O manganês é um elemento traço essencial, porém existe uma preocupação com seus potenciais efeitos neurotóxicos associados à exposição a níveis excessivos, podendo provocar uma síndrome conhecida como manganismo, cujos sintomas são semelhantes aos da doença de Parkinson. A maioria dos trabalhos envolvendo manganês usa espécies de Mn(II), mas sabe-se que Mn(III) é acumulado em maior quantidade no cérebro. Nesse sentido, foi feito um estudo dos danos oxidativos e de toxicidade provocados por três complexos de Mn(III): citrato, pirofosfato e salicilenodiamina (respectivamente MnCit, MnPPi e EUK8). Para tanto, as três espécies foram sintetizadas e caracterizadas por métodos espectroscópicos. Em seguida foram determinadas suas capacidades pró-oxidantes sobre os seguintes marcadores: dihidrorodamina (DHR), tirosina (Tyr), albumina (BSA) e dopamina (DA). Finalmente, seu efeito sobre células cerebelares e da cepa HeLa estressada por meio de irradiação UV também foi avaliado, e foi usado ascorbato na tentativa de tratar o dano sobre células HeLa. O teste com a DHR também foi feito em presença de H2O2 e ascorbato. A capacidade pró-oxidante testada por fluorescência da DHR sugere que o ascorbato atua como anti-oxidante. Além disso, MnCit e MnPPi (mas não EUK8), quando na presença de H2O2, são menos oxidantes. O mesmo comportamento foi percebido nas medidas de fluorescência de Tyr. A carbonilação da BSA, verificada pela absorbância do seu marcador (DNPH), seria indício de capacidade oxidante dos complexos, mas não percebeu-se variação significativa de grupos C=O na proteína após tratamento com espécies de Mn(III), mesmo em amostras com H2O2, embora notem-se as mesmas tendências apresentadas pelos complexos com DHR e Tyr. Estudos de oxidação de DA por luminescência tiveram resultados inconclusivos, mas dados mais concretos em testes com medidas de absorbância de soluções de DA e fluorescência de misturas de DA com DHR indicaram que DA é preferencialmente oxidada por todos os compostos. A viabilidade celular de culturas de células neuronais granulares (CGC) mostrou pouca diferença entre as toxicidades dos compostos, mas verifica-se uma relação inversamente proporcional entre as toxicidades e lipofilicidades dos complexos. O mesmo não ocorre nos experimentos com HeLa, cuja viabilidade foi avaliada por contagem de colônias após fixação e coloração das células, pois nesse caso o EUK8 se mostrou o mais tóxico dos três. Além disso, ao contrário do observado com a DHR, o ascorbato teve ação pró-oxidante, e, aparentemente, houve um efeito sinérgico negativo entre os complexos e a radiação UV. Tratamento com o quelante p-aminossalicilato só foi eficaz na recuperação das culturas para amostras não irradiadas. / Manganese is an essential trace element, however there is considerable concern regarding its neurological effects when in excess, giving rise to a condition termed manganism which is characterized by Parkinson disease-like symptoms. Most evaluations of manganese toxicity use poorly defined Mn(II) species, although Mn(III) is known to accumulate preferentially in the brain. Therefore, in this work we proposed a study of oxidative damage and citotoxicity of Mn(III) derivatives of citrate, pyrophosphate and salycilenediamine (respectively, MnCit, MnPPi and EUK8). The species were synthesized and characterized by spectroscopic methods. Their pro-oxidant abilities were assessed over markers of oxidant activity dihydrorhodamine (DHR), tyrosine (Tyr), albumin (BSA) and dopamine (DA). In addition, their effect over granular cerebral cells (CGC) and HeLa cells stressed by ultraviolet irradiation was studied, and treated with ascorbate. Tests with DHR were repeated treating the samples with H2O2 and ascorbate. Pro-oxidant ability tested by both DHR and Tyr fluorescence suggest that ascorbate is antioxidant towards Mn(III)-induced oxidative damage. MnCit and MnPPi (but not EUK8), when in presence of peroxide, are less oxidants. An analogous trend was observed for BSA, although without statistical significance. Evaluation of DA formation by luminescence was inconclusive, but competition studies of DA+DHR mixtures indicated that DA is preferentially oxidized by all the complexes. To CGC, little difference was observed for the toxicities of the complexes. An inverse relationship of toxicity and lipophilicity has been observed. However this was not observed for HeLa cells, to which EUK8 was more toxic. In addition, and in opposition to the DHR solution study, ascorbate was found to be pro-oxidant. A negative synergic effect was observed between complex doses and irradiation. Treatment of the cells with paraaminosalicylate was beneficial only for non-irradiated cells.

