Discoveries made with KCl-induced contractions have elucidated the more complex reactions involved in GPCRs signaling; once the mechanisms of smooth muscle Ca2+ sensitization and desensitization are fully understood, then the development of advanced treatments for vascular disorders such as hypertension, cerebral and coronary vasospasm, and vascular hyporeactivity following hemorrhagic shock may be possible. Studies have shown that KCl-induced contractions induce Ca2+-sensitization. Therefore, we tested the hypothesis that KCl induced Ca2+-sensitization is due to ROK activation by the increase in [Ca2+]i. To test this hypothesis, rabbit femoral arteries were permeabilized with 20µg/ml α-toxin and 1% Triton X-100 and subjected to different calcium concentrations in the presence or absence of various ROK inhibitors. For a comparison we also used various PKC and MLCK inhibitors and repeated these experiments in intact tissues. We found that either [Ca2+]i alone does not directly activate ROK or the permeabilization technique itself disrupts the normal ROK signaling system. Secondary findings revealed that α-toxin activates PKC pathways; in both chemically permeabilized preparations proteases also appear to be activated and MLCK is the primary kinase responsible for contraction.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-08-16T20:34:44Z No. of bitstreams: 1 CynthiaKerziaCostaDeAraujo_DISSERT.pdf: 2646231 bytes, checksum: 4f582aa34d0b6a7e4961cbd1db1b17a2 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-08-18T22:50:18Z (GMT) No. of bitstreams: 1 CynthiaKerziaCostaDeAraujo_DISSERT.pdf: 2646231 bytes, checksum: 4f582aa34d0b6a7e4961cbd1db1b17a2 (MD5) / Made available in DSpace on 2016-08-18T22:50:18Z (GMT). No. of bitstreams: 1 CynthiaKerziaCostaDeAraujo_DISSERT.pdf: 2646231 bytes, checksum: 4f582aa34d0b6a7e4961cbd1db1b17a2 (MD5) Previous issue date: 2016-03-18 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / ?No Brasil s?o gerados diversos tipos de subprodutos e res?duos agroindustriais como: baga?o de ped?nculo de caju, baga?o de cana-de-a??car, baga?o de coco e outros. A disposi??o final desses res?duos, ou seja, sua elimina??o causa s?rios problemas ambientais. Apesar de uma parte ser utilizada para fins diversos, uma grande quantidade ainda permanece sem utiliza??o. Neste sentido, os res?duos de conte?do lignocelul?sico, como a casca do coco verde, constituem uma mat?ria-prima renov?vel e abundante cujo aproveitamento ? desej?vel e tem provocado um interesse crescente para o uso na cadeia de produ??o de etanol de 2? gera??o. Neste trabalho foi utilizada como mat?ria-prima a casca de coco verde, a qual foi submetida a dois pr?-tratamentos com o objetivo de melhorar a acessibilidade das enzimas ? celulose. Os pr?-tratamentos aplicados ao baga?o de coco foram: ?cido/Alcalino utilizando H2SO4 0,6M e, em seguida, NaOH 1M e, o pr?-tratamento com Per?xido de Hidrog?nio Alcalino (PHA) a uma concentra??o de 7,35% (v/v) e pH 11,5. O objetivo principal do estudo em quest?o, foi avaliar a influ?ncia do biossurfactante (ramnolip?deo produzido pela Pseudomonas aeruginosa), durante a hidr?lise da casca de coco verde. Para efeito de compara??o, foram realizadas hidr?lises utilizando surfactante qu?mico, nesse caso o Triton X-100, e hidr?lises sem adi??o de surfactantes. Dessa forma, observou-se que, com a aplica??o de ambos os pr?