Nitric oxide (NO) and S-nitrosothiols (SNOs) are widespread signalling molecules that regulate immunity in animals and plants (Wendehenne et al., 2001). Previously, we have reported that Arabidopsis thaliana S-nitrosoglutathione reductase, (AtGSNOR1) modulates the extent of total cellular SNO formation, which subsequently regulates multiple modes of plant disease resistance (Feechan et al., 2005). Loss-of-function mutations in AtGSNOR1, leading to increased SNO levels, have recently been shown to result in S-nitrosylation of the key defence regulators NPR1 and AtSABP3, blunting their activity and subsequently leading to increased pathogen susceptibility (Tada et al., 2008; Wang et al., 2009). Thus, inhibiting AtGSNOR1 function leading to increased SNOs, would potentially provide a good strategy for bacterial effector proteins, delivered by the type III secretion system (TTSS), to promote infection. AtGSNOR1 is constitutively expressed in all organs in Arabidopsis and its expression is induced by wounding stress avirulent and non-host pathogen. Using gas phase chemiluminescence, we show that infection with Pseudomonas syringae pv. tomato strain DC3000 (PstDC3000) resulted in increase SNO levels which is TTSS. At the same time, RT-PCR and GUS analysis indicated that AtGSNOR1 expression was transiently suppressed by PstDC3000 which is also TTSS-dependent. Therefore, PstDC3000 infection suppresses denitrosylase function of AtGSNOR1 to increase SNO levels and this virulence effect is delivered by at least one of the effector protein secreted through TTSS. Several putative cis-acting elements were identified in AtGSNOR1 promoter through deletion analysis including GT-box, W-box and MYB/MYC binding motif. These elements comprise of positive and negative regulators which are critical for the induction and suppression of AtGSNOR1 in response to pathogen infection. A few transgenic plants expressing effector proteins were selected and tested for their suppressive effect on AtGSNOR1 expression during PstDC3000 infection. HopAM1 effector proteins showed the ability to suppress AtGSNOR1 when expressed in planta.

O NO regula diversos sistemas orgânicos. No sistema cardiovascular participa ativamente na regulação do tônus vascular entre outras funções. Disfunções na produção ou disponibilidade de NO podem comprometer sua atuação fisiológica. No âmbito vascular isto pode participar da hipertensão. Além da produção de NO pelas óxido nítrico sintases, outras vias paralela de produção fisiológica de NO a partir do nitrito tem sido descritas. O nitrito é o produto inicial da oxidação do NO, sendo posteriormente oxidado a nitrato. Sabe-se que estas três moléculas formam um ciclo dentro do organismo, onde o nitrato é excretado na saliva e convertido a nitrito pelas bactérias bucais.Este nitrito é deglutido com a saliva e exerce seus efeitos, através da conversão a NO. A conversão de nitrito a NO pode ocorrer de forma enzimática ou não enzimática. Como forma não enzimática, o nitrito é convertido a NO pela reação com H+. Esta redução ocorre principalmente no estômago. Esta tese visa elucidar possíveis mecanismos responsáveis pelos efeitos anti-hipertensivos do nitrito de sódio. Avaliamos ratos 2 rins 1 clipe (2R1C) tratados com nitrito e nitrato e verificamos os efeitos anti-hipertensivo destes. De forma Interessante, o aumento do pH gástrico com omeprazol impediu o efeito anti-hipertensivo tanto do nitrito quanto do nitrato. O omeprazol não gerou qualquer diferença na concentração plasmática de nitrito e nitrato. Foi verificado que o tratamento com nitrito e nitrato resultou em aumento das espécies nitrosiladas no plasma e este aumento foi impedido pelo omeprazol. Também testamos a influência do ciclo entero-salivar no efeito do nitrito e nitrato. Verificamos que o tratamento com enxaguante bucal levou ao bloqueio do efeito anti-hipertensivo do nitrato, mas não alterou este efeito nos animais tratados com nitrito. Em todas as abordagens experimentais o efeito anti-hipertensivo do nitrito apenas ocorreu quando houve aumento da concentração plasmática de espécies nitrosiladas. / Nitric Oxide plays many functional roles in physiological systems. In the cardiovascular system it participates in a unique way in the regulation