Master of Science / Department of Kinesiology / David C. Poole / Introduction: Dietary nitrate(NO[subscript]3[superscript]-) supplementation, via its reduction to nitrite (NO [subscript] 2 [superscript]-) and subsequent conversion to nitric oxide (NO) and other reactive nitrogen intermediates, reduces blood pressure and the O[subscript]2 cost of submaximal exercise in humans. Despite these observations, the effects of dietary NO [subscript]3 [superscript]- supplementation on skeletal muscle vascular control during locomotory exercise remain unknown. We tested the hypotheses that dietary NO [subscript]3 [superscript]- supplementation via beetroot juice (BR) would reduce mean arterial pressure (MAP) and increase hindlimb muscle blood flow in the exercising rat. Methods: Male Sprague-Dawley rats (3-6 months) were administered either NO [subscript]3 [superscript]- (via beetroot juice; 1 mmol · kg[superscript]-[superscript]1 · day[superscript]-[superscript]1, BR n=8) or untreated (control, n=11) tap water for 5 days. MAP and hindlimb skeletal muscle blood flow and vascular conductance (radiolabeled microsphere infusions) were measured during submaximal treadmill running (20 m · min[superscript]-[superscript]1, 5% grade). Results: BR resulted in significantly lower exercising MAP (control: 137 ± 3, BR: 127 ± 4 mmHg, P<0.05) and blood [lactate] (control: 2.6 ± 0.3, BR: 1.9 ± 0.2 mM, P<0.05) compared to control. Total exercising hindlimb skeletal muscle blood flow (control: 108 ± 8, BR: 150 ± 11 ml · min[superscript]-[superscript]1 · 100 g[superscript]-[superscript]1, P<0.05) and vascular conductance (control: 0.78 ± 0.05, BR: 1.16 ± 0.10 ml · min[superscript]-[superscript]1 · 100 g[superscript]-[superscript]1 · mmHg[superscript]-[superscript]1, P<0.05) were greater in rats that received beetroot juice compared to control. The relative differences in blood flow and vascular conductance for the 28 individual hindlimb muscles and muscle parts correlated positively with their percent type IIb + d/x muscle fibers (blood flow: r=0.74, vascular conductance: r=0.71, P<0.01 for both). Conclusion: These data support the hypothesis that NO [subscript]3 [superscript]- supplementation improves vascular control and elevates skeletal muscle O [subscript]2 delivery during exercise predominantly in fast-twitch type II muscles, and provide a potential mechanism by which NO [subscript]3 [superscript]- supplementation improves metabolic control.

Molecular nitrogen is a highly abundant element in the atmosphere; it is stable and not very reactive. Anthropogenic activities have caused greater concentrations of nitrogen-containing compounds that are highly reactive and ultimately toxic. Reactive nitrogen concentrations have become a growing concern on the South African Highveld, with satellite images indicating very high nitrogen dioxide concentrations in the region. This study investigates the nitrogen budget on the Highveld through the analysis of the nitrogen species emitted into the atmosphere on a temporal scale as well as the atmospheric conversion, transport and removal of these species. Data was collected at Elandsfontein monitoring site, which is centrally located on the industrialised Highveld. The formation and interaction of nitric oxide (NO), nitrogen dioxide (NO2), and nitrate (NO3) are a major focus in the study. NOx concentrations are higher in winter (6.5 to 8.5 μg.m-3) as a result of stable atmospheric conditions. NO3 concentrations also peak during winter (3.5 to 5.5 μg.m-3), with a distinct biomass burning peak during July and August. Diurnally, NOx concentrations indicate a tall-stack industrial source, with concentrations peaking at midday. NO3 concentrations are higher at night and lower during the day, as during the day the NO3 radical is rapidly photolysed and nitrates cannot be produced. Case studies indicate that the conversion rate of NO to NO2 is highly variable as a result of varying atmospheric factors. These rates range from 11% to 59% per hour. Rates of dry deposition of NO, NO2 and NO3 are generally higher during winter as a result of higher concentrations and increased atmospheric stability, which prevents transport out of the region. Nitrogen is predominantly deposited as NO2 throughout the year, except during spring when NO3 deposition dominates. The total amount of nitrogen deposited to the Mpumalanga Highveld region is in the range of 6.7 to 13.1 kg ha-1 yr-1, which is well below the stipulated critical load value. Such deposition therefore does not pose significant threats to the natural environment on the Highveld. Between 4% and 14% of the total emitted nitrogen on the Highveld is deposited to the surface via wet and dry deposition. The remainder stays in the atmosphere and is advected out of the region.

