Este trabalho propôe o desenvolvimento de micropartículas para veiculação deste fármaco na pele, aumentando a estabilidade da molécula e proporcionando uma liberação sustentada, o que resulta na otimização da terapia, visto que ocorre a redução dos efeitos colaterais. As micropartículas foram produzidas por três métodos diferentes, os dois primeiros utilizando a quitosana como polímero e o último utilizando uma associação de alginato com quitosana. As micropartículas de quitosana resultaram em interação do fármaco veiculado com o polímero e desta forma a sua utilização como sistema de liberação para veiculação do fármaco estudado foi descartada. As micropartículas de alginato/quitosana encapsularam efetivamente o fármaco, resultando em partículas irregulares com diâmetro médio de 148m. Elas apresentaram liberação sustentada do ácido retinóico por um período compatível com sua utilização tópica e por isso, parecem ser adequados para garantir estabilidade ao fármaco. Além disso, elas diminuíram a retenção do fármaco no estrato córneo quando comparado ao fármaco livre, mantendo seus níveis nas outras camadas mais profundas da pele. Esse direcionamento sítio-específico poderia diminuir a sua irritação dérmica, possibilitando, dessa forma, juntamente com o aumento de sua estabilidade, a obtenção de efeitos terapêuticos com a utilização de doses menores. Também não foram observadas interações entre o fármaco e o polímero demonstrando que a matriz de alginato foi capaz de proteger o fármaco do contato e da interação com a quitosana. Além disso, o método utilizado mostrou ser simples e rápido, realizado em condições amenas, sem o inconveniente da utilização de agentes cross-linking químicos tóxicos, como o glutaraldeído. / This study proposes the development of microparticles for drug delivery into the skin, thus increasing molecule stability and providing sustained release that results in therapy optimization as a result of reduction in side-effects. The microparticles were produced by three different methods. The two first methods used chitosan as a polymer, and the third utilized a chitosanalginate association. The chitosan microparticles resulted in the interaction of the delivered drug with the polymer; hence, its use as a release system for delivery of the studied drug was disregarded. The alginate/chitosan microparticles effectively encapsulated the drug, resulting in irregular particles with a mean diameter of 148m. They exhibited sustained release of retinoic acid for a period of time that was compatible with topical application and, therefore, seem to be suitable to ensure drug stability. Additionally, the microparticles decreased drug retention in the stratum corneum as compared to the free drug, thus keeping its levels in other deeper layers of the skin. Such site-specific direction could reduce dermal irritation, consequently enabling, conjointly with stability increase, the achievement of therapeutic effects with the use of smaller doses. Drug-polymer interactions were also not observed, showing that the alginate matrix was capable of protecting the drug from the contact and interaction with chitosan. Besides, the applied method proved to be simple and fast. It can be performed in mild conditions without the inconvenience of using toxic cross-linking chemical agents, such as glutaraldehyde.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A biossorção é o termo aplicado à tecnologia de adsorção de íons metálicos em solução, por meio de materiais de natureza biológica inativa, comparável à adsorção utilizando-se adsorventes convencionais. A biossorção depende da temperatura, pH, concentração inicial do íon metálico, e tipo de biosorvente. O objetivo principal dessa dissertação é estudar e comparar os processos de adsorção de cobre iônico a partir de soluções aquosas utilizando Sargassum sp., alginato de cálcio úmido e desidratado e carvão ativo em pó e granular, em regime de batelada e contínuo. O alginato de cálcio foi preparado através de uma solução 4% (m/v) de alginato de sódio gotejada em solução 37% (m/v) de cloreto de cálcio dihidratado. O alginato de cálcio apresentou uma umidade de 89% após secagem em estufa a 100C por 24 horas. Os parâmetros da biossorção foram fixados em: a dose de adsorventes de 2 g/L; a temperatura em 30C; pH da solução em 5,0; e velocidade de rotação da chapa rotatória em 150 rpm. Para os estudos de cinética e equilíbrio, os ensaios foram feitos em regime de batelada. Nos ensaios cinéticos para duas concentrações medias (0,16 e 3,15 mmol/L) variou-se o tempo de contato (1 a 120 min) para se atingir o equilíbrio. Nos ensaios de equilíbrio variou-se as concentrações (0,16 a 15,72 mmol/L) utilizando o tempo de equilíbrio determinado nos ensaios cinéticos. Foram utilizadas duas modelagens cinéticas (a de pseudo-primeira ordem e a de segunda ordem) e duas modelagens do equilíbrio (Langmuir e Freundlich). O modelo cinético de segunda ordem ajustou melhor os resultados. O tempo de equilíbrio para adsorção do cobre foi de 60 minutos para Sargassum sp. e pellets de alginatos de cálcio úmido e desidratado. Para os carvões ativos os tempos de equilíbrio para a adsorção do cobre foram mais rápidos, mas