Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2016-08-05T11:24:12Z No. of bitstreams: 2 Tese - Roberta Dias da Silva - 2014.pdf: 1832703 bytes, checksum: 26c93ff4297c9cfb09966a7e7c8c4b6a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-05T14:48:55Z (GMT) No. of bitstreams: 2 Tese - Roberta Dias da Silva - 2014.pdf: 1832703 bytes, checksum: 26c93ff4297c9cfb09966a7e7c8c4b6a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-05T14:48:55Z (GMT). No. of bitstreams: 2 Tese - Roberta Dias da Silva - 2014.pdf: 1832703 bytes, checksum: 26c93ff4297c9cfb09966a7e7c8c4b6a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-11-01 / Oxidative stress is related to the development of different pathological processes. Interventions that reduce the generation or the effects of free radicals have shown controversial results in animal models. In this study, we evaluated the effects of supplementation with different antioxidants through assessment of oxidative stress biomarkers in erythrocytes (thiobarbituric acid reactive substances, superoxide dismutase, glutathione full, glutathione peroxidase and catalase) in hepatocytes (malondialdehyde), clinical laboratory tests and histological exam. 40 Nelore bovines were sorted into five groups, as follows: one control without supplementation, three groups individually supplemented with vitamin E, selenium and zinc and one group supplemented with the combination of selenium and vitamin E. For 105 days, all animals were fed roughage (brachiaria hay) and concentrate (feed) in a 70:30 ratio. The Biomarkers of oxidative stress evaluation in erythrocytes did not indicate the presence of lipid peroxidation. In the liver, supplementation with antioxidant selenium and vitamin E reduced the number of foamy macrophages in the parenchyma, as well as tissue lipid peroxidation. The animals did not develop clinical liver abnormalities throughout the experimental period, since aspartate aminotransferase and gamma glutamyltransferase serum activity remained within normal range. There was positive correlation between the concentration of the biomarker malondialdehyde and foamy macrophages. In conclusion, feedlot cattle ingesting Brachiaria sp present lipid peroxidation in hepatocytes and the combination of the antioxidants selenium and vitamin E reduces the effects of oxidative stress. / O estresse oxidativo está relacionado ao desenvolvimento de diferentes processos patológicos. Intervenções que diminuem a geração ou os efeitos dos radicais livres têm apresentado resultados controversos em modelos animais. Neste estudo, avaliou-se os efeitos da suplementação com diferentes antioxidantes, por meio de avaliação de biomarcadores de estresse oxidativo em eritrócitos (substâncias reativas ao ácido tiobarbitúrico, superóxido dismutase, glutationa total, glutationa peroxidase e catalase), em hepatócitos (malondialdeído) e de exames laboratoriais e histológicos. Foram avaliados 40 bovinos da raça Nelore, distribuídos em cinco grupos, o controle sem suplementação, três grupos suplementados individualmente com vitamina E, selênio e, zinco e um grupo suplementado com a associação de selênio e vitamina E. Durante o período de 105 dias de confinamento experimental, os bovinos foram alimentados com volumoso (feno de braquiária) e concentrado (ração), na proporção de 70:30. A avaliação dos biomarcadores de estresse oxidativo nos eritrócitos não indicou presença de lipoperoxidação nos eritrócitos. Na avaliação de estresse oxidativo em fragmentos hepáticos, a suplementação com o antioxidante selênio associado à vitamina E reduziu as alterações histopatológicas (número de macrófagos espumosos) no parênquima hepático e a lipoperoxidação tecidual. No período do confinamento, os bovinos não desenvolveram alterações hepáticas clínicas e laboratoriais, apresentando atividade sérica de gama glutamiltransferase e aspartato aminotrasferase dentro dos valores de normalidade. Houve correlação positiva entre a concentração do biomarcador malondialdeído e a quantidade de macrófagos espumosos. Em conclusão, bovinos confinados ingerindo feno de Brachiaria sp apresentam lipoperoxidação de hepatócitos e a associação dos antioxidantes selênio e vitamina E reduz os efeitos do estresse oxidativo.

