Submitted by Luciana Sebin (lusebin@ufscar.br) on 2016-09-27T12:08:38Z No. of bitstreams: 1 DissLP.pdf: 10003873 bytes, checksum: 89fb02c36205a1922829c70d0aa75ff4 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-27T19:56:19Z (GMT) No. of bitstreams: 1 DissLP.pdf: 10003873 bytes, checksum: 89fb02c36205a1922829c70d0aa75ff4 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-27T19:56:24Z (GMT) No. of bitstreams: 1 DissLP.pdf: 10003873 bytes, checksum: 89fb02c36205a1922829c70d0aa75ff4 (MD5) / Made available in DSpace on 2016-09-27T19:56:30Z (GMT). No. of bitstreams: 1 DissLP.pdf: 10003873 bytes, checksum: 89fb02c36205a1922829c70d0aa75ff4 (MD5) Previous issue date: 2016-02-25 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Schistosomiasis mansoni is a disease caused by Schistosoma mansoni, it affects about 8 million people in Brazil and it is a neglected disease. Moreover, it is necessary to develop new tools for its control, since for over 40 years the same drug has been used to treat it. Thus, the search for vaccines is of great value for the control of schistosomiasis. In this study, we evaluated two recombinant enzymes of S. mansoni, the MTAP and APRT as immunogens, and checked the antiparasitic and anti-inflammatory effects in the schistosomiasis murine model. By means of prior immunization with MTAP and/or APRT, we analyzed, after the infection with S. mansoni, the host response to parasite board determination, immunological cytokines and histopathological changes in the liver of these animals. Our data showed that the MTAP enzyme was able to decrease the number of adult worms in previously immunized animals; APRT enzyme and/or the mix of the two enzymes induced reduction of inflammation in the liver of animals and promoted collagen deposition control; and MTAP enzyme and the mix stimulated a humoral immune response with production of IgG1 and IgE antibodies. Hence we suggest that the enzymes have potential for new studies that would seek a future vaccine target for the control of schistosomiasis. / A esquistossomose mansônica é uma doença causada pelo agente etiológico Schistosoma mansoni, acomete cerca de 8 milhões de brasileiros, sendo considerada uma doença negligenciada. Assim, faz-se necessário o desenvolvimento de novas ferramentas para o seu controle, visto que há mais de 40 anos usa-se o mesmo fármaco para o seu tratamento. Dessa forma, a busca por vacinas é de grande valia para o controle da esquistossomose mansônica. Neste estudo, avaliamos duas enzimas recombinantes de S. mansoni, a MTAP e a APRT, como imunógenos e verificamos o efeito antiparasitário e anti-inflamatório no modelo murino da esquistossomose mansônica. Por meio da imunização prévia com MTAP e/ou APRT e após a infecção com S. mansoni, analisamos a resposta do hospedeiro com determinação da carga parasitária, as citocinas imunológicas e as alterações histopatológicas no fígado desses animais. Dessa forma, nossos dados mostraram que a enzima MTAP foi capaz diminuir o número de vermes adultos nos animais previamente imunizados. A imunização com a enzima APRT e/ou com o mix das duas enzimas induziu redução do processo inflamatório no fígado dos animais e promoveu controle do depósito de colágeno. E durante a avaliação da resposta humoral a imunização com a enzima MTAP e o mix estimulou uma resposta específica com produção de anticorpos IgG1 e IgE. Assim, sugerimos que, as enzimas apresentam potencial para novos estudos que buscam um futuro alvo vacinal para o controle da esquistossomose mansônica.

