8-oxo-7,8-dihydroguanine (8oxoG) is one of the most important base lesions formed during oxidative damage of DNA. The aim of the present research was to investigate the effects of DNA concentration, G content, and the nature of oxidizing species on the kinetics of 8oxoG in model DNA solutions by using HPLC. The experimentally obtained yields of 8oxoG were typically in the range of 2-2.5% of total concentration of guanine. The ratios of the rate constant of hole diffusion in DNA to the rate constant of conversion of the hole into 8oxoG (kd/kr) were calculated from the experimental data using the diffusion model of charge transfer in DNA to be in the range of 200-300, in agreement with previously reported kd/kr ratios in the duplex DNA oligonucleotides (GGA)n or (GGTT)n. Our current diffusion model cannot satisfactorily explain the absence of the G content dependence of the 8oxoG yields, which indicates that a more advanced model is required.

Methamphetamine (METH) is a highly addictive substance that is highly prevalent in today’s society, with over 1 in 20 adults over 26 having taken it at least once. While it is known that METH, a common psychostimulant, acts on both the mesolimbic dopamine (DA) and nigrostriatal DA systems by affecting proteins involved in DA reuptake and vesicular packaging, the specific mechanism of what is known as METH neurotoxicity remains obscure, but has been shown to involve oxidative stress. Studies have shown that reactive oxygen species act on the same proteins that METH affects. Oxidative species have also been known to catalyze the formation of melanins in dopaminergic cells. We explore this link more fully here. In an in vitro system, oxidative species (including Fe3+, an inorganic catalyst for oxidative stress), enhance the rate of melanization of DA. Methamphetamine increased oxidative stress in an in vivo model. Additionally, METH enhanced phasic (stimulated) DA release and caused an electrically-independent efflux of DA. Lidocaine abolished phasic DA release, but did not affect METH-induced DA efflux, indicating action-potential dependent and independent mechanisms behind METH’s effects. The sigma-1 receptor antagonist BD 1063 significantly attenuated METH’s effect on DA release. Depletion of intracellular calcium (Ca2+) reserves also attenuated METH-enhancement of DA release. We investigated the role of oxidative species in METH-induced DA efflux. Reduced glutathione (the substrate for glutathione peroxidase) and 4-hydroxy-TEMPOL (a superoxide dismutase mimetic) blocked METH’s effect on DA release, suggesting that a reactive oxygen species (ROS), most likely superoxide, is necessary for METH-induced DA efflux. Finally, oxidative stress as well as acute METH impairs the vesicular monoamine transporter 2 (VMAT2) by S-glutathionylation modification of Cys-488, highlighting VMAT2 as a likely regulator of METH’s effects on electrically independent DA release. These findings help outline a model in which METH induces DA release in the NAc through a signaling cascade involving the sigma receptor and ROS signaling molecules.

L’ATP est synthétisée par les mitochondries à partir de réactions d’oxydoréduction catalysées les complexes de la chaîne respiratoire. Ces réactions impliquent des transferts d’électrons intra-protéine. Une capacité de production de l’anion superoxyde, formé par la réaction de l’oxygène avec un électron, a été identifiée pour les complexes I et III. Les espèces actives de l’oxygène (EAOs) sont des molécules dérivées de l’anion superoxyde. Si elles ne sont pas correctement régulées par les défenses antioxydantes de la cellule, ces EAOs peuvent réagir avec les composants de la cellule et nuire à son fonctionnement : ce déséquilibre est appelé stress oxydatif. L’altération d’un ou plusieurs complexes respiratoires associée à un stress oxydatif cellulaire est un mécanisme commun à de nombreuses maladies neurodégénératives. Dans ce travail nous nous intéressons plus particulièrement à l’atrophie optique autosomique dominante de type 1 (ADOA-1). L’ADOA-1 est une maladie neurodégénérative principalement causée par des mutations du gène codant la protéine mitochondriale OPA1 impliquée dans la dynamique mitochondriale. Les tableaux cliniques et l’âge de début de la maladie sont variables. Il n’existe pas de corrélation claire entre génotypes et phénotypes permettant d’expliquer cette variabilité ni de traitement à cette pathologie. L’hypothèse d’un stress oxydatif a été proposée pour expliquer la variabilité de ces symptômes. C’est pourquoi notre objectif est d’améliorer la compréhension des mécanismes