a capacidade de remoção do cobre foram muito baixas. Com base nos resultados desfavoráveis obtidos para os carvões ativos eles foram descartados para se prosseguir com os ensaios de equilíbrio. A isoterma de Freundlich melhor ajusta os dados experimentais para Sargassum sp. e alginato de cálcio úmido. A capacidade de adsorção máxima calculada pelo modelo de Freundlich: na concentração de equilíbrio de 11,66 0,06 mmol/L foi de 1,97 0,07 mmol/g para Sargassum sp.; e na concentração de equilíbrio de 12,12 0,03 mmol/L foi de 1,69 0,04 mmol/g para alginato de cálcio úmido. O processo de biossorção em regime contínuo, com alturas de leito variável de 10 a 40 cm, teve melhor desempenho com a altura de 40 cm. Em regime de batelada, o desempenho da Sargassum sp. foi superior ao dos alginatos de cálcio (úmido melhor que o desidratado), que por sua vez foram superiores ao desempenho dos carvões ativos em pó e granular (em pó melhor que o granular). O sistema contínuo com concentração inicial de cobre de 8,5 mmol/L deve ser operado com altura de leito igual ou superior a 40 cm, ou com sistemas multicolunas para soluções mais concentradas / Biosorption is the term applied to the adsorption technology of metallic ions in solution than through materials of inactive biological nature. Its close to adsorption using conventional adsorbents. Biosorption depends on temperature, pH, initial concentration of metal ion and type of biosorbents. The main objective of this dissertation is to study and compare the adsorption of copper ions from aqueous solution in batch and continuous system using Sargassum sp.; wet and dehydrated calcium alginate; powder and granular activated carbon. Calcium alginate was prepared by 4% w/v sodium alginate solution dropwise into 37% w/v calcium chloride dehydrate. Calcium alginate presented 89% of moisture after dry in an oven at 100C for 24 hours. Biosorption parameters were set as follow: dose of adsorbent 2 g/L; temperature at 30C, pH at 5.0, and stirring speed of the rotating plate at 150 rpm. For the kinetic and equilibrium studies, the tests were done in a batch system. In kinetic assays for two concentrations (0.16 and 3.15 mmol/L) was varied contact time (1-120 min) to reach equilibrium. In equilibrium assays were varied concentrations (0.16 to 15.72 mmol/L) using the time determined in kinetic experiments. It was used two kinetic modeling (the pseudo-first-order and second-order) and two equilibrium modeling (Langmuir and Freundlich). The second-order kinetic model better fitted the results. The equilibrium time for adsorption of copper was 60 minutes for Sargassum sp. and wet and dehydrated calcium alginate pellets. For the activated carbon, equilibrium times for the adsorption of copper were obtained faster, but the capacity of copper removal were extremely low. Based on the unfavorable results obtained for the activated carbon they were discharged to proceed with tests of equilibrium. The Freundlich isotherm fitted the experimental data for Sargassum sp. and wet calcium alginate. The maximum adsorption capacity calculated using Freundlich model: at equilibrium concentration of 11.66 0.06 mmol/L was 1.97 0.07 mmol/g for Sargassum sp. and at equilibrium concentration of 12.12 0.03 mmol/L was 1.69 0.04 mmol/g for wet calcium alginate. Biosorption in a continuous system, with bed heights from 10 to 40 cm, had the best performance with a height of 40 cm. In a batch system, the performance of Sargassum sp. was higher than calcium alginate (wet rather than dehydrated), which in turn were higher than the performance of powder and granular active carbon (powder better than granular). The continuous system with initial copper concentration of 8.5 mmol/L, must be operated with a bed height equal or greater 40 cm, or with multicolumn systems for more concentrated solutions

Made available in DSpace on 2014-12-17T15:42:14Z (GMT). No. of bitstreams: 1 MarciaSL_TESE.pdf: 4842135 bytes, checksum: b5c017ecd503f8ac8f797fb07359e645 (MD5) Previous issue date: 2011-06-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The alginates are copolymers of 1→4-linked β-D-mannuronic acid (M) and α-Lguluronic acid (G) residues that are arranjed in a block structure along a linear chain. Titanium dioxide, TiO2, is a ceramic material and can exist in three distinct crystallography forms: anatase, brookite and rutile. composites of organic and inorganic materials have better properties than the components alone. Thus, this study aims to synthesize, characterize and analyze the composite NaAlg-TiO2 in the form of powder and film. The synthesis of composite powders was performed using the sol-gel process and obtain the composite film was performed using the slow evaporation process, then the composites were analyzed by infrared spectroscopy, fluorescence x ray, thermal analysis, attenuated total reflection (ATR), x ray diffraction and impedance spectroscopy. The X ray diffraction patterns of composite powders show that with increasing calcination temperature, there were no complete transition of rutile-anatase crystalline phase, since at all temperatures studied (300, 500, 700, 900 and 1100?C) were observed peaks of anatase phase. Thermal