A resistência de bovinos Bos taurus taurus a ambientes tropicais é menor quando comparada com a de Bos taurus indicus. Essa característica influencia os aspectos reprodutivos e vem sendo amplamente descrita na literatura. No que diz respeito aos touros, esta influência torna-se ainda mais significativa, visto que uma menor fertilidade destes impõe enorme impacto na produção animal. Vários são os estudos que comprovam o efeito negativo do estresse térmico sobre os parâmetros seminais de touros europeus provocando diminuição da fertilidade, porém a patogenia deste quadro não está confirmada. Uma hipótese para esta menor fertilidade seria um maior nível de estresse oxidativo nos touros europeus, no qual haveria um desequilíbrio entre a produção de espécies reativas ao oxigênio (EROs) e os níveis de proteção antioxidante. Para testar esta hipótese, foram utilizados 20 touros Simental (Bos taurus taurus) e 20 touros Nelore (Bos taurus indicus), criados a campo, na região de Dourados, Mato Grosso do Sul, submetidos a duas coletas anuais, durante o verão e inverno, no período de dois anos consecutivos. As amostras foram analisadas através da análise espermática padrão, dos níveis das enzimas antioxidantes, catalase e superóxido dismutase (SOD), e dos níveis de malondialdeído (MDA), produto da peroxidação lipídica, que foi utilizado como índice de estresse oxidativo. Os dados foram analisados pela análise de variância ONEWAY, levando em conta as classes raça (Nelore e Simental) e estação do ano (verão e inverno), através do programa SAS for Windows. As variáveis defeitos menores, defeitos totais e motilidade não apresentaram diferenças quanto a raça ou estação do ano. Observou-se efeito de estação do ano sobre as variáveis defeitos maiores (20,32±15,30% e 12,17±12,27%, verão e inverno respectivamente; p=0,02) e MDA (842,66±527,98 e 419,30±434,14 ng/ml, verão e inverno respectivamente; p=0,006), apenas no sêmen dos touros de raça Simental. As concentrações das enzimas antioxidantes SOD e catalase, não diferiram entre as raças e tampouco entre as estações do ano. Observou-se correlação entre MDA e formas anormais de cabeça (r=0,39, p=0,0035) e entre SOD e defeitos primários (r= - 0,31, p=0,0075). Os resultados obtidos sugerem que houve um maior nível de estresse oxidativo nos animais de origem européia criados a campo em clima tropical em relação aos animais de origem indiana, provavelmente relacionado a uma produção excessiva de EROs sem a devida compensação antioxidante. / Resistance to tropical environments is lower in Bos taurus taurus than in Bos taurus indicus cattle. This trait influences reproductive aspects, and is greatly discussed in publications worldwide. This negative influence is much more significant in bulls, since lower fertility in them highly impacts animal production. There are many studies demonstrating that exposure of European bulls to heat stress will negatively affect their seminal parameters, leading ultimately to lower fertility rates, but the pathogenic pathway has not been clarified. One hypothesis is that this lower fertility is due to higher levels of oxidative damage in these bulls, in which there would be an imbalance between reactive oxygen species (ROS) production and antioxidant capacity. In order to test this hypothesis, 20 Simmental bulls (Bos taurus taurus) and 20 Nelore bulls (Bos taurus indicus) kept under extensive breeding in the Dourados region, in Mato Grosso do Sul state were used. Semen collection occurred twice a year, during summer and winter, for a two year period. Samples were submitted to standard semen analysis and the levels of the antioxidant enzymes catalase and superoxide dismutase (SOD) dismutase were measured. To evaluate oxidative damage to the cells the levels of the lipid peroxidation product malondialdehyde (MDA) were measured. Data were analyzed for breed (Nelore or Simmental) and season (summer or winter) by the ONEWAY analysis of variance, using the SAS for Windows. Minor defects, total defects and motility did not differ in breed or in season. Season showed an effect on major defects (20.32±15.30% and 12.17±12.27%, summer and winter respectively; p=0.02) and MDA (842.66±527.98 and 419.30±434.14 ng/mL, summer and winter respectively; p=0.006), only in the Simmental animals. SOD and catalase levels did not differ between breeds or seasons. Correlations were observed between MDA and sperm head defects (r=0.39, p=0.0035) and between SOD and primary defects (r= -0.31, p=0.0075). Results suggest a higher level of oxidative stress in European bulls raised in field conditions, when compared to Indian bulls under the same conditions, and this is probably due to a higher ROS production, without a concomitant increase in antioxidant levels.

