A desnutrição acomete 925 milhões de pessoas em todo o mundo, independentemente da idade e classe social, os mais acometidos são indivíduos hospitalizados, crianças e idosos. A desnutrição causa alterações fisiológicas em diversos tecidos. O tecido hematopoiético é afetado na desnutrição protéica, por ser um tecido de elevada e constante necessidade de proteínas, levando a alterações hematológicas como anemia e leucopenia. Estudos do nosso laboratório têm demonstrado in vivo, alterações do microambiente hematopoiético, em camundongos submetidos à desnutrição protéica, bem como hipoplasia medular, mudanças quantitativas da matriz extracelular (MEC) como o aumento do deposito de fibronectina na região subendosteal (local da fixação das células tronco/ progenitoras hematopoiéticas), alterações do ciclo celular das células tronco/ progenitoras hematopoiéticas e alteração na expressão de VLA5; principal integrina na interação das células a fibronectina. Sendo assim propõe-se neste projeto avaliar possíveis isoformas da molécula de fibronectina, para melhor compreender o seu papel biológico no ciclo celular as células tronco/ progenitoras hematopoiéticas em um modelo de desnutrição protéica. Para isso foram utilizados camundongos C57BLI/6J machos, adultos, mantidos em gaioleiros metabólicos, separados em dois grupos. O grupo controle recebeu uma ração normoproteíca com 12% de proteína e o grupo desnutrido, uma ração hipoprotéica com 2% de proteína, num período de 5 semanas. Após este período os animais foram sacrificados para avaliação hematológica, celularidade da medula óssea, quantificação e pesquisa de isoformas da fibronectina, análise do perfil protéico no estroma medular em culturas de longa duração bem como a análise do estabelecimento do estroma medular em culturas de longa duração. Os animais desnutridos apresentaram uma menor celularidade e uma diminuição significativa de células jovens na medula óssea. O estroma medular estabelecido em culturas de longa duração dos animais do grupo desnutrido apresentaram uma menor confluência em relação ao grupo controle. Também foi observado nas culturas de longa duração um aumento significativo da fibronectina no 28° dia de cultura, mas com uma diminuição da fibronectina no 35° dia, porém com uma menor quantidade de região EDA nos dois períodos analisados (sítio de ligação das integrinas a fibronectina) quando comparados aos animais do grupo controle. A pesquisa de isoformas de fibronectina no estroma medular, por meio de RT-PCR, revelou que tanto os animais do grupo controle quanto os animais do grupo desnutrido apresentaram diferentes isoformas de fibronectina, porém não foi possível fazer uma análise quantitativa das regiões de splicing alternativo. O perfil protéico das culturas de longa duração analisado por meio de eletroforese bidimensional demonstrou que os animais do grupo desnutrido possuem um perfil protéico diferente dos animais do grupo controle, também foi observado uma diferença do perfil protéico entre os 28° e 35° dias de cultura. Portanto a alteração quantitativa da molécula de fibronectina e da região EDA, bem como a presença de diferentes isoformas de fibronectina, juntamente com as alterações do perfil protéico podem ser devido a um aumento e ou degradação das proteínas de matriz extracelular. E estas alterações podem ser responsáveis pela hipoplasia medular e alteração do ciclo celular das células tronco/ progenitoras hematopoiéticas e estromais, talvez pela menor interação com as integrinas, sendo esta interação fundamental para a modulação de diversas funções celulares tais como proliferação e diferenciação e para a regulação do remodelamento da matriz extracelular. Mas ainda se faz necessário a quantificação das regiões de splicing alternativo, seqüênciamento das proteínas da matriz extracelular e a identificação das possíveis metalaproteinases presentes no estroma medular para melhor elucidar as funções da fibronectina e outras proteínas da matriz extracelular na manutenção da hemopoese. / Malnutrition affects 925 million people worldwide, regardless of age and social class, the most affected individuals are hospitalized, children and elderly.Malnutrition causes physiological changes in various tissues.The hematopoietic tissue is affected in protein malnutrition, because it is a tissue of high and constant need of proteins, leading to hematological abnormalities such as anemia and leucopenia. Studies from our laboratory have demonstrated in vivo changes in the hematopoietic microenvironment in mice submitted to protein malnutrition, and bone marrow hypoplasia, quantitative changes of the extracellular matrix (ECM) as the increase in deposit fibronectin in the region subendosteal (site of attachment of the cells stem / progenitor), changes in cell cycle of stem cells / progenitor and changes in the expression of VLA5; main integrin interaction of cells to fibronectin. Therefore this project proposes to evaluate possible isoforms of fibronectin molecule, to better understand its biological role in cell cycle stem cells / progenitor in a model of protein malnutrition. Mice were used for this C57BLI/6J