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Este trabalho demonstrou a construção de minibibliotecas de \"Expressed Sequence Tags\" (EST) com o uso de RT-PCR de baixa estringencia e \"primer\" consenso-degenerado. Através do estudo de parâmetros críticos como concentração de sais, temperatura e velocidade de ciclagem, composição dos \"primers\" e qualidade do RNA mensageiro, foi possível padronizar um protocolo. Tal protocolo permitiu um aumento do numero de seqüências por minibiblioteca em relação a protocolos similares existentes na literatura (Dias neto et al., 1997 e 2000) sem perda de características desejáveis como amplificação preferencial do centro dos genes e nomalização das mensagens. As seqüências produzidas levaram a um significativo aumento das seqüências de EST de S. mansoni disponíveis publicamente e permitiu a detecção da existência de novos fragmentos de genes expressos na fase adulta do parasita. Neste trabalho também clonamos uma apirase de S. mansoni cuja a seqüência não havia sido descrita anteriormente. O gene possui cerca de 2,7 mil pares de bases e codifica para uma proteína de 544 aminoácidos. Esta foi expressa em sistema heterologo e utilizada para obtenção de anticorpos policlonais contra esta proteína. Utilizando estes anticorpos foi possível detectar a proteína no tegumento do parasita. / This work demonstrates the construction of EST minilibraries employing low stringency RT-PCR and consensus-degenerate primers. Through the study of critical parameters such as salt concentration, cycling temperature and ramp speed, composition of primers and quality of messenger RNA a standard protocol was obtained. Such protocol allowed an increase in the number of sequences per minilibrary in relation to similar protocols in the literature (Dias Neto et al., 1997 and 2000) without no loss in desirable characteristics such as preferential amplification of the central portion of messages and normalization of messages. The sequences produced allowed a significant increase in the publicly available EST sequences of S. mansoni and permitted discovery of new gene fragments expressed in adult stage of the life cycle of the parasite. In this work we also cloned an apyrase of S. mansoni whose sequence had never been described previously. The gene has about 2.7 kilobases and codes for a protein of 544 aminoacids. This protein was expressed in a heterologous system and was used for production of a polyclonal antibody. This antibody was used for detection of this protein in the tegument of the parasite.

Infection with the helminth parasite Schistosoma mansoni leads to chronic inflammation and Th2 mediated fibrosis, which result in severe pathology characterised by hepatosplenomegaly. Dendritic cells (DCs) are adept initiators of CD4+ T cell responses, but their fundamental importance in this regard in Th2 settings remains to be demonstrated. Indeed, the role of DCs at different stages of infection with S. mansoni is also yet to be determined. In addition, the importance of the interaction of DCs with tissue factors in the tissue microenvironment on the development of Th2 response to S. mansoni antigens is an area of active research and debate. This thesis is comprises of four studies. The first study tackles the involvement and importance of DCs in the induction and development of Th2 responses against S. mansoni using CD11c–diphtheria toxin receptor mice to deplete CD11c+ cells during the priming stage of the CD4+ Th2 response against S. mansoni. Diphtheria toxin treatment significantly depleted CD11c+ DCs from all tissues tested, with 70-80% efficacy. Even this incomplete depletion resulted in dramatically impaired CD4+ T cell production of Th2 cytokines, altering the balance of the immune response and causing a shift towards IFN-γ production. In contrast, basophil depletion using Mar-1 antibody had no measurable effect on Th2 induction in this system. These data underline the vital role that CD11c+ antigen presenting cells can play in orchestrating Th2 development against helminth infection in vivo, a response that is ordinarily balanced so as to prevent the potentially damaging production of inflammatory cytokines. The second study addresses whether the exposure of DCs to the cercarial stage of the parasite is critical for either parasite survival or the subsequent development of the Th2 immune response against later stages of infection. It was found that CD11c depletion prior to infection resulted in increased parasite survival, but did not impair the development of CD4+ T cell Th2 response later in infection. The third study asked whether DCs continue to be necessary for the maintenance of the chronic immune response during infection with S. mansoni. In contrast, depletion of CD11c+ cells during the initiation (4 to 6 weeks) or maintenance (6 to 8 weeks or 12 to 14 weeks) of Th2 response to eggs, resulted in severely impaired Th2 cytokine production. Interestingly, depletion during the later stages of infection led to dramatic weight loss and mortality, coincident with impaired CD4+ T cell responses. These data suggest that CD11c+ antigen presenting cells, in addition to being important in the early priming phase, also play a vital role in the maintenance and homeostasis of chronic CD4+ T cell responses in a Th2 infection setting, the disruption of which can have lethal consequences. The final study in this thesis aimed to establish whether the tissue factor thymic stromal lymphopoietin (TSLP) is able to enhance or modulate the Th2 responses initiated by DCs stimulated with SEA. Contrary to previous studies, it was found that BMDCs do not become phenotypically activated by TSLP, in particular, they do not up-regulate the costimulatory molecule OX40L, nor does TSLP suppress the production of IL-12p40 or IL-12p70 in response to LPS or CpG. Further, exposure to TSLP had no impact on DC cytokine production or survival. Irrespective of this unaltered profile in vitro, TSLP exposed DCs transferred in vivo induced the production of significantly more Th1 and Th2 cytokines from polyclonally restimulated splenocytes than DCs exposed to medium alone. In addition to this, TSLP altered the kinetic of the immune response induced by DCs stimulated with the soluble egg antigen (SEA) of S. mansoni. This was characterised by the antigen specific production of T cell cytokines starting more rapidly than with non-TSLP treated control DCs. The alteration in the kinetics of the immune response was not restricted to Th2 antigens and was also seen to some extent in Propionibacterium acnes stimulated DCs. This suggests a possible role for TSLP in either inducing faster DC migration or greater production of T cell chemoattractants and thus, enhancing the rate of DC interaction with T cells.

