Thesis (M.Sc.), Memorial University of Newfoundland, School of Pharmacy, / Typescript. Bibliography: p. 141-166.

La thérapie cellulaire et les médicaments de thérapie innovante sont des solutions prometteuses pour la régénération des tissus ou organes présentant des défauts fonctionnels ou organiques. Avant le stade de l'insuffisance cardiaque terminale (stade IV NYHA) suite à un infarctus du myocarde, l'implantation d'un patch de fibrine cellularisé avec des progéniteurs myocardiques sur le site de nécrose de l'infarctus, est l'une des perspectives qui permettrait de régénérer un muscle cardiaque fonctionnel et apparait comme étant une alternative nouvelle avec notamment un essai clinique de phase I en cours (ESCORT : NCT02057900). Cependant, cette thérapie innovante présente de réelles contraintes, parmi lesquelles, un protocole nécessitant, i) une utilisation pour la production de cellules progénitrices myocardiques CD15+, de DMSO, de sérum foetal bovin, de trypsine porcine, de fibroblastes murins pouvant être la source d'une contamination chimique ou microbiologique, ii) une caractérisation importante des cellules produites, pour déterminer leur viabilité, leur pureté, leur état de différenciation, iii) d'implanter le patch de fibrine cellularisé dans un délai limité avant l'obtention des résultats de stérilité et d'endotoxines, iv) d'inciser le péricarde et de former une poche, geste chirurgical très invasif, afin d'implanter le patch cellularisé. Avec l'objectif de limiter ces contraintes et de renforcer la sécurisation pharmaceutique de ce médicament de thérapie innovante, les différents axes de ce travail ont porté sur i) l'ajout, juste avant l'implantation, d'une étape de cryoconservation des cellules dans un milieu sans sérum et sans DMSO, mais avec des agents cryoprotectants de qualité pharmaceutique. L'avantage apporté par la cryoconservation étant de rendre possible une production par lot, et la réalisation des contrôles sans contrainte de temps avant l'implantation, ii) la vectorisation des cellules par une encapsulation dans des microsphères formant une suspension injectable et permettant une implantation directement au travers du péricarde et immédiatement après la décongélation, iii) l'utilisation de polymères bioadhésifs afin de maintenir les microsphères au site d'implantation. Dans un premier temps, ce travail a permis d'identifier l'alginate de sodium de faible viscosité à 1,2% comme polymère pour réaliser l'encapsulation à l'aide d'une buse vibrante de 120 µm de diamètre. La nature et la concentration d'agents cryoprotectants ont également été définies. Les agents cryoprotectants ont été sélectionnés parmi les oses (glucose, saccharose, tréhalose), les polyols (glycérol, mannitol, sorbitol) et l'urée, à une concentration permettant d'atteindre une osmolarité totale de 500 mOsm/L pour abaisser le point de congélation de l'eau. Enfin le chitosane de faible viscosité à 0,5% a été utilisé comme polymère bioadhésif de surface pour maintenir les propriétés mécaniques et la forme des microsphères après la congélation. Dans un second temps, une évaluation biologique a permis de mesurer l'impact des étapes du procédé d'encapsulation et de cryoconservation, sur des cellules souches mésenchymateuses humaines utilisées comme modèle. Il a ainsi été possible d'optimiser le protocole ce qui a eu pour effet d'augmenter la viabilité, évaluée après encapsulation et congélation par une analyse en cytométrie de flux avec le 7AAD, de moins de 5% à environ 35%. / Cell therapy and advanced therapy medicinal products are promising solutions for the regeneration of tissues or organs with functional or organic defects. Before the terminal heart failure stage (stage IV NYHA) following a myocardial infarction, the implantation of a cellularized fibrin patch with myocardial progenitors at the location of the infarct necrosis, is one of the perspectives that would allow a functional heart muscle to regenerate and appears to be a new alternative, in particular, with an ongoing Phase I clinical trial (ESCORT : NCT02057900). However, this innovative therapy presents real constraints, among which, a protocol requiring, i) the use for the production of CD15+ myocardial progenitor cells of, DMSO, bovine fetal serum, porcine trypsin, and murine fibroblasts which may be the source of chemical or microbiological contamination, ii) an important characterization of the produced cells, to determine their viability, purity, and state of differentiation, iii) to implant the cellularized fibrin patch within a limited time frame before getting the results of sterility and endotoxins, iv) to incise the pericardium and to form a pouch, a very invasive surgical gesture, in order to implant the cellularized patch inside. With the objective of limiting these constraints and strengthening the pharmaceutical safety of this innovative therapy medication, different axes of this work have focused on i) the addition, just before the implantation, of a step of cryopreservation of the cells in a medium without serum and without DMSO, but with pharmaceutical-grade cryoprotectants. The advantages of cryopreservation is to allow production in batches, and controls to be carried out without time constraints before the implantation, ii) the vectorization of the cells by encapsulation in microspheres forming an injectable suspension and allowing direct implantation through the pericardium immediately after thawing, iii) the use of bioadhesive polymers to maintain the microspheres at the location of the implantation. This study initially enabled to identify a low-viscosity sodium alginate at 1.2% as a polymer being used for the encapsulation with the use of a vibrating nozzle which diameter is of 120 µm. The nature and the concentration of the cryoprotectants have also been defined. The cryoprotectants were selected from oses (glucose, sucrose, trehalose), polyols (glycerol, mannitol, sorbitol) and urea, at a concentration which achieves a total osmolarity of 500 mOsm/L in order to lower the freezing point of water. Finally, low viscosity chitosan at 0.5% was used as a bioadhesive polymer at the surface of the microspheres to maintain their shapes and mechanical properties after freezing. In a second step, a biological evaluation allowed to measure the impact of the encapsulation and the cryopreservation processes, on human mesenchymal stem cells used as a model. It was thus possible to optimize the protocol, which in return increased the viability ; evaluation made after encapsulation and freezing by a flow cytometry analysis with 7AAD ; from less than 5% to about 35%.

