Les smectites riches en fer appelées nontronite jouent un rôle majeur dans de nombreux processus biogéochimiques à la surface de la Terre. La compréhension de ces processus requiert une caractérisation cristallochimique détaillée de ces minéraux, avec notamment une connaissance précise du statut du fer. Les signatures spectrales du fer structural des nontronites ont été étudiées par spectroscopie infrarouge (IR), Mössbauer et XPS. La mise en évidence de ces signatures a été possible grâce à la synthèse hydrothermale à 150°C d’une série de nontronites avec une large gamme de teneur en [4]Fe(III). Les caractéristiques spectrales IR permettent d'effectuer une estimation robuste de la quantité de [4]Fe(III) tétraédrique présente dans la structure de la nontronite. Ces synthèses ont également permis de faire le lien entre la cristallochimie des nontronites synthétiques et la spéciation aqueuse du Si, Fe et Na. Les calculs de la spéciation aqueuse du Si montrent que l'augmentation des espèces anioniques H3SiO4-(aq) et H2SiO42-(aq) liée à l'augmentation du pH, favorise l'incorporation du Fe(III) dans les sites tétraédriques de la nontronite. La stabilité de ces nontronites dans les conditions de synthèse a ensuite été étudiée en fonction du temps. Une transformation minéralogique a été mise en évidence, amenant à la dissolution de la nontronite au profit d'un assemblage aegirine et hématite. Ces résultats montrent clairement que les nontronites synthétisées sont transitoires dans le système étudié. / Iron-rich smectites named nontronite are playing a great role in lot of biogeochemical processes at the Earth's surface. To better understand these processes a characterization of iron status in nontronites is required. Spectral iron features in the nontronite structure were studied by infrared (IR), Mössbauer, and XPS spectroscopies. IR characteristic fingerprints of Fe(III) allowed to obtain a detailed overview of the crystal-chemistry of this minerals, notably a robust estimation of the tetrahedral [4]Fe(III) content of the nontronite. This study was possible thanks to an experimental approach using hydrothermal synthesis (150°C) of a series of nontronites with a wide range of [4]Fe(III) content. These syntheses allowed to assess the link between the crystal-chemistry of synthetic nontronites and the aqueous speciation of Si, Fe, and Na. The aqueous speciation calculations indicated that the increase in anionic species H3SiO4-(aq) and H2SiO42-(aq) linked to the increase in pH, favored the incorporation of Fe(III) in the tetrahedral sites of nontronites. The stability of nontronites versus time was also studied. A mineral transformation was evidenced with the formation of the aegirine and hematite through the dissolution of nontronite. These results revealed that the synthetic nontronite are transitional in this experimental syntheses.

The objective of this study was to evaluate the functional and structural properties of different bovine collagen hydrolysates obtained through ultrasound assisted enzymatic hydrolysis. Six different samples of commercial bovine collagen were used in the hydrolyses: fiber, powder fiber, hydrolysate1, hydrolysate 2, gelatin 1 and gelatin 2 along with three different enzymes: Alcalase®, Flavourzyme® and pepsin. The analyses carried out were: physical-chemical, structural composition (Fourier transform infrared spectroscopy), hydrolysis degree, antioxidant and antimicrobial activity. Significant difference (p<0.05) was found for humidity, ashes, proteins, lipids, pH, hydroxyproline and collagen in the different crude samples. The use of previous or simultaneous ultrasound strengthened the enzymatic hydrolysis of the samples, generating larger number of bands in wave numbers (cm-1). Regarding the use of the enzyme Alcalase®, the enzymatic hydrolysis (16% enzyme) and previous ultrasound, applied to the samples (except for the fiber and powder fiber samples) resulted in lower residual protein content, higher hydrolysis degree and higher antioxidant activity. The hydrolysate 1 sample presented the lowest residual protein content (7.06 mg/mL), while hydrolysate 2 presented the highest hydrolysis degree (10.3%) and the gelatin 1 sample had the highest antioxidant activity (66.2%). When using the enzyme Flavouzyme®, the enzymatic hydrolysis and simultaneous ultrasound led to lower residual protein content and higher hydrolysis degree, and the hydrolysate 1 sample was the one with the highest antioxidant activity (52.2%) in the treatment that used enzyme only. When the enzyme pepsin was employed, the hydrolysate 1 sample presented the lowest residual protein content (4.50 mg/mL), while the Fiber sample had the highest hydrolysis degree value (21.7%) and the gelatin 2 sample showed the highest antioxidant activity value (53.7%). The lowest