>Magister Scientiae - MSc / The production of reactive oxygen species (ROS) is prominent in all aerobic metabolisms including plants. For this reason, the redox homeostasis of the production and scavenging of these intermediates is imperative for growth, development and survival during unfavourable conditions. In this study, a putative glutathione peroxidase gene (Glyma17g34110) from Glycine max (soybean) was identified and analyzed. The successful characterisation of Glyma17g34110 provided evidence of it being a glutathione peroxidase using glutathione as its preferred electron donor and substrate. Furthermore, it is known that antioxidant enzymes such as GPX exist in various tissues, performing a diverse set of functions. By a bioinformatic analysis of Glyma17g34110 and its promoter region, it was indicated that Glyma17g34110 could be a putative chloroplast protein that could play an important role in photosynthesis.One of the major factors affecting plant growth and development worldwide is abiotic stresses such as salinity. In the presence of salinity the production of harmful ROS is increased, resulting in detrimental reactions with important biological features (DNA, protein and lipid membranes), leading to cell death. The analysis of Glyma17g34110 under salt stress revealed that it is a salt sensitive gene and thus, the down-regulation of Glyma17g34110 could be due to the lack of known defence and response cis-acting elements present in the promoter region. Furthermore, it was proven in previous studies that the application of exogenous nitric oxide (NO) increases the activity of antioxidant enzymes. In this thesis it was observed that the presence of exogenously applied NO increased the expression of Glyma17g34110 tremendously in all soybean tissues (leaves, roots and nodules) investigated.Studies have found numerous cis-acting elements to be NO responsive, however, none of these elements were found in the promoter region upstream of glyma17g34110. This suggests that novel cis-acting elements could be present in the promoter region of Glyma17g34110.Thus, increasing the expression of Glyma17g34110 during salinity in the presence of NO, as well as the identification of these novel cis-acting elements, could lead to the enhancement of the defence mechanisms against ROS, which could lead to increasing plant tolerance to stress.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Introduction: manganese dependent superoxide dismutase (SOD2), is an important antioxidant enzyme, superoxide dismutase to anion produced in mitochondria in hydrogen peroxide, which in turn is catalyzed by glutathione peroxidase (GPX) into water and oxygen. Although be crucial for healthy cell, the role of SOD2 in cancer is highly controversial because in some kinds of cancers this enzyme exhibits a marked antitumor activity, while in others have a pro-tumor role. Previous investigations involving a polymorphism in codon 16 of SOD2 gene in which there is an exchange of a valine with an alanine (Val16Ala-SOD2) have associated increased efficiency of enzyme SOD2 at high risk of some cancers. However, in certain types of tumors, such as colorectal cancer are conflicting results. Studies suggest that high levels in tumor cells SOD2 colorectal cancers are associated with tumor progression. Perhaps this difficulty in defining the role of SOD2 in colorectal cancer biology is linked to great influence of environmental factors on the gastrointestinal system, especially the diet. For this reason, the development of an unbalance pharmacological model to investigate the role of superoxide-anion imbalance hydrogen peroxide (Superoxide Anion Hydrogen Peroxide imbalance, AS-HP) in colorectal cancer may be relevant. Objective: This study investigated the in vitro effect of drug-AS-HP imbalance caused by exposure to paraquat and the porphyrin in the viability and proliferative rate of commercial line of colorectal cancer cells (HT-29) and the response of these cells to chemotherapy oxaliplatin. The study also assessed the effect of AS-HP unbalance in the modulation of the expression of apoptotic genes, cell cycle and oxidative in HT-29 cells. Methods. HT- 29 obtained from American Type Cell Culture Collection (ATCC) were grown in DMEM, 10% fetal bovine serum, 1% antibiotic and antifungal in an oven with 5% CO2 and 37 ° C temperature. After 24 h the transfer of cells to 96-well plates at a concentration of 10 5 cells per well were exposed to these concentrations of 0.1 uM paraquat which is a superoxide-generating molecule or porphyrin which is a molecule with a similar effect SOD2 enzyme. Part of the cells was treated with oxaliplatin at a concentration of 20um and the other not. The effect on the viability, cell proliferation, cell cycle, apoptosis and modulation of genes of the cell cycle, apoptosis and oxidative metabolism (SOD1, SOD2, CAT, GPX, Caspase 3, Caspase 8, BAX, BCL-2 and P53colocar the name gene) was also evaluated. Assays were done in triplicate and compared by analysis of variance followed via test post hoc Tukey. Results: pharmacological imbalance AS-HP obtained via exposure of colorectal cancer cells to paraquat and porphyrin changed the standard of viability, cell cycle and in the modulation of gene expression. Both paraquat as nna porphyrin concentration 0.1 uM reduced the viability and proliferation rate of HT-29 cells. However, this effect was more pronounced in cells exposed to