Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T17:21:25Z No. of bitstreams: 1 TES_BIANCA_BARBIERI_COGNATO_COMPLETO.pdf: 2150300 bytes, checksum: 6c0ebf29cb36a3ae7a825cf9c0060dc4 (MD5) / Made available in DSpace on 2017-06-30T17:21:25Z (GMT). No. of bitstreams: 1 TES_BIANCA_BARBIERI_COGNATO_COMPLETO.pdf: 2150300 bytes, checksum: 6c0ebf29cb36a3ae7a825cf9c0060dc4 (MD5) Previous issue date: 2017-03-21 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Conselho Nacional de Pesquisa e Desenvolvimento Cient?fico e Tecnol?gico - CNPq / Two nematode species belonging to the Metastrongyloidea superfamily are capable to produce disease in humans: Angiostrongylus cantonensis and Angiostrongylus costaricensis. Both are rodent parasites and human infection is considered accidental. Eosinophilic meningoencephalitis, caused by A. cantonensis, raises concern due to the expanding number of cases and geographical area of occurrence. Molecular and immunological methods for the diagnosis are crucial, however, after many studies with different antigenic molecules, has a specific and sensitive test to discriminate the angiostrongyliasis to others parasitoses is lacking. Cross-reactivity with other helminthes, which may cause similar symptoms, and eosinophilic meningitis, has been a problem for the satisfactory performance of specificity in serological tests. In order to improve the diagnosis of angiostrongyliasis, a comparative analysis of protein recognition from different extracts from A. cantonensis, Toxocara canis, Schistosoma mansoni and Strongyloides stercoralis against positive serum for Angiostrongylus.spp was performed. Through extraction kit, one-dimensional and two-dimensional electrophoresis, western blot and mass spectrometry, 149 proteins were identified. Among these, 34 were exclusive to A. cantonensis, COI being present only in the A. cantonensis extract, having no similarities with any other parasite compared in NCBI (nr database) and WormBase Database. Additionally, nine proteins were recognized by more than one parasite and extract, being important cross reactivity markers in parasitic infections. With the data obtained in this study, we suggest the use of the follow proteins as cross-reactivity markers: Galectin 1, HSPA-5 and Ifa1. In addition, immunogenic proteins of A. cantonensis ES-7, Lec-5 and 14-3-3, were recombinant expressed in two cell types, CHO and HEK. Their potential diagnostic values were verified by uni and bidimensional electrophoresis, western and dot blot, and N-glycosidase F (PNGase F) treatment using serum from patients infected with A. cantonensis, negative serum for parasites, and positive for other parasites. ES7 protein expressed in HEK and CHO cells and Lec-5 expressed in CHO were recognized only by Angiostrongylus-positive serum and not by negative control and specificity control in 2D and 1D tests, respectively. In the 2D analyzes Lec-5 showed a weak recognition with negative serum. However, the 14-3-3 protein didn?t show any specificity against A. cantonensis serum, since it was recognized by all tested sera. Antigen-antibody recognition was found to be dependent on the glycogenic portions, since when treated with N-glycosidase F (PNGase F), recognition between proteins and serum disappeared. The heterologous expression, using mammalian cells, as well as the identification of shared and/or specific molecules, may represent a promising source of antigens for the diagnosis of eosinophilic meningoencephalitis, and molecular diagnostic tests become necessary. / Duas esp?cies de nemat?deos pertencentes a superfam?lia Metastrongyloidea, s?o capazes de produzir doen?a em humanos: Angiostrongylus cantonensis e Angiostrongylus costaricensis. Ambos s?o parasitas pr?prios de roedores e a infec??o humana ? considerada acidental. A meningite eosinof?lica, causada por A. cantonensis, tem gerado preocupa??o na comunidade cient?fica pela expans?o da ?rea geogr?fica de ocorr?ncia. M?todos moleculares e imunol?gicos para o diagn?stico desta infec??o s?o cruciais para o diagn?stico, entretanto, ap?s muitos estudos com diferentes mol?culas