male adults were kept in metabolic gaioleiros, separated into two groups. The control group received a ration normoproteíca with 12% protein and the malnourished group, a low protein diet with 2% protein over a period of 5 weeks. After this period the animals were sacrificed for hematological evaluation, bone marrow cellularity, and quantification of fibronectin isoforms research, analysis of protein profiles in marrow stroma in long-term cultures and analysis of the establishment of bone marrow stroma in long-term cultures. The malnourished animals showed a lower cellularity and a significant decrease of young cells in the bone marrow.The bone marrow stromal cultures established in long-term animal malnourished group had a lower convergence in the control group. It was also observed in cultures long term a significant increase in fibronectin on the 28th day of culture, but with a decrease in fibronectin 35 th day, but with a smaller amount of EDA region in both periods analyzed (binding site of integrins to fibronectin) compared to the control group.The survey of isoforms of fibronectin in bone marrow stroma, by RT-PCR revealed that both control animals and animals in the malnourished group presented different isoforms of fibronectin, but it was not possible to make a quantitative analysis of the regions of alternative splicing.The protein profile of long-term cultures analyzed using two-dimensional electrophoresis showed that animals of the malnourished group have a protein profile different from the control group was also observed a difference in protein profile between 28° and 35° day of culture. Therefore, the quantitative change of the molecule fibronectin and the region of EDA, as well as the presence of different isoforms of fibronectin, together with changes in protein profile may be due to an increase, or degradation of extracellular matrix proteins. And these changes may be responsible for bone marrow hypoplasia and alteration of the cell cycle of stem cells / progenitor and stromal cells; perhaps because of less interaction with integrins, this interaction is essential for the modulation of various cellular functions such as proliferation and differentiation and to regulation of extracellular matrix remodeling. But it is still necessary to quantify the areas of alternative splicing, sequencing of the extracellular matrix proteins and identification of possible metalaproteinases present in bone marrow stroma to better elucidate the roles of fibronectin and other extracellular matrix proteins in maintaining hemopoese.

A desnutrição acomete 925 milhões de pessoas em todo o mundo, independentemente da idade e classe social, os mais acometidos são indivíduos hospitalizados, crianças e idosos. A desnutrição causa alterações fisiológicas em diversos tecidos. O tecido hematopoiético é afetado na desnutrição protéica, por ser um tecido de elevada e constante necessidade de proteínas, levando a alterações hematológicas como anemia e leucopenia. Estudos do nosso laboratório têm demonstrado in vivo, alterações do microambiente hematopoiético, em camundongos submetidos à desnutrição protéica, bem como hipoplasia medular, mudanças quantitativas da matriz extracelular (MEC) como o aumento do deposito de fibronectina na região subendosteal (local da fixação das células tronco/ progenitoras hematopoiéticas), alterações do ciclo celular das células tronco/ progenitoras hematopoiéticas e alteração na expressão de VLA5; principal integrina na interação das células a fibronectina. Sendo assim propõe-se neste projeto avaliar possíveis isoformas da molécula de fibronectina, para melhor compreender o seu papel biológico no ciclo celular as células tronco/ progenitoras hematopoiéticas em um modelo de desnutrição protéica. Para isso foram utilizados camundongos C57BLI/6J machos, adultos, mantidos em gaioleiros metabólicos, separados em dois grupos. O grupo controle recebeu uma ração normoproteíca com 12% de proteína e o grupo desnutrido, uma ração hipoprotéica com 2% de proteína, num período de 5 semanas. Após este período os animais foram sacrificados para avaliação hematológica, celularidade da medula óssea, quantificação e pesquisa de isoformas da fibronectina, análise do perfil protéico no estroma medular em culturas de longa duração bem como a análise do estabelecimento do estroma medular em culturas de longa duração. Os animais desnutridos apresentaram uma menor celularidade e uma diminuição significativa de células jovens na medula óssea. O estroma medular estabelecido em culturas de longa duração dos animais do grupo desnutrido apresentaram uma menor confluência em relação ao grupo controle. Também foi observado nas culturas de longa duração um aumento significativo da fibronectina no 28° dia de cultura, mas com uma diminuição da fibronectina no 35° dia, porém com uma menor quantidade de região EDA nos dois períodos analisados (sítio de ligação das integrinas a fibronectina) quando comparados aos animais do grupo controle. A pesquisa de isoformas de fibronectina no estroma medular, por meio de RT-PCR, revelou que tanto os animais do grupo controle quanto os animais