Oxidative stress is caused by overproduction and overaccumulation of ROS (reactive oxygen species). This state is responsible for cellular damage during unfavorable environmental conditions such as drought, low temperatures, salinity. In order to directly study oxidative stress at tobacco plants (Nicotiana tabacum cv. Xanthi) I used specific herbicides, MV (methyl viologen) and 3-AT (3- aminotriazole). There were several markers used for monitoring oxidative damage to cellular components: DNA damage detected by a comet assay, lipid peroxidation, carbonylated proteins and modification of activities of antioxidant enzymes CAT (catalase) and APX (ascorbate peroxidase). Fluorescent microscopy documented changes in a redox state of tobacco cells and a specific signal for peroxisomes was observed after treatment with higher concentrations of MV and 3-AT. Application of both herbicides caused significant DNA damage, while they worked in a different concentrations, MV in µM and 3-AT in mM. Another convincing oxidative stress marker for MV was protein carbonylation. The inhibition of antioxidant enzymes CAT and APX was less significant when compared to the effects of 3-AT. Decreasing membrane stability proved to be an universal oxidative stress marker for both herbicides. On the other hand, lipid...

The aim of this thesis was to study the changes that occur in cell line H9c2 after exposure to an oxygen level reduced to 2 % for 24 hours. We monitored changes in compartmentation of chosen members of β-adrenergic signaling system. We found an increase in expression of β1AR and β2AR. Only β2AR showed change in compartmentation after hypoxia, as they relocate from membrane rafts to non-rafts fractions of membrane. AC also showed an increase of expression and was located in membrane rafts. The next aim of this work was to monitore apoptotic markers to determine whether there are activated pro-apoptotic or anti-apoptotic signals under chosen conditions of hypoxia. There was an increase in expression of both pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2. We compare ratios of Bcl-2 to Bax and we found that there is a bigger increase in protein Bax expression. Another apoptotic marker, caspase 3, was tested and we also found that there was an increase in expression of caspase 3 in cells after hypoxia. Furthermore, we studied possible activation of kinase signaling pathways that may contribute to protective effects of hypoxia. Expression of Akt and ERK kinases was increased after hypoxia, but we did not confirm activation by phosphorylation of these kinases. Levels of phosphorylated Akt...

Cancer is a leading cause of death in the western world and is increasing in frequency world-wide. Although diagnosis, treatment and therapeutic approaches to cancer have improved, many types of cancer are still lethal due to the lack of radical treatment. One of the fatal neoplastic disease types with poor prognosis is represented by malignant mesothelioma (MM). MM is characterised by very high mortality rate and limited therapeutic options. The etiology of the disease is mainly associated with exposure to asbestos fibres. The incidence of MM is increasing in many countries. The search for novel molecular targets, anti-cancer strategies and drugs, which would considerably improve the treatment is of great importance. Certain new drugs, especially those with specific molecular targets, show high selectivity in their action to cancer cells, and have considerably increased the cure rate in some types of cancer. Mitochondria have recently emerged as a very promising target for anti-cancer agents. A group of compounds with anti-cancer activity that induce apoptosis by way of mitochondrial destabilisation, termed 'mitocans', have been a recent focus of research. Several mitocans have been shown to selectively induce apoptosis in cancer cells and suppress the growth of many types of carcinomas in...

