Systematic measurements of the magnetic moment in dependence on temperature and magnetic field of hexagonal 6H-BaTiO3 + 0.04 BaO + x/2 Fe 2 O 3 (0.005 x 0.05) ceramics were performed to study the influence of Fe ions on the magnetic properties. While the samples show Curie–Weiss paramagnetism for Fe concentrations 1.0 mol%, antiferromagnetic interactions become manifest for 2.0 and 5.0 mol% iron. With increasing Fe content the antiferromagnetic interaction, which is assumed to be caused by a superexchange mechanism Fe 3+ Ti(1) − O 2− O(2) − Fe3+ Ti(2) , becomes stronger. At external magnetic fields smaller than 1 T a further, ferromagnetic interaction between Fe 3+ ions is detected below 200 K. The interactions between Fe 3+ ions in the samples with 2.0 and 5.0 mol% iron are also manifest in the EPR spectra by numerous lines with low intensity. Q-band EPR investigations of 5.0 mol% Fe doped single crystals confirm the existence of only one type of Fe 3+ –V O associates in the samples.

No description available.

Spin dynamics simulations are used to gain insight into important magnetic resonance experiments in the fields of chemistry, biochemistry, and physics. Presented in this thesis are investigations into how to accelerate these simulations by making them more efficient. Chapter 1 gives a brief introduction to the methods of spin dynamics simulation used in the rest of the thesis. The `exponential scaling problem' that formally limits the size of spin system that can be simulated is described. Chapter 2 provides a summary of methods that have been developed to overcome the exponential scaling problem in liquid state magnetic resonance. The possibility of utilizing the multiple processors prevalent in modern computers to accelerate spin dynamics simulations provides the impetus for the investigation found in Chapter 3. A number of different methods of parallelization leading to acceleration of spin dynamics simulations are derived and discussed. It is often the case that the parameters defining a spin system are time-dependent. This complicates the simulation of the spin dynamics of the system. Chapter 4 presents a method of simplifying such simulations by mapping the spin dynamics into a larger state space. This method is applied to simulations incorporating mechanical spinning of the sample with powder averaging. In Chapter 5, implementations of several magnetic resonance experiments are detailed. In so doing, use of techniques developed in Chapters 2 and 3 are exemplified. Further, specific details of these experiments are utilized to increase the efficiency of their simulation.

Membrane proteins play a key role in numerous physiological processes such as transport, energy transduction in respiratory and photosynthetic systems, and signal transduction, and are of great pharmaceutical interest, comprising more than 60% of known drug targets. However, crystallisation of membrane proteins, and G protein-coupled receptors (GPCRs) in particular, still relies heavily on the use of protein engineering strategies, which have been shown to hamper protein activity. Here, a range of biophysical methods were used to study the structure and function of two membrane proteins, a prokaryotic peptide transporter, PepT_So and a GPCR, neurotensin receptor 1 (NTS1), using different membrane reconstitution methods to study the proteins in a native-like environment. Firstly, using the pulsed electron paramagnetic resonance (EPR) method of double electron-electron resonance (DEER) the conformation of PepT_So reconstituted into lipid bilayers was assessed and compared to previous structural data obtained from crystallography and modelling. The influence of the membrane potential and the presence of substrate on the conformational heterogeneity of this proton-coupled transporter were investigated. Secondly, NTS1 purification was optimized for biophysical study. Cysteine mutants were created and a labelling protocol was developed and optimized for fluorophore and nitroxide labelling studies. NTS1 was then studied by continuous-wave EPR, to assess the influence of ligand on local protein dynamics, and to assess the structure of a receptor segment known as helix 8, that was proposed to be an α-helix, but was only observed to be helical in one of the NTS1 crystallographic studies. Ensemble and single-molecule Förster resonance energy transfer (FRET), and DEER were combined to study the dimerisation behaviour of NTS1, showing novel dynamics of the interfacial associations. Finally, the signalling mechanism of NTS1 was also investigated using microscale thermophoresis (MST) to assess the affinity of the receptor for G protein in vitro in the absence of ligand, or in the presence of agonist or antagonist. MST measurements were performed in detergent and in nanodiscs of different lipid compositions, to assess the influence of the lipid environment on receptor function. In summary, this thesis demonstrates the potential of biophysical techniques to study various aspects of membrane protein structure and function in native-like lipid systems, complementing e.g. structural data obtained from crystallographic studies with functional data for membrane proteins in more native environments, as well as shedding light on protein dynamics. The work presented here provides novel insights into PepTSo transport, and in particular into NTS1 structure, signalling, and oligomerisation, opening up several avenues for future research.

