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Cellules endothéliales issues de progéniteurs humains : des acteurs pertinents en ingénierie vasculaire ?Thébaud-Aubry, Noélie-Brunehilde 14 December 2009 (has links)
L’incidence des maladies cardiovasculaires d’origine athéromateuse demeure un problème majeur en santé publique et malgré le développement de techniques curatives endovasculaires, la chirurgie demeure nécessaire chez de nombreux patients. Le remplacement vasculaire se fait par une veine autologue qui reste le « gold standard » ou, lorsque les patients n’ont pas le capital vasculaire suffisant, par une prothèse. Actuellement, si les techniques utilisant des prothèses synthétiques sont satisfaisantes pour le remplacement d’artères de gros calibre, celui des artères de petit calibre demeure toujours un défi du fait du caractère thrombogène des biomatériaux utilisés et de leurs mauvaises propriétés mécaniques. Depuis quelques années, le concept d’ingénierie tissulaire a émergé et évolué. Il pourrait permettre de proposer de nouveaux types de substituts vasculaires hybrides et/ou biologiques, grâce en particulier à l’utilisation de cellules souches et de leurs progéniteurs, ouvrant d’intéressantes perspectives dans le domaine de l’ingénierie vasculaire. Le but de ce travail a été d’obtenir de manière fiable et reproductible des cellules à phénotype endothélial mature à partir de progéniteurs endothéliaux issus de moelle osseuse et sang périphérique humains et de définir leurs réponses dans des conditions proches de celles observées dans un vaisseau natif. Des cellules (PDECs : Progenitor Derived Endothelial Cells) ont pu être amplifiées à partir de progéniteurs, elles présentent les marqueurs membranaires classiquement utilisés pour définir des cellules endothéliales matures. Elles sont capables, sur différents revêtements utilisés cliniquement tels le collagène de type I et la colle de fibrine ainsi que sur un revêtement plus expérimental (Multicouches de PolyElectrolytes), de former une monocouche confluente. Ces PDECs résistent à des contraintes mécaniques de cisaillement de type artériel et l’analyse de gènes et protéines impliqués dans la biologie de l’endothélium a montré qu’elles répondent à ces stimulations par l’expression d’un phénotype en lien avec une activité antithrombogène. De plus, les travaux préliminaires réalisés sur ces PDECs cocultivés avec des progéniteurs ostéoblastiques, ouvrent d’intéressantes perspectives concernant leur utilisation dans le cadre de l’ingénierie du tissu osseux vascularisé. / The incidence of atherosclerotic arterial disease is still a major public health problem and despite endovascular surgery therapies, surgical treatment is necessary for many patients. Vascular bypass is performed with an autologous vein which remains the gold standard, or when patients do not have appropriate blood vessels to be used as replacement, with a synthetic prosthesis. Nowadays, synthetic vascular grafts have been successfully used in the treatment of the pathology of large arteries, but the replacement of the smaller sized arteries is still a challenge because synthetic vascular grafts are known to be highly thrombogenic and have poor mechanical properties. Recently, the tissue engineering concept has emerged and advances. It can allow to propose development of new hybrid or biologic vascular substitutes, using stem cells and progenitor cells, holding great promise for vascular tissue engineering. The aim of the present study was to obtain reliably and reproducibly, cells with mature endothelial phenotype from endothelial progenitor cells isolated from human bone marrow and peripheral blood and investigate cell response in conditions similar to those observed in a native vessel. We were able to expand cells (PDECs: Progenitor Derived Endothelial Cells) from progenitors which exhibit markers conventionally used to define mature endothelial cells. They were able, on scaffolds currently used in clinic like collagen type I and fibrin glue or on more experimental scaffold (Polyelectrolytes multilayers films), to form a confluent monolayer. These PDECs are able to withstand arterial shear stress and analysis of genes and proteins implicated in endothelium biology shows that these cells respond to shear stress stimulation with a phenotype connected to an anti-thrombogenic activity. Moreover, preliminary studies using co-cultures of PDECs and osteoblastic progenitors, open interesting perspectives concerning PDECs to be used in the field of vascularized bone tissue engineering.
