CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Novos biomarcadores da funÃÃo renal estÃo sendo estudados com o propÃsito de detectar precocemente alteraÃÃes renais em portadores de AF, dentre eles encontra-se a proteÃna quimiotÃtica de monÃcitos 1 (MCP-1), uma quimiocina de monÃcitos e macrÃfagos, produzida por cÃlulas do sistema renal em resposta ao processo de isquemia-reperfusÃo. OBJETIVO: Avaliar o uso de MCP-1 como biomarcador de lesÃo renal precoce em pacientes adultos com anemia falciforme em uso ou nÃo de hidroxiureia (HU). METODOLOGIA: Participaram do estudo 50 pacientes: 30 em uso de (HU)-grupo SSHU e 20 sem HU-grupo SS. Um grupo controle foi composto por 20 indivÃduos com HbAA, sem complicaÃÃes renais. ProteinÃria, albuminÃria, creatinina e urÃia urinÃrias, marcadores do estresse oxidativo como MDA e NOx foram determinados por mÃtodos espectrofotomÃtricos. MCP-1 urinÃrio foi detectado por enzima imunoensaio (ELISA). Os dados clÃnicos e de hemograma, creatinina e ureia sÃricas foram retirados do prontuÃrio mÃdico. Foi coletada a primeira urina do dia. O programa Graph Pad Prism 5.0 foi utilizado para anÃlise estatÃstica. A comparaÃÃo das mÃdias entre os grupos foi realizada atravÃs do teste t de Student e anÃlise de variÃncia (ANOVA). RESULTADOS E DISCUSSÃO: Albumina urinÃria esteve maior nos pacientes em relaÃÃo ao grupo controle (Controle-3.12 Â 4.35; SSHU- 11.85 Â 9.16; SS- 14.13 Â 12.22; p <0.0001). A taxa de filtraÃÃo glomerular estimada apresentou-se significantemente menor no grupo controle (Controle- 95.9 Â 19.92; SSHU- 137.9 Â 40.7 e SS- 140.1 Â 53.9; p= 0.0024). Observaram-se nÃveis elevados de MCP-1 (Controle- 42.12 Â 27.6; grupo SSHU- 166.2 Â 88.37 e grupo SS- 219.7 Â 115.0; p<0.001; p=0.039); MDA (Controle- 2.29 Â 1.13; grupo SSHU-5.25 Â 2.33 e grupo SS- 6.93 Â 2.12; p<0.0001;p=0.006) e NOx (Controle-2.25Â1.9; grupo SSHU-56.54 Â 9.15 e grupo SS 39.12 Â9.02; p<0.0001; p=0.001) nos pacientes em comparaÃÃo aos controles saudÃveis, e mais elevados no grupo SS em relaÃÃo ao grupo SSHU. Os pacientes com haplÃtipo Bantu/Bantu apresentaram maior concentraÃÃo de MCP-1, independente do uso de HU, seguido de Bantu/ Benin e Benin/Benin (p=0.01). Observou-se correlaÃÃo positiva entre os uma correlaÃÃo entre os nÃveis de MCP-1 e contagem de monÃcitos (p=0.004; r= 0.42); proteinÃria (p=0.002; r=0.43); albuminÃria (p=0.0004; r=0.47); TFG (p=0.02; r=0.32); MDA (p=0.02; r=0.32) e NOx (p=0.007; r= 0.38). CONCLUSÃO: Os resultados indicam que MCP-1 foi preditivo na detecÃÃo de alteraÃÃo renal, e que pode estar correlacionado ao dano causado pelo estresso oxidativo nos rins, evidenciado pelos altos nÃveis de MDA. Ainda, a HU parece ter reduzido o dano renal, visto que os pacientes em uso do fÃrmaco apresentaram nÃveis reduzidos desses parÃmetros.