paraquat. The action of oxaliplatin was enhanced by the presence of paraquat when analyzed, the mortality rate, apoptosis, cell proliferation rate. Paraquat tamém induced cell cycle interruption phases S and G2 / M Any paraquat as porphyrin were able to modulate differentially markers of oxidative metabolism and expression of genes investigated. However, the results were quite heterogeneous. This heterogeneity may be associated with chromosomal instability in cancer cells that have high levels, and varied mutational. Conclusion: The results confirm the hypothesis that the AS-HP unbalance acts on the biology of colorectal cancer, and in particular increased levels of superoxide, not only increase the mortality rate but also inhibit cell proliferation enhancing so antitumor action of oxaliplatin. These results may be clinically relevant in the construction of pharmaceutical and / or nutritional strategies as the use of vitamins and other dietary supplements which operate in AS-HP sheet and to assist in the successful chemotherapeutic treatment of disease. / Introdução: a superóxido dismutase dependente de manganês (SOD2), é uma importante enzima antioxidante, que dismuta o ânion superóxido produzido na mitocôndria em peróxido de hidrogênio, que por sua vez é catalisado pela glutationa peroxidase (GPX) em água e oxigênio. Apesar de ser crucial para a célula saudável, o papel da SOD2 no câncer é bastante controverso, pois em alguns tipos de neoplasias apresenta uma clara ação antitumoral, enquanto que em outras tem um papel pró tumoral. Investigações prévias envolvendo um polimorfismo no códon 16 do gene da SOD2 no qual ocorre uma troca de uma valina por uma alanina (Val16Ala-SOD2), têm associado maior eficiência da enzima SOD2 com risco elevado de alguns tipos de câncer. Entretanto, em certos tipos de tumores, como o câncer colorretal os resultados são conflitantes. Estudos sugerem que os níveis elevados de SOD2 em células de tumores colorretais estão associados com a progressão do tumor. Possivelmente esta dificuldade em definir o papel da SOD2 na biologia do câncer colorretal esteja vinculado a grande influência de fatores ambientais sobre o sistema gastrointestinal, com destaque a dieta. Por este motivo, o desenvolvimento de um modelo farmacológico de desbalanço para investigar o papel do desbalanço ânion superóxido-peroxido de hidrogênio (Superoxide Anion Hydrogen Peroxide imbalance, AS-HP) no câncer colorretal pode ser considerado relevante. Objetivo: investigar o efeito in vitro do desbalanço farmacológico do AS-HP causado pela exposição ao paraquat e a porfirina na viabilidade e taxa proliferativa da linhagem comercial de células de câncer colorretal (HT-29) e na resposta destas células ao quimioterápico oxaliplatina. O estudo também avaliou o efeito do desbalanço AS-HP na modulação da expressão de genes apoptóticos, do ciclo celular e oxidativos nas células HT-29. Métodos. Células HT-29 obtidas da American Type Culture Collection (ATCC) foram cultivadas em meio DMEM, 10% de soro bovino fetal, 1% de antibióticos e antifúngicos em estufa com CO2 a 5% e temperatura de 37oC. Após 24 h da transferência das células para placas de 96 poços na concentração de 10 5 células por poço, estas foram expostas a concentração de 0,1 uM de paraquat, que é uma molécula geradora de superóxido, ou de porfirina, que é uma molécula com efeito similar a enzima SOD2. Parte das células foi tratada com oxaliplatina na concentração de 20uM e outra não. O efeito na viabilidade, proliferação celular, ciclo celular, apoptose, e na modulação dos genes do ciclo celular, apoptose e metabolismo oxidativo (β-actina, SOD1, SOD2, CAT, GPX, Caspase 3, Caspase 8, BAX, BCL-2 e P53) também foram avaliados. Os ensaios foram realizados em triplicatas e comparados por análise de variância de uma via seguido de teste post hoc de Tukey. Resultados: o desbalanço farmacológico AS-HP obtido via exposição das células de câncer colorretal ao paraquat e porfirina alterou o padrão de viabilidade, ciclo celular e na modulação da expressão gênica. Tanto o paraquat quanto a porfirina na concentração 0,1 uM diminuíram a viabilidade e a taxa de proliferação das células HT-29. No entanto, este efeito foi mais pronunciado em células expostas ao paraquat. A ação da oxaliplatina foi potencializada pela presença do paraquat quando foram analisadas a taxa de mortalidade, apoptose, taxa de proliferação celular. O paraquat também induziu interrupção do ciclo celular nas fases S e G2 / M. Tanto o paraquat quanto a porfirina foram capazes de modular diferencialmente marcadores do metabolismo oxidativo e a expressão dos genes investigados. Entretanto, os resultados foram bastante heterogêneos. Esta heterogeneidade pode estar relacionada com a instabilidade cromossômica de células tumorais que apresentam níveis mutacionais altos e variados. Conclusão: os resultados obtidos corroboram a hipótese de que o desbalanço AS-HP age sobre a biologia do câncer colorretal, e que em especial o aumento nos níveis de superóxido, não só aumentam a taxa de mortalidade mas também inibem a proliferação celular, potencializando assim, a ação antitumoral da oxaliplatina. Estes resultados podem ser clinicamente relevantes na construção de estratégias farmacológicas e/ou nutricionais, como um adjuvante ao tratamento o uso de vitaminas ou outros suplementos dietéticos, que atuem no balanço AS-HP e que auxiliem no sucesso do tratamento quimioterápico da doença.