Neutrophil activation is the most important initial immune defense against invading microbes in newborn infants. The reduced neutrophil migration and uncontrolled regulation of reactive oxygen species (ROS) production observed in neonates, could result in a diminished infectious response or in tissue damage. The aims were to study neutrophil chemotactic response towards IL-8 and fMLP in term neonates; to examine neutrophil receptor expression involved in adhesion, migration, phagocytosis and complement after stimulation with IL-8 and fMLP in term neonates; and to investigate neutrophil production of ROS, induced by PMA and E.coli, after preincubation with IL-8 and fMLP in term and preterm newborn infants. Comparisons were made to neutrophils from healthy adults. Chemotaxis was distinguished from randomly migrating neutrophils, and the neutrophil migration distance and the number of migrating neutrophils per distance was evaluated. Neutrophils were labeled with antibodies to cell surface antigens (CD11b, CD18, CD65, CD15S, CD162, CD44, CD35, CD88, CD181, CD182 and CD64) after stimulation with IL-8 and fMLP. After preincubation of neutrophils with fMLP or IL-8 and stimulation with PMA or E.coli, respiratory burst was detected. The same analyses were also made in preterm infants (median 25+3weeks GA; range 23+0–29+2) within 3 days postnatal age. Neutrophils from neonates exhibited different migratory and receptor responses to IL-8 and fMLP, with a diminished response towards IL-8 in term newborn infants in terms of reduced chemotaxis and modulation of receptors involved in adhesion, chemotaxis, complement and phagocytosis as compared to adults. fMLP reduced PMA- and E.coli-induced respiratory burst in neutrophils from term neonates and adults. The reduced respiratory burst by fMLP may be a mechanism for reducing the detrimental effects of uncontrolled inflammation. Although a similar burst reduction was observed in preterm infants born >25 weeks GA with fMLP, a diminished neutrophil respiratory burst modulation in very preterm infants cannot be excluded and requires further studies at different gestational and postnatal ages.

L’infection par le virus de l’hépatite C (VHC) évolue généralement vers le développement d’une hépatite C chronique, fréquemment associée à des perturbations métaboliques, (insulinorésistance et stéatose), et de la structure hépatique (fibrose et cirrhose), favorisant le développement d’un hépatocarcinome. Le stress oxydant semble étroitement lié à la progression de ces pathologies, notamment l’insulino-résistance. Or, les mécanismes moléculaires par lesquels le VHC module le stress oxydant restent confus, ainsi que l’effet réciproque du stress oxydant sur la réplication virale. Jusqu’à récemment, la modulation de ce stress a été étudiée in vitro dans des modèles non réplicatifs du VHC ou dans des modèles d’expression ectopiques des protéines virales. Peu d’études ont encore tenté de caractériser l’effet d’une infection complète et productive dans les cellules cibles naturelles du VHC, les hépatocytes. Mes travaux de recherche ont donc consisté à étudier la modulation de la balance redox cellulaire dans un modèle d’infection complet de type HCVcc, et dans les cellules hépatocytaires Huh7.5. Cette étude a permis de caractériser le stress oxydant dans ce modèle, mais surtout d’identifier un antioxydant à activité pro-virale, la glutathion peroxydase 4 (GPx4). En effet, nous avons observé une stimulation viro-induite de l’expression et de l’activité de GPx4, seule enzyme capable de détoxifier les lipides membranaires oxydés. L’expression de cette enzyme semble nécessaire à la réplication ainsi qu’à l’infectivité virale. Son importance pour le VHC résiderait dans son activité catalytique permettant de maintenir l’intégrité des lipides cellulaires nécessaire au cycle viral / Chronic infection with Hepatitis C virus (HCV) is frequently associated with metabolic disturbances (insulin-resistance and steatosis) as well as with changes to hepatic structure (fibrosis and cirrhosis) that favor hepatocellular carcinogenesis. Insulin resistance is in particular linked to oxidative