residual protein content resulted from the higher hydrolysis of the protein structure of the samples, due to the different treatment components. The samples that suffered highest rupture of their peptide bonds presented higher hydrolysis degree. The hydrolysates with the highest hydrolysis degree values not always presented good values of antioxidant activity, since the generation of high functionality peptides is what determines these values. Previous ultrasound worked better than the simultaneous one when the enzyme Alcalase® was used, applying a 16% concentration to the substrate. For the enzyme Flavourzyme®, the use of ultrasound did not result in significant difference in the results obtained. The enzyme Alcalase® provided the highest antioxidant activity values, while the enzyme Flavourzyme® presented the highest hydrolysis degree values, and pepsin showed the lowest residual protein average. The hydrolysates obtained from the treatments with the enzyme pepsin in the hydrolysis presented the highest inhibiting action against the bacteria Salmonella choleraesuis and Staphylococcus aureus in lower-than-10% concentrations. Therefore, enzymes and different ultrasound applications were concluded to provide higher structural rupture of samples and increase in the functionality of different bovine collagen hydrolysates (antioxidant and antimicrobial activities). / Foi avaliado as propriedades funcionais e estruturais de hidrolisados de diferentes colágenos bovinos obtidos por hidrólise enzimática assistida por ultrassom. Foram utilizadas nas hidrólises seis diferentes amostras comerciais de colágeno bovino: fibra, fibra pó, hidrolisado 1, hidrolisado 2, gelatina 1 e gelatina 2 e três diferentes enzimas: Alcalase®, Flavourzyme® e pepsina. Foram realizadas análises físico-químicas, de composição estrutural (espectroscopia na região do infravermelho com transformada de Fourier), grau de hidrólise, atividade antioxidante e atividade antimicrobiana. Houve diferença significativa (p<0,05) para umidade, cinzas, proteínas, lipídios, pH, hidroxiprolina e colágeno para as diferentes amostras brutas. A utilização do ultrassom prévio ou concomitante potencializou a hidrólise enzimática das amostras, gerou maior quantidade de bandas em número de onda (cm-1). Quando do uso da enzima Alcalase®, a hidrólise enzimática (16% de enzima) e o ultrassom prévio, aplicados as amostras (com exceção das amostras fibra e fibra pó) provocaram menores teores de proteínas residuais, maior grau de hidrólise e maior atividade antioxidante. A amostra hidrolisado 1 apresentou o menor teor de proteína residual (7,06 mg/mL), hidrolisado 2 o maior grau de hidrólise (10,3%) e gelatina 1 a maior atividade antioxidante (66,2%). Na aplicação da enzima Flavouzyme®, a hidrólise enzimática e o ultrassom concomitante levaram a menores teores de proteína residual e maior grau de hidrólise, onde a amostra hidrolisado 1 foi a que apresentou maior atividade antioxidante (52,2%), referente ao tratamento utilizando apenas enzima. Na aplicação da enzima pepsina, a amostra hidrolisado 1 apresentou o menor teor de proteína residual (4,50 mg/mL), a amostra fibra o maior valor de grau de hidrólise (21,7%) e a amostra gelatina 2 o maior valor de atividade antioxidante (53,7%). Os menores teores de proteína residual foram decorrentes da maior hidrólise da estrutura proteica das amostras, ocasionadas pelos diferentes componentes dos tratamentos. As amostras que sofreram maior rompimento de suas ligações peptídicas apresentaram maior grau de hidrólise. Os hidrolisados com maiores valores de grau de hidrólise nem sempre apresentaram bons valores de atividade antioxidante, pois a geração de peptídeos de alta funcionalidade é o que determina estes valores. O ultrassom prévio foi melhor quando da utilização da enzima Alcalase®, utilizando uma concentração de 16% sobre o substrato. Para a enzima Flavourzyme® a utilização do ultrassom que proporcionou melhores resultados foi a concomitante a hidrólise enzimática. Já para a enzima pepsina a utilização das diferentes aplicações do ultrassom não acarretou em diferenças significativas nos resultados obtidos. A enzima Alcalase® proporcionou os maiores valores de atividade antioxidante, enquanto que a enzima Flavourzyme® os maiores valores de grau de hidrólise, e a pepsina a menor média de proteína residual. Os hidrolisados obtidos dos tratamentos que utilizaram a enzima pepsina na hidrólise apresentam maior inibição das bactérias Salmonella choleraesuis e Staphylococcus aureus em concentrações menores que 10%. Diante do exposto, pode-se concluir que as enzimas e as diferentes aplicações do ultrassom proporcionaram maior rompimento estrutural das amostras e aumento da funcionalidade dos hidrolisados (atividade antioxidante e antimicrobiana) de diferentes colágenos bovinos.