-tratamentos, foi poss?vel atingir uma maior convers?o de celulose ao comparar com o material in natura. No entanto, ao analisar os resultados obtidos para o processo de hidr?lise com a aplica??o de surfactantes, verificou-se que o ramnolip?deo produzido melhora, consideravelmente, a convers?o celul?sica da casca de coco verde, atingindo uma convers?o de 33% ap?s 72 horas de hidr?lise, enquanto que, a convers?o m?xima atingida com o uso do Triton X-100 foi de 23%. Esses resultados mostram que o biossurfactante pode ser aplicado na hidr?lise enzim?tica de materiais lignocelul?sicos, de modo a apresentar uma boa influ?ncia no processo.? / In Brazil many types of bioproducts and agroindustrial waste are generated currently, such as cacashew apple bagasse and coconut husk, for example. The final disposal of these wastes causes serious environmental issues. In this sense, waste lignocellulosic content, as the shell of the coconut is a renewable and abundant raw material in which its use has an increased interest mainly for the 2nd generation ethanol production. The hydrolysis of cellulose to reducing sugars such as glucose and xylose is catalysed by a group of enzymes called cellulases. However, the main bottleneck in the enzymatic hydrolysis of cellulose is the significant deactivation of the enzyme that shows irreversible adsorption mechanism leading to reduction of the cellulose adsorption onto cellulose. Studies have shown that the use of surfactants can modify the surface property of the cellulose therefore minimizing the irreversible binding. The main objective of the present study was to evaluate the influence of chemical and biological surfactants during the hydrolysis of coconut husk which was subjected to two pre-treatment in order to improve the accessibility of the enzymes to the cellulose, removing this way, part of the lignin and hemicellulose present in the structure of the material. The pre-treatments applied to coconut bagasse were: Acid/Alkaline using 0.6M H2SO4 followed by 1M NaOH, and the one with Alkaline Hydrogen Peroxide at a concentration of 7.35% (v/v) and pH 11.5. Both the material no treatment and pretreated were characterized using analysis of diffraction X-ray (XRD), Scanning Electron Microscopy (SEM) and methods established by NREL. The influence of both surfactants, chemical and biological, was used at concentrations below the critical micelle concentration (CMC), and the concentrations equal to the CMC. The application of pre-treatment with coconut residue was efficient for the conversion to glucose, as well as for the production of total reducing sugars, it was possible to observe that the pretreatment fragmented the structure as well as disordered the fibers. Regarding XRD analysis, a significant increase in crystallinity index was observed for pretreated bagasse acid/alkali (51.1%) compared to the no treatment (31.7%), while that for that treated with PHA, the crystallinity index was slightly lower, around 29%. In terms of total reducing sugars it was not possible to observe a significant difference between the hydrolysis carried out without the use of surfactant compared to the addition of Triton and rhamnolipid. However, by observing the conversions achieved during the hydrolysis, it was noted that the best conversion was using the rhamnolip?d for the husk pretreated with acid/alkali, reaching a value of 33%, whereas using Triton the higher conversion was 23.8%. The coconut husk is a residue which can present a high potential to the 2nd generation ethanol production, being the rhamonolipid a very efficient biosurfactant for use as an adjuvant in the enzymatic process in order to act on the material structure reducing its recalcitrance and therefore improving the conditions of access for enzymes to the substrate increasing thus the conversion of cellulose to glucose.