Abstract About one-third of mammalian proteins are secreted proteins and membrane proteins. Most of these proteins contain disulfide bonds in their native state, covalent links formed between the thiol groups of cysteine residues. In many proteins, disulfide bonds play an essential role in folding, stabilizing structure and the function of the protein. Therefore, understanding the pathways of disulfide bond formation is crucial for a wide range of medical processes and therapies. Disulfide bond formation is catalyzed by the Protein Disulfide Isomerase (PDI) family. To date the mechanisms of the PDIs in disulfide bond formation and pathways for disulfide bond formation have not been fully characterized. Here the structure of the substrate binding <b>b’x</b> domain of human PDI was determined. The structure shows that the<b> b'</b> domain has a typical thioredoxin fold and that the <b>x</b> region can interact with the substrate binding site of the <b>b'</b> domain. Specifically, the <b>x</b> region of PDI can adopt alternative conformations during the functional cycle of PDI action and that these are linked to the ability of PDI to interact with folding substrates. In addition, this study showed that two human proteins, GPx7 and GPx8 are involved in disulfide bond formation. The addition of GPx7 or GPx8 to a folding protein along with PDI and peroxide allows the efficient oxidative refolding of a reduced denatured substrate protein. Finally, this thesis includes the development of a system for the efficient production of disulfide bond containing proteins in the cytoplasm of E. coli. It showed that the introduction of Erv1p, a sulfhydryl oxidase and FAD-dependent catalyst of disulfide bond, allows the formation of native disulfide bonds in the cytoplasm of E. coli even without the disruption of genes involved in disulfide bond reduction. Introduction of Erv1p and a disulfide isomerase, e.g. PDI, allows the efficient formation of natively folded eukaryotic proteins with multiple disulfide bonds in the cytoplasm of E. coli. This system is able to express high levels of complex disulfide bonded eukaryotic proteins. / Tiivistelmä Noin kolmasosa kaikista nisäkkäiden proteiineista on solun ulkopuolelle eritettäviä proteiineja ja kalvoproteiineja. Monet näistä proteiineista sisältävät natiivissa konformaatiossaan disulfidisidoksia, jotka ovat kovalenttisia sidoksia kysteiinitähteiden tioliryhmien välillä. Useissa proteiineissa näillä disulfidisidoksilla on keskeinen rooli proteiinin laskostumisessa, kolmiulotteisen rakenteen stabiloinnissa sekä proteiinin toiminnassa. Disulfidisidosten muodostumisen taustalla olevien mekanismien tunteminen onkin tärkeää monien lääketieteellisten prosessien ja hoitomenetelmien kannalta. Disulfidisidosten muodostumista katalysoivat proteiinidisulfidi-isomeraasi (PDI) -perheeseen kuuluvat entsyymit. PDI entsyymien toimintamekanismeja ja disulfidisidosten muodostumisen reaktioreittejä ei kuitenkaan vielä tunneta tarkasti. Tässä väitöskirjassa selvitettiin ihmisen PDI entsyymin substraattia sitovan <b>b’x</b> alayksikön rakenne. Rakenteesta voidaan todeta <b>b’</b> alayksikön laskostuminen tyypilliseen tioredoksiini muotoon sekä <b>x</b> alueen interaktio <b>b’</b> alayksikön substraattia sitovan kohdan kanssa. PDI entsyymin katalysoiman reaktioketjun aikana <b>x</b> alayksikkö voi muuttaa konformaatiotaan mahdollistaen PDI entsyymin interaktion laskostuvien substraattiproteiinien kanssa. Tässä tutkimuksessa osoitettiin myös kahden ihmisen proteiinin, GPx7 ja GPx8 osallistuminen disulfidisidosten muodostumista katalysoiviin reaktioihin. GPx7 ja GPx8 entsyymien lisäys laskostumisreaktioon yhdessä PDI:n ja vetyperoksidin kanssa mahdollistaa pelkistetyn, denaturoidun substraattiproteiinin tehokkaan, hapettaviin reaktioihin perustuvan uudelleenlaskostumisen natiiviin muotoonsa. Osana tätä väitöstutkimusta kehitettiin menetelmä, joka mahdollistaa disulfideja sisältävien proteiinien tehokkaan tuoton E.colin solulimassa. Menetelmässä sulfhydryylioksidaasina ja FAD:sta riippuvana disulfidisidosten muodostumisen katalysaattorina toimiva Erv1p mahdollistaa disulfidisidosten muodostumisen E.colin solulimassa myös ilman solun pelkistävien reaktioreittien geneettistä poistamista. Erv1p yhdessä disulfidi-isomeraasin, kuten PDI, kanssa mahdollistaa oikein laskostuneiden, useita disulfidisidoksia sisältävien eukaryoottisten proteiinien tehokkaan tuotannon E.colin solulimassa. Menetelmällä pystytään tuottamaan suuria määriä monimutkaisia disulfidisidoksellisia proteiineja.

