A lagarta da espécie Lonomia obliqua é medicamente importante, cujo veneno, presente nas espículas, causa uma síndrome hemorrágica caracterizada por equimoses, alterações da coagulação, dentre outros sintomas. Isto sugere a presença de peptídeos bioativos com potencial farmacêutico, devido à capacidade de modular o comportamento das células endoteliais. O objetivo deste estudo é analisar os potenciais efeitos do veneno de Lonomia obliqua na angiogênese. Uma linhagem celular endotelial (HUVEC) foi exposta a diferentes concentrações do extrato de espículas da Lonomia obliqua (Lonomia obliqua Bristle extract - LOBE) 5 μg/mL, 10 μg/mL, 20 μg/mL e 50 μg/mL. Empregando citometria de fluxo, observou-se que nenhuma das doses afetou o ciclo celular, viabilidade ou apoptose das células endoteliais após 24h de exposição. Os esferóides das células HUVEC foram plaqueados numa matriz 3D de colágeno e observou-se que LOBE (10 μg/mL, 20 μg/mL e 50 μg/mL) induz um aumento na migração celular, consistente com o processo de angiogênese. A análise da dinâmica da VE-caderina indica que a exposição imediata a LOBE (10 μg/mL) induz um desprendimento da junção célula-célula, o que corrobora com a hemorragia observada nas vítimas de envenenamento. Através de espectrometria de massa, observou-se que LOBE possui vários potenciais peptídeos bioativos. Grupos destes peptídeos foram isolados por fracionamento com metanol a partir do veneno bruto. Os peptídeos presentes, em cada uma das 10 frações, foram caracterizados por espectrometria de massa e foram analisados os efeitos de cada fração sobre a angiogênese. Os resultados sugerem que alguns dos efeitos do envenenamento por Lonomia obliqua são devidos à presença de peptídeos bioativos que modulam o comportamento das células endoteliais. / The caterpillar of the species Lonomia obliqua is medically important, whose venom present in the bristles leads to an hemorrhagic syndrome characterized by ecchymosis, coagulation disorders and others symptoms. This suggests the presence of bioactive peptides with pharmaceutical potencial due to the ability to modulate the behavior of endothelial cells. The aim of this study is to analyze the potential effects of Lonomia obliqua venom on angiogenesis. An endothelial cell line (HUVEC) was exposed to different concentrations (5 μg/mL, 10 μg/mL, 20 μg/mL and 50 μg/mL) of Lonomia obliqua bristle extract (LOBE). Using flow cytometry, it was observed that none of the doses affected endothelial cell cycle, cell viability or apoptosis after 24h of exposition. Spheroids of HUVEC cells were plated in a 3D-collagen matrix and it was observed that LOBE (10 μg/mL, 20 μg/mL and 50 μg/mL) induced an increase on cell migration consistent with the angiogenesis process. Analysis of VE-cadherin dynamics indicates that the immediate exposition to LOBE (10 μg/mL) induced a loosening of cell-cell junction, which corroborates with the hemorrhage observed in the victims. By mass spectroscopy, it was observed that LOBE possesses several potentially bioactive peptides. Groups of these peptides were isolated by a methanol-based fractioning of the crude venom. The peptides present in each of the 10 fractions were characterized by mass spectroscopy and it was analyzed the effects of each fraction on angiogenesis. The results suggest that some of the effects of Lonomia obliqua envenomation are due to the presence of bioactive peptides that modulate the behavior of endothelial cells.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A esporotricose é uma doença micótica, infecciosa e crônica, que envolve o tecido cutâneo e subcutâneo, e que pode afetar seres humanos e animais. Esta micose sempre foi atribuída a um único patógeno, o Sporothrix schenckii, um fungo termodimórfico, que cresce como levedura a 37 C e como micélio à temperatura ambiente. No entanto, nos últimos anos, foi demonstrado que isolados identificados como S. schenckii apresentavam grande variabilidade genética, sugerindo que este táxon consiste em um complexo de espécies. Esta doença é causada pela implantação traumática do patógeno fúngico, porém, os mecanismos de invasão e disseminação deste microorganismo, bem como as moléculas envolvidas nestes processos, ainda são pouco conhecidos. Com base nessas informações, este trabalho visa identificar moléculas de superfície deste patógeno envolvidas na interação deste fungo com proteínas matriciais, bem como analisar diferenças fenotípicas entre espécies do denominado complexo Sporothrix. Foram utilizados, neste estudo, cinco isolados de Sporothrix spp., sendo três isolados clínicos, um isolado ambiental e um isolado de gato. A virulência de cada isolado foi comparada à capacidade adesiva à proteína matricial fibronectina. Foi