Ácidos graxos polinsaturados Omega-3 (n-3 PUFA) tais como o ácido eicosapentaenóico (C20:5 n-3, EPA) e docosahexaenóico (C22:6 n-3, DHA) reduzem a concentração plasmática de triacilgliceróis em humanos. Entretanto, uma alta proporção desses ácidos graxos na dieta poderia favorecer a susceptibilidade das células à peroxidação, aumentando o risco de desenvolvimento de doenças cardiovasculares. Embora modelos animais não sejam recomendados para avaliar o efeito de n-3 PUFA nas lipoproteínas plasmáticas, estes têm sido amplamente utilizados como modelo para avaliação de dano oxidativo. Diferenças nos procedimentos metodológicos também têm gerado dificuldade na comparação de resultados. Desta forma, o objetivo deste estudo foi aplicar os mesmos procedimentos metodológicos para comparar o efeito da suplementação de n-3 PUFA nos biomarcadores plasmáticos de estresse oxidativo utilizando um modelo humano e um modelo animal. Indivíduos foram aleatoriamente distribuídos em dois grupos num delineamento paralelo duplo cego e receberam uma suplementação de 460,0 mg/dia de n-3 PUFA (OMEGA) contendo 240,0 mg de EPA + 160,0 mg de DHA + 60,0 mg de outros n-3 PUFAs, ou óleo de soja (PLACEBO) durante 6 semanas. Ratos Wistar também foram distribuídos em dois grupos e receberam uma dieta contendo 192,5 mg/dia de n-3 PUFA (FO) sendo 116,3 mg de EPA + 61,5 mg de DHA + 14,7 mg de outros n-3 PUFAs ou óleo de soja (SO) durante 3 semanas. Indivíduos do grupo OMEGA apresentaram maior concentração de malondialdeído (MDA) no plasma medido por TBARS quando comparado aos respectivos valores no baseline. A suplementação com n-3 PUFA não alterou a concentração plasmática de &#945;-tocoferol e a atividade antioxidante determinada pelo método DPPH. Apesar dos animais terem recebido doses 10 vezes maiores de n-3 PUFA (2,9 mg/kcal) quando comparadas aos humanos (0,3 mg/kcal) não foram observadas alterações entre os grupos FO e SO para as concentrações de MDA no plasma e no homogenato de cérebro. Em resumo, pode-se sugerir que o modelo animal usado neste estudo parece não ser o mais adequado para avaliar o estresse oxidativo após intervenções dietéticas com n-3 PUFAs em função de diferenças no metabolismo e nos mecanismos de proteção antioxidante observados entre os dois modelos. / Omega-3 polyunsaturated fatty acids (n-3 PUFA) such as eicosapentaenoic (C20:5 n-3, EPA) and docosahexaenoic (C22:6 n-3, DHA) reduce plasma triacylglycerol concentration in humans. However, higher proportion of these fatty acids in the diet could raise cells lipoperoxidation susceptibility, increasing the cardiovascular disease risk. Although animal models are not recommended to evaluate the effect of n-3 PUFA in plasma lipoproteins, they have been widely used as model for oxidative damage. Difference in methodological proceedings has also caused difficulties to compare data among assays. Thus, the objective of this study was to apply the same methodology to investigate the effect of n-3 PUFA supplementation on oxidative biomarkers in animal and human model. Individuals were randomly assigned in two groups in a parallel double blind design and received a supplement of 460.0 mg/day n-3 PUFA (OMEGA) containing 240.0 mg EPA + 160.0 mg DHA + 60.0 mg other n-3 PUFAs, or soybean oil (PLACEBO) during 6 weeks. Wistar rats were also assigned in two groups and received a diet containing 192.5 mg/day n-3 PUFA (FO) containing 116.3 mg EPA + 61.5 mg DHA + 14.7 mg other n-3 PUFAs or soybean oil (SO) for 3 weeks. Individuals in OMEGA group showed higher malondialdehyde (MDA) concentration in plasma measured by TBARS when compared to their baseline values. N-3 PUFA supplementation did not change plasma &#945;-tocopherol concentration and antioxidant activity determined by DPPH method. Although animals have received a 10-fold higher dose of n-3 PUFA (2.9 mg/ kcal) than humans (0.3 mg/kcal), no alteration was observed between FO and SO groups for plasma and brain homogenate MDA concentration. In summary, it can be suggested that the model used in this study doesn\'t seem appropriate to evaluate oxidative stress after dietetic interventions with n-3 PUFA due to physiological differences involved in lipid metabolism and antioxidant protection observed between both models.