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Caracteriza??o do efeito antinociceptivo e anti-inflamat?rio do polissacar?deo extra?do da levedura Kluyveromyces marxianusOliveira, Renata Freitas de Araujo 26 May 2015 (has links)
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Previous issue date: 2015-05-26 / The yeasts produce different types of metabolites and macromolecules with huge biotechnological potential. Among them, the Kluyveromyces marxianus, species attracts industrial and economic interests for presenting certain qualities, such as thermal tolerance, high growth rates, broad substrate spectrum and modulation of immune response. This work investigated the effects of treatment with the polysaccharide extracted from the Kluyveromyces marxianus yeast (poly322) in pain and inflammation experimental models. The poly322 pharmacological properties were evaluated with writhing, formalin, nociception (manifested by CFA), tail flick, hot-plate and paw edema tests. The cytokine levels were determined by ELISA and the effects on motor performance were evaluated by rotarod and open field tests. The pretreatment with poly322 resulted in reduced nociception induced by acetic acid, CFA and formalin (late phase). Furthermore, animals treated with poly322 exhibited a significant reduction in mechanical hypernociception, paw edema, and local IL-6 levels increase induced by carrageenan. In contrast, treatment with poly322 did not alter the thermal stimulus response threshold in the tail flick and hot plate tests, indicating no central action. Confirming the specificity of the action suggested by nociceptive tests, the treatment with poly322 did not induce motor impairment. These results demonstrate that the poly322 has a potent antinociceptive and anti-inflammatory effects, possibly mediated by the inhibition of the production and/or release of the IL-6 cytokine. / As leveduras produzem diferentes tipos de metab?litos e macromol?culas com enorme potencial biotecnol?gico. Dentre essas, destaca-se a Kluyveromyces marxianus, esp?cie que desperta interesse industrial e econ?mico por apresentar qualidades, como a toler?ncia t?rmica, altas taxas de crescimento, amplo espectro de substrato e modula??o da resposta imune. O presente estudo investigou os efeitos do tratamento com o polissacar?deo extra?do da levedura Kluyveromyces marxianus (poly322) em modelos experimentais de dor e inflama??o. As propriedades farmacol?gicas do poly322 foram avaliadas nos testes de contor??es abdominais, formalina, nocicep??o manifesta por CFA, retirada da cauda, placa quente e edema de pata. Os n?veis de citocinas foram determinados por ELISA e os efeitos sobre o desempenho motor foram avaliados pelos testes do cil?ndro girat?rio e campo aberto. O pr?-tratamento com o poly322 resultou na redu??o da hipernocicep??o induzida pelo ?cido ac?tico, CFA e formalina (segunda fase). Al?m disso, os animais tratados com o poly322 exibiram uma redu??o significativa no edema de pata e no aumento local dos n?veis de IL-6 induzidos pela carregenina. Em contraste, o tratamento com o poly322 n?o alterou o limiar de resposta a est?mulo t?rmico nos testes de retirada da cauda e placa quente, indicando aus?ncia de a??o central. Confirmando a especificidade da a??o sugerida pelos testes nociceptivos, o tratamento com o poly322 n?o induziu comprometimento motor. Os resultados demonstram que o poly322 possui um potente efeito antinociceptivo e anti-inflamat?rio, possivelmente mediado pela inibi??o da produ??o e/ou libera??o da citocina IL-6.
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Expressão e produção de monoxigenases bacterianas em komagataella phafii (pichia pastoris) para utilização como enzimas acessórias para desconstrução de biomassaSantos, Fernanda Pinheiro dos 26 April 2017 (has links)
A descoberta das monoxigenases de polissacarídeos líticas dependentes de cobre (LPMOs), que agem em sinergismo com outras enzimas na desconstrução da celulose, gerou um grande interesse da comunidade científica por seu potencial de aplicação na produção de biocombustíveis a partir de resíduos lignocelulósicos. A busca por essas proteínas auxiliares em microrganismos surgiu como uma estratégia promissora, pois há grande diversidade de isoformas e disponibilidade de sequências genômicas dessas proteínas. Diante disso, o presente trabalho teve como objetivo expressar LPMOs recombinantes de origem bacteriana em levedura Komagataella phafii. Foram obtidos clones de K. phafii transformados com 6 genes selecionados a partir de banco de dados. Análises da cinética de expressão proteica por técnica de western blot indicaram secreção apenas da LPMO de 25 kDa codificada pelo gene de Thermobifida fusca YX. Ensaios de produção da LPMO de T. fusca foram realizados em biorreator de 1L e Erlenmeyers de 250 mL e 1000 mL. Nos ensaios em Erlenmeyers houve a detecção da proteína, confirmada por SDS-PAGE e western blot, acompanhado de um alto crescimento microbiano. No ensaio em biorreator não houve detecção da proteína de interesse e o crescimento microbiano foi baixo. Com a confirmação da expressão da LPMO nos ensaios em Erlenmeyers, estes foram parcialmente purificados e avaliados por gel de proteínas e western blot. Os resultados obtidos mostram que o sistema de expressão de K. phafii foi eficiente, expressando a LPMO de T. fusca. / The discovery of copper-dependent lytic polysaccharide monooxygenase (LPMOs), auxiliary proteins which act in synergy with other enzymes on the cellulose degradation, generated a great interest from the scientific community due to their potential application in biofuels production from lignocellulosic residues. The search for these auxiliary proteins in microorganisms has emerged as a promising strategy because there is a great diversity of isoforms and availability of genomic sequences. Thus, the present work aimed to express bacterial LPMOs in Komagataella phafii. Clones of K. phafii transformed with 6 genes selected from database were obtained. Analysis of protein expression kinetics by western blot technique showed accumulation only of the LPMO of 25 kDa coded by the gene from Thermobifida fusca YX. Production of LPMO from T. fusca was tested in 1 L bioreactor and 250 mL and 1000 mL Erlenmeyer flasks. In Erlenmeyer flasks experiments, there was protein detection, confirmed by SDS-PAGE and western blot, and a high microbial growth. In the bioreactor assay, there was no target protein detection, and microbial growth was low. After confirmation of LPMO expression in Erlenmeyer flasks assays, protein were partially purified and confirmed by protein gel and western blot. Results obtained showed that the expression system of K. phafii was effective, expressing the LPMO from T. fusca.