analysis shows that at 400?C caused the decomposition of sodium alginate in sodium carbonate and above 600?C, we observe an exothermic peak related to the decomposition of sodium carbonate and in the presence of titanium dioxide becomes sodium titanate. The XRD results confirm the formation of sodium carbonate at 700?C and the formation sodium titanate in the temperature range 900-1100?C. The sodium titanate influenced the electrical properties of the material, because with increasing temperature there was a decrease in conductivity, probably due to the creation of Ti vacancies, since the sodium can induce the reduction of surface Ti4+ ions into Ti3+ species. The infrared spectra of the composites in the form of powder and film showed a small shift in the bands compared to the spectrum of pure alginate, indicating that these shifts, even small ones, have evidence of miscibility between the polymer and ceramic material / O alginato de s?dio, NaAlg, ? um polissacar?deo formado por liga??es do tipo 1-4 entre os ?cidos β-D-manur?nico e α-L-gulur?nico arranjados em bloco ao longo de uma cadeia linear. O di?xido de tit?nio, TiO2, ? um material cer?mico e existe em tr?s formas cristalinas: anat?sio, bruquita e rutilo. Os comp?sitos de materiais org?nicos e inorg?nicos apresentam melhores propriedades do que os componentes isoladamente. Dessa forma, o presente trabalho teve objetivo sintetizar, caracterizar e analisar os comp?sitos, NaAlg-TiO2, em formas de p? e de membrana. A s?ntese dos p?s comp?sitos foi realizada atrav?s do processo sol-gel e a obten??o dos comp?sitos em forma de membrana foi realizada atrav?s do processo de evapora??o lenta. Em seguida, os comp?sitos foram caracterizados por espectroscopia de infravermelho, difratometria de raios X, espectroscopia de imped?ncia, fluoresc?ncia de raios X, an?lise t?rmica e espectroscopia de reflect?ncia total atenuada-ATR. Os difratogramas de raios X dos p?s comp?sitos mostraram que, com o aumento da temperatura de calcina??o, n?o houve a completa transi??o de fase anat?sio-rutilo, uma vez que, em todas as temperaturas estudadas (300, 500, 700, 900 e 1100?C) foram observados picos da fase anat?sio. A an?lise t?rmica mostrou que em 400?C ocorreu ? decomposi??o do alginato de s?dio em carbonato de s?dio e, acima de 600?C, se observa um pico exot?rmico referente ? decomposi??o do carbonato de s?dio que, na presen?a do di?xido de tit?nio, transforma-se em titanato de s?dio, confirmados, tamb?m, atrav?s dos difratogramas de raios X, em que na temperatura de 700?C observa-se picos referentes ao carbonato de s?dio e em 900 e 1100?C picos relacionados ao titanato de s?dio. O titanato de s?dio influenciou nas propriedades el?tricas do material, pois com o aumento da temperatura houve um decr?scimo na condutividade, provavelmente, devido ? cria??o de vac?ncias de Ti, uma vez que, o Na+ induz a redu??o dos ?ons Ti+4 para esp?cies Ti+3. Os espectros de infravermelho dos comp?sitos em formas de p? e de membrana mostraram um pequeno deslocamento nas bandas, quando comparados ao espectro do alginato de s?dio puro, indicando que estes deslocamentos, mesmo que pequenos, t?m ind?cios de miscibilidade entre o pol?mero e o material cer?mico

A criopreservaÃÃo à a metodologia de escolha em muitos bancos de microrganismos pois conduz a uma situaÃÃo de dormÃncia metabÃlica e em conseqÃÃncia as culturas mantÃm-se estÃveis por longos perÃodos. Ao reduzir a temperatura pode todavia ocorrer a formaÃÃo de cristais de gelo que tendem a promover lesÃo celular A lesÃo celular por sua vez pode ser evitada pelo processo de vitrificaÃÃo que ocorre, combinando uma soluÃÃo concentrada com o congelamento rÃpido A literatura relata o uso de carboidratos como agentes vitrificadores e crioprotetores TambÃm informa que a tÃcnica de imobilizaÃÃo de cÃlulas em alginato de sÃdio permite o fracionamento da cultura e favorece a preservaÃÃo de microrganismos O objetivo deste trabalho consiste em avaliar a viabilidade dos fungos dos gÃneros Malassezia spp e do grupo dos Zigomicetos imobilizados em alginato de sÃdio utilizando glicose e lactose como crioprotetores Doze cepas de espÃcies de Malassezia spp (9 M furfur 2 M globosa 1 M simpodialis) e doze cepas de Zigomicetos (7 Mucor s e 5 Rhizopus sp) pertencentes à micoteca do Centro Especializado em Micologia MÃdica da Universidade Federal do Cearà O gÃnero Malassezia abrange leveduras lipodependentes associadas a vÃrias enfermidades dermatolÃgicas compreendem na atualidade 13 espÃcies Zigomicetos, por sua vez sÃo fungos saprÃfitas e ubÃquos cujas hifas por apresentarem elevado tamanho e reduzido conteÃdo protÃico estÃo mais vulnerÃveis a danos mecÃnicos Dessa forma constituem grupos fÃngicos de preservaÃÃo difÃcil em estoque portanto uma metodologia que mantenham a viabilidade de Malassezia spp e Zigomicetos tambÃm poderia ser empregada com eficiÃncia para a estocagem de outros fungos As estruturas fÃngicas foram removidas e transferidas para duas soluÃÃes de estoque, formadas por 3% de caldo Sabouraud 15% de alginato de sÃdio e diferenciadas nas concentraÃÃes de glicose e lactose de 9% ou 23% Em seguida, foram adicionadas esferas plÃsticas, medindo 07cm de diÃmetro com