Dissertations / Theses on the topic 'Thioredoxin; oxidative stress'

The experiments described in this thesis involve the expression and characterisation of recombinant truncated thioredoxin (tTrx) and the potential involvement that thioredoxin (Trx) has in the cellular responses to oxidative stress. Truncated Trx (80 amino acids) was expressed from a plasmid containing the ORF for tTrx that had been introduced into E.coli BL-21(DE3) cells. The protein was initially extracted using a combination of high concentrations of urea, high pH levels, and multiple sonification steps to remove the tTrx from inclusion bodies formed during expression. This procedure produced a stable solution of tTrx. Purification of tTrx from this protein solution required anion exchange chromatography followed by gel permeation in a HPLC system to obtain fully purified, recombinant tTrx which allowed further characterisation studies to be undertaken. An initial investigation into tTrx was performed to determine some basic physical, biochemical and functional aspects of this hitherto relatively undefined protein. Analysis by sedimentation equilibrium indicated that freshly prepared tTrx forms a single species with a molecular weight of 18.8kDa. This value indicates that recombinant tTrx naturally forms a dimer in solution that was shown to be non-covalent in nature and stable in solution. The capacity of tTrx to reduce protein disulphide bonds was determined using the insulin reduction assay. Results show that tTrx lacks this particular redox ability. The rate of oxidisation at 4 degrees C was analysed using free thiol determination, sedimentation equilibrium and SDS-PAGE patterning. Results indicated a steady rise in the degree of oxidation of tTrx over an eight day period. After six days the oxidated protein consistently displayed the presence of intramolecular disulphide bonds. Covalently-linked disulphide dimers and higher molecular weight oligomers were detectable after eight days oxidation. An investigation of the reducing capacity of the basic Trx system determined that fully oxidised tTrx was unable to act alone as a substrate for thioredoxin reductase (TR). However, when reduced Trx was added to the system, it appeared capable of acting as an electron donor to the oxidised tTrx in order to reduce disulphide groups. Recombinant tTrx was successfully radiolabelled with Trans 35S-methionine/cysteine for use in cell association studies. No evidence was found to indicate the presence of a receptor for tTrx on either MCF-7 or U-937 cells. Findings suggest that a low level of non-specific binding of tTrx to these cell lines rather than a classical ligand-binding mechanism occurs thus suggesting the absence of a cell surface receptor for tTrx. The role that Trx may play in the cellular responses to oxidative stress was also investigated. The chemical oxidants hydrogen peroxide (H2O2) and diamide were used to establish an in vitro model of oxidative stress for the choriocarcinoma cytotrophoblast cell line JEG-3. Cellular function was assessed in terms of membrane integrity, metabolic activity and the ability to synthesis new DNA following exposure to these oxidants. Results indicated that both agents were capable of causing cells to undergo oxidative stress without inducing immediate apoptosis or necrosis. Initially, JEG-3 cells exposed to 38μM or 75μM H2O2 or 100μM diamide were shown to display altered cell metabolism and DNA synthesis without loss to cell viability or membrane integrity. Cells were also shown to be capable of some short-term recovery but later lapsed into a more stressed state. Expression levels of Trx were studied to determine whether this type of chemical stress caused a change in intercellular protein levels. Both cELISA and western blotting results indicated that only cells exposed to 100μM diamide displayed any significant increase in Trx protein levels after 6 or 8hrs exposure to the oxidant. Further studies over a longer time-frame were also performed. These found that when JEG-3 cells were exposed to 18μM H2O2 or 200μM diamide over 12-48hrs, a positive correlation between increasing endogenous Trx protein levels and a decline in cell proliferation was observed. Cytotrophoblast cells, which are responsible for implantation and placentation, are susceptible to oxidative stress in vivo and their anti-oxidant capacity is fundamental to the establishment of pregnancy. The findings obtained during these studies suggest that Trx plays a role in this process.

Methanogens are a group of microorganisms that utilize simple compounds such as H2 + CO2, acetate and methanol for the production of methane, an end-product of their metabolism.  These obligate anaerobes belonging to the archaeal domain inhabit diverse anoxic environments such as rice paddy fields, human guts, rumen of ruminants, and hydrothermal vents.  In these habitats, methanogens are often exposed to O2 and previous studies have shown that many methanogens are able to tolerate O2 exposure.  Hence, methanogens must have developed survival strategies to be able to live under oxidative stress conditions.  The anaerobic species that lived on Earth during the early oxygenation event were first to face oxidative stress.  Presumably some of the strategies employed by extant methanogens for combating oxidative stress were developed on early Earth.  
Our laboratory is interested in studying the mechanism underlying the oxygen tolerance and oxidative stress responses in methanogenic archaea, which are obligate anaerobe.  Our research concerns two aspects of oxidative stress.  (i) Responses toward extracellular toxic species such as SO32-, that forms as a result of reactions of O2 with reduced compounds in the environment.  These species are mostly seen in anaerobic environments upon O2 exposure due to the abundance of reduced components therein.  (ii) Responses toward intracellular toxic species such as superoxide and hydrogen peroxide that are generated upon entry of O2 and subsequent reaction of O2 with reduced component inside the cell.  Aerobic microorganisms experience the second problem.  Since a large number of microorganisms of Earth are anaerobes and the oxidative defense mechanisms of anaerobes are relatively less studied, the research in our laboratory has focused on this area.  My thesis research covers two studies that fall in the above-mentioned two focus areas.
In 2005-2007 our laboratory discovered that certain methanogens use an unusual sulfite reductase, named F420-dependent sulfite reductase (Fsr), for the detoxification of SO32- that is produced outside the cell from a reaction between oxygen and sulfide.  This reaction occurred during early oxygenation of Earth and continues to occur in deep-sea hydrothermal vents.  Fsr, a flavoprotein, carries out a 6-electron reduction of SO32- to S2-.  It is a chimeric protein where N- and C-terminal halves (Fsr-N and Fsr-C) are homologs of F420H2 dehydrogenase and dissimilatory sulfite reductase (Dsr), respectively.  We hypothesized that Fsr was developed in a methanogen from pre-existing parts.  To begin testing this hypothesis we have carried out bioinformatics analyses of methanogen genomes and found that both Fsr-N homologs and Fsr-C homologs are abundant in methanogens.  We called the Fsr-C homolog dissimilatory sulfite reductase-like protein (Dsr-LP).  Thus, Fsr was likely assembled from freestanding Fsr-N homologs and Dsr-like proteins (Dsr-LP) in methanogens.  During the course of this study, we also identified two new putative F420H2-dependent enzymes, namely F420H2-dependent glutamate synthase and assimilatory sulfite reductase.
Another aspect of my research concerns the reactivation of proteins that are deactivated by the entry of oxygen inside the cell.  Here I focused specifically on the role of thioredoxin (Trx) in methanogens.  Trx, a small redox regulatory protein, is ubiquitous in all living cells.  In bacteria and eukarya, Trx regulates a wide variety of cellular processes including cell divison, biosynthesis and oxidative stress response.  Though some Trxs of methanogens have been structurally and biochemically characterized, their physiological roles in these organisms are unknown.  Our bioinformatics analysis suggested that Trx is ubiquitous in methanogens and the pattern of its distribution in various phylogenetic classes paralleled the respective evolutionary histories and metabolic versatilities.  Using a proteomics approach, we have identified 155 Trx targets in a hyperthermophilic phylogenetically deeply-rooted methanogen, Methanocaldococcus jannaschii.  Our analysis of two of these targets employing biochemical assays suggested that Trx is needed for reactivation of oxidatively deactivated enzymes in M. jannaschii.  To our knowledge, this is the first report on the role of Trx in an organism from the archaeal domain.
During the course of our work on methanogen Trxs, we investigated the evolutionary histories of different Trx systems that are composed of Trxs and cognate Trx reductases.  In collaboration with other laboratories, we conducted bioinformatics analysis for the distribution of one of such systems, ferredoxin-dependent thioredoxin reductase (FTR), in all organisms.  We found that FTR was most likely originated in the phylogenetically deeply-rooted microaerophilic bacteria where it regulates CO2 fixation via the reverse citric acid cycle.

Ph. D.

Thesis advisor: Clare O'Connor
Within cells, reactive oxygen species (ROS) are synthesized naturally and in response to environmental stimuli. However, ROS have deleterious effects on a wide range of cellular molecules. Oxidative stress, caused by the ROS generated by the partial reduction of oxygen, is a major cause of cell damage linked to the initiation and progression of numerous diseases. Thioredoxin peroxidase (Tpx1) plays important roles in cellular defense against ROS. Although homologous genes and their functions have been identified in other eukaryotes, the level of activity as well as the necessity of this protective enzyme in S. pombe exposed to oxidative stress has yet to be fully elucidated. To explore the role of the Tpx1 protein in oxidative stress resistance, novel strains were constructed in which the tpx1 gene was overexpressed. The polymerase chain reaction was used to amplify txp1, and the amplified sequence was cloned into the yeast overexpression plasmid, pNMT41, which allows overexpression under the control of the powerful promoter. DNA sequencing was used to determine that the sequences had been properly inserted into the vector. The plasmids were transformed into two leu- yeast strains: FWP6 and TP108-3C. Production of the Tpx1 protein was ensured using Western Blot techniques. Experimentation to test the responses of the tpx1 strain to oxidative stress will employ a variety of reactive oxygen generators, including hydrogen n peroxide, menadione, tert-butyl hydroperoxide, and paraquat. The results generally supported the proposed role of Tpx1 to confer additional resistance against the oxidative stress. In a complementary line of investigation, knockout strains are being constructed to reduce the levels of the Tpx1 in S. pombe. Gene deletion cassettes were constructed for tpx1. Currently, the strains are being analyzed for the successful replacement of the endogenous tpx1 gene by homologous recombination. If the absence of the protein results in decreased cell viability, the role of Tpx1 indicated by the overexpression experiments could be supported
Thesis (BS) — Boston College, 2006
Submitted to: Boston College. College of Arts and Sciences
Discipline: Biology
Discipline: College Honors Program

