Lantibiotics like mutacin 1140 are receiving a considerable amount of attention because of their broad spectrum of activity, high potency, low immunogenicity, and good structural stability. Mutacin 1140 is produced by Streptococcus mutans JH1140 and is a type A (I) lantibiotic. Lantibiotics are ribosomally synthesized bacteriocins that undergo post-translational modifications to form lanthionine or β-methyllanthionine rings as well as 2, 3-didehydroalanine (Dha), 2, 3-didehydrobutyrine (Dhb), and S-amino vinyl-D-cysteine (AviCys). Their mode of action is pore formation and/or abduction of lipid II from the site of new cell wall synthesis. In order to gain further knowledge of both the structural integrity and structureunction relationship of type A (I) lantibiotics, chemical modifications or site directed mutagenesis was utilized. In the first aim of this study, two type A (I) lantibiotics were used, nisin A produced by Lactococcus lactis and gallidermin produced by Staphylococcus gallinarium. They both share homology in rings A and B, the lipid II binding domain, with rings A and B of mutacin 1140. What was discovered was that oxidation of the lanthionine rings results in the complete loss of bioactivity due to the loss of affinity for lipid II. Interestingly, the lateral assembly ability of the oxidized variants is not affected. In the second aim, the dehydrated threonine residue (Dhb) at position 14 in gallidermin underwent chemical modification using several thiol-compounds. The results showed that this residue is amendable to modification through thiol chemistry with some loss of bioactivity. However, the MICs for the chemical variants were still in the nanomolar range. From this work the first ever in vivo images of gallidermin were produced. The last aim of the study utilized site directed mutagenesis of the mutA gene of mutacin 1140 to determine the role of various residues in ring A and the hinge region of the peptide. It was determined that neither Trp4, Dha5, nor Arg13 are important for bioactivity but a set distance between rings A and B is essential. The majority of the mutants constructed showed either similar or increased bioactivity.

Submitted by Juan Carlos Roldão null (36780115860) on 2016-04-28T18:17:40Z No. of bitstreams: 1 Dissertação-Versão-Final-Juan_Carlos_Roldao.pdf: 3885340 bytes, checksum: 0d245c89d075ddeea3f1fc7154e9c5a9 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-05-02T13:34:47Z (GMT) No. of bitstreams: 1 roldao_jc_me_bauru.pdf: 3885340 bytes, checksum: 0d245c89d075ddeea3f1fc7154e9c5a9 (MD5) / Made available in DSpace on 2016-05-02T13:34:47Z (GMT). No. of bitstreams: 1 roldao_jc_me_bauru.pdf: 3885340 bytes, checksum: 0d245c89d075ddeea3f1fc7154e9c5a9 (MD5) Previous issue date: 2016-03-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Atualmente existe uma intensa busca por novos materiais com propriedades ajustadas para utilização em células solares orgânicas de modo a obter um aumento em sua eficiência de conversão para que possam substituir os dispositivos de silício. O politieno[3,4-b]-tiofeno-co-benzoditiofeno (PTB7) é um polímero recentemente proposto na literatura e com propriedades muito interessantes em células solares orgânicas, o que o coloca como uma possível alternativa ao amplamente utilizado poli(3-hexiltiofeno) (P3HT). Tem sido relatadas modificações em diferentes posições da unidade monomérica deste copolímero, tanto na estrutura benzoditiofeno (BDT), quanto na estrutura tienotiofeno (TT), que o compõe. Estas modificações levaram a novos polímeros com propriedades diferentes e por vezes mais interessantes que aquelas do PTB7 sem substituições. O trabalho que será apresentado visou estudar as propriedades estruturais, eletrônicas e ópticas do PTB7 e possíveis alterações ocorridas devido às modificações químicas realizadas na estrutura do BDT de suas unidades monoméricas. Tal estudo utilizou ferramentas de otimização de estruturas como Mecânica Molecular, Dinâmica Molecular e o método semi-empírico Parametric Method 6 (PM6), assim como de cálculo de estrutura eletrônica de materiais, como a Teoria do Funcional da densidade (DFT) e de cálculos de propriedades ópticas como a Teoria do Funcional da Densidade Dependente do Tempo (TD-DFT). Concluímos que o PTB7 no estado sólido pode ser considerado planar. Com o nosso modelo para o PTB7, obtivemos uma diferença de energia ∆EHL entre o Último Orbital Molecular Ocupado HOMO (do inglês Highest Occupied Molecular Orbital) e o Primeiro Orbital Molecular Desocupado LUMO (do inglês Lowest Unoccupied Molecular Orbital) de aproximadamente 1,84 eV, sendo que este valor está em boa concordância com o valor experimental. Em relação às substituições químicas, estudamos teoricamente 8 derivados do PTB7 e os resultados mostraram que é possível obter compostos com uma diminuição significativa do ∆EHL e também que é possível obter compostos com valores de energia do HOMO e do LUMO