L’objectif de cette thèse est de mieux comprendre les réactions de craquage thermique des naphtènes (hydrocarbures saturés cycliques) se déroulant dans les réservoirs pétroliers. Les naphtènes, représentant une famille importante de composés dans les huiles « Haute Pression/Haute Température » des gisements profonds, ont fait l’objet de très peu d’études dans ces conditions. La pyrolyse du n-butylcyclohexane a été étudiée à haute pression (100 bar) dans des réacteurs fermés en tubes en or, entre 300°C et 425°C. Le n butylcyclohexane produit majoritairement des n-alcanes (C1 à C4), d’autres naphtènes et des composés aromatiques. Un modèle cinétique complexe a été développé (833 réactions essentiellement radicalaires) ; il rend bien compte de nos résultats expérimentaux jusque 60% de conversion. L’extrapolation du modèle dans les conditions géologiques (température initiale de 160°C, gradient thermique de 2°C/MA et temps de réaction en millions d’années), montre que ce composé commence à se décomposer vers 180°C et que son temps de demi-vie correspond à 200-210°C. D’autres systèmes réactionnels impliquant des naphtènes, ont été également étudiés à 400°C et 100 bar. L’étude de la pyrolyse de la n-butyldécaline montre que cette molécule bicylique possède une réactivité similaire à celle du n-butylcyclohexane. L’étude à 400°C du mélange binaire n-octane/n-butylcyclohexane ne met pas en évidence d’effet cinétique significatif du n-butylcyclohexane sur la décomposition thermique du n-octane, mais l’extrapolation du modèle aux conditions géologiques montre que les naphtènes inhibent la décomposition des n-alcanes à basse température (200°C) et à haute pression (100 bar) / This thesis aims at better understanding the thermal cracking reactions of naphthenes (saturated cyclic hydrocarbons), occurring in petroleum reservoirs. Naphthenes represent a significant fraction of “High Pressure/High Temperature” oils in deeply buried reservoirs; however, studies in these conditions are very limited in literature. The pyrolysis of n-butylcyclohexane is studied in a gold sealed tube reactor between 300 and 425°C, at 100 bar. n-Butylcylohexane mainly leads to n-alkanes (C1-C4), other naphthenes and aromatic compounds. A detailed model is developed (833 reactions, mainly free-radical reactions); a good agreement with our experimental results is reached up to a conversion of 60%. According to simulation results obtained by extrapolating to lower temperature, this compound starts to undergo thermal cracking at 180°C and its time of half-life corresponds to a temperature around 200-210°C, while considering the following burial scenario of an oil reservoir: initial temperature of 160°C and a heating rate of 2°C/MY (Million Years). Other chemical systems including naphthenes, are also studied at 400°C and 100 bar. The study of 1-n-butyldecaline shows, that this bicyclic compound and n-butylcyclohexane have a similar reactivity at 400°C. The study of the binary mixture n-butylcyclohexane/n-octane at 400°C does not provide any significant evidence of a kinetic effect of n-butylcyclohexane on the thermal decomposition of n-octane, but the extrapolation of our model at geological conditions shows that naphthenes are inhibitors of the decomposition of n-alkanes at low temperature (200°C) and at high pressure (100 bar)

A resistência de bovinos Bos taurus taurus a ambientes tropicais é menor quando comparada com a de Bos taurus indicus. Essa característica influencia os aspectos reprodutivos e vem sendo amplamente descrita na literatura. No que diz respeito aos touros, esta influência torna-se ainda mais significativa, visto que uma menor fertilidade destes impõe enorme impacto na produção animal. Vários são os estudos que comprovam o efeito negativo do estresse térmico sobre os parâmetros seminais de touros europeus provocando diminuição da fertilidade, porém a patogenia deste quadro não está confirmada. Uma hipótese para esta menor fertilidade seria um maior nível de estresse oxidativo nos touros europeus, no qual