Adsorption on solid surfaces from solution is a fundamental property of a surfactant. It might even be the most important aspect of surfactant behavior, since it influences many applications, such as cleaning, detergency, dispersion, separation, flotation, and lubrication. Consequently, fundamental investigations of surfactant adsorption are relevant to many areas. The main aim of this thesis has been to elucidate the adsorption properties, primarily on the solid/water interface, of a particular class of polyhydroxyl based surfactants: the alkyl glucosides. By the use of ellipsometry, the equilibrium and kinetic aspects of adsorption on titanium dioxide with respect to structural effects has been studied. Furthermore, the effects of small amounts of cationic surfactant additives on the adsorption on silica have been investigated. The results have been compared with similar studies for other nonionic surfactants. We have found that the surfactant structure has a strong effect on the adsorption properties. An increase in the surfactant chain length increases the cooperativity of the system. An increase in the head group polymerization decreases the cooperativity and the plateau adsorbed amount at equilibrium. The effect of surfactant structure on the adsorption kinetics depends on the concentration relative to the cmc, while the there is a decrease in the rate of desorption with increasing hydrophobic chain length independent of the concentration. The adsorption/desorption process is concluded to be diffusion driven, as suggested by the model used. When comparing these results with studies on ethylene oxide based surfactants, we conclude that the two types of surfactants exhibit similar trends on surfaces onto which they adsorb. Adsorption from binary surfactant solutions is even more interesting than adsorption from single surfactant solutions, since it brings us one step closer to the systems used in applications. In addition, adsorption from a mixture can be very different from adsorption from any of the single surfactants in the mixture. Alkyl glucosides alone do not adsorb on silica, but addition of small amounts of a cationic surfactant to the alkyl glucoside solution allows for adsorption on silica. A comparison between the adsorption and bulk properties has shown that mixed micellization explains most, but not all, effects of the coadsorption properties. Changing the pH in the mixed systems reveals that a surfactant with a pH-dependent charge and the ability to adapt its charge to the environment, e.g. a surface, enhances the adsorbed amount over a wider range of pH values than a purely cationic surfactant. It is well known that alkyl glucosides and ethylene oxides adsorb differently on different types of hydrophilic surfaces. As a consequence, replacing ethylene oxides with alkyl glucosides might not be all straight-forward; however, we have shown that the effect of the surface can be eliminated by the use of a cosurfactant. / <p>QC 20101018</p>

SOARES, Denise Josino. Efeitos antioxidante e antiinflamatório da polpa de pitanga roxa (Eugenia uniflora L.) sobre células bucais humanas, aplicando experimentos in vitro e ex vivo. 2014. 98 f. Tese (Doutorado em tecnologia de alimentos)- Universidade Federal do Ceará, Fortaleza-CE, 2014. / Submitted by Elineudson Ribeiro (elineudsonr@gmail.com) on 2016-07-07T18:44:53Z No. of bitstreams: 1 2014_tese_djsoares.pdf: 2572830 bytes, checksum: 5fcdb95b76b4c0c19e882eb36d8865b7 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-07-21T20:26:33Z (GMT) No. of bitstreams: 1 2014_tese_djsoares.pdf: 2572830 bytes, checksum: 5fcdb95b76b4c0c19e882eb36d8865b7 (MD5) / Made available in DSpace on 2016-07-21T20:26:33Z (GMT). No. of bitstreams: 1 2014_tese_djsoares.pdf: 2572830 bytes, checksum: 5fcdb95b76b4c0c19e882eb36d8865b7 (MD5) Previous issue date: 2014 / Pitanga (Eugenia uniflora L.) is a tropical fruit found in the region that covers the central part of Brazil to Northern Argentina. This fruit has low lipid content, and is rich in bioactive compounds, such as polyphenols and carotenoids. In view of the use of pitanga tree in folk medicine and the shortage of scientific works about the antioxidative and anti-inflammatory effect of the purple pitanga, the present work aimed to investigate these characteristics in the pulp and in the sweetened tropical juice of purple pitanga, using in