Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2016-06-28T19:56:24Z No. of bitstreams: 1 DIS_ANA_HELENA_BRETANHA_LOPES_TORT_COMPLETO.pdf: 1228563 bytes, checksum: 1ae15f2eb2fe5ed55580c78c418cb7be (MD5) / Made available in DSpace on 2016-06-28T19:56:24Z (GMT). No. of bitstreams: 1 DIS_ANA_HELENA_BRETANHA_LOPES_TORT_COMPLETO.pdf: 1228563 bytes, checksum: 1ae15f2eb2fe5ed55580c78c418cb7be (MD5) Previous issue date: 2016-02-15 / Gliomas are primary tumors of the central nervous system that are associated with a high mortality rate; they course with an average survival rate of 2 years after the diagnosis. Less than 5 % of glioma patients survive more than five years after diagnosis, even those treated with state of the art protocols, which include surgery, radiotherapy and chemotherapy. Tumors result from impairments of intracellular signaling pathways, including the p38/MAPK pathway, which, are responsible to control of cell proliferation and tumorigenesis, among other cellular responses. The goal of the present work was to investigate the effects of ML3403, an inhibitor of p38/MAPK, on the viability and proliferation of glioma cells, and to assess its effect when combined with bevacizumab (BVZ). BVZ already used in the clinical setting as anadjuvant for treating gliomas. It is a monoclonal antibody against VEGF-A receptor and thus reduces the signaling involved in tumor angiogenesis. U138 and U251 glioma cells were treated with ML3403 (0.1 to 200 ?M) and BVZ (1 to 200 ?g/mL) and later assessedfor cell viability, by MTT method and proliferation by cell counting. The results demonstrate that treatment with ML3403 reduces glioma cell viability and proliferation. Co-treatment with BVZ does not present any significant effect. The use of p38/MAPK inhibitors may constitute an interesting treatment against glioma progression. / Por serem tumores prim?rios localizados no sistema nervoso central, os gliomas apresentam altas taxas de mortalidade com sobrevida m?dia de dois anos. Menos de 5% dos pacientes com gliomas sobrevivem mais de cinco anos ap?s o diagn?stico, mesmo com os tratamentos mais avan?ados, os quais envolvem cirurgia, radia??o e quimioterapia. Os tumores resultam de diferentes defeitos em vias de sinaliza??o intracelulares, incluindo a via p38/MAPK. Entre as respostas celulares mediadas pela fam?lia de p38/MAPK, destaca-se a regula??o da produ??o do fator de crescimento endotelial vascular (VEGF). O objetivo deste trabalho foi investigar os efeitos de ML3403, um inibidor da p38/MAPK, sobre a viabilidade das c?lulas de glioma, e avaliar o seu efeito combinado com bevacizumab (BVZ). O BVZ, j? utilizado na cl?nica como adjuvante no tratamento de gliomas, ? um anticorpo monoclonal contra o receptor de VEGF-Ae reduz a sinaliza??o envolvida no processo de angiog?nese tumoral. As c?lulas de gliomas U138MG e U251MG foram tratadas com ML3403 (0,1 a 200?M) e BVZ (1 a 200 ?g/mL)e avaliadas para viabilidade celular, atrav?s do m?todo do MTT e prolifera??o celular, atrav?s da contagem do n?mero de c?lulas. Os resultados obtidos demonstraram redu??o da viabilidade e prolifera??o celular ap?s o tratamento com ML3403. O co-tratamento com BVZ n?o apresentou efeito aditivo. A utiliza??o de inibidores da via de sinaliza??o p38/MAPK pode ser considerada como um tratamento promissor na diminui??o do crescimento de gliomas.

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2016-10-20T16:27:57Z No. of bitstreams: 1 DIS_ELISAMAR_SANTOS_MARIN_WAGNER_COMPLETO.pdf: 909420 bytes, checksum: 855d83fc62f091978eab516fd6b1df35 (MD5) / Made available in DSpace on 2016-10-20T16:27:57Z (GMT). No. of bitstreams: 1 DIS_ELISAMAR_SANTOS_MARIN_WAGNER_COMPLETO.pdf: 909420 bytes, checksum: 855d83fc62f091978eab516fd6b1df35 (MD5) Previous issue date: 2016-03-01 / Gaucher disease, first described in 1882, is a rare autosomal recessive genetic disorder characterized by a deficiency of glucocerebrosidase, an enzyme that catalyses the hydrolysis of lysosomal glucocerebroside into glucose and ceramide. The presence of two mutant alleles located on chromosome 1 q21 region confirms the diagnosis of Gaucher disease, with N370S and L444P being the most common mutations. Clinical characteristics commonly associated with Gaucher disease include hepatosplenomegaly, anemia, thrombocytopenia and bone involvement, hematological and neurological impairment in type II and III. The accumulation of mutant enzyme and subsequent depletion of normal enzyme can be treated with enzyme replacement therapy using recombinant glucocerebrosidase. Enzyme replacement therapy is quite effective in the treatment of this disease, often reversing the cascade of biochemical events that lead to clinical manifestations and improving many of the patient's signs and symptoms. There are three recombinant enzymes for enzyme replacement therapy in Gaucher disease: Velaglucerase alfa, Alfataliglucerase and Imiglucerase. These enzymes differ from each other mainly in relation to the form of production, the amino acid sequence, and the glycosylation pattern. Currently, enzyme replacement therapy is administered by a drug called Cerezyme? (imiglucerase), produced by recombinant DNA technology using cell cultures obtained from Chinese hamster ovary. The