of vascular tone among other functions. Dysfunctions in the production or availability of NO may compromise their physiological activity and participate in hypertension. Besides the production of NO by the nitric oxide synthase, other physiological pathways of NO production from nitrite have been described. The nitrite and nitrate are oxidation products of NO. Further nitrite is oxidized to nitrate. These three molecules are known to forma cycle in the body. Nitrate is excreted in saliva and reduced to nitrite by oral bacteria. Nitrite then is swallowed with the saliva and exerts its effects through conversion to NO. The conversion of nitrite to NO may occur by enzymatic or non-enzymatic manner. As a non-enzymatic way nitrite is reduced to NO by reaction with H+.This reaction occurs mainly in the stomach. This thesis aims to elucidate possible mechanisms responsible for the antihypertensive effects of sodium nitrite. We studied 2K1C rats treated with nitrite and nitrate and checked anti-hypertensive effects of these molecules. The increased gastric pH by omeprazole prevented the anti-hypertensive effect of nitrite and nitrate. Omeprazole did not cause any differences in plasma nitrite and nitrate. It was found that treatment with nitrite and nitrate resulted inincreased nitrosylated species in the plasma, and this increase was blocked by omeprazole. We also tested the influence of the entero-salivarycycle effect of nitrite and nitrate. We found that treatment with mouthwash blunted the antihypertensive effect of nitrate but this effect did not change in animals treated with nitrite. Interestingly in all experimental approaches the anti-hypertensive effect of nitrite only occurred when there was an increase in the plasma concentration of nitrosylated species

O NO regula diversos sistemas orgânicos. No sistema cardiovascular participa ativamente na regulação do tônus vascular entre outras funções. Disfunções na produção ou disponibilidade de NO podem comprometer sua atuação fisiológica. No âmbito vascular isto pode participar da hipertensão. Além da produção de NO pelas óxido nítrico sintases, outras vias paralela de produção fisiológica de NO a partir do nitrito tem sido descritas. O nitrito é o produto inicial da oxidação do NO, sendo posteriormente oxidado a nitrato. Sabe-se que estas três moléculas formam um ciclo dentro do organismo, onde o nitrato é excretado na saliva e convertido a nitrito pelas bactérias bucais.Este nitrito é deglutido com a saliva e exerce seus efeitos, através da conversão a NO. A conversão de nitrito a NO pode ocorrer de forma enzimática ou não enzimática. Como forma não enzimática, o nitrito é convertido a NO pela reação com H+. Esta redução ocorre principalmente no estômago. Esta tese visa elucidar possíveis mecanismos responsáveis pelos efeitos anti-hipertensivos do nitrito de sódio. Avaliamos ratos 2 rins 1 clipe (2R1C) tratados com nitrito e nitrato e verificamos os efeitos anti-hipertensivo destes. De forma Interessante, o aumento do pH gástrico com omeprazol impediu o efeito anti-hipertensivo tanto do nitrito quanto do nitrato. O omeprazol não gerou qualquer diferença na concentração plasmática de nitrito e nitrato. Foi verificado que o tratamento com nitrito e nitrato resultou em aumento das espécies nitrosiladas no plasma e este aumento foi impedido pelo omeprazol. Também testamos a influência do ciclo entero-salivar no efeito do nitrito e nitrato. Verificamos que o tratamento com enxaguante bucal levou ao bloqueio do efeito anti-hipertensivo do nitrato, mas não alterou este efeito nos animais tratados com nitrito. Em todas as abordagens experimentais o efeito anti-hipertensivo do nitrito apenas ocorreu quando houve aumento da concentração plasmática de espécies nitrosiladas. / Nitric Oxide plays many functional roles in physiological systems. In the cardiovascular system it participates in a unique way in the regulation of vascular tone among other functions. Dysfunctions in the production or availability of NO may compromise their physiological activity and participate in hypertension. Besides the production of NO by the nitric oxide synthase, other physiological pathways of NO production from nitrite have been described. The nitrite and nitrate are oxidation