stress, which is thought to play a key role in driving disease progression. However, molecular mechanisms by which HCV regulates oxidative stress are still unclear and reciprocally, the effect of oxidative stress on viral life cycle is not well understood. Until recently, induction of oxidative stress by HCV has mainly been investigated in non replicative in vitro models or in cell systems expressing viral proteins alone. Few studies have yet investigated oxidative stress in the context of productive HCV infection. My work consisted of studying the modulation of the cellular redox system using the HCVcc infection model, based on a replicative HCV isolate and the hepatoma cell line Huh7.5. This work provided a broad characterization of how HCV induces and prevents oxidative stress and identified glutathion peroxidase 4 (GPx4) as a pro-viral antioxydant enzyme. Indeed, we observed an HCVinduced upregulation of expression and activity of GPx4. We also demonstrated that GPx4 expression is required for viral replication and infectivity. As GPx4 possesses a particular catalytic activity, which is the detoxification of oxidized membrane lipids, we investigated the impact of the accumulation of oxidized lipids on HCV replication. These studies showed that GPx4 is an important host factor for HCV life cycle by maintaining membrane lipid integrity in an oxidative cellular environment

A doença renal crônica (DRC) é a afecção renal mais frequente em cães e caracteriza-se pela progressiva redução do número de néfrons funcionais. Com a evolução da doença, os cães podem apresentar um conjunto de manifestações clínicas denominada uremia. O suporte nutricional objetiva atenuar os efeitos do estado urêmico, retardar a progressão da doença e melhorar a qualidade de vida dos animais. O presente estudo objetivou avaliar os efeitos da dieta coadjuvante e manejo dietético no metabolismo de cálcio e fósforo [fósforo, cálcio total (CaT), cálcio iônico (Cai), paratormônio (PTH) e FGF-23 séricos], escore de condição corporal (ECC), escore de massa muscular (EMM), concentrações séricas de aminoácidos (AAs) e citocinas inflamatórias (CIT), bem como a capacidade antioxidante total (CAT) de cães com DRC alimentados com dieta coadjuvante. Foram selecionados 10 cães com DRC estádios 3 e 4 (IRIS, 2015) provenientes do atendimento do Hospital Veterinário da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo. As variáveis PTH, FGF-23, AAs, CIT e CAT foram avaliadas no início do estudo (T0) e após 6 meses de manejo dietético (T6). As determinações séricas de ureia, creatinina, CaT, Cai e fósforo; ECC e EMM foram determinadas em T0 e a cada 30 dias, durante os 6 meses de estudo. Para a análise dos resultados, testes estatísticos paramétricos e não paramétricos foram empregados e valores de p&lt;0,05 foram considerados significativos. As concentrações séricas de ureia, fósforo, CaT, Cai, IL-6, IL-10, TNF-&#945;, CAT, PTH e FGF-23 não apresentaram diferença entre os momentos T0 e T6. A creatinina elevou-se em T6 (p=0,0022). Os AAs fenilalanina, triptofano e ornitina decresceram no tempo T6 (p=0,0273; p=0,0253; p=0,0443, respectivamente) e a hidroxiprolina aumentou em T6 (p=0,0073). As concentrações séricas de PTH apresentaram correlação com a creatinina e ureia (r=0,45, p&lt;0,05; r=0,67, p&lt;0,01; respectivamente). O Cai apresentou correlação negativa com a ureia (r=-0,59, p&lt;0,01) e o fósforo sérico apresentou correlação positiva com o FGF-23 (r=0,51; p&lt;0,05). A ureia e creatinina apresentaram correlação positiva (r=0,62; p&lt;0,01). De acordo com os resultados encontrados, conclui-se que a dieta e o manejo nutricional empregados foram eficazes no controle do hiperparatireoidismo secundário renal, estresse oxidativo, marcadores inflamatórios e na manutenção do escore de condição corporal, massa muscular e nutrição proteica dos cães avaliados. / Chronic kidney disease (CKD) is the most common kidney disease in dogs and characterized by progressive reduction in the number of functional nephrons. With the evolution of the disease the dogs may present a set of clinical manifestations denominated uremia. The efforts of