Mycobacterium avium is human and animal opportunistic pathogen responsible for disseminated disease in immunocompromised patients. Mycobacteria have a capacity to adapt to the environmental conditions by inducing enzyme activities and altering their metabolism. M. avium A5 cells were grown in a defined minimal medium (Nitrogen Test Medium) with glutamine, nitrite, nitrate, or ammonia as sole nitrogen source at a concentration of 2 mM at 370C aerobically. The strain grew well on all the nitrogen sources except nitrite. It grew slowly on nitrite with a generation time of 6 days and cultures were not viable after 4 weeks of storage. These data confirm that M. avium can utilize a single nitrogen source in a defined minimal medium as documented by McCarthy (1987). M. avium genome has been sequenced and contains genes sharing sequence similarities to respiratory nitrate reductase and dissimilatory nitrite reductases. Because, M. avium can use nitrate or nitrite as sole nitrogen source for growth (McCarthy, 1987), it must have assimilatory nitrate- and nitrite-reductases. Nitrate- and nitrite-reductase activities of M. avium cells growing aerobically or undergoing anaerobic shift in the presence of ammonia, nitrate or ammonia and nitrate in combination were measured. M. avium produced nitrate- as well as nitrite-reductase activity. Nitrite- and nitrate-reductases used either NADH or NADPH as an electron donor. Nitrite reductase activity was greater than nitrate reductase activity. This observation supports the rapid reduction of nitrite and slow reduction of nitrate in M. avium as documented by McCarthy (1987) and explained why M. avium gives a negative result by the standard nitrate reductase test. In addition to assimilatory enzyme activity, M. avium A5 also produced dissimilatory nitrate- and nitrite-reductase activities. / Master of Science

O presente trabalho inclui duas etapas da pesquisa. Na primeira fase do projeto, a oxidação eletroquímica do nitrito em eletrodo de carbono vítreo modificado com um filme obtido eletrostaticamente de Co-TRP/Zn-TPPS, foi investigada em pH 4,7. Um aumento de corrente foi detectado em 0,75V, cujo máximo foi proporcional à concentração do íons nitrito, exibindo uma dependência inversa com a espessura do filme. A velocidade de transferência de carga foi avaliada por cronoamperometria, sendo que a velocidade de difusão da carga eletroquímica através do filme é mais alta do que a velodicade de difusão do íon em solução. O processo catalítico envolvendo a oxidação do íon nitrito no filme de pares de depósitos alternados foi também investigado com as técnicas da voltametria cíclica e de disco rotativo. Em concentrações relativamente baixas de substrato, o processo mediado foi tratado como um sub-caso, SR, na notação de Saveant, permitindo o cálculo da constante de velocidade para a reação de troca na interface filme-solução, sendo que tal valor foi estimado em 3,4x103 dm3 mol-1 s-1. Na segunda parte da investigação, a potencialidade analítica do eletrodo modificado foi confirmada por experimentos de injeção em fluxo usando solução carregadora tampão pH 4,7. Devido à atividade catalítica da camada imobilizada, a determinação de nitrito foi realizada em potencial menos positivo do que aqueles obtidos em eletrodos polidos (0,75 V vs. Ag/AgCl) e os dados obtidos demostraram o melhor desempenho do eletrodo modificado, indicando excelente repetibilidade e maior sensibilidade. Na condição experimental otimizada, o nitrito foi determinado na configuração FIA com limite de detecção de 0,1 &#181mol L-1 e a faixa analítica de aplicabilidade do método foi de 1 a 1000 µmol L-1. Nitrato também foi quantificado após sua redução estequiométrica a nitrito pela passagem da solução através da coluna de cádmio/cobre. Ambos analitos foram quantificados em água mineral e amostras de saliva e os resultados foram concordantes com aqueles obtidos usando método oficial. / This present work includes two research steps. ln the first step the electrochemical oxidation on nitrite on a glassy carbon disc electrode, modified with an electrostatically assembled film of tetraruthenated cobalt porphyrin/(meso-tetra(4-sulphonatephenyl)porphyrinate zinc(II), was investigated at pH = 4.7. A current enhancement was observed by using the modified electrode, the signal being proportional to the concentration of nitrite ions and exhibiting an inverse dependence with the film thickness. The charge transfer rate was evaluated by chronoamperometry, showing that the rate of diffusion of electrochemical charge through the film is higher than the rate of diffusion of substrates in solution. The catalytic process involving the oxidation of the nitrite ions at the bilayered film was also investigated based on cyclic and rotating disc voltammetry. At relatively low substrate concentrations the mediated process has been dealt with in terms of the SR sub-case in Saveant\'s notation, allowing the calculation of the rate constant for the cross-exchange reaction at the film-solution interface as 3.4x103 dm3 mol-1 s-1. In the second part the analytical potentiality of the modified electrode was studied by flow injection experiments carried out by using a pH 4.7 buffer as carrier solution. Owing to the catalytic activity of the immobilized layer, the determination of nitrite was performed at less positive potentials (0.75 V versus Ag/AgCl) and the data obtained showed a better performance indicated by both an excellent repeatability and increased sensitivity. At the optimized experimental condition, nitrite was determined in the FIA configuration with a limit of detection of 0.1 µmol L-1. Nitrate was also quantified after its stoichiometric reduction to nitrite by passing the solution through a cadmium/copper column. Both analytes were quantified in mineral water and saliva samples and the results were in agreement with the ones obtained by using an official method.