vitro and ex vivo experiments. In the present study, purple pitanga pulp was divided into two fractions (volatile and non-volatile), and the main compound of each fraction was identified and quantified. Human gingival cells (from six volunteers) were exposed to purple pitanga sweetened tropical juice and its main volatile and non-volatile compounds and analyzed by the catalase activity, DNA damage and interleukin 8 (IL-8) releases. The experiment was also performed with human gingival fibroblast (HGF-1), where cells were exposed to the individual main compounds from purple pitanga pulp and the IL-8 release was analyzed. Purple pitanga pulp presented mean values of soluble solids (8.33 ± 0.06 °Brix), pH (3.12 ± 0.01), titratable acidity (1.76 ± 0.20 g citric acid/100 mL) and total sugars (9.28 ± 0.60 g glucose/100 mL) within the standards required by current Brazilian law. This pulp also showed significant levels of the bioactive compounds: anthocyanins (24.82 ± 0.46 mg/100 mL), yellow flavonoids (11.33 ± 0.66 mg/100 mL) and total extractable polyphenols (26.85 ± 0.30 mg GAE/100 mL), making this product a good source of natural antioxidants. With regard to the main compound from volatile and non-volatile fractions of purple pitanga pulp, oxidoselina-1,3,7(11)-trien-8-one (85 ± 4.01 µg/mL) was observed in the volatile fraction and cyanidin-3-glucoside (340 ± 4.19 µg/mL )was observed in the non-volatile fraction. The low pH of the purple pitanga sweetened tropical juice decreases catalase activity, while oxidoselina-1,3,7(11)-trien-8-one and cyanidin-3-glucoside did not interfere and were not able to inhibit the activity of this enzyme. Purple pitanga sweetened tropical juice prevented DNA damage in human gingival cells. Due to the low number of volunteers in the experiment with purple pitanga sweetened tropical juice and the main compounds from volatile and non-volatile fractions of purple pitanga pulp, the results regarding the IL-8 release are inconclusive. Cyanidin-3-glucoside and oxidoselina-1,3,7(11)-trien-8-one presented anti-inflammatory effects in HGF-1 cells. / A pitanga (Eugenia uniflora L.) é uma fruta tropical encontrada na região que compreende a parte central do Brasil e o Nordeste da Argentina. Este fruto possui baixo conteúdo de lipídios, sendo rico em vitaminas e compostos bioativos, como os polifenóis e carotenóides. Devido ao uso da pitangueira na medicina popular e escassez de trabalhos científicos sobre as propriedades antioxidantes e antiinflamatórias da pitanga roxa, o presente trabalho teve como objetivo investigar essas características na polpa e no suco tropical de pitanga roxa adoçado, usando experimentos in vitro e ex vivo. No presente estudo, a polpa de roxa foi separada em duas frações (volátil e não volátil), sendo o composto majoritário de cada fração identificado e quantificado. Células da gengiva humana (provenientes de seis voluntários) foram expostas ao suco tropical de pitanga e ao composto majoritário de cada fração e analisadas quanto a atividade da catalase, o dano do DNA e a liberação da interleucina 8 (IL-8). O experimento também foi realizado em células dos fibroblastos gengivais humanos (HGF-1), cujas células foram expostas aos compostos majoritários das duas frações da polpa de pitanga roxa e a liberação da IL-8 foi analisada. A polpa de pitanga roxa apresentou valores médios de sólidos solúveis (8,33 ± 0,06 °Brix), pH (3,12 ± 0,01), acidez titulável (1,76 ± 0,20 g ácido cítrico/100 mL) e açúcares totais (9,28 ± 0,60 g glicose/100 mL) dentro dos padrões exigidos pela legislação brasileira vigente. A referida polpa apresentou, ainda, níveis consideráveis dos compostos bioativos: antocianinas (24,82 ± 0,46 mg/100 mL), flavonóides amarelos (11,33 ± 0,66 mg/100 mL) e polifenóis extraíveis totais (26,85 ± 0,30 mg GAE/100 mL), fazendo deste fruto uma boa fonte de antioxidantes naturais. Como composto majoritário das frações volátil e não volátil da polpa de pitanga observa-se a oxidoselina-1,3,7(11)-trien-8-ona (85 ± 4,01 µg/mL) e a cianidina-3-glicosídeo (340 ± 4,19 µg/mL), respectivamente. O baixo pH do suco tropical de pitanga roxa adoçado provocou uma redução da atividade da catalase, enquanto a oxidoselina-1,3,7(11)-trien-8-ona e a cianidina-3-glicosídeo não interferiram e não foram capazes de inibir a atividade desta enzima. O suco tropical de pitanga roxa adoçado preveniu o dano do DNA em células da gengiva humana. Devido ao baixo número de voluntários no experimento com o suco tropical de pitanga roxa adoçado e os compostos majoritários das frações volátil e não volátil da polpa de pitanga roxa, os resultados referentes à liberação da IL-8 são inconclusivos. Cianidina-3-glicosídeo e oxidoselina-1,3,7(11)-trien-8-ona apresentaram efeito antiinflamatório em células HGF-1.