Ceriporiopsis subvermispora é um fungo filamentoso, muito seletivo na degradação de lignina e, por isso, tem sido uma das espécies mais estudadas no processo de biopolpação. A biopolpação consiste em um tratamento biológico da madeira que antecede etapas convencionais de polpação, proporcionando níveis de economia de energia elétrica no processo que podem atingir valores de 30 a 40%. Para degradar a lignina, esse fungo secreta a enzima manganês-peroxidase (MnP), a qual requer um ácido carboxílico para quelar e transportar íons Mn3+ oriundos do seu ciclo catalítico. O complexo quelante-Mn3+ degrada apenas frações fenólicas da lignina, porém pode também iniciar a peroxidação de lipídeos e com isso gerar radicais peroxila que apresentam capacidade oxidativa suficiente para degradar estruturas não-fenólicas da lignina. Com base nesses aspectos, o presente trabalho teve o objetivo de avaliar a degradação de lignina por reações que envolvem a peroxidação de ácido linoléico iniciadas por metabólitos extracelulares extraídos de cultivos de C. subvermispora. Também foram avaliados sistemas miméticos baseados nos íons Fe2+ e Mn3+ como iniciadores das mesmas reações. Essencialmente, foi estudada a degradação de lignina in vitro em reações iniciadas por sistemas compostos que incluíram, MnP/Mn+2/H2O2, Fe3+/agentes redutores de Fe3+ produzidos durante a biodegradação da madeira, íons Mn+3 ou íons Fe+2, todos adicionados ao ácido linoléico. Para realizar esse estudo foi necessário preparar, tanto MnP, quanto compostos redutores de Fe3+, em cultivos de C. subvermispora. Também foram preparados e caracterizados dois substratos para as reações em estudo. Esses substratos compreenderam um complexo lignina-carboidrato (CLC) e um modelo de um material lignocelulósico completo, porém moído e livrado de toda a fração de extrativos. Nos dois casos, o material de partida foi à madeira de Eucalyptus grandis. A caracterização química desses substratos indicou um teor de lignina de 44,8% e 29,0%, respectivamente. Reações de peroxidação de ácido linoléico iniciadas pelos sistemas em estudo mostraram que todos foram efetivos para esse propósito, sendo que as maiores taxas de consumo de oxigênio durante essas reações foram observadas nos meios reacionais que continham Fe2+ em solução. O CLC inibiu as reações de peroxidação quando adicionado ao meio reacional em concentraçãoes maiores do que 0,3 mg/mL. Entretanto, reações prolongadas por 72 h com o CLC numa concentração inicial de 1 mg/mL indicaram que ele sofreu despolimerização. As vias de degradação da lignina contida no CLC ou na madeira de E. grandis moída envolveram a despolimerização da molécula, ou simplesmente a oxidação das cadeias laterais e das estruturas fenólicas livres. Quando o sistema foi baseado na ação de MnP/Mn+2/H2O2/ácido linoléico, foram comprovadas vias de degradação da lignina que envolveram desde a simples oxidação do carbono-α até a quebra de ligações do tipo β-O-4 e/ou entre os carbonos α e β. Os resultados obtidos corroboraram dados anteriormente publicados para a ação de C. subvermispora in vivo. Uma exceção foi à observação da reação de simples oxidação do Cα nos sistemas in vitro, que havia sido descartada em trabalhos anteriores que se basearam na caracterização de lignina contida em madeira biotratada por C. subvermispora (sistema in vivo). Os resultados permitiram concluir que vários sistemas miméticos podem iniciar a peroxidação de ácido linoléico in vitro. Quando essas reações foram conduzidas na presença de lignina (CLC ou E. grandis moído) foi possível observar transformações importantes na estrutura da lignina que eventualmente poderiam ser exploradas, por exemplo, em etapas de processos de branqueamento de polpas kraft. / The white-rot fungus Ceriporiopsis subvermispora degrades lignin selectively, being one of the most studied species in biopulping. Biopulping consists of a biological treatment of wood that precedes conventional pulping stages. The process can provide up to 30-40% of energy savings in mechanical pulping. To degrade lignin, this fungus secretes the enzyme manganese-peroxidase (MnP), which needs carboxylic acids to chelate and transport Mn3+ ions formed in the catalytic cycle of the enzyme. The chelate-Mn3+ complex is able to degrade phenolic structures of lignin; however, can also initiate lipid peroxidation reactions generating peroxyl radicals that are able to degrade nonphenolic lignin structures. Based on this background, the aim of this work was to evaluate lignin degradation through linoleic acid peroxidation reactions initiated by extracellular metabolites recovered from C. subvermispora cultures. Some biomimetic systems based on Fe2+ and Mn3+ ions were also evaluated as initiators of such reactions. The lignin degradation was studied in reaction systems composed of MnP/Mn+2/H2O2, Fe3+-reducing compounds produced during wood biodegradation by C. subvermispora, Mn+3 or Fe+2 ions, all of them in the