products of NO. Further nitrite is oxidized to nitrate. These three molecules are known to forma cycle in the body. Nitrate is excreted in saliva and reduced to nitrite by oral bacteria. Nitrite then is swallowed with the saliva and exerts its effects through conversion to NO. The conversion of nitrite to NO may occur by enzymatic or non-enzymatic manner. As a non-enzymatic way nitrite is reduced to NO by reaction with H+.This reaction occurs mainly in the stomach. This thesis aims to elucidate possible mechanisms responsible for the antihypertensive effects of sodium nitrite. We studied 2K1C rats treated with nitrite and nitrate and checked anti-hypertensive effects of these molecules. The increased gastric pH by omeprazole prevented the anti-hypertensive effect of nitrite and nitrate. Omeprazole did not cause any differences in plasma nitrite and nitrate. It was found that treatment with nitrite and nitrate resulted inincreased nitrosylated species in the plasma, and this increase was blocked by omeprazole. We also tested the influence of the entero-salivarycycle effect of nitrite and nitrate. We found that treatment with mouthwash blunted the antihypertensive effect of nitrate but this effect did not change in animals treated with nitrite. Interestingly in all experimental approaches the anti-hypertensive effect of nitrite only occurred when there was an increase in the plasma concentration of nitrosylated species

Orientador: Marcelo Ganzarolli de Oliveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-06T15:15:13Z (GMT). No. of bitstreams: 1 Seabra_AmedeaBarozzi_D.pdf: 4765202 bytes, checksum: 40113b24d319fff86b3e9a746e7b21ad (MD5) Previous issue date: 2006 / Doutorado / Físico-Química / Doutor em Ciências

Orientador: Marcelo Ganzarolli de Oliveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-12T09:59:43Z (GMT). No. of bitstreams: 1 Gonzalez_RegianedaSilva_D.pdf: 9735531 bytes, checksum: 707cf7da6df6ade965d498be44562499 (MD5) Previous issue date: 2008 / Resumo: Hidrogéis compostos de redes poliméricas são de grande interesse como biomateriais. Neste trabalho, foram sintetizados hidrogéis de poli(Nisopropilacrilamida) copolimerizados com ácido acrílico (P(NIPAAm-co-AAc)) e reticulados com metileno-bisacrilamida (MBAAm) em diferentes graus de reticulação, interpenetrados (IPNs) e semi-IPNs com PVA. A presença de AAc permitiu elevar a temperatura crítica inferior de solução (LCST) para 37° C. Observou-se que as propriedades mecânicas dos hidrogéis melhoram significativamente com a presença de PVA na rede polimérica e com o aumento do grau de reticulação. Observou-se que o aumento de 3 para 5% no grau de reticulação leva a uma mudança da morfologia dos poros dos hidrogéis de esféricos a tubulares e que a difusão de azul de metileno a partir dos hidrogéis com poros tubulares é aumentada quando o eixo principal dos poros está orientado paralelamente ao fluxo de soluto. Demonstrou-se que tanto o aumento do grau de reticulação como a redução da temperatura de 37 para 25° C levam a uma redução das velocidades de difusão de S-nitrosoglutationa (GSNO) e S-nitroso-Nacetilcisteína (SNAC) incorporadas nos hidrogéis. Verificou-se que tanto a absorção como a difusão destes solutos dependem das interações entre suas cargas elétricas resultantes e a carga da matriz polimérica em um determinado pH. Hidrogéis contendo GSNO e SNAC foram capazes de causar vasodilatação local na pele em aplicações tópicas, através de suas ações como doadores de óxido nítrico (NO). A ação vasodilatadora mais intensa e de mais curta duração da SNAC comparada à GSNO foi correlacionada com seu maior coeficiente de difusão na matriz de hidrogel. A aplicação do hidrogel aderido com adesivo de fibrina em um modelo animal de gastrosquises promoveu uma proteção efetiva das alças intestinais herniadas, com redução da inflamação. / Abstract: Hydrogels composed of polymeric networks are of great interest as biomaterials. In this work hydrogels of poly(N-isopropilacrilamide) copolimerized with acrylic acid (P(NIPAAm-co-AAc)) and cross-linked with metilene-bisacrilamide (MBAAm) at different cross-linking degrees, interpenetrated (IPNs) and semi-IPNs with PVA. The presence of AAc allowed increasing the lower critical solution temperature (LCST) to 37° C. It was observed that the mechanical properties of the hydrogels are significantly improved with the presence of PVA in the polymeric network and with the increase in the cross-linking degree. It was observed that an increase from 3 to 5% in the crosslinking degree leads to a change in the pore morphology of the hydrogels from spherical to tubular and that the diffusion of methylene blue from the hydrogels with tubular pores is increased when the main axis of the pores are parallel oriented to the flow of the solute. The diffusion of S-nitrosothiols showed that the increase in the cross-linking degree from as well as the reduction in temperature from 37 to 25° C, leads to a reduction in the diffusion rates of S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylcysteine (SNAC) incorporated in the hydrogels and that both the absorption and diffusion of these solutes depend on the interactions between their net electrical charge and that of the polymeric matrix at a given pH. GSNO and SNAC containing hydrogels were able to cause local vasodilation in the skin in topical applications through their actions as nitric oxide (NO) donors. The more intense and less lasting vasodilation action of SNAC, compared to GSNO was correlated to its higher diffusion coefficient in the hydrogel matrix. Application of the hydrogel bound through fibrin adhesive in an animal model of gastroschisis, provided an effective protection of herniated bowel, with reduction of inflammation. / Doutorado / Físico-Química / Doutor em Ciências

Les systèmes in situ sont des liquides à base de polymère et de solvant organique pharmaceutiquement acceptable, contenant le principe actif. Après injection sous-cutanée, lors du contact avec les fluides corporels, le polymère précipite sous forme d'implant (ISI) ou de microparticules (ISM) qui se dégradent progressivement en libérant le principe actif. Dans ce travail, des ISI et des ISM réalisés à partir d'un copolymère d'acide lactique et glycolique ont été développés pour la libération prolongée de S-nitrosothiols (RSNO), des donneurs de monoxyde d'azote. L'influence du type de formulation, du solvant, de l'hydrophobie du principe actif et de l'environnement (in vitro ou in vivo) sur la solidification, la dégradation de la matrice polymérique et sur la libération du RSNO ont été étudiés. Les expériences in vivo ont prouvé la prolongation par la formulation de l'effet des RSNO sur la pression artérielle chez le rat (jusque 42 h). Néanmoins, le temps de dégradation des formulations est supérieur à 1 mois et doit donc être optimisé pour une application de longue durée. Le potentiel de ces formulations dans un modèle d'infarctus a été évalué. La faisabilité d'une injection directe dans le myocarde infarci a été démontrée. D'après les premiers résultats, ces implants chargés en RSNO permettraient d'améliorer la perfusion du coeur. Enfin, la porosité de ces systèmes augmente durant leur dégradation, ce qui rend la matrice susceptible de recruter et d'héberger des cellules. In vitro, des ISI ont permis l'adhésion et la prolifération de cellules musculaires. Ces formulations adaptées aux RSNO pourraient constituer un outil thérapeutique dans le cadre des maladies ischémiques / In situ forming injectable systems are liquids based of a polymer and a pharmaceutically acceptable organic solvent, to which drug is added. After subcutaneous injection, contact with aqueous body fluids triggers polymer precipitation as implant (ISI) or microparticles (ISM). This matrix degrades then slowly while releasing the drug. In this work, ISI and ISM made of a copolymer of lactic and glycolic acid were developed for sustained release of S-nitrosothiols (RSNO), prodrugs of nitric oxide. Influence of formulation type, solvent, drug hydrophobicity and environment (in vitro vs in vivo) on polymeric matrix solidification, degradation and on RSNO release was studied. In vivo experiments proved that formulation extend (until 42 h) RSNO effect on arterial pressure of rats. However, matrix life-span is higher than 1 month, thus need optimization in view of an application requiring a long lasting release. Evaluation of these formulations has begun in a model of cardiac infarction. First, the feasibility