La voie de sécrétion bactérienne de type V permet l’exportation à la surface cellulaire de protéines dont certaines ont été identifiées comme d’importants facteurs de la pathogénicité bactérienne. Le type V regroupe la sécrétion des autotransporteurs et la sécrétion à deux partenaires (TPS). Les autotransporteurs sont constitués d’un domaine en tonneau β; et d’un domaine passager. L’interaction de l’autotransporteur avec le complexe protéique Bam, dont la pièce centrale est le transporteur BamA, permet l’insertion dans la membrane externe du tonneau β; et la sécrétion du passager. En revanche, la sécrétion à deux partenaires fait intervenir deux protéines, l’une appelée TpsA correspondant à la protéine exportée et l’autre, TpsB, formant un tonneau β qui contrôle le transport à travers la membrane externe. Les protéines TpsB sont spécifiques à leur(s) TpsA associée(s), et font partie de la superfamille des transporteurs Omp85 qui effectuent l’insertion de protéines dans la membrane externe bactérienne comme BamA, et dans celles des organites eucaryotes dont les chloroplastes et les mitochondries. Au cours de mon doctorat, je me suis intéressé à la sécrétion de l’hémagglutinine filamenteuse (FHA), qui est l’adhésine majoritaire de Bordetella pertussis, l’agent étiologique de la coqueluche. Cette adhésine qui permet à la bactérie de coloniser le tractus respiratoire de l’hôte est une protéine TpsA de 220 kD. Elle est très efficacement sécrétée par la voie de sécrétion à deux partenaires grâce à son transporteur spécifique TpsB nommé FhaC. L’étude cristallographique de FhaC a révélé un tonneau β; à 16 brins qui forme un canal dans la membrane externe obstrué par l’hélice-α; amino-terminale, H1, partagée par la majorité des TpsB, et par une boucle de surface, L6, conservée dans la superfamille Omp85. Cette conformation suggère un état au repos dans lequel le canal bouché ne pourrait pas transporter son partenaire. Afin de comprendre comment la FHA transite à l’intérieur du pore, il est donc nécessaire de connaître les changements de conformations que subit FhaC. Durant mon travail de thèse, nous avons apporté une vision plus dynamique de la sécrétion à deux partenaires en utilisant le couple FHA/FhaC comme modèle d’étude. Pour cela nous avons utilisé principalement la Résonance Paramagnétique Electronique (RPE). Cette technique de biophysique permet d’étudier FhaC en solution ou réincorporée dans une bicouche lipidique et de rendre compte de la mobilité à un site donné par l’utilisation de sondes paramagnétiques. Ainsi nous avons pu montrer que FhaC est en équilibre entre plusieurs conformations, avec H1 dans le pore ou du côté périplasmique de FhaC. La présence de la FHA déplace cet équilibre, favorisant ainsi la sortie de l’hélice hors du pore. Nous avons, par ailleurs, pu démontrer expérimentalement que la FHA transitait bien à l’intérieur du pore formé par FhaC et que l’hélice H1 se trouvait alors dans le périplasme. L’étude de la boucle L6 nous a permis de montrer que la mobilité de cette boucle était fortement contrainte à l’intérieur du pore même lors de la reconnaissance avec la FHA. Ce ralentissement de mobilité est lié, en autre, à une interaction avec un résidu d’un motif conservé présent sur le brin β13 qui influence la taille du pore. De manière plus générale, cette étude de la dynamique de FhaC contribue à la compréhension des mécanismes moléculaires de la voie TPS et des transporteurs de la superfamille Omp85. / Type V secretion in bacteria mediates the export to the cell surface of proteins, some of which have been identified as important factors of pathogenicity. Type V includes the secretion of autotransporters and the ‘Two-partner Secretion’ (TPS) pathway. Autotransporters consist of a β barrel domain and a passenger domain. The interaction of autotransporters with the Bam complex, of which the BamA transporter is the central component, allows the insertion of the β; barrel in the outer membrane and the secretion of passenger domain. In contrast, the two-partner secretion involves two proteins, the exported ‘TpsA’ protein and its TpsB partner that controls its transport across the outer membrane. TpsB proteins are specific to their associated TpsA(s) and belong to the superfamily of the Omp85 transporters, which carry out the insertion of proteins into the bacterial outer membrane, like BamA, or in the outer membranes of eukaryotic organelles including chloroplasts and mitochondria. For my PhD work, I have been interested in the secretion of filamentous hemagglutinin (FHA), which is the major adhesin of Bordetella pertussis, the causative agent of whooping cough. This adhesin allows the colonization by this bacterium of its host’s respiratory tract. This protein corresponds to a 220kD TpsA protein efficiently secreted by its specific transporter TpsB named FhaC. Crystallographic studies have revealed that FhaC harbours a 16-stranded β;-barrel occluded by both the