observado que os isolados com maior capacidade infectiva eram os que apresentavam maior capacidade adesiva à fibronectina. Verificamos então a expressão de adesinas para fibronectina na superfície de cada isolado, por Western blot, e observamos que os isolados mais virulentos e com maior capacidade adesiva expressavam mais adesinas para fibronectina. Bandas reativas com o anticorpo monoclonal contra adesina gp70 (mAb P6E7) foram reveladas nos extratos de parede celular dos isolados estudados. Análises por microscopia confocal revelaram a co-localização da gp70 com a adesina para fibronectina na superfície dos isolados. Análises filogenéticas demonstraram que os isolados estudados possuíam diferenças genotípicas capazes de agrupá-los em duas espécies, S. schenckii e S. brasiliensis. Esta análise revelou que o isolado avirulento era S. brasiliensis e não S. schenckii, como se pensava. Este dado novo nos levou a verificar se a virulência e as características fenotípicas estariam relacionadas ao genótipo. A avaliação da virulência mostrou que outro isolado de S. brasiliensis era tão virulento quanto os isolados de S. schenckii. Além disso, as características morfológicas, como tamanho, forma e perfil de crescimento, das fases miceliana e leveduriforme, e características microscópicas da parede das leveduras também foram avaliadas. Porém, não foi possível correlacionar, de forma clara, a morfologia celular com a especiação do gênero Sporothrix. A expressão da gp70 na superfície das duas espécies foi verificada e foi observado que o isolado virulento de S. brasiliensis quase não expressa a gp70 na sua superfície em contraste com o isolado avirulento de S. brasiliensis, que além de expressar esta glicoproteína em grande quantidade ainda a libera para o meio extracelular. Este estudo mostra que há uma correlação direta entre virulência e expressão de adesinas, porém, sem qualquer relação entre características fenotípicas e genótipo. / Sporotrichosis is a chronic and infectious disease that involves the cutaneous and subcutaneous tissue, which can affect humans and animals. This mycosis has always been attributed to a single pathogen, the Sporothrix schenckii, a dimorphic fungus, that grows as yeast at 37 C and as mycelia at room temperature. However, in recent years, some isolates identified as S. schenckii showed considerable genetic variability, suggesting that this taxon consists of a complex of species. This disease is caused by the traumatic inoculation of the fungal pathogen, however, the molecules involved in the invasion and dissemination of this microorganism are still poorly understood. The aim of this study is to identify surface molecules involved in the interaction of this fungus with extracellular matrix proteins and to examine phenotypic differences between species in the Sporothrix complex. Five isolates were used throughout this study, three clinical isolates, an environmental and one cat isolate. The virulence of each isolate was compared to the adhesive capacity to fibronectin. We observed that the most virulent isolates exhibited the higher capacity to interact with fibronectin. The expression of adhesins for fibronectin on the surface of each isolate was verified by Western blot. This analysis showed that the most virulent isolates expressed more fibronectin adhesins than the avirulent ones. Positive bands for the monoclonal antibody raised against gp70 adhesin (mAb P6E7) were revealed in cell wall extracts of the isolates studied. Confocal microscopy confirmed the colocalization of fibronectin and mAb P6E7 on the yeast cell surface. Molecular analysis showed genotypic differences between isolates used in this study, that can cluster than them into two species, S. schenckii and S. brasiliensis. This phylogenetic analysis revealed that the avirulent isolate was S. brasiliensis and not S. schenckii as previously thought. This new data led us to determine whether the virulence and phenotypic characteristics were related to genotype. The virulence analysis showed that another S. brasiliensis isolate was as virulent as the S. schenckii isolates. Moreover, morphological characteristics, such as, size, shape and growth profile of mycelial and yeast were also evaluated. However, no connection was observed between cell morphology with the speciation of the genus Sporothrix. The cell wall expression of gp70 was evaluated in the twou species. We observed that the virulent isolate of S. brasiliensis almost do not express gp70 in contrast with the avirulent isolate of the same specie. This study shows that there is a direct correlation between virulence and adhesins expression, however, no relationship between genotype and phenotypic characteristics was observed.