Schistosoma mansoni is a member of a genus of platyhelminths whose members cause the disease schistosomiasis. Particularly prevalent in sub-Saharan Africa, it is thought to be directly responsible for approximately 5500 deaths per year, as well as contributing significantly to morbidity, being responsible for 3.3 million lost disability-adjusted life years. Schistosomes are dioecious and male and female worms find one another and pair in the blood vessels of the host's liver. This sets in motion a unique feature of schistosome biology, the pairing-dependent sexual maturation of the female worms. Over the course of the next three weeks, the females fully develop their reproductive organs, especially ovaries and vitellarian tissue, to allow for the production of large quantities of eggs, which not only play a crucial role in the transmission of the parasites but are also responsible for much of the pathology associated with schistosomiasis. This thesis aims to explore the changes in gene expression which take place following pairing and result in the sexual maturation of females. To do so, RNA-Seq data was produced from male and female worms from mixed sex as well as single sex infections at 18, 21, 28, 35, 38 and 49 days post infection and analysed to understand when and how gene expression changes in paired worms. Then gene expression was examined in worms that had been removed from their partner to examine the process of regression, where female worms lose much of their reproductive tissue. The last experiments describe examine gene expression in the testes and ovaries of schistosomes, to reveal differences between the gonads of worms from mixed and single sex infections and understand in more detail how these worms may regulate the growth of their reproductive organs, contributing to our knowledge of schistosome biology.

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 180-211).

Thesis (M. S. in Chemistry)--Vanderbilt University, Dec. 2009. / Title from title screen. Includes bibliographical references.

published_or_final_version / Microbiology / Master / Master of Philosophy

A G protein-coupled receptor with structural characteristics of a biogenic amine GPCR was cloned from Schistosoma mansoni (SmGPCR). SmGPCR was codon-optimized and double-tagged with FLAG and His epitopes at the N- and C-terminal ends, respectively. Immunofluorescence experiments targeting these epitopes revealed that the expression of codon-optimized SmGPCR was highly increased compared to wild-type in mammalian cells. These studies also demonstrated that SmGPCR has a typical GPCR topology, the N-terminus being extracellular and C-terminus intracellular. Functional assays revealed that codon-optimized SmGPCR was responsive only to histamine, which caused a dose-dependent increase in intracellular Ca2+ (EC50 = 0.54 +/- 0.05 muM), but not cAMP, consistent with a Gq pathway of signal transduction. In vitro behavioral studies showed that treatment of S. mansoni cercaria with exogenous histamine caused a dose-dependent increase in the motility of the parasite.

Thesis (M.Sc. (Tropical Medicine))--Mahidol University, 1982.

Tübingen, Univ., Diss., 2003.