physiopathologiques impliqués dans cette maladie en développant des modèles mathématiques de la production des EAOs par la chaîne respiratoire. Nous avons utilisé deux méthodes de modélisation. Dans le premier cas, nous modélisons l’activité des complexes respiratoires et la production d’anion superoxyde par les complexes I et III par des équations de vitesse que nous construisons en trois étapes. Nous analysons d’abord les données biochimiques disponibles dans la littérature. Nous proposons ensuite des interprétations physiques à ces comportements et les traduisons sous forme de règles floues. Nous modélisons enfin ces règles en utilisant des fonctions données par le formalisme de Michaelis-Menten. Les équations de vitesse sont fonction de variables chimiques telles que la concentration des espèces chimiques impliquées dans les réactions des complexes respiratoires et ne prennent pas en compte le détail des réactions intra-protéine impliquées dans le fonctionnement des complexes. Cette méthode permet de construire un modèle simple, permettant de simuler l’activité des complexes I et III et leur production de superoxyde dans différentes conditions, et qui est facilement modifiable ou intégrable dans un modèle plus complet de la mitochondrie. Le modèle du complexe I que nous avons créé, est capable de simuler l’activité catalytique et la production des EAOs en mode direct par le complexe I pour différentes configurations et concentrations de substrats et produits. / Mitochondria are cellular organelles involved in ATP (adenosine triphosphate) supply to cells. Mitochondrial ATP is produced by the oxidative phosphorylation which involves redox reactions catalysed by the four protein complexes of the mitochondrial respiratory chain. These redox reactions require intra-protein electron transfers. The complex I and complex III of the respiratory chain are able to generate superoxide anion, which is formed by the reaction of oxygen with one electron. Reactive oxygen species (ROS) are molecules derived from the superoxide anion. ROS which are not regulated by cellular antioxidant defences can react with the components of the cells and disturb its functioning: this imbalance between ROS and antioxidant defences has been termed “oxidative stress”. Dysfunctions of one or several respiratory complexes associated to an oxidative stress is a mechanism common to numerous neurodegenerative diseases. In this work, we focus on autosomal dominant optic atrophy 1 (ADOA-1 or DOA-1). DOA-1 is a neurodegenerative pathology mainly caused by mutations in the gene OPA1 which codes for a mitochondrial protein involved in mitochondrial dynamics. The symptoms and ages of onset of the disease are variable. There is no clear correlation between genotypes and phenotypes which can explain this variability and to date, there is no established medical treatment for the disease. The hypothesis of an oxidative stress has been proposed to explain the variability of symptoms observed in patients. Indeed, the mitochondrial energetic metabolism is altered in biological models (cell cultures and animal models) of DOA-1 and low levels of antioxidant defences have been measured in cells from patients suffering from severe forms of the pathology. Hence, our objective is to improve the understanding of the physio-pathological mechanisms involved in this disease by developing mathematical models of ROS production by the respiratory chain. We use two modelling methods. The first method consists in modelling the activities of respiratory complexes and the superoxide production by complexes I and III with rate equations that we build in three steps. We first analyse the biochemical data available in the literature. We subsequently interpret this data physically and translate them in the form of fuzzy rules. We then model these rules with mathematical functions provided by the formalism of Michaelis-Menten. The rate equations depend on chemical variables such as the concentrations of chemical species involved in the reactions catalysed by the respiratory complexes. They do not include the details of intra-protein electron transfers, occurring during the catalysis performed by the complexes. This method enables us to build a simple model simulating the activities and superoxyde productions of complexes I and III in different conditions and that can easily be modified or integrated in a more comprehensive model of the mitochondrion. Our model of complex I can simulate the forward and reverse activities and ROS productions of the enzyme for different concentrations of substrates and products.

