Alginate biosynthesis is not only a major contributor to pathogenicity of P. aeruginosa but also an important factor in colonization of adverse environmental habitats by biofilm formation. The requirement of proteins Alg8 and Alg44, encoded by their respective genes in the alginate biosynthesis gene cluster, for alginate biosynthesis of P. aeruginosa was demonstrated, since deletion mutants were unable to produce or polymerise alginate. AlgX deletion mutants failed to produce the alginate characteristic mucoid phenotype, but showed low concentrations of uronic acid monomers in the culture supernatants. Complementation experiments using PCR based approaches were used to determine the complementing ORF and all deletion mutants could be complemented to at least wildtype levels by introducing a plasmid harbouring the respective gene. Increased copy numbers of Alg44 did not impact on the amount of alginate produced, whereas increased copy numbers of the alg8 gene led to an at least 10 fold stronger alginate production impacting on biofilm structure and stability. Topological analysis using reporter protein fusions and subsequent subcellular fractionation experiments revealed that Alg8 is located in the cytoplasmic membrane and contains at least 4 transmembrane helices, 3 of them at its C terminus. Its large cytosolic loop showed similarities to inverting glycosyltransferases and the similarities were used to generate a threading model using SpsA, a glycosyltransferase involved in spore coat formation of B. subtilis, as a template. Site-directed mutagenesis confirmed the importance of identified motifs commonly detected in glycosyltransferases. Inactivation of the DXD motif, which has been shown to be involved in nucleotide sugar binding, led to loss-offunction mutants of Alg8 and further replacements revealed putative candidates for the catalytic residue(s). Contradicting the commonly reported prediction of being a transmembrane protein, Alg44 was shown to be a periplasmic protein. The highest specific alkaline phosphatase activity of its fusion protein could be detected in the periplasmic fraction and not in the insoluble membrane fraction. Bioinformatical analysis of Alg44 revealed structural similarities of its N terminus to PilZ domains, shown to bind cyclic-di-GMP, and of its C terminus to MexA, a membrane fusion protein involved in multi-drug efflux systems. Thus, it was suggested that Alg44 has a regulatory role for alginate biosynthesis in bridging the periplasm and connecting outer and cytoplasmic membrane components. AlgX was shown to interact with MucD, a periplasmic serine protease or chaperone homologue, and is suggested to exert its impact on alginate production via MucD interaction. In vitro alginate polymerisation assays revealed that alginate production requires protein components of the outer and cytoplasmic membrane as well as the periplasm, and these data were used to construct a model describing a multi-enzyme, membrane and periplasm spanning complex for alginate polymerisation, modification and export.

Alginate biosynthesis is not only a major contributor to pathogenicity of P. aeruginosa but also an important factor in colonization of adverse environmental habitats by biofilm formation. The requirement of proteins Alg8 and Alg44, encoded by their respective genes in the alginate biosynthesis gene cluster, for alginate biosynthesis of P. aeruginosa was demonstrated, since deletion mutants were unable to produce or polymerise alginate. AlgX deletion mutants failed to produce the alginate characteristic mucoid phenotype, but showed low concentrations of uronic acid monomers in the culture supernatants. Complementation experiments using PCR based approaches were used to determine the complementing ORF and all deletion mutants could be complemented to at least wildtype levels by introducing a plasmid harbouring the respective gene. Increased copy numbers of Alg44 did not impact on the amount of alginate produced, whereas increased copy numbers of the alg8 gene led to an at least 10 fold stronger alginate production impacting on biofilm structure and stability. Topological analysis using reporter protein fusions and subsequent subcellular fractionation experiments revealed that Alg8 is located in the cytoplasmic membrane and contains at least 4 transmembrane helices, 3 of them at its C terminus. Its large cytosolic loop showed similarities to inverting glycosyltransferases and the similarities were used to generate a threading model using SpsA, a glycosyltransferase involved in spore coat formation of B. subtilis, as a template. Site-directed mutagenesis confirmed the importance of identified motifs commonly detected in glycosyltransferases. Inactivation of the DXD motif, which has been shown to be involved in nucleotide sugar binding, led to loss-offunction mutants of Alg8 and further replacements revealed putative candidates for the catalytic residue(s). Contradicting the commonly reported prediction of being a transmembrane protein, Alg44 was shown to be a periplasmic protein. The highest specific alkaline phosphatase activity of its fusion protein could be detected in the periplasmic fraction and not in the insoluble membrane fraction. Bioinformatical analysis of Alg44 revealed structural similarities of its N terminus to PilZ domains, shown to bind cyclic-di-GMP, and of its C terminus to MexA, a membrane fusion protein involved in multi-drug efflux systems. Thus, it was suggested that Alg44 has a regulatory role for alginate biosynthesis in bridging the periplasm and connecting outer and cytoplasmic membrane components. AlgX was shown to interact with MucD, a periplasmic serine protease or chaperone homologue, and is suggested to exert its impact on alginate production via MucD interaction. In vitro alginate polymerisation assays revealed that alginate production requires protein components of the outer and cytoplasmic membrane as well as the periplasm, and these data were used to construct a model describing a multi-enzyme, membrane and periplasm spanning complex for alginate polymerisation, modification and export.