Métodos de Microbiologia de anaeróbios estritos e de Biologia Molecular foram empregados para se conhecer a diversidade de microrganismos relacionados à degradação de ascarel em reatores anaeróbios metanogênicos. A avaliação de potencial metanogênico foi realizada para a escolha da melhor condição nutricional, bem como, para a seleção de material suporte e solvente adequado a solubilização do ascarel. Nos ensaios em batelada, a produção de metano foi maior nos reatores contendo etanol (média de 0,22 - 0,46 molCH4/gSTV, 46h). Remoção de 85,6% (86,7 mg/L de PCB em Aroclor 1016 e 1260) foi obtida na condição com espuma de poliuretano, etanol (46g/L) e formiato (680 mg/L). Diferentes solventes e surfactantes, tais como, DMSO, dioxano, ácido acético, ácido fórmico, n-hexano, acetona, etano, metanol, Tween 80, SDS (10%) e Triton X-100 foram avaliados para a solubilização de ascarel. Dentre esses, metanol, Triton X-100 e ácido fórmico foram eleitos para a realização de ensaio em reatores em batelada contendo espuma de poliuretano, com o propósito de avaliar o potencial metanogênico na degradação de PCB. Os valores de produção de metano foram muito semelhantes (0,21 0,38 molCH4/mLgSTV, 45h) nas diferentes condições, no entanto, a remoção de PCB foi maior nos reatores com metanol 790 mg/L (86,6%), ácido Fórmico 600 mg/L (84,5%) e Triton X-100 1% (72,1%). Portanto, a melhor condição foi contemplada para a operação do reator anaeróbio horizontal de leito fixo (RAHLF) no tratamento do ascarel, ou seja, células imobilizadas em espuma de poliuretano, etanol e formiato (como fonte de carbono), Triton X-100 (0,1%) e metanol (como solvente). No RAHLF, a remoção média de matéria orgânica (DQO) foi de 91% para concentração afluente média de 1270 mg/L. A presença de morfologias semelhantes à Methanosarcina e bacilos fluorescentes foi confirmada em exames microscópicos. Na análise filogenética, por meio de PCR/DGGE e seqüenciamento das bandas recortadas, os grupos encontrados foram relacionados aos Filos Proteobacteria, Firmicutes, Spirochaetes, Chlorobi e Chloroflexi, sendo que neste último estão incluídos representantes relacionados a degradação de PCBs. Dentre as arquéias metanogênicas verificou-se similaridade de 99% e 97% com Methanosaeta sp. e Methanolinea sp., relacionadas com a metanogenese acetoclástica e hidrogenotrófica, respectivamente / Molecular biology and microbiology methods were used to study the microbial communities related to degradation of ascarel at methanogenic conditions in an anaerobic reactor. The methanogenic potential was evaluated to choose the better nutritional condition as well as to select the better support material and the most suitable solvent to favor the solubilization of ascarel. The methane production was higher (0.22 0.46 molCH4/mLgSTV, 46h) in batch reactors containing ethanol (46 g/L) and formate (680 mg/L), the PCB elimination attaining 85.6% (86.7 mg/L de PCB as Aroclor 1016 and 1260) when Polyuretane foam was used as support material. Different solvents, namely DMSO, dioxane, n-hexane, acetic acid, formic acid, acetone, ethane, methanol, and surfactants, such as 10% SDS,, Triton X-100, were evaluated aiming o determine the better condition to solubilize ascarel. According to the results of such experiments, methanol, formic acid and Triton X-100 were selected for carrying out the batch experiments in reactors containing polyurethane foam to evaluate the methane production during the PCBs degradation. Regardless of the operation conditions the methane production rates were similar (0.21 0.38 molCH4/gSTV, 45h), however the elimination of PCB was higher in the reactors containing methanol (790 mg/L), formic acid (600 mg/L) and Triton X-100 (1%). Therefore, the better condition for treating ascarel-containing residues in a bench-scale horizontal-flow immobilized biomass (HAIB) was attained with cells immobilized in polyurethane foam when ethanol and formate were used as carbon sources, and in presence of Triton X-100 and methanol, the average elimination of organic material attaining 91% for affluent concentration of 1270 mg/L. The presence of Methanosarcina and fluorescent rods was confirmed by microscopy analysis. According to the filogenetics analysis, which was carried out by PCR/DGGE and band-sequencing, the Bacteria domain are related to the Filos Proteobacteria, Firmicutes, Spirochaetes, Chlorobi and Chloroflexi, this latter being directly related to the degradation of PCB. Among the methanogenic Archea, a similiraty of 99% and 97% was observed to Methanosaeta sp. and Methanolinea sp. related to acetoclastic and hydrogenthrophic methanogenesis, respectively