Abstract Water in the coastal areas of Ostrobothnia typically contains high levels of humus, iron and manganese. Organic matter and iron contained in groundwater form compounds that make water treatment more difficult. The treatment process of such water usually resembles that used in a traditional chemical water treatment plant. This kind of chemical treatment process is, however, an expensive alternative in view of the resources of a small or a medium-sized treatment plant. The aim of this research was to find out whether ozonation and oxidation with hydrogen peroxide can be used for the treatment of water containing high amounts of organic matter. Ozone is rather widely used in surface water plants for the final treatment of water. The most common applications include the disinfection and oxidation of substances that cause unpleasant taste and odour in water. This research deals with the use of ozone for the treatment of groundwater containing iron and manganese. Since ozone is a powerful oxidizer, it is able to oxidize iron and manganese contained in water into an insoluble form. Also a noticeable amount of organic matter is precipitated in the same process. However, in some of the researched cases the precipitate was so fine that it was impossible to separate it with traditional methods. A combination of ozonation and microfiltration proved to be a successful form of treatment when dealing with this type of water. Ozonation increased the AOC of water in all of the researched cases. The content of bromide is reasonably low in Finnish groundwater. The median measured in coastal Northern Ostrobothnia was 0.025 mgl-1. With the used ozone dosage, the bromate formed in the process stayed below the limit value of 10 μgl-1. Therefore it can be stated that the formation of bromate does not hinder ozone treatment from becoming a more common form of water treatment in Finland. However, the formation of bromate must be taken into consideration, especially if ozonation is made in hig pH. Hydrogen peroxide can be used as an alternative chemical in the oxidation and precipitation processes of iron in groundwater. The chemical is used for the oxidation of iron before filtration. If the formed precipitate is fine, microfiltration or flocking chemical can be used to remove the precipitate. / Tiivistelmä Pohjanmaan rannikkoseudun pohjavesille on tyypillistä korkeat rauta- ja mangaanipitoisuudet yhdistettynä suureen humuspitoisuuteen. Orgaaninen aine muodostaa pohjaveden raudan kanssa yhdisteitä, jotka vaikeuttavat vedenkäsittelyn toimintaa. Kyseisten vesien käsittely lähestyy yleensä perinteistä kemiallista pintavesilaitosta. Kuitenkin pienten ja keskisuurten vesilaitosten resursseihin nähden täydellinen kemiallinen käsittely on kallis vaihtoehto. Tämän tutkimuksen tarkoituksena oli selvittää otsonoinnin ja vetyperoksidihapetuksen käyttökelpoisuutta näiden runsaasti orgaanista ainetta sisältävien vesien käsittelyssä. Otsonoinnin yleisimmät sovellutukset ovat hajua ja makua aiheuttavien aineiden hapettaminen ja desinfiointi. Tässä tutkimuksessa selvitettiin otsonin käyttöä rauta- ja mangaanipitoisten pohjavesien käsittelyssä. Otsoni saa voimakkaana hapettimena vedessä olevan raudan ja mangaanin hapettumaan ei liukoiseen muotoon. Samalla kerasaostui huomattava määrä orgaanista ainetta. Kuitenkin muodostuva sakka on joissain tapauksissa niin hienojakoista, että sen erottaminen perinteisin menetelmin on mahdotonta. Näiden ns. ongelmavesien käsittelyssä saatiin hyviä tuloksia otsonoinnin ja mikrosuodatuksen yhdistelmällä. Otsonointi nosti huomattavasti kaikkien tutkittujen vesien AOC-pitoisuutta. Bromidipitoisuudet suomen pohjavesissä olivat kohtuullisia. Pohjanmaan rannikkoseudun pohjavesien bromidipitoisuuksien mediaani oli 0,025 mgl-1. Kaikilla vesillä käytännön otsoniannostuksilla syntyvät bromaattimäärät alittivat 10 μgl-1 raja-arvon. Bromaattiongelma ei ole este otsonikäsittelyn yleistymiselle Suomessa. Se on kuitenkin tiedostettava, varsinkin, jos otsonointi tehdään korkeassa pH:ssa. Vetyperoksidi on vaihtoehtoinen kemikaali pohjaveden raudan hapettamiseen ja saostamiseen. Vetyperoksidia voidaan käyttää raudan saostamiseen ennen suodatusta. Mikäli muodostuva sakka on hienojakoista, voidaan sakanerotus tehdä mikrosuodatuksella tai käyttää hyväksi flokkauskemikaaleja.