of a direct injection into the infarct area has been established. Second, first results seem to indicate that these implants loaded with RSNO could enhance heart perfusion. Finally, porosity of these systems increases during their degradation, allowing cell recruitment and colonization of resulting matrix. An in vitro study conducted on implants with porosity artificially increased showed adhesion and proliferation of muscular cells seeded onto the systems. As a result, in situ formulations are suitable drug delivery systems for S-nitrosothiols, and represent a potential therapeutic tool, in particular in the field of ischemic diseases

Le développement de nouveaux donneurs d’oxyde nitrique (NO) dans le traitement chronique des maladies cardiovasculaires nécessite l’étude de leur biodisponibilité après administration par voie orale. Les S-nitrosothiols (RSNOs) apparaissent d’intéressants candidats médicaments pour ce faire, et l’étude de leur perméabilité intestinale est une première étape indispensable. Il est nécessaire de disposer d’une méthodologie analytique suffisamment sensible et sélective, en particulier permettant de différencier entre la production endogène de NO, l’apport alimentaire en ions nitrites et nitrate et le médicament lui-même. Nos travaux de thèse ont consisté à utiliser le S-nitrosoglutathion (GSNO) comme modèle après son marquage par l’isotope stable 15 de l’azote (15N). La dérivation du 15NO libéré par deux méthodes conventionnelles (méthode de Griess conduisant à la formation d’un adduit azoïque ; réaction avec le 2,3-diaminonaphtalène (DAN) formant l’adduit 2,3-naphtotriazole (NAT)) et l’étude de la fragmentation en spectrométrie de masse tandem (MS/MS) des deux adduits correspondants ont mené à sélectionner la dérivation par le DAN comme étant la plus sensible. Une transition originale résultant de la fragmentation du NAT en mode Higher-energy Collisional Dissociation (HCD) au lieu du mode conventionnel Collisionally Induced Dissociation (CID) a été mise en évidence ; elle permet d’atteindre une limite de quantification de 5 nM (soit 20 fois plus basse que celle offerte par la fluorescence). La méthode LC-MS/MS a été validée et appliquée à l’étude de la perméabilité intestinale du GS15NO par deux modèles : l’un in vitro (monocouche de cellules épithéliales type Caco-2), l’autre ex vivo (intestin de rat isolé (ileum) dans une chambre de Ussing). Les valeurs de perméabilité apparente calculées à partir des concentrations des métabolites du GS15NO (ions nitrites, nitrates et RSNOs) le classent comme un médicament de perméabilité intermédiaire. En outre, des études sur les mécanismes de dénitrosation du GSNO ont été menées sur intestin isolé, démontrant en particulier le rôle d’enzymes telles que la γ-glutamyltransférase et la protein disulfide isomerase / The development of innovative nitric oxide (NO) donors for the chronic treatment of cardiovascular diseases implies their bioavailability studies after oral administration. S-nitrosothiols (RSNOs) look interesting drug candidates for this purpose and evaluating their intestinal permeability appears the first step to be realized. Thus, an analytical method offering high sensitivity is needed; moreover this method should be selective by differentiating between the endogenous production of NO, the intake of nitrite and nitrate ions via the diet, and the drug itself. Our work consisted in using S-nitrosoglutathione (GSNO) labeled with the stable nitrogen isotope 15 (15N) as a model. Released 15NO species were derivatized by two conventional methods: Griess method leading to the formation of an azo adduct; reaction with 2,3-diaminonaphthalene (DAN) producing 2,3-naphtotriazole (NAT); fragmentation studies of the two adducts by tandem mass spectrometry (MS/MS) allow the selection of DAN method because it provides the highest sensitivity. An original transition resulting from the NAT fragmentation in Higher-energy Collisional Dissociation (HCD) mode instead of the conventional Collisionally Induced Dissociation (CID) mode was pointed out and permitted to reach a limit of quantification of 5 nM (20 fold less than when using fluorescence). The LC-MS/MS method was validated and applied to the GS15NO intestinal permeability studies with two models: in vitro (a monolayer of Caco-2 epithelial cells), and ex vivo (isolated intestine of rat (ileum) in an Ussing chamber). The apparent permeability values calculated with concentrations of GS15NO metabolites (nitrite, nitrate ions and RSNOs) classify it as a middle permeable drug. Studies on GSNO denitrosating processes using isolated rat intestine demonstrate that the enzymes γ-glutamyltransferase and protein disulfide isomerase play a pivotal role