haveria um desequilíbrio entre a produção de espécies reativas ao oxigênio (EROs) e os níveis de proteção antioxidante. Para testar esta hipótese, foram utilizados 20 touros Simental (Bos taurus taurus) e 20 touros Nelore (Bos taurus indicus), criados a campo, na região de Dourados, Mato Grosso do Sul, submetidos a duas coletas anuais, durante o verão e inverno, no período de dois anos consecutivos. As amostras foram analisadas através da análise espermática padrão, dos níveis das enzimas antioxidantes, catalase e superóxido dismutase (SOD), e dos níveis de malondialdeído (MDA), produto da peroxidação lipídica, que foi utilizado como índice de estresse oxidativo. Os dados foram analisados pela análise de variância ONEWAY, levando em conta as classes raça (Nelore e Simental) e estação do ano (verão e inverno), através do programa SAS for Windows. As variáveis defeitos menores, defeitos totais e motilidade não apresentaram diferenças quanto a raça ou estação do ano. Observou-se efeito de estação do ano sobre as variáveis defeitos maiores (20,32±15,30% e 12,17±12,27%, verão e inverno respectivamente; p=0,02) e MDA (842,66±527,98 e 419,30±434,14 ng/ml, verão e inverno respectivamente; p=0,006), apenas no sêmen dos touros de raça Simental. As concentrações das enzimas antioxidantes SOD e catalase, não diferiram entre as raças e tampouco entre as estações do ano. Observou-se correlação entre MDA e formas anormais de cabeça (r=0,39, p=0,0035) e entre SOD e defeitos primários (r= - 0,31, p=0,0075). Os resultados obtidos sugerem que houve um maior nível de estresse oxidativo nos animais de origem européia criados a campo em clima tropical em relação aos animais de origem indiana, provavelmente relacionado a uma produção excessiva de EROs sem a devida compensação antioxidante. / Resistance to tropical environments is lower in Bos taurus taurus than in Bos taurus indicus cattle. This trait influences reproductive aspects, and is greatly discussed in publications worldwide. This negative influence is much more significant in bulls, since lower fertility in them highly impacts animal production. There are many studies demonstrating that exposure of European bulls to heat stress will negatively affect their seminal parameters, leading ultimately to lower fertility rates, but the pathogenic pathway has not been clarified. One hypothesis is that this lower fertility is due to higher levels of oxidative damage in these bulls, in which there would be an imbalance between reactive oxygen species (ROS) production and antioxidant capacity. In order to test this hypothesis, 20 Simmental bulls (Bos taurus taurus) and 20 Nelore bulls (Bos taurus indicus) kept under extensive breeding in the Dourados region, in Mato Grosso do Sul state were used. Semen collection occurred twice a year, during summer and winter, for a two year period. Samples were submitted to standard semen analysis and the levels of the antioxidant enzymes catalase and superoxide dismutase (SOD) dismutase were measured. To evaluate oxidative damage to the cells the levels of the lipid peroxidation product malondialdehyde (MDA) were measured. Data were analyzed for breed (Nelore or Simmental) and season (summer or winter) by the ONEWAY analysis of variance, using the SAS for Windows. Minor defects, total defects and motility did not differ in breed or in season. Season showed an effect on major defects (20.32±15.30% and 12.17±12.27%, summer and winter respectively; p=0.02) and MDA (842.66±527.98 and 419.30±434.14 ng/mL, summer and winter respectively; p=0.006), only in the Simmental animals. SOD and catalase levels did not differ between breeds or seasons. Correlations were observed between MDA and sperm head defects (r=0.39, p=0.0035) and between SOD and primary defects (r= -0.31, p=0.0075). Results suggest a higher level of oxidative stress in European bulls raised in field conditions, when compared to Indian bulls under the same conditions, and this is probably due to a higher ROS production, without a concomitant increase in antioxidant levels.