En raison des propriétés redox réversibles du ferrocène et de son activité antibactérienne, les polymères à base de ferrocène sont intéressants pour synthétiser de nouveaux liants pour des revêtements anti-adhésifs bactériens. Cette étude rend compte de l’homopolymérisation et de la copolymérisation de monomères méthacryliques porteurs de groupes ferrocényles avec le méthacrylate de lauryle (LM). Le méthacrylate de méthylferrocène (FMMA) mais aussi quatre nouveaux monomères nommés méthacrylate de 2- ferrocénylméthoxyéthyle (FMOEMA), méthacrylate de 3-ferrocénylméthoxypropyle (FMOPMA), 4- ferrocénylméthoxybutyle (FMOBMA) et de 2-ferrocénylméthoxyméthyléthyle (FMOMEMA) ont d’ bord été synthétisés et ensuite polymérisés via le procédé RAFT. Les cinétiques ’homopolymérisation ont été étudiées par RMN 1H in situ. La polymérisation a été contrôlée en utilisant le 2-cyanoprop-2-yl dithiobenzoate (CPDB) comme agent de transfert de chaîne, à 70°C, dans le toluène deutéré. Ces monomères contenant le groupement ferrocényle se sont révélés très réactifs via le procédé RAFT, conduisant à des conversions de 96% et à des polymères de faibles indices de polymolécularité (Ð<1,6). La conversion des monomères suit une cinétique de premier ordre (jusqu’à 80%) avec une augmentation linéaire de la masse molaire en fonction de la conversion en monomère. En utilisant le monomère FMMA comme référence, l’ spacement entre la partie polymérisable et le groupement ferrocényle a été augmenté pour le FMOEMA, FMOPMA, FMOMEMA et FMOBMA afin d’ méliorer la mobilité des groupements latéraux. Cette mobilité se traduit par une diminution notable des températures de transition vitreuse des homopolymères entre le pFMMA et le pFMOBMA. De plus, les copolymères diblocs préparés par voie séquencée présentent deux températures de transition vitreuse spécifiques à chaque bloc, démontrant une incompatibilité de ces derniers. Les propriétés électrochimiques des monomères et celles des polymères ont été caractérisées par voltampérométrie cyclique. Enfin, les propriétés anti-adhésives de ces homopolymères et copolymères diblocs vis-à-vis d’une bactérie marine ont été évaluées. / Due to the reversible redox properties of ferrocene and its antibacterial activity, ferrocenyl-based polymers are useful for the synthesis of new anti-adhesive binders for marine antifouling coatings. This study reports the homopolymerization and copolymerization with lauryl methacrylate of ferrocenyl-based methacrylic monomers. Ferrocenylmethyl methacrylate (FMMA) as well as four novel monomers, namely 2- (ferrocenylmethoxy)ethyl methacrylate (FMOEMA), 3-(ferrocenylmethoxy)propyl methacrylate (FMOPMA),4-(ferrocenylmethoxy)butyl methacrylate (FMOBMA) and 2-(ferrocenylmethoxy)methylethyl methacrylate (FMOMEMA) were first synthesized, and subsequently polymerized by the RAFT process. The homopolymerization kinetics were investigated by in situ NMR. The radical polymerization was controlled by using 2-cyanoprop-2-yl dithiobenzoate (CPDB) as a chain transfer agent, at 70 °C in deuterated toluene. These monomers containing a ferrocenyl moiety with alcoxy linkers were found to be as reactive as FMMA in RAFT polymerization, resulting in conversions of 96% and in polymers with low dispersities (ÐM < 1.6). Monomer conversion follows a first order kinetics (up to 80%) with a linear increase in the molecular mass as a function of the monomer conversion. By using the FMMA monomer as a reference, the length of the alcoxy linker between the ferrocene unit and the backbone was increased for FMOEMA, FMOPMA, FMOMEMA and FMOBMA to improve the mobility of the side groups. This increase in macromolecular mobility led to a significant decrease of glass transition temperatures of the homopolymers. In addition, diblock copolymers exhibited two glass transition temperatures indicating that the two blocks are incompatible. The electrochemical properties of the monomers and those of the polymers were characterized using cyclic voltammetry. Finally, the anti-adhesive properties of these homopolymers and diblock copolymers toward marine bacteria were evaluated.