mais interessantes que os do PTB7 quando na camada ativa for empregado como material doador o fenil-C61-butírico ácido metil ester (PCBM). / Currently there is an intensive search for new materials with tuned properties for use in organic solar cells to obtain an increase in its conversion efficiency and replace silicon devices. The polythieno[3,4-b]-thiophene-co-benzodithiophene (PTB7) is a polymer recently proposed in the literature and with very interesting properties in organic solar cells, which places it as a possible alternative to the widely used poli(3-hexilthiophene) (P3HT). It has been reported changes in different positions of the monomeric unit of this copolymer, both in benzodithiophene (BDT) structure, as in the thienothiophene (TT) structure that compose it. These modifications led to new polymers with different properties and sometimes more interesting than those of PTB7 without substitutions. The work to be presented aimed to study the structural, electronic and optical properties of PTB7 and possible changes due to chemical changes made in the BDT structure of its monomeric units. This study employed optimization tools like Molecular Mechanics, Molecular Dynamics and Parametric Method 6 (PM6), as well as calculations of the electronic structures with the Density Functional Theory (DFT) method, and optical properties such as the Time Dependent Density Functional Theory (TD-DFT) calculations. We conclude that the PTB7 chains in the solid state can be considered planar. With our model for PTB7, we obtained a difference ΔEHL between the Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccupied Molecular Orbital (LUMO) of approximately 1.84 eV, and this value is in good agreement with the experimental value. Regarding chemical substitutions, we studied theoretically 8 derivatives of PTB7 and the results showed that it is possible to obtain compounds with a significant decrease in ΔEHL and that it is possible to obtain compounds with HOMO and LUMO energy values more adjusted to the widely employed acceptor material phenylC61-butyric acid methyl ester (PCBM).

Orientadores: Cesar Costapinto Santana, Marisa Masumi Beppu / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-07T05:42:19Z (GMT). No. of bitstreams: 1 Torres_MarcoAntonio_D.pdf: 1417582 bytes, checksum: 10045a785cc6ab8b5ff6c19e8efd00d0 (MD5) Previous issue date: 2006 / Resumo: Neste trabalho foram produzidas microesferas de quitosana com tamanho controlado e porosidade influenciada pela técnica de atomização e coagulação. As condições de produção foram definidas pelo planejamento de experimentos. As microesferas obtidas foram modificadas quimicamente com anidrido acético, epicloridrina e glutaraldeído com objetivos de melhorar suas características iniciais de resistência e estabilidade. As possibilidades de uso apresentadas por essas matrizes se devem ao reconhecimento observado entre adsorbato e adsorvente e à realização de modificações químicas e estruturais. Após essas modificações as microesferas obtidas foram analisadas quanto às suas propriedades estruturais, capacidade de adsorção e dessorção. As microesferas foram utilizadas em sistemas de adsorção em banho finito e coluna de leito fixo. Os adsorbatos utilizados foram as proteínas BSA, lisozima e um concentrado protéico (condição real) obtido do soro do leite. As proteínas BSA e lisozima apresentam pontos isoelétricos distintos de 4,8 e 11, respectivamente, permitindo assim avaliar o sistema adsorvente-adsorbato pelas isotermas de adsorção, cinética de equilíbrio, capacidade de dessorção e regeneração dos adsorventes, em condições distintas de pH. O modelo de Langmuir descreveu bem os valores de adsorção obtidos experimentalmente. As capacidades máximas de adsorção para as proteínas BSA e lisozima foram de 9,24 mg/g e 11,95 mg/g, respectivamente, utilizando as microesferas de quitosana reticuladas com glutaraldeído. Os maiores valores de adsorção foram encontrados próximos aos pontos isoelétricos, mostrando que as interações eletrostáticas, fundamentais para o processo em toda a faixa de pH estudada, não estão agindo isoladamente no sistema. Comparando-se os métodos de tanque agitado e coluna de leito fixo foi possível observar diminuição significativa na adsorção e dessorção da solução artificial de proteínas do primeiro para o segundo método. Esses resultados podem ser explicados por limitações no tamanho da coluna do leito, tempo de residência e conseqüentemente pela baixa transferência de massa. Com um extrato real ocorreu diminuição, mais significativa ainda, da capacidade de adsorção quando comparado com a solução artificial de proteínas. Estes resultados refletem a complexidade das interações e a existência de competição pelos sítios de adsorção da superfície interna e externa das microesferas de quitosana reticuladas com glutaraldeído. Essa competição ocorre possivelmente entre as proteínas do extrato e outros grupos