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No / The protein O6-alkylguanine-DNA alkyltransferase (Atase) is responsible for the repair of DNA lesions generated by several clinically important anti-cancer drugs; this is manifest as active resistance in those cancer cell lines proficient in Atase expression. Novel O6-substituted guanine analogues have been synthesized, bearing acidic, basic and hydrogen bonding functional groups. In contrast to existing O6-modified purine analogues, such as methyl or benzyl, the new compounds were found to resist repair by Atase even when tested at concentrations much higher than O6-benzylguanine, a well-established Atase substrate active both in vitro and in vivo. The inactivity of the new purines as covalent substrates for Atase indicates that agents to deliver these groups to DNA would represent a new class of DNA-modifying drug that circumvents Atase-mediated resistance.

La néphropathie aux plantes chinoises (CHN) est une maladie rénale grave qui a été décrite pour la première fois en 1993 chez des patientes ayant suivi un régime amaigrissant à base d’extraits de plantes chinoises (Aristolochia fangchi) contenant des acides aristolochiques (AA). Cette néphropathie se caractérise par une atrophie tubulaire et une fibrose interstitielle aboutissant à l’urémie terminale et se complique fréquemment de cancers des voies urinaires. Au moment d’initier ce travail, il subsistait toujours un large débat quant au rôle étiologique réel des acides aristolochiques dans la genèse de cette maladie. En effet, les gélules à visée amaigrissante contenaient d’autres substances potentiellement néphrotoxiques. Mais surtout, il n’existait aucune preuve expérimentale que les AA pouvaient induire une fibrose rénale interstitielle. Dans la première partie de ce travail, nous démontrons que l’injection par voie sous-cutanée d’AA à la dose de 10 mg/Kg/jour à des rats Wistar mâles en déplétion sodée entraîne l’apparition au 35ème jour d’une atrophie tubulaire, d’une fibrose interstitielle et d’une insuffisance rénale, reproduisant ainsi les anomalies caractéristiques de la CHN. Nous avons ensuite montré que la dexfenfluramine, substance anorexigène à action de type sérotoninergique prise concomitamment par les patientes atteintes de CHN, ne potentialise pas la toxicité rénale des AA. Enfin, la stimulation du système rénine angiotensine (SRA) par la déplétion sodée ou l’inhibition de celui-ci par un traitement pharmacologique ne modifie pas la fibrose interstitielle ni l’insuffisance rénale induite par les AA. En conclusion, nous avons réussi à développer un modèle in vivo de fibrose rénale interstitielle induite par les AA. Dès lors nous avons apporté la preuve expérimentale de l’implication des AA dans le développement de la CHN. Ce modèle a permis de démontrer que les autres éléments potentiellement néphrotoxiques contenues dans la cure d’amaigrissement (dexfenfluramine, diurétique, laxatif) n’influençaient pas l’évolution de la fibrose interstitielle, ce qui confirme que la prise isolée d’AA suffit à expliquer le développement de la CHN. Cette confirmation à d’importantes implications en santé publique dans la mesure où des plantes contenant des acides aristolochiques font toujours partie des phytothérapies traditionnelles. De plus, il est apparu que, dans ce modèle, les mécanismes de la fibrose rénale interstitielle pouvaient être largement indépendants du SRA. Enfin, de par sa durée limitée et sa grande reproductibilité, ce modèle constitue un outil expérimental d’avenir pour l’étude des mécanismes physiopathologiques de la fibrose rénale interstitielle en général.