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2015-11-26T08:33:48Z No. of bitstreams: 2 Dissertação - Marina alves Coelho Silva - 2015.pdf: 1699881 bytes, checksum: 12e78126a8f1072850d257b422de3155 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-11-27T07:19:50Z (GMT) No. of bitstreams: 2 Dissertação - Marina alves Coelho Silva - 2015.pdf: 1699881 bytes, checksum: 12e78126a8f1072850d257b422de3155 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-11-27T07:19:50Z (GMT). No. of bitstreams: 2 Dissertação - Marina alves Coelho Silva - 2015.pdf: 1699881 bytes, checksum: 12e78126a8f1072850d257b422de3155 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-04-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Was evaluated the hepatoprotective effect of hydro alcoholic extract of the agroindustrial waste of jaboticaba-paulista (HEJB (Myrciaria cauliflora O. Berg, Myrtaceae) and ethanolic extract of breadfruit leaves (EEBL) (Artocarpus altilis (Park.) Fosberg, Moraceae) in experimental model of hepatotoxicity carbon tetrachloride (CCl4). The jaboticaba tree is native to Brazil and largely grown. It has a potential antioxidant, with phenolic compounds present primarily in the peel of fruit, which is a waste from the manufacturing process jaboticaba wine. Breadfruit is originating to the Pacific Islands and is distributed throughout the world. Fruits and leaves of this plant species have compounds with pharmacological properties, such as flavonoids. Were used Swiss mice, male, divided in eight groups, with five being the control groups (I: Baseline, received no treatment; II: olive oil, 10 mL/kg, i.p.; III: Propylene glycol 50%, 10 ml/kg, v.o. IV: 0.3% CCl4 in olive oil 10 mL/kg, i.p., negative control; V: Silymarin 200 mg/kg, v.o., positive control) and four treated groups, v.o. (VI: HEJB 250 mg/kg, VII: HEJB 500 mg/kg, VIII: EEBL: 250 mg/kg). Except groups I and II, all others received 0.3% CCl4 in olive oil on the 7th day of treatment, 2 hours after administration v.o. Were monitored the weight gain and no had significant difference between groups. At the end of the treatments, blood and liver of the animals were removed for biochemical analysis, after the animals were submitted to euthanasia and macroscopic evaluation of organs and cavities. The potential hepatoprotective and antioxidant activity of plant extracts were observed through the hepatic enzyme L-alanine aminotransferase (ALT), L-aspartate aminotransferase (AST), glutathione peroxidase (GPx ), glutathione reductase (GR) and catalase (CAT) and the dosage of malondialdehyde (MDA) - which required validation analytical HPLC-PDA. The bioanalytical method has a linear, selective, accurate, precise, and without interfering with LOQ 0.5 nmol/mL and LOD of 0.25 nmol/ml, suitable for the dosage of MDA in plasma and liver. There was a decrease of MDA in liver tissue, for the two extracts, as well as decreased levels of AST / ALT and GR to post-treatment with EHJB. The agroindustrial waste of jaboticaba fruit peel and the ethanolic extract of breadfruit leaves showed antioxidant activity in vivo. / Estudou-se o efeito hepatoprotetor do extrato hidroalcólico do resíduo agroindustrial de Jabuticaba-paulista (EHJB) (Myrciaria cauliflora O. Berg, Myrtaceae) e do extrato etanólico das folhas de fruta-pão (EEFP) (Artocarpus altilis (Park.). Fosberg, Moraceae) em modelo experimental de hepatotoxicidade por tetracloreto de carbono (CCl4). A jabuticabeira é planta frutífera, nativa do Brasil e muito cultivada. Possui um potencial antioxidante, com compostos fenólicos presentes principalmente na casca dos frutos, que é um dos resíduos do processo de fabricação do vinho de jabuticaba. A fruta-pão é originária das Ilhas do Pacífico e se encontra distribuída por todo o mundo. Frutos e folhas, dessa espécie vegetal, possuem compostos com propriedades