L’oxydation troposphérique des composés organiques volatils (COV) constitue une contribution importante à la formation de photooxydants. L’évaluation de l’impact environnemental des COV rend indispensable d’acquérir une bonne compréhension des processus en jeu. Ce travail porte donc sur l’étude en atmosphère simulée de la réactivité troposphérique avec le radical nitrate (NO3) de COV oxygénés appartenant à une série homogène d’éthers vinyliques aliphatiques (méthyl, éthyl, propyl et butyl vinyl éthers), et à une série de trois esters (acétates d’isopropényle, de vinyle, et d’allyle), auxquelles s’ajoute le 2,3,2 méthylbutènol (MBO). Pour tous ces composés, des études cinétiques (destinées à mesurer la constante d’oxydation par NO3 des produits étudiés) ont été menées selon la méthode relative, et pour certains composés selon la méthode absolue. Des études mécanistiques (destinées à identifier et quantifier les produits de la réaction, et à élucider le mécanisme réactionnel) ont également été menées. Une réévaluation de la constante cinétique d’oxydation du propène par NO3, qui a été mesurée selon la méthode absolue, a également été faite. Les expériences ont été menées dans la chambre de simulation atmosphérique du LISA, à température ambiante et à pression atmosphérique. Les durées de vie des composés étudiés vis-à-vis de NO3 ont été calculées, et comparées à celles vis-à-vis du radical OH et de l’ozone. Les résultats montrent que NO3 peut constituer un puits majeur pour les COV les plus réactifs, comme les éthers vinyliques. Par ailleurs, les apports de nos résultats aux règles de réactivité des COV ont été discutés / The tropospheric oxidation of the volatile organic compounds (VOC) constitutes an important contribution to the formation of photooxydants. It is necessary to acquire a good comprehension of the concerned chemical processes in order to correctly evaluate the environmental impact of the VOC. This work thus concerns the study under simulated conditions of the tropospheric reactivity with the nitrate radical (NO3) of oxygenated VOC pertaining to a homologous series of aliphatic vinyl ethers (methyl, éthyl, propyl and butyl vinyl ethers), and to a series of three esters (allyl and vinyl, isopropenyl acetates). In addition, the 2,3,2 méthylbutènol (MBO) has been studied too. For all these compounds, kinetic studies (in order to measure the rate constant of NO3 oxidation of the studied products) were performed according to the relative rate method, and for some compounds according to the absolute method. Mechanistic studies (in order to identify and quantify the reaction products, and to elucidate the chemical mechanism) were also performed. A revaluation of the rate constant of NO3 oxidation of propene, which was measured according to the absolute method, was also performed. The experiments were carried out in the LISA atmospheric simulation chamber, at room temperature and atmospheric pressure. The lifetimes of the studied compounds with respect to NO3 were calculated, and were compared to those with respect to OH radical and to ozone. The results show that NO3 can be a major sink for the most reactive VOC, like the vinyl ethers. In addition, the contributions of our results to the rules of reactivity of the VOC were discussed

As atividades humanas vêm aumentando dramaticamente a quantidade de nitrogênio inorgânico liberado nos ecossistemas, seja através da aplicação de fertilizantes na agricultura, da descarga de dejetos humanos e de seus rebanhos, ou da queima de combustíveis fósseis. Os excessos de nitrogênio são eventualmente transportados para corpos d´água, onde podem, na forma de nitrato, nitrito e amônio, atingir concentrações tóxicas para organismos aquáticos. Nesta pesquisa tive dois objetivos principais. O primeiro foi testar em laboratório a toxicidade relativa dos íons nitrato, nitrito e amônio, e a variação interespecífica na sensibilidade a esses íons, em larvas de cinco espécies de anuros (Rhinella ornata, Hypsiboas faber, Hypsiboas pardalis, Physalaemus cuvieri e Physalaemus olfersii ). Para isso utilizei bioensaios seguindo protocolos internacionalmente padronizados para testes de ecotoxicidade com organismos aquáticos, e que portanto permitem máximas reprodutibilidade e comparabilidade de resultados entre compostos, espécies, e laboratórios. No entanto, estes bioensaios carecem de realismo uma vez que simulam um cenário de exposição aguda a altas concentrações de contaminantes quando na natureza o cenário de exposição tende a ser crônico e prolongado a baixas concentrações. Além disso, bioensaios usam mortalidade como principal variável de resposta, quando também efeitos subletais podem influenciar a persistência de populações ao modular o sucesso dos indivíduos. Por isso, meu segundo objetivo foi testar em laboratório se concentrações relativamente baixas e ecologicamente relevantes de nitrato, nitrito e amônio podem afetar