O presente trabalho inclui duas etapas da pesquisa. Na primeira fase do projeto, a oxidação eletroquímica do nitrito em eletrodo de carbono vítreo modificado com um filme obtido eletrostaticamente de Co-TRP/Zn-TPPS, foi investigada em pH 4,7. Um aumento de corrente foi detectado em 0,75V, cujo máximo foi proporcional à concentração do íons nitrito, exibindo uma dependência inversa com a espessura do filme. A velocidade de transferência de carga foi avaliada por cronoamperometria, sendo que a velocidade de difusão da carga eletroquímica através do filme é mais alta do que a velodicade de difusão do íon em solução. O processo catalítico envolvendo a oxidação do íon nitrito no filme de pares de depósitos alternados foi também investigado com as técnicas da voltametria cíclica e de disco rotativo. Em concentrações relativamente baixas de substrato, o processo mediado foi tratado como um sub-caso, SR, na notação de Saveant, permitindo o cálculo da constante de velocidade para a reação de troca na interface filme-solução, sendo que tal valor foi estimado em 3,4x103 dm3 mol-1 s-1. Na segunda parte da investigação, a potencialidade analítica do eletrodo modificado foi confirmada por experimentos de injeção em fluxo usando solução carregadora tampão pH 4,7. Devido à atividade catalítica da camada imobilizada, a determinação de nitrito foi realizada em potencial menos positivo do que aqueles obtidos em eletrodos polidos (0,75 V vs. Ag/AgCl) e os dados obtidos demostraram o melhor desempenho do eletrodo modificado, indicando excelente repetibilidade e maior sensibilidade. Na condição experimental otimizada, o nitrito foi determinado na configuração FIA com limite de detecção de 0,1 &#181mol L-1 e a faixa analítica de aplicabilidade do método foi de 1 a 1000 µmol L-1. Nitrato também foi quantificado após sua redução estequiométrica a nitrito pela passagem da solução através da coluna de cádmio/cobre. Ambos analitos foram quantificados em água mineral e amostras de saliva e os resultados foram concordantes com aqueles obtidos usando método oficial. / This present work includes two research steps. ln the first step the electrochemical oxidation on nitrite on a glassy carbon disc electrode, modified with an electrostatically assembled film of tetraruthenated cobalt porphyrin/(meso-tetra(4-sulphonatephenyl)porphyrinate zinc(II), was investigated at pH = 4.7. A current enhancement was observed by using the modified electrode, the signal being proportional to the concentration of nitrite ions and exhibiting an inverse dependence with the film thickness. The charge transfer rate was evaluated by chronoamperometry, showing that the rate of diffusion of electrochemical charge through the film is higher than the rate of diffusion of substrates in solution. The catalytic process involving the oxidation of the nitrite ions at the bilayered film was also investigated based on cyclic and rotating disc voltammetry. At relatively low substrate concentrations the mediated process has been dealt with in terms of the SR sub-case in Saveant\'s notation, allowing the calculation of the rate constant for the cross-exchange reaction at the film-solution interface as 3.4x103 dm3 mol-1 s-1. In the second part the analytical potentiality of the modified electrode was studied by flow injection experiments carried out by using a pH 4.7 buffer as carrier solution. Owing to the catalytic activity of the immobilized layer, the determination of nitrite was performed at less positive potentials (0.75 V versus Ag/AgCl) and the data obtained showed a better performance indicated by both an excellent repeatability and increased sensitivity. At the optimized experimental condition, nitrite was determined in the FIA configuration with a limit of detection of 0.1 µmol L-1. Nitrate was also quantified after its stoichiometric reduction to nitrite by passing the solution through a cadmium/copper column. Both analytes were quantified in mineral water and saliva samples and the results were in agreement with the ones obtained by using an official method.