Melatonin, a nontoxic natural antioxidant, is of interest as a possible spin trap for use in spectroscopic methods to observe and identify short-lived free radicals, which have been linked to oxidative stress that may result in serious health problems. However, the reaction mechanisms for the oxidation of melatonin to form the product N1-acetyl-N2-formyl-5-methoxykynuramine are still not well understood. Computational quantum chemistry studies have been done on four proposed reaction mechanisms, involving the following major intermediate structures: a dioxetane, an epoxide, a melatonin radical cation, and a spin radical adduct. Molecular geometries were optimized at the DFT/B3LYP/cc-pVTZ level of theory, and single point energies were extrapolated to the complete basis set limit at the Hartree-Fock and second-order Møller-Plesset perturbation levels of theory using the cc-pVXZ (X = D, T, Q) basis sets. The lowest energy pathway was found to be the single electron transfer pathway, involving the melatonin radical cation intermediate.

Dans le cadre de cette thèse, nous avons étudié plusieurs processus radicalaires et en particulier l'addition de radicaux libres sur des systèmes insaturés tels que les alcènes et les hétérocycles aromatiques. Nous avons ainsi montré la réactivité unique des radicaux carbamoyles, issus de la décarboxylation des acides oxamiques. La carbamoylation "sans métal" photocatalysée des hétérocycles a donc été réalisée en présence d'un réactif iodé hypervalent, conduisant à des bases hétéroaromatiques fonctionnalisées, généralement avec de bons rendements. Le processus a été étendu aux acides oxamiques préparés à partir d'acides aminés homochiraux, la réaction se déroulant sans racémisation du substrat initial.Dans la recherche d'un groupe fonctionnel équivalent à un aldéhyde et compatible avec les groupes fonctionnels résidents, nous avons mis au point un nouveau procédé de carbo-cyanation des oléfines par voie radicalaire, permettant l'incorporation, sur un squelette oléfinique, d'un fragment portant un groupe électroattracteur et un groupe cyano. Des conditions photochimiques ont également été développées afin d'éviter l'utilisation d'amorçeurs coûteux tels que le di-tert-butyl hyponitrite (DTBHN). Cette réaction clé a ensuite été utilisée afin de construire, en seulement 4 étapes, un modèle du squelette bicyclique de l’alcaloïde leuconoxine.Enfin, dans la dernière partie de la thèse, nous avons décrit une synthèse simple de naphthalénones, portant un stéréocentre quaternaire benzylique tout carboné. La réaction entre un halogénure de phénacyle et un cyclopropène substitué se déroule sous photocatalyse en utilisant un catalyseur à l'iridium et de la lumière visible. Une carbo-arylation du cyclopropène est ainsi réalisée conduisant au cyclopropane correspondant, lequel s’ouvre dans les conditions de la réaction pour conduire à la naphthalénone souhaitée avec des rendements modérés, mais avec rétention de la chiralité du cyclopropène homochiral initial. / In the context of this thesis we studied several radical processes and in particular the addition of free-radicals onto unsaturated systems such as alkenes and aromatic heterocycles. We have thus shown the unique reactivity of carbamoyl radicals, issued from the decarboxylation of oxamic acids. Photocatalyzed “metal-free” carbamoylation of heterocycles was thus performed in the presence of an hypervalent iodine reagent, leading to -functionalized heteroaromatic bases, generally in good yields. The process was extended to oxamic acids prepared from homochiral amino acids and was shown to proceed without racemization.In the search for a functional group equivalent to an aldehyde and compatible with resident functional groups, we have devised a new free-radical olefin carbo-cyanation process, allowing the incorporation, on an olefin backbone, of a fragment bearing an electron-withdrawing group and a cyano group. Photochemical conditions have also been developed in order to avoid the use of costly initiators such as the di-tert-butyl hyponitrite (DTBHN). This key reaction was then used to construct, in only 4 steps, a bicyclic skeleton model for the alkaloid leuconoxine.Finally, in the last part of the thesis, we have described a straightforward synthesis of naphthalenones, bearing an all-carbon benzylic quaternary stereocenter. The reaction between a phenacyl halide and a substituted cyclopropene proceeds under photocatalysis using an iridium catalyst and visible light. A carbo-arylation of the cyclopropene is thus carried out leading to the corresponding cyclopropane, which is opened under the reaction conditions to afford the desired naphthalenone in moderate yields, with retention of the chirality of the starting homochiral cyclopropene.

