Le développement de la phase SIDA (Syndrome de l'ImmunoDéficience Acquise) chez les patients infectés par le virus VIH-1 (Virus de l'Immunodéficience Humaine) se caractérise par une diminution progressive du nombre de cellules T CD4. Orla majorité des cellules constituant cette déplétion sont non-infectées et appelées "bystander". En 2006, notre équipe a montré que le contact entre les cellules infectées exprimant les glycoprotéines de l’enveloppe (Env) et les cellules noninfectées exprimant les récepteurs CD4 et CXCR4 déclenche au sein de ces dernières la voie d’autophagie, ce qui mène à une mort cellulaire par apoptose. L'autophagie est un processus impliqué dans la dégradation de matériel cytoplasmiqueaprès sa séquestration au sein de vacuoles dans lesquelles il sera dégradé puis recyclé. Ce processus peut être hautement sélectif par l'action de protéines réceptrices tels que p62 ou NBR1.L’objectif de mon projet de thèse vise à comprendre comment l'autophagie induite par Env mène les cellules T CD4 bystander à leur mort par apoptose. Une précédente étude menée par notre équipe a démontré que les changements induits par Env au sein de ces cellules T CD4 non infectées comprenaient la production d’espèces oxygénées réactives (ROS) menant à un état de stress oxydatif. Nous montré que le stress oxydatif induit par Env est impliqué dans la mort des cellules T CD4 bystander par apoptose. Nous avons également observé que l’autophagie doit être dégradative pourmener ces cellules T CD4 à leur mort par apoptose. De plus, nous avons observé une dégradation des protéines peroxysomales par l’autophagie induite par Env dans le même modèle. Les peroxysomes sont des organelles essentielles de la cellule qui sont en partie responsables de la détoxification des ROS dans la cellule. Leur nombre est régulé par une dégradation sélective autophagique que l’on appelle la pexophagie.Dès lors, nous étudions l’hypothèse de l'induction par Env d’une dégradation sélective des systèmes antioxydants de la cellule par autophagie dans les cellules T CD4 bystander. Les peroxysomes étant des organites responsables de la réponse au stress oxydatif, leur dégradation sélective pourrait empêcher la cellule de faire face au stress oxydatif qu’elle subit et la mener vers une mort cellulaire par apoptose. En conclusion, nous montrons que l’autophagie induite par Env dégrade un système antioxydant important qui est un facteur clé nécessaire à la survie des cellules T CD4 bystander pour réduire le stress oxydatif induit par Env. / The development of AIDS (Acquired ImmunoDeficiency Syndrome) in HIV-1 (Human Immunodeficiency Virus)-infected patients is characterized by a progressive decrease in the number of CD4 T cells. The majority of dying cells are noninfected and called bystander CD4 T cells. In 2006, our team demonstrated that the contact between infected cells (expressing the envelope glycoproteins (Env)) and non-infected cells (expressing the CD4 and CXCR4 receptors) was responsible for enhancing the autophagic pathway which lead to their cellular death by apoptosis. The autophagicpathway is involved in the degradation of cytoplasmic material after its sequestration into vacuoles wherein it will be degraded and then recycled. This process can be highly selective through the involvement of receptor proteins such asp62 or NBR1.We aim at understanding how Env-mediated autophagy can lead to apoptosis in bystander CD4 T cells. A precedent workof our team showed that the changes induced by Env in bystander CD4 T cells included the production of reactive oxygen species (ROS) leading to an oxidative stress state. We showed that the oxidative stress induced by Env is involved in thecellular death by apoptosis of bystander CD4 T cells. We also show that the autophagic process involved has to be a degradative process to lead these CD4 T cells to their death by apoptosis. Moreover, we have observed that Env-mediatedautophagy was degrading peroxisomal proteins. Peroxisomes are essential organelles in the cell responsible partly for the detoxification of ROS in the cell. Their number is regulated through a selective autophagic degradation known aspexophagy.Therefore, we hypothesized that Env induced a selective degradation by autophagy of the cell antioxidant system in bystander CD4 T cells. Since peroxisomes are responsible for regulating the cellular response to an oxidative stress state, their selective degradation could prevent the cell from overcoming this event and eventually lead to its death by apoptosis. In conclusion, we are showing that Env-mediated autophagy degrades important antioxidant systems which are a key survival factor necessary to the bystander CD4 T cells to reduce the oxidative stress induced by Env.

