The Gram negative bacterium Thauera selenatis is able to respire with selenate as the sole terminal electron acceptor, utilising a periplasmic selenate reductase enzyme to reduce selenate to selenite. Previous characterisation of this enzyme has shown that it is a heterotrimeric molybdo-enzyme (SerABC) of the dimethylsulfoxide reductase family, containing a Mo-bis molybdopterin guanine dinucleotide co-factor, Fe-S clusters and a b-type haem (Schroder et al., 1997, J Biol Chem, 272: 23765-68, Dridge et al., 2007, Biochem J, 408: 19-28). In order to elucidate the electron transport pathway to selenate reductase, and how it can generate a proton motive force, detailed study was required. Firstly, the redox potential of the b-haem of SerC was determined by optical redox titration to be +234 mV. The serC gene was cloned and expressed heterologously in E. coli, but the protein was incorrectly folded into inclusion bodies, and attempts to refold and reconstitute SerC with haem were unsuccessful. A profile of c-type cytochromes in T. selenatis was undertaken, and characterisation of a number of cytochromes was carried out. Two cytochromes were purified, cytc7 and cytc4, and cytc4 was shown to be able to donate electrons to SerABC in vitro. Protein sequence was obtained by N-terminal sequencing and LC-MS/MS, and assigned cytc4 to the cytochrome c4 family of dihaem cytochromes. Redox potentiometry combined with UV-visible and electron paramagnetic spectroscopy showed that cytc4 is a dihaem cytochrome with a redox potential of +282 mV and both haems are predicted to have His-Met ligation. To investigate the role of membrane bound cytochromes in selenate respiration, PCR with degenerate primers amplified a partial gene coding for quinol: cytochrome c oxidoreductase (QCR). A microplate growth method was developed to monitor growth of T. selenatis under reproducible conditions, and used to analyse the effect of respiratory chain inhibitors on growth under different conditions. Aerobic metabolism was unaffected by QCR inhibitors, while nitrite reduction was totally inhibited, linking nitrite reduction to the generation of a proton motive force by the QCR. The QCR inhibitor myxothiazol partially inhibited selenate respiration, showing that some electron flux is via the QCR, but total inhibition of selenate respiration was achieved by combining myxothiazol with the more general inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO). These data suggest that electron transfer to selenate reductase occurs via a branched pathway, in which one route is inhibited by myxothiazol and the other by HQNO. Electron transfer via a QCR and a dihaem cytochrome c4 is a novel route for a member of the dimethylsulfoxide reductase family of molybdo-enzymes.

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Orientador: Priscila Lupino Gratão / Resumo: As plantas estão expostas a adversidades no ambiente que as circundam, como a contaminação por cádmio (Cd). Este metal pesado tem aumentado na atmosfera devido a atividades humanas. As plantas podem absorver o Cd, causando sérias alterações estruturais, fisiológicas e bioquímicas. As plantas desenvolveram sistemas de defesa complexos, incluindo mecanismos não enzimáticos e enzimáticos para evitar uma cascata de oxidação descontrolada causada pelo estresse oxidativo. Alguns elementos, como o selênio (Se), se utilizados em concentração adequadas, podem induzir uma melhora no sistema antioxidante, no crescimento e nos atributos fotossintéticos. Ainda é pouco conhecido o papel do Se nas respostas das plantas ao estresse. O objetivo deste trabalho foi obter novas informações sobre o papel do selenato e selenito no sistema de desintoxicação das plantas, incluindo a avaliação da nutrição mineral, atividade de enzimas antioxidantes e conteúdo de compostos não enzimáticos, pigmentos, alterações estruturais e o papel do Se na modulação do etileno, com o uso de mutantes hormonais como ferramenta. Nossos dados indicam que o Se é uma estratégia interessante para melhorar o metabolismo da planta sob condições normais ou estressantes. O selênio pode induzir aumento da ação do metabolismo de defesa antioxidante, provavelmente devido a alterações na sinalização do etileno. Além disso, em condições normais, o Se induz alterações estruturais nas células, o que pode contribuir para o desenvolvim... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Cadmium (Cd) contamination is a worldwide concern and one of the most severe causes of abiotic stress in plants, triggering losses in crop production and contamination risks to human health. This heavy metal increased in atmosphere due to human activities. Plants can uptake Cd, causing serious changes in structural, physiological and biochemical processes. Plants developed a complex defence systems including non-enzymatic and enzymatic mechanism to avoid oxidative stress and prevent an uncontrolled oxidation cascade. Some elements, such as selenium (Se), if used in adequate concentration, may induce an improvement in antioxidant system, growth and photosynthetic attributes. It is still unknown the mechanisms of Se in stress responses. The aim of this work was get new insights about the role of selenate and selenite-mediated detoxification strategies, including the evaluation of mineral nutrition, the activity of antioxidant enzymes and non enzymatic compounds, pigments, structural alterations and the role of Se in modulate ethylene, with the use of hormonal mutants as a tool. Our data indicates that Se is an interesting strategy to improve plant metabolism under normal or Cd stressful-condition. Selenium may induce enhancement in antioxidant defence metabolism, probably due to alterations in ethylene signalling. Moreover, under normal condition Se induce structural alterations in cells, which may contribute to plant development. Thus, the information available in this work is... (Complete abstract click electronic access below) / Doutor