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CHAPTER I - The aqueous extract prepared from Schizolobium parahyba (Sp) leaves, a native plant from Mata Atlantica forest (Brazil), was tested to analyze its ability to inhibit some biological and enzymatic activities induced by Bothrops alternatus and Bothrops moojeni snake venoms. Sp chromatography in Sephadex LH 20 resulted in 3 fractions: F1 (methanolic fraction); F2 (methanol:water fraction, 1:1 v/v); and F3 (aqueous fraction). These fractions were analyzed in terms of its capacity of inhibit the venoms fibrinogenolytic activity. Sp inhibited 100% of lethality, blood incoagulability, hemorrhagic and indirect hemolytic activities for a 1:10 ratio (venom/extract, w/w), and coagulant activity in a 1:5 ratio (venom/extract, w/w) induced by venoms. Venoms fibrinogenolytic activity were also neutralized by Sp in a 1:10 ratio, resulting in total protection of B&#946; chain and partial protection for the fibrinogen A&#945; chain. However, larger ratios of extract/proteins have caused a complete vanishing of all fibrinogen chains possibly caused by Sp precipitation. Interaction tests have demonstrated that for certain extract/proteins ratios Sp precipitates proteins non-specifically suggesting the presence of tannins, which are very likely responsible for the excellent inhibiting effects of the analyzed ophidian activities. Therefore, the fractioning of Sp was carried out aiming at separating these compounds that mask the obtained results. F1 inhibited 100% the venom fibrinogenolytic activity without presenting protein precipitation effect; F2 demonstrated only partial inhibition of these venoms activities. Finally, F3 did not inhibit fibrinogen proteolysis, but presented strong protein precipitating action. We conclude that Sp, together with tannins, also contains other compounds which can present specific toxin inhibition action of snake venoms. CHAPTER II - Do extrato aquoso liofilizado das folhas de Schizolobium parahyba foram isolados quatro compostos ativos, isoquercitrina, miricetina-3-O-glicosídeo, catequina e galocatequina, por cromatografia em Sephadex LH 20 seguido de HPLC semipreparativo em coluna C-18 e identificados por RMN do 1H e 13C. Essa é a primeira vez que estas substâncias são relatadas nesta espécie assim como suas atividades antiofídicas. Após isolamento foram todas testadas frente a atividade hemorrágica e fibrinogenolítica da peçonha de Bothrops alternatus (Bal). Na proporção 1:30 (Peçonha/fração, m/m) a isoquercitrina inibiu parcialmente a atividade hemorrágica; a miricetina-3-O-glicosideo e a catequina não foram capazes de inibir a hemorragia induzida pela Bal e a galocatequina, por sua vez, apresentou uma eficiente neutralização desta atividade da peçonha. Na proporção de 1:100 (Peçonha/fração, m/m) ficou evidenciado o efeito de inibição total da ação fibrinogenolítica da peçonha pela miricetina-3-O-glicosideo; a isoquercitrina não protegeu a proteólise da cadeia A&#945; do fibrinogênio e inibiu parcialmente a degradação da cadeia B&#946; enquanto a catequina e a galocatequina inibiram parcialmente a degradação da cadeia A&#945; e totalmente a da cadeia B&#946;. Dessa forma concluímos que a galocatequina e a miricetina-3-O-glicosideo são excelentes inibidores de toxinas hemorrágicas e fibrinogenolíticas respectivamente. Estudos vindouros como o de modelagem molecular e cristalização de raio-X usando toxinas isoladas e estas substâncias poderão ser de grande importância para o desenho de novas drogas auxiliares do tratamento com soro antiofídico ou para melhor elucidação da relação estruturafunção das toxinas destes venenos. / CAPÍTULO I - O extrato aquoso preparado das folhas de Schizolobium parahyba (Sp), uma planta nativa da Mata Atlântica (Brasil), foi testado para avaliar sua habilidade de inibir algumas atividades biológicas e enzimáticas induzidas pelas peçonhas brutas de Bothrops alternatus e Bothrops moojeni. Cromatografia de Sp em coluna de Sephadex