orifÃcio central e 0,5cm de comprimento ApÃs a gelificaÃÃo iÃnica com cloreto de cÃlcio, cada cepa foi estocada à temperatura de -20 ÂC e -80 ÂC distribuÃdas em cinco tubos criogÃnicos de 15mL contendo cinco esferas cada uma sendo avaliados em cinco perÃodos ApÃs nove meses foi verificado que nÃo houve recuperaÃÃo de nenhuma cepa de Malassezia spp Com relaÃÃo Ãs espÃcies de Zigomicetos observou-se que apÃs nove meses de estoque quatro cepas mostraram viÃveis a -80 ÂC empregando glicose a 9%, seis cepas foram preservadas a -80 ÂC, utilizando glicose a 23% finalmente seis e sete exemplares foram recuperados a -80 ÂC utilizando lactose a 9% e 23% respectivamente. Dessa forma, o uso de carboidratos como crioprotetores em conjunto com a tÃcnica de imobilizaÃÃo de cÃlulas em alginato de sÃdio constitui alternativa à manutenÃÃo de algumas espÃcies fÃngicas / Cryopreservation is the method of choice in many banks of microorganisms because it leads to a situation of metabolic dormancy and consequently cultures are stable for long periods. By reducing the temperature may however be formed of ice crystals which tend to cause cell damage to cell damage in turn can be avoided by vitrification process which takes place by combining a solution with the quick freezing the literature reports the use of carbohydrates vitrificadores cryoprotectants agents and also advises that the technique of immobilization of cells in sodium alginate allows the fractionation of culture and promotes the preservation of microorganisms The objective of this study is to assess the viability of the fungi of the genus Malassezia spp and the Zygomycetes group of assets in sodium alginate and lactose using glucose as cryoprotectants Twelve strains of species of Malassezia spp (9 M furfur 1 M 2 M globosa simpodialis) and twelve strains of zygomycetes (seven Mucor Rhizopus sp is 5) belonging to the mycology collection of the Specialized Center for Medical Mycology Federal University of Cearà The genus Malassezia comprises yeasts lipodependent associated with various dermatologic diseases currently comprise 13 Zygomycetes species, in turn, are ubiquitous and saprophytic fungi whose hyphae because they have high protein content and reduced size are more vulnerable to mechanical damage this way are groups of fungi in stock so hard to preserve a methodology to maintain the viability of Malassezia spp and Zygomycetes could also be used effectively for the storage of other fungi fungal structures were removed and transferred to two stock solutions, formed by 3% broth Sabouraud 15% sodium alginate and different concentrations of glucose and 9% lactose and 23% were then added plastic spheres, measuring 07cm in diameter and 0.5 cm center hole length After ionic gelation with calcium chloride Each strain was stored at -20  C and -80  C cryogenic distributed in five tubes containing 15 mL each of five levels being evaluated in five periods after nine months it was found that there was no recovery of any strain of Malassezia spp Regarding Zygomycetes species showed that after nine months of inventory four strains were viable at -80  C using glucose 9%, six strains were preserved at -80  C, using 23% glucose last six seven specimens were recovered at -80  C using a 9% lactose and 23% respectively. Thus, the use of cryoprotectants such as carbohydrates in conjunction with the technique of immobilization of cells in sodium alginate is an alternative to maintaining some fungal species

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O objetivo deste trabalho foi estudar a preparaÃÃo de biocatalisadores utilizando lipase de Candida antarctica tipo B (CALB) imobilizada covalentemente em quitosana, uma matÃria-prima abundante e de baixo custo no CearÃ, em quitosana-alginato e em agarose, com o intuito de utilizÃ-los na sÃntese de Ãsteres de vitamina A. Diversas estratÃgias de imobilizaÃÃo foram realizadas com o intuito de obter um derivado com elevada atividade enzimÃtica e com alta estabilidade tÃrmica e operacional. TrÃs tipos de suportes (agarose, quitosana e quitosana-alginato) foram preparados a partir de tais estratÃgias, sendo que um estudo aprofundado foi realizado com dois desses suportes (quitosana e quitosana-alginato). Apenas uma estratÃgia de imobilizaÃÃo foi realizada com agarose para testÃ-lo na sÃntese de palmitato de retinila, juntamente com dois derivados comerciais (lipase imobilizada de Thermomyces lanuginosus (Lipozyme TL IM) e lipase imobilizada de Mucor miehei (Lipozyme RM IM)), com o objetive de definir algumas condiÃÃes operacionais. Uma condiÃÃo avaliada que apresentou bons resultados na sÃntese foi o uso de peneira molecular para a retirada de Ãgua no meio reacional, sendo, portanto, utilizada nos estudos posteriores. ApÃs os estudos de imobilizaÃÃo e estabilidade tÃrmica a 60 ÂC, dois derivados (J8: quitosana ativada com glicidol seguido de etilenodiamina (EDA) e glutaraldeÃdo, e G10: quitosana-alginato ativada com glutaraldeÃdo) foram escolhidos, por apresentarem maiores atividades especÃficas (422,44 Â 50,4 U/g e 378,30 Â 34,7 U/g, respectivamente) e melhores estabilidades tÃrmicas (fatores de estabilizaÃÃo de 10,25 e 29,00, respectivamente), para estudos