Schizosaccharomyces pombe, com tots els organismes aeròbics, requereix l’oxigen per viure i té que lidiar amb la conseqüent toxicitat associada dels productes derivats de l’oxigen. Per mantenir l’ambient intracel·lular, S. pombe té sistemes antioxidants que són activats de manera ràpida i reversible. Segons la severtitat de l’estrès sofert, S. pombe té dos importants rutes antioxidants, les vies Pap1 i Sty1, per nivells de peròxid d’hidrogen (H2O2) sub-tòxiques i tòxiques, respectivament. Aquestes cascades inclouen proteïnes sensores que contenen cisteïnes anomenades “thiol switches”, les qual poden “encendre i apagar” les seves activitats. Per aquest motiu, tots els components involucrats en les cascades de senyalització antioxidant han de ser reciclades per poder estar disponibles de nou. Amb aquesta finalitat, els sistemes de reducció, els sistemes tioredoxina i glutatió/glutaredoxina, tenen com a rol principal ser el sistema de “backup” de tots els components de la cascada redox de senyalització en resposta a l’H2O2 en llevats de fissió. Al llarg de la tesis he estudiat la importància d’una correcta activitat dels dos sistemes de reducció en el reciclatge de proteïnes “redox” per evitar toxicitat cel·lular i per assegurar una progressió normal del cicle cel·lular. D’altra banda, he caracteritzat com els llindars de tolerància a l’estrès oxidatiu són important per desencadenar una resposta de senyalització o de toxicitat en S. pombe.
Schizosaccharomyces pombe, as all aerobic organisms, needs oxygen to live and have to deal with the side effects of the toxicity associated to oxygen by-products. To maintain intracellular environment, S. pombe has antioxidant systems that are activated fast and in a reversible manner. Depending on the severity of the oxidative stress suffered, S. pombe has two main antioxidant response pathways, Pap1 and Sty1 pathways, for sub-toxic and toxic hydrogen peroxide (H2O2) levels, respectively. These cascades involve sensor proteins that contain cysteine residues called thiol switches, which can switch on and off their activities. Therefore, all the components involved in the antioxidant signaling cascades have to be recycled in order to be ready to act again. For these reason, the reduction systems, thioredoxin and glutathione/glutaredoxin systems, have an essential role as backup systems of the components of the H2O2 signaling redox relays in fission yeast. Along this thesis I have studied the importance of a proper activity of both reduction systems in the recycling of redox proteins to avoid cell toxicity and to assure a normal cell cycle progression. On the other hand, we characterized how thresholds of oxidative stress tolerance are important to trigger signaling or toxicity responses in S. pombe.

Thioredoxin (Trx) and thioredoxin reductase (TrxR) along with an electron donor form a thioredoxin system. Such systems are widely distributed among the organisms belonging to the three domains of life. It is one of the major disulfide reducing systems, which provides electrons to several enzymes, such as ribonucleotide reductase, methionine sulfoxide reductase and glutathione peroxidase to name a few. It also plays an important role in combating oxidative stress and redox regulation of metabolism. Trx is a small redox protein, about 12 kDa in size, with an active site motif of Cys-X-X-Cys. The reduction of the disulfide in Trx is catalyzed by TrxR. Two types of thioredoxin reductases are known, namely NADPH thioredoxin reductase (NTR) with NADPH as the electron donor and ferredoxin thioredxoin reductase (FTR) which depends on reduced ferredoxin as electron donor. Although NTR is widely distributed in the three domains of life, it is absent in some archaea, whereas FTRs are mostly found in plants, photosynthetic eukaryotes, cyanobacteria, and some archaea. The thioredoxin system has been well studied in plants, mammals, and a few bacteria, but not much is known about the archaeal thioredoxin system. Our laboratory has been studying the thioredoxin systems of methanogenic archaea, and a major focus has been on Methanocaldococcus jannaschii, a deeply rooted archaeon that has two Trxs and one TrxR. My thesis research concerns the thioredoxin system of the late evolving members of the group which are exposed to oxygen more frequently than the deeply rooted members of the group, and have several Trxs and TrxRs. Methanosarcina mazei is one such organism, whose thioredoxin system is composed of one NTR, two FTRs, and five Trx homologs. Characterization of the components of a thioredoxin system sets the basis to further explore its function. I have expressed in Escherichia coli and purified the five Trxs and three TrxRs of M. mazei. I have shown the disulfide reductase activities in MM_Trx1 and MM_Trx5 by their ability to reduce insulin with DTT as the electron donor, and that in MM_Trx3 through the reduction of DTNB by this protein with NADPH as the electron donor, and in the presence of NTR as the enzyme. MM_Trx3 was found to be the only M. mazei thioredoxin to accept electrons through the NTR, and to form a complete Trx - NTR system. The Trx - FTR systems are well studied in plants, and such a system is yet to be defined in archaea. I have proposed a mechanism of action for one of the FTRs. FTR2 harbors a rubredoxin domain, and this unit is the only rubredoxin in this organism. Superoxide reductase, an enzyme that reduces superoxide radical to hydrogen peroxide without forming oxygen, utilizes rubredoxin as the direct electron source and this enzyme is found in certain anaerobes, including Methanosarcina species. Thus, it is possible that FTR2 provides electrons via a Trx to the superoxide reductase of M. mazei. This activity will define FTR2 as a tool in combating oxidative stress in M. mazei. In my thesis research I have laid a foundation to understand a complex thioredoxin system of M. mazei, to find the role of each Trx and TrxR, and to explore their involvement in oxidative stress and redox regulation.
Master of Science

Maintainence of appropriate redox homeostasis is crucial for processes such as protein folding in the endoplasmic reticulum (ER) and to minimise genesis of oxidative stress. Previous studies have indicated a possible link between ER stress and production of reactive oxygen species (ROS) although the cellular mechanisms involved were not fully elucidated. To investigate the cellular mechanisms involved in tolerance to oxidative stress and ER stress, genome-wide screens were performed to identify mutants sensitive to chronic ER stress induced by dithiothreitol and tunicamycin. These screens identified the Cu,Zn superoxide dismutase (SOD1) and genes involved in NADPH generation (RPE1, TKL1) as important for chronic ER stress tolerance. Superoxide anion has been identified as one of the ROS generated during ER stress. The ER oxidoreductase Ero1p, previously implicated in ROS production in vitro, did not appear to be a source of superoxide when the protein was over-expressed. It was also found that cellular NADP(H) levels affected induction of the unfolded protein response (UPR), since cells lacking TKL1 or RPE1 exhibited decreased UPR induction. These data indicate an important role for superoxide dismutase and cellular NADP(H) in survival of cells during ER stress. Subsequent analysis determined that NADPH generation was also required for adaptation to H2O2. Mutants affected in NADPH production were chronically sensitive to H2O2 but resistant to an acute dose. These mutants over-accumulated reduced glutathione (GSH) but maintained normal cellular redox homeostasis. This over- production of GSH was not regulated at the transcriptional level of GSH1 encoding ??- glutamyl cysteine synthetase. These data raise the important question as to how cells maintain cellular glutathione redox balance. To better understand how cells respond to perturbations in glutathione redox homeostasis, cells deleted for GLR1, encoding GSSG reductase, were exposed to extracellular oxidised glutathione (GSSG) and intracellular GSH and GSSG were monitored over time. Intriguingly cells lacking GLR1 showed increased levels of GSH accumulation upon GSSG treatment in a manner independent of GSH synthesis. It was subsequently found that the cytosolic thioredoxin-thioredoxin reductase system contributes to the reduction of GSSG in vivo.

La formation de ponts disulfure constitue une modification post-traductionnelle des protéines importante pour de nombreux processus physiologiques, jouant un rôle particulier dans le repliement, la catalyse et la régulation de leur activité. Ce travail concerne l'étude des relations structure-fonction d'oxydoréductases de peuplier appartenant à deux familles de la superfamille des thiorédoxines, les glutathion peroxydases (Gpxs) et les protéine disulfure isomérases (PDIs).L'étude biochimique fine de la Gpx5 a permis de montrer que cette peroxydase réduit le peroxynitrite, propriété inconnue pour ce type de Gpx et de détailler plusieurs étapes du mécanisme catalytique (formation de l'acide sulfénique, changement structural entre formes réduites et oxydées, régénération par les Trxs). La dimérisation de la Gpx5 n'est pas requise pour son activité mais pourrait jouer un rôle dans la reconnaissance de certains substrats. Enfin, l'inactivation de la cystéine peroxydatique par suroxydation suggère que les Gpxs pourraient également avoir une fonction dans la signalisation en réponse aux peroxydes.Concernant les PDIs, suite à une analyse phylogénétique détaillée amenant à proposer une nouvelle classification en 9 classes chez les organismes photosynthétiques, la caractérisation biochimique de plusieurs isoformes présentant des organisations modulaires distinctes et appartenant à trois classes de PDIs a été entreprise. Aucune activité enzymatique typique n'a été identifiée pour la PDI-A, alors que les PDI-L1a et -M possèdent à la fois une activité oxydase et réductase. Les deux modules a de la PDI-M catalysent des réactions spécifiques, de réduction ou d'oxydation
Protein activity and folding can be regulated by post-translational modifications that can impact on their physiological functions. One of these is the formation/reduction of disulfide bridges. The aim of the present work is to study the structure-function relationship of protein members of the thioredoxin superfamily, the protein disulfide isomerases (PDI) and the glutathione peroxidases (Gpx).A precise biochemical study has allowed us to demonstrate that this enzyme is an efficient peroxynitrite scavenger, a new finding for this type of protein and allowed investigating several steps of the Gpx5 catalytic mechanism (i.e. sulfenic acid formation, structural changes between reduce dand oxidized forms, Trx-mediated recycling). We also demonstrate that the dimer form of Gpx5 is not absolutely required for peroxide reduction but probably involved in peroxide specificity. Finally, the capability of the peroxidatic cysteine to be overoxidized brings some new clues in favor of an additional signaling function for Gpx5.Concerning PDIs, a detailed phylogenetic analysis of photosynthetic organisms allowed us to identify 9 classes of PDIs and to propose a new nomenclature that fits all these organisms. The biochemical characterization of isoforms of interest has allowed us to highlight some specificity of PDI-L1a and PDI-M in terms of reduction or oxidation reactions catalyzed. A detailed analysis of PDI-M isoform also indicates that the two Trx modules of this protein show differential oxidation or reduction capacities. We could not detect any activity for PDI-A isoforms, leaving us to wonder whether this enzyme is simply active or possesses highly specific protein partners