L'attention de la communauté scientifique, ainsi que le développement, pour les bioprocédés dédiés à la culture et à l'expansion de cellules souches mésenchymateuses (MSCs) pour la thérapie cellulaire et la médecine régénérative a considérablement grandi pendant ces dernières décennies. Une plus ample compréhension du lien entre la structure, la fonction et les propriétés des suspensions de cellules mésenchymateuses est devenue de première importance. Dans cette thèse, nous présentons tout d'abord les résultats d'une étude expérimentale portant sur l'écoulement de suspensions concentrées de cellules vivantes d'origine mésenchymateuse pour une grande gamme de concentration cellulaire. Nous caractérisons l'évolution de la viscosité relative en fonction de la contrainte de cisaillement appliquée pour des fractions volumiques cellulaires allant de 20 à 60%. Ces matériaux ont des empreintes rhéologiques compliquées mais très reproductibles, incluant des comportements de fluide à seuil, rhéofluidifiants ainsi que des fractures liées à la contrainte de cisaillement. Les propriétés rhéologiques de la suspension sont ensuite étudiées avec l'addition d'acide hyaluronique (HA), une biomolécule avec des séquences d'adhésion pour des récepteurs à la surface des cellules étudiées. Nous montrons que l'addition d'acide hyaluronique modifie substantiellement le comportement de la suspension et nous permet de contrôler les propriétés d'écoulement de la suspension à toutes les fractions volumiques. Cytométrie de flux et imagerie confocal à l'appui, nous montrons que l'effet observé est dû à un important changement dans la formation d'agrégats cellulaires dans la suspension, et donc dans l'envergure du réseau correspondant. La troisième partie de cette thèse porte sur l'ajout de polyéthylène glycol, une molécule qui n'est pas naturellement présente dans l'organisme mais fréquemment utilisée dans la formulation d'hydrogel. En utilisant trois types de PEG, l'influence de la charge des molécules est étudiée. Les résultats montrent que la charge est un paramètre important dans le contrôle des propriétés d'écoulement de suspensions cellulaires, car déterminant dans la formation et la compacité des agrégats. En considérant les agrégats comme des objets fractals, nous montrons qu'en prenant en compte les modifications de fractions volumiques avec le cisaillement, nous pouvons obtenir une courbe maitresse pour l'ensemble des conditions testées, et en extraire la force d'adhésion moyenne entre les cellules, au travers une population de plusieurs millions de cellules. Cette étude livre de nouveaux aspects sur la complexité des propriétés en écoulement de suspensions de cellules méchymateuses, adhérentes et concentrées, sur leur sensibilité à l'ajout de molécules, qu'elles soient naturellement présentes dans les tissues ou non, ainsi qu'une nouvelle méthode pour mesurer la force d'adhésion entre les cellules. / With the rapidly growing interest in the development of bioprocess systems to culture and expand mesenchymal stromal cells (MSCs) for cell therapy and regenerative medicine applications, greater understanding of the structure-function-property characteristics of mesenchymal cell suspensions is required. In this thesis, the results of a detailed experimental study into the flow behaviour of concentrated suspensions of living mesenchymal cells over a wide range of cell concentrations and in the presence of two macromolecules (hyaluronic acid and polyethylene glycol) often used in cellular therapy applications are presented. The change in the shear viscosity as a function of shear stress and shear rate for cell volume fractions varying from 20 to 60% are firstly presented, showing that these suspensions exhibit highly complex but reproducible rheological footprints, including yield stress, shear thinning and shear-induced fracture behaviours. The rheological properties of the suspension with the addition of hyaluronic acid (HA), a biomolecule with adhesion sequences for receptors on these types of cells, was then investigated. With the addition of HA, the rheology of these cell suspensions is significantly modified at all volume fractions. Using FACS and confocal imaging, we show that the observed effect of HA addition is due to it significantly modulating the formation of cellular aggregates in these suspensions, and thus the resultant volume spanning network. This understanding permits the rheology of concentrated mesenchymal cell suspensions to be tailored to suit particular processing scenarios. The third part of this project focused on the addition of polyethylene glycol, a molecule which is not naturally present in tissues but commonly utilised in hydrogels as injectable delivery vehicles for cells to sites of tissue damage. Using three different kinds of PEG, the influence of the charge of the molecules is investigated. The results show the charge is also a crucial parameter to tailor the flow behaviour of cell suspension when biomacromolecules are added, influencing the formation and the compactness of the cellular aggregates. Considering the aggregates as fractal structures, and by taking into account the changes in volume fractions with shear, a master curve for the range of conditions investigated was successfully achieved through the use of an analytical model. Critically, this model also permitted the estimation of the average adhesion force between cells, across a population of millions of cells. The outcomes of this study not only provide new insight into the complexity of the flow behaviours of concentrated, dynamically adhesive mesenchymal cell suspensions, and their sensitivity to associative biomolecule and synthetic molecule addition, but also a novel, rapid method by which to estimate adhesion forces between cells.