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Substitut osseux injectable, antibactérien et résorbable : études physico-chimiques et biologiques d'un ciment composite à base d'apatite / Injectable, antibacterial and resorbable bone substitute : a physico-chemical and biological study of an apatite-based composite cementJacquart, Sylvaine 01 October 2013 (has links)
Ce travail porte sur la recherche et le développement d'un matériau de substitution osseuse permettant une implantation par chirurgie mini invasive, limitant les infections post-opératoires et dont la résorbabilité serait adaptée à la cinétique de régénération osseuse. Nous nous sommes intéressés à un ciment à base de carbonate et de phosphate de calcium (CaCO3 – CaP) dont la réaction de prise conduit à la formation d'une apatite nanocristalline analogue au minéral osseux. Dans une première partie la cinétique de prise et le produit de réaction ont été caractérisés par différentes techniques, notamment la diffraction des RX et les spectroscopies FTIR et RMN du solide. Un sel d'argent – Ag3PO4 ou AgNO3, choisis pour leurs propriétés antibactériennes – a été ensuite introduit dans la formulation. Son effet sur la cinétique de la réaction chimique de prise a été mis en évidence par traitement des spectres FTIR et RMN et un mécanisme réactionnel original impliquant les ions argent et nitrate dans la formation de l'apatite a été proposé. L'ajout d'un polysaccharide, la carboxyméthylcellulose (CMC), dans la phase solide du ciment a montré une très nette amélioration de l'injectabilité de la pâte, avec la disparition du phénomène de séparation des phases qui limite généralement l'injectabilité des ciments minéraux. La résistance à la compression et le module élastique des ciments composites ont été par ailleurs augmentés, parallèlement à une diminution de leur porosité. Différentes études in vitro en présence de cellules ou de bactéries ont enfin été réalisées et ont mis en évidence respectivement la cytocompatibilité des différentes compositions de ciments étudiées et le caractère antibactérien de ces matériaux à partir d'une certaine concentration en argent. L'implantation in vivo de compositions choisies a présenté des résultats très prometteurs quant à la résorbabilité d'un ciment composite CaCO3 - CaP/CMC/Ag et à la néoformation osseuse. / The present work concerns research and development of a material for bone substitution, enabling implantation through a mini-invasive surgery, limiting post-operative infections and whose resorbability is adapted to bone regeneration kinetics. This study focused on a calcium carbonate and phosphate based cement, whose setting reaction leads to the formation of a nanocrystalline apatite, similar to bone mineral. First, the setting kinetics and the reaction products were characterised using different techniques, especially X-ray diffraction and FTIR and solid-state NMR spectroscopies. A silver salt – Ag3PO4 or AgNO3, chosen for their antibacterial properties – was then introduced in the formulation. Its effect on the setting reaction kinetics was revealed by data processing of FTIR and NMR spectra and an original reaction mechanism which involves silver and nitrates in the formation of apatite was proposed. The addition of a polysaccharide, carboxymethylcellulose (CMC), in the solid phase of the cement showed a clear improvement of the injectability of the paste, preventing the occurrence of filter-pressing phenomenon, often limiting the injectability of mineral cements. The resistance to compressive strength and elastic modulus of the composite cement were also improved together with a decrease in their porosity. Different in vitro studies were carried out in the presence of cells or bacteria and demonstrated the cytocompatibility of different cement compositions and their antibacterial properties starting at a certain silver concentration, respectively. In vivo implantation of selected compositions showed promising results concerning resorbability of a composite CaCO3 - CaP/CMC/Ag cement and the associated bone neoformation.