Les polymères à empreintes moléculaires sont des matériaux aux propriétés de reconnaissance spécifiques qui peuvent être mis à profit pour la détection d’une large gamme d’analytes. Ainsi, depuis quelques années, des travaux décrivent leur utilisation dans des capteurs en raison de leur capacité à piéger une cible définie.L’objectif de ce travail est d’ajouter des propriétés redox à des polymères à empreintes moléculaires pour détecter le Bisphénol A (BPA) par des méthodes électrochimiques simples. Ces polymères électroactifs sont synthétisés par polymérisation par précipitation d’une sonde redox, le méthacrylate deméthylferrocène (Fc), et du diméthacrylate d’éthylène glycol (EDMA) en présence du BPA pour le polymère imprimé (e-MIP-Fc) et en son absence pour le polymère non-imprimé (e-NIP-Fc).L’introduction d’un deuxième monomère fonctionnel, la 4-vinyl pyridine (4-VP), conduit à deux autres polymères imprimé (e-MIP-Fc-VP) et non-imprimé (e-NIP-Fc-VP). Les propriétés d’adsorption des polymères ainsi obtenus sont caractérisés en batch à l’aide de la LC-MS et présentent une capacité de reconnaissance du BPA avec un facteur d’empreinte de 2,5 et 1,3 respectivement pour l’e-MIP-Fc-VP et e-MIP-Fc justifiant de l’efficacité de l’empreinte. Leurs caractérisations par voltampérométrie cyclique confirment d’une part la bonne intégration du monomère ferrocényle dans les e-MIP/e-NIP et d’autre part la capacité de ces polymères à révéler la présence ou pas de la cible. Les particules e-MIP-Fc ont ensuite été intégrées dans des dispositifs type micro électrode ou transistor OECT (Organic ElectroChemical Transistor). Les premiers résultats, mêmes s’ils doivent être confirmés, s’avèrent encourageants avec,comme attendu, des modifications des propriétés électriques en présence du BPA. L’e-MIP-Fc-VP après mélange avec de la pâte de carbone, a été utilisé en sérigraphie pour obtenir une électrode de travail modifiée dans des électrodes sérigraphiées (Screen Printed Electrode). Ces électrodes permettent la reconnaissance du BPA avec des limites de détection et de quantification de 60 pM et 190 pM respectivement pour une gamme de concentrations comprise entre 0,15 et 1,84 nM, ouvrant ainsi des perspectives intéressantes pour la détection du BPA en milieu aqueux. / Molecularly imprinted polymers are materials with specific recognition properties that can be used for the detection of a wide range of template. In recent years, many works have been reported on their use in sensors because of their capability to specifically bind a defined analyte.The aim of this work is to assign to the molecularly imprinted polymers redox properties in order to detect Bisphenol A (BPA) by using easy electrochemical techniques. These electroactive polymers are synthesizedby precipitation polymerization of ferrocenylmethyl methacrylate (Fc) and ethylene glycol dimethacrylate(EDMA) in the presence of BPA for the imprinted polymer (e-MIP-Fc) and in its absence for the nonimprinted polymer (e-NIP-Fc). The copolymerization of the previous monomer with 4-vinylpyridine (4-VP) leads to two other imprinted (e-MIP-Fc-VP) and non-imprinted (e-NIP-Fc-VP) polymers. The resulting polymers are characterized in batch using LC-MS and have ability to recognize BPA with an imprinting factor of 2.5 and 1.3 respectively for e-MIP-Fc-VP and e-MIP-Fc the proving the recognition efficiency ofthese polymers. Their cyclic voltammetry recording confirm first, the good integration of the redoxferrocenyl monomer inside the polymers e-MIP/e-NIP during the polymerization, and on the other hand,the capability of these polymers to reveal the presence of BPA in the solution. The e-MIP-Fc particles were then integrated inside devices like microelectrode and OECT (Organic ElectroChemical Transistor). The first results, even if they must be confirmed, are positive regarding the modification of the electrical properties of these devices in the presence of BPA. The e-MIP-Fc-VP particles, after mixing with a carbon paste, were screen-printed to obtain a modified working electrode in a screen-printed electrode device. This electrode enable the recognition of BPA with limits of detection and quantification of 60 pM and 190 pM respectively, for a concentration range between 0.15 and 1.84 nM, thus opening up interesting perspectives for the detection of BPA in aqueous medium.