nutritional support are to mitigate the effects of uremic state, slow the progression of the disease and improve the quality of life of the animals. The aim of this study was to evaluate the effects of prescription diet and dietary management on the metabolism of calcium and phosphorus [phosphorus, total calcium (CaT), ionized calcium (Cai), parathyroid hormone (PTH) and FGF-23 in the serum], body condition score (BCS), muscle mass score (MME), serum concentrations of amino acids (AAs) and inflammatory cytokines (CYT), as well as the total antioxidant capacity (TAC) of dogs with CKD fed with a coadjuvant diet. Were selected 10 dogs with CKD stage 3 and 4 (IRIS, 2015), from the Veterinary Hospital at School of Veterinary Medicine and Animal Science, University of São Paulo. The PTH, FGF-23, AAs, CYT and TAC variables were evaluated at baseline (T0) and after 6 months of dietary management (T6). The serum determinations of urea, creatinine, CaT, Cai and phosphorus; BCS and MMS were determined at T0 and every 30 days, for 6 months. For the analysis of the results, parametric and non-parametric statistical tests were used and values of p<0.05 were considered significant. Serum concentrations of urea, phosphorus, CaT, Cai, IL-6, IL-10, TNF-&#945;, CAT, PTH, FGF-23 did not differ between T0 and T6. Serum creatinine increased in T6 (p=0.0022). The AAs phenylalanine, tryptophan and ornithine decreased in T6 (p=0.0273; p=0.0253; p=0.0443, respectively) and hydroxyproline increased in T6 (p=0.0073). Serum PTH concentrations correlate positively with the concentrations of creatinine and urea (r=0.45, p&lt;0.05; r=0.67, p&lt;0.01; respectively). The Cai was negatively correlated with urea (r = -0.59, p&lt;0.01) and serum phosphorus was positively correlated with FGF-23 (r = 0.51; p&lt;0.05). The urea and creatinine were positively correlated (r=0.62; p&lt;0.01). It was concluded that diet and nutritional management were effective in the control of renal secondary hyperparathyroidism, oxidative stress, inflammatory markers and maintenance of body condition score, muscle mass and protein nutrition in the evaluated dogs.

O amendoim (Arachis hypogaea L.) é um grão muito apreciado em todo o mundo, sendo cultivado em vários países desenvolvidos e em desenvolvimento. A planta é uma oleaginosa com alto valor nutricional e rica em compostos bioativos, cultivada largamente em zonas de clima semiárido, onde prevalece a seca, que pode afetar a produção e a composição dos grãos. O objetivo deste trabalho foi avaliar a composição química, atividade antioxidante, compostos fenólicos, tocoferóis e ácidos graxos de genótipos de amendoim contrastantes quanto à adaptação a ambientes com limitação hídrica, bem como definir quais atributos são capazes de caracterizar esses genótipos. Os genótipos utilizados foram oriundos do programa de melhoramento genético de amendoim da Embrapa Algodão (Campina Grande - PB), os quais são adaptados ao estresse hídrico. Foram analisados grãos com e sem película, uma vez que seu consumo pode ocorrer de ambas as formas. Para os grãos sem a película a caracterização da fração oleosa foi realizada por meio da identificação e quantificação dos ácidos graxos e tocoferóis, enquanto que a avaliação química da fração fenólica foi feita por espectrometria de massas de alta resolução (LC-ESI-LTQ-Orbitrap-MS). A atividade antioxidante foi determinada por meio da desativação de espécies reativas de oxigênio (superóxido, ácido hipocloroso e peroxila) e do método on-line (HPLC-ABTS). Os resultados da caracterização da fração oleosa e fenólica dos grãos despeliculados foram ainda avaliados por análise multivariada para se definir quais atributos eram capazes de caracterizar os diferentes genótipos de amendoim. Para os grãos com a película foi realizada a caracterização química dos polifenóis por LC-ESI-LTQ-Orbitrap-MS, e a avaliação da atividade antioxidante pelo sequestro dos radicais livres DPPH e ABTS, além da análise multivariada dos resultados. De maneira geral, os genótipos tolerantes à seca foram os que apresentaram maior capacidade antioxidante. Os genótipos de amendoim avaliados apresentaram de 