Ce mémoire rapporte les études de protéines amyloïdes en interaction avec des membranes modèle afin d’établir une relation structure toxicité. Nous avons choisi différents modèles membranaires (monocouches, bicouches) de composition lipidique et charges différentes et utilisé différentes méthodes physico-chimiques afin de caractériser les interactions des protéines amyloïdes avec les membranes.Nous avons montré l’importance de la contribution électrostatique dans les interactions de la protéine amyloïde HET-s (218-289) et ses mutants avec les membranes modèles.L’ellipsométrie a démontré que les mutants toxiques de HET-s (218-289) (M8, WT.Y1Y2V2) perturbentfortement les monocouches lipidiques à l’interface air-eau. La structure riche en feuillets β antiparallèles des protéines àl’interface air-eau et dans l’interaction avec les monocouches de lipides a été démontrée par la spectroscopie PMIRRAS (Polarization Modulation – Infrared Reflection Absorption Spectroscopy). Nous avons établie que l’interface air-eau peut modifier l’agrégation des protéines amyloïdes. A l’aide de la spectroscopie de fluorescence, la spectroscopie PWR (Plasmon-Waveguided Resonance) et la spectroscopie ATR-FTIR (Attenuated Total Reflection – Fourier Transform Infrared), nous avons mis en évidence que la protéine toxique M8 adopte une structure riche en feuillets β antiparallèles en altérant fortement l’intégrité des bicouches lipidiques. Au contraire, la protéine non toxique WT se structure en feuillets β parallèles dans ces interactions et elle ne perturbe pas l’homogénéité des membranes. La toxicité de la protéine M8 semble liée à son organisation différente et à sa capacité à réorganiser les membranes.Nos résultats confortent également l’hypothèse de la toxicité des oligomères amyloïdes.Une étude sur la fabrication d’une cellule microfluidique pour la séparation de différents types d’autoassemblage afin de les détecter et de les étudier en interaction avec des liposomes par spectroscopie infrarouge est présentée. Une cellule microfluidique de CaF2 de 8 μm d’épaisseur de canaux est obtenue et est utilisée pour la détection d’une protéine de test. / This manuscript reports the studies of amyloid proteins in interaction with membrane models in order to establish their structure-toxicity relationship.Membrane models (monolayer, bilayer) of different charge and lipid composition were used. We used various physico chemical methods to characterize the interaction of these amyloid proteins with membranes.We showed the importance of the electrostatic contribution in the interactions of the amyloid protein HET-s(218-289) and its mutants with model membranes.Ellipsometry showed that the toxic mutants of HET-s (218-289) (M8, WT.Y1Y2V2) strongly disturbed thelipid monolayers at the air-water interface. The structure rich in antiparallel β sheets of auto-assembled proteins at theair-water interface and in interaction with lipid monolayers at the air-water interface has been demonstrated by the PMIRRAS spectroscopy (Polarization Modulation - Infrared Reflection Absorption Spectroscopy). We established that theair-water interface can change the aggregation properties of amyloid proteins.By using fluorescence spectroscopy, PWR spectroscopy (Plasmon Resonance-Waveguided spectroscopy) and ATR-FTIR spectroscopy (Attenuated Total Reflection - Fourier Transform Infrared spectroscopy), we found that thetoxic protein (M8) adopted a structure rich in antiparallel β sheets greatly altered the integrity of lipid bilayers. Incontrast, the protein non-toxic (WT) organized in a structure rich in parallel β sheets in these interactions and it did notdisturb the homogeneity of the membranes. The toxicity of the protein M8 appears to be related to its differentorganization and its ability to rearrange membranes.Our results also support the hypothesis of the toxicity of amyloid oligomers.A study on the fabrication of a microfluidic cell for the separation of different aggregation states of amyloidproteins in order to detect these assemblies and to study their interaction with liposomes by infrared spectroscopy is presented. A CaF2 microfluidic cell with channels of 8 μm of thickness was obtained and was used for the detection of atested protein.

