Made available in DSpace on 2015-04-14T14:51:05Z (GMT). No. of bitstreams: 1 430842.pdf: 2935464 bytes, checksum: 65b5bce8dd872e39bf663fce3ac30b27 (MD5) Previous issue date: 2011-01-27 / A natureza como fonte de inspira??o provou ter grande impacto ben?fico no desenvolvimento de novas metodologias computacionais. Neste cen?rio, an?lises de intera??es entre uma prote?na alvo e um ligante podem ser simuladas por algoritmos inspirados biologicamente (BIA). Neste trabalho, os algoritmos inspirados biologicamente, especialmente algoritmos evolutivos, s?o aplicados a simula??es de docking molecular, que podem ser usadas na busca de novos f?rmacos. Estes algoritmos de docking foram aplicados a sirtu?nas, que comp?em uma importante fam?lia de prote?nas - desacetilases dependentes de NAD - que regulam o silenciamento gen?tico, inibi??o transcricional, estabilidade cromoss?mica, ciclo de divis?o celular e apopt?tico e resposta c?lular a agentes causadores de danos no DNA. Essas prote?nas tem sido alvos moleculares emergentes no desenvolvimento farmac?utico de medicamentos para o tratamento de doen?as humanas. Em fun??o da import?ncia estrutural das prote?nas, neste trabalho foi realizada a modelagem por homologia molecular de SIRT1 humana, utilizando-se a estrutura cristalogr?fica de Sir2 de Thermotoga maritima como modelo. Al?m disso, foi aplicado o procedimento de virtual screening para a SIRT1 modelada contra duas bases de dados. Uma foi baseada em estruturas derivadas da nicotinamida e outra composta por mol?culas da Sigma-Aldrich. Com base nos resultados obtidos no virtual screening, foram utilizados os acoplamentos com valores mais baixos de energia livre de liga??o (Plant Score) para a sele??o das melhores mol?culas. Foi empregada a Similarity Ensemble Approach Tool (SEA), uma abordagem estrat?gica para analisar as rela??es entre as estruturas e a atividade farmacol?gica. Os resultados indicam que algumas mol?culas apresentaram alta afinidade com a SIRT1, sugerindo novos inibidores de SIRT1, e essas mol?culas mostram um elevado n?mero de refer?ncias de atividades farmacol?gicas. Nossos resultados sugerem novos compostos inibidores de SIRT1 que apresentam potencial aplica??o terap?utica

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2015-06-09T11:33:18Z No. of bitstreams: 1 470158 - Texto Completo.pdf: 900834 bytes, checksum: c9822e05947627069fb5b4fc9d1f6005 (MD5) / Made available in DSpace on 2015-06-09T11:33:18Z (GMT). No. of bitstreams: 1 470158 - Texto Completo.pdf: 900834 bytes, checksum: c9822e05947627069fb5b4fc9d1f6005 (MD5) Previous issue date: 2015-03-17 / The international market for essential oils has an annual turnover of 1.8 billion dollars, yet the Brazilian participation in this market is only 0.1%. The vast biodiversity found in Brazil, although still little explored in relation to the chemical composition of its flora, places the country in a very promising position in terms of increasing its future participation in the essential oils market. Basil (Ocimum basilicum L.) is a widely cultivated aromatic plant due to its medicinal properties. The importance of this species lies in the plant architecture, coloration and morphology of its leaves and flowers, in addition to the chemical composition of its essentials oils, giving specific aromas and having various uses in the culinary, perfume and pharmaceuticals industries. In this study, two experiments were performed with the aim of studying germination in simulated hypergravity and open-air cultivation, to evaluate the growth of the basil, its yield and essential oil composition. The extraction of the essential oils was carried out by steam distillation in a Clevenger apparatus. Quantitative and qualitative evaluation of the compounds was performed by gas chromatography coupled to a mass spectrometer detector (GC/MS). The mean values for germination under the effect of hypergravity simulation in protocols 1, 2 and 3 ranged from 81% to 87%. After this stage, the seeds exposed to simulated hypergravity were cultivated in the open-air and the fresh basil leaves were collected after 90 days to obtain the essential oils. The overall yield of oil was 0.2mL to 0.3mL and the essential oil was 0.3% to 0.4%. The largest and most predominant compound in this research was eugenol, ranging from 46% to 62%, with the second largest being linalool, which ranged from 22% to 28%. Further research in this area is essential to understand the real impact of simulated hypergravity on basil, and its effect on auxins and gene expression. / O mercado internacional de ?leos essenciais movimenta anualmente 1,8 bilh?es de d?lares e a participa??o brasileira nesse mercado ? de apenas 0,1%. A grande biodiversidade brasileira, ainda pouco explorada em rela??o ? composi??o qu?mica de sua flora, coloca o Brasil numa situa??o muito promissora para aumentar sua participa??o futura no mercado de ?leos essenciais. O manjeric?o (Ocimum basilicum L.) ? uma planta arom?tica largamente cultivada, devido ? suas propriedades medicinais. A import?ncia dessa esp?cie est? na arquitetura da