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Polissacarídeo capsular do Streptococcus agalactiae como antígeno vacinal: desenvolvimento de um modelo vacinal para mucosas com Nanopartícula de quitosana / Capsular polysaccharide of Streptococcus agalactiae as vaccine antigen: development of a mucosal vaccine model with chitosan nanoparticleSibylle Sophie Hacker 19 December 2018 (has links)
A bactéria gram-positiva Streptococcus agalactiae do grupo B (GBS) faz parte da microbiota normal do trato geniturinário humano, sendo um organismo comensal do corpo da mulher. No entanto, em mulheres grávidas, quando há alterações na composição microbiana do canal vaginal, pode ocorrer a proliferação e a infecção pelo GBS. Este microrganismo, em sua forma patogênica oportunista, pode infectar o neonato durante o parto natural, assim como contribuir para infecções urinárias e uterinas durante a gestação. O GBS já foi identificado como um dos responsáveis pela alta taxa de mortalidade neonatal, sendo um dos principais agentes de infecção em recém-nascidos no mundo. Ele também pode ser a causa de infecções nas gestantes, levando a várias complicações, como corioamnionite, endometrite e infecções do trato urinário e do sítio cirúrgico. Pode haver comprometimento da gestação e do feto, com abortamento, morte fetal intrauterina e ruptura da membrana coriônica, levando a parto prematuro - que pode resultar em outras consequências graves. Este trabalho foi desenvolver um modelo vacinal para mucosa sublingual, utilizando o polissacarídeo capsular do Streptococcus agalactiae como antígeno, encapsulado em Nanopartículas de quitosana. Para o estudo de otimização dos parâmetros de fermentação, para aumentar a produtividade de cápsula polissacarídica (PS) presente na superfície celular, utilizou-se o Banco de Dados Kegg (Kyoto Encyclopedia of Genes and Genomes). A adição do suplemento L-Prolina foi o que propiciou a principio, maior relação entre crescimento bacteriano e formação de cápsula polissacarídica. A purificação e extração da cápsula polissacarídica foi realizada com etapas sucessivas de ultra filtração tangencial e precipitação alcóolica dos contaminantes. As caracterizações físico-químicas: difração de raios-X (DRX), cromatografia gasosa (CGMS), ressonância magnética (NMR) e determinação de açúcares pelo método fenol-sulfúrico, foram realizadas para identificação da composição e estrutura monossacarídica de açucares. O PS isolado apresenta ramificações de fucose, manose, glicose, galactose e N-acetil-glucosamina, apresentando estrutura amorfa. A liofilização do polissacarídeo foi realizada para fins de concentração e conservação. A encapsulação do polissacarídeo acoplada quimicamente com OVA, em uma Nanopartícula de quitosana, teve como finalidade aumentar a mucoadesividade e possibilitar maior absorção do antígeno entre as células da junção epitelial das mucosas sublinguais. A partir da análise de DLS (Espalhamento dinâmico de luz), as Nanopartículas apresentaram dimensões entre 200 a 400 nm e o Potencial Zeta acima de 20. O índice de polidispersão (PDI) está dentro do esperado (abaixo de 0.3). A capacidade de encapsulamento em relação à OVA foi de 92,8% dos grupos que continham PS. O teste IgG sérica total mostrou que o grupo G2 (Nanopartícula com Polissacarídeo e Proteína acoplados) foi o que teve maior reatividade no teste de ELISA, pela Análise de Variância (ANOVA) com ferramenta de Bonferrone. O teste sIgA mostrou que o grupo G2 (Nanopartícula com Polissacarídeo e Proteína acoplados) foi o que teve maior concentração de anticorpo sIgA total. Como resultado e conclusão, o polissacarídeo capsular do Streptococcus agalactiae é um bom candidato a antígeno vacinal. / Gram-positive bacteria Streptococcus agalactiae group B (GBS) is part of the normal microbiota of the human genitourinary tract, being a commensal organism of the female body. However, in pregnant women, when there are changes in the microbial composition of the vaginal canal, GBS proliferation and infection may occur. This microorganism, in its opportunistic pathogenic form, can infect the neonate during natural childbirth, as well as contribute to urinary and uterine infections during pregnancy. The GBS has already been identified as one of the responsible for the high neonatal mortality rate, being one of the main agents of infection in newborns in the world. It can also be the cause of infections in pregnant women, leading to various complications such as chorioamnionitis, endometritis, and urinary tract and surgical site infections. There may be pregnancy and fetal impairment, with abortion, fetal intrauterine death, and rupture of the chorionic membrane, leading to premature labor - which can result in other serious consequences. This work was to develop a vaccine model for sublingual mucosa using the capsular polysaccharide of Streptococcus agalactiae as antigen, encapsulated in chitosan nano particles. For the study of optimization of the fermentation parameters, the Kegg (Kyoto Encyclopedia of Genes and Genomes) database was used to