Les flavonoïdes sont étudiés depuis des années pour leurs propriétés préventives et leur potentiel comme agents thérapeutiques. Plusieurs mécanismes pourraient intervenir dans leur activité anti-cancéreuse dont une inhibition de l'adhérence et de l'étalement cellulaire et une inhibition des propriétés invasives des cellules cancéreuses. Les dérivés de 3-aryl-2-quinolones sont structurellement proches des flavonoïdes et ont été caractérisés comme étant des composés anti-migratoires (Joseph et Al., J.Med.Chem, 2002). Comme la migration cellulaire est hautement dépendante des structures adhésives assemblées par la cellule, nous avons étudié l'activité de ces composés sur les adhérences focales et fibrillaires. Ces larges complexes protéiques impliquent notamment les intégrines et leurs partenaires cytoplasmiques les liant au cytosquelette. Les intégrines permettent à la cellule de percevoir son microenvironnement et de s'y adapter. Les structures d'adhérence contenant les intégrines sont en retour capables de contrôler cet environnement (dégradation matricielle, fibrillogenèse…). Nos travaux montrent que les dérivés de 3-aryl-2-quinolone sont capables d'inhiber l'étalement cellulaire et de provoquer le désassemblage de structures adhésives préalablement établies de façon dose-dépendante et indépendamment de la composition de la matrice extracellulaire. L'activité des composés est finement liée à leur structure et de légères modifications de la composition chimique de leur chaîne latérale peuvent inhiber leur activité. Nous avons pu établir une relation structure-activité pour cette famille de composés et avons étudié les mécanismes moléculaires menant au désassemblage des structures d'adhérences quand les cellules sont traitées par ces molécules. Des études par RMN ont montré une interaction directe entre le chef de file de cette famille de composés et le domaine cytoplasmique des intégrines à chaîne béta 3 et nous avons pu montrer que cette interaction inhibait la liaison sur l'intégrine de l'un de ces activateurs, la kindline. L'agrégation plaquettaire est dépendante de l'activation des intégrines et constitue un excellent système d'étude physiologique pour les inhibiteurs de ces récepteurs. Le chef de file des dérivés de 3-aryl-2-quinolone est capable d'inhiber l'agrégation plaquettaire et la formation du thrombus, ce qui en fait un bon candidat médicament comme anti-thrombotique. / Flavonoïds have been studied for years for their potential chemopreventive and chemotherapeutic action. Several mechanisms might account for their anticancer activity, among which inhibition of cell adhesion and spreading, or inhibition of tumor cell invasion. 3-aryl-2-quinolone derivatives are chemical structures close to flavonoïds and were first designed as anti-migratory agents (Joseph et Al., J.Med.Chem, 2002). As cell migration is highly dependent on the cell adhesion machinery, we decided to investigate the action of these molecules on focal and fibrillar adhesions. These large protein complexes include heterodimeric transmembrane proteins, the integrins, and their cytoplasmic interactors able to link to the cytoskeleton. Integrins allow microenvironment sensing and cellular response to it. Adhesive structures containing integrins are also able to control cell microenvironment (matrix degradation, fibrillogenesis…). Our studies show that 3-aryl-2-quinolone derivatives are able not only to prevent cell spreading but also to disrupt already well-established focal adhesions in a reversible and ECM composition independent manner. The activity of the molecule is closely linked with its structure, as very slight modification of the lateral chain of the compound can totally impair its activity. Our work is focused on establishing a Structure-Activity Relationship of 3-aryl-2-quinolone derivatives and on investigating the molecular mechanisms underlying this activity. Osteoblasts treatment by 3-aryl-2-quinolone derivatives triggers a rapid disassembly of focal and fibrillar adhesions. NMR experiments show a direct interaction between the lead compound of the family and 3 integrin cytoplasmic tail and pull-down assay show that it is able to reduce the interaction between 3 integrin and kindlin, one of its coactivator. As platelet activation is an archetype of 3 integrin activation, we tested the activity of 3-aryl-2-quinolone on this physiological process. Under treatment, platelets failed to become activated and are unable to trigger thrombus formation, providing an interest to the 3-aryl-2-quinolone derivatives as potential anti-thrombotic agents.