planta, na colora??o e na morfologia das folhas e flores, al?m da composi??o qu?mica de seus ?leos essenciais, originando aromas espec?ficos e tendo utiliza??o variada na gastronomia, perfumaria e ind?stria farmac?utica. Nesse trabalho, foram realizados dois experimentos, com o objetivo de estudar a germina??o por hipergravidade simulada e o cultivo a c?u aberto para avaliar o crescimento do manjeric?o, rendimento e a composi??o do ?leo essencial. A extra??o do ?leo essencial foi feita por arraste a vapor em aparelho Clevenger. A avalia??o quantitativa e qualitativa dos compostos foi realizada por cromatografia gasosa acoplada a um detector de massas (CG/MS). As m?dias de germina??o deste trabalho foram de 81% a 87%. Ap?s essa etapa, as sementes sob efeito de hipergravidade simulada foram cultivadas a c?u aberto e, depois de 90 dias, foram coletadas folhas frescas de manjeric?o para obter ?leos essenciais. O rendimento total de ?leo foi de 0,2 mL a 0,3 mL e de ?leo essencial de 0,3%. a 0,4%. O composto majorit?rio e predominante em nossas pesquisas foi o eugenol, variando entre 46% a 62%, e o segundo maior composto foi o linalol, que ficou entre 22% a 28%. Pesquisas nessa ?rea s?o essenciais para entender o real impacto da hipergravidade simulada no manjeric?o, seu efeito na auxina e sua express?o g?nica.

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2015-06-11T19:45:55Z No. of bitstreams: 1 470271 - Texto Completo.pdf: 601264 bytes, checksum: 631a8bec7a15cd2f97fae4e0bb07768a (MD5) / Made available in DSpace on 2015-06-11T19:45:55Z (GMT). No. of bitstreams: 1 470271 - Texto Completo.pdf: 601264 bytes, checksum: 631a8bec7a15cd2f97fae4e0bb07768a (MD5) Previous issue date: 2015-03-24 / The esophageal and oral tumors are classified as the most frequent malignancies in Brazil. Esophageal cancer is the eighth most common in the world and oral cancer is ranked as the 5th among malignant neoplasms affecting man in Brazil. The lipopolysaccharide (LPS) are characteristic compounds of the cell wall of gram-negative bacteria. They are able to regulate gene expression of pro-inflammatory cytokines by binding to toll-like receptor 4 (TLR4) via NF-kB pathway. Recent studies show that LPS can increase the migration ability of cell lines of human esophageal cancer HKESC-2 by increasing its binding properties. In the meantime, it has not been tested the effect of LPS on esophageal cancer cells OE19 and OE21 and human oral carcinoma HN30. Thus, this study aimed to determine the action of LPS compounds on the proliferation and viability of cell lines of human esophageal cancer and human oral carcinoma HN30. Were used as treatment LPS for OE19 and OE21 cell lines and the PgLPS (lipopolysaccharide of Porphyromonas gingivalis) for HN30 cell line. Cell viability was assessed using the MTT assay and cell counting. The TLR4 expression by real-time PCR was also evaluated. LPS at higher concentrations decreased significantly cell viability in both cell lines, adenocarcinoma (OE19) and squamous esophageal carcinoma (OE21) at different times of treatment. In addition, both cell lines, OE19 and OE21, expressed TLR4 receptor. Taken together, our data demonstrated that LPS at high concentrations might contribute to tumor death, in agreement with previously data. / Os tumores de es?fago e de boca est?o classificados como as neoplasias malignas mais frequentes no Brasil. O c?ncer de es?fago ? o oitavo mais comum no mundo e o c?ncer de boca ? classificado como o 5? dentre as neoplasias malignas que afetam o homem no Brasil. Os lipopolissac?ridos (LPS) s?o compostos caracter?sticos da parede celular de bact?rias gram-negativas. Eles s?o capazes de regular a express?o de genes de citocinas pr?-inflamat?rias, atrav?s da liga??o ao receptor toll-like 4 (TLR4) via NF-kB. Estudos recentes mostram que o LPS pode aumentar a habilidade de migra??o de linhagens c?lulares de c?ncer de es?fago humano HKESC-2 atrav?s do aumento de suas propriedades de ades?o. Entretanto, ainda n?o foi testado o efeito do LPS sobre as c?lulas de c?ncer de es?fago e de carcinoma oral humano HN30. Deste modo, este estudo teve como objetivo determinar a a??o dos compostos de LPS (derivado de bact?rias) sobre a prolifera??o e viabilidade de linhagens celulares de c?ncer de es?fago humano, e de carcinoma oral humano. Foram utilizados como tratamento o LPS para as linhagens OE19 e OE21 e o PgLPS (lipopolissacar?deo da Porphyromonas gingivalis) para a linhagem HN30. A viabiliadade celular foi avaliada utilizando o ensaio MTT e contagem celular. Tamb?m foi avaliada a express?o do receptor TLR4 por PCR em tempo real. LPS em concentra??es mais elevadas reduziu significativamente a viabilidade celular em ambas as linhagens celulares de c?ncer de es?fago, o adenocarcinoma (OE19) e o carcinoma de c?lulas escamosas (OE21) em diferentes tempos de tratamento. Al?m disso, ambas as linhagens celulares, OE19 e OE21, expressaram o receptor TLR4. Avaliados em conjunto, os nossos dados demonstram que o LPS em concentra??es elevadas pode contribuir para a morte tumoral, de acordo com dados pr?vios.