do grupo desnutrido apresentaram diferentes isoformas de fibronectina, porém não foi possível fazer uma análise quantitativa das regiões de splicing alternativo. O perfil protéico das culturas de longa duração analisado por meio de eletroforese bidimensional demonstrou que os animais do grupo desnutrido possuem um perfil protéico diferente dos animais do grupo controle, também foi observado uma diferença do perfil protéico entre os 28° e 35° dias de cultura. Portanto a alteração quantitativa da molécula de fibronectina e da região EDA, bem como a presença de diferentes isoformas de fibronectina, juntamente com as alterações do perfil protéico podem ser devido a um aumento e ou degradação das proteínas de matriz extracelular. E estas alterações podem ser responsáveis pela hipoplasia medular e alteração do ciclo celular das células tronco/ progenitoras hematopoiéticas e estromais, talvez pela menor interação com as integrinas, sendo esta interação fundamental para a modulação de diversas funções celulares tais como proliferação e diferenciação e para a regulação do remodelamento da matriz extracelular. Mas ainda se faz necessário a quantificação das regiões de splicing alternativo, seqüênciamento das proteínas da matriz extracelular e a identificação das possíveis metalaproteinases presentes no estroma medular para melhor elucidar as funções da fibronectina e outras proteínas da matriz extracelular na manutenção da hemopoese. / Malnutrition affects 925 million people worldwide, regardless of age and social class, the most affected individuals are hospitalized, children and elderly.Malnutrition causes physiological changes in various tissues.The hematopoietic tissue is affected in protein malnutrition, because it is a tissue of high and constant need of proteins, leading to hematological abnormalities such as anemia and leucopenia. Studies from our laboratory have demonstrated in vivo changes in the hematopoietic microenvironment in mice submitted to protein malnutrition, and bone marrow hypoplasia, quantitative changes of the extracellular matrix (ECM) as the increase in deposit fibronectin in the region subendosteal (site of attachment of the cells stem / progenitor), changes in cell cycle of stem cells / progenitor and changes in the expression of VLA5; main integrin interaction of cells to fibronectin. Therefore this project proposes to evaluate possible isoforms of fibronectin molecule, to better understand its biological role in cell cycle stem cells / progenitor in a model of protein malnutrition. Mice were used for this C57BLI/6J male adults were kept in metabolic gaioleiros, separated into two groups. The control group received a ration normoproteíca with 12% protein and the malnourished group, a low protein diet with 2% protein over a period of 5 weeks. After this period the animals were sacrificed for hematological evaluation, bone marrow cellularity, and quantification of fibronectin isoforms research, analysis of protein profiles in marrow stroma in long-term cultures and analysis of the establishment of bone marrow stroma in long-term cultures. The malnourished animals showed a lower cellularity and a significant decrease of young cells in the bone marrow.The bone marrow stromal cultures established in long-term animal malnourished group had a lower convergence in the control group. It was also observed in cultures long term a significant increase in fibronectin on the 28th day of culture, but with a decrease in fibronectin 35 th day, but with a smaller amount of EDA region in both periods analyzed (binding site of integrins to fibronectin) compared to the control group.The survey of isoforms of fibronectin in bone marrow stroma, by RT-PCR revealed that both control animals and animals in the malnourished group presented different isoforms of fibronectin, but it was not possible to make a quantitative analysis of the regions of alternative splicing.The protein profile of long-term cultures analyzed using two-dimensional electrophoresis showed that animals of the malnourished group have a protein profile different from the control group was also observed a difference in protein profile between 28° and 35° day of culture. Therefore, the quantitative change of the molecule fibronectin and the region of EDA, as well as the presence of different isoforms of fibronectin, together with changes in protein profile may be due to an increase, or degradation of extracellular matrix proteins. And these changes may be responsible for bone marrow hypoplasia and alteration of the cell cycle of stem cells / progenitor and stromal cells; perhaps because of less interaction with integrins, this interaction is essential for the modulation of various cellular functions such as proliferation and differentiation and to regulation of extracellular matrix remodeling. But it is still necessary to quantify the areas of alternative splicing, sequencing of the extracellular matrix proteins and identification of possible metalaproteinases present in bone marrow stroma to better elucidate the roles of fibronectin and other extracellular matrix proteins in maintaining hemopoese.