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A esporotricose é uma doença micótica, infecciosa e crônica, que envolve o tecido cutâneo e subcutâneo, e que pode afetar seres humanos e animais. Esta micose sempre foi atribuída a um único patógeno, o Sporothrix schenckii, um fungo termodimórfico, que cresce como levedura a 37 C e como micélio à temperatura ambiente. No entanto, nos últimos anos, foi demonstrado que isolados identificados como S. schenckii apresentavam grande variabilidade genética, sugerindo que este táxon consiste em um complexo de espécies. Esta doença é causada pela implantação traumática do patógeno fúngico, porém, os mecanismos de invasão e disseminação deste microorganismo, bem como as moléculas envolvidas nestes processos, ainda são pouco conhecidos. Com base nessas informações, este trabalho visa identificar moléculas de superfície deste patógeno envolvidas na interação deste fungo com proteínas matriciais, bem como analisar diferenças fenotípicas entre espécies do denominado complexo Sporothrix. Foram utilizados, neste estudo, cinco isolados de Sporothrix spp., sendo três isolados clínicos, um isolado ambiental e um isolado de gato. A virulência de cada isolado foi comparada à capacidade adesiva à proteína matricial fibronectina. Foi observado que os isolados com maior capacidade infectiva eram os que apresentavam maior capacidade adesiva à fibronectina. Verificamos então a expressão de adesinas para fibronectina na superfície de cada isolado, por Western blot, e observamos que os isolados mais virulentos e com maior capacidade adesiva expressavam mais adesinas para fibronectina. Bandas reativas com o anticorpo monoclonal contra adesina gp70 (mAb P6E7) foram reveladas nos extratos de parede celular dos isolados estudados. Análises por microscopia confocal revelaram a co-localização da gp70 com a adesina para fibronectina na superfície dos isolados. Análises filogenéticas demonstraram que os isolados estudados possuíam diferenças genotípicas capazes de agrupá-los em duas espécies, S. schenckii e S. brasiliensis. Esta análise revelou que o isolado avirulento era S. brasiliensis e não S. schenckii, como se pensava. Este dado novo nos levou a verificar se a virulência e as características fenotípicas estariam relacionadas ao genótipo. A avaliação da virulência mostrou que outro isolado de S. brasiliensis era tão virulento quanto os isolados de S. schenckii. Além disso, as características morfológicas, como tamanho, forma e perfil de crescimento, das fases miceliana e leveduriforme, e características microscópicas da parede das leveduras também foram avaliadas. Porém, não foi possível correlacionar, de forma clara, a morfologia celular com a especiação do gênero Sporothrix. A expressão da gp70 na superfície das duas espécies foi verificada e foi observado que o isolado virulento de S. brasiliensis quase não expressa a gp70 na sua superfície em contraste com o isolado avirulento de S. brasiliensis, que além de expressar esta glicoproteína em grande quantidade ainda a libera para o meio extracelular. Este estudo mostra que há uma correlação direta entre virulência e expressão de adesinas, porém, sem qualquer relação entre características fenotípicas e genótipo. / Sporotrichosis is a chronic and infectious disease that involves the cutaneous and subcutaneous tissue, which can affect humans and animals. This mycosis has always been attributed to a single pathogen, the Sporothrix schenckii, a dimorphic fungus, that grows as yeast at 37 C and as mycelia at room temperature. However, in recent years, some isolates identified as S. schenckii showed considerable genetic variability, suggesting that this taxon consists of a complex of species. This disease is caused by the traumatic inoculation of the fungal pathogen, however, the molecules involved in the invasion and dissemination of this microorganism are still poorly understood. The aim of this study is to identify surface molecules involved in the interaction of this fungus with extracellular matrix proteins and to examine phenotypic differences between species in the Sporothrix complex. Five isolates were used throughout this study, three clinical isolates, an environmental and one cat isolate. The virulence of each isolate was compared to the adhesive capacity to fibronectin. We observed that the most virulent isolates exhibited the higher capacity to interact with fibronectin. The expression of adhesins for fibronectin on the surface of each isolate was verified by Western blot. This analysis showed that the most virulent isolates expressed more fibronectin adhesins than the avirulent ones. Positive bands for the monoclonal antibody raised against gp70 adhesin (mAb P6E7) were revealed in cell wall extracts of the isolates studied. Confocal microscopy confirmed the colocalization of fibronectin and mAb P6E7 on the yeast cell surface. Molecular analysis showed genotypic differences between isolates used in this study, that can cluster than them into two species, S. schenckii and S. brasiliensis. This phylogenetic analysis revealed that the avirulent isolate was S. brasiliensis and not S. schenckii as previously thought. This new data led us to determine whether the virulence and phenotypic characteristics were related to genotype. The virulence analysis showed that another S. brasiliensis isolate was as virulent as the S. schenckii isolates. Moreover, morphological characteristics, such as, size, shape and growth profile of mycelial and yeast were also evaluated. However, no connection was observed between cell morphology with the speciation of the genus Sporothrix. The cell wall expression of gp70 was evaluated in the twou species. We observed that the virulent isolate of S. brasiliensis almost do not express gp70 in contrast with the avirulent isolate of the same specie. This study shows that there is a direct correlation between virulence and adhesins expression, however, no relationship between genotype and phenotypic characteristics was observed.

