Succinate:ubiquinone oxidoreductase from Paracoccus denitrificans and particulate methane monooxygenase from Methylococcus capsulatus (Bath): experimental and theoretical EPR studies of the metal cofactors

This thesis summarizes the application of conventional and modern electron paramagnetic resonance (EPR) techniques to establish proximity relationships between paramagnetic metal centers in metalloproteins and between metal centers and magnetic ligand nuclei in two important and timely membrane proteins: succinate:ubiquinone oxidoreductase (SQR) from Paracoccus denitrificans and particulate methane monooxygenase (pMMO) from Methylococcus capsulatus. Such proximity relationships are thought to be critical to the biological function and the associated biochemistry mediated by the metal centers in these proteins. A mechanistic understanding of biological function relies heavily on structure-function relationships and the knowledge of how molecular structure and electronic properties of the metal centers influence the reactivity in metalloenzymes. EPR spectroscopy has proven to be one of the most powerful techniques towards obtaining information about interactions between metal centers as well as defining ligand structures. SQR is an electron transport enzyme wherein the substrates, organic and metallic cofactors are held relatively far apart. Here, the proximity relationships of the metallic cofactors were studied through their weak spin-spin interactions by means of EPR power saturation and electron spin-lattice (T_1) measurements, when the enzyme was poised at designated reduction levels. Analysis of the electron T_1 measurements for the S-3 center when the b-heme is paramagnetic led to a detailed analysis of the dipolar interactions and distance determination between two interacting metal centers. Studies of ligand environment of the metal centers by electron spin echo envelope modulation (ESEEM) spectroscopy resulted in the identication of peptide nitrogens as coupled nuclei in the environment of the S-1 and S-3 centers.

Finally, an EPR model was developed to describe the ferromagnetically coupled trinuclear copper clusters in pMMO when the enzyme is oxidized. The Cu(II) ions in these clusters appear to be strongly exchange coupled, and the EPR is consistent with equilateral triangular arrangements of type 2 copper ions. These results offer the first glimpse of the magneto-structural correlations for a trinuclear copper cluster of this type, which, until the work on pMMO, has had no precedent in the metalloprotein literature. Such trinuclear copper clusters are even rare in synthetic models.

Investigation of competitive antagonist binding to the nicotinic acetylcholine receptor using voltage-jump and light-flash techniques

1. The effect of 2,2’-bis-[α-(trimethylammonium)methyl]azobenzene (2BQ), a photoisomerizable competitive antagonist, was studied at the nicotinic acetycholine receptor of Electrophorus electroplaques using voltage-jump and light-flash techniques.

2. 2BQ, at concentrations below 3 μΜ, reduced the amplitude of voltage-jump relaxations but had little effect on the voltage-jump relaxation time constants under all experimental conditions. At higher concentrations and voltages more negative than -150 mV, 2BQ caused significant open channel blockade.

3. Dose-ratio studies showed that the cis and trans isomers of 2BQ have equilibrium binding constants (K _ᵢ) of .33 and 1.0 μΜ, respectively. The binding constants determined for both isomers are independent of temperature, voltage, agonist concentration, and the nature of the agonist.

4. In a solution of predominantly cis-2BQ, visible-light flashes led to a net cis→trans isomerization and caused an increase in the agonist-induced current. This increase had at least two exponential components; the larger amplitude component had the same time constant as a subsequent voltage-jump relaxation; the smaller amplitude component was investigated using ultraviolet light flashes.

5. In a solution of predominantly trans-2BQ, UV-light flashes led to a net trans→cis isomerization and caused a net decrease in the agonist-induced current. This effect had at least two exponential components. The smaller and faster component was an increase in agonist-induced current and had a similar time constant to the voltage-jump relaxation. The larger component was a slow decrease in the agonist-induced current with rate constant approximately an order of magnitude less than that of the voltage-jump relaxation. This slow component provided a measure of the rate constant for dissociation of cis-2BQ (k_ = 60/s at 20°C). Simple modelling of the slope of the dose-rate curves yields an association rate constant of 1.6 x 10⁸/M/s. This agrees with the association rate constant of 1.8 x 10⁸/M/s estimated from the binding constant (K_i). The Q₁₀ of the dissociation rate constant of cis-2BQ was 3.3 between 6° and 20°C. The rate constants for association and dissociation of cis-28Q at receptors are independent of voltage, agonist concentration, and the nature of the agonist.