a sobrevivência, o crescimento, o desenvolvimento e o comportamento das larvas de R. ornata, P. cuvieri e H. faber. Demonstrei através dos bioensaios de exposição aguda que nitrato, a forma mais abundante na natureza, é de baixa toxicidade quando comparada a nitrito e amônio. Demonstrei também que há significativa variação interespecífica na sensibilidade ao nitrogênio inorgânico, e que o ranqueamento de sensibilidade das espécies ao nitrato e ao nitrito foram similares, possivelmente por conta de mecanismos comuns de ação tóxica. Através de experimentos de exposição crônica demonstrei que concentrações relativamente baixas de nitrogênio inorgânico podem causar efeitos letais e subletais às larvas de anuros se houver exposição prolongada. O nitrato causou redução no desenvolvimento larval de P. cuvieri e o amônio na sobrevivência e nas taxas de atividade nos girinos de H. faber. A exposição crônica ao nitrito também reduziu significativamente a sobrevivência das três espécies testadas, o crescimento de H. faber e as taxas de atividade de R. ornata. Contudo, é improvável que as concentrações de nitrito que manipulei em laboratório sejam comuns na natureza, especialmente em condições aeróbicas. Esta pesquisa, além de fornecer importantes informações sobre os possíveis efeitos da poluição por nitrogênio em larvas de anuros, contribui para o avanço da ecotoxicologia no Brasil ao estabelecer as bases para o emprego de espécies nativas de anfíbios como sistema-modelo experimental. Estudos futuros que almejem avaliar o risco ambiental da contaminação por nitrogênio deverão por um lado monitorar concentrações em hábitats naturais e por outro avaliar as consequências das interações sinérgicas entre nitrogênio inorgânico e outros estressores físicos, químicos ou biológicos para larvas de anfíbios. / Human activities dramatically increased the amount of inorganic nitrogen released in ecosystems through the application of fertilizers in agriculture, the generation of human and livestock waste, and the combustion of fossil fuels. This nitrogen eventually reaches water bodies where it can, in the form of nitrate, nitrite and ammonium, be toxic to aquatic organisms. In this study I had two main objectives. The first was to test the relative toxicity of nitrate, nitrite and ammonium, and the interspecific variation in sensitivity to these ions, in tadpoles of five anuran species (Rhinella ornata, Hypsiboas faber, Hypsiboas pardalis, Physalaemus cuvieri and Physalaemus olfersii ). This objective was accomplished by laboratory bioassays following internationally standardized protocols for ecotoxicity tests with aquatic organisms, therefore allowing maximum reproducibility and comparability of results among compounds, species and laboratories. However, these bioassays lack realism for simulating a scenario of acute exposure to high concentrations of contaminants, while exposure in nature tends to be chronic and prolonged at low concentrations. Furthermore, bioassays use mortality as the main response variable, whereas sublethal effects may also influence the persistence of populations by modulating individual success. My second objective was therefore to test in the laboratory if low and environmentally relevant concentrations of nitrate, nitrite and ammonium affect survival, growth, development and behavior of R. ornata, P. cuvieri and H. faber larvae. Through acute exposure bioassays I demonstrated that nitrate, the most abundant N form in nature, has low toxicity when compared to nitrite and ammonium. I also demonstrated that there is significant interspecific variation in the sensitivity to inorganic nitrogen, and that the ranking of species sensitivity to nitrate and nitrite were similar, possibly due to common mechanisms of toxic action. Through chronic exposure I demonstrated that relatively low concentrations of inorganic nitrogen can cause lethal and sublethal effects on anuran larvae if there is extended exposure. Nitrate decreased developmental rate in P. cuvieri and ammonia decreased survival and activity rates in H. faber tadpoles. Chronic exposure to nitrite also significantly reduced survival of all three species tested, growth of H. faber and activity rates of R. ornata. However, it is unlikely that the concentrations of nitrite manipulated in the laboratory are common in nature, especially in aerobic conditions. This is the first study to document deleterious effects of nitrogen pollution to Brazilian amphibian species, and contributes to the development of ecotoxicology in Brazil by establishing the basis for the employment of native amphibians as model experimental system. Future studies that aim to assess the environmental risk of nitrogen contamination should monitor concentrations in natural habitats and evaluate the effects of synergistic interactions between inorganic nitrogen and other physical, chemical or biological stressors to amphibian larvae.