Axon initial segment (AIS) disruption has been described in a number of pathological environments where neuroinflammation is a contributing factor; however, whether this disruption is reversible in unknown. To address the principle of AIS structural recovery, we employed an acute neuroinflammatory model. Acute neuroinflammation induced disruption of AIS structural and functional domains and, importantly, upon resolution of neuroinflammatory conditions, was reversed. Consistent with other studies, we observed a close interaction of microglia with AISs, and utilized this acute neuroinflammatory model to investigate the relationship between reactive microglia and AIS integrity. Gene expression analysis of microglial transcription profiles identified reactive oxygen species (ROS)-producing enzymes as candidates in AIS pathogenesis. Experiments employing mice lacking the major ROS-producing enzyme NOX2, identified ROS as mediators of AIS disruption. Furthermore, we established calcium-dependent protease calpain as a disruptor of AIS protein clustering in inflammation-induced disruption. Since we observed an intimate interaction between microglia and the AIS, we conducted studies designed to identify a candidate in microglia that regulates microglial-AIS contact. During chronic inflammatory conditions, microglia enhance contact with AISs often completely surrounding the domain. Concomitant with this morphological change, neurofascin (Nfasc) expression increased in microglia. Nfasc is a cell adhesion molecule with cell-specific isoforms known to mediate glial-neuronal interactions, but until now, was not reported to be expressed by microglia. Here, I characterize the unique Nfasc isoform expressed by microglia and present evidence that suggests that microglial Nfasc may mediate microglial-AIS contact, a potentially pivotal interaction in the induction of AIS disruption by pro-inflammatory factors.

The brain renin-angiotensin system (RAS), of which angiotensin II (AngII) is the primary effector peptide, plays a critical role in the neurohumoral regulation of cardiovascular and body fluid homeostasis by modulating blood pressure, secretion of hypothalamic and pituitary hormones, and water intake. AngII produced locally in the brain or in the systemic circulation can act on brain regions called circumventricular organs (CVO), which lack the blood-brain-barrier. Dysregulation of central AngII signaling is implicated in the pathogenesis of hypertension; therefore, understanding the mechanisms of AngII in the CNS is an important area of investigation. Recently, a novel signaling mechanism for AngII in the periphery has been shown to involve NAD(P)H oxidase-derived reactive oxygen species (ROS). Although ROS are now known to be involved in numerous AngII-regulated processes in peripheral tissues, and are increasingly implicated in CNS neurodegenerative diseases, the role of ROS in central regulation of AngII-induced cardiovascular function remains unexplored. The hypothesis that ROS are critically involved in central AngII signaling and in AngII-dependent blood pressure and drinking behavior was tested by harnessing the power of an array of selective genetic tools, in combination with novel technologies for analysis of cardiovascular function in conscious mice. More specifically, central injections of adenoviruses encoding ROS-modulating molecules were used to examine the redox mechanisms in central AngII-mediated cardiovascular responses in vivo. Neuronal cell cultures were also used to investigate the involvement of NAD(P)H oxidase-derived ROS in AngII signaling, as well as to examine a link between calcium and ROS in intra-neuronal AngII signaling. Finally, in order to better understand the potential role of ROS in the brain in the pathogenesis of AngII-dependent hypertension, a mouse model that recapitulates the characteristics of human hypertension was employed in conjunction with genetic modulation of the redox state of the brain. These studies provide new evidence that ROS are involved in the intracellular signaling mechanism of AngII in the brain under normal and pathological conditions and offer new insight to how dysregulation of redox mechanisms in the brain may lead to the pathogenesis of AngII-dependent hypertension.

Particulate air pollution is known to exacerbate respiratory diseases, such as asthma and chronic obstructive pulmonary disease, in humans. It has been proposed that transition meta ls from inhaled particles may play a role in this exacerbation by generating radical species leading to damage in the lungs. The aim of this research was to determine the role that iron from particulate air pollution played in the generation of reactive oxygen species and subsequently the induction of inflammatory mediators in cells in culture. The production of reactive oxygen species by particulate air pollution was found to be dependent on the mobilization of iron from the particles by chelators, such as the physiologically relevant citrate. The amount of iron mobilized from the combustion particulate, coal fly ash, was dependent on the type of coal used to generate the fly ash and was greatest in the smallest size fraction collected for three different coal types. In addition, the amount of iron mobilized from coal fly ash by citrate correlated closely with the amount mobilized in human lung epithelial (A549) cells, as indicated by induction of the iron storage protein, ferritin. The amount of the proinflammatory cytokine, interleukin-8, secreted in response to coal fly ash treatment varied with the amount of iron mobilized intracellularly from the particles, with the greatest response to the smaller size fractions which released the largest amounts of iron. There was a direct relationship, above a threshold level of bioavailable iron, between the level of interleukin-8 and bioavailable iron in cells treated with coal fly ash. Tetramethyl thiourea and dimethyl sulfoxide prevented the increased production of interleukin-8 by human lung epithelial cells treated with coal fly ash, suggesting the role of a radical species in the induction of this inflammatory mediator. The mobilization of iron from coal fly ash by citrate or in human lung epihelial cells, as well as the induction of interleukin-8, did not correlate with the total amount of iron in the particles. Instead, preliminary results suggest that these measured values vary directly with the amount of iron contained in the aluminosilicate fraction of the fly ash.