La lipase pancréatique apparentée de type 2 du cobaye (GPLRP2) hydrolyse une grande variété de substrats lipidiques. Elle montre cependant une sélectivité selon l’organisation supramoléculaire du substrat et la présence de surfactants comme les sels biliaires (NaTDC). Nous avons utilisé la spectroscopie infrarouge (FTIR) pour étudier les interactions entres les phospholipides (DPPC), les surfactants et la GPLRP2 dans des conditions expérimentales proches de celles du tractus digestif. Pour étudier l’étape d’adsorption indépendamment de l’hydrolyse, un variant inactif de GPLRP2 (S152G) a été produit. Diverses dispersions aqueuses de phospholipides ont été préparées : des vésicules multilamellaires (MLV), unilamellaires (LUV) et des micelles mixtes DPPC-surfactant. GPLRP2 hydrolyse le DPPC présent dans des micelles mixtes DPPC-NaTDC mais n’a aucune activité sur le DPPC en phase lamellaire ou présent dans des micelles DPPC-Triton X100. L’analyse par FTIR de l’interaction de GPLRP2 S152G avec le système DPPC-NaTDC montre des changements importants dans le désordre conformationnel et la mobilité des chaînes acyles, la déshydratation de l’interface, l’orientation des têtes polaires et leurs liaisons hydrogène. Aucun effet n’est observé avec les MLV, les LUV ou le système DPPC-Triton X100. Il y a ainsi une reconnaissance spécifique du DPPC dans les micelles mixtes avec les sels biliaires, en accord avec l’activité enzymatique de GPLRP2. Les changements du spectre IR pendant l’hydrolyse du DPPC par la GPLRP2 ont été suivis. Certaines caractéristiques attribuées à la formation de produits de lipolyse peuvent être utilisées pour une étude quantitative de la lipolyse par FTIR. / Guinea pig pancreatic lipase-related protein type 2 (GPLRP2) hydrolyzes a large set of lipid substrates, but displays however some selectivity depending on the supramolecular structure of substrate and the presence of surfactants like bile salts (NaTDC). We used Fourier transform infrared (FTIR) spectroscopy to study the interactions between phospholipids (DPPC), surfactants and GPLRP2 under conditions close to those of the GI tract. To study the adsorption step independently from hydrolysis, a GPLRP2 inactive variant (S152G) was produced. Various phospholipid dispersions were prepared: multilamellar (MLV) and large unilamellar vesicles (LUV) and mixed micelles with surfactants. GPLRP2 was found to hydrolyze DPPC present in mixed DPPC-NaTDC micelles but was inactive on DPPC vesicles and DPPC-Triton X100 micelles. FTIR analysis of GPLRP2 S152G interaction with the DPPC-NaTDC system showed a decrease in the conformational disorder and mobility of the acyl chains, a dehydratation of the interface, and changes in the orientation and H-bonding of DPPC polar head-groups. These effects were not observed with MLV, LUV and DPPC-Triton X100 micelles, thus indicating a specific recognition of DPPC in mixed phospholipid-bile salt micelles, in agreement with phospholipase activity measurements. Changes in the IR spectra during DPPC hydrolysis by GPLRP2 were monitored. Specific spectral features were associated to the production of lipolysis products and could be used for quantifying phospholipid lipolysis by FTIR.