A nitrato redutase (NR) atua juntamente com a nitrito redutase (NiR) catalisando a primeira etapa da redução do nitrato. A NR, no citossol, é ativada, principalmente, pela luz e reduz o nitrato a nitrito. Em seguida, este é reduzido a amônio. Trabalhos anteriores demonstraram que a isoforma citossólica da NR está presente nas folhas e raízes do abacaxizeiro; já as associadas à membrana plasmática (NRMP) ainda não se tem registro. Sabe-se, no entanto, que a NRMP apresenta modos diferentes de ativação, como já constatados para outras espécies. Dentre os fatores que afetam sua atividade pode-se citar a temperatura. Pesquisas realizadas no Laboratório de Fisiologia Vegetal do IBUSP, acerca da influência do termoperíodo sobre os metabolismos nitrogenado e fotossintético de plantas de abacaxizeiro, aventaram a hipótese de que haveria uma nitrato redutase específica de membrana plasmática presente nas células radiculares, a qual seria regulada por termoperíodo, diferindo, portanto, da isoforma citossólica presente nas folhas. Assim, o presente trabalho teve como objetivo principal demonstrar a existência de uma isoforma da NR associada à membrana plasmática, a qual seria responsável pelo incremento da atividade dessa enzima registrada nas raízes de Ananas comosus, quando plantas cultivadas in vitro são submetidas ao termoperíodo (28°C dia/ 15°C noite). Para tanto, determinou-se o tempo mínimo de exposição das plantas de abacaxizeiro ao termoperíodo, necessário à indução da nitrato redutase radicular. Além disso, estudou-se a influência da idade das plantas na resposta ao tratamento com baixa temperatura noturna. Plantas cultivadas in vitro com 90 dias de idade ou com idades variadas foram transferidas para câmaras de crescimento com temperatura constante (28°C dia/noite controle experimental) ou com termoperíodo (28°C dia/ 15°C noite), fotoperíodo de 16 horas e intensidade luminosa de 55 moles m-2 s -1. Elas permaneceram nessas condições por 1, 3, 5, 7, 15, 30, 40, 50 ou 60 dias. Após cada período, a atividade in vivo da NR foi analisada durante a fase de ausência de luz. Para que fosse possível identificar uma possível NRMP nas células radiculares de abacaxizeiro, um método de ensaio in vitro foi padronizado e a melhor técnica de isolamento de frações de membrana plasmática foi selecionada. Após as plantas com 60 dias de idade serem submetidas por 30 dias ao tratamento termoperiódico ou à temperatura constante, as frações de membrana plasmática das células radiculares foram isoladas e o ensaio in vitro da NR foi realizado, utilizando-se NADH, NADPH ou succinato como doadores de elétrons. Os resultados indicaram que o tempo mínimo de exposição das plantas de abacaxizeiro ao termoperíodo foi de 30 dias. O método de ensaio enzimático in vitro foi padronizado para as plantas de abacaxizeiro e a técnica de isolamento de frações de membrana plasmática que se mostrou mais adequada para essa bromélia foi a de fracionamento por sistema de duas fases com Dextran T-500 e PEG 3350. O grau de pureza das frações, avaliado pela detecção da atividade da enzima citoplasmática malato desidrogenase (MDH), foi em média de 95%, evidenciando a eficácia da pradronização do método. Os resultados obtidos para as frações de membranas plasmáticas, extraídas das raízes das plantas que estiveram sob o tratamento termoperiódico, mostraram que a baixa temperatura noturna influenciou positivamente a atividade da nitrato redutase. O aumento da atividade foi observado quando NADH, NADPH ou succinato foram utilizados como doadores de elétrons. Isso significa que, provavelmente, mais de uma isoforma da NRMP está presente nas raízes de abacaxizeiro. Além disso, há indícios de que a isoforma que está ligada externamente à membrana por uma âncora glicosídica (que utiliza succinato como doador de elétrons) está presente nas células radiculares do abacaxizeiro e respondeu positivamente ao estímulo da baixa temperatura noturna. Em contrapartida, nenhuma diferença pôde ser observada quando as atividades da NR foram medidas nas frações de citoplasma das plantas controle e daquelas que foram tratadas com termoperíodo. Não foi detectada atividade nas frações citossólicas quando succinato foi oferecido como poder redutor da NR. Concluiu-se, portanto, que o incremento na atividade da NR, verificado nas plantas que foram tratadas com termoperíodo, deveu-se à indução pela baixa temperatura noturna da NRMP. Esta pesquisa trouxe contribuições importantes acerca da existência de uma nitrato redutase associada à membrana plasmática em abacaxizeiro, nunca antes detectada em uma bromélia e muito pouco estudada nos demais vegetais. As padronizações realizadas serão essenciais para aplicação em outras pesquisas do Laboratório de Fisiologia Vegetal do IBUSP, abrindo oportunidades para se aprofundar ainda mais o tema sobre o controle da ativação da NR. / The nitrate reductase (NR) acts together with the nitrite reductase (NiR) to catalyze the first step of the nitrate reduction. The NR localized in the cytosol is activated mainly by light and reduces nitrate to nitrite, followed by its reduction to ammonium. Previous work demonstrated that the cytosolic isoform of NR is present in leaves and roots of Ananas comosus, although the isoform associated with the plasma membrane (PM-NR) has not yet been registered in this species. The PM-NR has different modes of activation in comparison to the cytosolic NR, as already demonstrated in other species. Among the factors that affect its activity can be mentioned the temperature. Experiments developed in the Laboratory of Plant Physiology of IBUSP hypothesized that exists a specific plasma membrane NR in plant roots regulated by thermoperiod, differing from the cytosolic isoform present in leaves. The present work aimed to demonstrate the existence of