[pt] No Brasil está em larga expansão o uso de geração de eletricidade por termoeléctricas. Na geração de eletricidade por usinas térmicas são utilizados grandes quantidades de água e produtos químicos, que após utilização geram efluentes. A hidrazina é um produto químico usado para controle de corrosão em águas de caldeiras, sistemas de vapor e outros sistemas de usinas térmicas que após utilização acaba incorporada aos efluentes líquidos destas usinas. Com intuito de promover uma sistemática de controle de efluentes produzidos nas usinas, procurou-se, nesta dissertação, estudar efluentes contendo hidrazina com enfoque tecnológico. O processo abordado neste estudo consistiu no tratamento de efluentes contendo hidrazina, utilizando peróxido de hidrogênio com auxílio de catalisador de íons de cobre, para decomposição da hidrazina. Os ensaios foram feitos em laboratório, utilizando-se efluentes sintéticos com concentrações pré- determinadas de hidrazina que variaram entre 10 e 100 mg/L, com controle do pH que variou em 7 e 9,5, temperatura fixada em 220C, com adição de concentrações calculadas de peróxido de hidrogênio e catalisador de sulfato de cobre. Concluiu-se que o processo é viável para reduzir a concentração de hidrazina em efluentes a níveis inferiores aos limites da legislação (1 mg/L), utilizando-se quantidades estequiométricas de peróxido de hidrogênio em conjunto com sulfato de cobre em concentrações de 1 mg/L de Cu 2+ como catalisador, em efluentes com pH 9,5, a temperatura ambiente, em tempos inferiores a 30 minutos. Assim sendo, o trabalho mostrou-se adequado para satisfazer as condições de descarte de efluentes em águas brasileiras de acordo com a resolução CONAMA 20, carta P-031/01 cláusula 2 artigo V, de 9 de Fevereiro de 2001. / [en] In Brazil, the use of energy produced by power plant generators is in expansion. Power plants use large quantities of water and chemical products that after use end up in effluents. Hydrazine is used in water systems for corrosion control, because of its excelents oxygen scavenging capacity. The present work was conducted to study the treatment of effluents containing hydrazine, under a technological approach, with the purpose of contributing to a systematic of effluents control in power stations. The process studied in this work was the decomposition of hydrazine with hydrogen peroxid in presence catalyst cooper íon. The experiments were made in laboratory scale, using synthetic effluents with initial concentration of hydrazine at the levels 10 and 100 mg/L, with initial pH values 7 and 9,5, temperature fixed at 220C, with addition of st oichiometric amounts of hydrogen peroxide, with and without addition of cooper ion catalyst. It was conclued that the process its viable for reduction of hydrazine concentration in effluents with pH 9,5, below to levels under legislation (1mg/L), using stoichiometric amounts of hydrogen peroxide together with 1 mg/L of cooper ion, in times less that 30 minutes and ambient temperature. Therefore this work showed that the process is adequate in satisfying the Brazilian legislation for discharge of effluents into water bodies according to regulation CONAMA 20, letter P-031/01 clause 2 article V, 09 February 2001.

[pt] Estudos prévios relacionam a ocorrência de episódios de mortandade de peixes em corpos hídricos como a Lagoa Rodrigo de Freitas (LRF) com a rápida disponibilização de espécies poluentes e nutrientes naturais na coluna d água especialmente durante altas precipitações de chuva, quando ocorre transbordo dos rios poluídos da bacia sobre a água da Lagoa, ocasionando uma demanda de oxigênio dissolvido maior do que o normal para depuração de tais espécies. Nesse contexto existe interesse em evitar episódios críticos de insuficiência de OD na água dos corpos hídricos que possam advir de tais eventos. Estudos realizados no presente trabalho tiveram como objetivo caracterizar as águas de rios da Sub-bacia hidrográfica da LRF através de DBO, COT, SST, Ptotal no canal a montante de deságue para a Lagoa em períodos de chuva e de tempo seco; e avaliar um possível tratamento que proporcione a depuração acelerada dos poluentes utilizando peróxido de hidrogênio, fornecendo oxigênio para as águas poluídas através do processo de decomposição do oxidante. Avaliaram-se diferentes dosagens de H2O2 em tempo reacional de 24h de acordo com limites de ecotoxicidade conhecidos. Testes realizados em amostras de rio coletadas em dias de baixa precipitação contendo concentrações de DBO de até 2,2 mg/L mostraram uma velocidade de decaimento de H2O2 inferior ao para amostras tanto coletadas também em dia de baixa precipitação porém com elevada DBO (24,0 mg/L), quanto para dia de alta precipitação (13,2 mm em 24 h) com relevante concentração de material orgânico. Observou-se uma dosagem suficiente de 15,0 mg/L para as amostras coletadas em baixa precipitação e alta DBO, e dosagem suficiente de 3,0 mg/L para amostras coletas em maior evento de precipitação (13,2 mm em 24 h), acima das quais, não ocorre mais aumento significativo da velocidade de decaimento da [H2O2] e também de velocidade de contribuição de OD para a água. Concluiu-se que a adição de H2O2 nas águas de rios durante eventos de poluição causados por chuvas intensas ou lançamento de esgoto pode contribuir para evitar episódios críticos de insuficiência de OD em rios poluídos por material orgânico e na pluma de poluentes que pode ser formada por transbordo dos rios para a LRF. / [en] Previous studies have associated the occurrence of episodes of death of fish in water bodies such as the Lagoa Rodrigo de Freitas (LRF) to to the rapid availability of pollutants and natural nutrients in the water column species especially during high rain precipitation events, which occur when the rivers overflow and pollute the water of the lagoon, causing a biochemical oxygen demand higher than usual for the rate of natural depuration of the contaminating species. In this context there is interest in avoiding critical episodes DO deficiency in the water bodies that may arise from such events. Studies conducted in the present work aimed at characterizing the rivers of sub-basin of LRF through BOD, TOC, TSS, Ptotal on the canal that overflows into the lagoon in periods of rain and dry weather; and evaluate a possible treatment offering the accelerated depuration of pollutants using hydrogen peroxide, providing oxygen to the polluted water through the self-decomposition process. The study evaluated the effect of different doses H2O2 in 24 hours of reaction time according to known ecotoxicity limits. In tests on samples collected from rivers in days of little rain containing BOD concentrations up to 2.2 mg / L, H2O2 showed a decay rate lower than those of other samples also collected on days of low precipitation, but with high BOD (24, 0 mg / L), and days of high rainfall (24 hours 13.2 mm), with a significant concentration of dissolved organic contaminants. A maximum sufficient dose of 15.0 mg / L was found for the low and high samples precipitation BOD, and a maximum sufficient dose of 3.0 mg / L for most of the samples collected during the precipitation event (13.2 mm 24 hours), above which there is no significant increase over the rate of decomposition of [H2O2], and the rate of generation of DO in the water. It was concluded that the addition of H2O2 into the waters of rivers during pollution events caused by heavy rains or sewage release can help to avoid critical episodes of DO deficiency in polluted rivers by organic matter and pollutant plume that can be formed by overflow of those rivers to the LRF lagoon.