L'oxydase terminale plastidiale (PTOX) est présente uniquement chez les organismesphotosynthétiques. PTOX oxyde le plastoquinol (PQH2) et réduit l'oxygène en eau.PTOX est impliquée dans la synthèse des caroténoïdes, dans le transportphotosynthétique d'électrons et dans la chlororespiration. De plus, son activité estconsidérée comme pouvant jouer un rôle en tant que soupape de sécurité, permettant de maintenir oxydé le pool de plastoquinones (PQ) et d'éviter la surréduction duchloroplaste et ainsi la photoinhibition. Chez la majorité des plantes testées, les niveaux de PTOX sont plus élevés dans des conditions de stress (une exposition à forte intensité lumineuse, par exemple). D'autre part, la surexpression de PTOX chez Arabidopsis thaliana n'a pas rendu les plantes moins sensibles à la photoinhibition. Par ailleurs, il semble que PTOX surexprimée chez Nicotiana tabacum a induit la génération des espèces réactives de l'oxygène (ERO) et une photoinhibition importante sous forte lumière.Le but de cette thèse était la caractérisation de l'activité enzymatique de PTOX enutilisant la protéine purifiée et de comprendre pourquoi PTOX protège du stressphotooxydant dans certaines conditions et pourquoi elle augmente ce stress quand elle est surexprimée in planta.L'analyse biochimique de PTOX recombinante purifiée a démontré que l'enzymeexiste principalement sous forme tétramérique. Cette forme se dissocie partiellement,principalement en dimères. Le turnover maximal de l'enzyme purifié correspond à 320électrons par seconde et par molécule de PTOX. Nous avons démontré que PTOXgénère des ERO dans une réaction secondaire dépendante de la concentration dusubstrat (PQH2) et du pH de la solution. À pH 8 (représentant le pH du stroma deschloroplastes actifs), PTOX a une activité antioxydante quand la concentration de PQH2 est basse et prooxydante quand cette concentration est élevée.En mesurant la fluorescence de la chlorophylle a, nous avons démontré quePTOX est active lorsqu'elle est ajoutée aux membranes enrichies en PSII.L'attachement aux membranes dépend du pH et de cations de la solution: lorsque le pHdiminue ou lorsque la solution est riche en cations monovalents, la quantité de PTOXattachée à la membrane diminue.L'activité de PTOX in planta et son effet sur le transport des électronsphotosynthétiques ont été analysés en utilisant Arabidopsis thaliana surexprimant laphytoène désaturase bactérienne (CRTI) et Nicotiana tabacum surexprimant PTOX1 deChlamydomonas reinhardtii. Arabidopsis thaliana surexprimant CRTI a un niveau plusimportant de PTOX et de production d'ERO et le transport cyclique des électrons estsupprimé chez les transformants. Cela implique que PTOX est en compétition avec letransfert cyclique pour les électrons du pool PQ et que PTOX joue un rôle importantdans le contrôle de l'état rédox de ce pool. En utilisant Nicotiana tabacum surexprimant PTOX1, nous avons démontré que PTOX fait concurrence au transfert linéaire d'électrons photosynthétique, mais que PTOX est inactivée quand le pH du stroma est neutre. Grâce aux résultats obtenus, nous proposons un modèle où l'association de PTOX avec la membrane est contrôlée par le pH du stroma. Quand le pH est neutre, PTOX est soluble et n'est pas active, ce qui évite l'interférence avec le transfert linéaire d'électrons. Quand le pH du stroma est alcalin et la chaîne des transporteurs photosynthétiques est surréduite (lors des conditions du stress), PTOX s'attache à la membrane, devient active et joue le rôle de soupape de sécurité. / The plastid terminal oxidase PTOX is encoded by higher plants, algae and some cyanobacteria. PTOX is a plastid-localized plastoquinol (PQH2) oxygen oxidoreductase. PTOX was shown to be implicated in plant carotenoid biosynthesis, photosynthetic electron transport and chlororespiration and may act as a safety valve protecting plants against photo-oxidative stress. PTOX protein levels increase during abiotic stress indicating a function in stress acclimation. But overexpression of PTOX in Arabidopsis did not attenuate the severity of photoinhibition or, when overexpressed in tobacco, even increased the production of reactive oxygen species (ROS) and exacerbated photoinhibition.Biochemical analysis of recombinant