No description available.

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No description available.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

No description available.

Transglutaminase II (TG2) is a complex protein with five different reported activities. Increases in TG2 expression and TGase activity have previously been observed during wound healing in rat studies; however, it has been unclear whether these phenomena were directly involved in the healing process or if they were simply a by-product of it. The aims of this thesis were, thus, to determine if TG2 plays a role in wound healing in vivo and to elucidate the mechanism of any effects TG2 may have at the cellular level. TG2 ablation resulted in delayed wound healing. To gain mechanistic insight into this abnormality, primary fibroblast cultures from TG2-knockout and wildtype mouse embryos were analysed. TG2-null fibroblasts displayed decreased adhesion and integrin signalling during initial stages of adhesion. Intriguingly, TG2-null cells showed faster activation of Rac1 and RhoA in response to adhesion. Long-term adhesion of TG2-null fibroblasts resulted in increased basal phosphorylation of FAK and number of paxillin-stained focal adhesions, enhanced PI3-kinase signalling, faster actin dynamics and altered activation of p44/42 MAPK. These results are indicative of futile cycling of intracellular signalling pathways resulting from reduced focal adhesion turnover in the TG2-knockout fibroblasts. Rescue experiments demonstrated that TG2-mediated effects on cell adhesion occurred in the extracellular environment and that neither GTP-binding nor TGase activity is required for these effects. Results further showed that a ???compact??? conformation of TG2 was not required for this role of TG2. Interestingly, addition of recombinant TG2 to the extracellular environment increased cell spreading of TG2-null cells to a level far greater than that seen in wildtype cells, which did not increase their spreading in response to exogenous TG2. Demonstration of faster activation of the small GTPases in the TG2-null MEFs, and the apparent inhibition of exogenous TG2???s extracellular effects on cell spreading by endogenous protein in the wildtype cells, provide tantalising evidence for a role for intracellular TG2 in regulating activation of the small GTPases to promote efficient fibroblast migration. This work identifies TG2 as a facilitator of efficient wound closure through extracellular effects on integrin-mediated signalling and intracellular effects on activation of the small GTPases.

Coeliac disease is a chronic enteropathy triggered by gluten. Patients produce antibodies to gliadin and the autoantigen tissue transglutaminase (tTG). These anti-tTG autoantibodies are disease specific and used in diagnosis. The autoantibodies can be detected by immunofluorescence (the endomysial antibody tests) or by ELISA using recombinant tTG. In vivo tTG associates with fibronectin, which may account for the greater sensitivity of the endomysial antibody assay compared to the ELISA. This project had two aims:  to determine whether GST-tagged tTG bound fibronectin and then, using the fibronectin bound tTG, whether a two-tiered ELISA increased anti-tTG binding in coeliac disease patients. First fibronectin was coupled to a solid support and then incubated with tTG. This was then analysed using SDS-PAGE. Secondly, an ELISA with a two-tiered antigen coating was created by coupling tTG to Fn. This mimics the in vivo orientation of the antigen and could theoretically increase anti-tTG binding. Comparative ELISAs were then run to see if anti-tTG binding differed between tTG and fibronectin-coupled tTG antigen coatings. Results showed GST-tagged tTG bound fibronectin. Coupling of tTG to fibronectin gave no improved binding of anti-tTG. On the contrary, most patients tested had decreased anti-tTG binding compared to the normal tTG based ELISA.