No description available.

The function of the endometrium is to mediate implantation of the embryo. During the early stages of implantation, the endometrial stroma undergoes differentiation known as decidualization, a process critical for successful embryo implantation. The precise mechanisms involved are not clearly understood but extracellular matrix (ECM) remodelling is a key feature. Fibronectins (FNs) are large glycoproteins abundant in the ECM of the human endometrium. Up to twenty isoforms of FNs are generated from alternative splicing, including the EDIIIA+ and EDIIIB+ variants. This thesis investigated changes in endometrial stromal ECM levels, in particular FN and its splice variants, during decidualization and in response to the endometrial cytokines and growth factors that drive the implantation process. Furthermore, the influence of these splice variants on the functional properties of FN was explored, including cell attachment, spreading and proliferation, integrin binding and focal adhesion kinase activation. Structural studies including crystallization trials were carried out to investigate how the insertion of EDIIIA modulates the conformation of FN and accessibility of its integrin binding sites. These combined studies allow us to test the hypothesis that the regulation of alternative splicing provides a biological mechanism for modulating function of FN in the endometrium. The main findings from this study can be summarized as follows: Immunocytochemistry and immunoblotting demonstrated reduced endometrial stromal levels of EDIIIA+FN, total FN and tenascin during in vitro decidualization. Substrate-associated FN production by endometrial stromal cells was reduced in response to the endometrial cytokine TNFalpha as detected by ELISA. Recombinant FIII7-12A±B± fragments were expressed, purified and mediated endometrial stromal cell adhesion. Inclusion of EDIIIA in the recombinant FIII7-12 fragment decreased binding affinities to integrin alpha5beta1. These findings suggest that production of FNs in the endometrial stroma is modified during in vitro decidualization and in response to endometrial TNFalpha. This modification in ECM composition is likely to result in modulation of cellular processes, perhaps to allow for cellular differentiation and migration that is required for invasion of the implanting embryo.

Diseases involving fibrosis cause tens of thousands of deaths per year in the US alone. These diseases are characterized by a large amount of extracellular matrix, causing stiff abnormal tissues that may not function correctly. To take steps towards curing these diseases, a fundamental understanding of how cells interact with their substrate and how mechanical forces alter signaling pathways is vital. Studying the mechanobiology of cells and the interaction between a cell and its extracellular matrix can help explain the mechanisms behind stem cell differentiation, cell migration, and metastasis. Due to the correlation between force, extracellular matrix assembly, and substrate stiffness, it is vital to make in vitro models that more accurately simulate biological stiffness as well as measure the amount of force and extracellular matrix assembly. To accomplish this, blends of two types of poly(dimethylsiloxane) (PDMS) were made and the material properties of these polymer blends were characterized. A field of 5µm or 7µm microscopic pillars (referred to as posts) with a diameter of 2.2µm were fabricated from these blends. Each combination of PDMS blend and post height were calibrated and the stiffness was recorded. Additionally, polymer attachment experiments were run to ensure cells survived and had a normal phenotype on the different blends of PDMS when compared to pure PDMS. Finally, cells were placed onto a field of posts and their forces were calculated using the new stiffness found for each blend of post. Varying the PDMS material stiffness using blends allow posts to be much more physiologically relevant and help to create more accurate in vitro models while still allowing easy and accurate force measurement. More biologically relevant in vitro models can help us acquire more accurate results when testing new drugs or examining new signaling pathways.

In the process of pre-mRNA splicing introns are removed from pre-mRNA and exons are joined together. Current studies show, that about 95 % of genes, which contain more than two exons, can undergo alternative splicing. In this process some exons are included in or excluded from the final mRNA. Majority of pre-mRNA splicing take place co- transcriptionaly at this time RNA polymerase II is still attached to pre-mRNA. Alternative splicing is complex process that takes place in a close proximity of DNA and histones that might modulate alternative splicing decisions. Futher studies have validated fibronectin gene (FN1) and his alternative exons EDA and EDB (extra domain A and B) as suitably model for studying alternative splicing. Study using FN1 minigene reporter system, which is composed from EDA exon and two surrounding introns and exons, has proved that insertion of transcription enhancer SV40 infront of promotor, the level of EDA inclusion is decreased. So far, has not been prooved if this mechanism can function in real genome context and if distal transcription elements can influence alternative splicing. In this study, we have predicted transcription enhancer for FN1 gene by using The Ensemble Regulatory Build and FANTOM 5. The predicted transcription enhancer, is located 23,5 kbp upstream of TSS...