purified PTOX (PTOX from rice fused to the maltose-binding protein) showed that the enzyme exists mainly as a tetramer, which dissociated to a certain extent during electrophoresis, mainly into a dimeric form. The PTOX activity was 320 electrons s−1 PTOX−1. It was also shown that PTOX generates ROS in a side reaction in a substrate (decylPQH2) and pH-dependent manner when liposomes were used: at the basic stromal pH of photosynthetically active chloroplasts, PTOX was antioxidant at low decylPQH2 gaining prooxidant properties with increasing quinol concentrations. It is concluded that PTOX can act as a safety valve when the steady state [PQH2] is low while a certain amount of ROS is formed at high light intensities.It was shown by chlorophyll a fluorescence that recombinant purified PTOX is active when added to photosystem II (PSII)-enriched membrane fragments. PTOX attached tightly to the PSII-enriched membrane fragments. The amount of PTOX attaching to the membrane depended on pH and salts: an alkaline pH and monovalent compared to divalent cations increased PTOX attachment.PTOX activity in planta and its effect on photosynthetic electron transport were investigated using Arabidopsis expressing bacterial phytoene desaturase and tobacco expressing PTOX1 from Chlamydomonas. Arabidopsis expressing bacterial phytoene desaturase (CRTI lines) showed a higher PTOX content and increased PTOX related ROS generation. Furthermore, cyclic electron flow was suppressed in these lines. This implicates that PTOX competes efficiently with cyclic electron flow for PQH2 in the CRTI-expressing lines and that it plays a crucial role in the control of the reduction state of the plastoquinone pool. Using tobacco expressing PTOX1 from Chlamydomonas, it was shown that PTOX competes efficiently with photosynthetic electron flow, but gets inactive when the stromal pH is neutral. Based on the in vitro and in vivo results, a model is proposed, where the association of PTOX to the membrane is controlled by the stromal pH: When the stromal pH is neutral, PTOX exists as a soluble form and is enzymatically inactive avoiding the interference of PTOX with linear electron flow. When the stromal pH is alkaline and the photosynthetic electron chain is highly reduced under stress conditions as high light, PTOX binds to the membrane, gets enzymatically active and can serve as safety valve.

Cox11 (cytochrome c oxidase 11) is an ancient and conserved protein family present in most respiring organisms. Studies of several family members, mainly in yeast and bacteria, have revealed that these proteins are in charge of Cu+ delivery to the respiratory complex IV (COX). Absence of Cox11 leads to a non-functional COX complex and a complete respiratory deficiency. Although it is assumed that homologues in other species perform the same function, experimental data supporting this notion are lacking. The aim of this work was to characterize the putative Arabidopsis homologue AtCox11 (encoded by locus At1g02410) and to determine its functions. Comparison of AtCox11 with the well-studied ScCox11 in yeast revealed that the two proteins share high similarity in their sequences (32% amino acid identity) and in the predicted secondary structures. Surprisingly, despite this high similarity AtCox11 proved not to be able to functionally replace the yeast protein in ΔSccox11 yeast deletion strains. As presumed, AtCox11 is localized to mitochondria, probably tethered to the inner mitochondrial membrane with its C-terminus facing the intermembrane space. The subsequent experimental work addressed the functions of AtCox11. To this end AtCOX11 knock-down (KD) and overexpression lines (OE) were generated and their impact on plant phenotype was investigated. KD lines that were obtained by artificial micro RNA technology, possess approximately 30% of the WT AtCOX11 mRNA levels. Overexpression resulting in 4-6 fold higher AtCOX11 mRNA levels, was achieved by placing AtCOX11 under the control of the 35S promoter. Remarkably, both KD and OE plants had reduced levels of COX complex activity (~45% and ~80%, respectively) indicating that AtCox11 is, as expected, involved in COX complex assembly. The KD and OE plants exhibited reduced root lengths and