this isoform of NR present in the plasma membrane, which would be responsible for the increase of its activity in Ananas comosus roots when plants were cultivated in vitro under thermoperiod of 28°C day/ 15°C night. Initially, it was important to determine the minimum time of exposure to thermoperiod necessary for the induction of nitrate reductase in roots of pineapple plants. Furthermore, it was analyzed the influence of age in the response of plants to low night temperature treatment. For this purpose, plants with different ages cultivated in vitro were transferred to growth chambers either with constant temperature (28°C day/night experimental control) or with thermoperiod (28°C day/ 15°C night). The plants were cultivated in these conditions during 1, 3, 5, 7, 15, 30, 40, 50 or 60 days and then the in vivo NR activity was analyzed in the shoot and root tissues during the dark period. In order to identify a probable PM-NR in the pineapple root cells, it was also necessary to develop a NR in vitro assay protocol specific for Ananas comosus and the appropriate technique for plasma membrane isolation (Dextran T-500 and PEG 3350). The purity of the fractions, determined by the activity of cytoplasmic enzyme malate dehydrogenase (MDH), was on average 95%, indicating the effectiveness of the method. The next step was to evaluate the NR activity in citoplasmic and plasma membrane fractions of root tissues of Ananas comosus. Using 60 daysold plants exposed either to 30 days under the thermoperiodic treatment or to constant temperature, the plasma membrane fractions of roots were isolated and the in vitro NR assay was performed using NADH, NADPH or succinate as electron donators. The results indicated that the minimum thermoperiod exposure time necessary to induce NR activity was 30 days. Furthermore, it was demonstrated that low temperatures during the dark period positively influenced the activity of nitrate reductase in plasma membrane fractions and that the increase in its activity was observed when NADH, NADPH or succinate were used as electron donors. On the other hand, no difference in NR activity was observed in the cytoplasmic fraction of control plants and those which were treated with thermoperiod. Moreover, no NR activity was detected in cytosolic fractions when succinate was provided as electron donor. All together, this results showed that probably diferents isoforms are presents in pineapple roots. The extracellular isoform that is attached to the plasma membrane by a lipophilic anchor (using succinate as electron donor) can be present in root cells of pineapple and responded positively to the low night temperature stimulus. This study has made important contributions to the knowledge of the metabolism and physiology of Ananas comosus. This is the first time that the existence of a nitrate reductase associated with the plasma membrane, a very little studied enzyme, is documented in Bromeliaceae.

A eletroredução de íons nitrato foi estudada em eletrodos de Paládio suportados em carbono, preparados através de ultra-som, com diferentes tamanhos de partículas. Para tanto, foi necessária a caracterização do catalisador de Pd/C através de DR-X, MET, EDX. Após a caracterização dos catalisadores, preparou-se os eletrodos através de pintura dos catalisadores em tecido de carbono que foram caracterizados por voltametria cíclica. Os estudos de redução de íon nitrato foram realizados utilizando a técnica de DEMS. Foi realizada a normalização dos eletrodos através da porcentagem de paládio obtidas em análise de EDX, o que possibilitou realizar comparações entre os eletrodos. Os estudos foram realizados alterando condições como a temperatura e a concentração de íon nitrato e verifico-se a formação de N2O e NO. Durante os experimentos também foram observadas formações de produto m/z = 33 que no início eram atribuídos a hidroxilamina, mas resultados com compostos marcados isotopicamente mostraram que este produto é atribuído a espécies não nitrogenadas. Os eletrodos de Paládio mostraram-se eficientes para a redução de íons nitrato, embora não seletivo para a formação de nitrogênio, destacando o eletrodo preparado com catalisador de menor tamanho de partícula. Deste modo espera-se maiores valores de corrente para a formação dos produtos monitorados pois para nanopartículas de menor tamanho há aumento significativo da área do catalisador. / The eletroredução of nitrate ions was studied on carbon supported Palladium electrodes, prepared by the ultrasound method, with different sizes of particles. The characterization of the Pd/C catalysts was carried out by DR-X, MET, EDX. After the characterization of the catalysts, the electrodes were prepared through the painting procedure in carbon cloth that had been characterized by cyclic voltammetry. The studies of ion reduction nitrate have been carried through the DEMS technique. The normalization of the electrodes was performed by the percentage of palladium determined by EDX analysis. The studies have been carried out modifying experimental conditions such as temperature and concentration of nitrate ions and verifying the formation of N2O and NO. During the experiments, the product formation were detected by m/z = 33 that in the beginning it was attributed to hydroxylamine, but resulted with composites marked had isotopes shown that this substance is attributed the species without not nitrogenous. The electrodes with Palladium have revealed efficient for the reduction of nitrate ions, however do not selective for the nitrogen formation, detaching the electrode prepared with smaller particle size. In this way, it is expected larger values of current for the formation of the monitored products, because for smaller nanoparticles, there is a significant increase of the catalyst surface area.