moleculares / Abstract: This work is concerned with production of chitosan microspheres with sizes controlled and porosity influenced by spraying and coagulation process. The production conditions were defined through experimental planning. The microspheres were modified chemically with glutaraldehyde, epichlorohydrin and acetic anhydride in order to improve its initial characteristics of resistance and stability. The possibilities of use exhibited by these matrices are due to recognition adsorbate-adsorbent and the accomplishments these chemical and structural modifications. Soon after the gotten microspheres their structural, adsorption and dessorption properties were analyzed. The microspheres were used in adsorption system in two methods: stirred tank and fixed bed. The adsorbates used were the BSA and lysozyme proteins and a proteinic extract from milk serum. The BSA and lysozyme proteins have different isoeletric points, 4.8 and 11, respectively. This allowed study the adsorbent­absorbate system by adsorption isotherms, equilibrium kinetics, dessorption capability and regeneration of adsorbents, in different conditions of pH. The Langmuir described well the experimental values of capacity of adsorption. The maximum adsorption capacities were 9,24 mg/g and 11,95 mg/g for BSA and lysozyme proteins, respectively. The higher values of adsorption were found close to the isoelectric points, showing that the electrostatic interactions, important to the process during all pH range studied, it are not acting alone in the system. Comparing the methods of stirred tank with fixed bed happened significant reduction in the adsorption and dessorption from proteins artificial solution. These results may be explained by the limitations in the size of column, time of residence and consequently in the mass transfer. With a real extract occurred important reduction of the adsorption capacity when compared with synthetic proteins. These results show the complexity of the interactions and the competition between the extract proteins and others chemical groups by adsorption sites in the internal and external surface of chitosan microspheres crosslinked with glutaraldehyde / Doutorado / Desenvolvimento de Processos Biotecnologicos / Doutor em Engenharia Química

Le projet vise à développer de nouvelles méthodologies exploitant le caractère intensif et éco-compatible de l'extrusion réactive appliquée à des polymères issus de la biomasse. Les polymères naturels possèdent des propriétés physico-chimiques à l'état natif qui limitent leur utilisation dans un grand nombre de secteurs industriels. Des modifications chimiques même limitées permettent de modifier radicalement les propriétés de ces objets macromoléculaires. Les modifications chimiques couramment effectuées en solution ou en batch posent des problèmes de mise en œuvre, de contrôle réactionnel, de coût énergétique et environnemental (coproduits, utilisation de réactifs posant problèmes). Le projet vise à étudier et mettre au point d'une part les paramètres de composition (polymères, plastifiants, réactifs...) et d'autre part les conditions opératoires de la modification chimique réalisée en extrudeuse : taux d'hydratation, pression et température. Trois familles des biopolymères d'intérêt pour les grandes filières des bioraffineries seront considérées, avec des réactions de modification spécifiques visant à répondre aux spécifications d'applications : amidon et maltodextrines, lignines et dérivés thermoplastiques de la cellulose / The project involves developing new chemical modifications methodologies using the ecologically compatible and intensive characteristics of reactive extrusion applied to polymers from biomass. With the prospect of worldwide exhaustion of oil supply, the use of renewable natural polymers as potential substitution candidates for oil based application polymers appears as a sustainable alternative. Natural polymers possess physicochemical properties in the native state, which limit their use in many industrial sectors. Even limited chemical modifications radically change the properties of these macromolecular entities. Chemical modifications commonly performed at industrial level in solution or in batches are often very laborious to implement, control, and in addition suffer from energy and environmental cost problems (co-products, use of reagents posing problems). The project aims at studying and developing the composition parameters (polymers, plasticizers, reagents ...), and secondly examining the operating conditions of the chemical modification carried out in an extruder: hydration rate, pressure and temperature kinetics and other parameters with reactions of specific modifications to meet the requirements of industrial applications. Three families of biopolymers of interest to large sectors of biorefineries will be considered: starch and maltodextrins, thermoplastic lignin and cellulosic derivatives.