Cette thèse démontre qu'une approche expérimentale systématique de complexité croissante permet de réévaluer le sens du potentiel redox (EH), et fournit une mise à jour sur l'interprétation de sa valeur dans les assemblages complexes de matrices minérales, des consortiums microbiens, des nutriments et des contaminants sous dynamique, oxydoréduction oscillant conditions. Pour étudier l'utilité des mesures EH dans les systèmes environnementaux saturés d'eau une cascade complète redox 500 à -350 mV (pH ~7.4) a été reproduit dans le laboratoire. Les expériences ont révélé que l'électrode de Pt classique redox répond à des processus microbiens dans une mesure différente en fonction de l'oxygénation et de la présence d'un tampon d'oxydo-réduction physique, chimique et. Les mesures du EH dans des matrices argileuses appauvris dans le tampon redox, tels que le couple électroactif Fe3 + / Fe2 +, par conséquent, se révèlent avoir une utilité limitée. Dans de tels environnements, les couples redox sensible abondantes, cependant, non électroactif, tels que O2 / H2O, CrO42- / Cr (OH) 3, NO3- / NO2- / NH 4 +, Sb (OH) 6- / Sb2O3, et HAsO42 - / H3AsO3 ne pas d'impact mesuré EH. Pour quantifier l'effet de l'oxydation des perturbations sur la mobilité des oxyanions dans la matrice argileuse, j'ai effectué des expériences de traitement par lots sous oscillations redox contrôlées. cycles successifs de conditions oxiques et anoxiques ont été imposées sur les suspensions argileuses modifiées avec un mélange d'oxyde As (V), Sb (V), Cr (VI) et N (V). la mobilité des oxyanions a été étudiée dans des conditions stériles, avec l'addition de carbone organique labile (éthanol), et avec l'addition de microbienne du sol inoculum. Spéciation analyses ont révélé des réactions irréversibles de réduction avec ou sans ajouts d'éthanol. Fraîchement réduite As (III), Sb (III), Cr (III) et N (III) ne sont pas ré-oxydée pendant les périodes subséquentes oxiques démontrant un comportement non-oscillant. Microbiologiquement induit des transformations de réduction a diminué les concentrations aqueuses de Sb et Cr par précipitation, enlevés N par volatilisation, alors préservé Comme dans la solution. En fonction de la diversité microbienne, altérées par l'addition d'inoculum dans le sol, deux types de contaminants imbrications sont caractérisés comme inhibiteurs de réduction et de non-inhibiteurs. Ces données, le représentant de l'environnement souterrain saturé (sous-sol> 20 m), est en outre par rapport à oxyanion mobilité dans l'environnement proche de la surface (la couche arable <0,15 m). Les principales différences entre les systèmes de la couche arable et du sous-sol sont dans la fraction d'oxyhydroxyde Fe-, Mn- et minéraux Al-, la diversité microbienne, pCO2, et la gamme des valeurs EH développées au cours des cycles d'oxydo-réduction. Par exemple, la gamme EH de plus de 900 mV (500 à -300 mV) dans la suspension de la couche arable est opposée à la gamme EH de 100 mV (350-250 mV) dans la suspension du sous-sol. En outre, dans la suspension de la couche arable, fort cycle redox de Fe et Mn coïncide avec la mobilité d'oscillation de As et Sb. Cette corrélation suggère le rôle crucial des minéraux oxyhydroxyde agissant non seulement comme principaux sorbants, mais aussi comme catalyseurs pour des réactions d'oxydation éventuellement contrôlant la réversibilité de la séquestration des contaminants. Par conséquent, appauvri en minéraux oxyhydroxyde, matrice argileuse est révélée environnement propice à la