presence of linoleic acid. To perform this study, MnP and Fe3+-reducing compounds were initially produced in C. subvermispora cultures. Two different reaction substrates were also prepared. One was a lignin-carbohydrate complex (LCC) and, the other, was a complete lignocellulosic material that was milled and extracted to remove the extractive fraction. Both substrates were prepared from Eucalyptus grandis wood. The chemical characterization of the substrates showed 44.8 % and 29.0 % of total lignin, respectively. Linoleic acid peroxidation reactions initiated by the studied systems showed that all of them were efficient on this purpose. The highest oxygen consumption rates during these reactions were observed in the Fe2+ initiated reactions. The LCC inhibited the peroxidation reactions when added to the reaction medium at concentrations higher than 0.3 mg/mL. However, prolonging the reactions up to 72h with LCC at 1 mg/mL showed that it was depolymerized. The lignin degradation routes involved depolymerization or simple side chain and free-phenolic structure oxidations. When the reactive system was based on the use of MnP/Mn+2/H2O2/linoleic acid, some lignin degradation routes were demonstrated and they included Cα-oxidation, as well as β-O-4 and/or Cα-Cβ cleavages. These results corroborate previous findings published for the action of C. subvermispora in vivo. One exception was the simple Cα oxidation that was observed for the in vitro reactions, but was ruled out by previous works that were based on the characterization of residual lignins extracted from wood samples biotreated by C. subvermispora (in vivo system). The current results permitted to conclude that several mimetic systems were able to initiate linoleic acid peroxidation in vitro. When these reactions were performed in the presence of lignin (LCC or milled E. grandis) it was possible to show the occurrence of several lignin transformation reactions that could be exploited, for example, in pulp bleaching processes.

Vários adutos resultantes da reação de aldeídos α,β-insaturados, ou de seus epóxidos, com bases do DNA têm sido caracterizados nos últimos anos. Esses adutos podem levar à incorporação errada de bases durante a replicação ou a transcrição, resultando, se não reparados, em mutações que podem contribuir para a carcinogênese. O trans,trans-2,4-decadienal (DDE) é um dos aldeídos mais citotóxicos gerados endogenamente a partir da peroxidação lipídica. Verificamos que este aldeído afeta a viabilidade e altera o nível de glutationa de células CV1-P, além de induzir fragmentação do DNA e formação de diferentes produtos com o mesmo. Além da sua formação endógena, DDE é também encontrado em alguns alimentos, contribuindo para o aroma dos mesmos. Neste trabalho detectamos e isolamos 6 produtos formados a partir da reação de DDE com 2\' -desoxiguanosina (dG), sendo que fizemos a caracterização química completa de 3 desses produtos. Através desses estudos mostramos que um deles é o aduto 1,N2-eteno-2\' -desoxiguanosina (1,N2-εdGuo), também formado a partir da reação de dG com compostos carcinogênicos conhecidos, como o c1oreto de vinila. Os outros dois produtos são dois diastereoisômeros correspondentes a 1,N2-εdGuo com uma cadeia lateral substituinte. São produtos inéditos descritos pela primeira vez neste nosso trabalho. A detecção e caracterização desses produtos, assim como os mecanismos de fonnação propostos, podem contribuir para um melhor entendimento da genotoxicidade associada à peroxidação lipídica. Em paralelo desenvolvemos uma técnica muito sensível baseada em LC/ESI/MSMS para detecção desses adutos em sistemas biológicos. A detecção dos adutos caracterizados em DNA incubado com DDE in vitro e em DNA de células expostas ao aldeído aponta para a importância biológica desses compostos. Existe um crescente interesse no uso de adutos exocíclicos de DNA como marcadores de exposição a diversos carcinógenos ambientais e também a processos endógenos envolvendo a peroxidação lipídica. O mecanismo de formação e a estrutura química desses adutos são importantes para revelar as características estruturais queinfluenciam a alquilação do DNA por aldeídos insaturados e para que se possa estimar o papel desses aldeídos na carcinogênese. A formação dos adutos por produtos da peroxidação lipídica sugere a necessidade de estudos epidemiológicos para avaliar riscos de exposição ambiental, via alimentação ou mesmo via fatores indutores de lipoperoxidação. Os resultados aqui obtidos podem contribuir para o estabelecimento de relações entre estrutura química e função e para a avaliação do possível papel dessas lesões na toxicidade associada à exposição a essa classe de compostos. / Vários adutos resultantes da reação de aldeídos α,β-insaturados, ou de seus epóxidos, com bases do DNA têm sido caracterizados nos últimos anos. Esses adutos podem levar à incorporação errada de bases durante a replicação ou a transcrição, resultando, se