Background: Insulin resistance may play a pathogenic role in cardiovascular disease (CVD). Resistance to insulin has been associated with obesity, hypertension, and abnormal glucose and lipid metabolism. The constellation of these features among insulin resistant subjects has been called the metabolic syndrome. Prevalence of the metabolic syndrome increases with age and is most common in the elderly. Different criteria have been proposed to define the metabolic syndrome (ATP, WHO, AACE, EGIR). Current management of metabolic syndrome focuses on the specific risk factors that the patient may have without targeting the underlying insulin resistance. Angiotensin Converting Enzyme Inhibitors (ACEI) and Angiotensin Receptor Blockers (ARB) are widely used antihypertensive medications that may improve insulin sensitivity. We hypothesize that they can be used to reduce the long term cardiovascular complications in elderly hypertensive subjects with evidence of insulin resistance. In this study, we determined the effect of ACEI/ARB on the long term development of CVD in hypertensive non-diabetic elderly patients with the metabolic syndrome, as well as in patients with insulin resistance. Methods: Our research project utilizes the Cardiovascular Health Study (CHS) dataset. This dataset is a community based observational study where elderly participants were randomly selected and followed up for 11 years and the time to any cardiovascular event was recorded. In our project, we included hypertensive, non-diabetic individuals, with evidence of metabolic syndrome or insulin resistance, but had not experienced cardiovascular events at baseline. Cox regression model was used to evaluate the effect of ACEI/ARB on the time to the first cardiovascular event compared to the other antihypertensive medications adjusting for possible confounders such as age, race, gender, smoking status, triglycerides, LDL levels, systolic blood pressure, development of diabetes, congestive heart failure (CHF) and the number of anti-hypertensives. Results: In elderly hypertensive non-diabetic subjects with the metabolic syndrome according to the ATP and the WHO criteria, the hazard ratio for CVD associated with the use of ACEI/ARB was 0.65 or 0.68 (with 95 % C.I. of [0.45, 0.98], and [0.48, 0.96]) respectively when compared to the group exposed to the other anti-hypertensives. When the metabolic syndrome was defined according to the AACE and EGIR, the use of ACE/ARB was associated with hazard ratios for CVD equal to 0.74 and 0.899, respectively (with 95 % C.I. of [0.54, 1.09] and [0.61, 1.34]) compared to the use of the other anti-hypertensives. Hypertensive non-diabetic elderly subjects who were insulin resistant as evidenced by a HOMA-IR in the upper quartile, had a hazard ratio for CVD of 0.78 (95 % C.I. [0.56, 1.09]) associated with the use of ACEI/ARB compared to the use of other anti-hypertensives. Conclusions: The effect of ACEI/ARB on the development of cardiovascular events differs according to the definition of the metabolic syndrome. Elderly hypertensive patients with the metabolic syndrome, defined by ATP and WHO, seem to have lower risk of CVD with ACEI/ARB compared to the other antihypertensive medications. However, this association is not significant in elderly hypertensive patients in the upper quartile of HOMA and in patients with the metabolic syndrome as defined by AACE and EGIR criteria.

Xylella fastidiosa é uma bactéria patogênica encontrada em várias plantas. Esta bactéria secreta proteases extracelulares detectadas em gel de eletroforese, sendo a gelatina usada como substrato co-polimerizado. Três principais bandas protéicas foram detectadas com massa molar (MM) de 122, 84 e 65 kDa produzidas pelo isolado de citros (X0) e duas bandas de aproximadamente 84 e 65 kDa de isolado de videira (9713). Estas bactérias produziram zonas de hidrólise em meio sólido contendo gelatina, caseína e hemoglobina. Os resultados usando a gelatina como substrato foram os melhores para a atividade das proteases. A atividade enzimática das proteases de X. fastidiosa de citros e videira foi completamente inibida por PMSF e parcialmente inibida por EDTA, podendo ser visualizado em gel de eletroforese nativo. A temperatura ótima de atividade protéica foi de 30oC e o pH ótimo de 7,0. Além das proteases secretadas por este fitopatógeno, quitinase e β-1,3-glucanase foram também detectadas no sobrenadante das culturas. Os resultados sugeriram que estas proteases produzidas pela X. fastidiosa de citros e videira pertencem ao grupo das serina e metalo proteases. / Xylella fastidiosa is a pathogenic bacterium found in several plants. These bacteria secrete extracellular proteases into the culture broth as visualized in sodium-dodecyl-sulfate polyacrylamide activity gels containing gelatin as a co-polymerized substrate. Three major protein bands were produced by strain X0 (citrus) with molar masses (MM) of 122, 84 and 65 kDa. Grape strain 9713 produced two bands of approximately 84 and 64 kDa. These organisms produced zones of hydrolysis in agar plates amended with gelatin, casein and hemoglobin. Gelatin was the best substrate for these proteases. SDS-PAGE activity gel indicated that the protease activities of X. fastidiosa from citrus and grape were completely inhibited by PMSF and partially inhibited by EDTA. The optimal temperature for protease activity was 30oC with an optimal pH of 7.0. Among the proteolytic enzymes secreted by the phytopathogen, chitinase and β-1,3-glucanase activities were also detected in cultures of X. fastidiosa (citrus). From these results, it is suggested that these proteases produced by strains of X. fastidiosa from citrus and grape, belong to the serine- and metallo-protease group.