A lagarta da espécie Lonomia obliqua é medicamente importante, cujo veneno, presente nas espículas, causa uma síndrome hemorrágica caracterizada por equimoses, alterações da coagulação, dentre outros sintomas. Isto sugere a presença de peptídeos bioativos com potencial farmacêutico, devido à capacidade de modular o comportamento das células endoteliais. O objetivo deste estudo é analisar os potenciais efeitos do veneno de Lonomia obliqua na angiogênese. Uma linhagem celular endotelial (HUVEC) foi exposta a diferentes concentrações do extrato de espículas da Lonomia obliqua (Lonomia obliqua Bristle extract - LOBE) 5 μg/mL, 10 μg/mL, 20 μg/mL e 50 μg/mL. Empregando citometria de fluxo, observou-se que nenhuma das doses afetou o ciclo celular, viabilidade ou apoptose das células endoteliais após 24h de exposição. Os esferóides das células HUVEC foram plaqueados numa matriz 3D de colágeno e observou-se que LOBE (10 μg/mL, 20 μg/mL e 50 μg/mL) induz um aumento na migração celular, consistente com o processo de angiogênese. A análise da dinâmica da VE-caderina indica que a exposição imediata a LOBE (10 μg/mL) induz um desprendimento da junção célula-célula, o que corrobora com a hemorragia observada nas vítimas de envenenamento. Através de espectrometria de massa, observou-se que LOBE possui vários potenciais peptídeos bioativos. Grupos destes peptídeos foram isolados por fracionamento com metanol a partir do veneno bruto. Os peptídeos presentes, em cada uma das 10 frações, foram caracterizados por espectrometria de massa e foram analisados os efeitos de cada fração sobre a angiogênese. Os resultados sugerem que alguns dos efeitos do envenenamento por Lonomia obliqua são devidos à presença de peptídeos bioativos que modulam o comportamento das células endoteliais. / The caterpillar of the species Lonomia obliqua is medically important, whose venom present in the bristles leads to an hemorrhagic syndrome characterized by ecchymosis, coagulation disorders and others symptoms. This suggests the presence of bioactive peptides with pharmaceutical potencial due to the ability to modulate the behavior of endothelial cells. The aim of this study is to analyze the potential effects of Lonomia obliqua venom on angiogenesis. An endothelial cell line (HUVEC) was exposed to different concentrations (5 μg/mL, 10 μg/mL, 20 μg/mL and 50 μg/mL) of Lonomia obliqua bristle extract (LOBE). Using flow cytometry, it was observed that none of the doses affected endothelial cell cycle, cell viability or apoptosis after 24h of exposition. Spheroids of HUVEC cells were plated in a 3D-collagen matrix and it was observed that LOBE (10 μg/mL, 20 μg/mL and 50 μg/mL) induced an increase on cell migration consistent with the angiogenesis process. Analysis of VE-cadherin dynamics indicates that the immediate exposition to LOBE (10 μg/mL) induced a loosening of cell-cell junction, which corroborates with the hemorrhage observed in the victims. By mass spectroscopy, it was observed that LOBE possesses several potentially bioactive peptides. Groups of these peptides were isolated by a methanol-based fractioning of the crude venom. The peptides present in each of the 10 fractions were characterized by mass spectroscopy and it was analyzed the effects of each fraction on angiogenesis. The results suggest that some of the effects of Lonomia obliqua envenomation are due to the presence of bioactive peptides that modulate the behavior of endothelial cells.