Made available in DSpace on 2015-04-17T14:49:12Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1503208 bytes, checksum: b782febd7d14045e41ee0edccb77000e (MD5) Previous issue date: 2009-08-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Maximum Spirulina is an unicellular microalga, that she has form of helical filament and that she grows in waters strongly alkaline; she belongs to the cianobacterial group, cianofíceas or algae green-blued and they have been used since times many old. The microalgae presented a biomass growth in summer of 1,07 g/L and a 23,63% of rental. Present a high contend of proteins (54.93%) and low oils content (6,84%) and ashes (13,05%), this content of proteins rises in the flour degreased for 61,14%. being Fractioned the proteins was obtained majority incomes of albumins (36,61%), basic glutelina (31,29%) and globulin (21,03%), and minority of prolaminas (3,95%) and acid glutelina (0,29%). In the obtaining of the isolated proteic, the index in extracted total protein in the three extractions was 54,36%, the index of precipitation isoelectric in the extract of pH 10,0 was 46,81% and the index of total protein in the isolated proteic was of 95,44% in pH 10,0. the analysis of the molecular weight of the proteins of the flour in PAGE-SDS - she revealed from 8 to 13 bands. The subunidades of 16,21 and 15,74 kDa appeared prominent in the sample. The term-stability (Td, ºC) and the variation entálpica (H, KCal/g), examined by DSC, respective the degreased flour and isolated proteic were of 104,58°C and 102,47°C and 0,65 Kcal.g-1 and 0,59 Kcal.g-1, respectively. The nutritional characterization of the protein of maximum Spirulina, they demonstrated a content of tannins of 17,5 mg.g-1 and absence of the lectina inhibitors and tripsina. The composition of amino acids revealed her witnesses of all of the essential amino acids in superior amounts to the pattern FAO/WHO/UNU, with predominance of the leucina (3,96g/100g protein), fenilalanina (2,92 g/100g proteins) and valina (2,71 g/100 g proteins). The results of the chemical modifications revealed that the acetilation was shown more it executes than the succinylation in the different modification degrees (1; 2.5; 5; 10 and 15%). the solubility of the isolated not modified indicated a point isoelectric (low solubility) in pH 5,0, for the isolated ones modified the point isoelectric was observed in the pH 4,0. the solubility increased with the chemical modification, being observed a larger increase in the succinylation. The capacity of absorption of water was influenced positively with the chemical modification, being observed a larger increase in the acetilation in relation to succinylation. In the same way the capacity of oil absorption increases with the modification, being observed a larger increase of the capacity in the acetilation. The emulsion capacity increased with the chemical modification in all of the modification degrees, being observed a light increase in the succinylation. The emulsion activity was increased with the chemical modification, being observed a light increase in the succinylation. The stability of the emulsion increased with the chemical modification, being observed a larger increase in the succinylation. The relative viscosity of the isolated modified proteic increased with the concentration of the protein solution, with to room temperature and with the heating (90ºC), in the measure that increases the modification degree, it also increases the viscosity. The floaming capacity increased with the chemical modification, being observed a larger index in the succinylation, in the espumação stability is noticed that the decrease in the stability was less accentuated in the acetilation than in the succinylation, in all of the analyzed times and all of the modification degrees tested. The protein of maximum Spirulina presented a chemical and nutritional characteristic satisfactory in relation to minimum demands established by the international organisms. / A Spirulina maxima é uma microalga unicelular, que tem forma de filamento helicoidal e que se desenvolve em águas fortemente alcalinas; pertence ao grupo de cianobactérias, cianofíceas ou algas verde-azulada e têm sido utilizados desde tempos muitos antigos. A microalga apresentou um crescimento de biomassa durante o verão de 1,07 g/L e um 23,63% de rendimento. Apresenta um elevado teor de proteinas (54.93%) e baixo conteúdo de lipídeos (6,84%) e