N-terminal α;-helix, H1, shared by the majority of TpsB proteins, and by a surface loop, L6, that carries a conserved, hallmark motif of the Omp85 superfamilly. This conformation suggests that FhaC is in a resting state in which the channel does not transport its partner. To understand how the FHA passes through the FhaC pore, it is necessary to address the conformational changes undergone by FhaC. During my thesis work, we provided a more dynamic view of the TPS pathway using the FHA/FhaC couple as study model. For this we used Electron Paramagnetic Resonance (EPR). This biophysical technique allows to study of FhaC in solution or reincorporated into a lipid bilayer and it reports the mobility at specific sites of the protein by using paramagnetic probes. Thus we have shown that FhaC is in equilibrium between multiple conformations, with H1 in the pore or at the periplasmic side of FhaC. The presence of FHA displaces the conformational equilibrium, promoting the exit of the helix going from the pore. We have also experimentally demonstrated that FHA does transit through the pore formed by FhaC while helix H1 is then in the periplasm. The study of the L6 loop enabled us to show that the mobility of this loop is highly constrained in the pore and remains so upon the recognition of FHA. Its slow mobility is linked to an interaction between an invariant L6 residue and a conserved motif present on the β; strand 13 of the barrel. This interaction affects the size of the FhaC pore.More generally, the study of the dynamics of FhaC contributes to the understanding the molecular mechanisms of the TPS pathway and of transporters of the Omp85 superfamily.

The research in low-dimensional (low-D) quantum spin systems has become an arduous challenge for the condensed matter physics community during the last years. In systems with low dimensional magnetic interactions the exchange coupling is restricted to dimensions lower than the full three-D exhibited by the bulk real material. The remarkable interest in this field is fueled by a continuous stream of striking discoveries like superconductivity, quantum liquid and spin gap states, chiral phases, etc, derived from the strong effect of quantum fluctuations on the macroscopic properties of the system and the competition between electronic and magnetic degrees of freedom. The main goal of the current studies is to reach a broad understanding of the mechanisms that participate in the formation of those novel ground states as well as the characteristic dependence with respect to relevant physical parameters. In this thesis we present the results of an Electron Spin Resonance (ESR)-based study on different quasi-1D spin systems, exemplifying the realization of 1D-magnetic spin-chains typically containing transition metal oxides such as Cu2+ or V4+. The local sensitivity of the ESR technique has been considered useful in exploring magnetic excitation energies, dominant mechanisms of exchange interactions, spin fluctuations and the dimensionality of the electron spin system, among others. Aside from ESR other experimental results, e.g., magnetization and nuclear magnetic resonance besides some theoretical approaches were especially helpful in achieving a proper understanding and modeling of those low-D spin systems. This thesis is organized into two parts: The first three chapters are devoted to the basic knowledge of the subject. The first chapter is about magnetic exchange interactions between spin moments and the effect of the crystal field potential and the external magnetic field. The second chapter is a short introduction on exchange interactions in a 1D-spin chain, and the third chapter is devoted to ESR basics and the elucidation of dynamic magnetic properties from the absorption spectrum parameters. The second part deals with the experimental results. In the fourth chapter we start with the magnetization results from the zero-dimensional endohedral fullerene Dy3N@C80. This system is seemingly ESR “silent” at the frequency of X-band experiments. The fifth chapter shows an unexpected temperature dependence of the anisotropy in the homometallic ferrimagnet Na2Cu5Si4O14 containing alternating dimer-trimer units in the zig-zag Cu-O chains. In the sixth chapter different magnetic species in the layer structure of vanadium oxide nanotubes (VOx-NT) have been identified, confirming earlier magnetization measurements. Moreover the superparamagnetic-like nature of the Li-doped VOx-NT samples was found to justify its ferromagnetic character at particular Li concentration on the room temperature scale. In the seventh chapter the Li2ZrCuO4 system is presented as a unique model to study the influence of additional interactions on frustrated magnetism. The eighth chapter highlights the magnetic properties of the pyrocompound Cu2As2O7. The results suggest significant spin fluctuations below TN. The thesis closes with the summary and the list of references.