pollen germination rates (compared to WT). As both processes are dependent on respiratory energy, these phenotypic changes seemingly result from the reduced COX activity. Interestingly, the short-root phenotype in OE plants was rescued by a surplus of copper in the media, whereas copper deficiency intensified the phenotype. By contrast, KD plants did not respond to changes of the copper concentration. This difference in the copper response between KD and OE plants hints at a different cause for the reduced COX activity. It is proposed that the concentration of AtCox11 in KD plants limits the efficient insertion of Cu+ into COX, independent of the available copper concentration. In OE plants, binding of the limited copper by the high AtCox11 level may lead to a copper deficiency for the copper chaperone AtHcc1 that is required to load copper to subunit AtCoxII. Indeed, addition of copper to the media was able to rescue the phenotype. In line with these data, the analysis of the expression pattern of AtCOX11 revealed that it is expressed in tissues which require substantial mitochondrial and COX biogenesis to sustain their high metabolic and/or cell division rates. Furthermore AtCOX11 was shown to be up-regulated as part of the plant’s response to increased oxidative stress induced by the addition to the plant media of peroxides or inhibitors of respiratory complexes. The up-regulation of AtCOX11 in response to oxidative stress was corroborated with publicly available RNA microarray data and analysis of the AtCOX11 promoter, which revealed the presence of a number of potential oxidative stress responsive elements. Taken together, the experimental results presented in this thesis support the conclusion that AtCox11 is a member of the conserved Cox11 protein family. Most probably, this mitochondrial protein participates in the assembly of the COX complex by inserting Cu+ into the CuB center of the AtCoxI subunit. In addition to this expected role, the data indicate that AtCox11 might participate in cellular oxidative stress response and defense via a yet unknown mechanism.

Stereo vision is one of the most popular elements in the field of mobile robots and significantly contributes to their autonomous behaviour. The aim of the diploma thesis was to design and implement a camera module as a hardware sensor input, which is independent, with the possibility of supplementing the system with other cameras, and to create a depth map from a pair of cameras. The diploma thesis consists of theoretical and practical part, including the conclusion of results. The theoretical part introduces the ROS framework, discusses methods of creating depth maps, and provides an overview of the most popular stereo cameras in robotics. The practical part describes in detail the preparation of the experiment and its implementation. It also describes the camera calibration and the depth map creating. The last chapter contains an evaluation of the experiment.

Francisella tularensis is capable of infecting numerous cell types, including professional phagocytes. Upon phagocytosis, F. tularensis resides within the phagosome before escaping into the cytosol to replicate. Phagocytes constitute a hostile environment rich in ROS, which are employed as a means of killing pathogens. ROS interact with and disrupt the function of vital molecules such as DNA, proteins and bacterial structures. Iron potentiates the danger of ROS through the Fenton reaction where ferrous iron reduces H2O2 causing the formation of highly reactive hydroxyl radicals and anions. Low levels of ROS are formed during normal aerobic metabolism and pathogens thus have a need for defense mechanisms to handle the ever present levels of ROS but even more so to combat the onslaught of ROS experienced within a host. This thesis was focused on the investigation of the iron status and oxidative stress response of F. tularensis; thereby identifying key players controlling the bacterial iron content, its adaptation to oxygen-rich environments and defense against ROS. We identified subspecies-specific differences in iron content, where F. tularensis subsp. tularensis was found to contain significantly less iron than strains of subsp. holarctica. The reduced iron