Les travaux présentés dans ce mémoire concernent la caractérisation par infrarouge à transformée de Fourier (FTIR) et par spectroscopie d’émission optique (SEO) des réactions chimiques entre post-décharges et précurseurs organosiliciés, avec comme exemple le cas du 3-aminopropyltriéthoxysilane (APTES). Le but est d’obtenir la rétention la plus élevée possible de fonctions amine -NH2 dans les revêtements ou dans les nanoparticules synthétisées. Tout d’abord, un état de l’art des post-décharges Ar-N2 et Ar-O2 et leurs applications est présenté ainsi que la cinétique d’interaction de l’APTES dans ces post-décharges, mettant en évidence le rôle des mélanges plasmagènes sur la décomposition du précurseur et sur la nature des films déposés. Ensuite l’étude de la décomposition de l’APTES dans une post-décharge Ar-N2 est réalisée. Les analyses par SEO et par FTIR in situ montrent le rôle des atomes d’azote N dans la formation des différents sous-produits, à savoir HCN, CO et C=O. Les nanoparticules synthétisées contiennent peu d’amine primaire, et présentent une concentration non négligeable d’azote sous forme d’amide secondaire. Dans le cas de l’étude de la décomposition de l’APTES dans la post-décharge Ar-O2 en mode pulsé, une tendance se dessine : les nanoparticules synthétisées en phase gazeuse lorsque les rapports cycliques augmentent ont une composition qui s’appauvrit en azote et en carbone mais s’enrichit en oxygène. Les groupements NH2 initiaux sont fortement convertis en groupement amide. Les nanoparticules synthétisées avec des rapports cycliques élevés ont des compositions différentes de celles des films minces déposés sur les parois, plus proches de la silice et ce en raison de la gravure par l’oxygène atomique qui les affecte davantage. Le comportement spécifique des atomes d’oxygène et d’azote en post-décharge rend difficile la rétention des amines dans les polymères plasmas. Enfin nous avons terminé par une étude de l’hydrodynamique dans le cas de l’interaction entre l’acétylène et une post-décharge Ar-O2 et établi l’importance de l’écoulement sur toute approche visant à faire des mesures FTIR résolues temporellement / The present work deals with the characterization by Fourier transform infrared spectroscopy (FTIR) and by optical emission spectroscopy of chemical reactions between a post-discharge and an organosilicon precursor: the 3-aminopropyltriethoxysilane (APTES). The aim is to keep the highest retention of amine functions -NH2 in coatings or in the synthesized nanoparticles. First, a state of the art of Ar-N2 and Ar-O2 post-discharges and their applications is presented as well as the kinetics of the interaction of APTES in these post-discharges, highlighting the role plasma gases on the decomposition of the precursor and the nature of the deposited films. Then, the study of the decomposition of APTES in an Ar-N2 post-discharge is carried out. Analysis by optical emission spectroscopy (OES) and in situ FTIR show the role of the nitrogen atoms N in the formation of various main by-products, namely HCN, CO and C=O. The synthesized nanoparticles contain few primary amines, and have a significant concentration of nitrogen in the form of secondary amide. In the case of interaction APTES with pulsed Ar-O2 afterglow, there is one main trend: the nanoparticles synthesized in the gas phase when the duty cycle increases have a composition that decreases in nitrogen and carbon but increases in oxygen. The -NH2 groups are efficiently converted into amide groups. The nanoparticles synthesized with high duty cycle exhibit compositions that are different from those of thin films deposited on the walls, the latter being close to silica because of the etching by atomic oxygen, which affects them more. The specific behavior of oxygen and nitrogen atoms in post-discharge makes difficult the retention of a high level of amines in plasma polymers. Finally, we finished with a study of the hydrodynamics in the case of the interaction of acetylene with an Ar-O2 post-discharge and proved the key role of the flow for any approach aiming at getting time-resolved FTIR measurements

L'une des particularités de Mycobacterium tuberculosis, agent pathogène de la tuberculose, est sa capacité à accumuler des lipides dans son cytoplasme, ce qui favorise son entrée en dormance. Le séquençage du génome de M. tuberculosis a permis d'identifier certains gènes codant pour des enzymes lipolytiques, parmi ceux-ci : le gène codant pour la protéine Rv3097c aussi appelée LipY (composée d'un domaine PE et d'un domaine lipase relié par un Linker). Dans la première partie de ce travail de thèse, nous avons procédé à la caractérisation biochimique de LipY, mais aussi de ses formes mutantes LipY(∆PE), LipY(∆149) et LipY(∆170), et à l'étude d'inhibition des membres de la famille Lip apparentés à la lipase hormono-sensible humaine (Lip-HSL). Nous avons pu déterminer les propriétés cinétiques de l'activité lipase de LipY et de ses différentes formes mutantes.Dans la seconde partie, nous nous sommes intéressés à la compréhension du rôle des différents domaines de LipY, et du linker dans l'hydrolyse des lipides au cours de l'infection en utilisant des macrophages spumeux (macrophage riche en lipides) infectés au préalable par des souches de M. bovis BCG recombinantes. Ces résultats et les hypothèses posées durant ce travail de thèse, pourraient être appuyés par l'obtention de la structure tridimensionnelle de LipY. Pour cela, nous avons initié et procédé à la cristallogenèse de LipY. La poursuite des études d'optimisation des cristaux obtenus pourrait permettre d'aller plus en profondeur dans l'élucidation du rôle et du mécanisme d'action de LipY. / Mycobacterium tuberculosis is a pathogenic agent, responsible of the tuberculosis, which can store lipids into the cytoplasm. This accumulation allows the bacteria to enter in the dormancy phase. The sequencing of M. tuberculosis genome, allows to identify some genes coding for lipolytic enzymes, among which a gene coding for Rv3097c protein, also called LipY. (possessing PE domain linkto a lipase domain). During my PhD thesis, we first biochemically characterized LipY and its mutant forms LipY(ΔPE); LipY(Δ149) and Lip(YΔ170) and studied the inhibition of Lip family members related to the human hormone-sensible lipase (Lip-HSL). We determined the kinetic properties for the lipase activity of LipY and its mutants. In the second part, based on these previous results, we studied the role of these different domains and the linker on the hydrolysis of lipids during the infection phase, in infected foamy macrophages (lipids rich). For these studies, we used foamy macrophages infected by recombinants strains of M. bovis BCG (LipY(wt) and its mutants.These results and hypothesis can be confirmed and supported by resolving the tridimensional structure of LipY. Crystallogenesis tests allowed us to have some crystals of LipY(wt), which after optimization would allow us to have a better understanding of the role and action mechanism of LipY.