Alginate biosynthesis is not only a major contributor to pathogenicity of P. aeruginosa but also an important factor in colonization of adverse environmental habitats by biofilm formation. The requirement of proteins Alg8 and Alg44, encoded by their respective genes in the alginate biosynthesis gene cluster, for alginate biosynthesis of P. aeruginosa was demonstrated, since deletion mutants were unable to produce or polymerise alginate. AlgX deletion mutants failed to produce the alginate characteristic mucoid phenotype, but showed low concentrations of uronic acid monomers in the culture supernatants. Complementation experiments using PCR based approaches were used to determine the complementing ORF and all deletion mutants could be complemented to at least wildtype levels by introducing a plasmid harbouring the respective gene. Increased copy numbers of Alg44 did not impact on the amount of alginate produced, whereas increased copy numbers of the alg8 gene led to an at least 10 fold stronger alginate production impacting on biofilm structure and stability. Topological analysis using reporter protein fusions and subsequent subcellular fractionation experiments revealed that Alg8 is located in the cytoplasmic membrane and contains at least 4 transmembrane helices, 3 of them at its C terminus. Its large cytosolic loop showed similarities to inverting glycosyltransferases and the similarities were used to generate a threading model using SpsA, a glycosyltransferase involved in spore coat formation of B. subtilis, as a template. Site-directed mutagenesis confirmed the importance of identified motifs commonly detected in glycosyltransferases. Inactivation of the DXD motif, which has been shown to be involved in nucleotide sugar binding, led to loss-offunction mutants of Alg8 and further replacements revealed putative candidates for the catalytic residue(s). Contradicting the commonly reported prediction of being a transmembrane protein, Alg44 was shown to be a periplasmic protein. The highest specific alkaline phosphatase activity of its fusion protein could be detected in the periplasmic fraction and not in the insoluble membrane fraction. Bioinformatical analysis of Alg44 revealed structural similarities of its N terminus to PilZ domains, shown to bind cyclic-di-GMP, and of its C terminus to MexA, a membrane fusion protein involved in multi-drug efflux systems. Thus, it was suggested that Alg44 has a regulatory role for alginate biosynthesis in bridging the periplasm and connecting outer and cytoplasmic membrane components. AlgX was shown to interact with MucD, a periplasmic serine protease or chaperone homologue, and is suggested to exert its impact on alginate production via MucD interaction. In vitro alginate polymerisation assays revealed that alginate production requires protein components of the outer and cytoplasmic membrane as well as the periplasm, and these data were used to construct a model describing a multi-enzyme, membrane and periplasm spanning complex for alginate polymerisation, modification and export.