increase the productivity of polysaccharide capsule (PS) present on the cell surface. The addition of the L-Proline supplement gave rise to a higher ratio between bacterial growth and polysaccharide capsule formation. The purification and extraction of the polysaccharide capsule was performed with successive stages of tangential ultrafiltration and alcoholic precipitation of the contaminants. The physicochemical characterization of X-ray diffraction (XRD), gas chromatography (CGMS), magnetic resonance (NMR) and determination of sugars by the phenol-sulfuric method were performed to identify the composition and monosaccharide structure of sugars. The isolated PS presents branches of fucose, mannose, glucose, galactose and N-acetyl-glucosamine, presenting amorphous structure. Lyophilization of the polysaccharide was performed for concentration and conservation purposes. The encapsulation of the polysaccharide coupled chemically with OVA in a chitosan nano particle was aimed at increasing mucoadhesiveness and allowing greater absorption of the antigen between the cells of the sublingual mucosal epithelial junction. From the analysis of DLS (dynamic light scattering), the nanoparticles presented dimensions between 200 to 400 nm and the Zeta potential above 20. The polydispersity index (PDI) is within the expected range (below 0.3). The encapsulation capacity for OVA was 92.8% of the groups containing PS. The total serum IgG test showed that the G2 group (Nano particle with Polysaccharide and Protein coupled) was the one that had the highest reactivity in the ELISA test, by Analysis of Variance (ANOVA) with Bonferrone tool. The sIgA test showed that the G2 group (Nanoparticle with Polysaccharide and Protein coupled) had the highest concentration of total sIgA antibody. As a result and conclusion, the capsular polysaccharide of Streptococcus agalactiae is a good candidate for vaccine antigen.
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Polissacarídeo capsular do Streptococcus agalactiae como antígeno vacinal: desenvolvimento de um modelo vacinal para mucosas com Nanopartícula de quitosana / Capsular polysaccharide of Streptococcus agalactiae as vaccine antigen: development of a mucosal vaccine model with chitosan nanoparticleHacker, Sibylle Sophie 19 December 2018 (has links)
A bactéria gram-positiva Streptococcus agalactiae do grupo B (GBS) faz parte da microbiota normal do trato geniturinário humano, sendo um organismo comensal do corpo da mulher. No entanto, em mulheres grávidas, quando há alterações na composição microbiana do canal vaginal, pode ocorrer a proliferação e a infecção pelo GBS. Este microrganismo, em sua forma patogênica oportunista, pode infectar o neonato durante o parto natural, assim como contribuir para infecções urinárias e uterinas durante a gestação. O GBS já foi identificado como um dos responsáveis pela alta taxa de mortalidade neonatal, sendo um dos principais agentes de infecção em recém-nascidos no mundo. Ele também pode ser a causa de infecções nas gestantes, levando a várias complicações, como corioamnionite, endometrite e infecções do trato urinário e do sítio cirúrgico. Pode haver comprometimento da gestação e do feto, com abortamento, morte fetal intrauterina e ruptura da membrana coriônica, levando a parto prematuro - que pode resultar em outras consequências graves. Este trabalho foi desenvolver um modelo vacinal para mucosa sublingual, utilizando o polissacarídeo capsular do Streptococcus agalactiae como antígeno, encapsulado em Nanopartículas de quitosana. Para o estudo de otimização dos parâmetros de fermentação, para aumentar a produtividade de cápsula polissacarídica (PS) presente na superfície celular, utilizou-se o Banco de Dados Kegg (Kyoto Encyclopedia of Genes and Genomes). A adição do suplemento L-Prolina foi o que propiciou a principio, maior relação entre crescimento bacteriano e formação de cápsula polissacarídica. A purificação e extração da cápsula polissacarídica foi realizada com etapas sucessivas de ultra filtração tangencial e precipitação alcóolica dos contaminantes. As caracterizações físico-químicas: difração de raios-X (DRX), cromatografia gasosa (CGMS), ressonância magnética (NMR) e determinação de açúcares pelo método fenol-sulfúrico, foram realizadas para identificação da composição e estrutura monossacarídica de açucares. O PS isolado apresenta ramificações de fucose, manose, glicose, galactose e N-acetil-glucosamina, apresentando estrutura amorfa. A liofilização do polissacarídeo foi realizada para fins de concentração e conservação. A encapsulação do polissacarídeo acoplada quimicamente com OVA, em uma Nanopartícula de quitosana, teve como finalidade aumentar a mucoadesividade e possibilitar maior absorção do antígeno entre as células da junção epitelial das mucosas sublinguais. A partir da análise de DLS (Espalhamento dinâmico de luz), as Nanopartículas apresentaram dimensões entre 200 a 400 nm e o Potencial Zeta acima de 20. O índice de polidispersão (PDI) está dentro do esperado (abaixo de 0.3). A capacidade de encapsulamento