The Type Three Secretion Systems (T3SSs) of Gram-negative bacteria, including Shigella, Salmonella, and Enteropathogenic/Enterohaemorrhagic Escherichia coli (EPEC/EHEC), pass virulence factors directly into the host to mediate invasion. Prior to secretion down the narrow T3SS channel, effector proteins associate with chaperone proteins. The binding enables the T3SS to keep effectors soluble and partially unfolded for secretion. In the first part of this thesis, the association of one promiscuous chaperone, Spa15 of Shigella flexneri, with three of its cognate effectors has been studied. In addition to the role this plays in secretion, the binding of one particular substrate leads to Spa15 being involved in the regulation of the T3SS. The oligomerisation and impact of substrate binding upon Spa15 has been determined by crystallography and EPR. Once secreted, T3SS effectors subvert the host cytoskeleton for the benefit of the bacteria. Soluble homologues of Spa15 effectors from EHEC and Salmonella have been purified, and their interactions with host GTPases which lead to stress fibre phenotypes observed. The Twin Arginine Translocation (Tat) pathway provides a contrasting view of bacterial secretion. Instead of preventing folding in the cytoplasm, it is a criterion of transport that the protein be folded. One of the reasons for internal folding is the necessity to insert cofactors which could not be incorporated externally. In the second part of this thesis, a protein which exemplifies this necessity is studied. This is PhoD, the model protein for Tat export from Bacillus subtilis. PhoD is an alkaline phosphodiesterase expressed to scavenge phosphate in times of phosphate deficiency. The structure of PhoD has been solved, and the protein is shown to be able to cleave a component of its own cell wall. It uses an unusual catalytic site more reminiscent of the eukaryotic purple acid phosphatases than of other currently known alkaline phosphatases. Furthermore this site appears to require metal binding before export from the bacterial cytoplasm.

Les triterpènes sont des composés en C30 issus de la cyclisation du 2,3-epoxysqualène. L’utilisation industrielle et l’intérêt thérapeutique des triterpènes représentent un enjeu capital dans le domaine de la recherche des substances naturelles. Quant au cholestérol, il a été largement connu dans la littérature par son rôle modulateur des membranes naturelles aussi bien que synthétiques. Dans notre travail de thèse, nous nous sommes intéressés à étudier l’effet des triterpènes et du cholestérol sur la fluidité et la perméabilité des liposomes de dipalmitoylphosphatidylcholine. Deux approches sont utilisées pour réaliser ces études par le biais des liposomes charges ou des liposomes préformés. Les études de perméabilité sont réalisées par la spectroscopie de fluorescence à travers le suivi d’un fluorophore hydrophile, la sulforhodamine B en fonction du temps. Les études de la fluidité sont réalisées par la résonance paramagnétique électronique. Ces sondes permettent d’évaluer la fluidité à l’interface de la membrane ainsi qu’au cœur hydrophobe de la membrane. Les données obtenues de la cinétique de libération de la sulforhodamine sont quantifiées par des modèles mathématiques. Cers derniers serviront comme des outils pour prédire les mécanismes de libération. Nos résultats montrent que la perméabilité ainsi que la fluidité de la membrane sont réduites suite à l’ajout du cholestérol. L’effet des triterpènes sur les propriétés physiques des membranes dépend étroitement de la composition de la membrane, le taux du cholestérol, la structure du principe actif et le temps d’incubation / Triterpenes are C30 compounds derived from the cyclization of 2,3-epoxysqualene. The industrial use and the therapeutic interest of the triterpenes represent a capital importance in the field of the research of the natural substances. As for cholesterol, it has been widely known in the literature by its modulating role of natural as well as synthetic membranes. In our thesis work, we were interested in studying the effect of triterpenes and cholesterol on the fluidity and permeability of dipalmitoylphosphatidylcholine liposomes. Two approaches are used to carry out these studies via liposomes fillers or preformed liposomes. Permeability studies are carried out by fluorescence spectroscopy through the monitoring of a hydrophilic fluorophore, sulforhodamine B as a function of time. Studies of fluidity are carried out by electronic paramagnetic resonance. These probes make it possible to evaluate the fluidity at the interface of the membrane as well as at the hydrophobic core of the membrane. The data obtained from the release kinetics of sulforhodamine are quantified by mathematical models. These latter will serve as tools to predict the release mechanisms. Our results show that the permeability as well as the fluidity of the membranes are reduced following the addition of cholesterol. The effect of triterpenes on the physical properties of membranes is highly dependent on membrane composition, cholesterol level, the drug structure and incubation time