36 a 58% de ácidos graxos monoinsaturados em sua constituição, e relação oleico/linoleico mais elavada para os genótipos rasteiros. Foram também encontradas quantidades significativas de tocoferóis nos materiais analisados. O ácido p-cumárico, ácido cafeíco e a rutina foram identificados e quantificados por cromatografia líquida de alta resolução. Foram identificados, por meio da técnica de espectrometria de massas de alta resolução, 26 compostos fenólicos nos genótipos de amendoim despeliculados. Os grãos com película apresentaram perfil fenólico mais complexo, sendo identificados 58 compostos pertencentes a cinco classes distintas. Os isômeros do ácido coutárico foram os principais polifenóis, seguidos pela isorhamnetina-3-O-rutinosideo e os isômeros de ácido cumárico. De acordo com os resultados obtidos e a literatura, foram identificados 9 compostos nunca relatados em amendoim. As variáveis analisadas neste estudo mostraram ser possível o agrupamento dos genótipos de amendoim contrastantes quanto à adaptação aos ambientes com restrição hídrica. / Peanuts (Arachis hypogaea L.) are a widely appreciated grains throughout the world and it has been grown both in developed and developing countries. The plant is an oleaginous with high nutritional value and rich in bioactive compounds that has been cultivated in semi-arid zones - where drought prevails and it affects grain yield and composition. Thus, the aim of this work was in evaluating the chemical composition, antioxidant activity, phenolic compounds, tocopherols and fatty acids of contrasting peanut genotypes in relation to the adaptation to water limitation environments, as well as defining which attributes are able to characterize these genotypes. The adapted peanuts to water stress used here came from the breeding program of Embrapa Algodão (Campina Grande - PB). Grains with and without skin were analyzed, since their consumption can occur in both forms. In relation to the grains without the skin, the characterization of the oily fraction was made through the identification and quantification of the fatty acids and tocoferols, whereas the chemical evaluation of the phenolic fraction was performed by high-resolution mass spectrometry (LC-ESI-LTQ-Orbitrap-MS). The antioxidant activity was determined by reactive oxygen species deactivation (superoxide, hypochlorous acid and peroxyl) and on-line method (HPLC-ABTS). The results obtained through characterization of the oily and phenolic fraction of the grains without skin were further evaluated by multivariate analysis to determine which attributes were able to characterize the different peanut genotypes. In relation to the grains with skin, it was made the chemical characterization of the polyphenols by LC-ESI-LTQ-Orbitrap-MS and the evaluation of the antioxidant activity by the sequestration of the radicals DPPH and ABTS; besides, it was made the multivariate analysis of the results. In general, the samples tolerant to drought were the ones that presented greater antioxidant capacity than the others. The evaluated peanut genotypes presented 36 to 58% of monounsaturated fatty acids in relation to the total lipids in its constitution and showed oleic/linoleic ratio classified in normal and mid-oleic, which is a higher ratio for non-drought tolerant genotypes. Significant amounts of tocopherols were also found in the analyzed materials. p-coumaric acid, caffeic acid and rutin were identified and quantified by high-performance liquid chromatography. Twenty-six phenolic compounds were also identified in peanuts without skin using the high-resolution mass spectrometry technique. The peanuts with skin showed a more complex phenolic characteristc by being identified 58 compounds belonging to five different classes. The isomers of coutaric acid were the major polyphenols, followed by isorhamnetin-3-O-rutinoside and the isomers of coumaric acid. According to the results obtained and the literature, it is been identified 9 new compounds which were never found in peanuts. The variables analyzed in this study showed that it is possible to group contrasting genotypes peanuts as to drought tolerance.