não reparados, em mutações que podem contribuir para a carcinogênese. O trans,trans-2,4-decadienal (DDE) é um dos aldeídos mais citotóxicos gerados endogenamente a partir da peroxidação lipídica. Verificamos que este aldeído afeta a viabilidade e altera o nível de glutationa de células CV1-P, além de induzir fragmentação do DNA e formação de diferentes produtos com o mesmo. Além da sua formação endógena, DDE é também encontrado em alguns alimentos, contribuindo para o aroma dos mesmos. Neste trabalho detectamos e isolamos 6 produtos formados a partir da reação de DDE com 2\' -desoxiguanosina (dG), sendo que fizemos a caracterização química completa de 3 desses produtos. Através desses estudos mostramos que um deles é o aduto 1,N2-eteno-2\' -desoxiguanosina (1,N2-εdGuo), também formado a partir da reação de dG com compostos carcinogênicos conhecidos, como o c1oreto de vinila. Os outros dois produtos são dois diastereoisômeros correspondentes a 1,N2-εdGuo com uma cadeia lateral substituinte. São produtos inéditos descritos pela primeira vez neste nosso trabalho. A detecção e caracterização desses produtos, assim como os mecanismos de fonnação propostos, podem contribuir para um melhor entendimento da genotoxicidade associada à peroxidação lipídica. Em paralelo desenvolvemos uma técnica muito sensível baseada em LC/ESI/MSMS para detecção desses adutos em sistemas biológicos. A detecção dos adutos caracterizados em DNA incubado com DDE in vitro e em DNA de células expostas ao aldeído aponta para a importância biológica desses compostos. Existe um crescente interesse no uso de adutos exocíclicos de DNA como marcadores de exposição a diversos carcinógenos ambientais e também a processos endógenos envolvendo a peroxidação lipídica. O mecanismo de formação e a estrutura química desses adutos são importantes para revelar as características estruturais queinfluenciam a alquilação do DNA por aldeídos insaturados e para que se possa estimar o papel desses aldeídos na carcinogênese. A formação dos adutos por produtos da peroxidação lipídica sugere a necessidade de estudos epidemiológicos para avaliar riscos de exposição ambiental, via alimentação ou mesmo via fatores indutores de lipoperoxidação. Os resultados aqui obtidos podem contribuir para o estabelecimento de relações entre estrutura química e função e para a avaliação do possível papel dessas lesões na toxicidade associada à exposição a essa classe de compostos.

O fungo de podridão branca Ceriporiopsis subvermispora é seletivo na degradação de lignina em estágios curtos de colonização de madeira e dessa forma tem sido uma das espécies mais estudadas em biopolpação, que consiste em um tratamento biológico de cavacos de madeira que antecede etapas convencionais de polpação. Para degradar a lignina na parede celular vegetal esse fungo secreta a enzima manganês-peroxidase (MnP), a qual necessita de ácido oxálico para transportar o íon Mn3+ oriundo do seu ciclo catalítico. O complexo quelante-Mn3+ gerado degrada apenas porções fenólicas da lignina. Porém, por meio da peroxidação de lipídeos por Mn3+, radicais livres são gerados e apresentam potencial de oxidação suficiente para degradar estruturas não-fenólicas da lignina. Com base nesses aspectos, o presente trabalho avaliou a importância desses mediadores da MnP, ácido oxálico e lipídeos, no processo degradativo quanto à adição ou à supressão dessas substâncias nos cultivos. Em cultivos com madeira in natura ou extraídas com etanol (provavelmente isento de lipídeos) não ocorreu diferença significativa quanto à produção de metabólitos extracelulares (enzimas, ácido oxálico), à degradação de lignina e à atividade redutora de Fe3+ (envolvida na geração de radicais OH pela reação de Fenton). A formação de TBARS (substâncias que reagem com o ácido tiobarbitúrico), que serve como indício de reações de peroxidação de lipídeos, também foi semelhante, demonstrando que mesmo a partir de madeira livre de extrativos essas reações ocorrem. Com o avanço do biotratamento, a lignina foi despolimerizada por meio da diminuição de ligações ?-O-4, os teores de OH alifáticas e fenólicas foram diminuídos, enquanto o teor de grupos carboxila aumentou. Embora a degradação de celulose em madeira tenha sido baixa, cartões de holocelulose (livres de lignina) adicionados nesses cultivos foram despolimerizados. Nos cultivos em que houve a adição de uma fonte extra de lipídeos (óleo de soja), a produção de enzimas e degradação de lignina foram similares, enquanto a produção de ácido oxálico e TBARS foi estimulada por esse co-substrato. A maior concentração de óleo de soja adicionado (10,4 g/kg de madeira) fez com que a lignina presente na madeira residual apresentasse o mesmo teor de ligações ?