farmacológicas, tais como flavonóides. Utilizaram-se camundongos Swiss, machos, divididos em 8 grupos, sendo 5 grupos controles (I: Basal, não recebeu tratamento; II: Azeite, 10 mL/kg, i.p.; III: Propilenoglicol 50%, 10 mL/kg,v.o. IV: CCl4 0,3% em azeite, 10 mL/kg,i.p., controle negativo; V: Silimarina 200 mg/kg, v.o., controle positivo) e quatro grupos tratados, v.o. (VI: EHJB 250 mg/kg; VII: EHJB 500 mg/kg; VIII: EEFP: 250 mg/kg). Exceto os grupos I e II, todos os outros receberam CCl4 0,3% em azeite, no 7º dia de tratamento, 2 horas após as administrações, v.o. Monitorou-se a evolução ponderal e não houve diferença significativa entre os grupos. Ao final dos tratamentos, sangue e fígado dos animais foram colhidos para análise bioquímica, posteriormente estes foram submetidos a eutanásia e avaliação macroscópica dos órgãos e cavidades. O potencial hepatoprotetor e a atividade antioxidante dos extratos vegetais foram observados através da dosagem das enzimas hepáticas L-alanina aminotransferase (ALT), L-aspartato aminotransferase (AST), glutationa peroxidase (GPx), glutationa redutase (GR) e catalase (CAT), assim como da dosagem de malondialdeído (MDA) – que necessitou de validação analítica em HPLC-PDA. O método bioanalítico apresentou-se linear, seletivo, exato, preciso e sem interferentes, com LIQ de 0,5 nmol/mL e LD de 0,25 nmol/mL, adequados para a dosagem de MDA no plasma e no fígado. Houve redução de MDA no tecido hepático, para os dois extratos, assim como houve redução dos níveis de AST/ALT e GR para EHJB pós-tratamento. Os resíduos agroindustriais de cascas de frutos de jabuticaba e o extrato etanólico de folhas de fruta-pão apresentaram atividade antioxidante in vivo.

Malondialdehyde (MDA) is the commonly accepted biomarker of lipid peroxidation. We reported the surface-enhanced Raman (SERS) detection of MDA using Thiobarbituric acid (TBA) as a molecular probe. The lowest concentration of HPLC purified TBA-MDA adduct that can be determined with reasonable signal to noise ratio is 0.45 nM. The specificity of the SERS technique has been demonstrated by comparing the SERS spectrum of TBA-MDA adduct with other TBA-aldehyde adducts. As a small organosulfur compound, TBA exhibits tremendous structural complexity. Discussed in this thesis is the drastic pH and concentration dependence of TBA SERS spectral features. To understand the origins of the TBA SERS spectral variations, UV-Vis spectra of TBA were also acquired under same experimental conditions of SERS. Density function calculations (DFT) were performed for different TBA tautomers with different charge states to facilitate the SERS spectral interpretation, allowing us to speculate the type of tautomers dominating the nanoparticle surfaces.

This thesis deals with optimization and application of assays for determination of ecotoxicity of nanomaterials based on nanoscale zero-valent iron (nZVI), which are used in remedial technologies. After in situ application of nZVI, a significant decrease in toxicity of polluted environment was detected; however, a potential negative effect of nanoparticles has not been sufficiently investigated yet. Standard used tests were found to be incompatible with nZVI for toxicity determination. Specific characteristics of nZVI, such as high reactivity and sorption, complicate determining the toxicity by routinely used ecotoxicity tests. Concentration ranging from 0,1 to 10 g/l that are used in practise for decontamination were tested. These concentrations resulted in formation of turbidity, which prevented the use of standard tests. In this work, a new method has been optimized for in vitro toxicity testing of nZVI and derived nanomaterials using bacteria. The principle of this assay is determination of oxidative stress (OS). The disbalance between formation and degradation of reactive oxygen species (i.e. OS) leads to irreversible changes in biomolecules of organisms and formation of undesirable products. A toxic and mutagenic product - malondialdehyde (MDA) is formed during lipid peroxidation and it is a...