Les S-nitrosothiols (RSNOs) sont considérés comme des stocks circulant de monoxyde d'azote (NO) et qui ont de nombreux rôles in vivo. Une variation de la proportion des taux de RSNOs a été démontrée dans de nombreuses maladies. Il est donc important de pouvoir identifier et quantifier chaque RSNO dans les fluides biologiques pour la réalisation de diagnostics médicaux. Il devient alors intéressant de développer des outils analytiques pour la détermination des RSNOs, en utilisant de faibles volumes d'échantillons biologiques. Ce travail de thèse a ainsi été orienté vers le développement d'outils analytiques miniaturisés pour l'analyse des RSNOs dans les fluides biologiques, en se focalisant sur la conception de micro-dispositifs (laboratoires sur puce), intégrant toutes les étapes de l'analyse, à savoir l'injection, la séparation, la décomposition et la détection sur un seul et même dispositif pour l'identification et la quantification des RSNOs. Pour cela, chaque étape a dû être optimisée. Ainsi, une meilleure compréhension de la réactivité des RSNOs, en terme de voies de décomposition et de cinétique, a été étudiée en développant deux méthodologies basées sur l'électrophorèse capillaire (CE) couplée soit à la spectrométrie de masse (MS) soit à une détection par mesure de conductivité sans contact à couplage capacitif (C4D). Par la suite, les conditions de décomposition et la détection sensible du NO libéré ont été réalisées en utilisant des microcapteurs électrochimiques à NO. Sur la base des résultats obtenus, deux stratégies originales ont été développées pour la détection de la totalité des RSNOs présents dans le plasma (i) via la décomposition des RSNOs en utilisant des nanoparticules d’or couplées à des microcapteurs NO et (ii) via la conception d’un dispositif miniaturisé de diagnostic sur papier. Finalement, grâce à l’optimisation des étapes de décomposition, de séparation et de détection, une étude préliminaire a été menée pour concevoir une micropuce d’électrophorèse intégrant la décomposition des RSNOs et une détection électrochimique afin de quantifier indépendamment différents RSNOs. / S- nitrosothiols (RSNOs) are considered as biological circulating stock of nitric oxide (NO) that have many roles in vivo. The variation of RSNOs proportion has been recognized in many diseases, so that the identification and quantitation of each RSNO in biological fluids is of prime importance. There is thus interest for the development of analytical tools for their determination, using low biological sample volumes. This PhD work was thus orientated towards the development of miniaturized analytical tools for the analysis of RSNOs in biological fluids, with a focus on microdevices (lab-on-a-chip), by integrating the injection, separation, decomposition and detection steps for the simultaneous identification and quantitation of various RSNOs. To this aim, a better understanding of RSNO reactivity, in terms of decomposition, was necessary and was assessed by developing two methodologies based on capillary electrophoresis (CE) coupled to different detection techniques: mass spectrometry (MS) and capacitively coupled contactless conductivity detection (C4D). Then, the conditions for RSNOs decomposition and further sensitive detection of released NO by miniaturized electrochemical NO-sensors were determined. Finally, two original strategies were developed for the detection of the total amount of RSNOs in plasma (i) decomposition using gold nanoparticles and (ii) conception of miniaturized paper-based point of care device. Thanks to the optimization of decomposition, separation and detection steps, preliminary work was conducted to develop a microchip electrophoresis coupled to RSNOs decomposition to quantify separately the different RSNOs.