content resulted in an increased tolerance to H2O2, despite simultaneously causing a decrease in the activity of catalase - the iron-dependent enzyme responsible for degrading H2O2 in F. tularensis. This strongly suggests that the restricted iron uptake and storage by subsp. tularensis strains is beneficial by rendering the bacteria less susceptible to H2O2, thereby evading the toxic effects of the iron-driven Fenton reaction. This evasion is likely to be an important part of the higher virulence displayed by subsp. tularensis as compared to subsp. holarctica. We further identified that the global regulator, MglA, is important for the adaptation of LVS to oxygen-rich environments. Deletion of mglA from LVS resulted in a mutant, ΔmglA, with impaired defense to oxidative stress, as manifested by an inability to grow to wild-type levels under aerobic conditions, an accumulation of proteins with oxidative damage, a suppressed expression of iron-uptake related genes, an increased catalase activity, and an increased tolerance to H2O2. This phenotype was reversed in a microaerobic environment. We therefore conclude that MglA is an important factor for the defense of LVS to oxidative damage under aerobic conditions and speculate that MglA is of greatest importance in oxygen-rich foci. We also studied the role of OxyR in LVS by creating a ΔoxyR mutant as well as a double mutant, ΔoxyR/ΔkatG. The in vitro response of these mutants, as well as of ΔkatG, to defined ROS was assessed using H2O2, the O2- generating agent paraquat, and the ONOO- generator SIN-1. ΔoxyR was more susceptible to all ROS than LVS as was ΔkatG, with the exception of O2- Strikingly, ΔoxyR/ΔkatG was significantly more susceptible to all ROS tested compared to either single deletion mutant. LVS, ΔoxyR and ΔkatG replicated efficiently in bone marrow-derived macrophages whereas ΔoxyR/ΔkatG showed no replication. In mice, the ΔoxyR mutant displayed impaired replication in liver, but intact replication vs. LVS in spleen. Collectively, our results demonstrate an important role of OxyR in the oxidative stress response and virulence of F. tularensis, and further reveal overlapping roles of OxyR and catalase in the defense against ROS. The results thus shed new light on the complexity of ROS defense in F. tularensis.

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide (O2 • −) have been shown to serve as messengers in biological signal transduction, and many prokaryotic and eukaryotic proteins are now known to have their function controlled via ROS-mediated oxidation reactions occurring on critical cysteine residues. The tumour-suppressor protein p53 is involved in the regulation of a diverse range of cellular processes including apoptosis, differentiation, senescence, DNArepair, cell-cycle arrest, autophagy, glycolysis and oxidative stress. However, little is understood about the specific molecular mechanisms that allow p53 to discriminate between these various different functions. p53 is a multiple cysteine-containing protein and there is mounting evidence to suggest that redox-modification of p53 Cys residues participate in control of its biological activity. Furthermore, p53 activity has been linked to intracellular ROS levels. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) offers superior mass resolving power and mass measurement accuracy, which is beneficial for the study of intact proteins and the characterisation of their posttranslational modifications (PTMs). The primary goal of the work described in this thesis was to employ FT-ICR mass spectrometry to investigate the molecular details of p53 redox-regulation. The relative reactivity of each of the ten cysteine residues in the DNA-binding core domain of recombinant human p53 was characterised by treatment with the Cys-alkylating reagent N-ethylmaleimide (NEM) under various conditions. A combination of top-down and middle-down FT-ICR MS was used to unambiguously identify Cys182 and Cys277 as sites of preferential alkylation. These results were confirmed by site-directed mutagenesis. Interestingly, Cys182 and Cys277 have previously been implicated in p53 redox-regulation. Alkylation beyond these two residues was found to trigger rapid alkylation of the remaining Cys