cinzas (13,05 %). Este conteúdo de proteinas elevou-se na farinha desengordurada para 61,14%. Fracionando-se as proteinas obteve-se rendimentos majoritários de albuminas (36,61%), glutelina básica (31,29%) e globulina (21,03%), e minoritário de prolaminas (3,95%) e glutelina ácida (0,29%). Na obtenção do isolado protéico, o índice em proteina total extraída nas três extrações foi 54,36%, o índice de precipitação isoelétrica no extrato de pH 10,0 foi 46,81% e o índice de proteina total no isolado protéico foi de 95,44% em pH 10,0. A análise do peso molecular das proteinas da farinha em PAGE-SDS-bMe revelou de 8 a 13 bandas. As subunidades de 16,21 e 15,74 kDa apareceram proeminentes na amostra. A termoestabilidade (Td, ºC) e a variação entálpica (DH, Kcal por g), examinadas por DSC, respectivas a farinha desengordurada e isolado protéico foram de 104,58°C e 102,47°C, 0,65 Kcal.g-1 e 0,59 Kcal.g-1, respectivamente. A caracterização nutricional da proteina da Spirulina máxima, demonstrou um conteúdo de taninos de 17,5 mg.g-1 e ausência dos inibidores de lectina e tripsina. A composição de aminoácidos revelou a presencia de todos os aminoácidos essenciais em quantidades superiores ao padrão FAO/WHO/UNU, com predominância da leucina (3,96g por 100g proteina), fenilalanina (2,92 g por 100g proteinas) e valina (2,71 g por 100 g proteinas). Os resultados das modificações químicas revelaram que a acetilação se mostrou mais efetiva que a succinilação nos diferentes graus de modificação (1; 2.5; 5; 10 e 15 %). A solubilidade do isolado não modificado indicou um ponto isoelétrico (mínima solubilidade) em pH 5,0, para os isolados modificados o ponto isoelétrico se observou no pH 4,0. A solubilidade aumentou com a modificação química, detectando-se um maior aumento na succinilação. A capacidade de absorção de água foi influenciada positivamente com a modificação química, observando-se um maior aumento na acetilação em relação a succinilação. Da mesma forma a capacidade de absorção de óleo aumentou com a modificação, observando-se um maior aumento da capacidade na acetilação. A capacidade de emulsão aumentou com a modificação química em todos os graus de modificação, detectando-se um leve aumento na succinilação. A atividade de emulsão foi aumentada com a modificação química, observando-se um leve aumento na succinilação. A estabilidade da emulsão aumentou com a modificação química, detectando-se um maior aumento na succinilação. A viscosidade relativa dos isolados protéicos modificados aumentou com a concentração da solução de proteina, com a temperatura ambiente e com o aquecimento (90ºC), na medida que aumenta o grau de modificação, também aumenta a viscosidade. A capacidade de espumação aumentou com a modificação química, observando-se um maior índice na succinilação, na estabilidade de espumação percebe-se que o decréscimo na estabilidade foi menos acentuado na acetilação do que na succinilação, em todos os tempos analisados e todos os graus de modificação testados. A proteina da Spirulina máxima apresentou uma característica química e nutricional satisfatória em relação a exigências mínimas estabelecidas pelos organismos internacionais.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biophysic and Physical Chemistry Candidate: Bc. Martina Dvořáková Supervisor: Doc. Ing. Alice Lázníčková, CSc. Consultant: Mgr. Martina Hermannová, Ph.D. Title of diploma thesis: Optimization of preparative LC-MS method for fractionation of oligosaccharides of hyaluronanu This diploma thesis deals with optimization of LC-MS method for analysis of hyaluronan oligosaccharides in preparative mode. The theoretical part summarizes available information about biological and chemical properties of hyaluronic acid. Hyaluronic acid is easily enzymatically degradable by mammalian hyaluronidases that produce hyaluronan oligosaccharides. The biological function of these degradation products depend on their molecular weight. High-performance liquid chromatography is mainly used for separation and purification of hyaluronan oligosaccharides. A new method for the determination of hyaluronan oligosaccharides is based on a combination of separation techniques and mass spectrometry. The experimental part deals with optimization of ionisation conditions for electrospray ionization mass spectrometry in positive and negative ion mode. In the first step, we focused on setting of capillary voltage, cone voltage, desolvation temperature, flow...