rétention des contaminants, car il peut supporter des oscillations périodiques redox sans libérer les contaminants de retour à la phase aqueuse sur l'échelle de temps expérimental. / This thesis demonstrates that a systematic experimental approach of increasing complexity allows reassessing the meaning of the redox potential (EH), and provides an update on the interpretation of its value in complex assemblages of mineral matrices, microbial consortiums, nutrients, and contaminants under dynamic, redox-oscillating conditions. To study the usefulness of EH measurements in water-saturated environmental systems a full redox cascade from +500 to -350 mV (pH ∼7.4) was reproduced in the laboratory. The experiments revealed that conventional Pt redox electrode responds to physical, chemical, and microbial processes to a different extent depending on oxygenation and on the presence of a redox buffer. The measurements of EH in argillaceous matrices depleted in the redox buffer, such as the electroactive Fe3+/Fe2+ couple, thus, are shown to have limited usefulness. In such environments, the abundant redox-sensitive couples, yet non-electroactive, such as O2/H2O, CrO42-/Cr(OH)3, NO3-/NO2-/NH4+, Sb(OH)6-/Sb2O3, and HAsO42-/H3AsO3 do not impact measured EH. To quantify the effect of oxidizing perturbations on the mobility of oxyanions in the argillaceous matrix, I performed batch experiments under controlled redox oscillations. Successive cycles of oxic and anoxic conditions were imposed on the argillaceous suspensions amended with a mixture of oxidized As(V), Sb(V), Cr(VI), and N(V). Oxyanion mobility was investigated under sterile conditions, with the addition of labile organic carbon (ethanol), and with the addition of soil microbial inoculum. Speciation analyses revealed irreversible reduction reactions with and without ethanol additions. Freshly reduced As(III), Sb(III), Cr(III), and N(III) were not re-oxidized during subsequent oxic periods demonstrating non-oscillating behavior. Microbially induced reduction transformations decreased aqueous concentrations of Sb and Cr via precipitation, removed N via volatilization, while preserved As in the solution. Depending on microbial diversity, altered by the addition of soil inoculum, two types of contaminant interplays are characterized as inhibitory and non-inhibitory reductions. These data, the representative of saturated subsurface environment (subsoil > 20 m), is further compared to oxyanion mobility in the near-surface environment (topsoil < 0.15 m). The key differences between the topsoil and subsoil systems are in the fraction of oxyhydroxide Fe-, Mn-, and Al- minerals, microbial diversity, pCO2, and the range of EH values developed during redox cycles. For example, the EH range over 900 mV (from +500 to -300 mV) in the topsoil suspension is contrasted to the EH range of 100 mV (from +350 to +250 mV) in the subsoil suspension. Furthermore, in the topsoil suspension, strong redox cycling of Fe and Mn is coincident with the oscillating mobility of As and Sb. This correlation suggests the crucial role of oxyhydroxide minerals acting not only as major sorbents, but also as catalysts for oxidation reactions eventually controlling the reversibility of contaminant sequestration. Therefore, depleted in oxyhydroxide minerals, argillaceous matrix is shown to be suitable environment for contaminant retention, as it can stand periodical redox oscillations without releasing contaminants back to the aqueous phase on the experimental time scale.