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2015-06-25T11:34:05Z No. of bitstreams: 1 471145 - Texto Completo.pdf: 583122 bytes, checksum: b9760b9161356a851110fa2f73635187 (MD5) / Made available in DSpace on 2015-06-25T11:34:05Z (GMT). No. of bitstreams: 1 471145 - Texto Completo.pdf: 583122 bytes, checksum: b9760b9161356a851110fa2f73635187 (MD5) Previous issue date: 2015-03-09 / Glioblastoma multiform (GBM) is a highly aggressive tumor of the central nervous system and has a few available therapies. In GBM, proinflammatory signaling pathways, such as STAT3 and NF- ?B are aberrantly activated and associated with cell proliferation, survival, invasion and resistance to chemotherapy. Therefore, inhibition of these pro-inflammatory pathways has been suggested as a strategy to combat malignant cells. Resolvins of D-series promote resolution of inflammation by decreasing the activation of NF-?B. The Resolvin D2 (RvD2) is derived from a double sequence of docosahexaenoic acid lipoxygenation, which is derived from the essential fatty acid Omega-3. Our hypothesis is that the resolvins, by direct or indirect inhibition of pro-inflammatory pathways, could compromise the survival and growth of glioma. Our goal is to assess the role of RvD2 and its precursor 17 (R) HDHA (HR17) on tumor cell proliferation and survival in murine glioma model. For this, murine glioma cell line (GL261) was treated in vitro with RvD2 and HR17, and cell death and proliferation were evaluated. Furthermore, the effect of RvD2 and HR17 was evaluated in vivo after GL261 brain implantation; histopathological characteristics and immunohistochemistry for activated caspase-3 were evaluated to. It was found that RvD2 GL261 induces apoptosis in vitro. Furthermore, in vivo treatment of RvD2 and HR17 increased the number of tumor cells positive for activated caspase-3, although not alter tumor area. Interestingly, the HR17 treatment reduces tumor mitotic index in vivo. These results suggest that RvD2 and HR17 have a potential role in the induction of apoptosis in murine glioma cells. / Glioblastoma multiforme (GBM) ? um tumor altamente agressivo do Sistema Nervoso Central e possui poucas terapias dispon?veis. Em GBM, vias de sinaliza??o pr?-inflamat?rias, como da STAT3 e NF-?B, s?o aberrantemente ativadas e associadas com a prolifera??o celular, sobreviv?ncia, invas?o e resist?ncia ? quimioterapia. Por conseguinte, a inibi??o dessas vias pr?inflamat?rias tem sido sugerida como uma estrat?gia para combater as c?lulas malignas. As resolvinas da s?rie D promovem resolu??o da inflama??o diminuindo a ativa??o de NF-?B. A Resolvina D2 (RvD2) deriva de uma sequencia de dupla lipoxigena??o do ?cido docosahexaen?ico, o qual ? derivado do ?cido graxo essencial Omega-3. Nossa hip?tese ? que as resolvinas, por inibi??o direta ou indireta de vias pr?-inflamat?rias, poderiam comprometer a sobreviv?ncia e o crescimento do glioma. Nosso objetivo consiste em avaliar o papel da RvD2 e de seu precursor 17(R)HDHA (HR17) sobre a prolifera??o de c?lulas tumorais e sobreviv?ncia em um modelo de glioma murino. Para isso, a linhagem de c?lulas de glioma murino (GL261) foi tratada in vitro com RvD2 e HR17, e amorte celular e prolifera??o foram avaliadas. Al?m disso, o efeito de RvD2 e HR17 foi avaliado in vivo ap?s o implante de GL261 no c?rebro, avaliando-se caracter?sticas histopatol?gicas e a imunohistoqu?mica para caspase-3 ativada. Verificou-se que RvD2 induz apoptose de GL261 in vitro. Al?m disso, o tratamento de RvD2 e HR17 in vivo aumentou o n?mero de c?lulas tumorais positivas para caspase-3 ativada, embora n?o alterassem a ?rea do tumor. Curiosamente, o tratamento HR17 reduz o ?ndice mit?tico tumoral in vivo. Estes resultados sugerem que RvD2 e HR17 t?m um potencial papel na indu??o de apoptose em c?lulas de glioma murino.