La législation européenne impose la recherche d'entérotoxines staphylococciques dans les fromages si le niveau de S. aureus est supérieur à 105 ufc/g. Dans cette optique, l'objectif de cette thèse était de fournir des éléments scientifiques pour contrôler S. aureus par biopreservation. Dans un premier temps, la croissance de S. aureus a été suivie dans des fromages fermiers AOC Saint-nectaire. Dans ces fromages les populations de S. aureus se multiplient essentiellement durant les six premières heures de fabrication pour atteindre un maximum à 24 h. En fromages au lait cru, leur croissance et leur niveau maximal dépendraient de la concentration initiale dans le lait, du pH (en particulier pH à 6 h), de la température, mais le rôle des communautés microbiennes n'a pas pu être mis en évidence. Dans un deuxième temps, les capacités inhibitrices de souches de Lactococcus lactis et de Lactococcus garvieae ont été évaluées en lait microfiltré. L. lactis subsp. lactis inhiberait la croissance de S. aureus par abaissement du pH mais aussi par production d'H2O2 pour certaines souches. Par contre, le pH ne serait pas responsable de l'inhibition de S. aureus par L. garvieae. La compétition pour des acides aminés ne semblerait pas impliquée dans l'inhibition par L. lactis puisque cette espèce en synthétise de grande quantité en lait. Même si la thréonine, la phénylalanine, la méthionine, l’isoleucine et la valine deviennent limitants dans la co-culture de S. aureus avec L. garvieae, cette carence ne serait pas responsable de l'inhibition puisque leur ajout ne lève pas l'inhibition. Cependant du peroxyde d'hydrogène (3 mmol/l) produit par L. garvieae en co-culture en milieu BHI serait responsable de l'inhibition. En milieu de laboratoire, l'inhibition de S. aureus par L. garvieae est effective durant les premières heures de culture et diminue en cours d'incubation. Elle augmente avec la concentration de L. garvieae. Elle est plus forte à 24°C qu'à 30°C. Le rôle de la catalase de S. aureus dans l'interaction avec L. garvieae reste à préciser / The European legislation imposes the staphylococcal search for enterotoxins in cheeses if the level of S. aureus is higher than 105 ufc/g. Accordingly, the objective of this thesis was to provide scientific data to control S. aureus by biopreservation.In a first, the growth of S. aureus was monitored in farm cheeses AOC Saint-Nectaire. In these cheeses the populations of S. aureus multiplied mainly during the first six hours of manufacture to reach a maximum to 24 h. Their growth and their maximum level would depend on the initial concentration in milk, on the pH (in particular pH at 6 h), on the temperature, but the role of the microbial communities could not be highlighted. In the second step, the inhibiting capacities of strains of Lactococcus lactis and Lactococcus garvieae were evaluated in microfiltrated milk. L. lactis subsp. lactis would inhibit the growth of S. aureus by lowering the pH but also by the production of H2O2 for certain strain. On the other hand, the pH would not be responsible for the inhibition of S. aureus by L. garvieae. The competition for amino acids does not seem to be implied in inhibition by L. lactis since this species synthesis a great amount in milk. Even if threonine, phenylalanine, methionine, isoleucine and valin become limiting in the co-culture of S. aureus with L. garvieae, this would not be responsible for inhibition since their addition in milk does not raise inhibition. In laboratory medium the inhibition of S. aureus by L. garvieae was effective during the first hours of culture and decreases during incubation. It increased with the concentrations of L. garvieae. It was stronger at 24°C than at 30°C. It would be due to hydrogen peroxide (3 mmol/l) produced by L. garvieae in Co-culture in medium BHI. The role of the catalase of S. aureus in the interaction with L. garvieae remains to be specified

[pt] O presente trabalho teve como objetivo estudar o tratamento de efluentes contendo cianeto livre através do sistema H2O2/UV e selecionar as condições operacionais mais adequadas para uma maior eficiência do processo. Para isso, foram empregadas soluções sintéticas de KCN com características de pH e concentração similares às condições de um efluente industrial real. O fotorreator utilizado nos testes de oxidação foi um reator cilíndrico de seção anular, equipado com uma lâmpada de baixa pressão de 28 W concêntrica com emissão em 254 nm, onde a solução ficava diretamente em contato com a mesma. Este fotorreator foi acoplado a um sistema de refrigeração que mantinha a temperatura de operação em 25oC.As variáveis avaliadas foram concentração inicial de cianeto em solução, pH inicial da solução, potência de UV irradiada e razão molar [H2O2]/[CN-]. Para soluções contendo uma concentração inicial de cianeto igual a 100 ppm, foi possível atingir uma eficiência remoção de 99,9 por cento em 25 minutos, em pH igual a 9,5, com uma razão molar [H2O2]:[CN-] igual a 3. Para efluentes contendo uma concentração inicial de cianeto igual a 300 ppm, nas mesmas condições operacionais, alcançou-se a mesma eficiência em 30 minutos. / [en] The present work had the objective of studying the treatment of effluents containing free cyanide through the system H2O2/UV, and of selecting the best operational conditions for best efficiency of the process. For that, it was employed synthetic solutions of KCN with characteristics of pH and concentration similar to those of a real effluent. The photoreactor employed in the oxidation tests was a cylindrical reactor of annular section, equipped with a concentrical low pressure lamp of 28 W with emission in 254 nm, where the solution was in direct contact with the lamp. This photoreactor was coupled with a cooling system which kept the operation temperature at 25oC. The evaluated variables were initial cyanide concentration in solution, initial pH of the solution, power of radiated UV and molar ratio [H2O2]/[CN-]. For solutions containing an initial concentration of cyanide equal to 100 ppm, it was possible to reach a removal efficiency of 99.9 per cent in 25 minutes, in pH equal to 9.5, with a molar ratio of [H2O2]:[CN-] equal to 3. For effluents containing an initial concentration of cyanide equal to 300 ppm, at the same operational conditions, it was possible to achieve the same removal efficiency in 30 minutes.