Mecanismos redox estão envolvidos em diversos processos, como sobrevivência, proliferação e diferenciação celular, pela modulação da atividade de quinases, fosfatases e fatores de transcrição, entre outros, através da modificação oxidativa e reversível de resíduos de cisteína. Neste trabalho, nós estudamos processos redox subjacentes a diferenciação osteogênica induzida por BMP2, utilizando linhagens de células MC3T3-E1. Nosso objetivo foi investigar modificações redox como possíveis moduladores do processo de diferenciação osteogênica. Para isso, nós primeiramente caracterizamos a diferenciação osteogênica nas células MC3T3-E1 após o tratamento com BMP2, através da expressão do marcador osteogênico Osteocalcina, da fosforilação do complexo Smad 1/5/8 e da deposição de matriz extracelular calcificada. Análises de expressão gênica por qPCR mostraram que o tratamento com BMP2 resultou no aumento de expressão de NOX4, o que provavelmente leva ao aumento na produção de peróxido de hidrogênio intracelular. Nós investigamos também a modulação de peroxiredoxinas nesse processo e análises de expressão gênica mostraram que não há alterações nos níveis de expressão de Prx1 e 2 durante a diferenciação, mas os ensaios de western blot redox indicam que a Prx1 pode ser oxidada após o tratamento com BMP2, de maneira dose dependente. Outras análises in vitro mostram que células expostas a N-acetilcisteína (NAC) e PEG-catalase apresentam diferenciação osteogênica prejudicada, detectada por baixos níveis de deposição de matriz extracelular calcificada, comparado com células não-tratadas. Além disso, a fosforilação de Smad 1/5/8 são reduzidas nessas condições. Nossos dados sugerem que processos redox podem modular a sinalização celular durante o processo de diferenciação osteogência / Redox mechanisms are involved in several processes, such as cell survival, proliferation and differentiation, among other ways by modulating kinases, phosphatases and transcription factors activity that can occur through reversible and oxidative modification of cysteine residues. We were interested in studying redox processes underlying osteogenic differentiation induced by BMP-2, using MC3T3-E1 cell lineage. Our objective was to investigate redox modifications as possible modulators of the osteogenic differentiation process. We first characterized osteogenic differentiation in MC3T3-E1 cells upon BMP2 treatment, through gene expression of the osteogenic marker Osteocalcin, Smad 1/5/8 (belonging to the BMP-2 pathway) protein phosphorylation and extracellular matrix calcification. Gene expression analysis by qPCR showed that BMP2 treatment resulted in NOX4 upregulation, which probably also leads to hydrogen peroxide production. We have investigated peroxiredoxin modulation in this process, and gene expression analysis shows no significant change in peroxiredoxin 1 and 2 expression levels, but redox western blotting assays indicate that Prx1 can be oxidized after BMP2 treatment, in a dose dependent manner. In vitro analysis shows that cells exposed to N-acetyl-L-cysteine (NAC) and PEG-catalase display impaired osteogenic differentiation, detected by lower levels of calcified extracellular matrix deposition compared with non-treated cells. Moreover, phosphorylation of Smad 1/5/8 complex is reduced under these redox treatments. Our data suggest that redox pathways can modulate cell signaling during the osteogenic differentiation process

O proteassomo é o componente do sistema Ubiquitina-Proteassomo (UPS), responsável pela degradação de proteínas intracelulares marcadas com cauda de ubiquitina. No entanto, a unidade catalítica do proteassomo (20SPT), destituída de unidades regulatórias, é capaz de degradar proteínas de maneira ubiquitina-independente. Diversas modificações pós-traducionais já foram descritas para o 20SPT, incluindo a S-glutatiolação. De acordo com Demasi e col., (2003) o 20SPT da levedura Saccharomyces cerevisiae possui a atividade tipo-quimiotripsina modulada por glutationa e o mecanismo de glutatiolação implica na formação do intermediário ácido sulfênico. No presente trabalho, identificamos por espectrometria de massas (MS/MS) um total de sete resíduos diferentes de cisteína glutatiolados no 20SPT, sendo seis in vitro por incubação com GSH e três in vivo, extraído de células crescidas até atingir fase estacionária tardia em meio rico. Analisando a estrutura 3D do 20SPT, observou-se que os resíduos de cisteína glutatiolados não estão localizados na entrada da câmara catalítica nem próximos aos sítios-ativos, indicando um mecanismo alostérico da modulação da atividade proteassomal. O proteassomo glutatiolado extraído de leveduras é capaz de degradar proteínas oxidadas de maneira mais eficiente que o proteassomo reduzido por DTT, e ainda, esta degradação gera perfis peptídicos diferenciados por utilizar distintamente as atividades sítio-especificas, como visualizado por análises de HPLC e MS/MS. Por microscopia eletrônica verificamos a conformação aberta da câmara catalítica do proteassomo glutatiolado, sendo esta imediatamente fechada pela remoção da glutationa do 20SPT na presença de DTT. Caracterizamos ainda, enzimas reponsáveis pela desglutatiolação do 20SPT, capazes de recuperar as atividades proteassomais que haviam sido