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Methylmercury (MeHg) is an important environmental toxicant that may cause irreversible neurobehavioral and neuropsychological disorders in humans and experimental animals. The major mechanisms of MeHg-induced toxicity currently being explored are the disruption of intracellular calcium homeostasis, the induction of oxidative stress, inhibition of neuronal Na+/K+ -ATPase activity and change the status of antioxidant systems. In addition, recent data reported the involvement of MeHg toxicity with damage in thioredoxin system. On the other hand, flavonoids have been reported to possess divalent metal chelating properties, antioxidant activities and to readily permeate the blood brain barrier. They can also provide neuroprotection in a wide array of cellular and animal models of neurological diseases, including protection against MeHg toxicity. However, the exact mechanism of MeHg toxicity remain unclear and limited data on the interaction of MeHg with flavonoids are avaliable in literature. In view of this, our study evaluated the mechanisms of MeHg toxicity in vivo and in vitro models and evaluated the performance of different flavonoids: quercetin, quercitrin and rutin in diferent models of MeHg toxicity. Our study showed that MeHg (100 μM) caused lipid peroxidation and reactive oxygen species (ROS) generation in brain cortical slices. Quercitrin and quercetin protected against this toxicity and mitochondria from MeHg (5 μM)-induced ROS generation. In contrast, rutin did not afford a significant protective effect against MeHg (100 μM)-induced lipid peroxidation and ROS production in cortical brain slices. MeHg-generated ROS in cortical slices was dependent upon an increase in intracellular calcium levels. In vivo studies with mice treated during 30 days with MeHg (5mg/Kg) orally, presented a marked increase in toxicity parameters (loss in body weight gain, increased in micronucleis frequencies, nefrotoxicity), decrease in motor system performance (locomotor activity and motor coordination) and spatial memory deficiency as well as alteration in some biochemical parameters (decrease in glutathione peroxidase and Na+/K+ ATPase activity, increase in lipid peroxidation). The co-treatment with quercitrin (10mg/kg) intraperitoneally, decreased the behavior alterations manly by decreased lipid peroxidation levels, maintained the Na+/K+ ATPase and GPx activities. In addition, our study demonstrated, for the first time, that MeHg inhibited the activity of thioredoxin reductase. A single oral MeHg administration (1, 5 and 10 mg/Kg) caused a marked inhibition of kidney TrxR, while in liver a significant inhibition was observed after exposure to 5 and 10 mg/Kg of MeHg (TrxR was determined 24 hours after MeHg). In brain, MeHg did not inhibit TrxR. In vitro results demonstrated that MeHg inhibited brain (0.05 1 μM) , liver (0.05 1 μM) and kidney (0.025 1 μM) TrxR in a dose dependent manner Here, we have extended the characterization of mechanisms associated with the neuroprotective effects of flavonoids quercetin and quercitrin against MeHg-induced toxicity. In addition, we provided novel data establishing that (1) calcium plays a central role in MeHg toxicity, (2) in brain slices MeHg induces mitochondrial oxidative stress both via direct interaction with mitochondria as well as via mitochondria- indirect mechanisms. In addition (3) MeHg (5mg/kg) caused a number of behavioural alterations that are related with an inhibition of cerebelar and cerebral GPx and Na+/K+ ATPase activities and (4) increased in lipid peroxidation.The higly affinity of MeHg to selenol groups of endogenous molecules can lead to (5) inhibition of thioredoxin reductase that can contribute to MeHg toxicity. We conclude that MeHg lead to increase in mitochondria ROS generation that contributes to increase in lipid peroxidation. In addition, the inhibition of important antioxidant enzymes such as GPx ans TrxR can contribute to oxidative damage that can be related to development of behavioral damage. In this view the antioxidant activity of flavonoids quercetin and quercitrin seems to be direct associate with the capacity of flavonoids to confere protection against MeHg toxicity.
O metilmercúrio (MeHg) é um importante agente tóxico ambiental que pode causar desordens neurocomportamentais e neurofisiológicas irreversíveis em humanos e animais experimentais. Os principais mecanismos pelos quais o MeHg induz toxicidade são: a ruptura na homeostase do cálcio intracelular, a indução de estresse oxidativo, a inibição da atividade da Na+/K+ ATPase neuronal e mudanças nos níveis das enzimas antioxidantes. Adicionalmente, dados recentes relatam o envolvimento do sistema da tiorredoxina como um dos alvos de toxicidade do MeHg. Por outro lado, os flavonóides possuem propriedades quelantes para metal divalente, atividade antioxidante e são permeáveis a barreira cérebro-sangue. Além disso, eles podem oferecer neuroproteção a uma variedade de modelos animais e celulares de doenças neurológicas, incluindo proteção contra a toxicidade do MeHg. Considerando que o exato mecanismo pelo qual o MeHg exerce toxicidade permanece desconhecido e que poucos e controversos dados sobre a interação do MeHg com flavonóides são encontrados na literatura, este estudo avaliou os mecanismos de toxicidade do MeHg em modelos in vitro e in vivo bem como, o desempenho de diferentes flavonóides: quercetina, quercitrina e rutina em diferentes modelos de toxicidade induzidos pelo MeHg. Nosso estudo mostrou que o MeHg (100μM) causou aumento na peroxidação lipídica e na produção de espécies reativas de oxigênio (EROs) em fatias de córtex de ratos. Os flavonóides quercitrina (25 μg/mL) e quercetina (5, 10 e 25 μg/mL) protegeram contra esta toxicidade, e contra o aumento de ERO produzidas pelo MeHg (5μM) nas mitocôndrias. Diferentemente, o flavonóide rutina não obteve efeito protetor contra a indução da peroxidação lipídica e produção de ERO induzidas pelo MeHg em fatias corticais de cérebro. O aumento na produção de ERO, geradas pelo MeHg, foi dependente do aumento dos níveis intracelulares de cálcio (artigo 1). Já, estudos in vivo com camundongos tratados oralmente com MeHg (5mg/kg), durante 30 dias, mostraram um marcado aumento nos parâmetros de toxicidade (diminuição no ganho de peso, aumento na freqüência de micronúcleos e nefrotoxicidade), diminuição no desempenho do sistema motor (atividade locomotora e coordenação motora), e deficiência na memória espacial, bem como alterações em vários parâmetros bioquímicos (diminuição na atividade da glutationa peroxidase (GPx) e Na+/K+ ATPase e aumento na peroxidação lipídica). O co-tratamento com quercitrina (10mg/kg) pela via intraperitoneal, diminuiu as alterações comportamentais principalmente por diminuir os níveis de peroxidação lipídica e manter a atividade da GPx e da Na+/K+ ATPase iguais aos níveis do controle (manuscrito 1). Além disso, nosso estudo demonstrou, pela primeira vez, que o MeHg inibe a atividade da tiorredoxina redutase (TrxR). Uma única administração oral de MeHg (1, 5, 10 mg/kg), causou uma marcada inibição na atividade da TrxR renal, enquanto no fígad observou-se uma inibição significativa após exposição a 5 e 10 mg/kg (a atividade da TrxR foi determinada 24 horas após a administração de MeHg). No cérebro, o MeHg não inibiu a atividade da TrxR in vivo (artigo 2). Já os resultados in vitro revelaram que o MeHg causou uma inibição concentração dependente na atividade da enzima TrxR isolada de cérebro (0,05 1 μM) fígado (0,05 - 1 μM) e rim (0,025 1 μM). Assim, nós ampliamos a caracterização dos mecanismos associados com os efeitos neuroprotetores dos flavonóides quercetina e quercitrina na toxicidade induzida pelo MeHg. Adicionalmente, outros dados sobre a toxicidade do MeHg, foram obtidos, tais como: (1) o cálcio desempenha um papel central na toxicidade do MeHg, (2) em fatias de cérebro de ratos o MeHg induz estresse oxidativo mitocondrial via interação direta com as mitocôndrias, bem como via mecanismos mitocondriais indiretos. Além disso, (3) o MeHg (5mg/kg) pode levar a inúmeras alterações comportamentais que podem estar relacionadas à inibição da atividade das enzimas GPx e Na+/K+ ATPase e (4) aumento na peroxidação lipídica. A alta afinidade do MeHg por grupos selenóis das moléculas endógenas pode levar (5) a inibição da TrxR o que pode contribuir para a toxicidade do MeHg. Podemos concluir que o MeHg leva a um aumento na produção de ERO pelas mitocôndrias, o que contribui para um aumento na peroxidação lipídica induzida pelo MeHg. Além disso, a inibição de importantes enzimas antioxidantes como a GPx e a TrxR podem contribuir para aumentar o dano oxidativo, que parece estar relacionado com o aparecimento dedanos comportamentais. Desta forma a atividade antioxidante dos flavonóides quercetina e quercitrina parece estar diretamente associada à capacidade destes compostos emproteger contra a toxicidade do MeHg.

Le stress oxydatif et l'inflammation jouent un rôle pathogène dans l'athérosclérose. La Thiorédoxine-1 (Trx-1) est une protéine anti-oxydante et anti-inflammatoire ayant des effets athéroprotecteurs. Cependant, le clivage in vivo de la Trx-1 génère une protéine tronquée pro-inflammatoire, la Trx-80, qui compromet l'utilisation thérapeutique de la Trx-1. L'objectif de ma thèse est de caractériser une nouvelle stratégie thérapeutique basée sur l’utilisation des peptides mimétiques de la Trx-1 (TxMP) pour le traitement de l'athérosclérose. Nous avons synthétisé un petit peptide basé sur le site actif de la Trx-1 appelé CB3. D’une part, le peptide CB3 a été validé sur des macrophages murins péritonéaux en culture (viabilité cellulaire, réponses anti-oxydantes et anti-inflammatoires). D’une autre part, un modèle de souris athérosclérotique (ApoE2.Ki) nourri avec un régime alimentaire riche en graisse a reçu une injection de CB3 par voie intrapéritonéale afin de mesurer son effet antioxydant, anti-inflammatoire et anti-athérogène. Nos résultats ont clairement montré que le peptide CB3 exerce des effets protecteurs similaires à la Trx-1, à savoir une réduction de l'inflammation et du stress oxydatif chez les macrophages et les souris ApoE.Ki. L'effet athéroprotecteur du CB3 ouvre des perspectives thérapeutiques prometteuses pour le traitement de l'athérosclérose
Oxidative stress and inflammation play a pathogenic role in atherosclerosis. Thioredoxin-1 (Trx-1) is an anti-oxidative, anti-inflammatory protein with atheroprotective effects. However, in vivo cleavage of Trx-1 generates a truncated pro-inflammatory protein, Trx-80, which compromises the therapeutic use of Trx-1. The aim of my thesis is to characterize a new therapeutic strategy based on Trx-mimetic peptides (TxMP) for the treatment of atherosclerosis. We synthesized a small peptide based on the active site of Trx-1 named CB3. Firstly, CB3 was validated on cultured peritoneal murine macrophages (cellular viability, anti-oxidant and anti-inflammatory responses). Secondly, the atherosclerotic mouse model (ApoE2.Ki) fed a high fat diet was intraperitoneally injected with CB3 to measure their anti-oxidant, anti-inflammatory and anti-atherogenic effects. Our results clearly showed that, similar to the full length Trx-1, CB3 exerts protective effects by reducing inflammation and oxidative stress in macrophages and in ApoE.Ki mice. The atheroprotective effect of CB3 opens promising therapeutic approaches for treatment of atherosclerosis