-O-4 que o controle, enquanto maior degradação de OH alifáticas e menor formação de grupos carboxila foram observadas nessas ligninas. Em outro ciclo de cultivos, íons Ca2+ foram adicionados para precipitar o ácido oxálico produzido pelo fungo. Nos cultivos com a mais alta carga de cálcio (1400 mg/kg) houve diminuição na formação de ácido oxálico e consequentemente uma inibição na degradação de todos os componentes da madeira. Ao serem realizados cultivos com ácido oxálico exógeno, o fungo atuou de forma a igualar a concentração de ácido oxálico livre em relação a um cultivo sem esse suplemento, tanto por catabolismo quanto por precipitação desse ácido. Para as máximas cargas de cálcio, ácido oxálico e óleo de soja foram realizados outros cultivos sobre madeira em biorreatores para realização de ensaios de polpação. A polpação quimiotermomecânica sulfito alcalino dessas amostras de madeira biodegradada mostrou que os cultivos adicionados de Ca2+ e os não suplementados foram os que proporcionaram os maiores benefícios atribuídos ao biotratamento. Por outro lado, a adição de ácido oxálico anulou o benefício oriundo do biotratamento. A busca de correlações entre os níveis de metabólitos extracelulares com os benefícios do biotratamento para a polpação quimiotermomecânica não apresentaram tendências claras que indiquem a relevância de um metabólito em especial. Pelo contrário, aparentemente deve haver um compromisso entre todas as atividades extracelulares para que um determinado benefício seja obtido. / The white-rot fungus Ceriporiopsis subvermispora degrades lignin selectively during the initial stages of wood colonization and in this way it has been one of the most studied species in biopulping. This process consists of a biological treatment of wood chips that precedes conventional pulping stages. To degrade lignin in the plant cell wall this fungus secretes the enzyme manganese-peroxidase (MnP), which needs oxalic acid to transport the Mn3+ ion formed in the catalytic cycle of the enzyme. The oxalate-Mn3+ complex degrades only lignin phenolic portions. However, through lipid peroxidation intiated by Mn3+, free radicals are generated and they present enough oxidation potential to degrade nonphenolic lignin structures. With basis in these aspects, the present work evaluated the importance of these mediators of MnP, oxalic acid and lipids, in the degradative process either by their addition or suppression in wood-containing cultures. In cultivations with in natura wood or ethanol extracted wood (probably free of lipids) thre was no significant difference in the production of extracelular metabolites (enzymes, oxalic acid), or in the lignin degradation and the Fe3+-reducing activity (involved in the OH radicals generation by the Fenton´s reaction). The formation of TBARS (thiobarbituric acid reactive substances), that ii indicative of lipid peroxidation reactions, was also similar in both cultivations systems, demonstrating that even starting from extractives-free wood, these reactions can occur. With the progress of the biotreatment, the lignin was depolymerized through the decrease of ?-O-4 bonds, the contents of aliphatic and phenolic OH decreased, while carboxyl groups content increased. Although the cellulose degradation in wood has been low, holocellulose cards (free from lignin) added in these cultures were depolymerized. In the cultures where lipids were added (soy-bean oil), the enzyme production and lignin degradation were similar, while the oxalic acid and TBARS productions was stimulated by this co-substrate. With the highest concentration of soy-bean oil added (10,4 g/kg wood), the lignin in the residual wood presented the same content of ?-O-4 bonds as compared to the control, while higher degradation of aliphatic OH and lower formation of carboxyl groups were observed in these lignins. In another cycle of cultivations, Ca2+ ions were added to precipitate oxalic acid produced by the fungus. In the cultivations with the highest load of calcium (1400 mg/kg) there was a decrease in the oxalic acid formation and consequently an inhibition in the degradation of all the wood components. To the cultivations accomplished with exogenous oxalic acid, the fungus acted to equalize the concentration of free oxalic acid either by catabolism or by precipitation of this acid. For the highest loads of calcium, oxalic acid andsoy-bean oil, other cultivations were accomplished on 20 L-biorreactors to produce biotreated wood samples suitable for pulping experiments. The alkaline-sulfite chemitermomechanical pulping of these samples showed that the biotreated wood in Ca2+-ammended and nonsupplemented cultures were the ones that provided the highest benefits. On the other hand, the oxalic acid addition annulled the benefit originated from the biotreatment. The search for correlations among the levels of extracelular metabolites with the benefits of the biotreatment for the chemitermomechanical pulping did not present clear tendencies to indicate the relevance of a metabolite in special. On the contrary, probably there is a commitment among all of the extracellular activities, so that a certain benefit would be obtained.