Ce travail s’inscrit dans la continuité des études menées au Laboratoire de Chimie des Substances Naturelles sur la bioremédiation des éléments-traces, et vise à utiliser les écorces de sapin de Douglas pour éliminer l’arsenic et le césium des eaux. Des tests effectués en bain agité ont permis d’établir des isothermes d’adsorption. Leur traduction par le modèle mathématique de Langmuir a démontré que les écorces brutes possèdent de bonnes propriétés intrinsèques pour la biosorption des éléments étudiés. Afin d’augmenter ces propriétés pour le césium, l’écorce a été oxydée pour faire apparaître des fonctions acide carboxylique et/ou imprégnée par l’hexacyanoferrate de nickel. Pour améliorer la biosorption de l’As(V), des fonctions ammonium ont été intégrées à la structure de l’écorce grâce au greffage de la bétaïne et de polyéthylèneimine méthylée. En parallèle, des fonctions thiol ont été introduites via la fixation del’acide lipoïque et de la N-acétylhomocystéine thiolactone, pour accroître l’adsorption de l’As(III). Les modifications entreprises pour la biosorption du césium et de l’As(III) permettent de conserver l’affinité des écorces pour ces éléments tout en augmentant significativement leur capacité d’adsorption. Enfin, les propriétés de biosorption du césium par Biosorb, un biosorbant à base d’écorce développé et commercialisé par la société Pe@rl, ont été confirmées, y compris en colonne à lit fixe. Les données recueillies lors de ces expériences sont cohérentes avec celles issues du logiciel de simulation OPTIPUR, ce qui permet d’envisager l’industrialisation du procédé. / This work follows several studies conducted in the “Laboratoire de Chimie des Substances Naturelles” about bioremediation of trace elements, and aims to use Douglas fir bark to remove arsenic and cesium from water. Tests carried out in batch allowed the establishment of adsorption isotherms. Their interpretation following Langmuir adsorption model showed that crude Douglas fir bark has good intrinsic properties for biosorption of studied elements. In order to improve these properties for cesium sorption, bark was oxidised to form carboxylic acid functional groups and/or impregnated with nickel hexacyanoferrate. So as to increase As(V) biosorption, ammonium functional groups were incorporated in the bark structure by grafting of betaine and methylated polyethyleneimine. In parallel, thiol functional groups were introduced through the fixation of lipoic acid and N-acetylhomocysteine thiolactone, in order to enhance As(III) sorption. Chemical modifications carried out for cesium and As(III) biosorption led to keep the bark affinity for these elements while increasing significantly their adsorption capacity. Finally, the biosorption properties of cesium onto Biosorb, a bark-based biosorbant developed and marketed by Pe@rl society, were confirmed, including in fixed-bed column. Data collected during these experiments are consistent with those from OPTIPUR simulation software, making possible the process industrialisation.

La thèse est intitulée « Modifications chimiques de polysaccharides pour l'obtention de nouveaux systèmes polymère-principe actif ». Le thème de la recherche porte sur trois axes principaux, dont l’objectif majeur est la synthèse et la caractérisation de nouveaux systèmes pour la libération contrôlée de principes actifs. Le premier chapitre traite de l’optimisation de la méthode pour préparer des émulsions stables simples inverses (eau/huile) et multiples (eau/huile/eau) à base de polysaccharides (amidon et 2-hydroxyethyle cellulose). On a étudié le processus de relargage de la caféine, l’influence de la présence d’amidon dans la phase aqueuse sur la stabilité et cinétique de libérations. Le second chapitre a pour objet l’obtention de nouveaux principes actifs à partir d’aminoacides et leur immobilisation sur polysaccharides par couplage chimique covalent. Les conjugues polymère-médicament obtenus ont été caractérisés par spectroscopie, physico-chimie. Leur activité antimicrobienne, leur toxicité in vivo, l’efficacité de l’immobilisation et la cinétique de relargage in vitro de médicaments couplés ont été étudiées. Le dernier chapitre traite de la synthèse des dérivés de chitosane avec l’anhydride maléique et l’acide acrylique, ainsi que leur utilisation dans la préparation d’hydrogels sous forme de films et de nanoparticules par copolymérisation avec le méthacrylate de 2-hydroxyethyle et