Soluções de nitrato de prata são freqüentemente usadas em testes de micro e nanoinfiltração, apesar de nem todas as suas características estarem definidas claramente na literatura. Neste estudo foram avaliadas soluções aquosas de nitrato de prata quanto ao pH e à quantidade de prata iônica (ppm) em várias concentrações, bem como o desempenho dessas soluções em testes de microinfiltração realizados em dentes decíduos e permanentes. Numa primeira fase foi analisado o pH (pHmetro digital) de solução a 50% (p/v), tendo como variáveis a pureza da água, a marca comercial do sal, a cor do frasco, e a idade pós-preparo. Posteriormente, avaliou-se a quantidade de prata iônica, por espectrometria de emissão atômica, presente nas soluções (1%, 5%, 25% e 50%) ao longo de 168 horas de armazenagem. Em cavidades de classe V, confeccionadas nas faces vestibular e lingual/palatina de molares, foram aplicados dois sistemas adesivos (OptiBond FL ou OptiBond Solo Plus SE). Após a restauração (Filtek Z-250) foi determinado o valor médio de microinfiltração (mm) para diversas concentrações e idade pós-preparo das soluções. Os dados obtidos foram tratados por análise de variância e teste de Tukey (a=0,05). As soluções analisadas na primeira etapa apresentaram pH médio entre 7,9±2,2 a 11,8±0,9, e houve diferenças significativas para todas as variáveis. O teor médio de prata iônica apresentou diferenças significativas para o fator Concentração (4,75±0,5 a 1% e 293±15,3ppm a 50%); porém, não houve diferença para o fator Idade. Nos testes de microinfiltração houve diferença significante apenas para o fator Adesivo (p<0,01); os demais fatores e as interações não apresentaram diferenças significativas. Com base nos resultados obtidos neste estudo pode-se concluir que: 1) o pH de soluções aquosas de nitrato de prata varia de neutro a alcalino; 2) a quantidade de prata iônica e o pH se mantêm estáveis por até 168h; 3) a concentração e a idade pós-preparo das soluções não interferiram nos valores médios de microinfiltração; 4) o sistema adesivo OptiBond FL apresentou menores valores de microinfiltração, em dentes permanentes e decíduos / Silver nitrate solutions are frequently used in micro and nanoleakage tests, although not all of their characteristics are clearly defined in the literature. In this study an assessment was made of aqueous silver nitrate solutions as regards pH and the amount of ionic silver (ppm) in various concentrations, as well as the performance of these solutions in microleakage tests performed in primary and permanent teeth. In the first phase the pH (digital pH meter) of a 50% (w/v) solution was analyzed, having water purity, commercial brand of the salt, the color of the flask, and the storage time as variables. Afterwards, the amount of ionic silver present in the solutions (1%, 5%, 25% and 50%) was evaluated by atomic emission spectrometry, throughout 168 hours of storage. In Class V cavities made on the vestibular and lingual/palatal faces of molars, two adhesive systems were applied (OptiBond FL or OptiBond Solo Plus SE). After restoration (Filtek Z-250) the mean microleakage value (mm) was determined for the various concentrations and post-preparation time of the solutions. The data obtained were submitted to analysis of variance and the Tukey test (a=0.05). The solutions analyzed in the first stage had a mean pH ranging between 7.9±2.2 and 11.8±0.9, and there were significant differences for all the variables. The mean ionic silver content presented with significant differences for the factor Concentration (4.75±0.5 at 1% and 293±15.3 ppm at 50%); but there was no difference for the factor Time. In the microleakage tests there was significant difference only for the factor Adhesive (p<0.01); the other factors and the interactions did not present with any significant differences. Based on the results obtained in this study, it may be concluded that: 1) the pH of aqueous silver nitrate solutions varies from neutral to alkaline; 2) the amount of ionic silver and pH remained stable for up to 168h; 3) the concentration and the post-preparation time of the solutions did not interfere with the mean microleakage values; 4) the adhesive system OptiBond FL presented with the lowest microleakage values in both permanent and primary teeth

Recently, it has emerged that the NO metabolites, nitrite and nitrate can be chemically reduced in vivo to biologically active nitric oxide (NO). This generation of NO is dependent on reduction of nitrate to nitrite by facultative anaerobes on the dorsal surface of the tongue, entry of the nitrite into the enterosalivary circuit, transit to the stomach, and absorption through the gut wall into the circulation. Conversion of nitrite to NO is then facilitated by vascular nitrite reductase enzymes. This nitrate-nitrite-NO pathway has been shown to exert a number of beneficial effects in healthy volunteers e.g. lowering of blood pressure, however whether this pathway is affected by cardiovascular disease (CVD) is currently unknown. Ozone chemiluminescence was used to determine and compare nitrite and nitrate levels in 2 models of CVD. To study atherosclerosis wild type (WT) and apolipoprotein E knock out (ApoE KO) mice were used and for hypertension wistar kyoto (WKY) rats as controls vs. spontaneously hypertensive rats (SHR). Assessment of nitrite reductase activity was conducted in the compartment which showed the most consistent differences in distribution, the red blood cell (RBC) and in homogenates of liver tissue. The impact of dietary nitrite and nitrate on distribution of the 2 anions throughout the cardiovascular system was assessed to determine the utility of this approach in restoring levels of these anions in CVD. Finally, using flow cytometry I investigated whether dietary nitrate supplementation could be used to influence inflammatory responses as a mechanism to improve CVD. Compared to WT mice, nitrate levels were reduced in ApoE KO mice in the plasma and across most of the tissues. In contrast in SHRs, reduction of the anions was only apparent in RBCs with no differences compared to WKY in all other tested tissues. Furthermore I have demonstrated that the most efficient way to restore nitrate levels back up to baseline is through a dietary nitrate strategy and that a dose of 15mM nitrate in the drinking water is sufficient to achieve this. In addition I have shown that nitrite reductase activity is enhanced in CVD particularly at the level of the RBC in both atherosclerosis and hypertension and that this enhanced activity is due, in part, to upregulation of xanthine oxidoreductase (XOR). Finally I have shown that dietary nitrate is an effective way to modulate an acute inflammatory response. This modulation is mediated through interfering with the ability of the neutrophil to firmly adhere to the vascular endothelium. These changes were shown to be dose-dependent and concomitant with dose-dependent increases in plasma nitrite and plasma nitrate. These data suggest that utilization of the nitrate-nitrite-NO pathway with dietary nitrate may represent an effective approach for the treatment of CVD.