This thesis describes investigations into the role of non-stoichiometry in the surface and bulk properties of SrTiO₃ single crystals. A family of (n×n) reconstructions, where n = 2, 3, 4, 5, 6 are produced by argon ion sputtering of the SrTiO₃ (111) single crystals and subsequent annealing in UHV or in an oxygen rich environment. The sputtering process introduces defects or oxygen vacancies in the surface region of the sample, whilst the annealing gives rise to surface reconstructions. The surface preparation conditions such as sputtering time, annealing temperature and environment are optimized to obtain various reconstructions in a controlled and reproducible manner. High resolution STM images of these reconstructions are also obtained and utilized in the investigation of the surface reactivity. Fullerene molecules are deposited on the reconstructed surfaces to elucidate the surface reactivity through template assisted growth. Fullerene molecules are first deposited with substrate surfaces held at room temperature. Being the most highly reduced among the (n×n) family, the 5×5 reconstruction significantly influenced the growth of fullerenes. Both C₆₀ and C₇₀ adsorb as individual molecules and produce clusters with magic numbers. The 4×4 and 6×6 reconstructed surfaces encourage the formation of close-packed structures upon the deposition at room temperature. When the surface covered with fullerenes is heated to a temperature of around 200 °C, epitaxial islands are observed. The 6×6 reconstructed surface appeared to be less reactive than the 4×4. Electrical transport, cathodoluminescence (CL) and electron spin resonance (ESR) experiments are also carried out to investigate the effect of oxygen vacancies on the bulk properties of UHV annealed SrTiO₃ single crystals. Thermal reduction leads to carrier doping of the material, which not only gives rise to electrical conduction but also induces room temperature luminescence. Both the electrical conductivity and CL intensity increases with annealing time. The work presented in this thesis provides insight into the defect driven properties in both the surface and bulk of SrTiO₃ single crystals, which could play an important role in the development of oxide-based electronic devices.

Brazil nuts (Bertholletia excelsa, H.B.K.) are seeds of high nutritional value collected from South American rainforests and its productive chain is one of the most important non-timber economic activities in Brazilian amazon. The main objectives of this research were: 1) characterize the occurrence of chemical changes in Brazil nut kernels (BNK), cold-pressed Brazil nut oil (BNO) and Brazil nut flour obtained by water extraction (BNF); and 2) investigate strategies of control and monitoring these changes during storage. For this, consolidated techniques, such as spectrophotometry and chromatography, and a relatively new analytical technique, the electron spin resonance (ESR) spectroscopy, were employed. As major results, it was found that different combinations of storage temperatures and atmosphere packages have differently affected the tendency of radical formation and off-flavor volatile aroma compounds generation in BNK, and that the combination of refrigeration with vacuum packing was able to keep BNK at their best. It was demonstrated that a spin-trapping ESR spectroscopy method would be suitable to monitor oxidative changes in BNO with known history stored either in clear or in brown glass bottles under retail conditions. For BNF, it was demonstrated that minor variations on water activity (aw) might significantly affect the rates of both lipid oxidation and nonenzymatic browning reactions during storage. There was indication that for BNF with initial aw of 0.196, but not for BNF with initial aw of 0.101, under the studied conditions, secondary products from lipid oxidation might be substrates for nonenzymatic browning products formation. As a conclusion, these results may help to better understand chemical deteriorative processes in BNK and its co-products, according to the storage conditions, and that the use of less sample-demanding, fast and solvent-free analytical method to monitor these changes in BNO is feasible. / A castanha do Brasil (Bertholletia excelsa, H.B.K.) é uma semente de boa qualidade nutricional coletada em florestas tropicais da América do Sul, cuja cadeia produtiva é uma das mais importantes atividades econômicas não madeireiras da Amazônia brasileira. Os principais objetivos desta pesquisa foram: 1) caracterizar a ocorrência de alterações químicas em castanhas do Brasil (CB), óleo de castanha do Brasil obtido por prensagem a frio (OCB) e farinha de castanha do Brasil obtida por extração aquosa (FCB); e 2) investigar estratégias para controlar e monitorar essas alterações ao longo do armazenamento. Para isso, técnicas consolidadas como a espectrofotometria e a cromatografia, e uma técnica relativamente recente, a espectroscopia de ressonância de spin eletrônico (RSE), foram empregadas. Dentre os principais resultados obtidos, foi possível constatar o efeito de diferentes combinações de temperaturas e atmosferas de embalagem sobre a tendência de formação de radicais e sobre a geração de compostos voláteis de aroma relacionados a odor indesejável em CB, e que a temperatura de