Lesão do DNA mitocondrial (mtDNA) promove disfunção desta organela, contribuindo para a gênese do envelhecimento e fisiopatologia de doenças como aterosclerose e diabetes. A mitocôndria é a principal fonte quantitativa de espécies reativas de oxigênio (ROS) em células, e o complexo NAD(P)H oxidase a principal fonte de ROS envolvidas na sinalização celular. A possível inter-relação entre estas duas importantes vias produtoras de ROS não está definida. O objetivo deste estudo foi investigar o perfil de alterações na expressão e atividade da NAD(P)H oxidase de células musculares lisas vasculares (VSMC) em resposta a perturbações mínimas da função mitocondrial análogas às esperadas em doenças crônico-degenerativas vasculares. Inicialmente, validamos modelo in vitro de disfunção mitocondrial induzida por incubação de VSMC com brometo de etídio (24 - 72 h). Lesões mínimas do mtDNA foram documentadas por alterações nos produtos de amplificação (PCR) da região repetitiva da D-loop e redução da taxa de consumo de oxigênio total em ~15% vs. basal (p<0,05). Este grau de lesão não foi suficiente para induzir alterações morfológicas evidentes ou apoptose, e foi associado ao retardo de 25 - 30% no aumento de população celular induzido por soro fetal bovino. Nestas condições, não se detectou aumento da produção basal de superóxido ou mudanças nos níveis de glutationa, óxidos de nitrogênio, ou da atividade superóxido dismutase. A produção basal de peróxido de hidrogênio aumentou ~15%. Após disfunção mitocondrial, houve significativo aumento (30 - 45%) na atividade basal do complexo NAD(P)H oxidase em fração de membrana de VSMC. Entretanto, a ativação da oxidase pela AII, conhecido agonista da oxidase vascular, foi essencialmente abolida, indicando dependência funcional da ativação da oxidase com a integridade da mitocôndria. Em sintonia com esses dados, na condição basal, ocorreu aumento de expressão da isoforma Nox4 da oxidase, enquanto o aumento do mRNA da Nox1 normalmente visto após AII foi minimizado. Por outro lado, o aumento da atividade da NADPH oxidase causado pelo estressor do RE tunicamicina (indutor de Nox4) foi também abolido pela disfunção mitocondrial, entretanto, ocorreu aumento do mRNA da Nox4, indicando que as alterações funcionais da oxidase nesta situação não decorrem apenas de mudanças da expressão. Dissociação semelhante entre expressão e atividade ocorreu após exposição de 72 horas ao EtBr (i.e., durante adaptação). Nesta, ocorreu maior expressão do mRNA de Nox1 e Nox4 com AII, sem aumento da atividade da oxidase em membranas. Incubação do EtBr por 24 horas não induziu per se aumento consistente nos índices de estresse do RE e induziu inversão do padrão do tráfego subcelular da dissulfeto isomerase protéica (PDI), uma chaperona redox descrita recentemente como reguladora da NADPH oxidase. Após 72 horas de incubação com EtBr, a expressão de chaperonas marcadoras de estresse do RE foi bastante diminuída e o tráfego da PDI teve o padrão restaurado. Demonstramos por microscopia confocal evidências preliminares de possível co-localização entre Nox1 e mitocôndria. Estes dados sugerem uma relevante inter-relação funcional entre mitocôndria e complexo NAD(P)H oxidase, associada pelo menos a alterações de expressão e/ou tráfego subcelular de subunidades catalíticas e reguladoras desse complexo. / Mitochondrial DNA (mtDNA) damage induces dysfunction of this organelle, contributing to the genesis of aging and to the pathophysiology of diseases such as atherosclerosis and diabetes. Mitochondria are the main quantitative source of reactive oxygen species (ROS) in cells, while NAD(P)H oxidase complex is a major source of cell signaling-associated ROS. The possible crosstalk between these two relevant sources of ROS is unclear. The aim of this study was to investigate changes in activity and/or expression of vascular smooth muscle cell (VSMC) NAD(P)H oxidase in response to minor perturbations of mitochondrial function similar to those expected to occur in chronic degenerative vascular diseases. Initially, we validated an in vitro model of mitochondrial dysfunction in VSMC, through incubation with ethidium bromide (24 - 72 h). Minimal mtDNA damage after EtBr was shown by distinct amplification patterns (at PCR) of D-loop repetitive region and by ~ 15% oxygen consumption decrease vs. basal (p<0.05). Such mtDNA damage was not sufficient to induce morphologic changes or apoptosis, whereas serum-stimulated increase in cell number was prevented by 25-30%. Under those conditions, baseline