de estabilidade operacional de hidrÃlise e para sÃntese de palmitato de retinila. O derivado que apresentou melhor estabilidade tÃrmica a 60ÂC foi o G10, CALB imobilizada em quitosana-alginato, sendo aproximadamente 29 vezes mais estÃvel que a enzima solÃvel, e mais de 2 vezes mais estÃvel do que a enzima comercial Novozyme 435. PorÃm, o derivado J8 apresentou melhor estabilidade operacional de hidrÃlise, semelhante ao derivado comercial Novozyme 435. Um planejamento experimental 22 foi realizado para se avaliar a sÃntese de palmitato de retinila. Avaliou-se a influÃncia da temperatura (37 ÂC e 45 ÂC) e da razÃo entre os substratos, retinol:Ãcido palmÃtico (1:3 e 1:5), no rendimento de sÃntese, catalisada pelo derivado J8. Uma reaÃÃo utilizando o derivado G10 utilizando a melhor condiÃÃo do planejamento experimental foi realizada para ver o comportamento desse derivado. Com uma anÃlise estatÃstica dos resultados, pÃde-se observar que a razÃo entre os substratos teve efeito significativo no rendimento de sÃntese. Maiores foram obtidos quando a razÃo entre substratos foi igual a 1:5. Como os resultados nas temperaturas de 37 ÂC e 45 ÂC foram semelhantes, selecionou-se a temperatura de 37 ÂC para reaÃÃes posteriores, por necessitar de um menor gasto de energia para atingi-la / The objective of this work was to study the preparation of biocatalysts using lipase of Candida antarctica type B (CALB) covalently immobilized in agarose, chitosan, an abundant and low cost raw material, to be used in the synthesis of ester of Vitamin A. Several strategies of immobilization were studied in order to obtain a biocatalyst with good enzymatic activity and high thermal and operational stabilities. Three types of supports (agarose, chitosan and chitosanalginate) were activated by different strategies, but most of attention was given to the supports chitosan and chitosan-alginate. Only one derivative was prepared by immobilizing CALB in agarose and results of synthesis were compared to commercial derivatives (immobilized lipase of Thermomyces lanuginosus - Lipozyme TL IM - and immobilized lipase of Mucor miehei - Lipozyme RM IM), for the definition of some operational conditions. The operational condition that presented good results in the synthesis was used in further studies, such as removal of water from the reacional media by molecular sieves. After immobilization and thermal stabilities at 60 ÂC tests, two derivatives (J8: chitosan actived with glicidol follow by EDA and glutaraldehyde; G10: chitosan-alginate actived with glutaraldehyde) were selected: the ones that presented higher specific activities (422.44 Â 50.4 U/g and 378.30 Â 34.7 U/g, respectively) and best thermal stabilities (factors of stabilization of 10.25 and 29.0, respectively). Operational hydrolytic stabilities and the performance of these biocatalysts on the synthesis of retinyl palmitate were evaluated. One factorial design 22 was carried out to evaluate the synthesis of retinyl palmitate. The influence of the temperature (37 ÂC and 45 ÂC) and ratio between substrates concentration, retinol: palmitic acid (1:3 and 1:5), in the yield of synthesis, catalyzed for the J8 derivative, were evaluated. A statistical analysis of the results showed that the the most significant effect was the rate of substrates concentration. Higher yields of synthesis were obtained when the ratio of substrates concentration was equal to 1:5. Results of reaction yields at 37ÂC and 45 ÂC were very similar. Therefore, 37 ÂC was selected for further studies. Best results for thermal stability at 60ÂC were obtained for G10, CALB immobilized in chitosan-alginate, being approximately 29-fold more stable than soluble enzyme, and 2-fold more stable than the commercial enzyme (Novozyme 435). On the other hand, J8, CALB immobilized in chitosan, presented higher operational hydrolysis stability, with a similar deactivation profile to Novozyme 435

As leveduras de Candida spp. fazem parte da microbiota humana, entretanto podem causar tanto micoses superficiais quanto sistêmicas. As opções terapêuticas para as candidíases incluem três principais classes, os agentes poliênicos, azólicos e equinocandinas; no entanto, a maioria deles apresentam desvantagens de toxicidade e/ou farmacocinética, custos relativamente altos e aumento de isolados resistentes aos antifúngicos. A miltefosina (MFS), um composto alquilfosfocolina, é atualmente usada no tratamento de câncer de mama e de leishmanioses. Estudos prévios apontam a MFS como potencial alternativa para o tratamento das infecções fúngicas, porém possui elevada toxicidade. Dessa forma, este trabalho visa avaliar alternativas para o tratamento de candidíases. MFS e seus análogos sintéticos; nanopartículas de alginato e microemulsões como sistemas de liberação de MFS e/ou anfotericina B (AMB) foram avaliados quanto a atividade antifúngica in vitro e in vivo usando modelos de Galleria mellonella e modelo murino de candidíase vaginal. Dos análogos estruturais da MFS o composto 1 foi o mais ativo, apresentando efeito fungicida de maneira dose e tempo dependente; com a presença de ergosterol exógeno os valores de CIM de MFS e do composto 1 aumentam quando a concentração