The feasibility of FTIR-based spectroscopy as a tool to measure cellular response to therapeutics was investigated. Fourier transform mid-infrared spectroscopy has been used in conjunction with multivariate analysis (MVA) to assess the chemistry of many clinically relevant biological materials; however, the technique has not yet found its place in a clinical setting. One issue that has held the technique back is due to the spectral distortions caused by resonant Mie scattering (RMieS), which affects the ability to confidently assign molecular assignments to the spectral signals from biomaterials. In the light of recently improved understanding of RMieS, resulting in a novel correction algorithm, the analytical robustness of corrected FTIR spectra was validated against multi-discipline methods to characterise a set of renal cell lines which were selected for their difference in morphology.After validation of the FTIR methodology by discriminating different cell lines, the second stage of analyses tested the sensitivity of FTIR technique by determining if discrete chemical differences could be highlighted within a cell population of the same origin. The renal carcinoma cell line 2245R contains a sub-population to contain a sub-population of cells displaying 'stem-cell like' properties. These stem-like cells, however, are difficult to isolate and characterise by conventional '-omic' means. Finally, cellular response to chemotherapeutics was investigated using the established renal cell lines CAKI-2 and A-498. For the model, 5-fluorouracil (5FU), an established chemotherapeutic agent with known mechanisms of action was used. Novel gold-based therapeutic compounds were also assessed in parallel to determine their efficacy against renal cell carcinoma. The novel compounds displayed initial activity, as the FTIR evidence suggested compounds were able to enter the cells in the first instance, evoking a cellular response. The long-term performance, tracked with standard proliferation assays and FTIR spectroscopy in the renal cancer cell model, however, was poor. Rather than dismissing the compounds as in-active, the compounds may simply be more effective in cancer cell types of a different nature. The FTIR-based evidence provided the means to suggest such a conclusion. Overall, the initial results suggest that the combination of FTIR and MVA, in the presence of the novel RMieS-EMSC algorithm can detect differences in cellular response to chemotherapeutics. The results were also in-line with complimentary biological-based techniques, demonstrating the powerful potential of the technique as a promising drug screening tool.

A transmissão de dados e informação na fibra óptica vem aumentando nos últimos anos. O interesse para avaliar melhor a confiabilidade mecânica tomou-se uma necessidade, depois que uma fibra, de um cabo instalado, estava se partindo com muita facilidade. O foco principal deste trabalho foi voltado para estabelecer metodologias para avaliar o estado de degradação dos polímeros usados. Através destas técnicas, buscou-se encontrar parâmetros (E\', E\", tan &#948, Tg, etc.) que pudessem indicar o estado de degradação dos revestimentos. Diferentes técnicas foram empregadas para avaliar o revestimento das fibras tais como: calorimetria exploratória diferencial (DSC), diferencial fotocalorimetria (DPC), análise térmica dinâmico mecânico (DMTA) e espectroscopia na região do infravermelho (FIIR). A partir da técnica desenvolvida, DMTA, esta pode ser empregada para avaliar a confiabilidade mecânica de várias fibras comercias. As técnicas DMTA e FIIR mostraram ser muito promissoras, pois consegue-se, através delas, obter muitos dados a respeito das características dos polímeros. / The datas and information transmission in the optical fiber have been increasing in the last years. The interest to evaluate the mechanical reliability became necessary, after that a optical fiber, from a cable installed, was cracking very easily. The principal aim of this work was establishing methodologies to evaluate the polymer degradation state. Through of this techniques we search to find out parameters (E\', E\", tan &#948, Tg. etc) that could denote the degradation state of the coatings. Different techniques were utilized to evaluate the coating of the optical fibers such as: differential scanning calorimeter (DSC), differential photocalorimeter (DPC), dynamic mechanic thermal analyzer (DMTA) and infrared spectroscopy (FIIR). From the method development (DMTA), it was possible to evaluate the mechanical realiability of many commercial fibers. DMTA and FTIR techniques shown to be very promising to obtain very dates to respect of polymer characteristics.