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2015-10-09T19:45:36Z No. of bitstreams: 1 475655 - Texto Completo.pdf: 1136903 bytes, checksum: 1a2ad879662fb2a0203c2418ab7dc36f (MD5) / Made available in DSpace on 2015-10-09T19:45:36Z (GMT). No. of bitstreams: 1 475655 - Texto Completo.pdf: 1136903 bytes, checksum: 1a2ad879662fb2a0203c2418ab7dc36f (MD5) Previous issue date: 2015-05-15 / Liver fibrosis is a complex disease that is caused by inappropriate tissue repair due to the deposition of connective tissue. When a chronic lesion affects the liver, regenerative response fails and hepatocytes are replaced with abundant extracellular matrix (ECM). The imbalance between production and degradation of ECM will result in the accumulation of proteins that change normal liver architecture, and thus its functionality. The main source of ECM is the activated hepatic stellate cell (HSC). In order, to clarify possible therapeutic approaches to the disease, the this work aimed to evaluate the possible antifibrotic action of Pluchea sagitallis (Lam.) Cabrera on an activated HSC immortalized lineage (GRX). Our results demonstrated that the P. sagittalis aqueous extract at 0.039 and 0.078 mg/mL concentrations was able to reduce cell growth and proliferation. Regarding to oxidative stress evaluation, there was no statistically significant difference between the treated group and the control. Staining with OilRed-O (ORO) showed a statistically significant increase in intracellular lipid content after 5 days of treatment, exerting in vitro effect on the GRX phenotypic change of activated towards the quiescent state. These results were confirmed by colorimetric quantification of lipid content. Regarding the TGF-?1 and collagen production, there were no statistically significant differences observed between the groups. In conclusion, the P. sagittalis aqueous extract reduces the growth and proliferation of GRX cells and induces the reversal of activated towards a quiescent phenotype. There was no decrease in cell proliferation either by necrosis or by apoptosis via activation of the senescence. Thus, our data suggest that the extract showed an antifibrotic effect, possibly by activating phenotype reversal. / A fibrose hep?tica apresenta uma patog?nese complexa causada por reparo tecidual inadequado devido ? deposi??o de tecido conectivo. Quando um dano cr?nico acomete o f?gado, a resposta regenerativa falha e os hepat?citos s?o substitu?dos por matriz extracelular (ECM) excedente. Assim, o desequil?brio entre a degrada??o e a produ??o de ECM acarretar? no ac?mulo dessas prote?nas que alteram a arquitetura normal do f?gado e, consequentemente, sua funcionalidade. A principal fonte de ECM ? a c?lula estrelada hep?tica (HSC) ativada. Sendo assim, na tentativa de elucidar poss?veis abordagens terap?uticas para a doen?a, o objetivo desse trabalho foi avaliar a poss?vel a??o antifibr?tica do extrato aquoso de Pluchea sagitallis (Lam.) Cabrera sobre uma linhagem imortalizada de HSC ativadas (GRX). Nossos resultados demonstraram que as concentra??es de 0,039 e 0,078 mg/mL do extrato aquoso de P. sagitallis foram capazes de diminuir o crescimento e a prolifera??o celular. Quanto ? avalia??o do estresse oxidativo, n?o foi observada diferen?a estatisticamente significativa entre o grupo tratado e controle. A colora??o com oil red (ORO) mostrou aumento significativo do conte?do lip?dico intracelular ap?s 5 dias de tratamento, indicando efeito in vitro sobre a mudan?a fenot?pica em linhagem GRX, do estado ativado para o estado quiescente. Esses resultados foram confirmados pela quantifica??o colorim?trica de lip?dios. Em rela??o ? produ??o de TGF-?1 e col?geno total, n?o foram observadas diferen?as estatisticamente significativas entre os grupos. Concluindo, o extrato aquoso da P. sagittalis diminuiu o crescimento e a prolifera??o das c?lulas GRX e induziu a revers?o do fen?tipo ativado para quiescente. A diminui??o na prolifera??o celular n?o ocorreu nem por necrose nem por ativa??o da apoptose e senesc?ncia. Sendo assim, nossos resultados sugerem que o extrato apresenta um efeito antifibr?tico, possivelmente pela via que ativa a revers?o do fen?tipo.