de ergosterol aumenta no meio de cultura, sugerindo que MFS e o composto 1 possam interagir com o ergosterol resultando em alteração da permeabilidade de membrana. Em ensaios de susceptibilidade in vitro a MFS livre foi mais efetiva que a MFS incorporada em nanopartículas de alginato (MFS.Alg) para todas as cepas de Candida spp. testadas. Em contrapartida, larvas de G. mellonella infectadas com C. albicans (SC 5314 ou IAL-40) foram protegidas da infecção quando tratadas com MFS.Alg (100 mg/Kg) em que houve redução das taxas de mortalidade e de morbidade, bem como redução da carga fúngica e do processo de filamentação do fungo. A AMB e a MFS foram efetivas no modelo larvário em doses de 1 e 20 mg/kg, respectivamente. Importantemente, a MFS em nanocarreador de alginato foi menos tóxica quando comparado com a sua forma livre. Adicionalmente, microemulsões (ME) foram produzidas para a incorporação de AMB e MFS. A formação de um sistema monofásico semelhante a um gel é visualizada com absorção de água após 4 horas de incubação em que agregados bem estruturados são formados indicando a fase hexagonal. Em modelo murino de candidíase vaginal as formulações de AMB e MFS em sistemas de liberação [MFS.Alg (1x), MFS-ME (3x), AMB-ME (3x)] foram comparadas com a formulação em creme [MFS-CR (6x) e AMB-CR (6x)], e todas foram capazes de reduzir a carga fúngica do tecido vaginal, porém as formulações em sistema de liberação foram as mais vantajosas comparadas com a formulação em creme tendo em vista a redução do custo e do número de aplicações levando a maior comodidade do paciente durante o tratamento da candidíase vaginal. Os resultados obtidos mostram dentre os análogos da MFS o Composto 1 foi o que melhor apresentou atividade antifúngica; e que a AMB e/ou MFS incorporadas em sistemas de liberação controlada (nanopartículas de alginato e microemulsões) foram capazes de reduzir efeitos colaterais (no caso da MFS) e de reduzir a carga fúngica nos tecidos infectados, podendo ser alternativas terapêuticas para o tratamento das candidíases. / The yeasts of Candida spp. are part of the human microbiota; however, they can cause both superficial and systemic mycoses. Therapeutic options for candidiasis include three main classes, polyenes, azoles and echinocandins; however, most of them exhibit disadvantages of toxicity and/or pharmacokinetics, relatively high costs and increase of antifungal resistant isolates. Miltefosine (MFS), an alkylphosphocholine compound, is currently used in the treatment of breast cancer and leishmaniasis. Previous studies indicate MFS as an alternative potential for the fungal infections treatment, but it has high toxicity. Thus, this study aims to evaluate alternatives for the treatment of candidiasis. MFS and its synthetic analogs; alginate nanoparticles and microemulsions such as release systems for MFS and / or amphotericin B (AMB) were evaluated for antifungal activity in vitro and in vivo using Galleria mellonella model and murine model of vaginal candidiasis. Among the analogs of MFS, compound 1 was the most active, showing a fungicidal effect in a dose-dependent manner; with the presence of exogenous ergosterol the MIC values of MFS and compound 1 increase when the concentration of ergosterol increases in the culture medium, suggesting that MFS and compound 1 may interact with ergosterol resulting in an altered membrane permeability. In vitro susceptibility assays the free MFS was more effective than the MFS incorporated in alginate nanoparticles (MFS.Alg) against all strains of Candida spp. tested. In contrast, C. albicans-infected G. mellonella larvae (SC 5314 or IAL-40) were protected from infection when treated with MFS.Alg (100 mg/kg) in which mortality and morbidity rates were reduced, as well as reduction of the fungal load and filamentation. AMB and MFS were effective in the larval model at doses of 1 and 20 mg/kg, respectively. Importantly, MFS in alginate nanocarrier was less toxic when compared to its free form. In addition, microemulsions (ME) were produced for the incorporation of AMB and MFS. The formation of a single-phase gel-like system is visualized with water absorption after 4 hours of incubation in which well-structured aggregates are formed indicating the hexagonal phase. In the murine model of vaginal candidiasis, formulations of AMB and MFS incorporated in release systems [MFS.Alg (1x), MFS-ME (3x), AMB-ME (3x)] were compared with the cream formulation [MFS-CR 6x) and AMB-CR (6x)], and all formulations were able to reduce the fungal load of vaginal tissue, but the release-system formulations were the most advantageous compared to the cream formulation in order to reduce the cost and number of applications leading to greater patient comfort during the treatment of vaginal candidiasis. The results obtained show that among the analogs of MFS, compound 1 was the one that presented the best antifungal activity; and that AMB and/or MFS incorporated in controlled release systems (alginate nanoparticles and microemulsions) were able to reduce side effects (in the case of MFS) and reduce fungal load in infected tissues, and they could be therapeutic alternatives for treatment of the candidiasis.