Three strains of the green alga Scenedesmus quadricauda were selected to be specifically resistant to the presence of high concentrations of inorganic Se. The strain SeIV was found to be resistant to high doses of selenite, the strain SeVI was resistant to selenate, and the strain Se(IV+VI) to the presence of both Se compounds. It has been shown that both the dose and chemical form of Se are the critical factors in the cellular response. In this PhD. thesis are results from experiments with the resistant strains (activity of thioredoxin reductase, content of selenium and selenomethionine, effect of sulphur deficiency).

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Organic ligands, such as EDTA, accelerate the dissolution of silicate and oxide minerals. In natural systems oxyanions can compete with organic ligands for mineral surface sites thereby affecting ligand-promoted dissolution rates. Oxyanions can either enhance or inhibit dissolution depending upon pH. It has been suggested that the type of surface complex formed by the oxyanion is responsible for differences in ligand-promoted dissolution rates with mononuclear being dissolution enhancing and bi- or multinuclear being dissolution inhibiting. However, recent research has demonstrated that the type of surface complex formed by the oxyanion is not responsible for differences in ligand-promoted dissolution rates of oxide minerals. Accordingly, this manuscript examines the influence of selenite, molybdate, and phosphate on the EDTA-promoted dissolution of goethite and proposes a mechanism for the observed differences in dissolution rates over a pH range of 4 - 8. We propose that the surface complex formed by EDTA is the controlling factor for the observed dissolution rate, with mononuclear EDTA surface complexes accelerating goethite dissolution compared to bi- or multinuclear complexes. These experimental results suggest that EDTA forms multinuclear complexes with goethite surfaces at pH values greater than or equal to 6 and mononuclear complexes with goethite surfaces at pH values < 6. Dissolution results show that when the oxyanion and the EDTA are present in the system at concentrations nearly equaling the surface sites available for adsorption, the oxyanion reduces the adsorption of EDTA and inhibits dissolution over the pH range of 4 - 8. However, a different mechanism occurs at pH values greater than or equal to 6 when the oxyanion is present at 0.1 mM. EDTA adsorbs but the number of carboxylic groups that can sorb to the surface is reduced causing the formation of mononuclear complexes. This shift from multi- to mononuclear surface complexes enhances the EDTA-promoted dissolution of goethite in the presence of the oxyanions compared to EDTA-promoted dissolution in their absence. / Master of Science