Nos últimos anos o tratamento da infecção crônica pelo HCV passou por importantes mudanças. Recentes avanços em biologia molecular proporcionaram o melhor conhecimento sobre a estrutura molecular do HCV e permitiram o desenvolvimento de moléculas que tem como alvo proteínas específicas integrantes do ciclo replicativo do vírus, denominados agentes antivirais de ação direta (DAAs). No Brasil, atualmente, os DAAs aprovados para o tratamento da Hepatite C são: Simeprevir (2ª geração de inibidor de protease), Daclatasvir (inibidor de NS5A) e o Sofosbuvir (inibidor análogo nucleotídeo de polimerase). A combinação dessas diferentes classes de DAAs permite maior eficácia no tratamento do HCV, reduz a duração do tratamento e o risco da emergência de resistência. Ao mesmo tempo em que o desenvolvimento de DAAs promete melhorar a chance de sucesso do tratamento dos pacientes crônicos infectados pelo HCV, a emergência de variantes associadas à resistência representa um grande desafio ao sucesso da terapia antiviral atualmente proposta e informações sobre a presença dessas mutações ainda são escassas. Este estudo tem como objetivo o mapeamento de variantes associadas à resistência (RAVs) primárias aos inibidores da polimerase (NS5B) do HCV em pacientes monoinfectados (HCV) e em pacientes coinfectados (HCV/HIV). Para tal, o rastreamento de substituições de aminoácidos foi realizado entre as posições 159 e 495 da proteína NS5B do HCV nas sequências de 244 pacientes infectados pelo HCV-1: 133 monoinfectados [1b (n=93); 1a (n=40)] e 111 coinfectados [1a (n=93); 1b (n=18)]. A ocorrência natural de RAVs nos resíduos S282, L320 e P495 não foi observada nas sequências analisadas neste estudo. As RAVs encontradas no grupo de monoinfectados foram: L159F (16,1% - 1b), C316N (16,3% - 1b) e A421V (21,4% - 1a; 3,2 - 1b) ; e no grupo de coinfectados foram: C316N (7,1% - 1b), V321A (1,6% - 1a), M414V (1,3% - 1a); A421V (23,7% - 1a; 6,3% - 1b), A421G (1,3% - 1a); Y448H (1,3% - 1a). Entre os pacientes monoinfectados, a região NS5B do HCV-1a apresentou menor número de variantes associadas à resistência (RAVs) quando comparada ao subtipo 1b, ao contrário do observado nos coinfectados. A variante C316N foi a única que ocorreu em combinação com outras variantes. Houve a ocorrência concomitante das variantes L159F e C316N em 8 pacientes monoinfectados pelo HCV-1b (8/56; 14,3%). Entre os coinfectados, foi observada a ocorrência concomitante das variantes C316N e A421V em apenas 1 paciente infectado pelo HCV-1b (1/14; 7,1%). A presença de RAVs foi detectada nas duas populações estudadas neste estudo. Contudo, outros estudos são necessários para que se possa avaliar o real impacto dessas mutacões na resposta ao tratamento / The treatment of chronic HCV infection has undergone important changes recently. Advances in molecular biology provided a better knowledge of the HCV molecular structure and allowed the development of molecules which target specific proteins that have important roles in the viral replicative cycle, known as direct action antiviral agents (DAAs). In Brazil, DAAs approved for treating hepatitis C are Simeprevir (2nd generation protease inhibitor), Daclatasvir (NS5A inhibitor) and sofosbuvir (nucleotide analogue NS5B polymerase inhibitor). The combination of these different DAAs classes leads to a greater efficacy in the treatment of HCV, reducing its duration and the risk of resistance associated variants (RAVs) emergence. DAAs increase the chance of successful treatment of HCV chronic infected patients. RAVs emergence represents a major challenge to the success of antiviral therapy. Nevertheless, data about the presence of these mutations are still scarce. The aim of this study was to verify the presence of primary RAVs associated to HCV polymerase inhibitors primary resistance in monoinfected (HCV) and coinfected (HCV / HIV) patients. For this purpose, amino acid substitutions identification was conducted between positions 159 and 495 of the HCV NS5B protein in the sequences of 244 patients with HCV-1: 133 monoinfected [1a (n=40); 1b (n=93)] and 111 coinfected [1a (n=93); 1b (n=18)]. Naturally occurring RAV in S282, L320 and P495 residues were not observed among the sequences analyzed in this study. RAVs found in monoinfected patients were L159F (16.1% - 1b), C316N (16.3% - 1b) and A421V (21.4% - 1a; 3.2% - 1b); while in co-infected group, the following RAVs were identified: C316N (7.1% - 1b), V321A (1.6% - 1a), M414V (1.3% - 1a); A421V (23.7% - 1a; 6.3% - 1b), A421G (1.3% - 1a); Y448H (1.3% - 1a). Among monoinfected patients, HCV-1a NS5B region showed fewer RAVs when compared to HCV-1b, conversely to what was observed in HIV coinfected patients. Variant C316N occurred in combination with other ones: there was the simultaneous occurrence of L159F and C316N variants 8/56 (14.3%) 8 HCV-1b monoinfected patients Among the coinfected patients, it was observed concomitant occurrence of C316N and A421V variant in only 1/14 (7.1%) HCV-1b infected patient. RAVs were detected in both HCV and HCV/HIV infected populations in this study. Further studies are required to assess the real impact of these changes in response to treatment

Não consta resumo na publicação. / An α-amylase inhibitor purified from black beans (Phaseolus vulgaris L.) prevents the utilization of starch either in normal or diabetic rats during short-time starch loading tests. It causes hypoglycaemia and alters accordingly the serum insulin and non esterified fatty acids levels. These effects are dose dependent and doses higher than 31 mg of the inhibitor can promote the complete smoothing on serum glucose level. The existence of starch in the presence of the inhibitor in the intestine doesn\'t alter the secretion or production of α-amylase by the pancreas.

