Veterinary Science - Theses
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ItemFunctional analysis of Schistosoma mansoni egg proteins( 2013)Schistosomiasis is a prevalent, socioeconomically important disease of humans worldwide. Despite major efforts, there is only one drug routinely used for effective treatment and no vaccine to combat schistosomiasis. The overall objective of this thesis was to functionally characterise some key proteins from eggs of Schistosoma mansoni and explore their involvement in the disease process in vivo. In spite of the utility of double-stranded RNA interference (RNAi) in S. mansoni, methods used to date have had severe limitations. Based on evidence from the literature (chapter 1), the lentiviral delivery of artificial miRNAs (shRNAmir) was likely to circumvent these limitations. The main aims of this thesis were to: (1) construct a lentiviral vector for the delivery of a miRNA cassette in S. mansoni, (2) validate the accessibility of the miRNA-pathway in S. mansoni for selective gene knock-down, and (3) investigate the role of selected target molecules in immune responses and immunopathology in mice. This thesis demonstrated, for the first time, successful lentiviral transduction of S. mansoni (chapter 3), enabling the targeting of dividing and non-dividing cells. Direct polymerase chain reaction (PCR)-based detection of the transgene confirmed the presence of provirus in the parasite’s genome. In addition, the transgene was transcriptionally active under the control of a mammalian (CMV) promoter, allowing the validation of the lentiviral system in mammalian cells prior to application to the parasite. Furthermore, this is the first study to show that the miRNA pathway is functional in S. mansoni and can be utilised effectively for RNAi studies (chapter 4). Semi-quantitative PCR results showed transcriptional down-regulation of target genes. A first screen for off-target effects indicated a high specificity of the shRNAmir sequences designed specifically to the target genes. Importantly, lentiviral transduction and shRNAmir-induced transcriptional gene knock-down had no effect on the vitality or maturation of larval eggs stages, enabling in vivo studies. Experimental studies in mice (chapter 5) demonstrated that this newly established technique can be employed effectively for the identification and characterisation of molecules involved in host responses. While Th2 cells are indispensable for the regulation of granuloma formation, the key mechanism underlying S. mansoni egg-induced pathological changes related to the secreted immune-modulating protein omega-1. Furthermore, a central role of macrophages and tissue cells in the initiation of pathological changes was observed, deflecting the focus from CD4+ T cells alone. Therefore, this study substantially enhances our understanding of processes leading to granulomatous disease in response to S. mansoni eggs.