O nitrato é uma das principais fontes de nitrogênio disponível para as plantas, sendo a nitrato redutase (NR) a enzima responsável pela sua redução a nitrito. O nitrito é considerado tóxico em altas concentrações e, por esse motivo, a atividade da NR possui uma regulação complexa, principalmente em nível transcricional e pós-traducional. Trabalhos anteriores do nosso grupo, utilizando plantas de abacaxizeiro cultivadas in vitro, demonstraram que, em condições de termoperíodo de 28ºC dia/15ºC noite, as raízes apresentaram um estímulo positivo de atividade da NR na ausência de luz quando comparado às plantas crescidas em temperatura constante de 28ºC, associado posteriormente à atividade da NR de membrana plasmática (NRPM). Baseado nesses resultados questionou-se qual seria a influência da aplicação do estímulo de frio associado ou não à presença de luz na atividade da NR em folhas e raízes de abacaxizeiro. Este trabalho teve como objetivos investigar os efeitos do frio na atividade da NR em folhas e raízes de abacaxizeiro em diferentes tempos de exposição, na presença ou ausência da luz e em diferentes fases do ciclo de 24 horas (claro/escuro). Buscou-se averiguar qual NR estaria envolvida nessas respostas: a NR citossólica (NRc) ou de membrana plasmática (NRPM), assim como verificar o envolvimento do NO na sinalização pela baixa temperatura. O ritmo diário de atividade da NR também foi avaliado, logo após a exposição ao frio, em diferentes fases do ciclo de claro/escuro. As plantas foram expostas a 1, 3, 6 ou 9 horas a 10ºC ou 25ºC (controle) na luz ou no escuro. A NR foi avaliada pelo método in vitro. O estímulo positivo na atividade da NR pelo frio ocorreu principalmente após 6 horas no claro, para as folhas, e após 6 horas no escuro, para as raízes. Novas plantas foram submetidas às mesmas condições para o fracionamento celular, mostrando que, tanto em folhas como em raízes, o incremento de atividade da NR observado a 10ºC foi associado à NR citossólica (NRc). Em ambos os casos, o estímulo ocorreu utilizando-se o NADPH como doador de elétrons, sugerindo o possível envolvimento de uma isoforma NAD(P)H biespecífica. A quantificação do NO foi realizada por leitura em espectrofluorímetro, apontando uma maior emissão induzida pelo frio para as folhas tanto na presença da luz (após 1 e 3 horas) como em sua ausência (1 e 9 horas) e em raízes apenas no escuro (9 horas), sugerindo o envolvimento do NO na sinalização da baixa temperatura. Para verificar a influência do frio em diferentes fases do dia, 4 horários foram selecionados (início da fase clara, meio da fase clara, início da fase escura, meio da fase escura) para início de cada experimento. A NR foi medida logo após a exposição ao frio (6 horas a 10ºC), pelo método in vitro e durante 24 horas em reaquecimento (25ºC), quantificada a cada 3 horas pelo método in vivo. As raízes apresentaram aumento da atividade da NR apenas quando o estímulo da baixa temperatura foi aplicado na fase escura, enquanto as folhas sofreram incremento da atividade da NR independente da condição luminosa. Em reaquecimento, a NR das folhas teve seu ritmo atrasado em todas as situações, com exceção quando o frio foi aplicado no início da fase escura, na qual houve perda quase completa de variação ao longo do dia. As raízes não mostraram grandes alterações no ritmo diário da NR. Este trabalho mostrou que a temperatura de 10ºC tem efeitos diferentes sobre folhas e raízes, sendo que as modificações na atividade da NR, em curto prazo, parecem ocorrer por alterações na NRc. O NO parece estar envolvido na sinalização do frio, mas não se determinou sua origem biossintética. As raízes tiveram um aumento da atividade da NR pela baixa temperatura, que foi dependente do escuro, enquanto as respostas das folhas dependeram da fase do ciclo na qual foram submetidas a 10ºC / Nitrate is the main nitrogen source available to plants, and nitrate reductase (NR) is the enzyme responsible for its reduction to nitrite. Because of its toxicity in high concentrations, nitrite production by NR has a complex regulation, especially at transcriptional and post-translational level. A previous work from our group, using pineapple plants cultivated in vitro, showed that, under thermoperiod of 28ºC day/15ºC night, NR activity increased in roots during absence of light compared to activity in plants grown under constant temperature of 28ºC. Based on these results it was questioned what would be the effect of cold stimulus application with or without light on NR activity in leaves and roots of pineapple plants. This study aimed to investigate the effects of low temperature on NR activity in leaves and roots of pineapple plants at different exposure times in the presence or absence of light and at different phases of a 24 hour cycle (light/darkness). We also investigated which NR was involved in these responses: cytosolic (cNR) or plasma membrane NR (PMNR), as well as verifying the role of nitric oxide (NO) signaling at low temperature. Furthermore, the NR daily rhythm activity was measured after cold exposure, in different phases of the light/dark cycle. Plants were exposed to 10ºC or 25ºC (control