Xylella fastidiosa é uma bactéria patogênica encontrada em várias plantas. Esta bactéria secreta proteases extracelulares detectadas em gel de eletroforese, sendo a gelatina usada como substrato co-polimerizado. Três principais bandas protéicas foram detectadas com massa molar (MM) de 122, 84 e 65 kDa produzidas pelo isolado de citros (X0) e duas bandas de aproximadamente 84 e 65 kDa de isolado de videira (9713). Estas bactérias produziram zonas de hidrólise em meio sólido contendo gelatina, caseína e hemoglobina. Os resultados usando a gelatina como substrato foram os melhores para a atividade das proteases. A atividade enzimática das proteases de X. fastidiosa de citros e videira foi completamente inibida por PMSF e parcialmente inibida por EDTA, podendo ser visualizado em gel de eletroforese nativo. A temperatura ótima de atividade protéica foi de 30oC e o pH ótimo de 7,0. Além das proteases secretadas por este fitopatógeno, quitinase e β-1,3-glucanase foram também detectadas no sobrenadante das culturas. Os resultados sugeriram que estas proteases produzidas pela X. fastidiosa de citros e videira pertencem ao grupo das serina e metalo proteases. / Xylella fastidiosa is a pathogenic bacterium found in several plants. These bacteria secrete extracellular proteases into the culture broth as visualized in sodium-dodecyl-sulfate polyacrylamide activity gels containing gelatin as a co-polymerized substrate. Three major protein bands were produced by strain X0 (citrus) with molar masses (MM) of 122, 84 and 65 kDa. Grape strain 9713 produced two bands of approximately 84 and 64 kDa. These organisms produced zones of hydrolysis in agar plates amended with gelatin, casein and hemoglobin. Gelatin was the best substrate for these proteases. SDS-PAGE activity gel indicated that the protease activities of X. fastidiosa from citrus and grape were completely inhibited by PMSF and partially inhibited by EDTA. The optimal temperature for protease activity was 30oC with an optimal pH of 7.0. Among the proteolytic enzymes secreted by the phytopathogen, chitinase and β-1,3-glucanase activities were also detected in cultures of X. fastidiosa (citrus). From these results, it is suggested that these proteases produced by strains of X. fastidiosa from citrus and grape, belong to the serine- and metallo-protease group.