refrigeração combinada com a embalagem a vácuo foi a mais eficiente na preservação da qualidade da CB. Demonstrou-se que o uso de um método de aprisionamento de spins de RSE pode ser eficiente para monitorar alterações químicas em OCB com histórico conhecido embalado em frascos de vidro transparente ou marrons sob condições de armazenamento comercial. Para FCB, foi demonstrado que pequenas variações na atividade de água (aw) podem afetar significativamente as taxas de oxidação lipídica e de reações de escurecimento não enzimático durante armazenamento. Obteve-se indicação de que para FCB com aw inicial de 0,196, mas não para FCB com aw inicial de 0,101, produtos secundários da oxidação lipídica podem ser substratos para a formação de produtos do escurecimento não enzimático. Como conclusão geral, os resultados obtidos podem ajudar a explicar melhor os processos de deterioração química em CB e seus coprodutos, conforme as condições de armazenamento, e que o uso de um método que requer menor quantidade de amostras, é rápido e não usa solventes é viável para o monitoramento da qualidade de OCB.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Brazil nuts (Bertholletia excelsa, H.B.K.) are seeds of high nutritional value collected from South American rainforests and its productive chain is one of the most important non-timber economic activities in Brazilian amazon. The main objectives of this research were: 1) characterize the occurrence of chemical changes in Brazil nut kernels (BNK), cold-pressed Brazil nut oil (BNO) and Brazil nut flour obtained by water extraction (BNF); and 2) investigate strategies of control and monitoring these changes during storage. For this, consolidated techniques, such as spectrophotometry and chromatography, and a relatively new analytical technique, the electron spin resonance (ESR) spectroscopy, were employed. As major results, it was found that different combinations of storage temperatures and atmosphere packages have differently affected the tendency of radical formation and off-flavor volatile aroma compounds generation in BNK, and that the combination of refrigeration with vacuum packing was able to keep BNK at their best. It was demonstrated that a spin-trapping ESR spectroscopy method would be suitable to monitor oxidative changes in BNO with known history stored either in clear or in brown glass bottles under retail conditions. For BNF, it was demonstrated that minor variations on water activity (aw) might significantly affect the rates of both lipid oxidation and nonenzymatic browning reactions during storage. There was indication that for BNF with initial aw of 0.196, but not for BNF with initial aw of 0.101, under the studied conditions, secondary products from lipid oxidation might be substrates for nonenzymatic browning products formation. As a conclusion, these results may help to better understand chemical deteriorative processes in BNK and its co-products, according to the storage conditions, and that the use of less sample-demanding, fast and solvent-free analytical method to monitor these changes in BNO is feasible. / A castanha do Brasil (Bertholletia excelsa, H.B.K.) é uma semente de boa qualidade nutricional coletada em florestas tropicais da América do Sul, cuja cadeia produtiva é uma das mais importantes atividades econômicas não madeireiras da Amazônia brasileira. Os principais objetivos desta pesquisa foram: 1) caracterizar a ocorrência de alterações químicas em castanhas do Brasil (CB), óleo de castanha do Brasil obtido por prensagem a frio (OCB) e farinha de castanha do Brasil obtida por extração aquosa (FCB); e 2) investigar estratégias para controlar e monitorar essas alterações ao longo do armazenamento. Para isso, técnicas consolidadas como a espectrofotometria e a cromatografia, e uma técnica relativamente recente, a espectroscopia de ressonância de spin eletrônico (RSE), foram empregadas. Dentre os principais resultados obtidos, foi possível constatar o efeito de diferentes combinações de temperaturas e atmosferas de embalagem sobre a tendência de formação de radicais e sobre a geração de compostos voláteis de aroma relacionados a odor indesejável em CB, e que a temperatura de refrigeração combinada com a embalagem a vácuo foi a mais eficiente na preservação da qualidade da CB. Demonstrou-se que o uso de um método de aprisionamento de spins de RSE pode ser eficiente para monitorar alterações químicas em OCB com histórico conhecido embalado em frascos de vidro transparente ou marrons sob condições de armazenamento comercial. Para FCB, foi demonstrado que pequenas variações na atividade de água (aw) podem afetar significativamente as taxas de oxidação lipídica e de reações de escurecimento não enzimático durante armazenamento. Obteve-se indicação de que para FCB com aw inicial de 0,196, mas não para FCB com aw inicial de 0,101, produtos secundários da oxidação lipídica podem ser substratos para a formação de produtos do escurecimento não enzimático. Como conclusão geral, os resultados obtidos podem ajudar a explicar melhor os processos de deterioração química em CB e seus coprodutos, conforme as condições de armazenamento, e que o uso de um método que requer menor quantidade de amostras, é rápido e não usa solventes é viável para o monitoramento da qualidade de OCB.