6. We have measured the molecular rate constants of a competitive antagonist which has roughly the same K _ᵢ as d-tubocurarine but interacts more slowly with the receptor. This leads to the conclusion that curare itself has an association rate constant of 4 x 10⁹/M/s or roughly as fast as possible for an encounter-limited reaction.

Measurement of polycyclic aromatic hydrocarbons in ambient air by fluorescence spectroscopic techniques

250 p. + anexos

Study of crystal structure and phase transition studies in perovskite-type oxides using powder-diffraction techniques and symmetry-mode analysis.

251 p.

Análise da resposta dinâmica experimental de uma passarela tubular mista, aço-concreto, submetida ao caminhar humano.

Este trabalho de pesquisa apresenta como objetivo principal o desenvolvimento de investigação experimental dinâmica sobre estrutura real de uma passarela tubular mista aço-concreto. O sistema estrutural objeto deste trabalho corresponde a uma passarela composta por três vãos (32,5m, 17,5m e 20,0m, respectivamente) e dois balanços (7,50m e 5,0m, respectivamente), com comprimento total de 82,5m. A passarela com estrutura contínua de aço com as ligações soldadas se apoia em quatro pórticos também de aço. Estruturalmente está constituída por duas treliças planas que se interligam através de contraventamentos horizontais fixados na corda superior e inferior da treliça e lajes de concreto, formando um sistema misto com interação completa. A estrutura está submetida correntemente à travessia de pedestres e ciclistas. Testes experimentais foram realizados sobre o sistema estrutural e confrontados com resultados numéricos. Para a modelagem numérica do sistema são empregadas técnicas usuais de discretização, via método dos elementos finitos (MEF), por meio do programa ANSYS. Os resultados experimentais são analisados de acordo com a metodologia desenvolvida, sendo realizada análise modal experimental para a determinação das propriedades dinâmicas: freqüências, modos e taxa de amortecimento, enquanto que os resultados da estrutura, em termos de aceleração de pico, são comparados com os valores limites propostos por diversos autores, normas e recomendações de projeto, para uma avaliação do desempenho da estrutura em relação a vibração quando solicitada pelo caminhar dos pedestres no que diz respeito a critério para conforto humano.

Influence of oxygen plasma treatment on properties of ZrO2 films prepared by e-beam evaporation techniques

Zirconia films were prepared by e-beam evaporation, and oxygen plasma treatment was used to modify film properties. Spectrophotometry, x-ray diffractometry (XRD), and atomic force microscopy were used to characterize refractive index, extinction coefficient, rnicrostructure, and surface roughness, respectively. The experimental results indicate that both refractive index and extinction coefficient of the films were reduced slightly after oxygen plasma treatment, with the decrease of intrinsic stress and surface roughness. From XRD spectra, the intensity decrease of the T(110) diffraction peak was clearly observed after the treatment, which was caused by the restructuring of the film atoms. (C) 2008 Elsevier Ltd. All rights reserved.

Influência do refluxo duodenogástrico nas alterações histológicas da mucosa gástrica em ratos infectados por Helicobacter pylori: modelo experimental

O Helicobacter pylori, é tido como o principal fator de risco para o carcinoma gástrico. Diferentes estudos experimentais em animais procuram relacionar essa carcinogênese a outros fatores carcinógenos sem sucesso. Neste estudo, procurou-se avaliar-se em ratos, se há correlação entre o refluxo duodenogástrico, o Helicobacter pylori e o desenvolvimento do câncer gástrico ou de seus precursores. Para tal, realizou-se nos três grupos de ratos (n de dez por grupo) as técnicas de: piloroplastia precedida de infecção, gastrectomia subtotal precedida de infecção e um grupo no qual foi praticada apenas a infecção. Apois seis meses, analisou-se as alterações da mucosa, comparando-se os três grupos. As alterações da mucosa pesquisadas foram as seguintes: gastrites, metaplasias, displasias e neoplasias epiteliais. Ao término do estudo, foi encontrado, no grupo submetido a piloroplastia precedida de infecção um alto percentual de alterações epiteliais. Conclui-se que, no rato, a operação de piloroplastia, levou ao maior desenvolvimento da população do Helicobacter pylori, que se relaciona com as lesões pré- malignas e o adenocarcinoma gástrico.