antig?nicas, at? hoje n?o foi desenvolvido um teste de diagn?stico que seja espec?fico e sens?vel o suficiente para discriminar as angiostrongil?ases de outras parasitoses. A reatividade cruzada tem sido o principal problema encontrado nos estudos j? desenvolvidos para este prop?sito. Com o objetivo de aprimorar o diagn?stico das angiostrongil?ases, foi realizado neste estudo an?lise comparativa do reconhecimento de prote?nas de diferentes extratos teciduais de A. cantonensis, Toxocara canis, Schistosoma mansoni e Strongyloides stercoralis contra soro positivo para Angiostrongylus spp Atrav?s de kit de extra??o, eletroforese unidimensional e bidimensional, western blot e espectrometria de massas foram identificadas 149 prote?nas. Dentre estas, 34 foram exclusivas para A. cantonensis, sendo que COI estava presente apenas no extrato de A. cantonensis n?o possuindo similaridades com nenhum outro parasito comparado no NCBI (nr database) e WormBase Database. Estas prote?nas podem ser consideradas promissoras como marcadores de reatividade humoral espec?fica para o parasito. Todavia, outras nove prote?nas foram reconhecidas por mais de um parasito em mais de um extrato testado, sendo importantes marcadores de reatividade cruzada em infec??es parasit?rias. Com os dados obtidos neste estudo, sugerimos como marcadores de reatividade cruzada o uso das prote?nas: Galectin 1, HSPA-5 e Ifa1. Al?m disso, prote?nas imunog?nicas de A. cantonensis ES-7, Lec-5 e 14-3-3, foram expressas de forma recombinante em dois tipos celulares, CHO e HEK, e o potencial uso diagn?stico destas prote?nas foi verificado atrav?s de eletroforese uni e bidimensional, western e dot blot, e tratamento por N-glycosidase F (PNGase F), utilizando soro positivo para Angiostrongylus spp., soro negativo e positivo para outras parasitoses. Prote?na ES-7 expressa em c?lulas HEK e CHO, e Lec-5 expressa em CHO foram reconhecidas apenas pelo soro positivo para Angiostrongylus spp. e n?o pelo soro negativo e controles de especificidade em testes 2D e 1D, respectivamente. J? nas an?lises em 2D, Lec-5 apresentou fraco reconhecimento com soro negativo. J? a prote?na 14-3-3 n?o apresentou nenhuma especificidade pelo soro de A. cantonensis, j? que foi reconhecida por todos os soros testados. O reconhecimento ant?geno-anticorpo se mostrou dependente das por??es glic?dicas, j? que quando tratadas com N-glycosidase F (PNGase F), o reconhecimento das prote?nas pelo soro desapareceu. A express?o heter?loga, utilizando c?lulas de mam?feros, assim como a identifica??o de mol?culas compartilhadas e/ou espec?ficas, podem representar uma promissora fonte de ant?genos para o diagn?stico da meningite eosinof?lica, requerendo aprimorados testes moleculares para seu diagn?stico.

Made available in DSpace on 2016-04-28T20:16:24Z (GMT). No. of bitstreams: 1 2007-Catia Marques da Costa.pdf: 899456 bytes, checksum: b8d00f84cefd48cf338e385b27121da0 (MD5) Previous issue date: 2007-02-28 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Anaplasma marginale (Ricktsialles: Anaplasmataceae) is a ricketsial hemoparasite responsible for causing great economic losses in cattle from tropical and subtropical regions. The objectives of this work were to produce the recombinant membrane proteins AM254, VirB9 and VirB10 and to evaluate its possible antigenicity. The genes am254, virB9 and virB10 were submitted to various experiments and were , linked to pET47 (b) plasmid. After the certification of the recombinant plasmid (pET-47-am254, pET-47-virB9 and pET-47- virB10), it was transformed into E. coli Rosetta cells for expression. The recombinant proteins produced, were analyzed by Western blot assay where the reaction of the anti-histidin monoclonal antibody against rAM254 (47kDa), rVirB9 (31kDa) and rVirB10 (60kDa) was observed. After the confirmation of the production of recombinant proteins the indirect Enzyme-Linked Immnunosorbent Assay (ELISA) was standardized with sera previously confirmed by PCR and ELISA (rMSP1 and rMSP5). The averages of the optic densities (OD) of the positive sera were of 1.339; 1.288 and 1.240 and of the negative sera of 