Although transition metals are essential for life, misregulation of redox-active metal uptake, delivery, storage, and excretion has been linked with a series of neurodegenerative disorders. Alzheimer's disease (AD) is considered an epidemic and is the most widespread of all forms of dementia. Copper ions found in large concentrations localized in amyloid-ß plaques in the brain of AD patients have been linked with the generation of reactive oxygen species which are suspected to be the culprits leading to neuronal cell death. Herein a series of mechanistic and spectroscopic studies elucidate the chemistry about the metal-centered oxidation of biomolecules, including catecholamine neurotransmitters and some analogues by copper-complexes of amyloid-ß peptide. Transition metals can also be useful tools for characterization of metalloproteins due to their unique chemical and spectroscopic features. Herein a series of studies of the native Zn²+ and Cu²+-derivative of recombinant Blastula Protease 10 (BP10) from the sea urchin Paracentrotus lividus are presented in order to elucidate its catalytic mechanism, with the use of enzymology, metal substitution, and electronic absorption spectroscopy.

Este estudo objetivou avaliar os efeitos da melatonina sobre a qualidade e as taxas de blastocistos bovinos PIV. A melatonina é um poderoso eliminador de radicais livres e tem efeitos benéficos na PIV de algumas espécies. Foram feitas sessões de PIV (n=12), sendo aspirados os folículos (2 a 7 mm) de ovários obtidos de abatedouro. Complexos cumulus oócito (COCs) que apresentavam cumulus compacto e oócito com citoplasma homogêneo foram selecionados e randomicamente distribuídos ente os grupos controle (GC) e tratado com Melatonina (GM). Submetidos a MIV por 22h em 400&micro;l de TCM199(Dia-1). Para a FIV COCs foram transferidos para 400&micro;l de meio de fertilização, inseminado com sêmen descongelado previamente submetido ao gradiente descontínuo de Percoll&reg;, e incubados por 18h (dia0). As células do cumulus dos presumíveis zigotos foram removidas com hialuronidase e pipetagem sucessiva, sendo então cultivados (CIV) em 400&micro;l de KSOM- BSA (Dia 1). No Dia 3, foi avaliada a taxa de clivagem em estereomicroscópio e adicionados 400&micro;l KSOM-20% SFB. No Dia 5, 200&micro;l de meio foram removidos e 400&micro;l KSOM-10% SFB adicionado. No dia 7 foi avaliado o estágio de desenvolvimento dos embriões, e as porcentagens calculadas em relação ao total de presumíveis zigotos. Todos os procedimentos foram realizados em placa de 4 poços (Nunc Multidishes&reg;) sem cobertura de óleo mineral Na MIV e no CIV, os grupos receberam ou 0ng/ml ou 50ng/ml de melatonina, GC e GM, respectivamente. No Dia 7, de 4 sessões de PIV, todos os blastocistos expandidos foram corados com Iodeto de Propídeo e Hoechst 33342. Os demais blastocistos expandidos, das outras sessões, foram vitrificados, assim como todos os presentes no Dia 8. Uma amostra deste meio foi retirada para avaliação da concentração de espécies reativas ao ácido tiobarbitúrico (TBARS). Posteriormente os embriões foram descongelados e cultivados para a avaliação do efeito da melatonina sobre os embriões no cultivo pós-descongelação. A análise estatística foi feita usando SAS system for Windows 9.2&reg;. Dados paramétricos foram submetidos ao teste T de Student e os não paramétricos ao