O estudo de macrointeraçães na matriz extracelular (ECM) foi abordado em três modelos; a interação colágeno/colagenase, a interação colágeno/fibronectina e finalmente, a evolução da família enzimática das metaloproteinases de matriz (MMP). As MMP se caracterizam por sua notável especificidade contra componentes da ECM. As colagenases intersticiais, os membros mais estudados, clivam os colágenos intersticiais em um único ponto da molécula produzindo dois fragmentos característicos. Usando a hipótese da hidropaticidade complementar, a qual estipula que peptídeos codificados por sequências nucleotídicas complementares podem interagir entre si, foi possível caracterizar a sequência SQNPVQP em colagenase de fibroblasto ou SSNPIQP em colagenase de neutrófilo como importantes na interação das respectivas enzimas com o colágeno nativo. O fato da fibronectina, outro componente da ECM, ligar-se no mesmo domínio do colágeno clivado pelas colagenases possibilitou a utilização da mesma abordagem acima (hidropaticidade complementar) no estudo da interação colágeno/fibronectina. Sendo assim, foi possível caracterizar a sequência TNEGVMY da fibronectina como importante na interação com o colágeno. Adjacente a este sítio na fibronectina, existe uma sequência (AAHEEIC) que é homóloga ao sítio de ligação ao zinco presente nas MMP. Isto originou a possibilidade de que zinco pudesse modular a interação colágeno/fibronectina. De fato, zinco aumentou especificamente a ligação entre colágeno e fibronectina. Finalmente, abordou-se alguns aspectos filogenéticos da família das MMP. Foi caracterizada uma relação filogenética entre o núcleo enzimático das MMP e o domínio correspondente tanto na protease de Serratia como na protease B de Erwinia chrysanthemi, membros da mesma família de metaloproteinases bacterianas. Sendo assim, a atividade catalítica das MMP pode ter sido herdada das metaloproteinases bacterianas, enquanto a especificidade ao substrato talvez se constitua em uma aquisição das MMP eucarióticas. / The study of extracellular matrix (ECM) macrointeractions was approached in three models: the collagen/collagenase interaction, the collagen/fibronectin interaction and the molecular evolution of the matrix metalloproteinase (MMP) family of enzymes. MMP is characterized by its remarkable specificity against ECM components. Interstitial collagenases, the best studied members, attack interstitial collagens at a unique site producing two fragments. Using the complementary hydropathy hypothesis that states that peptides encoded by complementary nucleotide sequence can interact one to another, it was possible to characterize the sequence SQNPVQP (in fibroblast collagenase) or SSNPIQP (in neutrophil collagenase) as important in the interaction of the respective enzymes with collagen. The fact that fibronectin, another ECM component, binds at the same domain on collagen that is cleaved by collagenases arose the possibility of using the same approach (complementary hydropathy) in the study of collagen/fibronectin interaction. It was possible to characterize the fibronectin sequence TNEGVMY as important in the interaction with collagen. Adjacent to this site, there is a sequence (AAHEEIC) that shows an homology to the zinc-binding site present in several MMP. Therefore, zinc could be a modulator the collagen/fibronectin interaction. Finnaly, some phylogenetic aspects of the MMP family were studied. It was characterized a phylogenetic relationship between the catalytic core of MMP and the corresponding domain in Serratia protease and protease B from E. chrysanthemi, members of the same family of bacterial metalloproteinases. The catalytic activity of MMP can have evolved from the bacterial metaloproteinases whereas the substrate specificity is an acquisition of eukaryotic MMP