Papel regulatório da proteína anexina-1 e de seu derivado Ac2-26 no modelo experimental de asma alérgica em camundongos

A asma é uma doença inflamatória crônica caracterizada por hiper-reatividade das vias aéreas, acúmulo de eosinófilos, secreção de muco e remodelamento. No decorrer do estabelecimento do processo inflamatório, há liberação de mediadores endógenos que atuam limitando a evolução do quadro patológico e garantindo a manutenção da homeostasia (COHN, ELIAS e CHUPP, 2004). Dentre estes recebem destaque os hormônios glicocorticóides, reconhecidos por sua atividade anti-inflamatória, dependente, em parte, da geração de fatores intermediários como a proteína anexina-1 (AnxA1) (KAMAL, FLOWER e PERRETTI, 2005; PERRETTI, 2003). Neste estudo investigou-se o papel regulatório da AnxA1 e do peptídeo derivado Ac2-26 (50 - 200 g/animal) no modelo experimental de asma alérgica murina. Camundongos BALB/c (AnxA1+/+) e depletados do gene codificante para AnxA1 (AnxA1-/-) foram sensibilizados com ovoalbumina (OVA 50 g) e hidróxido de alumínio (5 mg), por via subcutânea. Após 14 dias, foi feito reforço com OVA (25 g), por via intraperitoneal, e nos dias 19, 20 e 21 foram desafiados com OVA (25 g), por via intranasal. O tratamento consistiu na administração intranasal do peptídeo Ac2-26 (50 - 200 g), 1 h antes de cada desafio. As análises foram feitas 24 h após o último desafio e incluíram: i) função pulmonar (resistência e elastância) e hiper-reatividade das vias aéreas à metacolina (3 27 mg/ml) através de pletismografia invasiva; ii) alterações morfológicas através de histologia clássica; iii) quantificação de colágeno e iv) quantificação de mediadores inflamatórios através de ELISA. Verificou-se que camundongos AnxA1-/-, quando ativamente sensibilizados e desafiados com OVA apresentaram exacerbação do quadro de hiper-reatividade das vias aéreas, assim como do número de eosinófilos no lavado broncoalveolar e no infiltrado peribrônquico, deposição de colágeno e nos níveis de IL-13 em comparação aos controles AnxA1+/+. Em paralelo, observou-se que o peptídeo Ac2-26 levou a uma redução da hiper-reatividade das vias aéreas frente à estimulação com metacolina nos animais AnxA1+/+. O peptídeo Ac2-26 reduziu o infiltrado inflamatório no parênquima pulmonar e o número de eosinófilos peribronquiolares, além da produção de muco no tecido pulmonar e da geração IL-4, IL-13, eotaxina-1 e -2. Em conjunto, nossos achados mostram que os camundongos AnxA1-/- mostraram-se mais responsivos à estimulação antigênica, o que foi indicativo de que a AnxA1 parece exercer um papel regulatório importante sobre a resposta inflamatória alérgica murina. Além disso, o efeito inibitório do peptídeo Ac2-26 sobre a resposta alérgica pulmonar foi indicativo de que este se coloca como um composto anti-inflamatório e anti-alérgico promissor para utilização na terapia da asma.

Resultados do programa experimental de fumagen de pescado

The fish smoking is a traditional process in most african countries and has been gradually improved to the light of changes in forms, techniques and materials. In Mozambique, in particular, this method is not as developed as expected, because it has more advantages in wetlands during rainy seasons, where there is no shortage of salt and in order to give a special taste to the dry fish. In many parts of the country, mainly in coastal areas, the smoking process is made manually by families: a small fire with any type of wood, close to a small amount of fish on a stick, tilted on fire in order to take smoke and heat for several hours. This process is used in the south (Inhaca Island), in central and northern regions (Beira, Nampula) and was also seen in Niassa Lake. This paper presents a program aimed at the application of existing knowledge about fish smoking process and at technology improvement.

Analytical techniques for indentation of viscoelastic materials

Indentation of linearly viscoelastic materials is explored using elastic-viscoelastic correspondence analysis for both conical-pyramidal and spherical indentation. Boltzmann hereditary integrals are used to generate displacement-time solutions for loading at constant rate and creep following ramp loading. Experimental data for triangle- and trapezoidal-loading are examined for commercially-available polymers and compared with analytical solutions. Emphasis is given to the use of multiple experiments to test the fidelity and predictive capability of the obtained material creep function. Plastic deformation occurs in sharp indentation of glassy polymers and is found to complicate the viscoelastic analysis. A new method is proposed for estimating a material time-constant from peak displacement or hardness data obtained in pyramidal indentation tests performed at different loading rates.