superoxide production, as well as levels of glutathione or nitrogen oxides or superoxide dismutase activity were unchanged. Baseline hydrogen peroxide production increased ~15%. VSMC membrane fraction NADPH oxidase activity was increased by 30-45% after mitochondrial dysfunction. However, oxidase activation due to AII (100 nM, 4h) was markedly abrogated, indicating that A-II-driven oxidase activation requires integrity of mitochondrial function. Accordingly, there were increases in baseline mRNA expression of Nox4 oxidase isoform, while the expected increase in Nox1 by AII was minimized. On the other hand, the NADPH oxidase activity induced by the endoplasmic reticulum stressor tunicamycin (Nox4 inducer) after mitochondrial dysfunction was abrogated, however simultaneously with increased Nox4 mRNA, thus indicating that the observed functional alterations in the oxidase complex in these conditions cannot be associated only to mRNA expression changes. After VSMC EtBr incubation for 72 h, similar dissociation between expression and activity was observed, with increase in Nox 1 and Nox4 mRNA by AII, without parallel increase in membrane fraction oxidase activity. Although there was little change in ER stress markers after 24h EtBr, protein disulfide isomerase (PDI), a redox chaperone recently described by us as a novel NAD(P)H oxidase regulator, exhibited a reversal of its subcellular traffic pattern. After 72 h EtBr, the expression of ER markers was strongly decreased and normal PDI traffic was restored. Confocal microscopy suggested possible co-localization between Nox1 and mitochondria. These results suggest a functionally relevant crosstalk between mitochondria and NADPH oxidase complex associated at least to changes in expression and/or subcellular traffic of catalytic or regulatory subunits of this complex.

Neste trabalho alguns complexos diimínicos de cobre(II) com ligantes do tipo base de Schiff, já estudados em nosso laboratório, além de um novo complexo de zinco foram sintetizados e caracterizados por análise elementar, espectroscopias UV/Vis, Infravermelho e de Ressonância Paramagnética Eletrônica (EPR). Estudos sobre a reatividade destes complexos de cobre(II) frente à oxidação de carboidratos por oxigênio molecular foram então realizados. Estes estudos envolveram várias etapas, com variação das concentrações do catalisador, do substrato e do tampão e variação do pH, verificando-se a influência de cada um destes fatores na cinética de reação. Para tentar entender a interação da albumina bovina (BSA) com os complexos diimínicos de cobre(II) estudados, foram realizados experimentos utilizando-se a técnica de dicroísmo circular e espectroscopia eletrônica. A estabilidade termodinâmica relativa dos vários compostos foi assim determinada, estimando-se os respectivos valores das constantes de estabilidade. Experimentos sobre a atividade oxidante destes complexos frente à albumina bovina, causando danos oxidativos à proteína, também foram efetuados, determinando-se a formação de grupos carbonil na proteína, que foram monitorados espectrofotometricamente, através da obtenção das correspondentes dinitrofenil-hidrazonas, pela reação com dinitrofenil-hidrazina (DNPH). Através da técnica de EPR, foram realizados estudos para a detecção e identificação de espécies radicalares na interação da albumina com os complexos de cobre(II) em presença de peróxido de hidrogênio, com o uso de um captador de spin apropriado. Estudos sobre as possíveis interações dos complexos de cobre(II) com a albumina humana (HSA) e com o plasma sanguíneo foram realizados através das técnicas de EPR e SDS-PAGE. Além disso, a reatividade dos complexos de cobre(II) frente a glutationa, um agente redutor presente em alta concentração no citosol, foi investigada através de experimentos de fluorescência. Finalmente, a influência dos complexos imínicos de cobre(II) sobre a respiração mitocondrial foi investigada, já que estes complexos se mostraram indutores eficientes de apoptose frente a diferentes células tumorais. Para este estudo utilizou-se um oxígrafo para monitorar o consumo de oxigênio durante a respiração mitocondrial, em presença dos complexos de cobre estudados. / In this work some copper(II) complexes with Schiff base ligands, already studied in our laboratory, have been prepared, as well as a new similar zinc complex. After being characterized, especially by spectroscopic techniques (UV/Vis, IR, and EPR), these complexes had their reactivity as catalysts for the oxidation of