LH 20 resultou em 3 frações: F1 (fração metanólica); F2 (fração metanol : água, 1:1 v/v) e F3 (fração aquosa). Estas frações foram analisadas quanto a capacidade de inibir a atividade Fibrinogenolítica das peçonhas. Sp inibiu em 100% a letalidade, a incoagulabilidade sanguínea, a atividade hemorrágica e hemolítica indireta na proporção de 1:10 (peçonha/extrato, m/m) e atividade coagulante na proporção de 1:5 (peçonha/extrato, m/m) induzidas pelas peçonhas. A atividade fibrinogenolítica das peçonhas também foi neutralizada por Sp na proporção de 1:10, resultando em proteção total da cadeia B&#946; e parcial da A&#945; do fibrinogênio. No entanto, proporções maiores de peçonha/extrato mostraram desaparecimento de todas as cadeias do fibrinogênio numa possível precipitação causada por Sp. Os testes de interação extrato/proteínas demonstraram que em determinadas proporções de extrato/proteínas Sp precipita proteínas inespecificamente, sugerindo a presença de taninos, os quais muito provavelmente são os responsáveis pelo excelente efeito inibidor das atividades ofídicas analisadas. Assim, com o intuito de separar estes componentes que mascaram os resultados obtidos, foi realizado o fracionamento de Sp. F1 inibiu em 100% a atividade fibrinogenolítica das peçonhas e não apresentou efeito de precipitação de proteínas; F2 demonstrou inibição parcial desta atividade das peçonhas e F3 não inibiu a proteólise do fibrinogênio, mas apresentou forte ação precipitante de proteínas. Concluímos, assim que Sp, além de taninos, também contém outros compostos e que estes podem apresentar ação específica de inibição de toxinas de peçonhas de serpentes. CAPÍTULO II - Do extrato aquoso liofilizado das folhas de Schizolobium parahyba foram isolados quatro compostos ativos, isoquercitrina, miricetina-3-O-glicosídeo, catequina e galocatequina, por cromatografia em Sephadex LH 20 seguido de HPLC semipreparativo em coluna C-18 e identificados por RMN do 1H e 13C. Essa é a primeira vez que estas substâncias são relatadas nesta espécie assim como suas atividades antiofídicas. Após isolamento foram todas testadas frente a atividade hemorrágica e fibrinogenolítica da peçonha de Bothrops alternatus (Bal). Na proporção 1:30 (Peçonha/fração, m/m) a isoquercitrina inibiu parcialmente a atividade hemorrágica; a miricetina-3-O-glicosideo e a catequina não foram capazes de inibir a hemorragia induzida pela Bal e a galocatequina, por sua vez, apresentou uma eficiente neutralização desta atividade da peçonha. Na proporção de 1:100 (Peçonha/fração, m/m) ficou evidenciado o efeito de inibição total da ação fibrinogenolítica da peçonha pela miricetina-3-O-glicosideo; a isoquercitrina não protegeu a proteólise da cadeia A&#945; do fibrinogênio e inibiu parcialmente a degradação da cadeia B&#946; enquanto a catequina e a galocatequina inibiram parcialmente a degradação da cadeia A&#945; e totalmente a da cadeia B&#946;. Dessa forma concluímos que a galocatequina e a miricetina-3-O-glicosideo são excelentes inibidores de toxinas hemorrágicas e fibrinogenolíticas respectivamente. Estudos vindouros como o de modelagem molecular e cristalização de raio-X usando toxinas isoladas e estas substâncias poderão ser de grande importância para o desenho de novas drogas auxiliares do tratamento com soro antiofídico ou para melhor elucidação da relação estruturafunção das toxinas destes venenos. / Mestre em Genética e Bioquímica

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A pitanga (Eugenia uniflora L.) à uma fruta tropical encontrada na regiÃo que compreende a parte central do Brasil e o Nordeste da Argentina. Este fruto possui baixo conteÃdo de lipÃdios, sendo rico em vitaminas e compostos bioativos, como os polifenÃis e carotenÃides. Devido ao uso da pitangueira na medicina popular e escassez de trabalhos cientÃficos sobre as propriedades antioxidantes e antiinflamatÃrias da pitanga roxa, o presente trabalho teve como objetivo investigar essas caracterÃsticas na polpa e no suco tropical de pitanga roxa adoÃado, usando experimentos in vitro e ex vivo. No presente estudo, a polpa de roxa foi separada em duas fraÃÃes (volÃtil e nÃo volÃtil), sendo o composto majoritÃrio de cada fraÃÃo identificado e quantificado. CÃlulas da gengiva humana (provenientes de seis voluntÃrios) foram expostas ao suco tropical de pitanga e ao composto majoritÃrio de cada fraÃÃo e analisadas quanto