Transglutaminase II (TG2) is a complex protein with five different reported activities. Increases in TG2 expression and TGase activity have previously been observed during wound healing in rat studies; however, it has been unclear whether these phenomena were directly involved in the healing process or if they were simply a by-product of it. The aims of this thesis were, thus, to determine if TG2 plays a role in wound healing in vivo and to elucidate the mechanism of any effects TG2 may have at the cellular level. TG2 ablation resulted in delayed wound healing. To gain mechanistic insight into this abnormality, primary fibroblast cultures from TG2-knockout and wildtype mouse embryos were analysed. TG2-null fibroblasts displayed decreased adhesion and integrin signalling during initial stages of adhesion. Intriguingly, TG2-null cells showed faster activation of Rac1 and RhoA in response to adhesion. Long-term adhesion of TG2-null fibroblasts resulted in increased basal phosphorylation of FAK and number of paxillin-stained focal adhesions, enhanced PI3-kinase signalling, faster actin dynamics and altered activation of p44/42 MAPK. These results are indicative of futile cycling of intracellular signalling pathways resulting from reduced focal adhesion turnover in the TG2-knockout fibroblasts. Rescue experiments demonstrated that TG2-mediated effects on cell adhesion occurred in the extracellular environment and that neither GTP-binding nor TGase activity is required for these effects. Results further showed that a ???compact??? conformation of TG2 was not required for this role of TG2. Interestingly, addition of recombinant TG2 to the extracellular environment increased cell spreading of TG2-null cells to a level far greater than that seen in wildtype cells, which did not increase their spreading in response to exogenous TG2. Demonstration of faster activation of the small GTPases in the TG2-null MEFs, and the apparent inhibition of exogenous TG2???s extracellular effects on cell spreading by endogenous protein in the wildtype cells, provide tantalising evidence for a role for intracellular TG2 in regulating activation of the small GTPases to promote efficient fibroblast migration. This work identifies TG2 as a facilitator of efficient wound closure through extracellular effects on integrin-mediated signalling and intracellular effects on activation of the small GTPases.

A Transglutaminase (TGase; proteína-glutamina γ-glutamil-transferase; EC 2.3.2.13) é uma enzima que catalisa reações de acil-transferência introduzindo ligações cruzadas entre cadeias protéicas. Em função de suas características, as TGases microbianas têm ampla e crescente aplicação na indústria alimentícia e em outras áreas. Portanto, o objetivo desse trabalho foi aumentar o conhecimento existente sobre a produção e as aplicações dessas enzimas. A composição do meio de cultura para a produção da enzima em cultivo submerso (CSm) pelo Bacillus circulans BL32, um isolado Amazônico, foi otimizada através de uma estratégia em três etapas. A otimização do meio resultou numa atividade de TGase que é 60 % maior que a máxima obtida utilizando um meio de cultura previamente citado na literatura para a produção dessa enzima, além da redução dos custos dos constituintes do mesmo. Metodologias de planejamento experimental foram utilizadas para otimizar a temperatura de incubação e o pH do meio de cultura. As melhores condições de cultivo para a produção da TGase pelo B. circulans BL32 para a produção da enzima em CSm foram 30 °C e pH 8.5, sendo que a máxima produção foi obtida no final da fase estacionária de multiplicação. Os efeitos da agitação e aeração sobre a produção de TGase e esporulação do B. circulans BL32 em sistema de CSm também foram estudados. Os resultados demonstraram que as condições ótimas de processo para a formação de biomassa e esporulação são diferentes. Portanto, foi adotada uma estratégia de controle da taxa de aeração em dois estágios, com formação de biomassa, no primeiro estágio, nas condições ótimas de crescimento seguido por um segundo estágio sob as condições de esporulação. Também estudou-se a produção de TGase pelo B. circulans BL32 em cultivo no estado sólido (CES). Vários resíduos agroindustriais foram usados como substrato para crescimento do microrganismo e produção da enzima. As melhores condições de cultivo