Orientador: Prof. Dr. Tiago Rodrigues / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2017. / O melanoma e uma doenca causada pela proliferacao exacerbada dos melanocitos caracterizada por alta capacidade metastatica e alta mortalidade. Apesar de existirem drogas disponiveis para seu tratamento, existe uma busca por tratamentos alternativos, devido a toxicidade e resistencia tumoral. Com os avancos no campo da nanotecnologia, surgiram novas tecnicas e possibilidades para o desenvolvimento de drogas. O objetivo deste estudo foi avaliar a citotoxicidade de nanoparticulas de quitosana doadoras de oxido nitrico (¿PNO) em celulas de melanoma (B16-F10) e melanocitos (Melan-A), e investigar os mecanismos envolvidos neste processo. Os resultados obtidos mostram potentes efeitos citotoxicos das nanoparticulas de quitosana contendo S-nitroso-mercaptossuccinico (S-nitroso-MSA-CS) contra a linhagem tumoral B16-F10 em um modo concentracao dependente (EC50= 6,7 ¡Ó 0,7 £gg/mL), enquanto que as nanoparticulas de acido mercaptossuccinico (MSA-CS) apresentaram um EC50 seis vezes maior (> 40 £gg/mL). Em contraste, as nanoparticulas de S nitroso-MSA-CS nao foram significativamente citotoxicas nos melanocitos nao tumorais (Melan-A) com EC50 quatro vezes maior (32,0 ¡Ó 7,5 £gg/mL). De acordo com os resultados obtidos por citometria de fluxo (dupla marcacao com anexina V-FITC/PI e caspase-3 na forma ativa) pode-se associar a morte celular induzida pelas nanoparticulas de S-nitroso-MSA-CS a um perfil de apoptose tardia (com marcacao positiva para anexina V e PI), ativacao de caspase-3 sem liberacao de LDH. Observou-se tambem um aumento na producao de especies reativas de oxigenio e nitrogenio, aumento da geracao de superoxido mitocondrial e oxidacao de grupamentos tiolicos de proteinas celulares, o que mostra que o estresse oxidativo e um evento importante no processo de morte celular induzido pelas nanoparticulas de S-nitroso-MSA-CS nas celulas B16-F10. Alem disso, observou-se por imunofluorescencia indireta o aumento da nitracao dos residuos de tirosina e nitrosacao de cisteina das proteinas celulares. Nossos resultados apontam para o potencial uso destas nanoparticulas para a terapia antitumoral. / The cowpea (Vigna unguiculata (L.) Walp) is a legume of important economic and nutritional representativeness, especially in Brazil. Serine protease inhibitors, such as trypsin, have been described in many species, as well as in other plants. In this specie an inhibitor with activity on human neutrophil elastase (HNE) has not yet been identified. This protease is involved in many pathological processes, such as the onset and progression of chronic obstructive pulmonary disease (COPD). We purified and characterized an inhibitor from the protein extract of Vigna unguiculata presenting activity towards HNE. Firstly, we performed the alkaline extraction procedure for proteins followed by three different chromatographic steps using Hitrap Q (ion exchange), Source15RPC (Reversed-Phase) and ACE18 (Reversed Phase) columns. These steps were followed by the inhibitory activity tests using fluorogenic substrates, MeO-Suc-Ala-Ala-Pro-Val-MCA (elastase) and Z-Phe-Arg-MCA (trypsin), and quantitation assays of protein concentration. To determinate the size of the molecule, we used MALDI-TOF mass spectrometry and SDS-PAGE. The molecular mass of the inhibitor was 10,99 kDa. The dissociation constant (Ki) toward HNE was 9 pM. HNE inhibitor showed no inhibitory activities toward trypsin and thrombin. However, the inhibitor presented activity toward subtilisin and chymotrypsin. These datas indicate that this molecule is a novel inhibitor to HNE and we named it Vigna unguiculata Elastase Inhibitor (VuEI).