Kanalrhodopsine (ChRs) sind lichtgesteuerte Ionenkanäle aus einzelligen Grünalgen, die in der Optogenetik verwendet werden. Photonabsorption führt zur Isomerisierung des Retinal-Kofaktors, was eine Reihe von Reaktionen auslöst, die als Photozyklus bezeichnet werden und die Bildung des leitenden Zustands umfassen. In dieser Arbeit wurde der Photozyklus-Mechanismus ausgewählter ChRs mittels FTIR (Fourier Transform Infrarot)- und UV-Vis-Spektroskopie, sowie Retinalextraktion und HPLC (Hochleistungsflüssigkeitschromatographie)-Analyse untersucht. Photorezeptoren sind dafür optimiert, Lichtenergie zu nutzen, um Konformationsänderungen des Proteins hervorzurufen. Dafür wird ein Teil der Lichtenergie durch eine transiente Verdrillung des Chromophors gespeichert. In dieser Arbeit wird gezeigt, dass der Transfer der gespeicherten Energie zum Protein in ReaChR stark vom Protonierungszustand von Glu163 beeinflusst wird; er wird durch eine erhöhte Rigidität des aktiven Zentrums bei protoniertem Glu163 verlangsamt. In Chrimson hingegen relaxiert der Chromophor nach Photoisomerisierung, was auf einen verdrillten Chromophor im Dunkelzustand hinweist, was vermutlich für die bathochrome Verschiebung von Bedeutung ist. Zusätzlich zur Chromophorgeometrie beeinflusst der Protonierungszustand von Glu163 in ReaChR und dem homologen Glu165 in Chrimson die Stereoselektivität der Photoreaktion. Ein weiterer Faktor der Stereoselektivität ist Asp196 in ReaChR (Asp195 in C1C2), welches im Photozyklus deprotoniert. Die Bildung des leitenden Zustands in C1C2 und ReaChR geht mit einem Wassereinstrom ins Protein einher, welcher den Transport größerer Kationen erleichtert. Die Deprotonierung von Glu130 in ReaChR (Glu129 in C1C2) verändert die Ionenselektivität des Kanals, wie aus elektrophysiologischen Messungen bekannt ist. In Chrimson ist das Ausmaß des Wassereinstroms deutlich reduziert, was – in Übereinstimmung mit elektrophysiologischen Experimenten – den Transport von Protonen begünstigt. / Channelrhodopsins (ChRs) are light-gated ion channels found in single-cell algae and used in optogenetics. Photon absorption leads to isomerization of the retinal cofactor, initiating a number of reactions that are referred to as photocycle and involve formation of the ion-conducting state. In this thesis, the photocycle mechanism of selected ChRs was investigated using FTIR (Fourier Transform Infrared) and UV-Vis spectroscopy, as well as retinal extraction and subsequent HPLC (High Performance Liquid Chromatography) analysis. Photoreceptors are optimized to use photon energy to drive conformational changes of the protein. Therefore, a fraction of the photon energy is stored by a transient distortion of the chromophore. In this thesis, it is shown that in ReaChR the transfer of the stored energy to the protein is largely affected by the protonation state of Glu163, being decelerated by protonated Glu163 due to an enhanced rigidity of the active site. In contrast, the chromophore in Chrimson relaxes upon photoisomerization, hinting at a distorted retinal geometry in the dark state, which is probably essential for its unprecedented bathochromic absorption. In addition to the chromophore geometry, the protonation state of Glu163 in ReaChR and the homologue Glu165 in Chrimson affects the stereoselectivity of the photoreaction. Another factor for stereoselectivity is Asp196 in ReaChR (Asp195 in C1C2) which deprotonates in the photocycle. Formation of the ion-conducting state in C1C2 and ReaChR involves water influx into the protein, facilitating transport of larger cations. Deprotonation of Glu130 in ReaChR (Glu129 in C1C2) alters the ion selectivity of the channel as known from electrophysiological experiments. In Chrimson, the extent of water influx is drastically reduced which favors the conductance of protons in agreement with electrophysiological characterization.