A adesão celular está ligada à formação e disseminação de metástases, a principal causa de óbito de pacientes diagnosticados com câncer. O objetivo deste trabalho foi investigar in vitro o efeito de fotossensibilizadores na adesão celular. Foram utilizadas porfirinas comerciais (PpIX, CPpI, TSPP, TMPyP e Zn(II)TMPyP) e um fotossensibilizador sintetizado através da ligação de poli-L-lisina à protoporfirina IX (PLLPpIX). A adesão celular foi estudada por RICM, técnica que permite quantificar a área de contato entre uma célula e um substrato por binarização das imagens digitais utilizando limiares apropriados. A técnica foi padronizada e revelou dois regimes de adesão celular: um limitado e outro não limitado pela quantidade de proteína de adesão adsorvida na superfície. Neste último foi observada lise celular. Todos os fotossensibilizadores estudados foram capazes de aumentar a adesão celular na ausência de irradiação comparados ao controle sem fotossensibilizador, o que não havia sido observado nos ensaios de resistência à tripsinização normalmente utilizados para estudar o efeito de fotossensibilizadores na adesão celular. Quanto maior a anfifilicidade do fotossensibilizador, maior foi o efeito na adesão, o que é explicado pela capacidade das moléculas em se intercalarem na membrana, mudando a sua rigidez. Este aumento da adesão no escuro correlaciona com a diminuição da migração segundo ensaios de ferida. A análise do padrão de expressão de integrinas na superfície celular revela que o aumento da adesão correlaciona com o aumento na expressão de αV. Quando os fotossensibilizadores estão concentrados na região perimembranar (1 minuto de incubação) e as células são irradiadas, há um aumento da adesão em relação ao controle sem fotossensibilizador, mas uma diminuição em relação ao controle tratado com o fotossensibilizador e não irradiado, o que implica que a PDT leva a uma diminuição da adesão celular e não a um aumento como reportado na literatura. Com 3h de incubação, PLLPpIX impede a adesão celular, enquanto PpIX praticamente não muda a adesão comparado ao controle não irradiado. Esta ausência do efeito da irradiação sugere que a PpIX afeta a adesão celular principalmente devido a sua intercalação na membrana e não devido à formação de espécies reativas. Com 3h de incubação os fotossensibilizadores não se encontram na membrana e, portanto, o efeito na adesão celular é indireto e também não está relacionado à diferenças na eficiência de internalização. O comportamento observado deve ter relação com diferenças de citolocalização. Outro processo que pode alterar a adesão celular é a oxidação das proteínas do soro fetal bovino. Como observado nos estudos de fotossensibilização de células, PLLPpIX foi capaz de impedir a adesão celular, diferentemente da PpIX. A maior eficiência da PLLPpIX foi associada a presença do polímero, o qual força por questões estéricas que a interação da PLLPpIX com a albumina, o componente majoritário do soro, fique restrita à superfície da proteína, deixando o fotossensibilizador disponível para interagir com o oxigênio molecular e gerar oxigênio singlete. Assim, a funcionalização com um polímero tornou a PpIX capaz de modular a adesão celular tanto agindo dentro da célula quanto na matriz extracelular. / Cell adhesion is associated to the formation and spread of metastasis, the leading cause of death in cancer patients. The aim of this study was to investigate, in vitro, the effect of photosensitizers in cell adhesion. Five commercial porphyrins (PpIX, CPpI, TSPP, TMPyP e Zn(II)TMPyP) and Protoporphyrin IX covalently tethered to poli-L-lysine (PLLPpIX) were used. Cell adhesion was mainly studied by RICM, a technique that allows quantifying the contact area between a cell and a substrate for binarization of digital images using appropriate thresholds. The technique was standardized and disclosed two systems for cell adhesion: a system limited by the amount of adhesion proteina adsorbed on the surface and another one no limited, in which cell lysis was observed. All photosensitizers were able to enhance cell adhesion in the absence of irradiation compared to control without photosensitizer, which had not been observed in the trypsinization resistance tests usually used to study the effect of photosensitizers in cell adhesion. The greater the amphiphilicity of the photosensitizer, the greater was the effect on cell adhesion. This is explained by the ability of molecules to fit in the membrane, changing its tension. This increased adhesion correlates with the decrease in migration according to wound healing assays. Analysis of the integrin expression pattern on cell surface reveals that increased adhesion correlates with increased expression of alpha V. When photosensitizers are concentrated in the perimembranar region (1 minute of incubation) and cells are irradiated, there is an increase in adhesion when compared to control without photosensitizer, but a decrease relative to controls treated with