Experimental demonstration of the Rayleigh acoustic viscous boundary layer theory.

Amplitude and phase velocity measurements on the laminar oscillatory viscous boundary layer produced by acoustic waves are presented. The measurements were carried out in acoustic standing waves in air with frequencies of 68.5 and 114.5 Hz using laser Doppler anemometry and particle image velocimetry. The results obtained by these two techniques are in good agreement with the predictions made by the Rayleigh viscous boundary layer theory and confirm the existence of a local maximum of the velocity amplitude and its expected location.

Theoretical and experimental studies on Jc and AC losses of 2G HTS coils

This paper begins with introducing the winding techniques of two superconducting double-pancake coils wound using 2G coated conductors. These winding techniques are able to guarantee a good performance for the superconducting coils. Then the coil critical currents were measured and compared with a simulation model. The results were consistent. Finally the coil AC losses were measured using an experimental circuit including a compensation coil. The simulation results are close to the experiment results. © 2010 IEEE.

Experimental study on an SFCL using series-connected YBCO thin films

Superconducting Fault Current Limiters (SFCLs) are able to reduce fault currents to an acceptable value, reducing potential mechanical and thermal damage and allowing more flexibility in an electric power system's design. Due to limitations in current YBCO thin film manufacturing techniques, it is necessary to connect a number of thin films in different series and parallel configurations in order to realise a practical SFCL for electric power system applications. The amount of resistance generated (i.e. the degree of current limitation), the characteristics of the S-N transition, and the time at which they operate is different depending on their comparative characteristics. However, it is desirable for series-connected thin films to have an operating time difference as small as possible to avoid placing an excess burden on certain thin films. The role of a parallel resistance, along with the influence of thin film characteristics, such as critical current (Ic), are discussed in regards to the design of SFCLs using YBCO thin films. © 2008 IOP Publishing Ltd.

An experimental study on engine dynamics model based in-cylinder pressure estimation

The information provided by the in-cylinder pressure signal is of great importance for modern engine management systems. The obtained information is implemented to improve the control and diagnostics of the combustion process in order to meet the stringent emission regulations and to improve vehicle reliability and drivability. The work presented in this paper covers the experimental study and proposes a comprehensive and practical solution for the estimation of the in-cylinder pressure from the crankshaft speed fluctuation. Also, the paper emphasizes the feasibility and practicality aspects of the estimation techniques, for the real-time online application. In this study an engine dynamics model based estimation method is proposed. A discrete-time transformed form of a rigid-body crankshaft dynamics model is constructed based on the kinetic energy theorem, as the basis expression for total torque estimation. The major difficulties, including load torque estimation and separation of pressure profile from adjacent-firing cylinders, are addressed in this work and solutions to each problem are given respectively. The experimental results conducted on a multi-cylinder diesel engine have shown that the proposed method successfully estimate a more accurate cylinder pressure over a wider range of crankshaft angles. Copyright © 2012 SAE International.

Exploring the use of sensors to measure behavioral interactions: an experimental evaluation of using hand trajectories.

Humans appear to be sensitive to relative small changes in their surroundings. These changes are often initially perceived as irrelevant, but they can cause significant changes in behavior. However, how exactly people's behavior changes is often hard to quantify. A reliable and valid tool is needed in order to address such a question, ideally measuring an important point of interaction, such as the hand. Wearable-body-sensor systems can be used to obtain valuable, behavioral information. These systems are particularly useful for assessing functional interactions that occur between the endpoints of the upper limbs and our surroundings. A new method is explored that consists of computing hand position using a wearable sensor system and validating it against a gold standard reference measurement (optical tracking device). Initial outcomes related well to the gold standard measurements (r = 0.81) showing an acceptable average root mean square error of 0.09 meters. Subsequently, the use of this approach was further investigated by measuring differences in motor behavior, in response to a changing environment. Three subjects were asked to perform a water pouring task with three slightly different containers. Wavelet analysis was introduced to assess how motor consistency was affected by these small environmental changes. Results showed that the behavioral motor adjustments to a variable environment could be assessed by applying wavelet coherence techniques. Applying these procedures in everyday life, combined with correct research methodologies, can assist in quantifying how environmental changes can cause alterations in our motor behavior.