em relação à OVA foi de 92,8% dos grupos que continham PS. O teste IgG sérica total mostrou que o grupo G2 (Nanopartícula com Polissacarídeo e Proteína acoplados) foi o que teve maior reatividade no teste de ELISA, pela Análise de Variância (ANOVA) com ferramenta de Bonferrone. O teste sIgA mostrou que o grupo G2 (Nanopartícula com Polissacarídeo e Proteína acoplados) foi o que teve maior concentração de anticorpo sIgA total. Como resultado e conclusão, o polissacarídeo capsular do Streptococcus agalactiae é um bom candidato a antígeno vacinal. / Gram-positive bacteria Streptococcus agalactiae group B (GBS) is part of the normal microbiota of the human genitourinary tract, being a commensal organism of the female body. However, in pregnant women, when there are changes in the microbial composition of the vaginal canal, GBS proliferation and infection may occur. This microorganism, in its opportunistic pathogenic form, can infect the neonate during natural childbirth, as well as contribute to urinary and uterine infections during pregnancy. The GBS has already been identified as one of the responsible for the high neonatal mortality rate, being one of the main agents of infection in newborns in the world. It can also be the cause of infections in pregnant women, leading to various complications such as chorioamnionitis, endometritis, and urinary tract and surgical site infections. There may be pregnancy and fetal impairment, with abortion, fetal intrauterine death, and rupture of the chorionic membrane, leading to premature labor - which can result in other serious consequences. This work was to develop a vaccine model for sublingual mucosa using the capsular polysaccharide of Streptococcus agalactiae as antigen, encapsulated in chitosan nano particles. For the study of optimization of the fermentation parameters, the Kegg (Kyoto Encyclopedia of Genes and Genomes) database was used to increase the productivity of polysaccharide capsule (PS) present on the cell surface. The addition of the L-Proline supplement gave rise to a higher ratio between bacterial growth and polysaccharide capsule formation. The purification and extraction of the polysaccharide capsule was performed with successive stages of tangential ultrafiltration and alcoholic precipitation of the contaminants. The physicochemical characterization of X-ray diffraction (XRD), gas chromatography (CGMS), magnetic resonance (NMR) and determination of sugars by the phenol-sulfuric method were performed to identify the composition and monosaccharide structure of sugars. The isolated PS presents branches of fucose, mannose, glucose, galactose and N-acetyl-glucosamine, presenting amorphous structure. Lyophilization of the polysaccharide was performed for concentration and conservation purposes. The encapsulation of the polysaccharide coupled chemically with OVA in a chitosan nano particle was aimed at increasing mucoadhesiveness and allowing greater absorption of the antigen between the cells of the sublingual mucosal epithelial junction. From the analysis of DLS (dynamic light scattering), the nanoparticles presented dimensions between 200 to 400 nm and the Zeta potential above 20. The polydispersity index (PDI) is within the expected range (below 0.3). The encapsulation capacity for OVA was 92.8% of the groups containing PS. The total serum IgG test showed that the G2 group (Nano particle with Polysaccharide and Protein coupled) was the one that had the highest reactivity in the ELISA test, by Analysis of Variance (ANOVA) with Bonferrone tool. The sIgA test showed that the G2 group (Nanoparticle with Polysaccharide and Protein coupled) had the highest concentration of total sIgA antibody. As a result and conclusion, the capsular polysaccharide of Streptococcus agalactiae is a good candidate for vaccine antigen.
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Comparison of Anti-Pneumococcal Functions of Native and Modified Forms of C-Reactive ProteinNgwa, Donald Neba 01 May 2016 (has links)
The anti-pneumococcal function of native C-reactive protein (CRP) involves its binding to phosphocholine molecules present on Streptococcus pneumoniae and subsequent activation of the complement system. However, when pneumococci recruit complement inhibitory protein factor H on their surface, they escape complement attack. Non-native forms of CRP have been shown to bind immobilized factor H. Accordingly, we hypothesized that modified CRP would bind to factor H on pneumococci, masking its complement inhibitory activity, allowing native CRP to exert its anti-pneumococcal function. As reported previously, native CRP protected mice from lethal pneumococcal infection when injected 30 minutes before infection but not when injected 24 hours after infection. However, a combination of native and mutant CRP was found to protect mice even when administered 24 hours after infection. Therefore, it is concluded that while native CRP is protective only against early-stage infection, a combination of native and mutant CRP offers protection against late-stage infection.