Alginate biosynthesis is not only a major contributor to pathogenicity of P. aeruginosa but also an important factor in colonization of adverse environmental habitats by biofilm formation. The requirement of proteins Alg8 and Alg44, encoded by their respective genes in the alginate biosynthesis gene cluster, for alginate biosynthesis of P. aeruginosa was demonstrated, since deletion mutants were unable to produce or polymerise alginate. AlgX deletion mutants failed to produce the alginate characteristic mucoid phenotype, but showed low concentrations of uronic acid monomers in the culture supernatants. Complementation experiments using PCR based approaches were used to determine the complementing ORF and all deletion mutants could be complemented to at least wildtype levels by introducing a plasmid harbouring the respective gene. Increased copy numbers of Alg44 did not impact on the amount of alginate produced, whereas increased copy numbers of the alg8 gene led to an at least 10 fold stronger alginate production impacting on biofilm structure and stability. Topological analysis using reporter protein fusions and subsequent subcellular fractionation experiments revealed that Alg8 is located in the cytoplasmic membrane and contains at least 4 transmembrane helices, 3 of them at its C terminus. Its large cytosolic loop showed similarities to inverting glycosyltransferases and the similarities were used to generate a threading model using SpsA, a glycosyltransferase involved in spore coat formation of B. subtilis, as a template. Site-directed mutagenesis confirmed the importance of identified motifs commonly detected in glycosyltransferases. Inactivation of the DXD motif, which has been shown to be involved in nucleotide sugar binding, led to loss-offunction mutants of Alg8 and further replacements revealed putative candidates for the catalytic residue(s). Contradicting the commonly reported prediction of being a transmembrane protein, Alg44 was shown to be a periplasmic protein. The highest specific alkaline phosphatase activity of its fusion protein could be detected in the periplasmic fraction and not in the insoluble membrane fraction. Bioinformatical analysis of Alg44 revealed structural similarities of its N terminus to PilZ domains, shown to bind cyclic-di-GMP, and of its C terminus to MexA, a membrane fusion protein involved in multi-drug efflux systems. Thus, it was suggested that Alg44 has a regulatory role for alginate biosynthesis in bridging the periplasm and connecting outer and cytoplasmic membrane components. AlgX was shown to interact with MucD, a periplasmic serine protease or chaperone homologue, and is suggested to exert its impact on alginate production via MucD interaction. In vitro alginate polymerisation assays revealed that alginate production requires protein components of the outer and cytoplasmic membrane as well as the periplasm, and these data were used to construct a model describing a multi-enzyme, membrane and periplasm spanning complex for alginate polymerisation, modification and export.

Thesis (M.S.)--University of Alabama at Birmingham, 2008. / Title from PDF title page (viewed on Sept. 17, 2009). Includes bibliographical references (p. 46-49).

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Orientador: Theo Guenter Kieckbusch / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-16T09:52:42Z (GMT). No. of bitstreams: 1 Bierhalz_AndreaCristianeKrause_M.pdf: 2842843 bytes, checksum: cab45fbde17a0c38ebace1685ac8ac68 (MD5) Previous issue date: 2010 / Resumo: Pectinas e alginatos são polímeros naturais com grande potencial de aplicação no desenvolvimento de filmes biodegradáveis, por serem materiais renováveis e obtidos a partir de diversas fontes a baixo custo. Atualmente, diversos estudos estão voltados ao desenvolvimento de tecnologia que controle o crescimento indesejável de microorganismos, pelo uso de embalagens ativas. Na presente pesquisa, foram desenvolvidos filmes simples de pectina e filmes compostos de pectina e alginato em diferentes proporções, reticulados com cálcio e contendo glicerol como plastificante. Foi avaliada a influência das concentrações de cálcio e do plastificante e do tempo de reticulação sobre as propriedades características dos filmes. A adição de natamicina como agente antimicrobiano também foi investigada. Os filmes foram preparados por casting, isto é, alíquotas da solução filme-formadora foram vertidas em moldes de acrílico e submetidas à gelificação e secagem. Devido ao alto poder gelificante do Ca++, filmes com baixo grau de reticulação foram inicialmente confeccionados (1º estágio). Estes préfilmes foram submetidos a uma reticulação complementar com imersão em uma solução mais concentrada de íons cálcio contendo plastificante. O alginato e a pectina mostraram-se compatíveis em todas as proporções, resultando em filmes com bom aspecto visual. O aumento da concentração de alginato diminuiu a solubilidade em água e o grau de intumescimento e aumentou a espessura, a resistência à tensão, a flexibilidade, a opacidade e a temperatura de transição vítrea dos filmes. Filmes pré-reticulados com 1% CaCl2.2H2O (13,60 mg Ca++/g biopolímero) na solução do 1º estágio, imersos durante 20 minutos em uma solução contendo 5% CaCl2.2H2O e 3% glicerol (2º estágio) apresentaram um compromisso entre alta resistência mecânica, boa aparência