l’acrylamide de N- isopropyl. Les dérivés ont été évalués par leur degré de fonctionnalisation. Les nanosystèmes ont été caractérisés par leur potentiel zêta, leur capacité de gonflement, d’adhésion et de relargage in vitro. Tous les résultats expérimentaux ont été soutenus par importantes études bibliographiques / The thesis is entitled “Chemical modifications of polysaccharides for the obtaining of new drug-polymer systems“. The topic of research includes four main working axes, diverted from the major objective to synthesize and characterize new drug delivery systems. The first chapter treats the optimization of the preparation method of stable simple W/O emulsions and multiple W/O/W emulsions, based on polysaccharides (starch and 2-hydroxyethyle cellulose). We also studied the process of caffeine release outside the multiple emulsions particles and the influence of starch in the internal aqueous phase stage upon the stability and the drug release kinetics. The second chapter studies the obtaining of new active principles from aminoacid and their immobilization on polysaccharides by covalent coupling. The new drug-polymer conjugates were characterized by spectral, physicochemical properties, antimicrobial activity, in vivo toxicity, immobilization efficiency and I vitro drug release kinetics. The last chapter treats the synthesis of the chitosan derivatives with maleic anhydride or acrylic acid, and their use in the preparation of hydrogels as film and nanoparticles by copolymerization with 2-hydroxymethacrilate or N-isopropyl acrylamide. The derivatives were evaluated by determining the degree of functionalization and by spectral methods. The nanosystems were characterized by measurements of zeta potential, swelling and adhesion capacity and membership and in vitro drug release kinetics. All experimental results were supported by important bibliographic studies

Cyclotides are a family of small and macrocyclic proteins that have been found in Violacaee and Rubiaceae plant species. These proteins contain a cystine knot: two disulfides bonds together with their connecting peptide backbone form an embedded ring which is penetrated by a third disulfide bond. The cyclotides have been attributed a wide range of biological activities, which in combination with their chemical stability and structural plasticity have made them attractive tools for pharmaceutical applications. The sequence of eleven novel cyclotides, vibi A-K, from Viola biflora was determined by the use of both chemical (extraction and characterization) and molecular biology (cDNA analyses) approaches. A clear discrepancy in the results from the two methods was observed. Additionally, one novel cyclotide, vodo O, was isolated from Viola odorata. To correlate cytotoxic potency to sequence, vodo O and vibi D, E, G and H were tested on a lymphoma cell line. Based on the presence or absence of a cis-Pro bond, the cyclotides are divided into the Möbius and bracelet subfamilies. The bracelet proteins have a higher net charge and are more cytotoxic potent than the Möbius ones. To explore these differences, charged and hydrophobic residues in varv A (Möbius) and cycloviolacin O2 (bracelet) were chemically modified and tested for their cytotoxicity. The net-charge of the two proteins was not important for the potency. The Glu residue in cycloviolacin O2 was crucial, while this residue was of minor importance in varv A. Oxidation of the single Trp residue declined the potency significantly in both proteins. To evaluate how the surface properties correlate to the degree of cytotoxic potency, models of all cyclotides hitherto tested were constructed by homology modelling. Calculations showed that the membrane orientation of varv A and cycloviolacin O2 differed significantly, which might explain their difference in potency