Conselho Nacional de Desenvolvimento Científico e Tecnológico
The diphenyl ditelluride (PhTe)2 is one of the most toxic organic compounds of tellurium which can make their use unsafe. The mechanism(s) involved in (PhTe)2 toxicity is(are) elusive, but thiol oxidation of critical proteins are important targets. Consequently, the possible remedy of its toxicity by thiol-containing compounds is of experimental and clinical interest. Therefore, this study aimed to evaluate the toxicity of in vivo exposure to (PhTe)2 from oxidative stress biomarkers and behavioral parameters in adult mice and the possible protective effect of N-acetylcysteine (NAC). They evaluated parameters of oxidative stress and behavior in mice. In order to alleviate the toxicity, NAC was administered before (3 days) and simultaneously (PhTe)2 (7 days). Mice were separated into six groups receiving daily injections of (1) Potassium phosphate buffer (TFK) (2.5 ml/kg, intraperitonealy (i.p.)) plus canola oil (10 ml/kg, subcutaneously (s.c.)), (2) NAC (100 mg/kg, i.p.) plus canola oil s.c., (3) TFK i.p. plus (PhTe)2 (10 μmol/kg, s.c.), (4) TFK i.p. plus (PhTe)2 (50 μmol/kg, s.c.), (5) NAC plus (PhTe)2 (10 μmol/kg, s.c.), and (6) NAC plus (PhTe)2 (50 μmol/kg, s.c.). Treatment with (PhtE) started on day 2 of treatment with NAC. The results demonstrate that (PhTe)2 induced behavioral changes in locomotor activity at a concentration of 50 μmol/kg and NAC did not change the effect of (PhTe)2. Motor coordination and lift the bar were compromised and both showed severe motor abnormalities in test animals independent of concentration of (PhTe)2 . The (PhTe)2 also inhibited important selenoenzymes, thioredoxin reductase (at concentrations of 10 μmol/kg and 50 μmol/kg) and glutathione peroxidase (at concentration of 10 μmol/kg) but produced little or no effect on the antioxidant activity of catalase and glutathione reductase. Contrary to expectation, the co-administration of NAC did not protect against deleterious effects (PhTe)2. It was possible to establish high sensitivity of brain tissue compared to the damage (PhTe)2. Other low molecular weight thiols must be investigated to determine whether they may or may not be effective against ditellurides.
O ditelureto de difenila (PhTe)2 é um dos compostos orgânicos de telúrio mais tóxicos, o que pode tornar seu emprego pouco seguro. O mecanismo envolvido na toxicidade do (PhTe)2 ainda é incerto, mas a oxidação de tióis em proteínas são alvos importantes. A partir disso, compostos contendo tiol possívelmente poderiam solucionar ou minimizar a sua toxicidade. Portanto, este estudo teve como objetivo avaliar a toxicidade da exposição in vivo ao (PhTe)2 a partir de biomarcadores de estresse oxidativo e parâmetros comportamentais em camundongos adultos e o possível efeito protetor da N-acetilcisteína (NAC). Foram avaliados parâmetros de estresse oxidativo e comportamentais em camundongos. A fim de mitigar a toxicidade, foi administrado NAC antes (3 dias) e, simultaneamente ao (PhTe)2 (7 dias). Os camundongos foram separados em seis grupos que receberam injeções diárias de (1) Tampão fosfato de potássio (TFK) (2.5 ml/kg, intraperitonealmente (i.p.)) mais óleo de canola (10 ml/kg, subcutaneamente (s.c.)), (2) NAC (100 mg/kg, i.p.) mais óleo de canola s.c., (3) TFK i.p. mais (PhTe)2 (10 μmol/kg, s.c.), (4) TFK i.p. mais (PhTe)2 (50 μmol/kg, s.c.), (5) NAC mais (PhTe)2 (10 μmol/kg, s.c.), e (6) NAC mais (PhTe)2 (50 μmol/kg, s.c.). O tratamento com (PhTe)2 começou no quarto dia de tratamento com NAC. Os resultados demonstram que (PhTe)2 induziu alterações comportamentais na atividade locomotora na concentração de 50 μmol/kg e a NAC não modificou o efeito do (PhTe)2. A coordenação motora e a força de sustentação na barra foram comprometidas e ambas revelaram alterações motoras graves nos animais testados independente da concentração de (PhTe)2. O (PhTe)2 também inibiu selenoenzimas importantes, tiorredoxina redutase (nas concentrações de 10 μmol/kg e 50 μmol/kg) e glutationa peroxidase (na concentração de 10 μmol/kg), mas produziu mínimo ou nenhum efeito sobre a atividade antioxidante da catalase e glutationa redutase. Contrariamente ao esperado, a co-administração com NAC não protegeu contra os efeitos deletérios do (PhTe)2. Foi possível estabelecer grande sensibilidade do tecido cerebral frente aos danos causados pelo (PhTe)2. Outros tióis de baixo peso molecular devem ser investigados para determinar se eles podem ou não ser eficazes contra diteluretos.

Conselho Nacional de Desenvolvimento Científico e Tecnológico
Oxidative stress is an important molecular mechanism of lead (Pb) toxicity. The thioredoxin system (selenoenzyme thioredoxin reductase-TrxR, thioredoxin protein- Trx and NADPH) is essential for the antioxidant defense and cellular redox control. In our previous study, it was showed that cytosolic renal TrxR1 activity of rats increased after acute and long-term exposure to Pb and this was the only parameter that changed after both exposures to low Pb doses. Then, it was suggested that TrxR1 could operate in the early defense against Pb toxicity and it could also be used as a bioindicator of early effects of Pb. Thus, the main objective of this thesis was to investigate the role of thioredoxin system in Pb-induced changes, evaluating: I) in vitro the activity of purified TrxR1, as well as the activity and the protein expression of TrxR1 and Trx1 in renal HEK 293 culture cells exposed to Pb; II) in vivo, the effects of Pb exposure in rats and in occupationally-exposed humans on renal (only in rats) and blood TrxR1 activity (both rats and humans), comparing these effects to oxidative stress parameters, as well as to classical bioindicators of Pb effect and exposure. The results of the in vitro study showed that lead is a less potent inhibitor of the purified TrxR1 activity (IC50 = 0.27 TM) than its structural homologous glutathione reductase (IC50 = 0.048 TM). TrxR1 inhibition was independent on the selenocysteine residue of the active site and was reversible by bovine serum albumin and by the EDTA chelating. TrxR1 inhibition also occurred in HEK 293 cells exposed to the highest Pb acetate concentration (60 TM), without alterations in protein expression. However, under glutathione (GSH) depletion after pre-incubation of cells with L-buthionine-[S,R]-sulfoximine (BSO) and further exposure to Pb, the activity and expression of both TrxR1 and Trx1 increased in the absence of cytotoxicity and of changes in GR and glutathione S-transferase activities, which indicates Trx system as an important protective mechanism against Pb toxic effects in GSH-depleted cells. On the other hand, blood TrxR1 activity did not change either after acute exposure of rats or long-term exposure of humans to Pb. However, the increase of renal TrxR1 activity in rats exposed to the highest dose of Pb acetate (25 mg/kg) occurred concomitantly with the increase of blood and renal Pb levels over time (6, 24 e 48 h), whereas the erythrocyte δ-ALA-D inhibition, which is a classical indicator of Pb effects, occurred after 6 h of exposure and the activity was further recovered (at 24 and 48 h). Moreover, the increase of renal TrxR1 activity occurred without renal histopathologycal damage, which corroborates the increase of this enzyme as an early event of Pb toxicity. Overall, the results of the current study point out the thioredoxin system as a target for Pb, but mainly as a protective mechanism against Pb toxicity. However, the absence of changes in blood TrxR1 activity in Pb-exposed animals and humans indicates that this enzyme is not an appropriate bioindicator of the toxic effects of Pb in exposed populations.
O estresse oxidativo é um importante mecanismo molecular da toxicidade do chumbo (Pb). O sistema da tiorredoxina (selenoenzima tiorredoxina redutase -TrxR, proteína tiorredoxina -Trx e NADPH) é essencial na defesa antioxidante e no controle redox celular. Em nosso estudo prévio, foi demonstrado que a atividade da enzima TrxR1 (citosólica) renal de ratos aumentou na exposição aguda e prolongada ao Pb, sendo o único parâmetro alterado em ambas exposições a doses baixas de Pb. Assim, foi sugerido que a TrxR1 atuaria precocemente na defesa contra a toxicidade do metal, podendo também ser utilizada como um bioindicador dos efeitos precoces do Pb. Assim, o objetivo geral desta tese foi investigar o papel do sistema da tiorredoxina nas alterações induzidas pelo Pb, avaliando: I) in vitro a atividade da TrxR1 purificada, bem como a atividade e expressão protéica da TrxR1 e Trx1 em culturas de células renais HEK 293 expostas ao Pb e II) in vivo, os efeitos do Pb em ratos e em humanos ocupacionalmente expostos ao Pb sobre a atividade da TrxR1 renal (somente em ratos) e sanguínea (ratos e humanos), comparando esses efeitos com parâmetros de estresse oxidativo, bem como com indicadores clássicos de efeito e de exposição ao Pb. Os resultados do estudo in vitro mostraram que a atividade da enzima TrxR1 purificada foi inibida pelo Pb (IC50 = 0.27 TM) de forma menos potente que a sua homóloga estrutural glutationa redutase (IC50 = 0.048 TM). Essa inibição foi independente do resíduo de selenocisteína do sítio ativo da TrxR1 e foi revertida pela albumina sérica bovina e pelo quelante EDTA. A inibição da TrxR1 também ocorreu em células HEK 293 expostas à maior concentração de acetato de Pb (60 TM), sem alterações na expressão protéica. Entretanto, quando os níveis celulares de glutationa (GSH) foram depletados por pré incubação das células com L-butionina-[S,R]-sulfoximina (BSO) e posterior exposição ao Pb, a atividade e a expressão da TrxR1 e da Trx1 aumentaram na ausência de citotoxicidade e de alterações nas atividades da GR e glutationa S-transferase, apontando esse sistema como um importante mecanismo contra a toxicidade do Pb em células sob depleção de GSH. Por outro lado, a atividade da TrxR1 sanguínea não alterou na exposição aguda de ratos e prolongada de humanos ao Pb. No entanto, o aumento da atividade da TrxR1 renal em ratos expostos à maior dose de acetato de Pb (25 mg/kg) foi concomitante com o aumento dos níveis sanguíneos e renais de Pb ao longo do tempo (6, 24 e 48 h), enquanto que a inibição da enzima δ-ALA-D eritrocitária, um indicador clássico de efeito do Pb, ocorreu após 6 h de exposição, sendo sua atividade restabelecida posteriormente (24 e 48 h). Além disso, o aumento da atividade da TrxR1 renal ocorreu sem danos histopatológicos renais, confirmando essa alteração como um evento precoce da toxicidade do Pb. Em geral, os resultados do presente estudo apontam o sistema da tiorredoxina como alvo do Pb, mas principalmente como um mecanismo de proteção contra o metal. Entretanto, a ausência de alterações na atividade da TrxR1 sanguínea em animais e humanos expostos ao Pb, indica que essa enzima não é um bioindicador adequado dos efeitos tóxicos do Pb em populações expostas.