Made available in DSpace on 2017-07-21T19:59:45Z (GMT). No. of bitstreams: 1 Fernanda Gravina.pdf: 2149838 bytes, checksum: e6a1a4c198175fce83c0ce2c98c81a23 (MD5) Previous issue date: 2015-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Microorganisms are essential components in the maintenance of biogeochemical cycles and ecosystems. In agricultural environments, continuous use of herbicides to minimize the loss in productivity could damage growth inibition of micro-organisms. One cause of this inhibition is increased production of ROS (reactive oxygen species), such as superoxide radicals (O2 -) and hydroxyl (OH-) and hydrogen peroxide (H2O2), causing oxidative stress. The magnitude of this stress can be conditioned by the chemical group and mode of action of the herbicide. The cell responses against ROS involve an increase in the expression of enzymes such as superoxide dismutase and catalase, responsible for the dismutation of O2- and H2O2, respectively. The objective of this study was to evaluate the relationship between the effect of herbicides with different modes of action and answers of superoxide dismutase (SOD) isoenzymes in the bacterium Escherichia coli K-12 bacteria. For this, 2,4-D, paraquat and atrazine herbicides were used, and knockout strains of E. coli K-12 strains carrying mutations in the gene encoding Mn-SOD (sodA) and Fe-SOD (sodB). Herbicides were shown to be capable of promoting the imbalance of redox potential, increasing the production of H2O2 and malondialdehyde (MDA) above the rates observed in controls and differently between strains. Exposed to paraquat, which indices in vivo redox cycling, strains of E. coli wt and ΔsodB showed increased production of H2O2, and increase in the Mn-SOD activity, probably as a result from activation of the SoxR, which promotes the transcription of the gene sodA. In ΔsodA, the toxicity rates with paraquat were not higher than the control, indicating a possible regulation of the Fe-SOD expression by OxyR transcriptional factor. Our results indicated that deletion of genes encoding SOD enzymes originated patterns of antioxidative responses, according to the tested periods of time, regardless the herbicides. To ΔsodB, the damage was minor in time 9pm; but in ΔsodA, the damage was minor in time 5pm, demonstrating the important role of these genes in the defense against oxidative stress in different stages of growth. Cellular responses demonstrated despite the observed toxicity indices, the strains were able to grow at rates close to those verified in control, including an increase in the fitness value in ΔsodA, which indicates a wide plasticity of responses, and a potential for a quick fitting of E. coli K-12. Extending this hypothesis, considering an environment containing a toxic molecule, such as an herbicide, and a bacterium having a polymorphic system for SOD, if a mutation appears in a gene coding for isoforms, it may show an increase in cell viability and still maintain a functional antioxidative enzyme. We suggest that E. coli K-12, a strain created in a laboratory, and with probably low survivability in a natural environment, developed mechanisms of in vitro herbicide tolerance, even without previous selection. This phenotypic plasticity model might be found in other bacteria of agricultural land, with high turnover of cultures and intense use of herbicides, which could cause a considerable impact on the diversity and functionality of soil microbiota. / Microrganismos representam componentes essenciais na manutenção dos ciclos biogeoquímicos e de ecossistemas. Em ambientes agrícolas, o uso contínuo de herbicidas para minimizar a perda na produtividade pode acarretar danos inibindo o crescimento em microrganismos. Uma das causas desta inibição é o aumento da produção de ERO (espécies reativas de oxigênio), como os radicais superóxido (O2-) e hidroxila (OH-), e o peróxido de hidrogênio (H2O2), causando estresse oxidativo. A magnitude deste estresse pode ser condicionada pelo grupo químico e pelo modo de ação do herbicida. As respostas celulares contra ERO envolvem o aumento na expressão de enzimas como superóxido dismutase e catalase, responsáveis pela dismutação de O2- e H2O2, respectivamente. O objetivo deste trabalho foi avaliar a relação entre o efeito de herbicidas com diferentes modos de ação e as respostas das isoenzimas de superóxido dismutase (SOD) na bactéria Escherichia coli K-12. Assim, foram utilizados os herbicidas 2,4-D, atrazina e paraquat, e linhagens mutantes de E. coli K-12 nocauteadas para os genes codificantes das enzimas Mn-SOD (sodA) e Fe-SOD (sodB). Os herbicidas mostraram-se capazes de promover o desequilíbrio do potencial redox, aumentando a produção de H2O2 e malondialdeido (MDA), acima das taxas observadas nos controles e de forma distinta entre as linhagens. Expostas