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2015-10-20T10:56:16Z No. of bitstreams: 1 475810 Texto Completo.pdf: 380863 bytes, checksum: d1fcdd6e53d6287f6c48e59678eb0404 (MD5) / Made available in DSpace on 2015-10-20T10:56:16Z (GMT). No. of bitstreams: 1 475810 Texto Completo.pdf: 380863 bytes, checksum: d1fcdd6e53d6287f6c48e59678eb0404 (MD5) Previous issue date: 2015-05-15 / Conselho Nacional de Pesquisa e Desenvolvimento Cient?fico e Tecnol?gico - CNPq / After the proceeding of a transplant, it is of huge concem the use of all therapeutic resource available in order to prevent graft rejection. The incidence and severity of acute rejection have been reduced along time due to the development of new immunosuppressive agents such as cyclosporin, tacrolimus, mycophenolate mofetil (MMF), specific polyclonal and monoclonal antibodies. Mofetil mycophenolate (MMF) is a prodrug active only after its hydrolysis to mycophenolic acid (MPA). Once it, has a large variability on inter-individual response, an increasing need of therapeutic monitoring arises. This will permit an individualization of MMF therapy, optimizing immunosuppression and minimizing potential toxic effects.The aim of pharmacogenetics is to evaluate the association between individual genetic characteristics and different responses to the same therapeutic regimen. When considering immunosuppressive drugs, genetic changes on a single nucleotide (single nucleotide polymorphisms - SNPs) of genes encoding proteins involved in transport or drug metabolism may affect patient?s response to therapy. UDP-glucuronosyltransferases (UGTs) belong to a group of enzymes involved in phase II reactions, responsible for the detoxification of endogenous and exogenous substrates. UGT1A9 is of particular interest once it is the primary enzyme involved in the metabolism of the MPA. This enzyme is encoded by the UGT1A9 gene.In the present study, we investigated the effect of UGT1A9 c.98T>C (rs72551330; g. 87289T>C) allelic variants on MMF metabolism in 39 renal transplant volunteers. MPA levels were measured by high pressure liquid chromatography using ultraviolet detection (HPLC/UV). The analysis of c.98T>C polymorphism was performed by polymerase chain reaction (PCR), followed by fragment purification and sequencing. All investigated individuals showed having the same polymorphism genotype (c.98TT) evaluation of variants or genotype influence on plasma levels of MPA and MPAG was not possible, even though different levels were observed in the study. / Ao realizar um transplante, ? de fundamental import?ncia que, seja utilizado todo o arsenal terap?utico dispon?vel e cuidados para evitar a rejei??o do enxerto. A incid?ncia e a intensidade da rejei??o aguda t?m sido reduzidas gra?as ao uso de f?rmacos imunossupressores, como ciclosporina, tacrolimus, micofenolato de mofetil (MMF), anticorpos monoclonais e policlonais. O MMF ? um pr?-f?rmaco com atividade ap?s sua hidr?lise a ?cido micofen?lico (MPA). No entanto, possui grande variabilidade inter-individual de resposta e por isso ? crescente a import?ncia da realiza??o de seu monitoramento terap?utico, o qual permite individualizar a dose de MMF e otimizar a imunossupress?o, minimizando os potenciais efeitos t?xicos. A farmacogen?tica estuda a rela??o entre as caracter?sticas gen?ticas do indiv?duo e as diferentes respostas a uma mesma terapia farmacol?gica.No caso de f?rmacos imunossupressores, altera??es gen?ticas de um ?nico nucleot?deo (single nucleotide polymorphisms - SNPs) em genes que codificam prote?nas envolvidas no transporte ou no metabolismo do f?rmaco podem modificar a resposta do paciente ? terap?utica. As UDP-glucuronosiltransferases (UGTs) pertencem a um grupo de enzimas envolvidas na fase II, respons?vel pela detoxifica??o de subst?ncias end?genas e ex?genas. A UGT1A9 ? de especial interesse por ser a principal enzima envolvida no metabolismo do ?cido micofen?lico. Esta enzima ? codificada pelo gene UGT1A9. No presente estudo, foi investigado o efeito das variantes al?licas de UGT1A9 c.98T>C (rs72551330; g. 87289T>C) no metabolismo de MMF em 39 pacientes volunt?rios transplantados renais.Foram dosados os n?veis de MPA por cromatografia l?quida de alta efici?ncia utilizando detec??o com ultravioleta (HPLC/UV) e a an?lise do polimorfismo c.98T>C do gene UGT1A9 foi realizada utilizando rea??o em cadeia de polimerase (PCR), seguida de purifica??o do fragmento e sequenciamento. Todos os indiv?duos investigados apresentaram o mesmo gen?tipo (c.98TT) para este polimorfismo, n?o possibilitando a avalia??o da influ?ncia das variantes ou gen?tipos deste polimorfismo sobre os n?veis plasm?ticos de MPA e MPAG, apesar de n?veis diversos destes compostos terem sido identificados nos pacientes.