Cataract disease affects millions of people worldwide. It is characterized by the accumulation of light-scattering bodies within the lens that reduce visual acuity. Cataracts are effectively treated surgically, but at great expense, costing Medicare $3.4 billion in 1997. Development of an alternative therapy for this disease would provide medical and economic benefits. We have investigated a novel antioxidant, the superoxide scavenger Mn(III)-salophen, as a therapeutic agent in the selenite cataract model. Mn(III)-salophen has been shown to protect E. coli colonies against oxidative stress but was untested in a eukaryotic system. A total dose of 300 mmol/kg, given IP in four equal 75 mmol/kg doses spaced four hours apart, protects 75% of neonatal rats from nuclear cataract development five days after selenite injection. Selenite is toxic through its reaction with the endogenous antioxidant glutathione (GSH). The reduction of selenite to selenide through an intermediate, selenodiglutathione (GSSeSG) leads to generation of superoxide radical, one of several toxic oxygen species that can damage the lens. Mn(III)-salophen causes an in vitro preservation of the lifetime of GSSeSG by interrupting the reduction of selenite. We have established that the reduction of GSSeSG to selenide does not use GSH as a reducing agent, but rather depends upon electrons generated in the earlier reduction of selenite to selenodiglutathione. These electrons can be intercepted by known one-electron scavengers, arresting the metabolism of GSSeSG. Extensive proteolysis of lens crystallins and loss of calcium homeostasis occur in cataractous lenses from a rat treated with sodium selenite. The visual protection with Mn(III)-salophen is accompanied by a partial loss of the calcium homeostasis, a net increase in sodium, and calpain-mediated proteolysis of à -crystallins similar to lenses from animals treated with selenite alone. Although preservation of alpha-crystallins may contribute to the greater transparency in the protected lens, generalized à -crystallin proteolysis is insufficient for cataract formation. From these experiments we propose that Mn(III)-salophen minimizes the oxidative stress imposed upon the cell by interfering with the metabolism of selenodiglutathione. This allows the cell to compensate for the loss of cation homeostasis and prevents aggregation of proteolyzed crystallins into cataracts. / Ph. D.

Submitted by Eliza Barboza (agro_eliza@hotmail.com) on 2018-06-08T14:26:27Z No. of bitstreams: 1 Eliza Barboza TESE versão.pdf: 1545927 bytes, checksum: ac8fd976ca18df84595a993ec2a3e701 (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-06-12T16:57:44Z (GMT) No. of bitstreams: 1 barboza_e_dr_jabo.pdf: 1545927 bytes, checksum: ac8fd976ca18df84595a993ec2a3e701 (MD5) / Made available in DSpace on 2018-06-12T16:57:44Z (GMT). No. of bitstreams: 1 barboza_e_dr_jabo.pdf: 1545927 bytes, checksum: ac8fd976ca18df84595a993ec2a3e701 (MD5) Previous issue date: 2018-04-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo deste trabalho foi avaliar concentrações de selênio (selenato e selenito de sódio) no crescimento, produção e biofortificação de duas cultivares de repolho. Para isso foram realizados dois experimentos em casa de vegetação, em sistema hidropônico, no período de julho a novembro de 2014, na UNESP, campus Jaboticabal-SP. Os experimentos diferiram conforme a fonte de selênio (Se) utilizada (selenato e selenito de sódio). Em ambos os experimentos, avaliaram-se dois fatores, sendo: duas cultivares de repolho (‘Fênix’ e 'Red Dinasty’) e cinco concentrações de Se (0, 5, 15, 30 e 60 µmol L-1) na solução nutritiva. O delineamento experimental utilizado foi o de blocos casualizados, com esquema fatorial 2 x 5 e quatro repetições. As variáveis analisadas foram o teor e acúmulo de Se na raiz e cabeça de repolho, área, diâmetro, comprimento e densidade de raiz, produtividade de repolho, massa seca de raiz, caule e cabeça de repolho e o teor e acúmulo de nitrogênio, fósforo e enxofre na cabeça de repolho. As fontes selenato e selenito promoveram o crescimento das raízes de repolho devido aos aumentos verificados em área, diâmetro, comprimento e densidade de raiz, em baixas concentrações de Se aplicadas. O aumento da produção de massa seca de raiz provavelmente foi devido o maior crescimento da raiz, que foi influenciado pelo fornecimento de Se. A massa seca do caule foi diferente apenas entre as cultivares de repolho com aplicação de selenato ou selenito. O teor de nitrogênio apresentou diferenças apenas entre as cultivares quando utilizada a fonte selenito. Ambas as fontes de Se também proporcionaram aumentos na absorção de fósforo e enxofre quando baixas concentrações de Se foram fornecidas. Observou-se que a aplicação de até 16,77 e 7,54 µmol L-1 de Se com as fontes selenato e selenito, respectivamente, proporcionaram os maiores incrementos na produtividade do repolho, e mesmo com a redução da produtividade com concentrações acima destas, os valores foram maiores que o tratamento controle. Isso indica que o Se foi benéfico ao repolho, pois não reduziu a sua produção. Verificou-se que a biofortificação foi eficiente para ambas as cultivares de repolho, que absorveram e acumularam Se na cabeça de repolho em função do incremento de Se, tanto com selenato como selenito. / The objective of this research was to evaluate concentrations of selenium (sodium selenate and selenite) in the growth, production and biofortification of two cabbage cultivars. Two experiments were carried out in greenhouse under hydroponic system, from July to November, 2014, at UNESP, Jaboticabal-SP campus. The experiments differed according to the source of selenium (Se) used (sodium selenate and selenite). In both experiments two factors were evaluated: two cabbage cultivars ('Fênix' and 'Red Dinasty') and five concentrations of Se (0, 5, 15, 30 and 60 μmol L-1) in the nutrient solution. The experimental was a randomized block design, with 2 x 5 factorial and four replications. The variables analyzed were the content and accumulation of Se in the root and shoot cabbage, evaluating area, diameter, length, density and dry mass in the root, cabbage yield, stem and cabbage head, and the content accumulation of nitrogen, phosphorus and sulfur in the cabbage shoot. Selenate and selenite sources promoted the growth of cabbage roots due to increases in area, diameter, length and root density at low concentrations of Se applied. The increase in root dry mass production was probably due to higher root growth, which was influenced by the supply of Se. The dry mass of the stem was different only between the cabbage cultivars with application of selenate or selenite. The nitrogen content presented differences only among the cultivars when the selenite source was used. Both S sources also provided increases in phosphorus and sulfur absorption when low concentrations of if were provided. It was observed that the application of up to 16,77 and 7,54 μmol L-1 of Se with selenate and selenite sources, respectively, provided the greatest increases in cabbage yield, and even with the reduction of productivity with concentrations above these, the values were higher than the control treatment. These indicate that the Se was beneficial to the cabbage, since it did not reduce its production. It was verified that biofortification was efficient for both cabbage cultivars, which absorbed and accumulated Se in the cabbage head as a function of the increase of Se, with both selenate and selenite.