ao paraquat, o qual origina ciclismo redox in vivo, as linhagens de E. coli wt e ΔsodB apresentaram aumento na produção de H2O2, além de aumento na atividade de Mn-SOD, provavelmente como consequência da ativação do SoxR, o qual promove a transcrição do gene sodA. Em ΔsodA, os índices de toxicidade com o paraquat não foram maiores que o controle, indicando uma possível regulação da expressão da enzima Fe-SOD pelo fator transcricional OxyR. Nossos resultados indicaram que a deleção dos genes codificantes para SOD provocaram padrões nas respostas antioxidativas, de acordo com os tempos testados, independentemente dos herbicidas. Para ΔsodB, os danos foram menores no tempo de 9h; já em ΔsodA, os danos foram menores no tempo de 5h, demonstrando a importância do papel destes genes na defesa contra o estresse oxidativo em diferentes fases de crescimento. As respostas celulares demonstraram que, apesar dos índices de toxicidade observados, as linhagens foram capazes de crescer em taxas próximas às verificadas no controle, incluindo ainda um aumento do valor adaptativo em ΔsodA, o que indica uma plasticidade ampla de respostas e um potencial de adaptação rápida. Ampliando esta hipótese, considerando um ambiente contendo uma molécula tóxica, e uma bactéria que possua um sistema polimórfico para SOD, caso ocorra uma mutação no gene para uma de suas isoformas, ela poderá aumentar a viabilidade celular e ainda manter uma enzima antioxidativa funcional. Sugere-se que E. coli K-12, uma linhagem desenvolvida em laboratório e provavelmente com baixa capacidade de sobrevivência em ambiente natural, apresenta mecanismos de tolerância in vitro a herbicidas, mesmo sem seleção prévia. Tal modelo de plasticidade fenotípica poderia ser encontrado em outras bactérias de solo agrícola com alta rotatividade de culturas e intenso uso de herbicidas, podendo causar um impacto considerável na diversidade e funcionalidade desta microbiota.

As espécies reativas de oxigênio são fundamentais na fisiologia espermática. No entanto, um desequilíbrio entre a produção e a capacidade antioxidante caracteriza o estresse oxidativo (EO). O espermatozoide é extremamente suscetível ao EO pois, dentre outras características, a membrana plasmática é rica em ácidos graxos poli-insaturados responsáveis por promoverem a fluidez necessária em processos fisiológicos como motilidade e fertilização. Por outro lado, essas insaturações são mais facilmente oxidadas e vulneráveis à peroxidação lipídica. Em função desta susceptibilidade, estas células dependem fortemente de compostos presentes no plasma seminal (PS) para a proteção contra esse evento. Dessa forma, a carnosina, dipeptídeo presente no PS pode ser uma das responsáveis pela proteção contra o acúmulo do MDA. No entanto, durante a criopreservação do sêmen equino é necessário retirar o PS. Em estudo recente, verificamos que esta remoção, torna os espermatozoides sensíveis ao subproduto extremamente deletério da peroxidação lipídica, o malondialdeído (MDA). Como a carnosina é removida junto com o plasma seminal durante a criopreservação, foram desenvolvidos 2 experimentos sequenciais visando a melhora da qualidade do sêmen criopreservado com adição de carnosina. Amostras de sêmen de sete garanhões foram tratadas com concentrações crescentes de carnosina adicionadas ao diluidor (1mM, 50mM e 100mM). Após a descongelação, as amostras foram divididas retrospectivamente em grupos de alta congelabilidade (AC: motilidade maior que 30%) e baixa congelabilidade (BC: motilidade menor que 30%). Amostras tratadas com 50mM apresentaram menor porcentagem de células com lesão de membrana plasmática e, quando tratadas com 100mM, células com maior amplitude do deslocamento lateral de cabeça. Amostras controle BC apresentaram menor porcentagem de células com DNA íntegro em relação às amostras AC. No entanto, houve um leve aumento na porcentagem de células com DNA íntegro em amostras BC com 100mM, não diferindo das amostras AC. Por outro lado, amostras BC criopreservadas com 50mM apresentaram maiores porcentagens de células com escore calculado de potencial de membrana mitocondrial e mais suscetíveis ao EO em relação ao controle. Apesar da proteção parcial, a maior suscetibilidade à peroxidação lipídica torna-se um problema, especialmente pelo fato de que espermatozoides equinos são mais suscetíveis ao MDA. Um motivo para este efeito seria a afinidade da carnosina em reagir com açúcares, o que poderia influenciar negativamente a atividade mitocondrial e o status oxidativo, ao diminuir a produção de piruvato pela via glicolítica. Desta forma, no experimento 2, amostras BC foram tratadas com a combinação de carnosina (0 e 50mM) e piruvato (0 e 5mM) em arranjo fatorial 2x2. Verificou-se que o tratamento com piruvato (5mM) proporcionou menos células com baixa atividade mitocondrial. Por