The aquatic zooplankton crustacean Daphnia magna must be able to tolerate thermal stress in order to survive their native shallow ponds that are susceptible to drastic seasonal and diurnal temperature fluctuations as well as to globally increasing temperatures. Survival in such variable environments requires plastic responses that must include fundamental aspects of Daphnia biochemistry and physiology. Adaptive response to selection favoring such plastic phenotypes requires the presence of genetic variation for plastic response in natural populations. Adverse effects of elevated temperature on aquatic organisms are diverse and so are their plastic responses; among the most severe challenges aquatic organisms face when exposed to heat is the elevated oxidative stress. In this work we focused on short-term and long-term responses of Daphnia to temperature changes that increase its resistance to oxidative stress. Daphnia acclimated to stressful but non-lethal temperature (28ºC) show longer survive during exposure to a lethal temperature (37ºC) than those acclimated to the optimal temperature (18ºC). Short-term reciprocal switches between 18ºC and 28ºC result in intermediate temperature tolerance. These changes are accompanied by mirroring changes in total antioxidant capacity indicating the increased antioxidant capacity as a possible causative mechanism for heat tolerance gained from acclimation. The analysis of 6 geographically distinct genotypes representing a range of temperature tolerance levels shows a genetic difference in response to short-term and long-term acclimation as well as in the effect of antioxidant capacity on temperature tolerance. These results indicate a significant degree of local adaptation in heat and oxidative stress defenses in Daphnia and provide a better understanding of adaptive responses of this zooplankton crustacean to rising temperatures.

<p>Oxidative stress can be defined as a state of disturbance in the pro-oxidant/antioxidant balance in favour of the former, leading to potential damage. Processes associated with oxidative stress involve reactive oxygen species and radicals and can result in elevated levels of oxidatively modified or toxic molecules that can cause cellular malfunction, and even cell death. Destruction of membrane lipids, lipid peroxidation, caused by reactive oxygen species and radicals has been coupled to many diseases and also normal ageing. </p><p>The measurement of low molecular weight biomarkers of oxidative stress present in complex matrices such as brain tissue, plasma, urine or cerebrospinal fluid is a delicate and difficult task and there is a need for improved analytical tools in this field of research. </p><p>The major foci of this thesis and the work underlying it are the development of analytical techniques and methods for determining biomarkers for oxidative stress and lipid peroxidation. Aspects of particular concern include the effects of sample treatments prior to analysis, evaluation of the developed methods with respect to possible artefacts, and the scope for results to be misinterpreted. The specific research goals and issues addressed are detailed in five papers, which this thesis is based upon.</p><p><b>Paper I</b> focuses on malondialdehyde, describing and evaluating two new simplified sample pre-treatment regimes for the determination of malondialdehyde in rat brain tissue by capillary electrophoresis with UV detection. The effects of sample storing and handling are also considered.</p><p><b>Paper II</b> describes the synthesis, characterization and implementation of a new internal standard for the determination of malondialdehyde in biological samples using electrophoretic or chromatographic separation techniques. The usefulness of the internal standard is demonstrated in analyses of rat brain tissue samples.</p><p><b>Paper III</b> presents a method for the determination of 4-hydroxynon-2-enal in brain tissue from rats employing micellar electrokinetic chromatography separation and laser-induced fluorescence detection. </p><p><b>Paper IV</b> is focused on the development of a new methodology for determining the stereoisomeric F2-isoprostanes in human urine samples employing chromatographic separation on porous graphitic carbon and detection by electrospray ionization-tandem mass spectrometry. The results from this study conflict with the hypothesis that peripheral isoprostanes are elevated in patients with Alzheimer’s disease.</p><p><b>Paper V</b> describes porous graphitic carbon chromatography-tandem mass spectrometry for the determination of isoprostanes in human cerebrospinal fluid. A new simplified sample pre-treatment regime, involving a column switching technique, is presented that allows direct injection of a relatively large volume of CSF into the chromatographic system.</p>