Diferentes estratégias têm sido propostas a fim de melhorar o desempenho dos bioeletrodos utilizados nas biocélulas a combustíveis e nos biossensores. Por examplo, a funcionalização de eletrodos de carbono tem sido feita para esse fim. Neste estudo, propomos o desenvolvimento de fibras flexíveis de carbono (FFCs) funcionalizadas com grupos quinona e modificados com álcool desidrogenase (ADH) NAD-dependente para obter bioeletrodos para uma bio-eletrocatálise eficiente de etanol. Grupos quinona na superfície das FFCs foram obtidas utilizando o tratamento oxidativo com permanganato e também pelo ancoramento eletroquímico de antraquinona: ambas metodologias resultaram em bioeletrodos para a eletro-oxidação de NADH que pode aumentar a bio-eletrocatálise do etanol. De acordo dados espectroscópicos, microscópicos, e eletroquímicos, defeitos contendo grupos C=O nos eletrodos de FFCs são atribuídos à melhora na oxidação do NADH, aumentando a bio-eletrocatálise do etanol. Para se investigar o papel dos grupos quinona na eletro-oxidação do NADH, propomos uma configuração experimental baseado na espectroscopia de ressonância paramagnética de elétrons em modo operando (operando EPR). Com essa técnica, fomos capaz de mostrar a correlação entre o número de elétrons livres desemparelhados, a concentração superficial de quinonas e a oxidação do NADH com controle eletroquímico. Correlação para a concentração de spins revela um aumento no número de elétrons desemparelhados livres com o aumento do sobrepotencial aplicado e a oxidação do NADH, o que corrabora com a hipótese de que grupos quinona podem afetar a eletrocatálise rumo à oxidação do NADH a NAD+. É vislumbrado que operando EPR pode fornecer infromação útil para provar a dinâmica da transferência de elétrons em superfície de carbono e possa ser extendida a outros sistemas bioeletroquímicos. / There are several strategies to improve the performance of bioelectrodes applied in biosensors and biofuel cells. For instance, surface functionalization of the carbon-based electrodes has been used to this intend. Herein, we propose the development of flexible carbon fibers (FCFs) functionalized with quinone groups and modified with NAD-dependent alcohol dehydrogenase (ADH) to obtain bioelectrodes for efficient ethanol bio-electrocatalysis. Quinones groups on FCFs surfaces were obtained by using oxidative treatment with permanganate, and also by electrochemical grafting of anthraquinone: both these methodologies result in bioelectrodes for the electro-oxidation of NADH that can improve the ethanol bio-electrocatalysis. Based on spectroscopic, microscopic and electrochemical data, defects containing C=O groups on FCFs electrodes are attributed to improve the NADH oxidation, enhancing the ethanol bio-electrocatalysis. In order to investigate the role of quinone groups on the NADH electro-oxidation, we propose an experimental setup based on operando electron paramagnetic resonance spectroscopy (operando EPR). With this technique, we are able to show a correlation among the number of free unpaired electrons, surface concentration of quinones and NADH oxidation under electrochemical control. Correlation for the spin concentration reveals an increasing number of free unpaired electrons with increasing applied overpotential and NADH oxidation, which corroborates the hypothesis that quinone groups can act as electrocatalysts towards the oxidation of NADH to NAD+. It is glimpsed that operando EPR can provide useful information in probing the electron transfer dynamics on a carbon surface and may be extended to others bioelectrochemical systems.

Tritrpticina (TRP3) é um peptídeo antimicrobiano com 13 resíduos de amino ácidos com três Ws sequenciais. Com o objetivo de contribuir para a compreensão de seu mecanismo de ação, realizaram-se estudos funcionais e conformacionais da TRP3 e de dois análogos onde um (WLW) ou dois (LWL) W foram substituídos por L. Os peptídeos foram igualmente ativos contra bactérias Gram positivas e negativas. Sua atividade hemolítica requereu concentrações maiores, diminuindo na ordem TRP3>WLW>LWL. Os peptídeos permeabilizaram membranas modelo de E. coli ou contendo fosfolipídios carregados negativamente. Espectros de CD sugeriram que os peptídeos adquirem diferentes conformações ao se ligarem a bicamadas e micelas. Estudos de fluorescência mostraram que a ligação a membranas decresce na ordem: TRP3>WLW>LWL e que os peptídeos se localizam próximos à interface membrana-água. Espectros de RPE de marcadores de spin lipídicos indicaram que a ligação dos peptídeos altera a organização dos lipídios, aumentando o empacotamento molecular / Tritrpticin (TRP3) is a 13-residue antimicrobial peptide that contains three sequential Ws. With the aim of contributing to the understanding of its mechanism of action, functional and conformational studies were performed with TRP3 and two of its analogues where one (WLW) or two (LWL) of the W were replaced by L. The peptides were equally active against both Gram positive and Gram negative bacteria. Higher concentrations were required for hemolytic activity which varied in the order: TRP3>WLW>LWL. The peptides permeabilized membranes model membranes mimicking E. coli\'s lipid composition or containing different negatively charged phospholipids. CD spectra suggested the peptides acquired different conformations upon binding to bilayers or micelles. Fluorescence studies showed that membrane binding decreases in the order: TRP3>WLW>LWL and that the peptides are located close to the water-membrane interface. EPR spectra of lipid spin labels indicated that peptide binding alter lipid organization, increasing molecular packing