Métodos de Microbiologia de anaeróbios estritos e de Biologia Molecular foram empregados para se conhecer a diversidade de microrganismos relacionados à degradação de ascarel em reatores anaeróbios metanogênicos. A avaliação de potencial metanogênico foi realizada para a escolha da melhor condição nutricional, bem como, para a seleção de material suporte e solvente adequado a solubilização do ascarel. Nos ensaios em batelada, a produção de metano foi maior nos reatores contendo etanol (média de 0,22 - 0,46 molCH4/gSTV, 46h). Remoção de 85,6% (86,7 mg/L de PCB em Aroclor 1016 e 1260) foi obtida na condição com espuma de poliuretano, etanol (46g/L) e formiato (680 mg/L). Diferentes solventes e surfactantes, tais como, DMSO, dioxano, ácido acético, ácido fórmico, n-hexano, acetona, etano, metanol, Tween 80, SDS (10%) e Triton X-100 foram avaliados para a solubilização de ascarel. Dentre esses, metanol, Triton X-100 e ácido fórmico foram eleitos para a realização de ensaio em reatores em batelada contendo espuma de poliuretano, com o propósito de avaliar o potencial metanogênico na degradação de PCB. Os valores de produção de metano foram muito semelhantes (0,21 0,38 molCH4/mLgSTV, 45h) nas diferentes condições, no entanto, a remoção de PCB foi maior nos reatores com metanol 790 mg/L (86,6%), ácido Fórmico 600 mg/L (84,5%) e Triton X-100 1% (72,1%). Portanto, a melhor condição foi contemplada para a operação do reator anaeróbio horizontal de leito fixo (RAHLF) no tratamento do ascarel, ou seja, células imobilizadas em espuma de poliuretano, etanol e formiato (como fonte de carbono), Triton X-100 (0,1%) e metanol (como solvente). No RAHLF, a remoção média de matéria orgânica (DQO) foi de 91% para concentração afluente média de 1270 mg/L. A presença de morfologias semelhantes à Methanosarcina e bacilos fluorescentes foi confirmada em exames microscópicos. Na análise filogenética, por meio de PCR/DGGE e seqüenciamento das bandas recortadas, os grupos encontrados foram relacionados aos Filos Proteobacteria, Firmicutes, Spirochaetes, Chlorobi e Chloroflexi, sendo que neste último estão incluídos representantes relacionados a degradação de PCBs. Dentre as arquéias metanogênicas verificou-se similaridade de 99% e 97% com Methanosaeta sp. e Methanolinea sp., relacionadas com a metanogenese acetoclástica e hidrogenotrófica, respectivamente / Molecular biology and microbiology methods were used to study the microbial communities related to degradation of ascarel at methanogenic conditions in an anaerobic reactor. The methanogenic potential was evaluated to choose the better nutritional condition as well as to select the better support material and the most suitable solvent to favor the solubilization of ascarel. The methane production was higher (0.22 0.46 molCH4/mLgSTV, 46h) in batch reactors containing ethanol (46 g/L) and formate (680 mg/L), the PCB elimination attaining 85.6% (86.7 mg/L de PCB as Aroclor 1016 and 1260) when Polyuretane foam was used as support material. Different solvents, namely DMSO, dioxane, n-hexane, acetic acid, formic acid, acetone, ethane, methanol, and surfactants, such as 10% SDS,, Triton X-100, were evaluated aiming o determine the better condition to solubilize ascarel. According to the results of such experiments, methanol, formic acid and Triton X-100 were selected for carrying out the batch experiments in reactors containing polyurethane foam to evaluate the methane production during the PCBs degradation. Regardless of the operation conditions the methane production rates were similar (0.21 0.38 molCH4/gSTV, 45h), however the elimination of PCB was higher in the reactors containing methanol (790 mg/L), formic acid (600 mg/L) and Triton X-100 (1%). Therefore, the better condition for treating ascarel-containing residues in a bench-scale horizontal-flow immobilized biomass (HAIB) was attained with cells