diminuídas pela glutatiolação: as oxidoredutases glutarredoxina 2 e as tiorredoxinas citosólicas. O mecanismo ainda inclui a hidrólise dessas oxidorredutases, fenômeno também verificado para diversas proteínas da suprafamília tiorredoxina, provavelmente devido a propriedades estruturais desta família. A glutatiolação do proteassomo apresenta-se como uma nova modificação pós-traducional de ocorrência fisiológica dependente do estado redox celular. Esta modificação promove aumento da atividade proteolítica, sugerindo uma função antioxidante atuante na remoção de proteínas oxidadas durante desafios oxidativos / The proteasome is the protease of the Ubiquitin-Proteasome System (UPS) responsible for the breakdown of intracellular ubiquitin-tagged proteins. However, the catalytic particle of the proteasome (20SPT) is capable of hydrolyzing some substrates in an ubiquitin-independent fashion. The S-glutathiolation of the 20SPT was described among several post-translational modifications and according to Demasi et. al. (2003), the chymotrypsin-like activity of proteasome from yeast Saccharomyces cerevisiae is regulated by glutathione. The mechanism of S-glutathiolation is dependent on the formation of the sulfenic acid intermediate in the cisteine residues of the 20SPT. In this present work, we identified in vitro and in vivo, a total of seven different S-glutathiolated proteasomal cysteine residues by mass spectrometry studies (MS/MS) and, by analyzing the 3D structure of the 20SPT, the modified cysteine residues are not located either on the entrance of the catalytic core or near to the active sites, indicating an allosteric mechanism of proteasomal modulation. During protein degradation, the natively S-glutathiolated 20SPT produces different patterns of peptide products when compared to the DTT-reduced particle through distinct site-specific cleavage of the protein substrates, as herein demonstrated by HPLC and MS/MS analyses. Furthermore, by electron microscopy, we showed that the entrance of the natively glutathiolated 20SPT is in the open conformation that immediately shifts to the closed conformation in the presence of DTT. We have also characterized the deglutathiolase role of the oxidoreductases Glutaredoxin 2 and Citosolic Thioredoxins 1 and 2 which recover the partially inhibited 20SPT activities. The deglutathiolation mechanism also includes the oxidoreductase degradation dependent on the 20SPT activation. The proteasome Sglutathiolation emerges as a new physiological post-translational modification correlated to the cellular redox state. Moreover, the S-glutathiolation of the 20SPT increases its proteolytic activity suggesting an antioxidant role by removing oxidized proteins generated during oxidative challenges.

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The mobilization and transformation of arsenic within the critical zone is a major cause of human suffering worldwide. Microorganisms, as they grow and utilize organic matter, accelerate redox processes that can transform and mobilize arsenic within aquifers on a large scale. As such, naturally occurring groundwater arsenic is a particularly hazardous problem that is chronically poisoning over 100 million people annually. Historically, groundwater arsenic research has been focused on the two principal inorganic arsenic species: arsenate [As(V)] and arsenite [As(III)]. Recently, organic arsenic species have garnered more attention due to their mobility, toxicity, and contemporary recognition of the ephemeral yet significant role they have in the global arsenic cycle. Here, I discuss laboratory and in situ experiments focused on exploring how microorganisms transform, mobilize, and sequester arsenic within a biogeochemically complex aquifer system. In my laboratory experiments, I collected aquifer sediments from a naturally contaminated bedrock aquifer and incubated a series of laboratory microcosms. Our results show that simultaneously robust iron and sulfate reduction temporarily mitigated arsenic contamination but then directed arsenic to an unstable adsorbed phase were it was later mobilized. Second, I discuss two aquifer injection experiments designed to examine in situ microbial redox processes and the further explore the potential to stimulate arsenic sequestration through arsenic-sulfide precipitation. Our results show that in situ stimulation of microbial metabolisms accelerated the reduction of arsenic bearing iron (oxy)hydroxides as well as sulfate and arsenic reduction. Within 3 weeks of these contemporaneously occurring redox reactions, 90% of the dissolved inorganic arsenic was removed (~2000 ppb) and an effective long-term, anaerobically stable, sequestration of arsenic was observed by way of a significant increase of arsenic-sulfide precipitate. Finally, using both the laboratory and field experiments, I explore the potential of organic arsenic production rates under stimulated conditions. We report new methylation rates that are consequential to the potential efficacy of enhanced, biologically-driven arsenic remediation and the reconsidered significance of biomethylation pathways in aquifers. These results expand our current understanding of the metabolic reach aquifer microorganisms potentially have over the fate of arsenic.