L’arthrose est une maladie dégénérative de l’articulation caractérisée par une dégradation du cartilage, une inflammation de la membrane synoviale et un remodelage de l’os sous-chondral. En conditions physiologiques, les chondrocytes, seul type cellulaire du cartilage, sont en hypoxie (≈ 2% d’oxygène). Le cartilage étant un tissu avascularisé, il existe un gradient de concentration en oxygène au sein des différentes couches du cartilage. Lors du développement de l’arthrose, la dégradation du cartilage provoque une rupture de ce gradient, exposant ainsi les cellules des couches profondes à des concentrations en oxygène beaucoup plus élevées et induisant des modifications de leur métabolisme, ce qui induit leur dysfonction. Le syndrome métabolique est défini par un ensemble de perturbations glucidiques, lipidiques et vasculaires menant au développement de maladies cardiovasculaires et au diabète de type 2. Récemment, un lien entre arthrose et syndrome métabolique a été suggéré, introduisant une notion d’arthrose métabolique. Au cours de cette étude, nous nous sommes intéressés au lien entre arthrose, syndrome métabolique et stress oxydant induit par de fortes concentrations de glucose. Dans la première partie de ce travail, nous avons étudié les effets in vitro de 25 mM de glucose sur une lignée de chondrocytes humains immortalisés (T/C28a2), en hypoxie (2% d’oxygène) et en normoxie (21% d’oxygène). Nous avons montré que le glucose à 25 mM induisait la production d’espèces réactives de l’oxygène (ERO) et de l’azote, l’activation de la caspase 3, la production d’interleukine 6 (IL-6), la diminution de l’activité lysyl oxydase (LOX), qui est impliquée dans les liaisons de pontage des fibres de collagène et d’élastine, ainsi que l’activation du système thiorédoxine (Trx). Ce dernier est un système de défense anti-oxydant endogène composé de la thiorédoxine, de la thiorédoxine réductase (TR) et de Txnip, qui intervient dans le maintien de l’homéostasie cellulaire en réduisant les protéines oxydées, contrôlant ainsi l’environnement redox des cellules. Les effets du glucose 25 mM ont été observés dans les deux conditions d’oxygène étudiées, cependant la réponse cellulaire en normoxie était plus précoce qu’en hypoxie. Nous avons également pu mettre en évidence un rôle de régulateur négatif de la Trx-1 sur la production d’IL-6 faisant intervenir la voie de signalisation p38MAPK. Dans la deuxième partie de ce travail, nous nous sommes intéressés aux effets de l’apport exogène de resvératrol sur les modifications induites par le glucose à 25 mM. Le resvératrol (3,4’,5-trihydroxystilbène) est un polyphénol de la famille des stilbènes, connu pour ses multiples propriétés anti-oxydantes, anti-inflammatoires, anti-diabétiques et anti-cancer. Nous avons pu observer que le resvératrol à 25 μM était capable de diminuer les effets délétères provoqués par le glucose à 25 mM. Cependant, la biodisponibilité du resvératrol est très limitée, empêchant son utilisation en thérapeutique humaine. Par conséquent, dans la troisième partie de cette étude, nous nous sommes intéressés au développement de nouvelles formulations galéniques de resvératrol (nano-émulsions (NE)) et à leurs effets sur un modèle de cellules endothéliales aortiques bovines (BAEC), sur les T/C28a2 ainsi que sur des chondrocytes humains en culture primaire provenant de cartilage de patients arthrosiques. Nous avons montré qu’une des NE permettait d’augmenter le passage intracellulaire de resvératrol dans les deux modèles étudiés et d’en potentialiser les effets protecteurs contre un stress oxydant. Cette NE s’est également montrée efficace dans le rétablissement de l’activité LOX dans les cellules de patients arthrosiques. En conclusion, nous avons montré que le glucose à 25 mM avait des effets délétères sur les chondrocytes de la lignée T/C28a2 et que l’apport exogène de resvératrol permettait de lutter contre ses effets. (...)
Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation, inflammation of synovial membrane and subchondral bone remodelling. Under physiological conditions, chondrocytes - the only cell type found in cartilage - are under hypoxia (around 2% of oxygen). As cartilage is an avascular tissue, an oxygen gradient is established from the superficial to the deeper layers. During OA development, cartilage degradation is responsible for a break in this gradient; consequently, cells from the deepest layers are exposed to higher oxygen concentrations inducing modifications in cell metabolism leading to their dysfunction. Metabolic syndrome (MetS) is defined by a cluster of factors (impairment of glucose and lipid metabolism, vascular dysfunctions…) leading to cardiovascular diseases and type 2 diabetes development. Recently, a link between OA and MetS has been suggested, introducing a notion of metabolic OA. We have focused our study on the link between OA, MetS and oxidative stress induced by high glucose concentrations. In the first part of this study, we have determined the in vitro effects of 25 mM glucose on an immortalized human chondrocyte cell line (T/C28a2), under hypoxia (2% oxygen) and normoxia (21% oxygen). We demonstrated that 25 mM glucose induced radical oxygen species (ROS) and nitric oxide production, caspase 3 activation, interleukin 6 (IL-6) production, decrease in lysyl oxidase (LOX) activity (involved in type II collagen crosslinks), and activation of the thioredoxin (Trx) system. Trx system is an endogenous anti-oxidant system, composed by thioredoxin, thioredoxin reductase (TR) and Txnip; it is involved in cellular homeostasis by reducing oxidized proteins, thereby controlling cellular redox environment. Effects of 25 mM glucose have been observed under both oxygen conditions; nevertheless, cellular response under normoxia underwent earlier than under hypoxia. We have also highlighted Trx-1 as a negative regulator of IL-6 production through p38MAPK signalling pathway. In the second part of this study, we have focused our work on the effects of the addition of an exogenous antioxidant, i.e. resveratrol, on the modifications induced by 25 mM glucose. Indeed, resveratrol (3,4’,5-trihydroxystilbene) is a polyphenol of the stilbene family, known for its multiple anti-inflammatory, anti-oxidative, anti-diabetes and anti-cancer properties. We have observed that 25 μM resveratrol was able to decrease deleterious effects induced by 25 mM glucose. However, resveratrol bioavailability is very low, avoiding its use in human therapeutic strategy. Consequently, in the third part of this study, we have developed new galenic formulations of resveratrol, i.e. nano-emulsions (NEs) and determined their effects on a bovine aortic endothelial cells (BAEC) model, on T/C28a2 cells and also on primary cultures of human chondrocytes from osteoarthritic cartilages. One of our NEs was able to increase resveratrol intracellular passage in both cellular models, and to increase the protective effects of resveratrol against oxidative stress. This NE was also efficient in the normalization of LOX activity in osteoarthritic chondrocytes. To conclude, we have demonstrated that 25 mM glucose induced deleterious effects on chondrocytes of the T/C28a2 cell line, and that an exogenous supply in resveratrol allowed to counteract these effects. Development of a new galenic formulation of resveratrol opens new interesting prospects in human therapeutic strategy against OA associated with MetS

Une meilleure compréhension de la physiologie de la semence des céréales constitue certainement un moyen pour améliorer et développer de nouvelles variétés capables de correspondre aux besoins économiques et écologiques du moment. Les rédoxines constituent des marqueurs intéressants pour appréhender la qualité technologique et germinative du grain de blé en particulier. Le criblage des banques de données a permis d’isoler des isoformes de ces rédoxines. Cette étude a confirmé l’implication des thiorédoxines dans la réduction des protéines de réserve du blé et de maïs. Elle a permis de mettre en évidence un autre rôle de certains isoformes de thiorédoxines h dans la formation de polymères de hauts poids moléculaires. L'inhibition de l’expression de gènes par interférence ADN montre que les thiorédoxines et les glutarédoxines sont impliquées dans la protection contre le stress oxydatif chez le blé. De même, l'application d’un stress biotique simulé par la laminarine a permis de discriminer l'implication de différents marqueurs du stress, et de montrer en particulier que la 1-Cys-Prx peut être considérée comme un indicateur de l'état redox du grain pendant la germination. La mise en place d’une méthode simple et efficace de transformation des céréales via Agrobacterium, constitue un moyen pour comprendre davantage le rôle de ces rédoxines dans la gestion des stress, et les éventuelles conséquences sur la qualité technologique du grain
A better understanding of the physiology of seed cereal constitutes certainly a means to improve and develop new varieties capable of corresponding to the actual economic and ecological needs. Redoxins are interesting markers to apprehend the technological and germinative quality of wheat seed in particular. The screening of data banks allowed isolating isoforms of these redoxins. This study confirmed the implication of thioredoxins in the reduction of storage proteins in wheat and corn seeds. It allowed to bring to light another role of some thioredoxins h isoforms in the formation of high molecular weights polymers. The inhibition of the expression of genes by DNA interference shows that thioredoxins and glutaredoxins are involved in the protection against oxidative stress in wheat. Also, the application of a biotic stress simulated by laminarin allowed to discriminate the implication of various stress markers, and to highlight in particular that the 1-Cys-Prx can be considered as an indicator of the redox state of the grain during germination and seedling. The implementation of a simple and effective method of transformation of cereal via Agrobacterium constitutes a means to understand more on the role of these redoxins in the management of the stress, and the possible consequences on the technological quality of the seed