a atividade da catalase, o dano do DNA e a liberaÃÃo da interleucina 8 (IL-8). O experimento tambÃm foi realizado em cÃlulas dos fibroblastos gengivais humanos (HGF-1), cujas cÃlulas foram expostas aos compostos majoritÃrios das duas fraÃÃes da polpa de pitanga roxa e a liberaÃÃo da IL-8 foi analisada. A polpa de pitanga roxa apresentou valores mÃdios de sÃlidos solÃveis (8,33  0,06 ÂBrix), pH (3,12  0,01), acidez titulÃvel (1,76  0,20 g Ãcido cÃtrico/100 mL) e aÃÃcares totais (9,28  0,60 g glicose/100 mL) dentro dos padrÃes exigidos pela legislaÃÃo brasileira vigente. A referida polpa apresentou, ainda, nÃveis considerÃveis dos compostos bioativos: antocianinas (24,82  0,46 mg/100 mL), flavonÃides amarelos (11,33  0,66 mg/100 mL) e polifenÃis extraÃveis totais (26,85  0,30 mg GAE/100 mL), fazendo deste fruto uma boa fonte de antioxidantes naturais. Como composto majoritÃrio das fraÃÃes volÃtil e nÃo volÃtil da polpa de pitanga observa-se a oxidoselina-1,3,7(11)-trien-8-ona (85  4,01 Âg/mL) e a cianidina-3-glicosÃdeo (340  4,19 Âg/mL), respectivamente. O baixo pH do suco tropical de pitanga roxa adoÃado provocou uma reduÃÃo da atividade da catalase, enquanto a oxidoselina-1,3,7(11)-trien-8-ona e a cianidina-3-glicosÃdeo nÃo interferiram e nÃo foram capazes de inibir a atividade desta enzima. O suco tropical de pitanga roxa adoÃado preveniu o dano do DNA em cÃlulas da gengiva humana. Devido ao baixo nÃmero de voluntÃrios no experimento com o suco tropical de pitanga roxa adoÃado e os compostos majoritÃrios das fraÃÃes volÃtil e nÃo volÃtil da polpa de pitanga roxa, os resultados referentes à liberaÃÃo da IL-8 sÃo inconclusivos. Cianidina-3-glicosÃdeo e oxidoselina-1,3,7(11)-trien-8-ona apresentaram efeito antiinflamatÃrio em cÃlulas HGF-1. / Pitanga (Eugenia uniflora L.) is a tropical fruit found in the region that covers the central part of Brazil to Northern Argentina. This fruit has low lipid content, and is rich in bioactive compounds, such as polyphenols and carotenoids. In view of the use of pitanga tree in folk medicine and the shortage of scientific works about the antioxidative and anti-inflammatory effect of the purple pitanga, the present work aimed to investigate these characteristics in the pulp and in the sweetened tropical juice of purple pitanga, using in vitro and ex vivo experiments. In the present study, purple pitanga pulp was divided into two fractions (volatile and non-volatile), and the main compound of each fraction was identified and quantified. Human gingival cells (from six volunteers) were exposed to purple pitanga sweetened tropical juice and its main volatile and non-volatile compounds and analyzed by the catalase activity, DNA damage and interleukin 8 (IL-8) releases. The experiment was also performed with human gingival fibroblast (HGF-1), where cells were exposed to the individual main compounds from purple pitanga pulp and the IL-8 release was analyzed. Purple pitanga pulp presented mean values of soluble solids (8.33  0.06 ÂBrix), pH (3.12  0.01), titratable acidity (1.76  0.20 g citric acid/100 mL) and total sugars (9.28  0.60 g glucose/100 mL) within the standards required by current Brazilian law. This pulp also showed significant levels of the bioactive compounds: anthocyanins (24.82  0.46 mg/100 mL), yellow flavonoids (11.33  0.66 mg/100 mL) and total extractable polyphenols (26.85  0.30 mg GAE/100 mL), making this product a good source of natural antioxidants. With regard to the main compound from volatile and non-volatile fractions of purple pitanga pulp, oxidoselina-1,3,7(11)-trien-8-one (85  4.01 Âg/mL) was observed in the volatile fraction and cyanidin-3-glucoside (340  4.19 Âg/mL )was observed in the non-volatile fraction. The low pH of the purple pitanga sweetened tropical juice decreases catalase activity, while oxidoselina-1,3,7(11)-trien-8-one and cyanidin-3-glucoside did not interfere and were not able to inhibit the activity of this enzyme. Purple pitanga sweetened tropical juice prevented DNA damage in human gingival cells. Due to the low number of volunteers in the experiment with purple pitanga sweetened tropical juice and the main compounds from volatile and non-volatile fractions of purple pitanga pulp, the results regarding the IL-8 release are inconclusive. Cyanidin-3-glucoside and oxidoselina-1,3,7(11)-trien-8-one presented anti-inflammatory effects in HGF-1 cells.