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2016-07-26T14:25:23Z No. of bitstreams: 1 DIS_MARCOS_VINICIUS_FORTES_ALBA_COMPLETO.pdf: 1333852 bytes, checksum: c687fbcabc052faa5ed3567c986640e9 (MD5) / Made available in DSpace on 2016-07-26T14:25:23Z (GMT). No. of bitstreams: 1 DIS_MARCOS_VINICIUS_FORTES_ALBA_COMPLETO.pdf: 1333852 bytes, checksum: c687fbcabc052faa5ed3567c986640e9 (MD5) Previous issue date: 2016-02-15 / Nuclear Medicine enables, among other things, the visualization, characterization and quantification of biochemical processes, metabolism, biomarkers and receptors in vivo by molecular imaging. One of the methods of functional imaging available in this area is Positron Emission Tomography (PET), which depends on the administration of a radiopharmaceutical in the studied organism. One disease that can be studied by PET scan is multiple sclerosis (MS) through the access to the TSPO (Translocator protein) receptors density. The radiopharmaceutical [11C]-(R) -PK11195 has high affinity and selectivity for these receptors, which are increased in activated microglia cells in the case of MS. Therefore, the objective of this study was to develop and to validate the production process (synthesis, purification and quality control) of the radiotracer [11C]-(R)-PK11195 in the Radiopharmaceutical Production Center of the Brain Institute of Rio Grande do Sul (BraIns), allowing performing PET scans for the study of MS. The radioisotope was produced in the 16 MeV PETrace cyclotron (GE Healthcare) as [11C]CO2 via the nuclear reaction 14N (p, ?)11C after the bombardment of a gaseous target containing N2 and 0.5% of O2 with protons. The production was performed in the equipment TRACERlab FX-C Pro (GE Healthcare) to meet the requirements of Good Manufacturing Practices (GMP). The radiopharmaceutical quality control was carried out at the BraIns Quality Control Laboratory and the final product was tested for Identity and Radionuclidic Purity, Radiochemical Purity, Specific Activity, Residual Solvents, pH, Sterility, the Filtration Membrane Integrity and Bacterial Endotoxins. The synthesis was performed through the methylation of the precursor (R)-[N-desmethyl]PK11195 using [11C]CH3I (methyl iodide labeled with carbon-11) under the defined optimal conditions: 400 uL of dimethyl sulfoxide (DMSO), approximately 1 mg of precursor, approximately 10 mg of potassium hydroxide (KOH) at 40 ?C for 1 minute. The reaction mixture was then neutralized with 1 mol.L-1 hydrochloric acid and diluted with mobile phase (76% acetonitrile and 24% Milli-Q water). The diluted mixture was injected into a semi-preparative HPLC system for purification. Solid phase extraction (through Sep-Pak C18 cartridges - Waters) was used to remove the solvent. Product elution and formulation was conducted with 0.7 mL of ethanol and 6.3 ml of 0.9% saline (sterile and pyrogen free). Sterilization was performed by filtration on a 0.22 ?m pore membrane and, as well as the filling process, was carried out under laminar flow. The process steps could be optimized and fitted to the equipments and laboratories structure. Radiochemical yield of the process in relation to the [11C ]CH3I start activity was 15.5 ? 2.4 % ( 7471.5 ? 1283.8 MBq ; n = 39 ) . All batches were approved according to the specifications defined and tested in the quality control, reaching Radiochemical Purity levels greater than 99% and a Specific Activity of 39.7 ? 