immobilized in polyurethane foam when ethanol and formate were used as carbon sources, and in presence of Triton X-100 and methanol, the average elimination of organic material attaining 91% for affluent concentration of 1270 mg/L. The presence of Methanosarcina and fluorescent rods was confirmed by microscopy analysis. According to the filogenetics analysis, which was carried out by PCR/DGGE and band-sequencing, the Bacteria domain are related to the Filos Proteobacteria, Firmicutes, Spirochaetes, Chlorobi and Chloroflexi, this latter being directly related to the degradation of PCB. Among the methanogenic Archea, a similiraty of 99% and 97% was observed to Methanosaeta sp. and Methanolinea sp. related to acetoclastic and hydrogenthrophic methanogenesis, respectively

Les membranes modèles représentent un outil indispensable pour l'étude des membranes biologiques, elles ont en effet grandement contribué à leur description. Dans ce travail, nous nous sommes intéressés à l'étude de la coexistence de phases liquide-ordonnée (lo) et liquide-désordonnée (ld) au sein de membranes modèles de type LUV (« Large Unilamellar Vesicle »). Nous avons cherché en premier lieu à mettre au point une méthodologie permettant de détecter la formation de la phase lo et d'estimer quantitativement la fraction membranaire Φo en phase lo dans des LUVs de composition ternaire PC/SM/Chol (phosphatidylcholine / sphingomyéline / cholestérol), capable d'induire une coexistence de phase. Pour cela, les propriétés d'auto-extinction de fluorescence et de distribution sélective en fonction de la phase lipidique d'une sonde fluorescente unique (C12NBD-PC) ont été mises à profit. La deuxième partie de notre travail a été consacrée à l'étude de la solubilisation par le détergent Triton X-100 des membranes de LUVs présentant une coexistence de phase lo/ld. Nous avons cherché à démontrer qu'il était possible d'extraire la fraction membranaire se trouvant strictement en phase lo. Pour cela, les transitions de structure induites par l'interaction du Triton X-100 avec des LUVs à 4°C ont été étudiées par une procédure de séparation par gradient de densité. Nous avons tenté d'évaluer le rapport effectif approprié détergent/lipides nous permettant d'isoler les fractions résistantes correspondant aux domaines en phase lo existant au niveau de la membrane des LUVs avant l'addition de détergent.

In Chapter I is a general description of novel metal complexes which hydrolytically cleave peptides, proteins, DNA, and other biological molecules. These reagents are becoming the more important as potential therapeutic agents. A panel of ligands was investigated for coordination to ZrIV and other metals in groups 4, 5, and 6 to effect the greatest degree of hydrolysis. Chapter II describes a ZrIV complex which is capable of hydrolyzing a 30 amino acid peptide, insulin chain B, with amino acid specificity. Oxidized insulin chain B peptide was hydrolyzed after only 4 h of treatment at pH 7.0 and 60 °C using ZrCl4 in the presence of 4,13-diaza-18-crown-6. MALDI-TOF and ESI LC-MS mass spectra indicated that insulin chain B was hydrolyzed by ZrIV at the Gly8-Ser9, Ser9-His10, and Gly20-Glu21 amide bonds within the oligopeptide. To our surprise, the cysteine sulfonic acid sequences Cys(SO3H)7-Gly8 and Cys(SO3H)19-Gly20 were also cleaved. To the best of our knowledge, this constitutes the first example of metal-assisted hydrolysis of a Cys(SO3H)-Xaa amide bond. This is significant in light of the fact that cysteine sulfonic acid formation in proteins is triggered by oxidative stress and has been associated with amyloid fibril formation, Parkinson’s disease, and other deleterious, physiological processes. Chapter III describes the metal-assisted hydrolysis of sphingomyelin which is a principle phospholipid component of animal cell membranes. The sphingomyelin assays showed evidence of metal-assisted hydrolysis after 20 h of treatment at lysosomal pH 4.8 and cytosolic pH 7.0 at both physiological temperature 37 °C and 60 °C. The metal ion CeIV was the most reactive, followed by ZrIV, and then HfIV. The goal of this work is to develop metal-based reagents to reverse the lethal build-up of sphingomyelin that occurs in lysosomes of patients suffering from Niemann-Pick disease.