Die hier vorliegende Arbeit beschäftigt sich mit Eignung bakterieller Hüllproteine als Bindungsmatrix für die Kopplung funktionaler Moleküle mit dem Ziel, sensorische Schichten zu erzeugen. Bakterielle Hüllproteine sind biologische SAMs, anderen Oberfläche sich modifizierbare COOH-, NH2- und OH-Gruppen befinden. Die Ausbildung polymerer Strukturen erfolgt dabei in wässrigen Systemen und auf Oberflächen. Im Zuge der boomenden Entwicklung von Biosensoren werden insbesondere Biotemplate gesucht, die zwischen biologischer Komponente und Sensoroberfläche vermitteln. Bakterielle Hüllproteine stellen eine solche Zwischenschicht dar. Als Anwendungsbeispiel wurden die Proteine daher mit einem FRET-Paar und Thrombin und Kanamycin-Aptameren modifiziert. Hierbei wurden das FRET-Paar H488 und H555 an die bakteriellen Hüllproteine der beiden Haldenisolate A12 und B53 mittels EDC mit einer Modifizierungsrate von 0,54 molFarbstoff/molProtein kovalent gebunden. Bei der vorhandenen p4-Symmetrie bedeutet dies, dass ein FRET-Paar pro Einheitszelle vorhanden war. Der Nachweis eines Energietransfers zwischen den beiden am Protein gebundenen Fluoreszenzfarbstoffen H488 und H555 erfolgte mittels statischer und zeitaufgelöster Fluoreszenzmessung. Die Ergebnisse zeigten, dass ein Energietransfer nur möglich war, wenn die Proteine in polymerer Form vorlagen, unabhängig davon, ob sich die Proteine immobilisiert an einer Oberfläche oder in wässriger Lösung befanden. Mittels Variieren des Donor-Akzeptor-Verhältnisses konnte ein maximaler Energietransfer von 40 % generiert werden, wenn das Verhältnis der Fluoreszenzfarbstoffe von Donor und Akzeptor 4 betrug. Die Fluoreszenzintensität der Fluorophore wurde durch die Bindung an die Proteine nicht verringert oder gelöscht. Dies legt nahe, dass die Farbstoffe in den hydrophoben Poren immobilisiert wurden und die Poren die Fluoreszenzfarbstoffe schützen. Um weitere Aussagen über die Lage der gebundenen Fluoreszenzfarbstoffe zu erhalten, wurden die bakteriellen Hüllproteine der Stämme A12 und B53 enzymatisch verdaut und die Fragmente mittels SEC und SDS-PAGE untersucht. Dabei zeigten sich je nach Enzym und Protein unterschiedliche Bandenmuster bezüglich modifizierter und nativer Hüllproteine. Dies belegt, dass die Fluoreszenzfarbstoffe an NH2-und COOH-Gruppen der Proteine gebunden wurden und so teilweise den enzymatischen Verdau hinderten. Die SEC deutet an, dass die Fluoreszenzfarbstoffe an verschiedenen Stellen am Protein gebunden wurden. In einem zweiten Beispiel wurde das bakterielle Hüllprotein von A12 mit einem Aptamer modifiziert. Aptamere sind kurze einzelsträngige Oligonukleotide, die u.a. mittels ihrer ausgebildeten 3D-Struktur spezifisch Zielstrukturen reversibel binden können. Die hier verwendeten Aptamere binden spezifisch Thrombin und Kanamycin. Die Aptamere wurden mit Hilfe einer der beiden Vernetzer PMPI oder Sulfo-SMCC an die bakteriellen Hüllproteine kovalent gebunden. Nach dem Modifizieren der Proteine wurden diese auf entsprechenden Sensorchips immobilisiert und die Aktivität des gekoppelten Aptamers mittels Affinitätsmessungen, SPR-Spektroskopie und QCM-D-Messungen analysiert. Die Funktion des gebundenen Thrombinaptamers konnte mittels Affinitätsmessungen und QCM-D nachgewiesen werden und entspricht in beiden Fällen einer Bindung von 2 nmol Thrombin pro Quadratzentimeter. Die Funktionalität des Kanamycinaptamers sollte mittels SPR bestimmt werden, jedoch konnte keine Funktionalität des gekoppelten Kanamycinaptamers nachgewiesen werden. Alle Messungen bestätigten jedoch, dass die Bindungsmatrix aus bakteriellen Hüllproteinen keinerlei oder nur ein sehr geringes Hintergrundsignal liefert. Werden nun beide Komponenten, FRET-Paar und Aptamere, an das Protein gebunden, ist es möglich, eine sensorische Schicht zu erzeugen. Die Zielstruktur, welche detektiert werden soll, wird an das Aptamer gebunden und so in räumliche Nähe zur Sensorfläche gebracht. Stell die Zielstruktur einen Fluoreszenzlöscher dar, so wird der Energietransfer durch die räumliche Nähe des Fluoreszenzlöscher gestört. Die Detektion des Zielmoleküls erfolgt nun über die Änderung von Fluoreszenzintensitäten. Die hier vorgelegte Arbeit soll einen Grundstein legen für die Entwicklung eines solchen Sensors und insbesondere die Detektion eines Energietransfers optimieren und Schwachstellen in der Detektion nachweisen. Die systematische Untersuchung der Fluoreszenzfarbstoffe auf dem Protein ermöglichen es, in zukünftigen Arbeiten einen FRET zweifelsfrei zu detektieren. Die Modifizierung von bakteriellen Hüllproteinen von A12 mit Aptameren und die Detektion der Funktionalität der Aptamere mittels verschiedener Methoden zeigte auf, dass die bakteriellen Hüllproteine als universelle Bindungsmatrix für sensorische Moleküle dienen können, bei denen Affinitätsmessungen, SPR- oder QCM-D-Messungen genutzt werden. Besonders hervorzuheben ist, dass bakterielle Hüllproteine nahezu kein Hintergrundsignal liefern und aufgrund ihrer dünnen Monolage von etwa 6 - 9 nm die Sensitivität der Messungen nur gering beeinträchtigen.