Clostridium difficile est une bactérie entéropathogène responsable de diarrhées nosocomiales post-antibiotiques et de colites pseudomembraneuses. Ces dernières années, l'incidence et la gravité des infections à C. difficile ont significativement augmenté en Amérique et en Europe. Cette évolution semble être liée à l'émergence puis à la dissémination très rapide d'un clone particulièrement virulent de PCR-ribotype 027. Les facteurs de virulence majeurs de C. difficile sont les toxines TcdA et TcdB qui sont responsables des lésions intestinales. Cependant, l’étape de colonisation de l’intestin par la bactérie est considérée comme un pré-requis à l’infection. Afin de mieux comprendre les mécanismes d’hypervirulence de la souche 027, nous nous sommes focalisés sur l’étude du processus de colonisation intestinal de cette souche en le comparant à celui de la souche non épidémique 630∆erm. Dans un premier temps, nous avons étudié le rôle de la protéine de liaison à la fibronectine FbpA. La caractérisation in vitro et in vivo d’un mutant d’inactivation de fbpA, nous a permis de montrer l’implication de cette protéine dans le processus de colonisation de la souche non épidémique 630∆erm. La difficulté à obtenir un mutant dans la souche épidémique 027 R20291 ne nous a pas permis de comparer les propriétés adhésives de FbpA entre les deux souches. Dans un deuxième temps, nous avons étudié les caractéristiques des protéines flagellaires FliC, FliD, FlgE et MotB. Nous avons montré que les flagelles agissent en tant qu’adhésines chez la souche 027 et que ce rôle est moins important chez la souche 630∆erm. Nous avons également montré que les flagelles sont impliqués dans des processus cellulaires autres que l’adhésion et la colonisation. Selon une étude transcriptomique d’un mutant ∆FliC de la souche 027 R20291, il s’est avéré que la flagelline est aussi impliquée dans la production de toxines, la sporulation et dans l’adaptation de la bactérie aux conditions de stress. Une étude complémentaire serait nécessaire afin de mieux comprendre le système de régulation qui régi ces différents processus cellulaires.Finalement, nous avons effectué une analyse transcriptomique de la cinétique de colonisation in vivo de la souche 027. L’étude a révélé l’expression précoce des gènes de toxines et de sporulation au cours du processus d’infection. Elle nous a également permis d’identifier des gènes spécifiques à la souche 027 qui sont exprimés lors du processus infectieux. Ces gènes pourraient éventuellement être impliqués dans la virulence de C. difficile 027 et pourraient constituer de nouvelles pistes d’étude. / Clostridium difficile is an enteropathogenic bacterium that causes post-antibiotic nosocomial diarrhea and pseudomembranous colitis. During the last decade, the incidence and the severity of C. difficile infections have significantly increased in America and Europe. This evolution seems to be related to the emergence and to the rapid dissemination of a particularly virulent clone of PCR-ribotype 027. The main virulence factors of C. difficile are the TcdA and TcdB cytotoxins which are responsible for intestinal lesions. However, the intestinal colonization by the bacterium is considered as an indispensible step for infection.To better understand the hypervirulence mechanisms of strain 027, we focused on the study of intestinal colonization process of this strain compared to the colonization process of the non-epidemic strain 630Δerm. First, we studied the role of the fibronectin binding protein FbpA. In vitro and in vivo characterization of a mutant FbpA showed the involvement of this protein in the colonization process of the non-epidemic strain 630Δerm. The difficulty of obtaining a mutant in the epidemic strain R20291 027 does not allow us to compare the adhesive properties of FbpA between the two strains.In a second step, we studied the characteristics of flagellar proteins FliC, FliD, FlgE and MotB. We showed that the flagella have a role in the adhesion and colonization of strain 027 and that this role is less important in strain 630Δerm. We also showed that flagella are involved in other cellular processes than adhesion and colonization. A transcriptomic study of a FliC mutant in 027 R20291 shows that flagellin is also involved in toxin production, sporulation and in the adaptation of bacteria to stress conditions. Further study should be performed to better understand the regulation system that governs these different cellular processes. Finally, we performed a transcriptomic analysis of the kinetic of in vivo colonization of the 027 R20291 strain. The study revealed a very early expression of toxin and sporulation genes during the first stages of the infection process. This analysis also allowed us to identify some genes, specific to 027 strains, which appeared regulated during the infection process. These genes could be involved in the virulence of C. difficile 027 strains and could provide new issues of study to better understand C. difficile virulence.