La BSP1, principale protéine du plasma séminal bovin, interagit avec les membranes des spermatozoïdes et joue un rôle crucial dans les événements qui conduisent à la fécondité des spermatozoïdes, lors du phénomène de la capacitation. Le but de cette recherche est d’investiguer la nature de ces interactions. Ce travail vise à démontrer l’influence des lipides qui composent les membranes sur l’action de la protéine BSP1. À l’aide de la fluorescence intrinsèque de la protéine, l’affinité de la protéine a été caractérisée pour quatre systèmes lipidiques. Les résultats montrent que la composition lipidique affecte significativement l'affinité de la protéine pour les membranes. Nous avons observé l'ordre suivant : 1-palmitoyl-2-oléoyl-sn-glycéro-3-phosphocholine (POPC) > POPC/1-palmitoyl-2-oléoyl-sn-glycéro-3-phosphoéthanolamine (POPE) ≈ POPC/1-palmitoyl-2-oléoyl-sn-glycéro-3-phospho-L-sérine (POPS) > POPC/cholestérol. La protéine interagit préférentiellement avec POPC. La présence de POPE, POPS, ou cholestérol dans la membrane diminue systématiquement l’affinité. Il est connu que la présence de POPE ou cholestérol augmente l’empilement des lipides dans les membranes. Cet effet de condensation des chaînes pourrait être défavorable à l’insertion de la partie hydrophobe de la protéine dans les membranes et réduire ainsi l'affinité. La diminution de l’affinité de la protéine induite par la présence de POPS, un lipide chargé négativement, pourrait être associée aux interactions électrostatiques répulsives car la protéine porte une charge globale négative. La littérature mentionne que la BSP1 extrait sélectivement les phospholipides de type choline et le cholestérol lors de son association avec les membranes de spermatozoïdes. Un efflux lipidique est aussi observé avec des membranes modèles. Nous avons désiré caractériser la « solubilisation » des membranes par la BSP1, par diffusion dynamique de la lumière. Comme étape préliminaire, nous avons étudié comment le détergent Triton X-100 solubilise les membranes en utilisant cette technique. Les mesures démontrent que la composition lipidique des membranes (POPC, POPC/POPE, POPC/1-palmitoyl-2-oléoyl-sn-glycéro-3- [phospho-rac-(1-glycérol)] (POPG)) n’affecte pas le mécanisme général de solubilisation/reconstitution des membranes modèles. Il a été montré qu'il existe trois régions lors des processus de solubilisation pour les différents systèmes lipidiques : i) le détergent se distribue dans les membranes, ii) une coexistence de membranes saturées en détergents et de micelles mixtes de phospholipides/Triton X-100 et iii) exclusivement des micelles mixtes de phospholipides/Triton X-100. Nos résultats montrent que la forme conique de POPE augmente la résistance des membranes à la solubilisation. La présence de POPG, apportant une charge négative à l’interface des membranes, n’induit aucun changement aux processus de solubilisation/reconstitution des membranes par Triton X-100. La diffusion dynamique de la lumière a également permis d’observer si la protéine BSP1 induit des modifications morphologiques des membranes suite à son interaction avec les membranes de POPC. Nos observations n'ont montré aucune variation significative de la taille des particules lors du titrage des vésicules de POPC par la protéine, sur une gamme de rapport molaire de POPC/BSP1 variant de 20 à 0.6. Avec des compositions aussi différentes, on suppose une transition des vésicules saturées en protéine à des complexes de protéine avec un peu de lipides. Cependant, il semble impossible avec la diffusion dynamique de la lumière de différencier ces particules. / BSP1, the main protein in bovine seminal plasma, interacts with sperm membranes and plays a crucial role in events that lead to sperm fertility, during the capacitation. The purpose of this research is to investigate the nature of these interactions. This work aims to demonstrate the influence of the lipids that compose membranes on the action of the BSP1 protein. Using the intrinsic fluorescence of the protein, the affinity of the protein was characterized for four lipid systems. The results show that the lipid composition significantly affects the affinity of the protein for membranes. We observed