0.470, 0.324 and 0.414 for AM254, VirB9 and VirB10 respectively with significant difference to the level of 5% between positives and negatives for each recombinant protein. The study demonstrated that the proteins rAM 254, rVirB9 and rVirB10 of A. marginale are recognized by sera of bovines immunized by different Brazilian isolates of the ricketsia (homologous and heterologous), showing the antigenicity potential of these proteins. / Anaplasma marginale (Ricktsialles: Anaplasmataceae) ? uma riquetsia intraeritroc?tica, respons?vel por ocasionar grandes perdas econ?micas na pecu?ria bovina das regi?es tropical e sub-tropical. O objetivo desse trabalho foi produzir as prote?nas de membrana recombinantes AM254, VirB9 e VirB10 e avaliar sua poss?vel antigenicidade. Os genes am254, virB9 e virB10 foram submetidos a v?rios procedimentos experimentais at? serem ligados ao plasm?deo pET-47b(+). Ap?s a certifica??o do plasm?deo recombinante (pET-47bam254, pET-47b-virB9 e pET-47bvirB10) este foi transformado em E. coli Rosetta para express?o. As prote?nas recombinantes produzidas foram analisadas pelo ensaio Western blot onde foi observada a rea??o do anticorpo monoclonal anti-histidina contra rVirB9 (31kDa), rVirB10 (60kDa) e rAM254 (47kDa). Ap?s a confirma??o da produ??o das prote?nas recombinantes realizou-se a padroniza??o do ensaio de imunoadsor??o enzim?tica (ELISA) indireto com soros oriundos de sangue total previamente confirmados por PCR; os mesmos soros tamb?m foram testados por ELISA (rMSP1 e rMSP5). As m?dias das densidades ?pticas (DO) dos soros positivos foram de 1,339; 1,288 e 1,240 e dos soros negativos de 0,470, 0,324 e 0,414 para AM254, VirB9 e VirB10 respectivamente com diferen?a significativa ao n?vel de 5% entre positivos e negativos para cada prote?na recombinante. O estudo demonstrou que as prote?nas rAM 254, rVirB9 e rVirB10 de A. marginale s?o reconhecidas por soros de bovinos imunizados com diferentes isolados brasileiros da riqu?tsia (hom?logo e heter?logos), revelando o potencial antig?nico dessas prote?nas.

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-09-04T20:22:35Z No. of bitstreams: 1 LucasLuizVieiraDissertacao2016.pdf: 2717272 bytes, checksum: b547db9dacaad5a2da23826b5d215ccb (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-09-04T20:23:00Z (GMT) No. of bitstreams: 1 LucasLuizVieiraDissertacao2016.pdf: 2717272 bytes, checksum: b547db9dacaad5a2da23826b5d215ccb (MD5) / Made available in DSpace on 2017-09-04T20:23:00Z (GMT). No. of bitstreams: 1 LucasLuizVieiraDissertacao2016.pdf: 2717272 bytes, checksum: b547db9dacaad5a2da23826b5d215ccb (MD5) Previous issue date: 2016-04-14 / Invasive fungal infections are a major public health problem in the world, since they increase morbidity and patients??? hospitalization time. In Brazil, the highest mortality rate among the systemic mycoses is caused by an endemic disease named paracoccidioimycosis, which the etiologic agent is the dimorphic fungus Paracoccidioides spp. The worldwide increased resistance to the commercially available antifungal agents, their limited spectrum of activity against some fungal pathogens and concerns with their toxic side effects are reasonable evidence of the necessity of novel therapeutic strategies, especially the development of new antifungal agents. Thus, the essential gene rim8 was identified by comparative genomics as an orthologous sequence in the genome of human pathogenic fungi absent in the human genome. In filamentous fungi and yeasts, gene expression regulation by the ambient pH involves components of a signaling pathway that mediate proteolytic activation of the transcription factor PacC/Rim101 in response to alkaline environmental pH. The rim8 gene in yeasts, also called palF in filamentous fungi, performs an essential step in this signaling pathway. It is important for host-pathogen interaction, leading to increased virulence and pathogen survival. Thus, Rim8 is a very interesting and promising drug target. The aim of this work is to optimize heterologous expression and purification of Rim8 protein from P. luzii to further perform its structural