogues of the unique, non-lipidic synthetic CD1d-restricted effector, PPBF, to explore structure activity relationships for T cell activation. In collaboration with immunologists, the synthetic glycolipids and non-lipidic antigens have been studied for their ability to activate CD1d-restricted natural killer T cells or for their ability to stimulate signalling through Mincle.
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ItemCharacterisation of cytokines involved in porcine hematopoiesis( 2014)The prophylactic of use in-feed antibiotics and chemical based medicines such as anthelmintic are commonly used in order to maintain health and promote growth in food production animals. The inclusion of low regular or sub-therapeutic doses of antimicrobials in animal feed increases their efficiency to digest food by suppressing sensitive gut flora and reducing infection caused by opportunistic pathogenic organisms which could be detrimental to both the animal’s health and productivity. This practice has been linked to the emergence of drug resistant organisms, many of which are resistant to antibiotics commonly used in human medicine, posing a threat to both animals and humans (Hu). To reduce this threat, an alternative method of therapeutic treatment in food production animals must be developed. One suggestion is to enhance the animal’s own immune system, Hematopoiesis is the process of developing mature WBC from hematopoietic stem cells (HSC) within the bone marrow (BM). Therefore an understanding of hematopoiesis in pigs may uncover new therapeutic treatments. Cytokines are signalling molecules released by stimulated white blood cells (WBC), which activate essential cells and pathways essential in an innate or adaptive immune response to infection. These proteins initiate proliferation, differentiation and maturation of immature immune cells and precursors within the BM. Cytokines may therefore provide a viable alternative to current therapeutic practices. Although, many cytokines have been identified in Hu and mice (Mo), few have been studied in pigs. The cytokines of the common β subunit (CD131), interleukin-3 (IL-3), interleukin-5 (IL-5) and granulocyte, macrophage colony stimulating factor (GM-CSF) may prove to be valuable therapeutics. This unique group of cytokines not only share the same receptor subunit, but are involved in the production of several cells types. With this in mind, these cytokines, along with CD131, were analysed in silico, in vitro and in vivo. In silico phylogenetic analysis confirmed these cytokines in pigs developed similarly to other mammals. Molecular modelling suggests that structural changes due to altered genetic sequences, along with differences in CD131 binding residues, may result in a lack of cross species activity. The recombinant (rec) form of each porcine (Po) cytokine was produced and biological activity confirmed. In vitro analysis for rPoGM-CSF and rPoIL-3 showed maturation of immature CD90+ and CD172a+ BM cells resulting in monocyte and macrophage development. Moreover, rPoIL-5 induced proliferation and maturation for eosinophils. To assess the activity of each cytokine in vivo, pigs were administered the chemotherapeutic drug, 5-flurouricil (5-FU) to produce a state of myelosuppression. The in vivo activity was examined on BM and peripheral white blood cells (WBC). rPoGM-CSF induced proliferation of CD90+ and CD172a+ cells within the BM resulting in increased platelets whilst rPoIL-3 was also shown to significantly increase basophil levels. More importantly, all 3 cytokines increased levels of eosinophils at different stages throughout the trial, with rPoIL-5 inducing the highest eosinophil production. These results suggest that these Po cytokines may have potential as therapeutics to enhance immunity in pigs, specifically in parasite infections. This report has analysed the activity of rPoIL-3, rPoIL-5 and rPoGM-CSF. In doing so, we have identified some of the roles of these cytokines in hematopoiesis. Furthermore, we have demonstrated the involvement of all three cytokines in the regulation of eosinophils and confirmed the role of PoIL-5 as the major eosinophilopoietin in the production of eosinophils in pigs