La mucoviscidose est la maladie génétique la plus fréquente dans la population caucasienne. Le genre Scedosporium se situe au deuxième rang parmi les champignons filamenteux isolés des expectorations dans ce contexte. Au niveau pulmonaire, les colonisations/infections entraînent le recrutement de phagocytes qui induisent un stress oxydatif normalement délétère pour les pathogènes. Pour se défendre, ceux-ci ont développé des systèmes antioxydants, notamment diverses enzymes. Ce travail de thèse visait à étudier la réponse au stress oxydatif chez Scedosporium. Tout d’abord, la capacité à germer en présence d’oxydants a été évaluée. Par la suite, trente-trois gènes potentiellement impliqués dans la défense contre le stress oxydatif ont été identifiés. Leur expression en présence d’oxydants et en co-cultures avec des phagocytes suggère un rôle majeur, notamment pour une catalase, une peroxyrédoxine et deux thiorédoxine réductases. Par ailleurs, un mutant défectif pour un gène codant une superoxyde dismutase (SOD) pariétale et spécifique des spores a été produit. L’auranofin, un inhibiteur des thiorédoxine réductases, présente une activité vis-à-vis des Scedosporium et un effet additif avec des triazolés. Un test ELISA a été développé pour le sérodiagnostic des scédosporioses, utilisant une catalase et une Cu/Zn-SOD recombinantes. Ce test sensible et spécifique permet de distinguer les infections à Scedosporium de celles à Aspergillus fumigatus et des colonisations à Scedosporium. Au final, ces résultats indiquent un rôle majeur des enzymes antioxydantes chez Scedosporium, qui pourraient être de véritables facteurs de virulence et donc de nouvelles cibles thérapeutiques
Cystic fibrosis (CF) is the most common genetic disease in Caucasian populations. The Scedosporium genus ranks the second among the filamentous fungi colonizing the airways of CF patients. In the respiratory tract, colonizations/infections lead to the recruitment of phagocytes which produce an oxidative stress, usually deleterious for pathogens. To defend themselves, pathogens have developed protective antioxidant systems, especially various enzymes. This thesis aimed to study the oxidative stress response in Scedosporium species. First, capacity of several Scedosporium isolates to germinate upon oxidative stress conditions was evaluated. Then, thirty-three genes potentially involved in protection against the oxidative stress were identified. Their overexpression in response to oxidants and in co-cultures with phagocytes suggested a crucial role, especially for one catalase, one peroxiredoxin and the two thioredoxin reductases. A mutant defective for the gene encoding a superoxide dismutase (SOD) anchored to the cell wall and specific for the conidia was produced. Auranofin, a thioredoxin reductase inhibitor, exhibits little anti-Scedosporium activity and an additive effect with triazole drugs. An ELISA was developed for serodiagnosis of scedosporiosis, using recombinant proteins derived from one catalase and a Cu/Zn-SOD. This sensitive and specific assay allows to differentiate Scedosporium infections from Aspergillus fumigatus infections and Scedosporium colonizations. Finally, these results indicate a crucial role of antioxidant enzymes in Scedosporium species, which could therefore be considered as virulence factors and as possible new therapeutic targets

Abstract Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) are two of the most common lymphomas in the Western world. DLBCL is an aggressive disease with a good response to treatment; about 75% of patients achieve permanent remission after first-line treatment. In patients with relapses or primary refractory disease, prognosis is dismal; only 10–20% of them can be cured, even with aggressive treatments. FL is an indolent lymphoma with a very good response to treatment and slow progression. Median survival with modern treatments is over 15 years. Nevertheless, some patients have short remissions and succumb to disease. Oxidative stress, TP53 mutations, and translocations of MYC, Bcl-2 and Bcl-6 have been linked in many neoplasms to aetiology and poor prognosis. This thesis concerns oxidative stress and redox-state-regulating enzymes in DLBCL and FL, and TP53 mutations and translocations of MYC, Bcl-2 and Bcl-6 in DLBCL. High expression levels of the antioxidant enzyme thioredoxin and a marker of oxidative stress, nitrotyrosine, were related to poor prognosis in DLBCL. In FL, high-level expression of peroxiredoxin was associated with good prognosis. TP53 mutations in specific regions LSH and L3 and concurrent translocation of Bcl-2 were associated with poor prognosis in DLBCL. Not all TP53 mutations predicted survival. High expression levels of Bcl-2 and MYC were associated with poor prognosis in DLBCL. Based on the results presented here, antioxidant function may have protective roles, but also may cause resistance to treatment. TP53 mutations have prognostic roles in DLBCL, but should be further defined. Novel therapies could be developed in connection with these mechanisms
Tiivistelmä Diffuusi suurisoluinen B-solulymfooma (DLBCL) ja follikulaarinen lymfooma (FL) ovat kaksi yleisintä lymfoomaa länsimaissa. DLBCL on aggressiivinen syöpä, joka reagoi hyvin hoitoihin, jopa 75 % paranee. Kuitenkin potilailla, joilla syöpä uusiutuu hoitojen jälkeen tai etenee hoidon aikana, on erittäin huono ennuste, noin 10-20 % näistä potilaista voidaan parantaa. FL on hyväennusteinen lymfooma, joka yleensä reagoi hyvin hoitoihin. Mediaani elossaoloaika kaikilla FL potilailla on yli 15 vuotta taudin toteamisesta. Osalla potilaista FL kuitenkin on aggressiivisempo. Oksidatiivinen stressin, TP53- mutaatioiden, MYC, Bcl-2 ja Bcl-6 -translokaatioiden on todettu olevan huonoon ennusteeseen yhteydessä olevia tekijöitä monissa syövissä, kuten lymfoomissa. Tämä väitöskirja tutki oksidatiivisen stressin ja hapetus-pelkistys reaktioon liittyvien entsyymien osuutta R-CHOP-hoidetuissa DLBCL:ssa ja FL:ssa immunohistokemian (IHC) avulla. DLBCL:ssa tutkittiin lisäksi TP53 mutaatioita, MYC, Bcl-2 ja Bcl-6 translokaatioiden roolia taudin kulussa. Korkea ekspressio oksidatiivisen stressin merkkiainetta nitrotyrosiinia ja antioksidantti thioredoksiinia olivat yhteydessä huonoon ennusteeseen DLBCL:ssa. FL:ssa runsas ilmentyminen antioksidativiisiin entsyymeihin kuuluvia peroksiredoksiineja olivat yhteydessä hyvään ennusteeseen. TP53 mutaatiot LSH ja L3 alueella ja Bcl-2 -translokaatiot yhdessä olivat yhteydessä huonoon ennusteeseen DLBCL:ssa. Kaikki TP53-mutaatiot eivät olleet assosioituneet huonoon ennusteeseen. DLBCL:ssa Bcl-2 ja MYC –proteiinien runsas ilmentyminen IHC:llä arvioituna liittyi huonoon ennusteeseen. Tulosten perusteella solujen hapetus-pelkistystilaa säätelevillä entsyymeillä voi olla dualistinen rooli, osittain suojeleva ja osittain vahingoittava lymfoomissa. TP53 -mutaatioilla voi olla ennusteellista merkitystä, mutta tämä vaatii lisää tutkimuksia

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The temperature-dependent dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of Paracoccidioidomycosis (PCM), a human systemic mycosis highly prevalent in countries of Latin America. P. brasiliensis is subjected to different insults from human host, such as oxidative stress caused by reactive oxygen species produced by the host during the infection. Thioredoxin (TRX) is an intracellular redox protein that is required to maintain redox homeostasis in response to both reductive and oxidative stress conditions in several organisms. We report here the characterization of a 811 bp cDNA Pbtrx1, encoding a PbTRX1 of 116 amino acids, with a predicted molecular mass of 12 kDa and pI 5.2. This putative protein presented one highly conserved active site motif (WCGPC) between TRXs from several organisms. The phylogenetic analysis performed with PbTRX1 and TRXs from other organisms, putted P. brasiliensis in the fungi clade. We also performed the prediction of the secondary structure of PbTRX1 that shows a pattern characteristic of the open twisted alpha/beta, similar to TRX secondary structures described in other fungus. In order to obtain the recombinant PbTRX1, the expression construct pGEX-4T-3-trx1 was introduced into Escherichia coli cells and the expression and purification of the recombinant protein was obtained. The recPbTRX1 and PbTRX1 from yeast cells extract were found to catalyze the reduction of insulin. However the PbTRX1 from yeast cells extract treated with H2O2 showed highly insulin reduction activity than the yeast cells no treated. PbTRX1 was detected by Western blotting in the extracts from yeast cells growth and from mycelium to yeast transition. The yeast cells growth was significantly inhibited by H2O2; however the mycelium to yeast transition was little affected by this oxidant. Semi-quantitative RT-PCR was employed to analysis the expression of Pbtrx1 gene in response to H2O2. The level of Pbtrx1 transcripts was higher in yeast cells treated with H2O2 than in yeast cells no treated. To realize how P. brasiliensis deals with oxidative stress is essential to understand the mechanisms involved in its survival in the host. It may be possible that PbTRX1 enhances survival of P. brasiliensis in the host, protecting the fungus against the reactive oxygen species and allowing, in this way, the progress of the infection.
O fungo termodimórfico, Paracoccidioides brasiliensis, é o agente etiológico da paracoccidioidomicose (PCM), uma micose sistêmica humana, com alta prevalência na América Latina. No hospedeiro humano, o fungo P. brasiliensis está sujeito a vários insultos, tais como o estresse oxidativo causado pelas espécies reativas de oxigênio, que são produzidas pelas células de defesa do hospedeiro durante a infecção. A tiorredoxina (TRX) é proteina redox intracelular que participa da manutenção da homeostase redox da célula, tanto em condições de estresse oxidativo quanto redutor. Neste trabalho apresentamos a caracterização de um cDNA de 811 pb, designado como Pbtrx1, que codifica para uma proteína, PbTRX1, de 116 resíduos de aminoácidos com massa molecular predita de 12 kDa e pI de 5,2. PbTRX1 apresentou um motivo de sítio ativo conservado (WCGPC) entre as TRXs de vários organismos. Análise filogenética com PbTRX1 e TRXs de outros organismos colocou P. brasiliensis no clado de fungos. Foi também realizada a predição da estrutura secundária da PbTRX1, que apresentou um padrão característico formado por cadeias-β que estão envolvidas por α-hélices. Para obter a proteína recombinante, recPbTRX1, foi realizada a construção do pGEX-4T-3-trx1 e este foi introduzido nas células de Escherichia coli. Assim, a expressão e purificação da proteína recombinante foi obtida. A proteína recPbTRX1 e a PbTRX1, presente no extrato protéico de células leveduriformes, apresentaram atividade redutora de insulina. Entretanto, a PbTRX1 presente no extrato protéico de células leveduriformes tratadas com H2O2, mostrou maior atividade redutora de insulina quando comparada com extrato de células leveduriformes não tratadas. A PbTRX1 foi detectada, por Western blotting, em extratos de células leveduriformes em crescimento e durante a transição de micélio para levedura. O crescimento das células leveduriformes foi inibido por H2O2, entretanto a transição de micélio para levedura foi pouco afetada por este oxidante. A técnica de RT-PCR semi-quantitativo foi empregada para análise da expressão do Pbtrx1 em resposta ao H2O2. O nível de transcritos de Pbtrx1 foi maior nas células leveduriformes tratadas com H2O2 do que nas células não tratadas. Para compreender como P. brasiliensis lida com estresse oxidativo é essencial entender os mecanismos envolvidos em sua sobrevivência no hospedeiro. É possível que PbTRX1 aumente a sobrevivência de P. brasiliensis no hospedeiro, protegendo o fungo contra espécies reativas de oxigênio e, desta maneira, permitindo o progresso da infecção.