Wilcoxon, diferenças foram consideradas significativas quando p<0,05. Blastocistos expandidos do GM apresentaram maior número de células, sendo que estas apresentaram porcentagem menor de ruptura de membrana que os do GC. A melatonina não influenciou as taxas de clivagem, de blastocistos no Dia 7 nem a taxa de blastocistos passíveis de vitrificação. Não foi observado efeito da melatonina sobre a tolerância dos embriões à vitrificação nem no período de cultivo após o descongelamento. Inesperadamente, a melatonina aumentou a concentração de TBARS no meio. A melatonina melhorou a qualidade embrionária, contudo suas propriedades antioxidantes não foram evidenciadas. / This aimed to evaluate melatonin effect over bovine embryo quality and rates on in vitro embryo production procedures (IVP). Melatonin is an effective free radical scavenger and beneficial effects of melatonin have been demonstrated in IVP for some species. Embryo production sessions (12) were performed; follicles (2 to 7 mm) were aspirated from slaughterhouse-derived ovaries. Cumulusoocyte complexes (COCs) who had a compact cumulus and oocyte with homogeneous cytoplasm were selected and randomly allocated (35 to 55 per group) on either control Group (CG) or Melatonin Treatment Group (MG). COCs underwent in vitro maturation (IVM) for 22 hours in 400&micro;l of TCM199 (Day -1). For in vitro fertilization (IVF) COCs were moved to fertilization medium 400&micro;l and were inseminated with previously submitted to discontinuous PercollTM gradient, frozenthawed semen, incubated for 18 hours (Day 0). After IVF presumptive zygotes were striped from remaining cumulus cells by incubation with hyaluronidase and gentile pipetting, then moved to 400&micro;l of KSOM- BSA (Day 1). On Day 3 embryos were inspected under a stereomicroscope to evaluate cleavage rate, and 400&micro;l KSOM-20% FCS was added. On Day 5 200&micro;l of medium were removed and 400&micro;l KSOM-10% FCS was added. On Day 7 embryos were evaluated regarding their developmental stage, percentages (%) were calculated over the total of presumptive zygotes, expanded blastocysts were vitrified. All procedures were performed on a Nunc MultidishesTM 4 well dish without mineral oil overlay. During IVM and IVC groups received either 0ng/ml or 50 ng/ml of melatonin, CG and MG respectively. On Day 7 out of 4 replicates all expanded blastocysts were dyed with Propidium Iodide and Hoechst. Remaining expanded blastocysts ass well as all the expanded blastocysts on Day 8 were vitrified. A medium sample was withdrawn for thiobarbituric acid assay (TBARS). Latter embryos were thawed and cultured aiming to evaluate melatonin effect over cryotolerance and post thaw culture system. Statistical analysis was performed using SAS system for Windows 9.2TM parametric data was submitted to student T-test and non parametric to Wilcoxon. Differences were considered meaningful when p<0,05. Expanded blastocysts from MG presented more cells and less cells with ruptured membrane than the ones from CG. But melatonin treatment neither influenced cleavage nor Blastocyst rates on Day 7, nor the rate of blastocysts that can be vitrified. Neither over embryos cryotolerance nor over post thaw culture system. Unexpectedly melatonin increased TBARS levels on medium. Melatonin improves bovine embryo quality however its antioxidant properties were not demonstrated.