O transporte de &#945;-metil glicosídio ( &#945;-MG) em Saccharomyces cerevisiae foi recentemente reportado como transporte ativo, do tipo simporte de &$945;-MG com H+ mediado pela permease Agt1p. A cepa AP77-11B (cepa selecionada em nosso laboratório) 14C-&#945;-MG pelo mecanismo descrito como difusão facilitada porque não existe co-transporte de H+ durante o transporte de &#945;-MG. Os genes HXT1-HXT17 pertencem à família dos transportadores de hexoses em Saccharomyces cerevisiae. Então, nós decidimos investigar a possibilidade que o transporte de &#945;-MG poderia ser mediado pelos transportadores de hexoses. Nós demonstramos que cepa MC966A (tipo selvagem), KY73 (isogênica com MC966A mas deletada nos HXT1-7), BSY08 (isogênica com KY73 com o AGT1 deletado), BSY09 (isogênica com MC966A com o AGT1 deletado) e a EBY.VW4000 (hxt1-17 agt1 gal2-null), não cresceram em &#945;-MG como fonte de carbono. Além disso, estas cepas não transportaram &#945;-MG por difusão facilitada quando as células foram cultivadas em meio com maltose, levando-nos a concluir que os transportadores de hexoses não estavam envolvidos no transporte de &#945;-MG. Nós observamos que a cepa AP77-11B apresentou alta atividade de &#945;-metilglicosidase periplásmica quando as células foram cultivadas em &#945;-MG. Esta atividade enzimática foi ensaiada usando um método descrito primeiramente para invertase periplásmica, no qual as células eram incubadas com fluoreto de sódio, um inibidor da enolase, antes da incubação com &#945;-MG. Então, a glicose produzida durante a hidrólise do -MG poderia ser determinada. A atividade extracelular só está presente em células cultivadas em -&#945;MG. Células de-reprimidas não mostraram atividade de alpha-metilglicosidase. Os parâmetros cinéticos determinados para &#945;-metilglicosidase, indicaram que esta enzima tem baixa afinidade para o alpha-MG. Além do mais, a atividade específica da alpha-metilglicosidase periplásmica aumentou ao longo da curva de crescimento em &#945;-MG. Os resultados reportados mostraram que existem duas vias de utilização de &#945;-MG em Saccharomyces cerevisiae. Uma via é mediada pela Agt1p, responsável pelo transporte ativo de &#945;-MG. Na outra via, a &#945; -metilglicosidase é secretada para o espaço periplásmico das células. Então, a glicose produzida pela hidrólise do &#945;-MG é transportada pelos transportadores de hexoses por difusão facilitada. / Alpha-Methyl glucoside ( alpha-MG) transport in Saccharomyces cerevisiae was previously reported to be an active transport, a H+ -symport mediated by the Agt1p permease. Strain AP77-11B (a strain obtained in our laboratory) takes up 14C- alpha-MG by a mechanism which was ascribed to be facilitated diffusion since there is no H+-cotransport during the alpha-MG uptake. The HXT1-HXT17 there is no H genes belong to a family of hexose transporters in Saccharomyces cerevisiae. Therefore, we decided to investigate the possibility that -MG transport could be mediated by hexose transporters. We demonstrated that strains MC966A (w.t.), KY73 (isogenic to MC966A but hxt1-hxt7-null), BSY08 (isogenic to KY73 with AGT1 deleted), BSY09 (isogenic to MC966A with AGT1 deleted) and even strain EBY.VW4000 (hxt1-hxt17 agt1 gal2-null), were not able to grow on alpha-MG as the sole carbon source. Moreover, none of them presented alpha-MG transport by facilitated diffusion when the strains were grown on maltose leading us to conclude that the HXT glucose transporters were not involved in alpha-MG transport. We found that strain AP77-11B displayed a high periplasmic alpha-methylglucosidase activity when cells were grown on alpha-MG. This enzymatic activity was assayed using a method first described for periplasmic invertase in which cells were incubated with sodium fluoride, an inhibitor of enolase, prior to the incubation with alpha-MG. Then the glucose produced during alpha-MG hydrolysis could be accurately measured. The extracellular activity was present only in cells grown on alpha-MG. Glucose derepressed cells did not show periplasmic alpha-methylglucosidase activity.

We have studied the inhibitory effect of five polyphenols namely, resveratrol, piceatannol, quercetin, quercetrin, and quercetin-3-β-d glucoside on Escherichia coli ATP synthase. Recently published X-ray crystal structures of bovine mitochondrial ATP synthase inhibited by resveratrol, piceatannol, and quercetin, suggest that these compounds bind in a hydrophobic pocket between the γ-subunit C-terminal tip and the hydrophobic inside of the surrounding annulus in a region critical for rotation of the γ-subunit. Herein, we show that resveratrol, piceatannol, quercetin, quercetrin, or quercetin-3-β-d glucoside all inhibit E. coli ATP synthase but to different degrees. Whereas piceatannol inhibited ATPase essentially completely (∼0 residual activity), inhibition by other compounds was partial with ∼20% residual activity by quercetin, ∼50% residual activity by quercetin-3-β-d glucoside, and ∼60% residual activity by quercetrin or resveratrol. Piceatannol was the most potent inhibitor (IC50 ∼14 μM) followed by quercetin (IC50 ∼33 μM), quercetin-3-β-d glucoside (IC50 ∼71 μM), resveratrol (IC50 ∼94 μM), quercitrin (IC50 ∼120 μM). Inhibition was identical in both F1Fo membrane preparations as well as in isolated purified F1. In all cases inhibition was reversible. Interestingly, resveratrol and piceatannol inhibited both ATPase and ATP synthesis whereas quercetin, quercetrin or quercetin-3-β-d glucoside inhibited only ATPase activity and not ATP synthesis.