Danos induzidos por hiperglicemia em tecidos no diabetes são caracterizados por quatro mecanismos conectados: aumento do fluxo metabólico através da via do poliol, ativação da proteína quinase C (PKC), aumento da atividade da via das hexosaminas e aumento da produção intracelular dos precursores dos produtos finais de glicação avançada (AGEs). Entre eles, os derivados de metilglioxal, um potente agente de modificação de proteínas e DNA, têm sido associados a complicações microvasculares no diabetes: nefropatia, retinopatia e neuropatia. O metilglioxal é produzido a partir das trioses fosfato, acetona e aminoacetona, um catabólito de treonina e glicina, gerado na matriz mitocondrial. A aminoacetona sofre oxidação enzimática, catalisada por aminoxidase sensível a semicarbazida (SSAO), ou química, catalisada por íons de cobre e ferro, produzindo metilglioxal, H2O2 e NH4 +. Sabendo que metilglioxal e H2O2 são capazes de induzir apoptose e/ou necrose em células produtoras de insulina (RINm5f) propomos uma possível atividade pró-oxidante da aminoacetona sobre células beta do pâncreas. O tratamento destas linhagens com aminoacetona/Cu(II) aumentou a morte celular, fluxo de Ca2+ intracelular, produção de NO, fragmentação do DNA, depleção dos níveis de glutationa reduzida (GSH), expressão gênica da proteína apoptótica Bax, enzimas antioxidantes - glutationa peroxidase (GPx), glutationa redutase (GRd), catalase e isoformas de superóxido dismutases (CuZnSOD e MnSOD) - e óxido nítrico sintase induzida (iNOS). Embora as concentrações normais e patológicas da aminoacetona, provavelmente seja muito menores que as usadas nos experimentos, sugerimos que, em tecidos de diabéticos, um acúmulo da aminoacetona em longo prazo pode conduzir a danos oxidativos e eventualmente morte das células beta do pâncreas / Tissue damages induced by hyperglycemia in diabetics are characterized by four linked mechanisms: increased flux through the polyol pathway, protein kinase C (PKC) activation, increased hexosamine pathway activity and intracellular production of advanced glycation end product (AGE) precursors. The production of AGEs by modifying proteins and DNA agent, such as methylglyoxal, has been implicated in microvascular complications in diabetes: nephropathy, retinopathy and neuropathy. Methylglyoxal is putatively produced in vivo from trioses phosphate, acetone and aminoacetone, a catabolite of threonine and glycine synthesized in the mitochondrial matrix. Aminoacetone has been reported to undergo semicarbazide sensitive amine oxidase- catalyzed and copper- and iron-catalyzed oxidations by molecular oxygen to methylglyoxal, NH4 + ion and H2O2. Considering that methylglyoxal and H2O2 have been found to promote apoptosis/necrosis to insulin-producing cells (RINm5f), we propose a possible pro-oxidant role of aminoacetone in pancreatic beta-cells. Treatment of RINm5f cells with aminoacetone plus Cu(II) ion promotes an increase of non-viable cells, influx of Ca2+ ions, NO production, DNA fragmentation, depletion of reduced glutathione (GSH) levels, and increased mRNA expression of pro-apoptotic protein (Bax), antioxidant enzymes - glutathione peroxidase (GPx), glutathione reductase (GRd), MnSOD, CuZnSOD and catalase - and inducible nitric oxide synthase (iNOS). Although both normal and pathological concentrations of aminoacetone are probably much lower than those used here, it is tempting to propose that excess aminoacetone in diabetic patients, at long term, may drive oxidative damage and eventually death of pancreatic beta-cells

Made available in DSpace on 2016-05-02T13:55:25Z (GMT). No. of bitstreams: 1 Dissertacao Guilherme Zolini1.pdf: 7663 bytes, checksum: 2f28c5d53aef01e6521ccd46bac0339d (MD5) Previous issue date: 2006-09-14 / This work was carried out in the Microorganism Fisiology Biology and Phytopharmacy laboratories of the José do Rosário Vellano University (UNIFENAS) The aim of the study was to evaluate some aspects related to the immunitary activity of neutrophils in rats treated with kefir a probiotic made from various microorganisms The tests were conducted to determine the level of cytokine TNF-alpha cell recruiting cellular metabolism neutrophils oxygen consumption hydrogen peroxide formation by neutrophils screening for myeloperoxidase positive neutrophils and assay of redox titration The tested groups consisted of albino male Wistar rats the test group received 