e baixa solubilidade em água. Esta formulação foi selecionada para a confecção dos filmes ativos contendo natamicina. A adição da natamicina provocou um aumento na permeabilidade ao vapor de água, na solubilidade em água e na opacidade, e uma redução na tensão na ruptura em relação aos filmes sem antimicrobiano. Determinações experimentais da massa de natamicina liberada em ensaios nos quais os filmes eram imersos em água apresentaram bom ajuste ao modelo difusional da segunda Lei de Fick, com valores de difusividade efetiva variando entre 9,53.10-9 e 9,22.10-12 cm2/s. A difusividade aumentou com a espessura e diminuiu com o aumento da concentração de alginato no filme / Abstract: Pectin and alginate are natural polymers with potential applications in the development of biodegradable package films, since they can be obtained at low cost from a large variety of renewable sources. There is a growing interest concerning the control of microbial activity in prepared food using active packaging. In this work, single and composite films based on alginate and pectin crosslinked with calcium ions and containing glycerol as plasticizer were prepared. The influence of calcium and glycerol concentrations and of the time of reticulation on the film characteristics was evaluated and the addition of natamycin as an active agent was investigated. The strong gelling power of Ca++ ions hinders smooth casting procedures so that a film with low degree of reticulation has to be initially confectioned by casting (1st stage). These pre-films are further crosslinked in a second contact with a more concentrated Ca++ solution containing plasticizer (2nd stage). Alginate and pectin were compatible in all proportions studied resulting in visually attractive films. Increasing the alginate concentration decreased film solubility in water and the swelling degree and increased the thickness, resistance to tensile stress, flexibility, water vapor permeability, opacity and glass transition temperature. Films pre-reticulated with 1% CaCl2.2H2O (13.60 mg Ca++/g biopolymer) in the solution of the 1st stage, immersed for 20 minutes in a 5% CaCl2.2H2O and 3% glycerol solution (2nd stage) showed a good compromise between high mechanical resistance, attractive appearance and low solubility in water. This formulation was selected for the manufacture of active films containing natamycin. Addition of natamycin promoted an increase in water vapor permeability, solubility in water and opacity and decreased the tensile strength when compared to films without the added anti-microbial agent. Experimental data of mass of natamycin released by immersion of the film in water were well fitted to Fick's second law diffusional model, with effective diffusivity values ranging from 9.53. 10-9 e 9.22.10-12 cm2/s. The diffusivity increased with the thickness and decreased with alginate concentrations in the film / Mestrado / Engenharia de Processos / Mestre em Engenharia Química

Orientadores: Rubens Maciel Filho, Paulo Jorge da Silva Bártolo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-17T08:33:25Z (GMT). No. of bitstreams: 1 Rezende_RodrigoAlvarenga_D.pdf: 29746268 bytes, checksum: 24e72154795e591bf70b4fc339cf0127 (MD5) Previous issue date: 2010 / Resumo: A busca por soluções que resgatem a saúde e a dignidade de vítimas de acidentes ou com problemas de saúde, como perda ou danos de órgãos, tem motivado uma evolução acelerada da biofabricação. Novos materiais e técnicas de fabricação vêm sendo desenvolvidos rapidamente com o apoio dos recursos computacionais, que se torna parte imprescindível deste processo. O objeto de estudo desta tese vem ao encontro do desenvolvimento de ferramentas computacionais e procedimentos para a biofabricação, o que requer a utilização de um material biocompatível. O material em questão é o alginato, um hidrogel proveniente de algas pardas marinhas e que tem sido amplamente utilizado em diversos setores por mais de cinquenta anos. Uma caracterização mecânica do alginato é realizada, com auxílio de um reômetro de pratos e de um analisador dinâmico mecânico (DMA), bem como uma caracterização do comportamento químico, tal como o seu poder de inchamento (swelling) e o seu processo de reticulação (gelação), a fim de servirem como dados de alimentação para as aplicações das ferramentas computacionais, nomeadamente, o Ansys e a técnica de otimização dos algoritmos genéticos. O Ansys é utilizado na simulação do comportamento de escoamento do alginato puro. Já os algoritmos genéticos, como ferramentas de otimização de propriedades físicas de estruturas em alginato. / Resumo: A busca por soluções que resgatem a saúde e a dignidade de vítimas de acidentes ou com problemas de saúde, como perda ou danos de órgãos, tem motivado uma evolução acelerada da biofabricação. Novos materiais e técnicas de fabricação vêm sendo desenvolvidos rapidamente com o apoio dos recursos computacionais, que se torna parte imprescindível deste processo. O objeto de estudo desta tese vem ao encontro do desenvolvimento de ferramentas computacionais e procedimentos para a biofabricação, o que requer a utilização de um material biocompatível. O material em questão é o alginato, um hidrogel proveniente de algas pardas marinhas e que tem sido amplamente utilizado em diversos setores por mais de cinquenta anos. Uma caracterização mecânica do alginato é realizada, com auxílio de um reômetro de pratos e de um analisador dinâmico mecânico (DMA), bem como uma caracterização do comportamento químico, tal como o seu poder de inchamento (swelling) e o seu processo de reticulação (gelação), a fim de servirem como dados de alimentação para as aplicações das ferramentas computacionais, nomeadamente, o Ansys e a técnica de otimização dos algoritmos genéticos. O Ansys é utilizado na simulação do comportamento de escoamento do alginato puro. Já os algoritmos genéticos, como ferramentas de otimização de propriedades físicas de estruturas em alginato. / Doutorado / Desenvolvimento de Processos Químicos / Doutor em Engenharia Química