the following order: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) > POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) ≈ POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) > POPC/cholesterol. The protein interacts preferentially with POPC. The presence of POPE, POPS, or cholesterol in membranes decreases systematically the affinity. It is established that the presence of POPE or cholesterol increases the packing of lipids in membranes. This condensation effect could be detrimental to the insertion of the hydrophobic part of the protein into the membranes and reduces, as a consequence, the affinity. The decrease in protein affinity induced by the presence of POPS, a negatively charged lipid, could be associated with repulsive electrostatic interactions as the protein global charge is negative. The literature mentions that BSP1 selectively extracts choline phospholipids and cholesterol when combined with sperm membranes. A lipid efflux is also observed with model membranes. We characterized membrane "solubilisation" by BSP1, using dynamic light scattering. As a preliminary step, we studied how Triton X-100 detergent solubilizes membranes using this technique. The measurements showed that the lipid composition of the membranes does not affect the general solubilization/reconstitution mechanism of the model membranes (POPC, POPC/POPE, POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1-rac-glycerol) (POPG)). It is known that three different regions exist during the solubilization process for the different lipid systems: i) the detergent is distributed in the membranes, ii) a coexistence of membranes saturated with detergents and mixed phospholipid/Triton X-100 micelles and iii) exclusively mixed phospholipid/Triton X-100 micelles. Our results show that the conical shape of POPE increases the resistance of the membranes to solubilization. The presence of POPG, bringing a negative charge at the membrane interface, does not induce any change in solubilization/reconstitution processes. Dynamic light scattering also made it possible to observe if the BSP1 protein induces morphological changes in the membranes following its interaction with POPC membranes. Our observations showed no significant variation in particle size during the titration of POPC vesicles by the protein, over a molar ratio range of POPC/BSP1 from 20 to 0.6. Considering such different compositions, a transition from vesicles saturated with protein to protein complexes with some lipids is assumed. However, it appeared impossible with dynamic light scattering to differentiate these particles.

In this diploma thesis was studied time-resolved fluorescence in polymer-surfactant system. At first aggregation numbers of cationic (cetyltrimethylammonium bromide), anionic (sodium dodecylsulfate) and nonionic (Triton X-100) surfactants were studied by steady-state and time-resolved fluorescence spectroscopy. These two methods were compared. Aggregation numbers by steady-state method were always lower than aggregation numbers measured by time-resolved method. Steady-state method of determination aggregation numbers is useless for surfactants with high aggregation number and for aerated samples. Addition of hyaluronan to surfactant system was studied. There was observed change in aggregation number after addition of hyaluronan and change in percentage of dynamic quenching after addition of hyaluronan. Hyaluronan affected aggregation number of cetyltrimethylammonium bromide and Triton X-100. Hyaluronan increased percentage of dynamic quenching in cetyltrimethylammonium bromide and in Triton X-100. Pyren in sodium dodecylsulfate was quenched by sphere of action with negligible percentage of dynamic quenching and addition of hyaluronan had no effect on quenching. As next goal of this thesis, the determination of the position of fluorescence probe pyrene in cetyltrimethylammonium bromide, sodium dodecylsulfate and Triton X-100 micelles was chosen. Position of pyrene changed with charge and structure of micelles. Next was studied how percentage of dynamic quenching by iodide compounds changes with different charge of micelle. In all cases majority of dynamic quenching was calculated.