outro lado, a carnosina (50mM), promoveu maior motilidade total, progressiva e células rápidas. Houve uma tendência de aumento nas células com velocidade progressiva e atividade mitocondrial na combinação de tratamentos. Não houve diferença entre os grupos na suscetibilidade ao EO que, no entanto, correlacionou-se negativamente com células móveis, rápidas e integridade de membrana plasmática e acrossomal. Estes resultados indicam que subprodutos da peroxidação lipídica, sendo o principal deles o MDA, podem causar danos ao DNA, às mitocôndrias e à cinética espermática. Neste contexto, a carnosina (100mM) parece ter um leve efeito protetor ao DNA contra o acúmulo de MDA. Além disto, 50mM de carnosina parece auxiliar na manutenção da velocidade progressiva e atividade mitocondrial quando associada ao piruvato (5mM). Assim, a carnosina e o piruvato podem ser utilizados na prevenção de crioinjúrias em amostras de baixa congelabilidade. / Reactive oxygen species (ROS) plays a key role in the sperm physiology. However, an imbalance between ROS production and antioxidant capacity characterize the oxidative stress (OE). The spermatozoa are extremely susceptible to EO because, among other characteristics, the plasma membrane is rich in polyunsaturated fatty acids responsible for promoting fluidity necessary in physiological processes such as motility and fertilization. However, these unsaturations are more easily oxidized and vulnerable to lipid peroxidation. Due to this susceptibility, these cells strongly depend on compounds present in the seminal plasma (SP) to protect against this event. Thus, carnosine, a dipeptide present in SP of stallions, may be a key factor on the protection against MDA accumulation. Nevertheless, during the equine sperm cryopreservation process, SP is removed. In a recent study, we observed that seminal plasma removal led to an increased susceptibility of equine spermatozoa to extremely deleterious product of lipid peroxidation, malondialdehyde (MDA). As the carnosine is removed together with the seminal plasma during cryopreservation, two sequential experiments were developed aiming to improve the quality of stallion cryopreserved semen by means of carnosine therapy. Samples from seven stallions were treated with increasing concentrations of carnosine added to the extender (1mM, 50mM and 100mM) and submitted to cryopreservation. After thawing, samples were classified as high freezeability (HF: total motility greater than 30%) and low freezeability (LF: total motility lower than 30%). Samples treated with 50mM presented lower percentage of sperm showing plasma membrane damage and, when treated with 100mM, a greater amplitude of the lateral head displacement was observed. Untreated LF samples showed a lower percentage of cells showing intact DNA in relation to HF samples. By contrast, when LF samples were treated with 100mM, there was an increase in the percentage of cells with intact DNA, which was similar to the HF samples. On the other hand, LF samples cryopreserved with 50mM had a higher percentage of cells showing high calculated mitochondrial membrane potential score and increased susceptibility to OE in relation to the control. Despite the partial protection, the increased susceptibility to lipid peroxidation is a concern since equine spermatozoa is highly vulnerable to the MDA. Those results could be due to the affinity of carnosine to react with sugars, which could negatively influence mitochondrial activity and an oxidative state by decreasing pyruvate production. Hence, in experiment 2, LF samples were treated with a combination of carnosine (0 and 50mM) and pyruvate (0 and 5mM) in a 2x2 factorial arrangement. We observed that samples treated with pyruvate (5mM) had decreased percentage of cells with low mitochondrial activity. On the other hand, carnosine (50mM) increased total motility, progressive motility and fast cells. We also observed a tendency to increased progressive velocity and mitochondrial activity in the combination of treatments. There was no difference on sperm susceptibility to OE between treatments. However, this variable correlated negatively with the percentage of motile and rapid cells as well as those showing intact membrane and acrosome. These results indicate that the byproduct of lipid peroxidation (MDA) may cause damage to DNA, mitochondria and sperm kinetics. In this context, carnosine (100mM) appears to have a mild protective effect on DNA against the accumulation of MDA. Furthermore, 50mM of carnosine seems to improve progressive velocity and mitochondrial activity when associated with pyruvate (5mM). Thus, carnosine and pyruvate can be used on cryoinjuries prevention in low freezeability samples.

<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.

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