Orientador: Ângela Maria Moraes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-18T04:16:35Z (GMT). No. of bitstreams: 1 Girata_AnaKelly_M.pdf: 3155026 bytes, checksum: c60ce299846f01bde6490d2ea47e303f (MD5) Previous issue date: 2011 / Resumo: A quitosana é um polissacarídeo obtido pela desacetilação da quitina e o alginato, um biopolímero extraído de algas. Ambos são atóxicos, biocompatíveis e facilitam a cicatrização de feridas. Por estas razões, podem ser empregados para a produção de curativos para o tratamento de lesões de pele. Além disso, muitos curativos têm incorporado compostos contendo prata para a prevenção e o controle de infecções bacterianas. Diante disso, este trabalho teve por objetivo o desenvolvimento de metodologia de produção de membranas constituídas por quitosana e alginato contendo um agente antimicrobiano à base de prata estabilizada (AlphaSan® RC2000) para aplicação na terapia de lesões de pele. As membranas produzidas foram caracterizadas quanto à morfologia de superfície, à espessura, à absorção de fluidos, à perda de massa quando em contato com diferentes fluidos, à capacidade de drenagem de água, à resistência mecânica, à citotoxicidade a fibroblastos, ao desempenho quanto à atividade antimicrobiana, à sensibilidade e irritabilidade dérmica. Os resultados mostraram que o aumento da quantidade de fármaco causa o aumento da opacidade e da espessura das membranas. A eficiência de incorporação do AlphaSan® RC2000 foi de até 99%. As membranas contendo o agente antimicrobiano foram capazes de absorver de 15 g a 42,3 g de fluido por grama de membrana. A maior perda de massa das membranas ocorreu em água e correspondeu a 27,5% em um intervalo de 168 horas. Para a capacidade de drenagem foram obtidos valores na faixa de 4.458 a 7.198g/m2dia. Os valores de resistência à tração variaram de 2 a 9 MPa. As membranas apresentaram eficácia contra Pseudomonas aeruginosa e Staphylococus aureus, citotoxicidade in vitro moderada a fibroblastos e foram classificadas como não irritantes e não sensibilizantes. As membranas foram capazes de liberar até 100% de prata presente em sua estrutura em 168h. O modelo que melhor se ajustou ao perfil de liberação foi o que combinou o modelo de cinética de primeira ordem e o de Lei das Potências. Diante desses resultados sugere-se que as membranas de quitosana-alginato obtidas apresentam bom potencial de uso como curativos na terapia de lesões de pele / Abstract: Chitosan is a polysaccharide obtained by deacetylation of chitin and alginate is a polymer extracted from algae. Both are nontoxic, biocompatible and facilitate wound healing. Because of these characteristics, these compounds can be employed for the production of dressings to treat skin lesions. Moreover, many dressings incorporate in their composition compounds containing silver, to prevent and control bacterial infections. Thus, this study aimed at developing a methodology for the production of membranes composed of the polysaccharides chitosan and alginate containing an antimicrobial agent based on stabilized silver (AlphaSan® RC2000) for application in the therapy of skin lesions. The produced membranes were characterized for surface morphology, thickness, absorption of fluids, mass loss in different fluids, water drainage capacity, mechanical resistance, cytotoxicity to fibroblasts and performance regarding antimicrobial activity. The attained results showed that increasing the amount of drug, the membranes became more opaque and thicker. The efficiency of incorporation of AlphaSan® RC2000 was up to 99%. The membranes containing the antimicrobial agent were able to absorb from 15 g to 42.3 g of fluid per gram of membrane. The highest membrane mass loss occurred in water and was correspondent to 27.5% in 168 hours. The water drainage capacity decreased as the concentration of the antimicrobial compound was increased, and values in the range of 4,458 to 7,198g/m2day were obtained. The values of tensile strength ranged from 2 to 9 MPa. The membranes showed efficacy against Pseudomonas aeruginosa and Staphylococcus aureus, moderate in vitro cytotoxicity to fibroblasts and were classified as non-irritating and non-sensitizing. The membranes containing the drug were able to release up to 100% of the silver present in its structure in 168h. The model that best fitted the drug release profile consisted of a combination of a first-order kinetic model and of a Power Law model. These results suggest that the alginate-chitosan membranes obtained have good potential to be used as a curative in the therapy of skin lesions / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

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