carbohydrates by molecular oxygen verified. The influence of different factors on the kinetics of reaction, such as variation of the catalyst concentration, substrate concentration, buffer concentration and pH of the solution, was investigated. In order to understand the possible interactions of the copper(II) complexes studied with the protein albumine, studies using CD and electronic spectroscopies were carried out by adding the copper complexes to bovine serum albumine (BSA), and determining the corresponding relative stability constants for each complex. The activity of such copper complexes as oxidant agents toward the protein was also verified, with the formation of carbonyl groups in the protein observed by monitoring spectrophotometrically the corresponding dinitrophenylhydrazones, after reaction with dinitrophenylhydrazine (DNPH). By using EPR spectroscopy and an appropriate spin scavenger, reactive oxygen species were detected and identified as a result of the oxidative damage to the protein occurring in the presence of the copper complexes and hydrogen peroxide. Interactions of the human serum protein (HSA) and human plasma with the copper complexes studied were verified by EPR and SDS-PAGE techniques. Additionally, the reactivity of these complexes toward the reductive agent glutathione, present in the citosol at high concentration, was investigated by fluorescence measurements. Finally, the influence of the complexes in the mitochondrial respiration was verified, since those compounds are able to induce efficiently apoptosis in the presence of different tumoral cells, monitoring the oxygen consumption with a selective oxygen electrode

INTRODUÇÃO: A ativação da NADPH oxidase de neutrófilos requer o acoplamento das subunidades citosólicas p47phox, p67phox, p40phox e Rac2 ao componente de membrana citocromo b558. Em trabalhos anteriores, nós mostramos que as isoformas vasculares da oxidase são reguladas pela proteína dissulfeto isomerase (PDI), uma chaperona redox. Neste trabalho, nós utilizamos um sistema cellfree semirecombinante como ferramenta para investigar o papel da PDI na ativação da NADPH oxidase do neutrófilo. RESULTADOS: Inibidores da PDI, scrambled RNAse (100g/mL) ou bacitracina (1mM), praticamente suprimiram a geração de superóxido. Para avaliação dos efeitos do estado redox da PDI sobre a atividade da oxidase, amostras de PDI foram previamente oxidadas (H2O2; 0,5 mM) ou reduzidas (DTT; 0,5 mM). A PDI oxidada (100 nM) aumentou a produção de superóxido em aproximadamente 30%, enquanto a mesma concentração de PDI reduzida promoveu efeito inverso, inibindo a atividade do complexo. A adição de um peptídeo contendo a seqüência peptídica do sítio ativo da PDI inibiu a produção de superóxido em 70%. Dados de imunolocalização e colocalização demonstraram que a interação da PDI com a subunidade p47phox parece ser intensificada pelo estímulo com PMA e envolvem modificações do estado redox de ambas as proteínas. CONCLUSÕES: Nossos dados confirmam a associação física e funcional entre a PDI e o complexo NADPH oxidase. Além disso, sugerem que a PDI exerça um importante papel como fator de regulação redox da ativação da oxidase. / Activation of the leukocyte NADPH oxidase requires the assembly of the cytosolic subunits p47phox, p67phox and p40phox and Rac2 with the membranebound cytochrome b558. We have previously shown that the vascular oxidase is regulated by the redox chaperone protein disulfide isomerase (PDI). Taking advantage of the semirecombinant cellfree system, we sought to investigate the role of PDI in the activation of neutrophil NADPH oxidase. The PDI thiol inhibitors scrambled RNase (100g/mL) or bacitracin (1mM), almost suppressed superoxide generation. In order to investigate if the redox status of PDI thiols could modulate superoxide generation, PDI was oxidized or reduced by treatment with H2O2 (0.5mM) or DTT (1mM), respectively. Oxidized PDI increased by 30% superoxide production, while reduced PDI diminished superoxide generation also in 30%. The addition of a peptide (1M) containing PDI´s exact active site sequence inhibited superoxide production by 70 %. Immuno and colocalization data demonstrated the interaction of PDI with the subunit p47phox to be intensified by PMA stimulation and to involve redox status exchange of both proteins. Our data confirm the physical and functional association between PDI and the oxidase complex. Moreover, we show a relevant role for PDI as a redoxdependent supportive factor for NADPH oxidase activation.