<p>Ispitana&nbsp; je&nbsp; mogućnost ekstrakcije fumonizina iz kukuruza&nbsp;sa vodom i fosfatnim puferom, umesto sa sme&scaron;om&nbsp;acetonitril&ndash;metanol&ndash;voda.&nbsp; Ekstrakcione metode&nbsp;neorganskim&nbsp; rastvaraĉima&nbsp; su&nbsp; uspe&scaron;no&nbsp; primenjene za&nbsp;odreĊivanje&nbsp; fumonizina&nbsp; B₁, B_2&nbsp;i&nbsp; B_3&nbsp;metodom&nbsp; tečne&nbsp;hromatografije sa fluorescentnom detekcijom, kao i&nbsp;ukupnih fumonizina pomoću&nbsp; imunohemijske metode.&nbsp;Analiziran&nbsp; je&nbsp; veći broj&nbsp; uzoraka na&nbsp; prisustvo i sadržaj&nbsp;fumonizina.&nbsp; Ispitani su uzorci&nbsp; kukuruza (235), sakupljeni&nbsp;tokom vi&scaron;egodi&scaron;njeg perioda (2005.&nbsp; i 2009&minus;2013.&nbsp; godine)&nbsp; i&nbsp;p&scaron;enice (83) roda 2010. i 2012. godine, sa područja&nbsp; severne&nbsp;Srbije.&nbsp; Ispitana je kontaminiranost kukuruza sa područja&nbsp;<br />severne Srbije fumonizinima, drugim mikotoksinima i&nbsp;plesnima, kao i mogući uticaj klimatskih faktora na stepen&nbsp;kontaminacije.&nbsp; Rezultati kontaminiranosti useva&nbsp;fumonizinima&nbsp; za svaku godinu&nbsp; pojedinačno&nbsp; su povezivani&nbsp;sa vremenskim prilikama koje su&nbsp; pratile istu. Različita&nbsp;hrana na bazi žitarica je analizirana ELISA metodama.&nbsp;Razvijena je ELISA i metoda&nbsp; tečne hromatografije sa&nbsp;fluorescentnom detekcijom&nbsp; za određivanje fumonizina u tri&nbsp;vrste lekovitog bilja sa područja Srbije.&nbsp; Ispitana je&nbsp;mogućnost&nbsp; primene infracrvene spektroskopije sa&nbsp;Furijeovim transformom&nbsp; za &nbsp;odredivanje&nbsp; fumonizina.&nbsp;Razvijen je&nbsp; ekspertni sistem za re&scaron;avanje problema izbora&nbsp;<br />optimalnog postupka određivanja fumonizina u kukuruzu.</p> / <p>Possibility of fumonisin extraction from maize using water and phosphate buffer instead of acetonitrile-methanol-water mixture was examined. The methods of extraction without organic solvents have successfully been applied for the determination of fumonisins&nbsp; B1, B2 and&nbsp; B3 using&nbsp; liquid chromatography-fluorescence detection&nbsp; method,&nbsp; as well as total fumonisins using enzyme-linked immunosorbent&nbsp; assay&nbsp; (ELISA).&nbsp; Large number of samples was analyzed for the presence and content of fumonisins. Maize samples (total 235) collected throughout several-year period (2005 and 2009-2013) and wheat samples (total 83) from 2010 and 2012 harvest originating from the territory of northern Serbia were analyzed. Contamination of maize originating from the territory of northern Serbia with&nbsp;fumonisins and other mycotoxins and moulds was examined, as well as the potential impact of climatic factors on contamination level.&nbsp; The results on the fumonisin-contamination of cereals obtained for each individual year are related with the climatic&nbsp; conditions characteristic for the relevant year. A variety of cereal-based food was analyzed using ELISA methods. Improved&nbsp; liquid chromatography-fluorescence&nbsp; detection method and ELISA for the determination of fumonisins in three &nbsp;medicinal plant&nbsp; species from the territory of Serbia were developed.&nbsp; Possible application of&nbsp; Fourier transform&nbsp; infrared spectroscopy&nbsp; (FTIR) for quantification of fumonisin was investigated.&nbsp; An expert system&nbsp; to solve the problem of selecting an optimal&nbsp;method for&nbsp; determination of fumonisins in&nbsp; maize has been developed.</p>