Doctor of Philosophy / Rice (Oryza sativa L.) is very susceptible to water deficit at any time during its life cycle as a semi-aquatic cereal crop. However, the consequential damage is particularly severe if water deficit occurs during reproductive phases. The conspicuous injury often observed in rice plants exposed to water stress during meiosis of the pollen mother cell is the reduction of grain set, which is attributed to the decline of male fertility. In spite of much research on drought-induced male sterility in rice, the underlying mechanisms of the problem are poorly understood. This project was therefore conducted to investigate the molecular mechanisms of water deficit-induced pollen sterility in rice. In this study three consecutive days of water deficit treatment at -0.5 MPa osmotic potential during anther development effectively reduced the leaf water potential (leaf) and the number of viable pollen which later led to a decrease in grain set. Moreover, this thesis demonstrates that the immediate deleterious effects of water deficit to plant fertility could be estimated using a young microspore viability index, which showed a strong correlation with mature viable pollen and grain set. The present work has also illustrated that oxidative stress appears to be a plausible cause for the decline of male fertility and grain set. Water deficit has induced the excessive production of reactive oxygen species (ROS) above the redox balance, which in turn caused detrimental effects to cellular DNA and might result in programmed cell death (PCD) in the anthers. Moreover, ROS accumulation effectively influenced ATP synthesis leading to a decrease in the level of ATP in the anthers. Excessive ROS accumulation after drought could be the consequence of insufficient activity of the antioxidant system, which has been illustrated by qRT-PCR expression analysis of major antioxidant genes. Down-regulation of those genes would increase the incidence of oxidative damage. In contrast, stable or up-regulated expression of these genes resulted in less oxidative damage. Detailed investigations of sugar metabolism in anthers has provided supplemental data to develop a model of sugar unloading and transport within anther using in situ hybridisation to mRNA techniques. Analysis of sugar transportation within the cellular compartments of anther has unveiled the role of sugar metabolism on pollen sterility in rice. qRT-PCR assays of genes associated with the sugar metabolic pathway has demonstrated that the supply of both sucrose and hexoses from the anther walls to the locules was not restricted after water deficit stress. The results indicate that water deficit might not cause sugar starvation for developing microspores as previously thought, nor inhibit the initial steps of sugar utilisation such as glycolysis. This thesis has suggested new ideas regarding the role of rising sugar levels to cope with oxidative stress in anthers. Sugar accumulation might have provided protection against oxidant damage by strengthening the antioxidant system. However, the interplay between sugar and oxidative stress is not straightforward and needs to be further characterised. In-depth investigations on the interaction between sugar signalling and oxidative stress responses may help indentify the role of sugars in protecting anthers under water deficit. Although many studies on drought and chilling stresses in rice anthers have been performed, the causal mechanism of male sterility still remains to be elucidated. Findings presented in this thesis may contribute to understanding molecular mechanisms of male sterility in rice as a response to drought stress. A more detailed investigation of mitochondrial respiration in rice anthers is required to further examine this problem. Finally, this thesis suggests that signalling molecules such as 14-3-3 proteins and abscisic acid (ABA) might act upstream of ROS production and antioxidant defence in plants. Further work on these molecules might therefore further illustrate how they influence plant fertility under water shortage conditions.

S100A8 is a highly-expressed calcium-binding protein in neutrophils and activated macrophages, and has proposed roles in myeloid cell differentiation and host defense. Functions of S100A8 are not fully understood, partly because of difficulties in generating S100A8 knockout mice. Attempts to silence S100A8 gene expression in activated macrophages and fibroblasts using RNA interference (RNAi) technology were unsuccessful. Despite establishing validated small interfering RNA (siRNA) systems, enzymaticallysynthesized siRNA targeted to S100A8 suppressed mRNA levels by only 40% in fibroblasts activated with FGF-2+heparin, whereas chemically-synthesized siRNAs suppressed S100A8 driven by an S100A8-expression vector by ~75% in fibroblasts. Suppression of the gene in activated macrophages/fibroblasts was low, and some enzymatically-synthesized siRNAs to S100A8, and unrelated siRNA to GAPDH, induced/enhanced S100A8 expression in macrophages. This indicated that S100A8 may be upregulated by type-1 interferon (IFN). IFN-β enhanced expression, but did not directly induce S100A8. Poly (I:C), a synthetic dsRNA, directly induced S100A8 through IL-10 and IFN-dependent pathways. Induction by dsRNA was dependent on RNA-dependent protein kinase (PKR), but not cyclooxygenase-2, suggesting divergent pathways in LPS- and dsRNA-induced responses. New mechanisms of S100A8 gene regulation are presented, that suggest functions in anti-viral defense. S100A8 expression was confirmed in lungs from influenza virus-infected mice and from a patient with severe acute respiratory syndrome (SARS). Multiple pathways via mitochondria mediated S100A8 induction in LPS-activated macrophages; Generation of reactive oxygen species via the mitochondrial electron transport chain and de novo synthesis of ATP may be involved. This pathway also regulated IL-10 production, possibly via PKR. Extracellular ATP and its metabolites enhanced S100A8 induction. Results support involvement of cell stress, such as transfection, in S100A8 expression. A breast tumor cell line (MCF-7) in which the S100A8 gene was silenced, was established using micro RNA technology; S100A8 induction by oncostatin M was reduced by >90% in stably-transfected cells. This did not alter MCF-7 growth. The new approach to investigate the role of S100A8 in a human tumor cell line may assist in exploring its functions and lead to new studies concerning its role in cancer.