INTRODUÇÃO: evidências sugerem a participação de fatores genéticos na susceptibilidade para o desenvolvimento das complicações renais em pacientes portadores de diabetes mellitus tipo 1 (DM1). Vários genes relacionados às vias bioquímicas induzidas pela hiperglicemia têm sido investigados e o estresse oxidativo foi reconhecido como o principal mecanismo patogênico responsável pelo dano celular causado pela hiperglicemia no DM. Assim, genes que codificam enzimas que participam de vias antioxidantes endógenas são candidatos a conferirem susceptibilidade, ou proteção, contra as complicações renais. Os sistemas da glutationa, glutarredoxina, tiorredoxina e a enzima transcetolase são importantes mecanismos de defesa celular contra o estresse oxidativo. OBJETIVOS: avaliar a associação entre os seguintes polimorfismos de um único nucleotídeo (SNP) e a doença renal em pacientes diabéticos tipo 1: -2030 T/G (rs34071297) e +718C/T (rs713041) no gene que codifica a glutationa peroxidase 4 (GPX4); -3310 G/C (rs10427424) no gene que codifica a glutationa sintetase (GSS); -247 A/G (rs2978668) no gene que codifica a glutationa redutase (GSR); -2763 A/G (rs6556885) no gene que codifica a glutarredoxina (GLRX); -224 T/A (rs2301242) no gene que codifica a tiorredoxina (TXN); +402 T/C (rs7211) no gene que codifica a proteína de interação com a tiorredoxina (TXNIP); -192 G/A (rs3788319) no gene que codifica a tiorredoxina redutase 2 (TXNRD2) e -3787 T/G (rs7637934) e -1410 T/C (rs11130365) no gene que codifica a transcetolase (TKT). CASUÍSTICA E MÉTODOS: 443 pacientes (192 do sexo masculino e 251 do sexo feminino) com DM1 com mais de 10 anos de diagnóstico foram classificados conforme a presença ou ausência das seguintes complicações: (1) nefropatia diabética franca (ND), definida por macroalbuminúria a proteinúria persistente; (2) nefropatia diabética estabelecida (NDE), definida por macroalbuminúria a proteinúria persistente ou RFGe < 60 mL/min/1,73 m2 ou pacientes em terapia de substituição renal e (3) ritmo de filtração glomerular estimado (RFGe) ou < que 60 mL/min/1,73 m2. O teste de Pearson 2 foi usado para comparar as frequências dos genótipos e a magnitude de associação foi estimada pelo cálculo do odds ratios (OR). A OR ajustada foi estimada por regressão logística para possíveis fatores de confusão (sexo, idade ao diagnóstico, tempo de diabetes, HbA1c, concentrações plasmáticas de colesterol e triglicérides e presença de hipertensão arterial). Pacientes controle não diabéticos também foram incluídos para avaliar se os SNPs não confeririam susceptibilidade para o DM1. RESULTADOS: A presença de pelo menos um alelo T do polimorfismo +718C/T no gene GPX4 conferiu proteção para a presença de ND estabelecida (OR=0,41; IC 95% 0,19-0,83, p= 0,0146) e ND franca (OR=0,37; IC 95% 0,15- 0,85; p= 0,021) na população masculina mesmo após ajuste para os fatores de confusão e a presença de dois alelos polimórficos A no polimorfismo -224 T/A no gene TXN conferiu risco para a presença de ND franca na população feminina após ajuste para os fatores de confusão (OR= 4,06; IC 95% 1,59-10,6, p= 0,0035). O genótipo TT para o SNP +402 T/C do gene da TXNIP foi mais frequente nos pacientes portadores de DM1 em relação aos controles não diabéticos. O genótipo CC do SNP TXNIP +402 T/C conferiu proteção para a presença de ND estabelecida em homens mesmo após ajuste para os fatores de confusão (OR=0,45; IC 95% 0,22-0,91; p= 0,02). CONCLUSÕES: Os SNPs +718C/T (rs713041) no gene GPX4, -224 T/A (rs2301242) no gene TXN e +402 T/C (rs7211) no gene TXNIP, modulam o risco para o comprometimento renal na população de portadores de DM1 estudada
INTRODUCTION: there is evidence suggesting that genetic factors are involved in the susceptibility to the development of renal complications in patients with type 1 diabetes mellitus (DM1). Several genes related to the mechanisms of hyperglycemia-induced cell damage have been investigated. Oxidative stress is recognized as a major pathogenic factor of cellular damage caused by hyperglycemia. Thus, genes that encode enzymes involved in endogenous antioxidant pathways may be candidates for conferring risk or protection against renal complications. The glutathione, glutaredoxin, and thioredoxin systems and transketolase enzyme are important mechanisms of cellular defense against oxidative stress. OBJECTIVES: to evaluate the association between the following single nucleotide polymorphisms (SNPs) and renal disease in type 1 diabetic patients: -2030 T/G (rs34071297) and +718C/T (rs713041) in the gene encoding glutathione peroxidase 4 (GPX4); -3310 G/C (rs10427424) in the gene encoding glutathione synthetase (GSS); -247 A/G (rs2978668) in the gene encoding glutathione reductase (GSR); -2763 A/G (rs6556885) in the gene encoding glutaredoxin (GLRX); -224 T/A (rs2301242) in the gene encoding thioredoxin (TXN); +402 T/C (rs7211) in gene encoding thioredoxin interacting protein (TXNIP); -192 G/A (rs3788319) in the gene encoding thioredoxin reductase 2 (TXNRD2); - 3787 T/G (rs7637934) and -1410 T/C (rs11130365) in the gene encoding transketolase (TKT). MATERIALS AND METHODS: 443 patients (192 males and 251 females) with type 1 diabetes duration > 10 years were grouped according to presence or absence of the following complications: (1) overt diabetic nephropathy (DN) defined by persistent macroalbuminuria to proteinuria; (2) established diabetic nephropathy (EDN), defined by persistent macroalbuminuria or proteinuria or estimated glomerular filtration rate (RFGe) < 60 mL/min/1.73 m2 or patients under renal replacement therapy and (3) RFGe or < 60 mL/min/1.73 m2. Pearsons 2 test was performed to compare the genotype frequencies and magnitude of association was estimated using odds ratios (OR). Adjusted OR was estimated by logistic regression for possible confounders (sex, age at diagnosis, diabetes duration, HbA1C, cholesterol and triglyceride concentrations and the presence of hypertension). Nondiabetic subjects were also included. RESULTS: The presence of at least one T polymorphic allele of the SNP GPX4 +718 C/T was protective against EDN (OR = 0.41, CI 95% 0.19- 0.83, p= 0.0146) and against overt DN (OR=0.37; IC 95% 0.15-0.85; p= 0.021) in the male population even after adjustment for possible confounders. The presence of two polymorphic alleles of the SNP TXN -224 T/A conferred independent risk for the presence of overt DN in the female population after adjustment for possible confounders (OR = 4.06, CI 95% 1.59- 10.6, p= 0.0035). The TT genotype for the SNP TXNIP +402 T/C was more frequent in patients with type 1 diabetes compared to nondiabetic controls. The genotype CC of the SNP TXNIP +402 T/C was protective against EDN in male population even after adjustment for possible confounders (OR=0.45; IC 95% 0.22-0.91; p= 0.02) CONCLUSIONS: The SNPs GPX4 +718C/T (rs713041), TXN -224 T/A (rs2301242) and TXNIP +402T/C (rs7211) modulate the risk for renal disease in the studied population of type 1 diabetes patients

Posttranslational modification (PTM) of proteins is essential to creating a diverse proteome with the complex functions necessary to regulate key cellular processes. Redox-based PTMs exhibit many desirable characteristics to finely modulate transcriptional regulators; they occur rapidly and can alter protein conformation, localisation and activity. The plant immune system offers an excellent model in which to study redox-based modifications due to the rapid accumulation of oxidising agents that occurs during immune invasion. This so-called “oxidative burst” causes spontaneous oxidation of cysteine residues that are present in many regulatory proteins. These modifications fine-tune the activities of proteins that harbour them, enabling them to act in a concerted effort to reprogram the transcriptome, prioritising the expression of immune-related genes over housekeeping genes. Disulphide bonds (S-S) and S-nitrosothiols (SNO, i.e. the addition of an NO group to a cysteine moiety) have been shown to play particularly important roles in plant immunity. However, what still remains unclear is how these redox-based PTMs are rendered reversible, enabling them to act as molecular signalling switches. The work presented in this thesis explores a class of enzymes that are responsible for controlling the cellular levels of protein oxidation: the Thioredoxins. In addition to their well-established role in reducing disulphide bonds, I demonstrate in Chapter 3 that Thioredoxins are able to reverse protein S-nitrosylation during plant immune signalling. Immune-inducible Thioredoxin-h5 (TRXh5) was shown to be unable to restore immunity in gsnor1 mutants that display excessive accumulation of the NO donor S-nitrosoglutathione, but rescued impaired immunity and defence gene expression in nox1-mutants that exhibit elevated levels of free NO. This data indicates that TRXh5 discriminates between protein-SNO substrates to provide previously unrecognized specificity and reversibility to protein-SNO signalling in plant immunity. Furthermore, data is presented to show that TRXh5 reversed the effects of S.nitrosylation on many immune-related transcriptional regulators in vitro, forming the initial stages of an investigation into which proteins and pathways might be controlled by reversible S-nitrosylation in plant immunity (Chapters 3 & 4). Although the majority of transcriptional regulators are likely modified at their site of action, the nucleus, very little is currently known about nuclear redox signalling in plants. Therefore, in Chapter 5 a subclass of theThioredoxin superfamily was studied, the Nucleoredoxins, which have previously been shown to display disulphide reduction activity and localise in part to the nucleus. Here it is revealed that the activity and nuclear accumulation of Nucleoredoxin 1 (NRX1) is induced by the plant leaf pathogen Pseudomonas syringae, suggesting a key role for this protein in immune signalling. Target-capture experiments and subsequent mass spectrometry analysis identified the first in vitro targets of NRX1 and revealed many proteins with roles in oxidative stress, including the hydrogen peroxide scavenger Catalase 2 (CAT2). Moreover, overexpression of NRX1 was shown to be able to rescue the enhanced cell death phenotype of cat2 knockout mutants in response to the oxidative stressor, methyl viologen. Accordingly, nrx1 knockout mutants also exhibited an enhanced cell death phenotype in response to methyl viologen treatment. Together, these data indicate that NRX1 plays a key role in the control of oxidative stress-mediated cell death, potentially through direct regulation of Catalase proteins. Taken together, the work in this thesis implicates members of the Thioredoxin family as key regulators of transcriptional reprogramming during plant immunity and uncovers a novel role for Thioredoxin superfamily member, NRX1, in the control of oxidative stress.