1,0 ml of kefir solution the negative control received 1,0 ml of saline solution (NaCl 0,9 %) and the positive control received 100 mg/kg of tocopherol (Vit E) except for those on TNF-alpha and cell recruiting in which the positive control received 0,5 mg/kg of dexamethasone by 7 days gavage except for redox assay where the groups did not consist of animals The results were analyzed by means +- SEM using comparison test of Student Newman Keuls (SNK) except for respirometry assay for which t Student test was used Significant differences were found in kefir and negative control groups in cell recruiting assays (P<0,05) hydrogen peroxide formation stimulated by forbol ester (P<0,05) and identification of myeloperoxidase (P<0,01) given that both previously mentioned had the same index shown by the positive control For the cell recruiting assay the kefir the positive control and the negative control groups presented 12,0 +- 1,0 x 106, 7,3 +- 1,4 x 106 and 17,2 +- 1,9 x 106 neutrophils/mL respectively On the other hand in the hydrogen peroxide formation stimulated by forbol ester the kefir and the positive control groups presented averages of 1,46 +- 0,16 e 1,50 +- 0,22 femtomol/cell respectively showing no difference The negative control at 2,14 +- 0,18 femtomol/cell was considered statistically different from the kefir group In the assay for myeloperoxidase identification kefir and positive control groups were considered identical with indexes of 54,8 +- 3,0 % 47,3 +- 5,7 % respectively different from the negative control group with index of 74,0 +- 1,9 % positive MPO The other assays did not show significant effects In conclusion the ingestion of kefir was capable of diminishing cell recruiting inhibiting H2O2 production and suggests the reduction of the activity of MPO in the neutrophils / Este trabalho foi realizado nos laboratórios de Fitofármacos e Biologia e Fisiologia de Microrganismos da Universidade José do Rosário Vellano (UNIFENAS) com objetivo de avaliar alguns aspectos relacionados à atividade imunitária de neutrófilos em ratos tratados com que fir um probiótico composto por vários microrganismos Os ensaios foram realizados para se determinar o nível da citocina TNF-alpha recrutamento celular metabolismo celular consumo de oxigênio pelos neutrófilos formação de peróxido de hidrogênio pelos neutrófilos pesquisa de neutrófilos mieloperoxidase positivos e ensaio de titulação redox Os grupos foram formados por ratos Wistar albinos machos onde o grupo teste recebeu 1,0 mL de solução de quefir controle negativo 1,0 mL de solução salina (NaCl 0,9%) e controle positivo recebeu tocoferol (vit E) na dose de 100 mg/kg; nos ensaios de dosagem de TNF-alpha e recrutamento celular, contudo o controle positivo recebeu dexametasona na dose de 0,5 mg/kg por gavagem durante 7 dias exceto em ensaio redox onde grupos não foram formados por animais Os dados obtidos foram analisados por médias +- EPM seguindo teste de comparação de Student Newman Keuls (SNK) exceto para ensaio de respirometria que foi feito teste t de Student Foram encontradas diferenças significativas entre os grupos quefir e controle negativo nos ensaios de recrutamento celular (P<0,05) formação de peróxido de hidrogênio estimulado com ester de forbol (P<0,05) e identificação da mieloperoxidase (P<0,01) observando que ambos anteriormente citados apresentaram índices iguais ao controle positivo Para o ensaio de recrutamento celular os grupos quefir controle positivo e controle negativo apresentaram 12,0 +- 1,0 x 106 7,3 +- 1,4 x 106 e 17,2 +- 1,9 x 106 neutrófilos/mL respectivamente Já no ensaio de formação de peróxido de hidrogênio estimulado com ester de forbol os grupos quefir e controle positivo apresentaram médias de 1,46 +- 0,16 e 1,50 +- 0,22 femtomol/cel respectivamente não evidenciando diferença o controle negativo apresentou valor de 2,14 +- 0,18 femtomol/cel sendo considerado estatisticamente diferente ao grupo quefir No ensaio de identificação da mieloperoxidase o grupo quefir e controle positivo foram considerados iguais com índices de 54,8 +- 3,0 % e 47,3 +- 5,7 % respectivamente e diferentes do grupo controle negativo com índice de 74,0 +- 1,9 % MPO positivo Os demais ensaios não apresentaram efeitos significativos Concluiu-se que a ingestão do quefir foi capaz de diminuir o recrutamento de neutrófilos inibir a produção de H2O2 e sugere a diminuição da atividade de MPO nos neutrófilos