group) during 1, 3, 6 or 9 hours. NR was quantified by in vitro method. In the leaves, the increase of NR activity by low temperature (10ºC) occurred mainly after 6 hours in the presence of light, while in the roots the highest NR activity occurred after 6 hours at 10ºC in darkness. Based on these results, other groups of plants were subjected to the same conditions for cell partitioning, showing that in both leaves and roots the increase of NR activity by cold was associated with cytosolic NR (NRc). In both cases, the positive stimulation occurred with NADPH as the electron donor, suggesting the possible involvement of a NAD(P)H bispecific isoform. NO quantification, measured by spectrofluorimetry, indicated a greater emission induced by cold in the leaves both in the presence (after 1 and 3 hours) and absence (1 and 9 hours) of light and in roots only in darkness (9 hours), suggesting an involvement of NO in low temperature signaling. To evaluate the influence of cold at different day phases, we performed 4 experiments beginning at different times of the 24-hour cycle (beginning of light phase, middle of light phase, beginning of dark phase, middle of dark phase). NR activity was measured immediately after cold exposure (6 hours at 10°C) by in vitro method and after rewarming at 25°C during 24 hours, quantified by in vivo method every 3 hours. In roots, NR activity showed an increase only when the cold stimulus was applied at dark phase, while in leaves, NR was independent of the light condition. Upon rewarming, leaves presented a delay in NR daily behavior in all situations, except when low temperature was applied at the beginning of dark phase, showing almost no variation throughout the day. This study demonstrated that the temperature of 10ºC affected leaves and roots differently, and the changes in NR activity after short exposure time could be associated with NRc. NO seemed to be involved in cold signaling, but its biosynthetic origin has not been determined yet. Roots showed an increment of NR activity by low temperature dependent of the dark condition, while the responses of leaves depended on the phase of the 24-hour cycle in which they were subjected to 10ºC

<p>Controlled drainage is a management strategy designed to mitigate water quality issues caused by subsurface drainage. To improve controlled drainage system management and better understand its hydrological and environmental effects, this study analyzed water table recession rate, as well as drain flow, nitrate and phosphorus loads of both free and controlled drainage systems, and simulated the hydrology of a free drainage system to evaluate surface runoff and ponding at the Davis Purdue Agricultural Center located in Eastern Indiana. </p> <p>Statistical analyses, including paired watershed approach and paired t-test, indicated that controlled drainage had a statistically significant effect (<i>p</i>-value <0.01) on the rate of water table fall and reduced the water table recession rate by 29% to 62%. The slower recession rate caused by controlled drainage can have negative impacts on crop growth and trafficability by causing the water table to remain at a detrimental level for longer. This finding can be used by farmers and other decision-makers to improve the management of controlled drainage systems by actively managing the system during storm events. </p> <p>A method was developed to estimate drain flow during missing periods using the Hooghoudt equation and continuous water table observations. Estimated drain flow was combined with nutrient concentrations to show that controlled drainage decreased annual nitrate loads significantly (p<0.05) by 25% and 39% in two paired plots, while annual soluble reactive phosphorus (SRP) and total phosphorus (TP) loads were not significantly different. These results underscore the potential of controlled drainage to reduce nitrate losses from drained landscapes with the higher level of outlet control during the non-growing season (winter) providing about 70% of annual water quality benefits and the lower level used during the growing season (summer) providing about 30%. </p> <p>Three different methods including monitored water table depth, a digital photo time series and the DRAINMOD model simulations were used to determine the generation process of surface ponding and runoff and the frequency of incidence. The estimated annual water balance indicated that only 7% of annual precipitation contributed to surface runoff. Results from both simulations and observations indicated that all of the ponding events were generated as a result of saturation excess process rather than infiltration excess.</p> <p>Overall, nitrate transport through controlled drainage was lower than free drainage, indicating the drainage water quality benefits of controlled drainage, but water table remained at a higher level for longer when drainage was controlled. This can have negative impacts on crop yields, when water table is above a detrimental level, and can also increase the potential of nutrient transport through surface runoff since the saturation excess was the main reason for generating runoff at this field.</p>