L'embolisation artérielle a récemment émergé sur le plan interventionnel comme un traitement sûr et efficace pour arrêter une hémorragie ou induire la dévascularisation d'un tissu cible. La visualisation directe des agents d'occlusions vasculaires n'est pas toujours possible. L'estimation de leur position dans la branche vasculaire en se basant sur la distribution de produit opaque aux rayons X est partiellement incorrecte. Un marquage des agents d'occlusion vasculaire est souhaitable pour leur suivi par IRM pendant l'intervention ou a posteriori, ce, notamment en raison de la diffusion de l'IRM comme modalité d'imagerie anatomique et interventionnelle. Dans ce travail, plusieurs protocoles ont été établis pour repérer des agents d'occlusion vasculaire marqués avec le SPIO, in situ, in vitro et dans des « conditions » in vivo, sous IRM 1,5T et 3T. Les agents d'occlusion étudiés sont des microsphères de trisacryl (Embosphère®), et des microparticules ou microcapsules utilisées éventuellement comme vecteurs de principes actifs : parmi celles-ci, des microsphères gonflables (Hépasphère™) non dégradables ont été marquées, et des microbilles d'alginate et des systèmes d'émulsions dégradables réalisés par nos soins. L'établissement de protocoles de détectabilité sous IRM a impliqué le choix de séquences et l'optimisation des paramètres de ces séquences, le choix de l'antenne, ainsi que la mise au point d'une quantité suffisante de marqueur dans les agents d'occlusion afin qu'ils soient visibles à l'IRM. Une méthodologie de mesure de faible variation de l'intensité du signal des images IRM a été développée. Un modèle expérimental in vitro a été conçu avec la présence des microparticules marquées dans un environnement simulant grossièrement une vascularisation tumorale et son drainage veineux. La fixation des immunoglobulines (IgG1), équivalentes aux anti-VEGF, sur des microsphères (Hépasphères™) a été étudiée. Enfin, les propriétés mécaniques et électriques des systèmes de microémulsions ont été explorées par ultrasons et par impédancemétrie.

The overall goal of this thesis is to better understand mechanisms governing associative phase separation within admixtures of plant proteins (e.g., pea and canola) and anionic polysaccharides (e.g., gum Arabic, alginate or é-carrageenan). The process involves the electrostatic attraction between two biopolymers of opposing charges, and typically results in the formation of both soluble and insoluble complexes during an acidic pH titration. If successful, polysaccharides could be triggered to coat the proteins surface to give novel, and hopefully improved functionality as ingredients for food and biomaterials.<p> In the first study, the effect of protein enrichment and pH on the formation of soluble and insoluble complexes in admixtures of pea legumin (Lg) and vicilin (Vn) isolates with gum Arabic (GA) was investigated by turbidimetric, surface charge and fluorometric measurements. The solubility of the protein isolates and mixed biopolymer systems was also studied as a function of pH. Enrichment of the crude Lg and Vn isolates by low pressure liquid chromatography led to a shift towards higher pHs at the onset of soluble complex formation in the presence of GA for both protein isolates, whereas the onset of insoluble complex formation was unaffected. Complexation of the Lg (or Vn) isolates with GA resulted in a shift in the pH where neutrality (zeta potential = 0 mV) occurred to lower pH values, relative to the Lg (or Vn) isolates alone. In the case of the enriched Vn isloate, changes to its tertiary structure were observed by fluorometry upon complexation with GA, whereas no changes were found for the enriched Lg isolate. Complexation of Lg and Vn isolates with GA also had little effect on their solubilities relative to protein alone solutions.<p> In the second study, the formation of soluble and insoluble complexes, and the nature of their interactions as determined by optical density analysis, were investigated in admixtures of canola protein isolate (CPI) and anionic polysaccharides (alginate and é-carrageenan) as a function of pH and biopolymer weight mixing ratio. The solubilities of formed complexes were also investigated versus protein alone. In both CPI-polysaccharide systems, critical pH associated with the onset of soluble and insoluble complexes shifted to higher pHs as the mixing ratios increased from 1:1 to 20:1 (CPI:polysaccharide), and then became constant. There complexes formed primarily through electrostatic attractive forces with secondary stabilization by hydrogen bonding. The solubilities of the CPI-alginate complexes were significantly enhanced relative to CPI alone or CPI-é-carrageenan, which were similar.