foram 0,6 L/min de ar, 33 °C e 10 log 10 UFC/g de substrato para a concentração celular do inóculo, em resíduo fibroso de soja como substrato. A determinação das condições de extração para a efetiva recuperação da TGase produzida em CES foi realizada através do emprego de ferramentas de planejamento experimental. A melhor condição de extração da enzima foi quando utilizou-se água a 7 ºC como solvente, por 5 minutos, 250 rpm e uma relação de sólidos/líquidos de 1:6. Caseína, proteína isolada de soja e proteína hidrolisada de carne foram tratadas com essa TGase microbiana. A redução do número de grupos aminos livres após o tratamento com a enzima, principalmente, na caseína, demonstrou a formação de ligações cruzadas catalisadas por essa TGase. As propriedades emulsificantes dessas proteínas foram melhoradas após o tratamento com a TGase do B. circulans BL32. Além disso, a fim de investigar o mecanismo de inativação térmica, incubou-se a enzima por diferentes períodos de tempo em temperaturas entre 30 e 70 °C. As cinéticas de termoinativação desta TGase seguiram o modelo de Lumry-Eyring e a enzima mostrou-se estável até 50 °C, sendo que após 12 h nessa temperatura a mesma ainda mantém 50 % da sua atividade enzimática. Os resultados sugerem que esta TGase microbiana apresenta um grande potencial de uso em aplicações alimentícias e não alimentícias. / Transglutaminase (TGase; protein-glutamine γ-glutamyltransferase; EC 2.3.2.13) is an enzyme capable of catalyzing acyl transfer reactions by introducing covalent cross-links between proteins. Microbial TGases have found widespread and growing applications in the food and non-food industry. In this work, an effort has been made in order to increase available knowledge about microbial TGases. Medium composition for TGase production on submerged cultivations by Bacillus circulans BL32, a recently isolated strain from the Amazon basin, was optimized using a stepwise strategy. The optimization of the medium resulted not only in a 60 % higher TGase activity than the obtained in a media previously cited in the literature but also in a reduction of constituents costs. Statistical experimental methods also were used to optimize the temperature and pH parameters. The best culture conditions for TGase production by B. circulans BL32 were 30 °C, pH 8.5 and the highest production was obtained in late-stationary culture phase. And besides, the effects of agitation and aeration on TGase production and cell sporulation on submerged cultivations were studied. The results demonstrated that the optimal process conditions are different for biomass and spore production. It was adopted a two-stage aeration rate control strategy with biomass production under the growth conditions in the first stage followed by the second stage under the conditions for sporulation. The present work also dealt with the TGase production in solid state cultivations. Several agro-industrial residues were used as substrates for microbial growth and enzyme production. The best culture conditions were determined as being 0.6 L air min-1, 33 °C and 10 log 10 CFU g-1 of dried substrate to the inoculum cell concentration, on industrial fibrous soy residue as substrate. The optimization of downstream processing parameters for the effective enzyme recovery of the cultivated solids was carried out. The optimal conditions for the extraction were: water as solvent at 7 ºC; 5 min of extraction time; agitation speed of 250 rpm; and 1:6 solid:liquid ratio. Casein, soy protein isolated, and hydrolysed animal protein were treated with this microbial TGase. The decrease in the amount of free amino groups after TGase treatment, mainly in the casein, demonstrated the cross-linking catalyzed by this enzyme. The emulsifying properties of these proteins were improved after treatment with B. circulans BL32 TGase. Furthermore, in order to investigate the mechanism of thermal inactivation, the enzyme was incubated at temperatures ranging from 30 to 70 °C. The thermoinactivation kinetics of this microbial TGase followed a Lumry-Eyring model. The enzyme presented good stability until 50 °C. About 50 % of the activity still remained after heating for 12 h in this temperature. Results presented in this work suggest that this microbial TGase exhibit some interesting properties for food and non-food industrial applications.