Made available in DSpace on 2017-07-21T19:26:05Z (GMT). No. of bitstreams: 1 Fabiano Meira Luz.pdf: 2485302 bytes, checksum: 8a6331dbf4508b235557d3d643c996a7 (MD5) Previous issue date: 2013-04-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The process of drainage of organic soils can significantly alter the dynamics of soil organic matter (SOM), as it promotes the oxygenation of the medium and accelerates microbial activity. The spectroscopic techniques of electron paramagnetic resonance (EPR), laser induced fluorescence (LIF) and absorption in the UV-Visible were used in the study of humification of soil organic matter (SOM) not physically or chemically fractionated.. Soil samples were collected in a low land area in the city of Ponta Grossa - PR submitted to three different historical uses. They are R: reforestation DCF: drainage, cultivation and fallow; DF: Drain and fallow. The results indicate differences in the degree of humification of samples from drained and non-drained areas. The data suggest that the non-drained soil of the area R has a lower decomposition rate in the deeper profiles where the availability of O2 is lower. In the drained areas, DF and DCF, the degree of humification of SOM profile at 0.0 - 0.1 m is always less humified, as verified by all the techniques. / O processo de drenagem dos solos orgânicos pode alterar significativamente a dinâmica da matéria orgânica do solo (MOS), pois promove a oxigenação do meio e acelera a atividade microbiana. As técnicas espectroscópicas ressonância paramagnética eletrônica (RPE), fluorescência induzida a laser (FIL) e absorção no UV-visível foram utilizadas no estudo da humificação da matéria orgânica do solo (MOS), não fracionadas física ou quimicamente. As amostras de solo foram coletadas em área de várzea localizada na cidade Ponta Grossa – PR e submetidas a três diferentes históricos de uso. A saber, reflorestamento – R, drenagem-cultivo e pousio – DCP; drenagem e pousio – DP. Os resultados obtidos apontam diferenças entre o grau de humificação das amostras das áreas drenadas e não drenadas. Os dados indicam que o solo não drenado da área R, tem menor taxa de decomposição nos perfis mais profundos, onde a disponibilidade de O2 é menor. Nas áreas drenadas, DP e DCP, o grau de humificação da MOS no perfil 0,0 - 0,1 m apresenta-se menos humificado, resultado encontrado por todas as técnicas.