Les biofilms sont des édifices macromoléculaires qui résultent de l'adhérence de microorganismes à une surface. Ils sont constitués de cellules enchâssées dans un réseau d'exopolymères d'origine biologique qui forment une matrice extracellulaire. Les biofilms posent des problèmes technologiques et sanitaires dans de nombreux domaines, aussi bien agroalimentaire, médical, ou industriel. Comprendre les mécanismes de formation de ces structures est donc un enjeu majeur. Malgré une grande diversité de la structure des biofilms, de grands principes semblent en régir la composition. Ainsi, la présence de polysaccharides comme l'alginate et la cellulose joue un rôle majeur dans leur formation et dans la définition de leurs propriétés mécaniques. Si la présence de polymères protéiques comme les fibres amyloïdes semble avoir un caractère universel au sein des biofilms, leur rôle dans la formation de la matrice et dans ses propriétés mécaniques restait à définir. Lors de cette étude, nous avons caractérisé la structure et la composition de trois biofilms monobactériens issus de trois espèces différentes P. aeruginosa NK 125502, S. enterica CIP 58.58 et S. epidermidis CIP 53.124. Nous avons décrit la formation de fibres amyloïdes par différents peptides issus de protéines bactériennes impliquées dans la formation de biofilm et montré par différentes techniques qu'ils sont capables de former des fibres amyloïdes. Nous avons notamment identifié un peptide amyloïde, suggérant la présence de ce type de fibre au sein des biofilms de Staphylococcus, mais aussi plus généralement dans les biofilms des espèces exprimant une protéine de type Bap (Biofilm associated protein). Enfin, nous avons analysé les propriétés mécaniques de différentes matrices synthétiques à base d'alginate et de méthyl-cellulose, en présence et en absence de protéines et de peptides amyloïdes afin de mieux comprendre l'apport qu'a ce type de fibre sur les propriétés de ces structures. Ainsi, les fibres amyloïdes modifient les propriétés mécaniques des gels synthétiques, permettant d'augmenter la déformation sous contrainte. En conclusion, ce travail apporte de nouveaux éléments pour la compréhension du rôle des fibres amyloïdes dans le renforcement de la matrice du biofilm. La capacité à former des fibres amyloïdes par un peptide issu de la protéine Bap de S. epidermidis suggère que cette propriété est plus largement présente au sein de cette famille de protéines. Les travaux menés au cours de cette thèse, et l'ensemble des techniques utilisées, avec notamment la mise au point de l'observation de la biréfringence du rouge Congo par microscopie confocale permettront de développer les études sur cette famille de protéines amyloïdes ainsi que sur les matrices complexes de type biofilm

The overall goal of this thesis is to better understand mechanisms governing associative phase separation within admixtures of plant proteins (e.g., pea and canola) and anionic polysaccharides (e.g., gum Arabic, alginate or é-carrageenan). The process involves the electrostatic attraction between two biopolymers of opposing charges, and typically results in the formation of both soluble and insoluble complexes during an acidic pH titration. If successful, polysaccharides could be triggered to coat the proteins surface to give novel, and hopefully improved functionality as ingredients for food and biomaterials.<p> In the first study, the effect of protein enrichment and pH on the formation of soluble and insoluble complexes in admixtures of pea legumin (Lg) and vicilin (Vn) isolates with gum Arabic (GA) was investigated by turbidimetric, surface charge and fluorometric measurements. The solubility of the protein isolates and mixed biopolymer systems was also studied as a function of pH. Enrichment of the crude Lg and Vn isolates by low pressure liquid chromatography led to a shift towards higher pHs at the onset of soluble complex formation in the presence of GA for both protein isolates, whereas the onset of insoluble complex formation was unaffected. Complexation of the Lg (or Vn) isolates with GA resulted in a shift in the pH where neutrality (zeta potential = 0 mV) occurred to lower pH values, relative to the Lg (or Vn) isolates alone. In the case of the enriched Vn isloate, changes to its tertiary structure were observed by fluorometry upon complexation with GA, whereas no changes were found for the enriched Lg isolate. Complexation of Lg and Vn isolates with GA also had little effect on their solubilities relative to protein alone solutions.<p> In the second study, the formation of soluble and insoluble complexes, and the nature of their interactions as determined by optical density analysis, were investigated in admixtures of canola protein isolate (CPI) and anionic polysaccharides (alginate and é-carrageenan) as a function of pH and biopolymer weight mixing ratio. The solubilities of formed complexes were also investigated versus protein alone. In both CPI-polysaccharide systems, critical pH associated with the onset of soluble and insoluble complexes shifted to higher pHs as the mixing ratios increased from 1:1 to 20:1 (CPI:polysaccharide), and then became constant. There complexes formed primarily through electrostatic attractive forces with secondary stabilization by hydrogen bonding. The solubilities of the CPI-alginate complexes were significantly enhanced relative to CPI alone or CPI-é-carrageenan, which were similar.