A Transglutaminase (TGase; proteína-glutamina γ-glutamil-transferase; EC 2.3.2.13) é uma enzima que catalisa reações de acil-transferência introduzindo ligações cruzadas entre cadeias protéicas. Em função de suas características, as TGases microbianas têm ampla e crescente aplicação na indústria alimentícia e em outras áreas. Portanto, o objetivo desse trabalho foi aumentar o conhecimento existente sobre a produção e as aplicações dessas enzimas. A composição do meio de cultura para a produção da enzima em cultivo submerso (CSm) pelo Bacillus circulans BL32, um isolado Amazônico, foi otimizada através de uma estratégia em três etapas. A otimização do meio resultou numa atividade de TGase que é 60 % maior que a máxima obtida utilizando um meio de cultura previamente citado na literatura para a produção dessa enzima, além da redução dos custos dos constituintes do mesmo. Metodologias de planejamento experimental foram utilizadas para otimizar a temperatura de incubação e o pH do meio de cultura. As melhores condições de cultivo para a produção da TGase pelo B. circulans BL32 para a produção da enzima em CSm foram 30 °C e pH 8.5, sendo que a máxima produção foi obtida no final da fase estacionária de multiplicação. Os efeitos da agitação e aeração sobre a produção de TGase e esporulação do B. circulans BL32 em sistema de CSm também foram estudados. Os resultados demonstraram que as condições ótimas de processo para a formação de biomassa e esporulação são diferentes. Portanto, foi adotada uma estratégia de controle da taxa de aeração em dois estágios, com formação de biomassa, no primeiro estágio, nas condições ótimas de crescimento seguido por um segundo estágio sob as condições de esporulação. Também estudou-se a produção de TGase pelo B. circulans BL32 em cultivo no estado sólido (CES). Vários resíduos agroindustriais foram usados como substrato para crescimento do microrganismo e produção da enzima. As melhores condições de cultivo foram 0,6 L/min de ar, 33 °C e 10 log 10 UFC/g de substrato para a concentração celular do inóculo, em resíduo fibroso de soja como substrato. A determinação das condições de extração para a efetiva recuperação da TGase produzida em CES foi realizada através do emprego de ferramentas de planejamento experimental. A melhor condição de extração da enzima foi quando utilizou-se água a 7 ºC como solvente, por 5 minutos, 250 rpm e uma relação de sólidos/líquidos de 1:6. Caseína, proteína isolada de soja e proteína hidrolisada de carne foram tratadas com essa TGase microbiana. A redução do número de grupos aminos livres após o tratamento com a enzima, principalmente, na caseína, demonstrou a formação de ligações cruzadas catalisadas por essa TGase. As propriedades emulsificantes dessas proteínas foram melhoradas após o tratamento com a TGase do B. circulans BL32. Além disso, a fim de investigar o mecanismo de inativação térmica, incubou-se a enzima por diferentes períodos de tempo em temperaturas entre 30 e 70 °C. As cinéticas de termoinativação desta TGase seguiram o modelo de Lumry-Eyring e a enzima mostrou-se estável até 50 °C, sendo que após 12 h nessa temperatura a mesma ainda mantém 50 % da sua atividade enzimática. Os resultados sugerem que esta TGase microbiana apresenta um grande potencial de uso em aplicações alimentícias e não alimentícias. / Transglutaminase (TGase; protein-glutamine γ-glutamyltransferase; EC 2.3.2.13) is an enzyme capable of catalyzing acyl transfer reactions by introducing covalent cross-links between proteins. Microbial TGases have found widespread and growing applications in the food and non-food industry. In this work, an effort has been made in order to increase available knowledge about microbial TGases. Medium composition for TGase production on submerged cultivations by Bacillus circulans BL32, a recently isolated strain from the Amazon basin, was optimized using a stepwise strategy. The optimization of the medium resulted not only in a 60 % higher TGase activity than the obtained in a media previously cited in the literature but also in a reduction of constituents costs. Statistical experimental methods also were used to optimize the temperature and pH parameters. The best culture conditions for TGase production by B. circulans BL32 were 30 °C, pH 8.5 and the highest production was obtained in late-stationary culture phase. And besides, the effects of agitation and aeration on TGase production and cell sporulation on submerged cultivations were studied. The results demonstrated that the optimal process conditions are different for biomass and spore production. It was adopted a two-stage aeration rate control strategy with biomass production under the growth conditions in the first stage followed by the second stage under the conditions for sporulation. The present work also dealt with the TGase production in solid state cultivations. Several agro-industrial residues were used as substrates for microbial growth and enzyme production. The best culture conditions were determined as being 0.6 L air min-1, 33 °C and 10 log 10 CFU g-1 of dried substrate to the inoculum cell concentration, on industrial fibrous soy residue as substrate. The optimization of downstream processing parameters for the effective enzyme recovery of the cultivated solids was carried out. The optimal conditions for the extraction were: water as solvent at 7 ºC; 5 min of extraction time; agitation speed of 250 rpm; and 1:6 solid:liquid ratio. Casein, soy protein isolated, and hydrolysed animal protein were treated with this microbial TGase. The decrease in the amount of free amino groups after TGase treatment, mainly in the casein, demonstrated the cross-linking catalyzed by this enzyme. The emulsifying properties of these proteins were improved after treatment with B. circulans BL32 TGase. Furthermore, in order to investigate the mechanism of thermal inactivation, the enzyme was incubated at temperatures ranging from 30 to 70 °C. The thermoinactivation kinetics of this microbial TGase followed a Lumry-Eyring model. The enzyme presented good stability until 50 °C. About 50 % of the activity still remained after heating for 12 h in this temperature. Results presented in this work suggest that this microbial TGase exhibit some interesting properties for food and non-food industrial applications.