and functional characterization and also, to use it as a molecular target for drugs development. The rim8 gene was chemically synthesized with a histidine tag and cloned into a pET-21a vector. Heterologous expression of the gene was made using two strains of E. coli, obtaining the best conditions using LB medium with 0.5% glucose at 30 ??C for 2 hours and 0.25 mM IPTG and adding protease inhibitor cocktail. The Rim8 heterologous protein was purified using a nickel affinity chromatography column. Expression and purification results were analyzed by SDS-PAGE and in some cases, confirmed by western blot. Immunization of BALB/c mice was performed with the purified protein to obtain anti- Rim8 antibodies. The antibody production was confirmed by ELISA test. The protein immunocitolocalization in P. lutzii cells showed a difuse protein-plasma membrane association at acidic pH. At neutral pH the fluorescence pattern is showed as localized foci in plasma membrane and later, with extracellular alcalinization, it migrates into the cytosol. Thus, it can be inferred that this work gives some contribution to the development of new antifungal drugs, but still must undergo further production steps, proteolysis reduction and protein purification to allow structural and functional characterization of Rim8. / Infec????es f??ngicas invasivas s??o um problema de sa??de p??blica no mundo, j?? que aumentam a morbidade e o tempo de interna????o de pacientes. A micose sist??mica com o maior ??ndice de mortalidade no Brasil, onde ?? considerada end??mica, ?? a paracoccidioidomicose, causada pelo fungo dim??rfico Paracoccidioides spp. A tend??ncia global de aumento da resist??ncia aos agentes antif??ngicos dispon??veis comercialmente, o espectro limitado de atividade contra alguns fungos patog??nicos e a preocupa????o com a toxicidade s??o evid??ncias da necessidade de novas estrat??gias terap??uticas, sobretudo do desenvolvimento de novas drogas antif??ngicas. Nesse sentido, o gene essencial rim8 foi identificado por gen??mica comparativa como uma sequ??ncia ort??loga no genoma de fungos patog??nicos humanos e ausente em humanos. Em fungos filamentosos e leveduras, a regula????o da express??o g??nica pelo pH envolve componentes de uma via de sinaliza????o que levam ?? ativa????o proteol??tica do fator de transcri????o PacC/Rim101 em resposta ?? alcaliniza????o do pH externo. O gene rim8 em leveduras ou palF em fungos filamentosos possui um papel essencial nessa cascata de sinaliza????o, sendo importante para a intera????o pat??geno-hospedeiro, aumento de virul??ncia e sobreviv??ncia do pat??geno. Por isso, Rim8 ?? um alvo molecular promissor e bastante interessante para o desenvolvimento de drogas. O objetivo deste trabalho ?? otimizar a express??o heter??loga e purifica????o da prote??na Rim8 de P. lutzii, visando realizar a caracteriza????o estrutural e funcional da prote??na para futuramente utiliz??-la como alvo molecular para o desenvolvimento de drogas. O gene rim8 foi sintetizado quimicamente com cauda de histidinas e foi clonado em vetor pET-21a. A express??o heter??loga do gene foi padronizada usando duas estirpes de E. coli, obtendo as melhores condi????es para purifica????o com o uso de meio LB contendo 0,5% de glicose a 30??C por 2 h e 0,25 mM de IPTG e coquetel de inibidores de proteases. Os resultados da express??o e purifica????o foram analisados por SDS-PAGE e/ou confirmados por western blot. Realizou-se a imuniza????o de camundongos BALB/c com a prote??na purificada para obten????o de anticorpos anti-Rim8. A produ????o dos anticorpos foi confirmada por ELISA. A imunocitolocaliza????o em c??lulas de P. lutzii mostrou que a prote??na se encontra associada ?? membrana plasm??tica de forma difusa em pH ??cido, em seguida apresenta focos localizados em resposta a um pH pr??ximo da neutralidade e, por fim, migra para o interior da c??lula em pH alcalino. Sendo assim, pode-se inferir que o trabalho apresenta contribui????es para o desenvolvimento de novas drogas antif??ngicas, al??m de auxiliar o entendimento do processo biol??gico no qual a prote??na RIM8 est?? envolvida.