Oxidative stress can be defined as a state of disturbance in the pro-oxidant/antioxidant balance in favour of the former, leading to potential damage. Processes associated with oxidative stress involve reactive oxygen species and radicals and can result in elevated levels of oxidatively modified or toxic molecules that can cause cellular malfunction, and even cell death. Destruction of membrane lipids, lipid peroxidation, caused by reactive oxygen species and radicals has been coupled to many diseases and also normal ageing. The measurement of low molecular weight biomarkers of oxidative stress present in complex matrices such as brain tissue, plasma, urine or cerebrospinal fluid is a delicate and difficult task and there is a need for improved analytical tools in this field of research. The major foci of this thesis and the work underlying it are the development of analytical techniques and methods for determining biomarkers for oxidative stress and lipid peroxidation. Aspects of particular concern include the effects of sample treatments prior to analysis, evaluation of the developed methods with respect to possible artefacts, and the scope for results to be misinterpreted. The specific research goals and issues addressed are detailed in five papers, which this thesis is based upon. <b>Paper I</b> focuses on malondialdehyde, describing and evaluating two new simplified sample pre-treatment regimes for the determination of malondialdehyde in rat brain tissue by capillary electrophoresis with UV detection. The effects of sample storing and handling are also considered. <b>Paper II</b> describes the synthesis, characterization and implementation of a new internal standard for the determination of malondialdehyde in biological samples using electrophoretic or chromatographic separation techniques. The usefulness of the internal standard is demonstrated in analyses of rat brain tissue samples. <b>Paper III</b> presents a method for the determination of 4-hydroxynon-2-enal in brain tissue from rats employing micellar electrokinetic chromatography separation and laser-induced fluorescence detection. <b>Paper IV</b> is focused on the development of a new methodology for determining the stereoisomeric F2-isoprostanes in human urine samples employing chromatographic separation on porous graphitic carbon and detection by electrospray ionization-tandem mass spectrometry. The results from this study conflict with the hypothesis that peripheral isoprostanes are elevated in patients with Alzheimer’s disease. <b>Paper V</b> describes porous graphitic carbon chromatography-tandem mass spectrometry for the determination of isoprostanes in human cerebrospinal fluid. A new simplified sample pre-treatment regime, involving a column switching technique, is presented that allows direct injection of a relatively large volume of CSF into the chromatographic system.

Intramyocellular lipid (IMCL), an ectopic fat depot found within skeletal muscle fibers, is highly associated with obesity and strongly correlated with insulin resistance. IMCL accumulation in sedentary individuals may contribute to insulin resistance by interfering with insulin signaling in skeletal muscle, leading to inadequate glucose uptake by the cell. Lipid peroxidation is also associated with both obesity and insulin resistance, and with IMCL, but a relationship has yet to be established among all of these variables. The purpose of this project is to study for the first time the relationships among lipid peroxidation, IMCL content, and glucose uptake in skeletal muscle. Nine insulin-sensitive adults (IS), 13 insulin-resistant adults (IR), 10 diabetic (DB) and 8 subjects pre- and post- 12-week intervention with insulin-sensitizing thiazolinedione (TZD) were assessed for soleus IMCL with nuclear magnetic resonance, insulin sensitivity by both hyperinsulinemic-euglycemic clamp (GDR) and homeostasis model assessment index (HOMA1), and anthropometrics, including body mass index (BMI), percent fat by DEXA scan, and waist circumference. Vastus lateralis biopsies of all subjects were homogenized and analyzed by immunoblotting for post-translational protein modifications occurring from lipid-peroxidation (HNE). GDR and HOMA were significantly different among IS, IR, and DB groups, as expected, as were waist circumference and BMI. IMCL was significantly higher in DB than in IS and IR. HNE was also higher in DB than in IS, although it did not differ from IR. HNE was significantly correlated to GDR, HOMA1, and BMI, but not to IMCL, WAIST, or percent fat measures. IMCL showed a strong, negative correlation with GDR and was the primary, independent predictor of GDR in stepwise multiple regression. HNE was the primary, independent predictor of HOMA in stepwise multiple regression. Paired t-tests revealed improvements in insulin sensitivity measures after 12 weeks of TZD intervention, but no significant differences were observed in IMCL or HNE after intervention. These data show that skeletal muscle HNE and IMCL are both determinants of insulin resistance in obese, sedentary adults. HNE and IMCL are not related and therefore impact insulin resistance independently. These results reveal, for the first time, a negative relationship between skeletal muscle HNE and insulin sensitivity in sedentary individuals and underscore the importance of lipid peroxidation in insulin resistance.