residues, presumably accompanied by protein unfolding. These observations have implications for the re-activation of mutant p53 with Cys-targeting compounds which result in the death of cancer-cells. Furthermore, the molecular interaction between p53 and the ROS hydrogen peroxide was investigated. p53 was found to form two disulfide bonds upon treatment with H2O2. An enrichment strategy was developed to purify oxidised p53 and top-down FT-ICR mass spectrometry revealed unambiguously that Cys176, 182, 238 and 242 were the oxidised residues. Interestingly, Cys176, 238 and 242 are Zn2+- binding residues suggesting that p53 contains a zinc-redox switch. The mechanism of H2O2 oxidation was investigated, and revealed that oxidation via an alternative pathway results in indiscriminate over-oxidation of p53. Moreover, Cys176, 238 or 242 was shown to act as a nucleophile, and the intracellular antioxidant glutathione (GSH) did not prevent oxidation of the Zn2+-binding Cys residues, providing further evidence for a role in p53 redox-regulation. This study has revealed hitherto unknown details regarding the chemistry of cysteine residues within the important tumour-suppressor protein p53. Furthermore, the analytical power of FT-ICR MS for the study of multiple Cys-containing proteins has been very clearly demonstrated.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

A cisplatina ainda é um dos agentes quimioterápicos mais efetivos. No entanto, em elevadas doses pode ocorrer hepatotoxicidade. Alguns antioxidantes têm sido mostrado amenizar a hepatotoxicidade induzida pela cisplatina mas o mecanismo molecular envolvido não está bem esclarecido.No presente estudo nós investigamos moleculares subjacente ao efeito protetor da dimetiltiuouréia (DMTU), um conhecido eqüestrador de radical hidroxil, contra a lesão oxidativamitocondrial hepática induzida pela cisplatina em ratos. Ratos Wistar machos adultos ( 200 a 220g) foram divididos entre 4 grupos de 8 animais cada. O grupo controle foi tratado apenas com uma injeção intraperitoneal (i.p.) de solução salina (1 ml/ 100g de peso). Ao grupo DMTU foi administrado apenas DMTU (500 mg/kg de peso, i.p., seguido de 125 mg/kg, i.p., duas vezes ao dia até o sacrifício). Ao grupo cisplatina foi administrado uma injeção única de cisplatina (10 mg/kg de peso, i.p.). Ao grupo DMTU + cisplatina foi administrado DMTU (500mg/kg de peso, i.p.), pouco antes da injeção da cisplatina (10 mg/kg de peso, i.p.), seguido por injeções de DMTU (125 mg/kg de peso, i.p.) duas vezes ao dia até o sacrifício ( 72 horas após o tratamento). A hepatotoxicidade foi evidenciada no grupo cisplatina pelo aumento dos níveis séricos de alanina (ALT) e aspartato (AST)aminotransferases. O mecanismo de hepatotoxicidade induzido pela cisplatina mostrou-se envolvido na rigidez de membrana; na redução da razão glutationa reduzida em relação a glutationa oxidada (GSH/GSSG); na redução dos níveis de ATP, GSH e NADPH; na lipoperoxidação; na lesão oxidativa da cardiolipina e de proteínas com grupos fidrílicos. Mais ainda, a morte celular por apoptose foi também demonstrada e os achados fortemente sugerem a participação do xi mecanismo sinalizador mitocondrial neste processo; o DMTU não apresentou nenhum efeito direto sobre a mitocôndria e inibiu substancialmente a lesão mitocondrial induzida pela cisplatina, prevenindo a hepatotoxicidade. Todos os seguintes efeitos induzidos pela cisplatina foram previnidos pelo DMTU: (a) elevação dos níveis séricos de AST e ALT; (b) redução dos níveis de ATP hepático;(c)peroxidação lipídica;(d)oxidação da cardiolipina; (e)oxidação de proteínas sulfidrílicas; (f) rigidez da membrana mitocondrial; (g) oxidação de GSH; (h)oxidação de NADPH e (i) morte celular por apoptose. Os resultados mostraram o papel principal da mitocôndria e dos radicais hidroxilas na proteção do fígado saudável contra a lesão hepática induzida pela cisplatina, delineando um número de etapas que podem ser consideradas no desenvolvimento de futuros agentes citoprotetores / Cisplatin is still one of the most effective chemotherapeutic agents. However, at higher doses hepatotoxicity may occur. Some antioxidants have been shown to ameliorate cisplatin-induced hepatotoxicity but the involved