La biodégradation du 2-aminobenzothiazole (ABT) a été comparée entre des bactéries en suspension dans l'eau et des bactéries immobilisées sur un support d'alginate. Trois processus de dégradation de l'ABT ont été étudiés : la photodégradation sous lumière solaire en présence du complexe Fe(III)-acide nitrilotriacétique (FeNTA), la biodégradation par la bactérie aérobie stricte Rhodococcus rhodochrous et la combinaison de ces deux processus. Le métabolisme de R. rhodochrous a été étudié par RMN in vivo du 31P et du 13C : des informations importantes sur le métabolisme phosphoré et carboné ont été obtenues. La réponse de la bactérie face à divers stress a été évaluée et a montré sa capacité d'adaptation aux variations environnementales. La spéciation du fer pour son rôle important dans l'activation de la biodégradation d'ABT a été etudiée : complexes organiques, oxydes et oxy(hydr)oxydes de fer ont été testés pour connaître les formes biodisponibles pour R. rhodochrous

Carragenanas, agaranas e alginatos são hidrocolóides largamente utilizados em todo tipo de produtos alimentícios como aditivos espessantes. Eles não são absorvidos pelo organismo e, portanto não introduzem calorias extras na dieta. A irradiação se apresenta com grande potencial como um método alternativo na preservação de alimentos pois não induz aumento da temperatura, e é, portanto, de grande eficácia na descontaminação de ingredientes alimentícios sensíveis ao calor. Neste trabalho, soluções dos hidrocolóides agararana, carragenana e aiginato de sódio, foram irradiadas com diferentes doses (0-10 kGy) de radiação gama de Co-60 na presença de antioxidantes também utilizados na indústria alimentícia: ácido ascórbico, extrato vegetal de rosela (Híbiscus sabdariffa L.) e isofiavona de soja. As soluções dos polissacarídeos comestíveis agarana, carragenana e alginato de sódio mostraram ser bons sistemas para avaliar o efeito da radiação ionizante por apresentarem radiossensibilidade característica medida pelas mudanças na viscosidade. Os resultados obtidos mostram que esses antioxidantes apresentam, no geral, ação radioprotetora o que pode ser de grande valia nas aplicações futuras da irradiação de alimentos em escala comercial. / Carrageenan, agaran e alginates are hydrocolloids largely employed in every kind of food products as stabilizing agent and viscosity builder. The human body does not absorb them, so they do not introduce extra calories in the diet. Irradiation is presented as an important alternative method in food preservation because do not induce temperature increase being of good efficiency in cold food ingredients decontamination. In this work aqueous solutions of carrageenan, agar e sodium alginate were gamma irradiated (0-10 kGy) in presence of ascorbic acid, roselle {Hibiscus sabdariffa L.) extract and soy isoflavone. Edible polysaccharide solutions showed to be suitable systems for the evaluation of ionizing radiation effects as they presented a singular radiosensitivity through viscosity changes. The results obtained showed that in general the antioxidants employed had a radioprotective action that can be of importance in the future commercial applications of food irradiation.

Carragenanas, agaranas e alginatos são hidrocolóides largamente utilizados em todo tipo de produtos alimentícios como aditivos espessantes. Eles não são absorvidos pelo organismo e, portanto não introduzem calorias extras na dieta. A irradiação se apresenta com grande potencial como um método alternativo na preservação de alimentos pois não induz aumento da temperatura, e é, portanto, de grande eficácia na descontaminação de ingredientes alimentícios sensíveis ao calor. Neste trabalho, soluções dos hidrocolóides agararana, carragenana e aiginato de sódio, foram irradiadas com diferentes doses (0-10 kGy) de radiação gama de Co-60 na presença de antioxidantes também utilizados na indústria alimentícia: ácido ascórbico, extrato vegetal de rosela (Híbiscus sabdariffa L.) e isofiavona de soja. As soluções dos polissacarídeos comestíveis agarana, carragenana e alginato de sódio mostraram ser bons sistemas para avaliar o efeito da radiação ionizante por apresentarem radiossensibilidade característica medida pelas mudanças na viscosidade. Os resultados obtidos mostram que esses antioxidantes apresentam, no geral, ação radioprotetora o que pode ser de grande valia nas aplicações futuras da irradiação de alimentos em escala comercial. / Carrageenan, agaran e alginates are hydrocolloids largely employed in every kind of food products as stabilizing agent and viscosity builder. The human body does not absorb them, so they do not introduce extra calories in the diet. Irradiation is presented as an important alternative method in food preservation because do not induce temperature increase being of good efficiency in cold food ingredients decontamination. In this work aqueous solutions of carrageenan, agar e sodium alginate were gamma irradiated (0-10 kGy) in presence of ascorbic acid, roselle {Hibiscus sabdariffa L.) extract and soy isoflavone. Edible polysaccharide solutions showed to be suitable systems for the evaluation of ionizing radiation effects as they presented a singular radiosensitivity through viscosity changes. The results obtained showed that in general the antioxidants employed had a radioprotective action that can be of importance in the future commercial applications of food irradiation.

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.

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.