Orientador: Marcelo Ganzarolli de Oliveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-09T16:40:14Z (GMT). No. of bitstreams: 1 Simplicio_FernandaIbanez_D.pdf: 1516606 bytes, checksum: 8ee850e83207e6e98a9aec046efdba5d (MD5) Previous issue date: 2007 / Resumo: Óxido nítrico (¿NO) produzido endogenamente em humanos é considerado um antioxidante efetivo na inibição da peroxidação lipídica. Todavia, no plasma e em células mamíferas, ¿NO circula principalmente como S-nitrosotióis primários (RSNOs). Neste trabalho, a peroxidação in vitro de comicelas do ácido linoleico-SDS (AL-SDS) e da lipoproteína de baixa densidade (LDL) catalisada por lipoxigenase de soja (SLO), íons Fe (II) e Cu (II), foram monitoradas na presença e na ausência de três RSNOs primários: S-nitrosocisteína (CISNO), S-nitroso-N-acetilcisteína (SNAC) e S-nitrosoglutationa (GSNO) a 37ºC. Medidas cinéticas e espectrofotométricas baseadas na formação de duplas conjugadas, adutos fluorescentes oxidados AL-lisina e na detecção eletroquímica de ¿NO livre, foram utilizadas para mostrar que RSNOs são antioxidantes mais potentes que seus tióis livres correspondentes (RSHs) em codições equimolar. Esses resultados são consistentes com o bloqueio da peroxidação do AL-SDS e LDL por RSNOs através da inativação dos radicais peroxil/alcoxil (LOO¿/LO¿) e pela transnitrosação com hidroperóxidos de AL pré-formado, levando a produtos nitrogenados de AL oxidado, que foram mostrados pela liberação de ¿NO livre por redução com ácido ascórbico. A ação antioxidante de SNAC e GSNO contra a peroxidação da LDL é refletida na quantidade reduzida de ¿NO livre detectado pela decomposição de RSNOs catalisados por Cu (II) na presença da LDL. Esses resultados indicam que RSNOs primários endógenos podem participar no bloqueio da peroxidação lipídica in vivo, não somente através da inativação primária dos radicais alcoxil/peroxil mas também através da inativação dos hidroperóxidos lipídicos pré-formados. A administração oral de SNAC previniu o princípio e progressão da doença não alcoólica do fígado gorduroso (NAFLD) em ratos Wistar alimentados com dieta deficiente em colina e reverteu a NAFLD em diferentes dietas com camundongos ob/ob. Esses efeitos foram correlacionados positivamente com um decréscimo na concentração de hidroperóxidos lipídicos no homogenatos de fígado e com habilidade dos RSNOs em previnir a peroxidação lipídica do ácido linoleico e da LDL in vitro / Abstract: Nitric oxide (¿NO) produced endogenously in humans is considered an effective chain-breaking antioxidant in the inhibition of lipid peroxidation. However, in the plasma and cells of mammals, ¿NO circulates mainly as primary S-nitrosothiols (RSNOs). In this work, the in vitro peroxidation of linoleic acid-SDS comicelles (LA-SDS) and of low density lipoprotein (LDL) catalyzed by soybean lipoxygenase (SLO), Fe (II) and Cu (II) ions, were monitored in the presence and absence of three primary RSNOs: S-nitrosocysteine (CySNO), S-nitroso-N-acetylcysteyne (SNAC) and S-nitrosoglutathione (GSNO) at 37 ºC. Kinetic and spectrophotometric measurements based on the formation of conjugated double bonds, fluorescent oxidized LA-lysine adducts and the electrochemical detection of free NO, were used to show that RSNOs are more potent antioxidants than their corresponding free thiols (RSHs) in equimolar conditions. These results are consistent with the blockage of LA-SDS and LDL peroxidation by RSNOs through the inactivation of peroxyl/alkoxyl (LOO¿/LO¿) radicals and through the transnitrosation with preformed LA hydroperoxides, leading to nitrogen-containing products of oxidized LA, which were shown to release free ¿NO upon reduction with ascorbic acid. The antioxidant actions of SNAC and GSNO against LDL peroxidation are reflected in a reduced amount of free NO detected upon Cu (II)-catalyzed decomposition of RSNOs in the presence of LDL. These results indicate that endogenous primary RSNOs may play a major role in blocking lipid peroxidation in vivo, not only through the primary inactivation of alkoxyl/peroxyl radicals but also through the inactivation of preformed lipid hydroperoxides. Oral administration of SNAC prevented the onset and progression of nonalcoholic fatty liver disease (NAFLD) in Wistar rats fed a choline-deficient diet and reversed NAFDL induced by different diets in ob/ob mice. These effects were positively correlated with a decrease in the concentration of lipid hydroperoxydes in liver homogenate and with the ability of RSNOs to prevent lipid peroxidation of linoleic acid and LDL in vitro / Doutorado / Físico-Química / Doutor em Ciências