碩士
國立中央大學
生命科學研究所
92
Abstract Thioredoxin peroxidase II (TPx II) is an oxidative stress-inducible enzyme that functions in cell proliferation and differentiation, and protects other proteins from oxidative damage. Arsenic is an environmental pollutant associated with human skin, lung, liver cancers and cardiovascular diseases. Recent studies suggested that arsenic-induced toxicity is associated with the generation of free radicals. This study investigates the mechanism of sodium arsenite (As(III))- and other oxidative stress-induced TPx II expression in murine fibroblast cells (3T3). The results showed that As(III), H2O2, CdCl2 and DPPH dose-dependently induced TPx II protein expression in 3T3 cell. The mRNA level of TPx II were also induced by As(III), H2O2, and DPPH, but not CdCl2. In addition, the phospho-ERK protein level was significantly enhanced in response to As(III) treatment. The As(III)-induced TPx II expression could be dose-dependently abolished by the presence of Rotterlin (PKC δ specific inhibitor); however, Rotterlin showed no effect on H2O2- and DPPH-induced TPx II expression. We further investigated the effect of As(III) on TPx II expression in transcription level. The results showed that a DNA response element located at TPx II promoter 269-169 pair was critical for As(III) inductory effect on TPx II gene expression. The As(III)-induced TPx II expression was further enhanced by another transcriptional factor FKHR that had no effect on H2O2-, CdCl2- and DPPH-induced TPx II expression. These results indicated that the mechanism of As(III) induces TPx II protein expression likely through oxidative stress pathway by which is dependent on FKHR and distinct from that induced by H2O2, CdCl2 and DPPH.

Thioredoxins are small, ubiquitous, disulfide oxidoreductase proteins characterised by a conserved dicysteine active site. Within the cell, they are believed to maintain the redox environment and participate in a broad range of biochemical processes. Plant thioredoxins are a diverse multigene family, primarily classified according to the system by which they are reduced and their subcellular localization. Thioredoxins located in the cytoplasm (type -h) are usually dependent on NADPH for reduction by NADPH-thioredoxin reductase. There are four cytosolic thioredoxins in grass species, with subclass 4 believed to be the most ancient. The highly conserved nature of thioredoxin-h4, in plant species as diverse as angiosperms and gymnosperms, implies a conservation of gene function. Discovery of thioredoxin-h4 function in barley (Hordeum vulgare) was the core focus of the research presented in this thesis. The characterisation of thioredoxin-h4 was approached from both, genetic, and protein biochemistry perspectives. To commence the research, the transcript profile of barley thioreodoxin-h4 (HvTrx-h4) was examined in barley reproductive tissues. As a direct consequence of findings, anther and stigma tissues were used in protein interaction studies employing a mono-cystenic active-site HvTrx-h4 affinity chromatography technique. HvTrx-h4 was mutated, recombinantly expressed, purified and immobilised in order to isolate and identify proteins with which it interacted. Identification of HvTrx-h4 protein targets sought to reveal the pathways in which thioredoxin-h4 is involved. To further characterise the expression of HvTrx-h4, the promoter and 5′ untranslated regions of genomic sequence were isolated and used to drive expression of green fluorescent protein in transgenically modified barley. This enabled examination of the temporal and spatial regulation of HvTrx-h4 under normal growth conditions, as well as in response to abiotic stress and plant hormone treatments. Through these studies it was discovered that HvTrx-h4 is likely to be the subject of post-transcriptional modifications. Subsequent investigations revealed HvTrx-h4 is also regulated at the post-translational level through glutathionylation. Previous studies have ascribed a role for thioredoxins in plant oxidative stress defence. The question of whether modulation of HvTrx-h4 expression could be manipulated to alter plant oxidative stress tolerance was considered. To investigate, transgenic tobacco plants (Nicotiana tabacum) containing altered amounts of thioredoxin-h4 protein were subjected to various stresses; abiotic, biotic and chemical, in nature. Tobacco constitutively over-expressing thioredoxin-h4 displayed increased tolerance to ultraviolet light B, heat and methyl viologen treatment. Knowledge acquired by this study and presented in this thesis, suggest a role for barley thioredoxin-h4 in the oxidative stress response. Furthermore, the description of both post-transcriptional and post-translational regulation of HvTrx-h4 constitutes the first report of this level of regulation for a plant cytosolic thioredoxin.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2010

Inhibition of disulfide bond formation in Escherichia coli implicates an intricate collaboration of proteins which comprise the glutathione and thioredoxin reducing pathways. Bioengineers have successfully engineered E. coli possessing mutated reducing pathways that promote, rather than inhibit, disulfide bond formation in the cytoplasm. The transcriptome of six such mutant E. coli strains have been characterized using Microarray technology. We find that all mutant strains, exhibit a unique response to oxidative stress, not observed in wild type. Statistical analyses revealed the expression of more than 200 genes that are affected by mutations within the reducing pathways. Significantly up-regulated biological processes include cysteine biosynthesis, histidine biosynthesis, NADH Dehydrogenase I biosynthesis, sugar catabolic processes, and activation of stress responses . The second part of this work describes the construction of an E. coli strain that promotes the complete conversion of glutathione into its seemingly dormant derivative, glutathionylspermidine. This engineered strain can be used in assays designed to evaluate the effectiveness of glutathionylspermidine as a substitute for glutathione and, hopefully, allude to its true metabolic function.
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of study named: Effects of selected natural substances on the antioxidant system of an organism Developed: Mgr. Anna Hodková Department of Pharmacology and Toxikology, Faculty of Medicine in Pilsen, Charles University in Prague Pilsen 2016 The aim of this study was to compare the effects of selected natural substances on the antioxidant defense system under comparable conditions, focusing on influencing the activity of selenoenzymes thioredoxin reductase (TrxR-1) and glutathione peroxidase (GPx-1). Experiments were performed in rats (Wistar, male). Livers, and in some cases kidneys were collected in all experiments. Homogenates were created from the collected organs and subsequently the activity of TrxR-1 and GPx-1, glutathione reductase (GR), catalase (CAT) and superoxide dismutase (SOD), and reduced glutathione (GSH) and lipid peroxidation (LP) levels were determined. We demonstrated significant effects of selected natural substances on the redox system, including influences of selenoenzymes thioredoxin reductase and glutathione peroxidase. The biggest influence on the activity of selenoenzymes thioredoxin reductase and glutathione peroxidase had hydroxytyrosol (HT) and oleuropein (OLEU). In rat liver tissue there was a significant decrease of the activity of both above mentioned enzymes after...

Exposure to toxic bile acids (BA) and retinoic acids (RA) is implicated in toxicities related to excessive oxidative stress. This thesis examined roles and mechanisms of the oxidative stress-responsive nuclear factor (erythroid 2-like) factor 2 (Nrf2) in adaptive cell defense against BA and RA toxicities. Using liver cells and mouse models, many antioxidant proteins known to be Nrf2 target genes, particularly the rate-limiting enzyme for glutathione (GSH) biosynthesis, i.e., glutamate-cysteine ligase subunits (GCLM/GCLC), were induced by BA [lithocholic acid (LCA)] or RA (all-trans, 9-cis and 13-cis) treatment. Evidence for increased Nrf2 transactivation by LCA and all-trans-RA was exemplified in HepG2 by: (1) reduced constitutive and inducible expression of GCLM/GCLC upon Nrf2 silencing via small-interfering RNA; (2) increased inducible expression of GCLM/GCLC genes by Nrf2 overexpression, but overexpression of dominant-negative Nrf2 decreased it; (3) increased nuclear accumulation of Nrf2 as signature event of receptor activation; (4) enhanced Nrf2-dependent antioxidant-response-element (ARE) reporter activity as indicative of increased Nrf2 transactivation; and (5) increased Nrf2 occupancy to AREs of GCLM and GCLC. Additionally, in BA-treated HepG2 cells, we observed concomitant increases of many ATP-binding cassette (ABC) transporters (MRPs 1-5, MDR1 and BCRP) in parallel with increased cellular efflux. Nrf2 silencing in HepG2 cells decreased constitutive and inducible expression of MRP2, MRP3 and ABCG2. However, Nrf2-silenced mouse hepatoma cells, Hepa1c1c7, and Nrf2-/- mice had decreased constitutive and/or inducible expression of Mrps 1-4, suggesting species differences in Nrf2-dependent regulation of hepatic ABC transporters. Protection by Nrf2 against BA and RA toxicities was confirmed by observations that Nrf2 silencing increased cell susceptibility to BA- and RA-induced cell death. Moreover, Nrf2-/- mice suffered more severe liver injury than the wildtype. Increased GSH and efflux activity following increased GCLM/GCLC and ABC transporters, respectively, can mitigate LCA toxicity. Activation of MEK1-ERK1/2 MAPK was shown to primarily mediate Nrf2 transactivation and LCA-induced expression of antioxidant proteins and Nrf2-dependent and -independent ABC transporters. In conclusion, Nrf2 activation by BA and RA led to coordinated induction of antioxidant and ABC proteins, thereby counteracting resultant oxidative cytotoxicity. The potential of targeting Nrf2 in management of BA and RA toxicities merits further investigation.