Human embryonic stem cells are pluripotent cells that have indefinite replicative potential and ability to differentiate into derivatives of three germ layers. HESCs are conventionally derived and grown on mitotically inactivated mouse embryonic fibroblasts and there are some alternative feeder types of human origin that have been used to replenish hESCs while trying to prevent cross-species contamination. The trophic factors that are secreted by the feeders are found to be important for long-term pluripotency but there are also supportive culture systems for hESCs lacking feeder cells which might suggest that not only the interactions with the feeders affect the behaviour of hESCs but also the components of the niche may take part in the decision of self-renewal or differentiation. Extracellular matrix components are known to exert their stimulatory or inhibitory effects by localising cells into a specific microenvironment in natural niches but have been relatively little investigated for hESCs. The aim of this study was to investigate ECM components which might have a role in the maintenance of hESCs. I have first investigated human placental stromal fibroblasts and immortalised human placental stromal fibroblasts for the support hESC pluripotency as an anlternative feeder type to conventional mouse embryonic fibroblasts. Secondly, the matrices derived from hPSFs and ihPSFs were assessed for their ability to support hESC pluripotency. Tandem mass spectrometry was used to identify ECM components released by human feeders in order to characterise the range of extracellular matrix proteins that support the growth of self-renewing hESCs. The majority of the molecules was shared between the cell types irrespective of hPSF cell derived matrix was not being supportive for hESC pluripotency, with some ECM components being unique ihPSFs. Collagen VI, tenascin C and versican were tested for hESC attachment and as substrates for feeder-free culture system in order to develop an optimised feeder-free system. Furthermore, integrin receptor profile of different hESC lines was also determined in order to identify the mechanisms of substrate attachment. Integrin attachment was shown to be vital for hESC engagement to fibronectin and vitronectin in feeder-free systems. The components of the integrin signalling machinery were identified in hESCs and the significance of integrin-mediated signalling in hESC self-renewal was demonstrated by blocking integrin β1 on fibronectin and integrin aVβ5 on vitronectin. Moreover, intracellular signalling mediator c-Src was shown to involve in ECMregulated signalling by affecting the phosphorylation of Focal Adhesion Kinase. Inhibition of Src led to a decrease in the expression of pluripotency-associated markers. Finally, the effects of growth factor supplementation on the maintenance of pluripotency in defined feeder-free conditions were studied by withdrawal of growth factors and blocking FGF Receptors. FGF-2 was shown to be essential for long-term self-renewal while the effects on pluripotency deteriorated in the absence of both FGF-2 and Activin A. Taken together this project highlighted the importance of substrate attachment and growth factors on the regulation of hESC self-renewal.

Estudamos a ação das proteases bothropasina e Bothrops protease A, do veneno da serpente Bothrops jararaca, sobre o fibrinogênio (FBG), fibronectina (FN) e cininogênio (HK), como ferramenta para geração de peptídeos bioativos. As sequências primárias dos produtos de digestão foram identificadas por espectrometria de massas, com as buscas direcionadas por peptídeos em comum gerados pelas duas proteases. Foram encontradas oito sequências em comum provenientes do FBG e onze, da FN. Apenas a bothropasina clivou o HK, liberando desArg9BK. Foram sintetizados peptídeos derivados do FBG (FBG1-6) e da FN (FN1-4), além de des-Arg9-BK. Oito peptídeos apresentam potencial atividade antiangiogênica predita in silico. Observamos a inibição da elastase (28-20%) causada por FBG1-2-5-6. A melhor inibição da trombina foi de 17%, por FBG1. Contudo, a maioria dos peptídeos intensificou sua atividade. Por fim, este trabalho sugere que as proteases de veneno de serpentes podem ser usadas como ferramentas para processar componentes do plasma, visando à busca por peptídeos bioativos. / We studied the action of the proteases bothropasin and Bothrops protease A purified from the venom of snake Bothrops jararaca upon fibrinogen (FBG), fibronectin (FN) and kininogen (HK), as a tool to generate bioactive peptides. The primary sequences of the digestion products were identified by mass spectrometry and we focused the search for common peptides released by both proteases simultaneously. Sequences in common released by both proteases were found, being eight peptides from FBG, and 11 from FN. Only bothropasin was able to cleave HK releasing des-Arg9-BK. Peptides from fibrinogen (FBG1-6) and from fibronectin (FN1-4), as well as the des-Arg9-BK were synthetized. Eight peptides have potential antiangiogenic predicted in silico. We observed the inhibition of elastase (28-20%) caused by FBG1-2-5-6. The best inhibition of thrombin was 17% by FBG1. However, most of the peptides intensified its activity. Finally, this work suggests that the snake venom protease can be used as tools to process plasma components in order to search for bioactive peptides.