O trabalho ora apresentado foi concebido como tendo dois objetivos. O primeiro, mais geral, foi implementar uma nova metodologia para o estudo de mudanças conformacionais em proteínas, ou seja, de sua dinâmica estrutural. A técnica de marcação de spin sítio dirigida aliada à ressonância paramagnética eletrônica (SDSL-RPE) são os pilares desse novo método que faz, agora, parte do conjunto de técnicas disponíveis no Grupo de Biofísica Molecular Sérgio Mascarenhas do Instituto de Física de São Carlos (USP). O segundo objetivo, mais específico, representou o caminho efetivamente tomado para que se alcançasse o objetivo geral. Para isso, foi proposto o estudo da correlação estrutura e função de dois sistemas biológicos muito interessantes. O primeiro deles envolveu o estudo do movimento das hélices que compõem a estrutura da proteína ligante de cálcio S100A12 humana (HS100A12) induzido pelos íons cálcio e zinco. Sabendo que a proteína S100A12 humana além de ligar íons Ca+2, apresenta afinidade por outros metais divalentes, como os íons Zn+2 e Cu+2, e que a formação de diferentes oligômeros da proteína é governada pela concentração dos íons Ca+2 e Zn+2, realizamos estudos espectroscópicos utilizando a técnica de dicroísmo circular a fim de investigarmos a estabilidade térmica da proteína HS100A12 na presença e ausência dos íons cálcio e zinco. Mudanças conformacionais na estrutura da HS100A12 foram monitoradas através da construção de uma série de mutantes (simples e duplos) em que resíduos nas hélices B, C e D foram trocados por cisteínas, subsequentemente marcadas com a sonda magnética MTSSL e submetidas às análises de SDSL-RPE. Estas consistiram na medida do espectro de RPE dos vários mutantes em temperatura ambiente para estudarmos os efeitos da presença dos íons sobre a dinâmica experimentada pela sonda nas diversas posições. Além disso, efetuamos medidas de distância entre duas sondas seletivamente inseridas na estrutura protéica, procurando assim complementar o entendimento acerca do efeito da presença dos íons sobre a proteína. Por fim, devido ao fato da proteína HS100A12 estar envolvida em alguns eventos de sinalização celular e interação com o receptor para produtos de glicosilação (RAGE), decidimos também, estudar a interação da proteína com modelos de biomembranas, utilizando monocamadas de Langmuir. O outro problema de interesse utilizou a lizosima do fago T4, uma proteína padrão, da qual uma variedade de mutantes é produzida rotineiramente a fim de obtermos mais detalhes a respeito da sua correlação estrutura e função e tornar mais sólido o entendimento da técnica SDSL. Inicialmente, realizamos um estudo com a suposta criação de uma cavidade no \"core\" hidrofóbico da porção C-terminal da enzima, quando mutamos a Leu 133 por Ala e/ou Gly, ou seja, quando trocamos um resíduo grande por um de menor volume, pois se acredita que a proteína sofra um reajuste estrutural com o intuito de preencher o espaço vazio criado por essa substituição. Para isso, propusemos estudar por SDSL o movimento da α-hélice H inserindo o marcador de spin na posição vizinha ao resíduo mutado. Adicionalmente, realizamos um experimento de \"transmutação\" com a enzima T4L, a fim de investigar a natureza das contribuições para os diferentes modos dinâmicos experimentados pelo marcador de spin quando introduzido em sítios topologicamente semelhantes. / The work presented here was conceived with two main objectives. The first one, more general, involved the implementation of a new methodology for the study of conformational changes in proteins, i.e., its structural dynamics. The technique of Site-directed Spin Labeling combined with Electronic Paramagnetic Resonance (SDSL-EPR) are the pillars of this new method, which is now part of the set of techniques available at the Grupo de Biofísica Molecular Sérgio Mascarenhas, Instituto de Física de São Carlos (USP). The second objective, more specific, represented the path actually taken to achieve the overall goal. Therefore, it was proposed to study the structure-function correlation in two interesting biological systems. The first involved the study of the movement of the helices that form the structure of the human calcium binding protein S100A12 (HS100A12) induced by calcium and zinc ions. Knowing that, besides Ca+2, human S100A12 has also affinity for other divalent metals, such as Zn+2 and Cu+2 ions, and that the formation of different protein oligomers is governed by the concentration of Ca+2 and Zn+2, we performed spectroscopic studies using circular dichroism (CD) to investigate the thermal stability of protein HS100A12 in the presence and absence of calcium and zinc. Conformational changes in the structure of HS100A12 were monitored by producing a series of mutants (singles and doubles) in which residues in helices B, C and D were replaced by cysteine and subsequently labeled with a magnetic probe MTSSL and then analyzed via SDSL-EPR. The latter consisted of the EPR spectra measurement of many mutants at room temperature to study the effects of the presence of ions on the dynamics experienced by the probe in different positions. In addition, we performed measurements of the distance between two probes inserted in the protein structure, thereby, seeking to improve the understanding of the effect of the ions presence on the protein. Finally, due to the fact that HS100A12 is involved in some events of cell signaling and interaction with the Receptor for Advanced Glycation End Products (RAGE), we also decided to study the interaction of protein with models of biomembranes using Langmuir monolayers. In the other problem of interest, we used a variety of mutants of the enzyme T4 lysozyme, a protein standard, in order to obtain more details about its structure-function correlation and make more solid the understanding of SDSL technique. Initially, we conducted a study about the alleged creation of a cavity in the hydrophobic C-terminal portion of the enzyme, when we replaced the Leu 133 by Ala and/or Gly, or when we changed a large residue for a smaller one, because it is believed that the protein undergoes a structural adjustment in order to fill the gap created by this substitution. For this, we studied by SDSL the α-helix H motion, inserting the spin label in a neighbor position of the mutated residue. Additionally, we performed an experiment of \"transmutation\" with the enzyme T4L in order to investigate the nature of contributions for different dynamic modes experienced by the spin label when it is introduced in topologically similar sites.