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Hydrogels de polygalacturonate réticulés par les ions Fe2+ : impact du mode d'association local sur les mécanismes de gélification, contrôle de la structure multi-échelle et des propriétés mécaniques. / Polygalacturonate hydrogels using Fe2+ as cross-linkers : impact of the local association mode on gelation mechanisms, control of the multiscale structure and the mechanical properties.Maire du Poset, Aline 24 September 2018 (has links)
Ce travail de thèse décrit la formulation d'hydrogels de polygalacturonate (polyGal) réticulés par les cations Fe2+, ainsi que leur caractérisation expérimentale depuis les échelles moléculaires jusqu'aux échelles macroscopiques, en utilisant notamment la spectroscopie d'absorption X (EXAFS), la diffusion de neutrons aux petits angles (DNPA) ainsi que des mesures de rhéologie. Nous avons élaboré un protocole de gélification robuste permettant d'obtenir des gels cylindriques reproductibles qui présentent des gradients de concentrations contrôlés depuis leur partie basale jusqu'à leur partie apicale. Le rapport R = [Fe]/[Gal] a une valeur constante de 0,25 tout au long du gel, ce qui prouve que les cations Fe2+ s'associent avec 4 unités galacturonate. La confrontation des résultats d’EXAFS et de dynamique moléculaire a démontré que ces associations se font via le modèle ''egg-box''. Les mécanismes de réticulation qui contrôlent la structure du réseau formée par les chaînes aux échelles locales sont donc les mêmes dans l'ensemble du gel, ce qui est confirmé par les mesures de DNPA. La formation des gradients de concentration macroscopiques provient des mécanismes de diffusion des cations à travers le gel lors de sa formation. Ces gradients de concentration contrôlent les propriétés mécaniques des gels. En outre, nous avons prouvé que le mode d'association "egg-box" permettait la protection des ions Fe2+ contre l'oxydation, ce qui confère à ces hydrogels un potentiel applicatif pour soigner l'anémie car ils pourraient permettre la vectorisation du fer sous cette forme réduite biodisponible jusqu’à l’intestin.Nous avons étendu notre étude à la formulation d'hydrogels avec d'autres cations (Ca2+ et Zn2+). Ces hydrogels présentent des propriétés macroscopiques proches de celles des hydrogels Fe2+-polyGal car les mécanismes de diffusion des cations régissant la formation des gradients macroscopiques lors de la formation des gels sont similaires. Les hydrogels présentent cependant des structures locales différentes car les modes d'associations locaux varient d"un cation à l’autre. L’ensemble de ces résultats nous a permis de proposer un mécanisme généralisé permettant de décrire les mécanismes de formation d"hydrogels de polygalacturonate pour les cations divalents, et ainsi de moduler finement leur structure sur plusieurs échelles. Ces hydrogels pourraient donc être des outils de choix pour la vectorisation de molécules actives et le contrôle de leur relargage. / This pHD thesis describes the design of polygalacturonate hydrogels (polyGal) cross-linked by the Fe2+ cations, and their experimental characterization from the molecular scales up to the macroscopic scales, by combining EXAFS spectroscopy, Small Angle Neutron Scattering (SANS) and rheological measurements. We designed a robust gelling protocol that allowed to obtain reproducible cylindrical gels with controlled concentration gradients from the lower side to the upper side of the gel. The ratio [Fe]/[Gal] has a constant value all along the gel, which demonstrate that the Fe2+ cations are associated with 4 galacturonate units. The comparison of EXAFS measurements and molecular dynamics simulation has shown that these associations followed the "egg-box" model. The crosslinking mechanisms that control the structure of the network made by the chains at local scale is therefore the same throughout the whole gel, which is confirmed by SANS measurements. The formation of the macroscopic concentration gradients comes from the mechanisms that drive the cations diffusion through the gel during the gelation process. These gradients control the gels mechanical properties. Besides, we proved that the “egg-box" association enables to protect Fe2+ against oxidation, which gives to these hydrogels an applicative potential to cure anemia as they could allow to target iron under its bioavailable form up to the gut.We have extended the study to the design of hydrogels with other cations (Ca2+ et Zn2+). The macroscopic properties of these hydrogels are very close to that of the Fe2+-polyGal hydrogels because the cation diffusion that govern the formation of macroscopic gradients during the gelationg process are similar. The hydrogels have however different local structures because the cation- polyGal local association varies from one cation to another. All these results allowed us to propose a generalized mechanism that describes the polygalacturonate hydrogels formation for divalent cations, and thus to tune their structure over several scales. These hydrogels could therefore be some promising tools for the vectorization of active molecules and the control of their release.