We have studied the inhibitory effect of natural and structurally modified polyphenols on Escherichia coli ATP synthase to test (I) if the beneficial dietary effects of polyphenols are related to their inhibitory actions on ATP synthase, (II) if inhibitory effects of polyphenolic compound could be augmented through structural modifications, and (III) if they can act as antimicrobial agent through their actions on ATP synthesis. X-ray crystal structures of polyphenol binding sites suggested that polyphenols bind at a distinct polyphenol binding pocket, at the interface of α,β,γ-subunits. We found that both natural and modified polyphenols inhibit E. coli ATP synthase to varying degrees and structural modifications resulted in augmented inhibition. Inhibition was reversible in all cases. Both natural and modulated compounds inhibited E. coli cell growth to varying degrees. We conclude that dietary benefits of polyphenols may be in part due to the inhibition of ATP synthase.

No

Les mycotoxines sont des métabolites secondaires de moisissures contaminant de façon naturelle de nombreuses denrées alimentaires, notamment les céréales. Le déoxynivalénol (DON), produit par Fusarium sp., est la mycotoxine la plus répandue dans le monde. Du fait de sa grande stabilité chimique, le DON est difficile à éliminer, et se retrouve dans les céréales et les produits finis ou il induit des effets toxiques pour l'homme et l'animal. De nouvelles stratégies de lutte sont mises en places, telle la transformation biologique utilisant des bactéries ou des plantes. En effet certaines bactéries possèdent des enzymes capables de transformer le DON en de nouveaux composés, le déepoxy-déoxynivalénol (DOM-1) et le 3-épi-déoxynivalénol (3-epi-DON). De plus, certaines plantes sont naturellement capables de transformer le DON dans le but de l'éliminer et de le détoxifier, formant ainsi le deoxynivalénol-3-ß-D-glucoside (D3G). L'objectif de cette thèse était d'évaluer la toxicité de ces dérivés du DON au niveau de l'intestin et du système immunitaire par le biais d'analyses in silico, in vitro, ex vivo et in vivo. Les tests de toxicité in vitro sur la lignée humaine intestinale cellulaire Caco-2 montrent que le DOM-1, le 3-epi-DON et le D3G n'étaient pas cytotoxiques, ils ne modifiaient ni la viabilité, ni la fonction de barrière des cellules, mesurée par la résistance électrique transépithéliale. Les tests de toxicité ex vivo sur des explants jéjunum porcin ont montré que le DOM-1, le 3-epi-DON ou le D3G n'induisaient pas de modifications histomorphologiques. En revanche, les explants exposés au DON montraient des lésions morphologiques et une régulation positive de l'expression des cytokines pro-inflammatoires. L'impact de ces trois dérivés a été également analysé sur l'expression de l'ensemble des gènes du tissu, avec une analyse microarray. Ceci a montré que ces dérivés du DON n'induisaient aucun changement dans l'expression des gènes par rapport au groupe contrôle. Le DON quand a lui exprimait différentiellement 747 sondes, correspondantes à 333 gènes impliqués dans l'immunité, la réponse inflammatoire, le stress oxydatif, la mort cellulaire, le transport moléculaire et la fonction mitochondriale. L'analyse in silico a montré que le D3G, contrairement au DON était incapable de se lier au site-A du ribosome, principale cible de la toxicité pour le DON. Les deux dérivés microbiens eux, étaient capables de se fixer au site-A au sein du ribosome, mais contrairement au DON ils ne formaient que deux liaisons hydrogènes au lieu de trois. De plus, ces trois dérivés n'induisaient pas de stress ribotoxique, d'activation des MAPKs (mitogen-activated protein kinases), et de réponse pro-inflammatoire. Une étude complémentaire a été menée in vivo pour évaluer la toxicité du DOM-1 chez le porc (gavage pendant 21 jours avec .0.14mg / kg de poids vif). Les résultats ont montré que le DOM-1, contrairement au DON n'induisait pas les effets toxiques du DON au niveau des paramètres zootechniques (pas de vomissements, aucune diminution de la consommation alimentaire ou de perte de poids), sur l'intestin et le foie (pas de dommages tissulaires), ou sur la réponse immunitaire (pas de réponse inflammatoire induite). En conclusion, nos résultats montrent l'efficacité de ces transformations enzymatiques. La déepoxydation et l'épimérisation bactérienne, ainsi que la glycosylation par les plantes permettent de sensiblement diminuer la toxicité du DON, passant par une absence de toxicité sur le ribosome avec une absence d'activation des MAPKs et de réponses inflammatoires. Dans ce contexte de contamination par les mycotoxines, ces méthodes de luttes alternatives semblent être des approches prometteuses. / The Fusarium sp. mycotoxin deoxynivalenol (DON) is one of the most frequently widespread mycotoxin worldwide. Due to its high structural stability, the elimination of DON, once present in cereals or feed materials, becomes difficult. Thereby, it is present in many cereals and final feed products, inducing several toxic effects on human and animals, and causing big economic losses. New strategies of to fight against mycotoxins were developed, as biological transformation, either by the use of bacteria or plants. Indeed, some microorganisms are able to transform DON in new products, by enzymatic reaction, forming the deepoxy-deoxynivalenol (DOM-1) and the 3-epi-deoxynivalenol (3-epi-DON). Moreover, some plants naturally own the capacity to glycosylate DON in the aim to detoxify it, forming the deoxynivalenol-3-ß-D-glucoside (D3G). The aim of this thesis