11.1 GBq / micromol , 10 minutes before the injection. All analytical methods were validated and met the requirements established by current regulations. Three consecutive batches were produced and approved by the quality control, so the process could be considered validated. The developed process was suitable for production of adequate doses of the radiopharmaceutical [11C]-(R)-PK11195 for clinical use at BraIns. The production of the radiopharmaceutical labeled with carbon-11 at the institute provides an additional tool for improving health services in the country and to improve the life quality of the patients. / A medicina nuclear possibilita, dentre outras coisas, a visualiza??o, caracteriza??o e quantifica??o de processos bioqu?micos, metab?licos, biomarcadores e receptores, in vivo, atrav?s da imagem molecular. Dentre as modalidades de imagem funcional dispon?veis nesta ?rea est? a Tomografia por Emiss?o de P?sitrons (PET), que depende da administra??o de um radiof?rmaco no organismo estudado. Uma das doen?as pass?veis de estudo atrav?s do exame PET ? a esclerose m?ltipla (EM), atrav?s do acesso ? densidade de receptores TSPO (Prote?na Translocadora). O radiof?rmaco [11C]-(R)-PK11195 possui alta afinidade e seletividade por estes receptores, aumentados nas c?lulas ativadas da micr?glia nos casos de EM. Portanto, o objetivo deste trabalho foi desenvolver e validar o processo produtivo (s?ntese, purifica??o e controle de qualidade) do radiof?rmaco [11C]-(R)-PK11195 no Centro de Produ??o de Radiof?rmacos do Instituto do C?rebro do RS (InsCer), possibilitando a realiza??o de exames PET para o estudo da EM. O radiois?topo foi produzido no C?clotron PETrace 16 MeV (GE Healthcare) na forma de [11C]CO2 atrav?s da rea??o nuclear 14N(p,?)11C, por meio do bombardeamento com pr?tons do alvo gasoso contento N2 e 0,5 % de O2. A produ??o foi realizada no equipamento TRACERlab FX-C pro (GE Healthcare) atendendo ?s exig?ncias de Boas Pr?ticas de Fabrica??o (BPF). O controle de qualidade do radiof?rmaco foi realizado no Laborat?rio de Controle de Qualidade do InsCer e o produto acabado foi submetido ?s an?lises de Identidade e Pureza Radionucl?dica, Pureza Radioqu?mica, Atividade Espec?fica, Solventes Residuais, pH, Esterilidade, Integridade da Membrana Filtrante e Teor de Endotoxinas. A s?ntese foi realizada atrav?s da metila??o do precursor (R)-[N-desmetil]PK11195 utilizando o [11C]CH3I (iodeto de metila marcado com carbono-11) sob as condi??es ?timas definidas: 400 ?L de dimetilsulf?xido (DMSO), aproximadamente 1 mg de precursor, aproximadamente 10 mg de hidr?xido de pot?ssio (KOH), a 40 ?C durante 1 minuto. A mistura de rea??o foi posteriormente neutralizada com ?cido clor?drico 1 mol.L-1 e dilu?da com fase m?vel (acetonitrila 76 % e ?gua Milli-Q 24 %). A mistura dilu?da foi injetada em sistema CLAE semi-preparativo para purifica??o. Extra??o de fase s?lida (atrav?s de cartuchos SepPak C18 ? Waters) foi utilizada para remo??o do solvente. A elui??o e formula??o do produto foi realizada com 0,7 mL de etanol e 6,3 mL de solu??o salina 0,9 % (est?ril e livre de pirog?nios). A esteriliza??o foi realizada atrav?s de filtra??o em membrana de poro de 0,22 ?m e, assim como o envase, ocorreu em fluxo laminar. As etapas do processo foram otimizadas e adequadas aos equipamentos e estrutura dos laborat?rios. O rendimento radioqu?mico do processo em rela??o ? atividade de [11C]CH3I de partida foi de 15,5?2,4 % (7471,5?1283,8 MBq; n=39). Todos os lotes produzidos foram aprovados quanto ?s especifica??es definidas e testadas no controle de qualidade, atingindo Pureza Radioqu?mica maior que 99 % e uma Atividade Espec?fica de 39,7?11,1 GBq/?mol, 10 minutos antes da inje??o. Todas as metodologias anal?ticas foram validadas e atenderam os crit?rios estabelecidos pela norma vigente. Tr?s lotes consecutivos foram produzidos e aprovados no controle de qualidade e o processo p?de ser considerado validado. O processo desenvolvido se mostrou adequado para produ??o do radiof?rmaco [11C]-(R)-PK11195 em doses suficientes para realiza??o de exames em pacientes no InsCer. A produ??o do radiof?rmaco marcado com carbono-11 no instituto disponibiliza mais uma ferramenta para melhoria dos servi?os de sa?de no pa?s e para melhoria da qualidade de vida dos pacientes.