Selenium (Se) in the aquatic environment is predominantly found as the soluble selenium oxyanions; selenate (SeO42-), and selenite (SeO32-). These oxyanions are toxic, and they readily bio-accumulate in the food chain. However, numerous studies have proven the viability of microbial remediation in reducing them to elemental selenium (Se0) which is considered to be biologically inert and relatively less toxic. Of the various microorganisms that have been employed in Se bioremediation, Pseudomonas stutzeri NT-I has shown great potential in removing high concentrations of SeO42- and SeO32-. Of these two selenium oxyanions, SeO32- is more toxic, is the most reactive and is usually found in mildly oxidising acidic environments. Therefore, the focus of this study is on selenite. In this study Pseudomonas stutzeri NT-I was used in aerobic batch reduction of various SeO32- concentrations (0.5 to 10 mM) to Se0 under already known optimum conditions of temperature 35±2 °C, pH 7 – 8 and salinity 5 g.L-1 NaCl. The selenium (Se) reduction efficiency of strain NT-I was assessed under varying conditions, such as the presence and absence added nitrogen and glucose substrates, inhibited metabolic activity and bacterial cells. Moreover, the variation in the different parameters such the oxidation reduction potential (ORP), metabolic activity (MA) and the concentrations of SeO32-, glucose and total organic carbon (TOC) were also monitored. Key results indicated that the rate and amount of SeO32 reduction was influenced by the concentration to be reduced. For an initial SeO32- concentration of 0.5 mM, reduction was gradual and after 36 h, approximately 75 % had been reduced to Se0 which was equivalent to 0.375 mM. In the reduction of 10 mM SeO32- however, the reduction rate was rapid and even though the overall percentage reduction averaged around 18 %, this was equivalent to 1.8 mM This indicated that the increased initial reduction rate was a result of increased biomass activity in response to increased selenite concentration. This response is likely a defence mechanism employed by strain NT-I to detoxify its surrounding in elevated SeO32- concentrations. The results of these biological experiments were modelled, and the non-growth kinetics were found to fit the adapted Monod equation with k_s and k_Se values of 4.723 mM and 2.869 mmol.(h.g)-1 respectively. Pseudomonas stutzeri NT-I’s capability of being able to survive in very high selenium concentrations make it an attractive and versatile microbial species suitable for the bioremediation of selenium laden industrial wastewater. / Dissertation (MSc (Applied Science: Environmental Technology))--University of Pretoria, 2020. / Chemical Engineering / MSc (Applied Science: Environmental Technology) / Restricted

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