Compared to normal cells, cancer cells tend to have higher concentrations of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) due to an accelerated cellular metabolism. The high ROS content leaves cancer cells increasingly susceptible to oxidative stress-induced cell death. This susceptibility can be manipulated in selective cancer therapy by further increasing production of ROS or inhibiting peroxide removal systems or a combination of the two. Pharmacological ascorbate (high-dose intravenous ascorbate) has been shown to sensitize pancreatic cancer to ionizing radiation (IR) by increasing production of ROS such as H2O2. Glutathione reductase (GR) and thioredoxin reductase (TrxR) are both important enzymes in peroxide removal systems. GR and TrxR function to recycle key electron donors in the cellular removal of H2O2. We hypothesized that inhibiting the peroxide removal systems via inhibition of GR and TrxR would enhance ascorbate-induced cytotoxicity in pancreatic cancer cells. Inhibition of TrxR activity enhanced ascorbate-induced cytotoxicity in MIA PaCa-2 pancreatic cancer cells. Additionally, knockdown of GR protein expression in combination with pharmacological ascorbate treatment increased MIA PaCa-2 pancreatic cancer cell sensitivity to IR. In MIA PaCa-2 and 403 F1 patient-derived pancreatic cancer cells, inhibition of both TrxR and GR activity combined with pharmacological ascorbate enhanced radiosensitivity. However, this effect was not seen in 339 patient-derived pancreatic cancer cells treated with the same dose of ascorbate. In conclusion, inhibition of TrxR activity, GR activity, or both enhances radiosensitivity and ascorbate-induced cytotoxicity in some, but not all, pancreatic cancer cell lines. Treatments combining ascorbate with inhibition of H2O2 removal may be an effective strategy for treatment of pancreatic adenocarcinoma.

A scientific and cultural revolution occurred with the sequencing of the human genome. The information provided by this accomplishment has provided tools for researchers to test new ideas in silico and on the bench. In redox biology many of the genes, transcripts, proteins, and redox active species have been well characterized. However, the vast majority have not been quantitated in an absolute manner. This is a necessary step to provide the tools for mathematical modeling and systems biology approaches for predicting changes in the cellular redox environment and the biochemical and biological consequences. Here we demonstrate techniques for the absolute quantitation of human catalase, glutathione peroxidase, peroxiredoxin, thioredoxin, and superoxide dismutase within cells. These techniques can be parsed into two groups: detection of activity and detection of total amount of species. Methods for the absolute quantitation of active catalase, peroxiredoxins, and superoxide dismutase have been developed by utilizing specific characteristics of each enzyme. Catalase generates oxygen in the presence of hydrogen peroxide that can easily be detected with a Clark electrode (oxygen monitor); the data are fit to a single-exponential to determine the observed pseudo-first-order rate constant. From this the effective number of fully active catalase enzymes in the sample can be determined. Peroxiredoxin in the disulfide state can be reduced by thioredoxin; thioredoxin from E. coli loses fluorescence upon oxidation. The loss of fluorescence over time is mathematically fit to a single-exponential to determine the observed pseudo first-order rate constant from which the number of active enzymes can be determined. Using an inhibition assay to detect superoxide dismutase activity along with the rate constants at which superoxide reacts with the dismutase and the competing superoxide-reacting-indicator-molecule, the concentration of active superoxide dismutase can be determined. To detect the total amount of protein of an enzyme in a biological sample, an immunoassay was first implemented. This method utilized Bio-Plex® beads from Bio-Rad; however, it was problematic because the antibodies applied did not perform satisfactorily not allowing sufficient signal-to-noise to be deployed. Quantitative mass spectrometry was then implemented to detect total catalase, glutathione peroxidase 1, peroxiredoxin 2, and thioredoxin 1 in human red blood cells. With the absolute concentration of these enzymes and proteins along with data for oxygen consumption rates and peroxisomal hydrogen peroxide concentration for several cell lines, we hypothesize that a reasonable model of hydrogen peroxide and superoxide flux can be constructed. Quantitative data such as these provide the foundation for the new redox biology of the 21st century. Presented here is a roadmap for the obligatory first steps to dissect quantitatively the cellular and tissue metabolic pathways and redox networks that are the basis of all of biology.