imiglucerase differs from natural glucocerebrosidase, from placental origin, by an amino acid at position 495, where histidine is replaced with arginine. However, the cost of Cerezyme? is very high, hindering access to therapy. In Brazil, there are nearly 600 carriers of the disease. Although the treatment is guaranteed by law to all patients, the expenses on the purchase of the medicine are very high. The cost could be considerably reduced if the enzyme could be produced in an alternative way. Studies have shown that advanced reproductive techniques allow transgenic animals to be used as bioreactors for production of recombinant proteins of high potential biological and pharmaceutical interest. The Molecular Biology Laboratory and the development of University of Fortaleza in partnership with Quatro G developed the first Latin America transgenic goat clone, whose mammary gland is used as a bioreactor for producing recombinant protein human glucocerebrosidase. This study presented an alternative of glucocerebrosidase enzyme purification. The alternative method was achieved by using milk obtained through an induced lactation of the transgenic and cloned goat. According to the results, one can create a positive expectation for glucocerebrosidase through the procedure cited throughout this study. The confirmation of the presence of glucocerebrosidase enzyme activity was carried out by fluorimetry test. Cerezyme? and goat?s milk control (not genetically modified) were used with positive and negative control, respectively. The objective of this study was to establish protocols for purification of glucocerebrosidase enzyme from the cloned and transgenic goat's milk and confirm the enzyme activity, to evaluate the expression of the enzyme by electrophoresis, and to characterize the recombinant protein by mass spectrometry. / A doen?a de Gaucher, descrita pela primeira vez em 1882, ? uma rara desordem gen?tica autoss?mica recessiva caracterizada pela defici?ncia da atividade da glucocerebrosidase, uma enzima lisossomal que catalisa a hidr?lise de glucocerebros?deo em glicose e ceramida. A presen?a de dois alelos mutantes localizado na regi?o q21 do cromossomo 1 confirma o diagn?stico, sendo as muta??es mais comuns s?o N370S e L444P. O ac?mulo desta enzima pode ser tratada com terapia de reposi??o enzim?tica, usando glucocerebrosidase recombinante, bastante eficaz no tratamento da doen?a, revertendo toda a cascata de eventos bioqu?micos que acabam por ocasionar as manifesta??es cl?nicas apresentadas pelos pacientes ou melhorando muitos dos seus sinais e sintomas. As caracter?sticas cl?nicas comumente associados com a doen?a de Gaucher incluem hepatoesplenomegalia, anemia, trombocitopenia e envolvimento ?sseo, hematol?gico e comprometimento neurol?gico no tipo II e III. H? tr?s enzimas recombinantes para a terapia de reposi??o enzim?tica na doen?a de Gaucher: Velaglucerase alfa, Alfataliglucerase e Imiglucerase que diferem entre si principalmente em rela??o ? forma de produ??o, ? sequ?ncia de amino?cidos e ao padr?o de glicosila??o. Atualmente, a terapia de reposi??o enzim?tica ? administrada por um medicamento chamado Cerezyme? (imiglucerase), produzido atrav?s da tecnologia do DNA recombinante utilizando culturas de c?lulas obtidas do ov?rio de hamster chin?s. A imiglucerase difere da glucocerebrosidase natural, de origem placent?ria, por um amino?cido na posi??o 495, onde a histidina ? substitu?da por arginina. Entretanto, o custo do medicamento dispon?vel ? muito alto, dificultando o acesso ? terapia. No Brasil, h? aproximadamente 600 portadores da doen?a e o tratamento ? garantido por lei a todos os pacientes, embora os gastos com a compra do medicamento sejam muito elevados. Este custo poderia ser consideravelmente reduzido se a enzima pudesse ser produzida de forma alternativa. Estudos demonstraram que t?cnicas reprodutivas avan?adas permitem que animais transg?nicos possam ser usados como biorreatores para a produ??o de prote?nas recombinantes de elevado potencial biol?gico e interesse farmac?utico. O Laborat?rio de Biologia Molecular e do Desenvolvimento da Universidade de Fortaleza em parceria com a Quatro G Pesquisa e Desenvolvimento desenvolveu o primeiro clone transg?nico de cabras da Am?rica Latina cuja gl?ndula mam?ria ? usada como um biorreator para a produ??o da prote?na glucocerebrosidase humana recombinante. Neste presente estudo, foi apresentado uma alternativa de purifica??o da enzima glucocerebrosidase a partir do leite obtido atrav?s da lacta??o induzida da cabra transg?nica e clonada. De acordo com os resultados, pode-se criar uma expectativa positiva para a obten??o da glucocerebrosidase atrav?s do procedimento citado ao longo deste estudo. A confirma??o da presen?a de atividade enzim?tica de glucocerebrosidase foi realizada pelo ensaio fluorim?trico, Cerezyme? e leite da cabra controle (n?o transg?nica) foram usados com controle positivo e negativo, respectivamente. O objetivo deste estudo foi estabelecer protocolos de purifica??o da enzima glucocerebrosidase a partir do leite da cabra clonada e transg?nica e confirmar a atividade enzim?tica, avaliar a express?o da enzima por eletroforese e caracterizar a prote?na recombinante atrav?s da espectrometria de massa.