Nos últimos anos o tratamento da infecção crônica pelo HCV passou por importantes mudanças. Recentes avanços em biologia molecular proporcionaram o melhor conhecimento sobre a estrutura molecular do HCV e permitiram o desenvolvimento de moléculas que tem como alvo proteínas específicas integrantes do ciclo replicativo do vírus, denominados agentes antivirais de ação direta (DAAs). No Brasil, atualmente, os DAAs aprovados para o tratamento da Hepatite C são: Simeprevir (2ª geração de inibidor de protease), Daclatasvir (inibidor de NS5A) e o Sofosbuvir (inibidor análogo nucleotídeo de polimerase). A combinação dessas diferentes classes de DAAs permite maior eficácia no tratamento do HCV, reduz a duração do tratamento e o risco da emergência de resistência. Ao mesmo tempo em que o desenvolvimento de DAAs promete melhorar a chance de sucesso do tratamento dos pacientes crônicos infectados pelo HCV, a emergência de variantes associadas à resistência representa um grande desafio ao sucesso da terapia antiviral atualmente proposta e informações sobre a presença dessas mutações ainda são escassas. Este estudo tem como objetivo o mapeamento de variantes associadas à resistência (RAVs) primárias aos inibidores da polimerase (NS5B) do HCV em pacientes monoinfectados (HCV) e em pacientes coinfectados (HCV/HIV). Para tal, o rastreamento de substituições de aminoácidos foi realizado entre as posições 159 e 495 da proteína NS5B do HCV nas sequências de 244 pacientes infectados pelo HCV-1: 133 monoinfectados [1b (n=93); 1a (n=40)] e 111 coinfectados [1a (n=93); 1b (n=18)]. A ocorrência natural de RAVs nos resíduos S282, L320 e P495 não foi observada nas sequências analisadas neste estudo. As RAVs encontradas no grupo de monoinfectados foram: L159F (16,1% - 1b), C316N (16,3% - 1b) e A421V (21,4% - 1a; 3,2 - 1b) ; e no grupo de coinfectados foram: C316N (7,1% - 1b), V321A (1,6% - 1a), M414V (1,3% - 1a); A421V (23,7% - 1a; 6,3% - 1b), A421G (1,3% - 1a); Y448H (1,3% - 1a). Entre os pacientes monoinfectados, a região NS5B do HCV-1a apresentou menor número de variantes associadas à resistência (RAVs) quando comparada ao subtipo 1b, ao contrário do observado nos coinfectados. A variante C316N foi a única que ocorreu em combinação com outras variantes. Houve a ocorrência concomitante das variantes L159F e C316N em 8 pacientes monoinfectados pelo HCV-1b (8/56; 14,3%). Entre os coinfectados, foi observada a ocorrência concomitante das variantes C316N e A421V em apenas 1 paciente infectado pelo HCV-1b (1/14; 7,1%). A presença de RAVs foi detectada nas duas populações estudadas neste estudo. Contudo, outros estudos são necessários para que se possa avaliar o real impacto dessas mutacões na resposta ao tratamento / The treatment of chronic HCV infection has undergone important changes recently. Advances in molecular biology provided a better knowledge of the HCV molecular structure and allowed the development of molecules which target specific proteins that have important roles in the viral replicative cycle, known as direct action antiviral agents (DAAs). In Brazil, DAAs approved for treating hepatitis C are Simeprevir (2nd generation protease inhibitor), Daclatasvir (NS5A inhibitor) and sofosbuvir (nucleotide analogue NS5B polymerase inhibitor). The combination of these different DAAs classes leads to a greater efficacy in the treatment of HCV, reducing its duration and the risk of resistance associated variants (RAVs) emergence. DAAs increase the chance of successful treatment of HCV chronic infected patients. RAVs emergence represents a major challenge to the success of antiviral therapy. Nevertheless, data about the presence of these mutations are still scarce. The aim of this study was to verify the presence of primary RAVs associated to HCV polymerase inhibitors primary resistance in monoinfected (HCV) and coinfected (HCV / HIV) patients. For this purpose, amino acid substitutions identification was conducted between positions 159 and 495 of the HCV NS5B protein in the sequences of 244 patients with HCV-1: 133 monoinfected [1a (n=40); 1b (n=93)] and 111 coinfected [1a (n=93); 1b (n=18)]. Naturally occurring RAV in S282, L320 and P495 residues were not observed among the sequences analyzed in this study. RAVs found in monoinfected patients were L159F (16.1% - 1b), C316N (16.3% - 1b) and A421V (21.4% - 1a; 3.2% - 1b); while in co-infected group, the following RAVs were identified: C316N (7.1% - 1b), V321A (1.6% - 1a), M414V (1.3% - 1a); A421V (23.7% - 1a; 6.3% - 1b), A421G (1.3% - 1a); Y448H (1.3% - 1a). Among monoinfected patients, HCV-1a NS5B region showed fewer RAVs when compared to HCV-1b, conversely to what was observed in HIV coinfected patients. Variant C316N occurred in combination with other ones: there was the simultaneous occurrence of L159F and C316N variants 8/56 (14.3%) 8 HCV-1b monoinfected patients Among the coinfected patients, it was observed concomitant occurrence of C316N and A421V variant in only 1/14 (7.1%) HCV-1b infected patient. RAVs were detected in both HCV and HCV/HIV infected populations in this study. Further studies are required to assess the real impact of these changes in response to treatment

Não consta resumo na publicação. / An α-amylase inhibitor purified from black beans (Phaseolus vulgaris L.) prevents the utilization of starch either in normal or diabetic rats during short-time starch loading tests. It causes hypoglycaemia and alters accordingly the serum insulin and non esterified fatty acids levels. These effects are dose dependent and doses higher than 31 mg of the inhibitor can promote the complete smoothing on serum glucose level. The existence of starch in the presence of the inhibitor in the intestine doesn\'t alter the secretion or production of α-amylase by the pancreas.