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2016-08-03T18:19:52Z No. of bitstreams: 1 DIS_GUSTAVO_BELLANI_MIGOTT_COMPLETO.pdf: 6201853 bytes, checksum: 70237f84b98539735536877e1ffbc4e7 (MD5) / Made available in DSpace on 2016-08-03T18:19:52Z (GMT). No. of bitstreams: 1 DIS_GUSTAVO_BELLANI_MIGOTT_COMPLETO.pdf: 6201853 bytes, checksum: 70237f84b98539735536877e1ffbc4e7 (MD5) Previous issue date: 2016-02-29 / Tuberculosis is an infectious disease responsible for about 1.3 million deaths annually worldwide. Despite the appearance of multi-resistant strains of Mycobacterium tuberculosis (Mtb), since the 80?s there is a gap in the development of new antimicrobials. With the advent of bioinformatics and computational molecular biophysics, became possible to test, from a established molecular target, numerous molecules, especially associated with the prediction of binding free energy. In the current dissertation, were selected 14 compounds with recognized activity against the enzyme 2-trans-enoil-ACP redutase (InhA, EC 1.3.1.9) of Mtb. These molecules were divided into three groups. Set 1: 5 compounds with distant values of binding free energy. Set 2: 9 compounds with close binding free energy values and similar molecular structures (derived from Genz 10850). Set 3: 14 ligands, corresponding to the sum of the set?s 1 and 2. Sampling obtained from molecular dynamics and 2 ns of simulations, in explicit solvent, allowed to estimate the free energy of bind associated with the methods MM/GBSA, MM/PBSA, (QM)MM/GBSA, LigScore, DrugScore, AutoDock and SQM. The ranking of the compounds were based in the correlation (R2) between the predicted and experimental values. Results showed similar values of R2 in all tested methods. More accurate methods, such as SQM and (QM)/MM/GBSA, not obtained better correlations in comparison with simplified methods, as LigScore and DrugScore. In general, Set 1 obtained a moderate correlation (R2 of 0.00-0.80). Set?s 2 and 3, showed weak correlation (R2 < 0.40). Despite the satisfactory results of Set 1, the tested methods presented limitations in the ranking of compounds with close values of experimental binding free energy and similar molecular structures, as Set 2. / A tuberculose ? uma doen?a infectocontagiosa respons?vel por cerca de 1,3 milh?o de mortes anualmente a n?vel mundial. Apesar do aparecimento de cepas multirresistentes de Mycobacterium tuberculosis (Mtb), desde a d?cada de 80 se observa uma lacuna no desenvolvimento de novos antimicrobianos. Com o advento da bioinform?tica e biof?sica molecular computacional, tornou-se poss?vel, a partir de um alvo molecular preestabelecido, testar in?meros candidatos a f?rmacos, com destaque para a predi??o da energia livre de liga??o. Nesta disserta??o, foram selecionados 14 compostos com conhecida atividade frente a enzima 2-trans-enoil-ACP redutase (InhA, EC 1.3.1.9) de Mtb. Estas mol?culas foram divididas em 3 grupos. Grupo 1: 5 compostos com valores esparsos de energia livre. Grupo 2: 9 compostos com valores similares de energia livre e derivadas do composto Genz-10850. Grupo 3: 14 compostos correspondendo ? uni?o dos Grupos 1 e 2. Amostragens por simula??es de din?mica molecular de 2 ns, em solvente expl?cito, permitiram estimar os valores da energia livre de liga??o destes compostos com os m?todos MM/GBSA, MM/PBSA, (QM)MM/GBSA, LigScore, DrugScore, AutoDock e SQM. O ranqueamento dos compostos foi baseado na correla??o (R2) entre valores experimentais e estimados de energia livre. Os resultados apontaram valores de R2 similares entre os m?todos testados. T?cnicas mais robustas, como SQM e (QM)MM/GBSA, n?o obtiveram resultados mais acurados em compara??o ?quelas mais simples, como LigScore e DrugScore. No geral, foram obtidos valores moderados de correla??o (R2 de 0,00-0,80) para o Grupo 1. Os Grupos 2 e 3 exibiram correla??es fracas (R2 <0,40). Apesar dos resultados satisfat?rios para o Grupo 1, os m?todos utilizados apresentaram limita??es e n?o foram capazes de predizer e ranquear corretamente compostos com valores pr?ximos de energia livre e estruturas moleculares similares, como os do Grupo 2.

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.