The gene for methylthioadenosine phosphorylase (MTAP) is absent in almost 30% of cancers, opening a door for selective chemotherapy. One strategy to target the absence of MTAP involves the design of a cytotoxic methylthioadenosine (MTA) analogue. Non-cancerous cells would break down the cytotoxic analogue, since they contain MTAP, but cancerous cells would not, since they do not have MTAP. However, before such a compound can be made, we need to better understand the types of substrates accommodated by MTAP. The purpose of this thesis was therefore to explore a series of MTA analogues, probing the structure-function relationships between MTAP and specific structural modifications of MTA. Nine phenylthioadenosine (PTA) derivatives bearing ortho-, meta-, or para- methyl carboxylate, carboxylate, and hydroxymethyl substituents were synthesized and tested for cytotoxicity and as substrates for MTAP. The biological results of these nine compounds suggested that addition of substituents to the ortho-position was not tolerated by MTAP, and substituents similar to the hydroxymethyl might be accommodated by MTAP. None of the compounds were cytotoxic. This informed the design of ten more PTA derivatives, most of which were synthesized and tested for cytotoxicity and as substrates for MTAP. The range of functionalities included an amine, an acetamide, a urea, an isovaleramide, and an N-nitrosourea group inspired by the known anticancer agent lomustine. The amine derivatives of PTA were the best substrates of all MTA analogues tested (including PTA). The meta-amine derivative and the meta-isovaleramide showed minor cytotoxicity. Finally, the urea derivatives were moderate substrates of MTAP, and this pointed towards the future creation of other nitrosoureas as potential cytotoxic MTAP substrates. / Graduate / 2017-08-25

Los cuerpos nucleares de Cajal (CBs) son estructuras nucleares implicadas en la biogénesis de ribonucleoproteínas nucleares y nucleolares de pequeño tamaño (snRNPs y snoRNPs) requeridas para el procesamiento nuclear de pre-mRNAs y pre-rRNAs, respectivamente. El CB concentra la proteína coilina, un marcador molecular de esta estructura, snRNPs, el factor de supervivencia de las neuronas motoras (SNM) y las proteínas que comparte con el nucleolo Nopp140 y fibrilarina. Los CB son estructuras dependientes de transcripción, pero los mecanismos de ensamblaje molecular de estos cuerpos nucleares son poco conocidos.En este estudio se utilizan métodos de inmunofluorescencia, expresión ectópica de proteínas del CB y métodos bioquímicos para analizar la importancia de dos modificaciones postraduccionales, la metilación de la coilina y la conjugación con SUMO1 del factor SMN para el ensamblaje molecular de los CBs. Se ha utilizado la línea celular MCF7 como un modelo de hipometilación endógena debido al déficit del gen MTAP. La hipometilación de la coilina conduce al desensamblaje de los CBs y a la relocalización nucleolar de la coilina no metilada. Este efecto revierte en células transfectadas que expresan el gen MTAPwt, indicando que el grado de metilación de la coilina marca su destino nuclear.Respecto a la importancia de la sumoilación en el ensamblaje de los CBs, hemos demostrado la existencia de un subtipo de CBs que concentran SUMO1 y la conjugasa de SUMO Ubc9. En neuronas, hemos detectado la presencia de SUMO durante la fase de reformación de CBs, en la respuesta al estrés. Los experimentos de inmunoprecipitación confirman la interacción de SUMO-1 con el factor SMN y demuestran que la lisina K119, portadora de una secuencia consenso de sumoilación, es esencial para la regulación del número de CBs. / Cajal bodies (CBs) are nuclear structures involved in the biogenesis of small nuclear and nucleolar ribonucleoproteins (snRNPs and snoRNPs) required for nuclear processing of pre-mRNAs and pre-rRNAs, respectively. CBs concentrate the protein coilin, a molecular marker of this structure, snRNPs, the survival of motor neurons factor (SMN) and proteins shared with the nucleolus Nopp140 and fibrillarin. CBs are transcription-dependent structures, but the mechanisms of molecular assembly of these structures are poorly understood.In this study we used inmunofluorescence, ectopic expresion of CB proteins and biochemical methods to analyze the importance of two posttranslational modifications, methylation of coilin and conjugation of SMN with SUMO1, for the molecular assembly of CBs. The cell line MCF7 has been used as a model of endogenous hypomethylation due to the lack of MTAP gene. Coilin hypomethylation leads to the disassembly of CBs and nucleolar relocation of unmethylated coilin. This effect reverses in transfected cells expressing the gene MTAPwt, indicating that the degree of methylation of coilin directs its nuclear destination.On the importance of sumoylation in the assembly of CBs, we have demonstrated the existence of a subset of CBs which concentrate SUMO1 and the SUMO1 conjugase Ubc9. In neurons, we detected the presence of SUMO1 during the reformation of CBs in response to stress. Immunoprecipitation experiments confirm the molecular interaction of SUMO1 with SMN and demonstrate that lysine 119, carrying the SMN sumoylation consensus sequence, is essential for regulating the number of CBs.