Nitric oxide is a small free radical generated by family of enzymes, the nitric oxide synthases. In a series of experiments performed over the last 15 years we showed that nitric oxide is induced by all three isoforms of nitric oxide synthase during tendon healing and that it plays a crucial beneficial role in restoring tendon function. In normal tendon we found very little nitric oxide synthase activity while in injured rat and human tendons nitric oxide synthase activity was expressed in healing fibroblasts in a temporal fashion. In healing rat Achilles tendon fibroblasts the first isoform to be expressed was endothelial nitric oxide synthase (eNOS), followed by inducible nitric oxide synthase (iNOS), and then brain or neuronal nitric oxide synthase (bNOS). Systemic inhibition of nitric oxide synthase activity decreased the cross sectional area and mechanical properties of the healing rodent Achilles tendons. Addition of nitric oxide via NO-flurbiprofen or NO-paracetamol enhanced rat Achilles tendon healing. Addition of nitric oxide to cultured human tendon cells via chemical means and via adenoviral transfection enhanced collagen synthesis, suggesting that one mechanism for the beneficial of nitric oxide on tendon healing might be via matrix synthesis. The final part of the work involved three randomized, double-blind clinical trials which evaluated the efficacy of nitric oxide donation via a patch in the management of the tendinopathy. In all three clinical trials there was a significant positive beneficial effect of nitric oxide donation to the clinical symptoms and function of patients with Achilles tendinopathy, tennis elbow and Achilles tendonitis.

Facile, one-step synthesis of self-assembling, cationic block copolymers of poly(2-N-(dimethylaminoethyl) methacrylate) (pDMAEMA) and PEO-PPO-PEO (Pluronic®) is developed. The copolymers are obtained via free-radical polymerization of DMAEMA initiated by Pluronic-radicals generated by cerium (IV). The copolymers possess surface activity, are polycationic at pH<7.1, and self-assemble into micelle-like aggregates when neutralized. Potential applications of the novel copolymers for DNA transfection in gene therapy are discussed. / Singapore-MIT Alliance (SMA)

M.S. / Biochemistry / The EPR spectrum of the Tyr-Cys· free radical in oxidized apoGAOX has been investigated, using a combination of approaches. Power saturation analysis has been used to resolve two unique spectra through Evolving Factor Analysis (EFA) global fitting, indicating the presence of two distinct free radical species in the sample. The component that dominates at low microwave power arises from the Tyr-Cys· side chain, while the high power component has not yet been assigned. The experimental results show that the EPR spectrum collected at low power includes approximately 7% of the high power component. EPR spectra have been collected for ten different isotope derivatives of GAOX, including ²H-labeled, ¹³C-labeled, 17[superscript]O-labeled, and ³³S-labeled forms. XSophe simulation of the EPR spectra has been performed for the isotopically labeled samples in order to determine the spectroscopic parameters - g-values, hyperfine coupling constants, and linewidths. The g-values and the methylene proton hyperfine coupling constants obtained for the isotopically labeled samples are consistent with the literature values. The magnitude of the hyperfine coupling constants associated with each of the nuclei confirms that significant electron spin density is found on the methylene protons, the alternating carbon atoms within the aromatic π system and the 2p[subscript]z orbital of both sulfur and oxygen. Moreover, the rotation angle of the methylene protons to the phenoxyl ring around the C1-C7 bond has been evaluated based on the experimentally defined hyperfine coupling constants of the two methylene protons.