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Etude de la coacervation complexe entre la beta-lactoglobuline et la gomme d'acacia en solution aqueuseSchmitt, Christophe 27 October 2000 (has links) (PDF)
L'influence de la concentration totale en biopolymères et de la polydispersité de la protéine sur la formation et la stabilité de coacervats a été étudiée dans un mélange b-lactoglobuline/gomme d'acacia/eau. Les mélanges de b-lactoglobuline (b-lg) native ou agrégée (56% d'agrégats insolubles à pH 4,75) et de gomme d'acacia à des pH compris entre 3,6 et 5,0 sont caractérisés par une séparation de phase par coacervation complexe. Une concentration totale élevée en biopolymères limite l'influence du pH et du ratio de mélange protéine : polysaccharide sur la formation des coacervats dans les deux systèmes. En revanche, une forte polydispersité de la b-lg permet d'étendre l'aire de la zone biphasique des diagrammes de phases ternaires obtenus à pH 4,2. Des coacervats et des précipités sont obtenus en présence d'agrégats de b-lg. Seuls des coacervats sont visibles avec la b-lg native. Leur taille est contrôlée par les agrégats protéiques qui interagissent spécifiquement avec une fraction de la gomme d'acacia. La structure des coacervats est caractérisée par une vacuolisation issue d'une coalescence partielle des plus petits coacervats. Après interaction avec la gomme d'acacia, des mesures de dichroïsme circulaire indiquent une modification de la structure en hélice a de la b-lg, caractérisée par une densité de charge positive. La stabilité et la structuration des coacervats formés résultent d'un équilibre entre des phénomènes de floculation, coalescence et sédimentation des coacervats comme indiqué par des mesure de diffusion de la lumière en milieu turbide et microscopie confocale à balayage laser. Enfin, l'étude de la cinétique de structuration des mélanges b-lg/gomme d'acacia/eau par diffusion de la lumière aux petits angles, révèle des phénomènes de diffusionnels et hydrodynamiques responsables de la croissance des domaines structuraux. Dans certains cas, la cinétique de coacervation complexe peut être décrite par le modèle théorique de décomposition spinodale.
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<i>In Vitro</i> Studies of the Substrate Specificities of Heparan Sulfate 2-<i>O</i>- and 6-<i>O</i>-sulfotransferasesSmeds, Emanuel January 2004 (has links)
<p>Heparan sulfate (HS), a linear negatively charged polysaccharide located at the cell surface and in the extracellular matrix, interacts with, and thereby regulates the functions of numerous proteins. HS-protein interactions depend on the fine structure of HS, especially its sulfation pattern. This thesis aimed to understand how differently sulfated domains in HS are generated. Specifically, the substrate specificities of HS hexuronic acid 2-<i>O</i>-sulfotransferase (2OST) and HS glucosaminyl 6-<i>O</i>-sulfotransferases (6OSTs) were investigated. </p><p>Three different 6OSTs (6OST1-3) have been cloned and characterized. To study the mechanisms controlling 6-<i>O</i>-sulfation we incubated the recombinant purified 6-OST isoforms with different 6-<i>O</i>-desulfated poly- and oligosaccharide substrates and the active sulfate donor 3'-phosphoadenosine 5'-phospho[<sup>35</sup>S]sulfate (<sup>35</sup>S-labeled PAPS). All three enzymes catalyzed 6-<i>O</i>-sulfation of both <i>N</i>-acetylated (GlcNAc) as well as <i>N</i>-sulfated (GlcNS) glucosamines next to a nonreducing iduronic acid (IdoA) or glucuronic acid (GlcA). Similar specificities were demonstrated, although some differences in substrate preferences were noted.</p><p>To understand how pre-existing 2-<i>O</i>-sulfates affects 6-<i>O</i>-sulfation, 6OST2 and 6OST3 were incubated with pair-wise mixed octasaccharide substrates with different contents of 2-<i>O</i>-sulfates. The specificities for substrates with two or three 2-<i>O</i>-sulfates were higher compared to octasaccharides with no or one 2-<i>O</i>-sulfate indicating that 2-<i>O</i>-sulfate groups substantially promote the subsequent 6-<i>O</i>-sulfation. </p><p>Overexpression of the 6OSTs in a mammalian cell line resulted in increased 6-<i>O</i>-sulfation of -GlcA-GlcNS- and -GlcA-GlcNAc- sequences. The results were not isoform specific, but affected by the overexpression level. </p><p>The 2OST catalyzes 2-<i>O</i>-sulfation of both IdoA and GlcA residues, with high preference for IdoA units. To study how 2-<i>O</i>-sulfation of GlcA and IdoA is regulated, we incubated the enzyme with different substrates and <sup>35</sup>S-labeled PAPS. Our findings revealed that the 2OST almost exclusively sulfated IdoA also with a ratio of GlcA to IdoA of 99:1, suggesting that 2-<i>O</i>-sulfation of GlcA occurs before IdoA is formed.</p>
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