was to assess the toxicity of these DON derivatives, on the intestine and immune response, using several approaches such as in silico, in vitro, ex vivo and in vivo models. On the human intestinal Caco-2 cell line, DOM-1, 3-epi-DON and D3G were not cytotoxic; they did not alter its viability and barrier function, as measured by the trans epithelial electrical resistance. The expression profile of DOM-1, 3-epi-DON and D3G-treated jejunal explants was similar to that of controls and these explants did not show any histomorphology alteration. On the other hand, the treatment of intestinal explants with DON, induced morphological lesions and upregulated the expression of proinflammatory cytokines. The impact of these three derivatives was also studied on intestinal explants with a pan-genomic transcriptomic analysis. Results show that the derivatives of DON did not induce any change on the gene expression in comparison to the control-treated explants. In contrary, DON-treated explants differentially expressed 747 probes, representing 323 genes involved in immune and inflammatory responses, oxidative stress, cell death, molecular transport and mitochondrial function. In silico analysis revealed that D3G, opposing to DON, was unable to bind to the A site of the ribosome, which is the main target for DON toxicity. Both DOM-1 and 3-epi-DON were able to fit into the pockets of the A site of the ribosome but only by forming two hydrogen bonds, while in this position, DON forms three hydrogen bonds. Moreover, the three derivatives do not elicit a ribotoxic stress, MAPKinase activation, and inflammatory response. Then, an in vivo study was carried out to assess the toxicity of DOM-1 on pig (feed forced during 21 days at 0.14 mg/Kg BW). The results showed that DOM-1 does not have as much toxic effects as DON on zootechnical parameters (no emesis induced, no decrease of food consumption or weight loss observed), on intestine and liver (no tissues damages), or on the immune response (no inflammatory response induced). Our data demonstrate that bacterial de-epoxidation or epimerization of deepoxy-DON modified its interaction with the ribosome, leading to an absence of MAPKinase activation and toxicity; and that the glycosylation of DON suppresses its ability to bind to the ribosome and decreases its intestinal toxicity. The mycotoxin deoxynivalenol (DON) remains an important challenge in many regions in the world. Thus, these biological detoxifications of DON seem to represent a new promising approach helping manage the problem of its contamination.

<p>Adsorption on solid surfaces from solution is a fundamental property of a surfactant. It might even be the most important aspect of surfactant behavior, since it influences many applications, such as cleaning, detergency, dispersion, separation, flotation, and lubrication. Consequently, fundamental investigations of surfactant adsorption are relevant to many areas.</p><p>The main aim of this thesis has been to elucidate the adsorption properties, primarily on the solid/water interface, of a particular class of polyhydroxyl based surfactants: the alkyl glucosides. By the use of ellipsometry, the equilibrium and kinetic aspects of adsorption on titanium dioxide with respect to structural effects has been studied. Furthermore, the effects of small amounts of cationic surfactant additives on the adsorption on silica have been investigated. The results have been compared with similar studies for other nonionic surfactants.</p><p>We have found that the surfactant structure has a strong effect on the adsorption properties. An increase in the surfactant chain length increases the cooperativity of the system. An increase in the head group polymerization decreases the cooperativity and the plateau adsorbed amount at equilibrium. The effect of surfactant structure on the adsorption kinetics depends on the concentration relative to the cmc, while the there is a decrease in the rate of desorption with increasing hydrophobic chain length independent of the concentration. The adsorption/desorption process is concluded to be diffusion driven, as suggested by the model used. When comparing these results with studies on ethylene oxide based surfactants, we conclude that the two types of surfactants exhibit similar trends on surfaces onto which they adsorb.</p><p>Adsorption from binary surfactant solutions is even more interesting than adsorption from single surfactant solutions, since it brings us one step closer to the systems used in applications. In addition, adsorption from a mixture can be very different from adsorption from any of the single surfactants in the mixture. Alkyl glucosides alone do not adsorb on silica, but addition of small amounts of a cationic surfactant to the alkyl glucoside solution allows for adsorption on silica. A comparison between the adsorption and bulk properties has shown that mixed micellization explains most, but not all, effects of the coadsorption properties. Changing the pH in the mixed systems reveals that a surfactant with a pH-dependent charge and the ability to adapt its charge to the environment, e.g. a surface, enhances the adsorbed amount over a wider range of pH values than a purely cationic surfactant.</p><p>It is well known that alkyl glucosides and ethylene oxides adsorb differently on different types of hydrophilic surfaces. As a consequence, replacing ethylene oxides with alkyl glucosides might not be all straight-forward; however, we have shown that the effect of the surface can be eliminated by the use of a cosurfactant.</p>