Oxidative stress, caused by increased levels of reactive oxygen species (ROS)and reactive nitrogen species (RNS) and/or a decrease in antioxidant capacity, can result in the oxidation of various bio-molecules, such as proteins, lipids and deoxyribonucleic acid (DNA). These oxidized bio-molecules may contribute to pathologies such as cardiovascular diseases, neurodegenerative disorders and cancer. The Sympathetic Activity and Ambulatory Blood Pressure in Africans (SABPA) study was initiated in 2008 to investigate the coping styles and catecholamine metabolic markers of Africans, contributing to their higher sympathetic output and poorer psychosocial wellbeing. This study forms part of the SABPA study, but with a specific aim to investigated lipid and protein oxidation markers in hypertensive Africans versus their normotensive counterparts. Analytical methods for the quantification of specific lipid and protein oxidation markers were optimized and validated. Urine samples from 172 urbanized black South Africans were collected and 3-nitrotyrosine (3NT) and thiobarbituric acid reactive substances (TBARS) were quantified in these samples, using the optimized spectrophotometric and LC-MS/MS methods. Statistical analyses showed that in both males and females, TBARS and 3NTcorrelated with each other. In males, 3NT also correlated with physical activity level (PAL) and C-reactive protein (CRP), while TBARS also correlated with body mass index (BMI). In females 3NT correlated with BMI, while TBARS correlates with PAL. These correlations meant that they could influence the calculations of the true effect of 3NT and TBARS levels between normotensive and hypertensive subjects. After analyses of covariance (ANCOVA) analyses it was determined that the hypertensive male subjects had higher TBARS values than the normotensive male subjects did (p-value = 0.03) and the normotensive female subjects had higher 3NT levels compared to the hypertensive female subjects (p-value = 0.04). These results partially supported the hypothesis that that elevated concentrations of specific urinary lipid and protein oxidation markers will be observed in the hypertensive test subjects compared to their normotensive counterparts. The results also indicated that there were indeed a difference in lipid and protein oxidation between hypertensive and normotensive subject. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013

Oxidative stress, caused by increased levels of reactive oxygen species (ROS)and reactive nitrogen species (RNS) and/or a decrease in antioxidant capacity, can result in the oxidation of various bio-molecules, such as proteins, lipids and deoxyribonucleic acid (DNA). These oxidized bio-molecules may contribute to pathologies such as cardiovascular diseases, neurodegenerative disorders and cancer. The Sympathetic Activity and Ambulatory Blood Pressure in Africans (SABPA) study was initiated in 2008 to investigate the coping styles and catecholamine metabolic markers of Africans, contributing to their higher sympathetic output and poorer psychosocial wellbeing. This study forms part of the SABPA study, but with a specific aim to investigated lipid and protein oxidation markers in hypertensive Africans versus their normotensive counterparts. Analytical methods for the quantification of specific lipid and protein oxidation markers were optimized and validated. Urine samples from 172 urbanized black South Africans were collected and 3-nitrotyrosine (3NT) and thiobarbituric acid reactive substances (TBARS) were quantified in these samples, using the optimized spectrophotometric and LC-MS/MS methods. Statistical analyses showed that in both males and females, TBARS and 3NTcorrelated with each other. In males, 3NT also correlated with physical activity level (PAL) and C-reactive protein (CRP), while TBARS also correlated with body mass index (BMI). In females 3NT correlated with BMI, while TBARS correlates with PAL. These correlations meant that they could influence the calculations of the true effect of 3NT and TBARS levels between normotensive and hypertensive subjects. After analyses of covariance (ANCOVA) analyses it was determined that the hypertensive male subjects had higher TBARS values than the normotensive male subjects did (p-value = 0.03) and the normotensive female subjects had higher 3NT levels compared to the hypertensive female subjects (p-value = 0.04). These results partially supported the hypothesis that that elevated concentrations of specific urinary lipid and protein oxidation markers will be observed in the hypertensive test subjects compared to their normotensive counterparts. The results also indicated that there were indeed a difference in lipid and protein oxidation between hypertensive and normotensive subject. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013