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Previous issue date: 2011-12-16 / The genus Angiostrongylus Kamensky, 1905 belongs to the Phylum Nematode, with round shape as its main feature. Two species have medical importance, A. costaricensis living in mesenteric arteries of wild mice and causing abdominal angiostrongyliasis in human and A. cantonensis which lives in pulmonary arteries of rats and may cause eosinophilic meningoencephalitis in humans. The diagnosis of both diseases is difficult due to absence of parasite in feces in case of the infection by A. costaricensis and seldom detected larvae in the cerebrospinal fluid in case of eosinophilic meningoencephalitis. Several studies have been performed to improve the diagnosis of angiostrongyliasis which should be able to differentiate in a specific and sensitive way among other parasitic infections. The 31kDa antigen has been considered the main antigen for eosinophilic meningoencephalitis diagnosis due to A. cantonensis infection. However this antigen is obtained from crude extracts of the worm by a laborious process of purification with low yielding and insufficient amount for large distribution to other diagnostic centers. In order to improve the serologic diagnostic of angiostrongyliasis and make the antigens widely available the present work aimed to identify new antigenic targets and also characterize the 31kDa antigen for further recombinant production. Besides that, essential molecules for parasite survival were investigated which in the future may be targets for disease treatment. Two sources of antigen from female worms were used: excretion and secretion products (ES) and total extract (TE). In ES, sample antioxidant enzymes activity were detected such as catalase and superoxide dismutase. Also was identified by Western blot and Mass spectrometry (MS), 17 proteins target for disease diagnosis and treatment like hemoglobinases, heat shock proteins and proteases inhibitors. In TE sample antioxidant enzymes as well as glutathione transferases (GST) which is another kind of defense enzyme were also detected. GSTs were purified by affinity chromatography and analyzed by MS. Peptide sequences from this experiment matched with homologous sequences of sigma class GST. In TE samples was possible to characterize the 31kDa and after two-dimensional electrophoresis was shown to be composed of four spots around 4.5 of isoelectric point (pI) and being recognized by sera from patients infected with Angiostrongylus spp. The spots were analyzed by MS and three different proteins were identified: 14-3-3 protein, NAC domain containing protein, and epsilon subunit of the coatomer protein complex isoform 2. The 31kDa antigen was characterized as a glycoprotein through studies of oxidation of carbohydrate where it was observed that the antigenicity of four spots was dependent on sugar residues. The DNA sequences of the antigens were obtained by random sequencing of the genome for 454 platform (Roche) and deposited in Genbank. The data generated in this study contribute significantly to the development of recombinant antigens that may be widely distributed for independent diagnostic validation / O g?nero Angiostrongylus Kamensky, 1905 agrupa animais pertencentes ao filo Nematoda, cuja caracter?stica marcante ? a forma corporal cil?ndrica. Duas esp?cies possuem import?ncia m?dica: A. costaricensis cujo habitat natural s?o as art?rias mesent?ricas de camundongos silvestres e na infec??o humana pode levar ao desenvolvimento de angiostrongil?ase abdominal; e A. cantonensis que habita as art?rias pulmonares de roedores e na infec??o humana pode causar meningoencefalite eosinof?lica. O diagn?stico de ambas as doen?as ? dificultado pela aus?ncia de formas parasit?rias nas fezes, no caso de infec??es por A. costaricensis e raramente encontradas no l?quido cefaloraquidiano no caso de meningoencefalite eosinof?lica. Muitos estudos v?m sendo desenvolvidos para o aprimoramento da detec??o das angiostrongil?ases visando testes que sejam capazes de discernir das diferentes infec??es parasit?rias de forma sens?vel e espec?fica. O ant?geno de 31kDa ? considerado atualmente o principal ant?geno para o diagn?stico da meningoencefalite eosinof?lica, causada por A. cantonensis, entretanto ? proveniente da purifica??o de extratos brutos do parasito o que acarreta num processo laborioso e dispendioso que em ?ltima an?lise gera quantidades insuficientes para que haja ampla distribui??o e compartilhamento entre os centros de diagn?stico. Com o intuito de aprimorar o diagn?stico sorol?gico das angiostrongil?ases e tornar os ant?genos dispon?veis globalmente o presente trabalho buscou identificar novos alvos antig?nicos e caracterizar o ant?geno de 31kDa para posterior propaga??o de formas recombinantes. Al?m disso, foram estudadas mol?culas que podem ser fundamentais na manuten??o do parasitismo, que futuramente poder?o ser alvos para o tratamento das angiostrongil?ases. Duas fontes de ant?genos a partir de vermes adultos f?meas foram empregadas: produtos de excre??o e secre??o (ES) e extrato bruto (TE). Nos ES foi detectada a atividade de enzimas antioxidantes como catalase e super?xido dismutase e identificadas, por western blot e espectrometria de massas (MS), 17 prote?nas alvo para o diagn?stico e tratamento das angiostrongil?ases dentre elas hemoglobinases, prote?nas de choque t?rmico e inibidores de proteases. Nas amostras de TE al?m da identifica??o de enzimas antioxidantes, estavam presentes glutationas transferases (GST), outra classe de enzimas de defesa. Estas prote?nas foram purificadas por cromatografia de afinidade e analisadas por MS o que revelou sequencias pept?dicas hom?logas a GST de classe sigma. Em TE tamb?m foi poss?vel a caracteriza??o do ant?geno de 31kDa que quando submetido a eletroforese bidimensional mostrou-se ser composto por 4 spots com ponto isoel?trico (pI) em torno de 4,5 sendo reconhecidos pelo soro de pacientes infectados com Angiostrongylus spp. Os spots foram analisados por MS e tr?s diferentes prote?nas foram identificadas: 14-3-3; prote?na com dom?nio NAC e a subunidade ?psilon do coatamero. O ant?geno de 31kDa foi caracterizado como uma glicoprote?na atrav?s de estudos de oxida??o de glic?deos, onde se observou que a antigenicidade dos 4 spots foi dependente de res?duos de a??car. As sequ?ncias de DNA dos ant?genos foram obtidas pelo sequenciamento aleat?rio do genoma pela plataforma 454 (Roche) e depositadas no Genbank. Os dados gerados no presente trabalho contribuem de forma significativa para o desenvolvimento de ant?genos recombinantes que poder?o ser amplamente distribu?dos para valida??o e aplica??o em diagn?stico
| Identifer | oai:union.ndltd.org:IBICT/oai:tede2.pucrs.br:tede/225 |
| Date | 16 December 2011 |
| Creators | Morassutti, Alessandra Loureiro |
| Contributors | Graeff-teixeira, Carlos |
| Publisher | Pontif?cia Universidade Cat?lica do Rio Grande do Sul, Programa de P?s-Gradua??o em Zoologia, PUCRS, BR, Faculdade de Bioci?ncias |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da PUC_RS, instname:Pontifícia Universidade Católica do Rio Grande do Sul, instacron:PUC_RS |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 2008925231902741151, 500, 600, 36528317262667714 |
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