molecular mechanism has not been clarified. In the present study we investigated the molecular mechanism underlying the protective effect of dimethylthiourea (DMTU), a known hydroxyl radical scavenger, against liver mitochondrial oxidative damage induced by cisplatin in rats.Adult male Wistar rats (200 to 220g) were divided into 4 groups of 8 animals each. The control group was treated only with an intraperitoneal (i.p.) injection of saline solution (1ml/100g body weight). The DMTU group was given only DMTU (500 mg/kg body weight, i.p, followed by 125 mg/Kg, i.p., twice a day until sacrifice). The cisplatin group was given a single injection of cisplatin (10 mg/kg body weight, i.p.). The DMTU+cisplatin group was given DMTU (500 mg/kg body weight, i.p.), just before the cisplatin injection (10 mg/kg body weight, i.p.), followed by injections of DMTU (125 mg/kg body weight, i.p.) twice a day until sacrifice (72 hours after the treatment). epatotoxicity was evidenced in the cisplatin group by the increased serum levels of alanine (ALT) and aspartate (AST) aminotransferases. The mechanism of cisplatininduced hepatotoxicity was found to involve membrane rigidification; decreased GSH/GSSG ratio, ATP, GSH and NADPH levels; lipid peroxidation; oxidative damage of cardiolipin and protein sulfhydryl groups. Moreover, cell death by apoptosis was also demonstrated and the findings strongly suggest the participation of the mitochondrial signaling pathway in this process; DMTU did not present any direct effect on mitochondria and substantially inhibited cisplatin-induced mitochondrial injury, therefore preventing the hepatotoxicity. All the following cisplatin-induced xiv effects were prevented by DMTU: (a) elevation of AST and ALT serum levels; (b) decreased hepatic ATP levels; (c) lipid peroxidation; (d)cardiolipin oxidation; (e) sulfhydryl protein oxidation; (f) mitochondrial membrane rigidification; (g) GSH oxidation; (h) NADPH oxidation and (h) apoptotic cell death. Results show the central role of mitochondria and hydroxyl radicals in the protection of healthy liver against cisplatin-induced injury, highlighting a number of steps that might be considered in the development of novel cytoprotective agents.

A produção in vitro de embriões expõe o concepto a condições diferentes do ambiente intra-tubárico-uterino. A alta tensão de oxigênio durante o cultivo in vitro induz estresse oxidativo mediante aumento dos níveis das espécies reativas de oxigênio. A condição de estresse oxidativo altera a expressão de RNAm e miRNAs, podendo comprometer vias de interação materno-embrionária. Recentemente, os exossomos têm sido descritos como um mecanismo complementar de transporte de RNAm e miRNAs, que beneficiam a comunicação bidirecional materno-embrionária. Portanto, além do estudo dos próprios embriões, a análise dos exossomos isolados do meio de cultivo da PIVE também é relevante. Comumente são utilizados dois modelos de cultivo na PIVE, a alta tensão (20%) e a baixa tensão (5%) de oxigênio. A hipótese deste estudo é que a alta tensão de oxigênio no cultivo embrionário gera uma condição de estresse oxidativo que causa alterações de expressão de RNAm e miRNAs presentes nos embriões e nos exossomos. Além disso, o estresse oxidativo exerceria efeito sobre o padrão de secreção destes exossomos. Para testar essa hipótese este estudo produziu embriões bovinos in vitro cultivados em diferentes tensões de oxigênio. Os embriões foram coletados no dia 7 e os meios de cultivo, para isolamento os exossomos, foram coletados nos dias 3 e 7 do cultivo. Os blastocistos cultivados em alta tensão de oxigênio apresentaram um aumentou dos níveis de EROs através de análise de fluorescência. Este resultado validou o modelo de estresse oxidativo para embriões. Os resultados iniciais indicaram que apesar do miR-199a-5p, descrito como possível regulador dos genes alvos ERBB2 e ERBB3, ter apresentado maior expressão nos embriões cultivados em alta tensão, os transcritos ERBB2 e ERBB3 e a proteína ERBB2 não apresentaram diferença significativa entre os grupos. Uma vez que não se estabeleceu uma relação de regulação entre o miR-199a-5p e o gene alvo ERBB2 para os embriões bovinos, este estudo se voltou para a análise de outros 96 RNAm