Made available in DSpace on 2015-05-14T13:21:29Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 3046844 bytes, checksum: 9fc0abd4ac9137e94ab4a27445373f2f (MD5) Previous issue date: 2013-08-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / In this study, a micro-flow-batch analyzer (μFBA) using an in-line cadmium reduction microcolumn for the photometric determination of nitrate and nitrite in dairy samples was developed. The method is based on the Griess-Llosvay reaction and measuring of the absorbance at 540 nm using a green LED integrated into the μFBA built in the urethane-acrylate resin. Initially, the natural nitrite ion content of natural dairy sample product is determined in the micro-mixing chamber, while the nitrate is reduced to nitrite in the micro-column coupled to the micro cadmium sponge and then determined as in the micro-chamber nitrite. content of nitrate ions was estimated from the difference between the first and second determination. The analytical curve for nitrate and nitrite was linear in the work range of 10.0 100.0 μg L 1 with a correlation coefficient of 0.992 and 0.998, respectively. The limit of detection and relative standard deviation were estimated at 0.39 μg L 1 and < 1.7 % (n = 5) for nitrite and, 0.41 μg L 1 and < 1.3 % (n = 5) for nitrate. Comparing with the reference methods, no statistically significant differences were observed when applying the paired t-test at a 95% confidence level. The accuracy was assessed through recovery test (97.7 to 102.9 %). The proposed microsystem employed in-line cadmium sponge microcolumn presented satisfactory portability, robustness, flexibility, low-cost device and reduced chemicals consumption compared to recent methods. / Neste estudo, um analisador micro-flow-batch (μFBA) utilizando uma micro-coluna redutora de cádmio em linha foi desenvolvido para a determinação fotométrica de íons nitrato e nitrito em amostras de laticínios. O método empregado baseia-se na reação de Griess-Llosvay e a medição da absorbância foi realizada a 540 nm, utilizando um LED verde integrado na micro-câmara construída em resina fotopolimerizável uretano-acrilato. Inicialmente, o teor de íons nitritos natural da amostra de lacticínio é determinado na micro-câmara de mistura, enquanto que o nitrato é reduzido a nitrito na micro-coluna de cádmio esponjoso acoplada ao microssistema e posteriormente determinado na micro-câmara como nitrito. O teor de íons nitrato foi estimado a partir da diferença entre a segunda e a primeira determinação. A curva analítica para nitrato e para nitrito foi linear na faixa de trabalho de 10,0 a 100,0 μg L-1, com um coeficiente de correlação (r2) de 0,992 e 0,998, respectivamente. O limite de detecção e o desvio padrão relativo foi calculado em 0,39 μg L-1 e <1,7% (n = 5) para o nitrito, e 0,41 μg L-1 e <1,3% (n = 5) para o nitrato. Comparando-se com os métodos de referência, não foram observadas diferenças estatisticamente significativas quando se aplica o teste t pareado com nível de confiança de 95%. A exatidão do método foi avaliada através do teste de recuperação (97,7 a 102,9%). O microssistema proposto empregando a micro-coluna de cádmio esponjoso em linha apresentou portabilidade satisfatória, robustez, flexibilidade, aparelhagem simples e consumo reduzido de produtos químicos em relação aos métodos automáticos mais recentes.

The temperature and/or entropy of electrically polarisable materials can be altered by changing electric field E. Research into this electrocaloric (EC) effect has focussed on increasing the size of the EC effects, with the long-term aim of building a cooler with an EC material at its heart. Materials and experimental methods are briefly reviewed. A ‘resetting’ indirect route to isothermal entropy change ∆S for hysteretic first-order transitions is described. An indirect route to adiabatic temperature change ∆T, without the need for field-resolved heat capacity data, is also described. Three temperature controllers were built: a cryogenic probe for 77-420 K with ∼5 mK resolution, a high-temperature stage with vacuum enclosure for 295-700 K with ∼15 mK resolution, and a low-temperature stage for 120-400 K with electrical access via micropositioners. Automation enables dense datasets to be compiled. Single crystals of inorganic salts (NH4)2SO4 , KNO3 and NaNO2 were obtained. Applying 380 kV cm−1 across (NH4)2SO4 , it was found that |∆S| ∼ 20 J K−1 kg−1 and |∆T | ∼ 4 K, using the indirect method near the Curie temperature TC = 223 K. Without the ‘resetting’ indirect method, |∆S| ∼ 45 J K−1 kg−1 would have been spuriously found. Preliminary indirect measurements on KNO3 and NaNO2 give |∆S| ∼ 75 J K−1 kg−1 for ∆E ∼ 31 kV cm−1 near TC = 400 K and |∆S| ∼ 14 J K−1 kg−1 for ∆E ∼ 15 kV cm−1 near TC = 435 K, respectively. A cation-ordered PbSc0.5Ta0.5O3 ceramic showing a nominally first-order transition at 295 K was obtained. The Clausius-Clapeyron phase diagram is revealed via indirect measurements where |∆S| ∼ 3.25 J K−1 kg−1 and |∆T | ∼ 2 K, and direct measurements where |∆T | ∼ 2 K. Clamped samples show broadening of the field-induced transition. Epitaxial, ∼64 nm-thick SrTiO3 films were grown by pulsed laser deposition on NdGaO3 (001) substrates with a La0.67Sr0.33MnO3 bottom electrode. The indirect method gives |∆S| ∼ 8 J K−1 kg−1 and |∆T | ∼ 3.5 K near 180 K with |∆E| = 780 kV cm−1. Finite element modelling (FEM) was used to optimise the geometry of multilayered capacitors (MLCs) for EC cooling. Intrinsic cooling powers of 25.9 kW kg−1 are predicted for an optimised MLC based on PVDF-TrFE with Ag electrodes.