e 378 miRNAs, destes 40 RNAm e 8 miRNAs apresentaram alterações significativas entre os embriões cultivados em alta e baixa tensão de oxigênio. Entre as principais funções alteradas estão associadas à resposta ao estresse oxidativo, proliferação e diferenciação celular, remodelação epigenética, apoptose, metabolismo e reconhecimento materno-fetal. Por fim, com o objetivo de compreender um panorama maior dos efeitos do estresse oxidativo, este estudo se propôs a analisar o padrão de expressão e os miRNAs do conteúdo dos exossomos do meio de cultivo. O estresse oxidativo alterou tanto a concentração dos exossomos quanto a expressão dos mRNAs, em ambos os dias do desenvolvimento embrionário (D3 e D7). Dentre os miRNAs, destaca-se o miR-210 que foi considerado por este trabalho como biomarcador não invasivo da condição de normoxia no cultivo in vitro de embriões bovinos. Em conclusão, esse estudo não elucidou como o estresse oxidativo altera a interação materno-embrionária em embriões produzidos in vitro em diferentes condições de tensão de oxigênio, mas gerou conhecimentos adicionais sobre do desenvolvimento embrionário bovino e os efeitos da alta tensão de oxigênio. / The in vitro embryo production exposes the concept to conditions different from the intra-tubal-uterine environment. The high oxygen tension during in vitro production induces the oxidative stress by increasing the concentration of reactive oxygen species. The oxidative stress condition changes the mRNA and miRNAs expression, it can compromise pathways of maternal-embryonic interaction. Recently, the exosomes have been described as a complementary mechanism of mRNA and miRNAs transport, which improve the bidirectional maternal-embryonic communication. Therefore, the studies of the embryos and exosomes isolated from the IVP culture medium are relevant. Two cultivation models are usually used in IVP, the high tension (20%) and the low tension (5%) of oxygen. The hypothesis of this study is that the high oxygen tension in the embryonic culture generates an oxidative stress condition that causes changes in the mRNA and miRNAs expression. In addition, this oxidative stress is able to modulate the secretion pattern of the exosomes isolated from IVP embryos. To test this hypothesis, this study produced in vitro bovine embryos developed at different oxygen tensions. The embryos were sampled on day 7 and the culture media, for exosomes isolation, were collected on days 3 and 7 of the embryo development. Blastocysts cultured in high oxygen tension showed increased levels of ROS through fluorescence analysis. This result validated the oxidative stress model for embryos. In order to understand the effect of oxidative stress on the pathways of maternal-embryonic interaction, this study aimed to analyze the ERBB signaling pathway. Despite the fact that miR-199a-5p, described as a possible regulator of the ERBB2 and ERBB3 target genes, showed higher expression in embryos cultured under high tension, the ERBB2 and ERBB3 transcripts and the ERBB2 protein showed no significant difference between high and low oxygen tension groups. Since a regulatory relationship between miR-199a-5p and the ERBB2 target gene was not established for bovine embryos, this study turned to the analysis of other 96 mRNAs and 378 miRNAs, out of 40 mRNAs and 8 miRNAs showed significant changes among the groups. The main altered functions are the response to oxidative stress, cell proliferation and differentiation, epigenetic remodeling, apoptosis, metabolism and maternal-fetal recognition. Finally, in order to understand the bigger picture of oxidative stress effect we analyze the secretion pattern and the miRNA content of the exosomes from culture medium of IVP embryos. Oxidative stress change both, the exosome concentration and the miRNA expression (at D3 and D7). Among the miRNAs, we highlight the miR-210 that was considered by this work as a non-invasive biomarker of the normoxia condition in the in vitro culture of bovine embryos. In conclusion, this study did not elucidate how oxidative stress changes the maternal-embryonic interaction in IVP embryos, but it generated additional knowledge about bovine embryonic development and the high oxygen tension effects.