the photosensitizer without irradiation, implying that PDT leads to a reduction of cell adhesion and not to an increase as reported in the literature. With 3h of incubation PLLPpIX prevents cell adhesion, while PpIX practically does not change the adhesion compared to dark control. This lack of effect of irradiation suggests that PpIX affects cell adhesion primarily because of its intercalation into the membrane and not due to the formation of reactive species. With 3h of incubation the photosensitizers are not on the membrane and therefore the effect on cell adhesion is indirect and it is not also related to differences in uptake efficiency. The observed behavior must be related to differences in subcellular localization arising from differences in molecular structure. Another process that can alter the cell adhesion is serum protein oxidation. As noted in the studies with cells, photosensitization of serum with PLLPpIX (but not with PpIX) was capable of preventing cell adhesion. The greater efficiency of PLLPpIX was associated with the presence of the polymer, which, by the steric hindrance, forces that interaction of PLLPpIX with albumin, the major serum component, is restricted to the protein surface, leaving the photosensitizer available to interact with molecular oxygen and generate singlet oxygen. Thus, the functionalization of a polymer has turned PpIX capable of modulating cell adhesion by acting both within and outside (in extracellular matrix) the cell

Hidrogéis são membranas formadas pela reticulação de cadeias poliméricas, empregados na área farmacêutica como produtos biomédicos. Dentre os principais polímeros selecionados para a síntese de hidrogéis, destaca-se a poliacrilamida (PAAM) devido às suas propriedades como hidrofilicidade e alto grau de intumescimento. Proteínas terapêuticas e enzimas são veiculadas em hidrogéis como carreadores de fármaco ou como dispositivos para tratamento de feridas e escaras na pele. Este trabalho teve como objetivo a síntese de uma membrana à base de PAAM favorável para veiculação de proteínas. As proteínas empregadas foram papaína e albumina de soro bovino (BSA) e as etapas do processo englobaram síntese da membrana, adição das proteínas no sistema, irradiação em condições específicas e caracterização da membrana. Ao utilizar temperaturas criogênicas na síntese e na irradiação das amostras, houve predomínio de reticulação da cadeia polimérica, fato que não ocorria em temperatura ambiente. As membranas foram obtidas com incorporação dos ativos na concentração de 0,2 a 1% (p/p), obtendo-se concentração de PAAM entre 4% a 10% (p/p), as quais receberam irradiação com raios gama provenientes de uma fonte 60Co, na dose de 25 kGy. Nas condições realizadas, as membranas não apresentaram citotoxicidade nem adesão celular, o perfil de liberação das proteínas foi adequado, a papaína manteve sua bioatividade preservada apesar do decaimento biológico e, segundo estudos de carga das moléculas, a membrana possui maior afinidade com a papaína, liberando-a mais lentamente. Desta forma, o método proposto e as membranas obtidas foram apropriados para a obtenção de um biomaterial. / The use of hydrogels for biomedical purposes has been extensively investigated. Polyacrylamide (PAAM) is widely used due to properties such as hydrophilicity and swelling degree. Pharmaceutical proteins correspond to highly active substances which may be applied for distinct purposes. This work concerns the development of radio-synthesized hydrogel for protein release using papain and bovine serum albumin (BSA) as model proteins. The polymer was solubilized (1% w/v) in water and lyophilized. The proteins were incorporated into the lyophilized polymer and the hydrogels were produced by simultaneous crosslinking and sterilization using gamma radiation at 25 kGy under frozen conditions. The produced systems were characterized in terms of swelling degree, gel fraction, crosslinking density, fluid handling capacity, determination pH at point of polymer zero charge and evaluated according to protein release, bioactivity, cytotoxicity and cell adhesion. The hydrogels developed presented different properties as a function of polymer concentration and the optimized results were found for the samples containing 4-10% polyacrylamide. Protein release was controlled by the electrostatic affinity of acrylic moieties of polymer and proteins. This selection was based on the release of the proteins during the experiment period (up to 50 hours), maintenance of enzyme activity and the nanostructure developed. The system was suitable for protein loading and release and according to the cytotoxic assay and cell adhesion it was also adequate for biomedical purposes and this method was able to generate a matrix to protein release.

L’objectif de mon travail de thèse a été de caractériser l’adhésion de vésicules géantes lipidiques et de cellules vivantes. Dans le but d’obtenir des informations quantitatives sur l’adhésion, j’ai développé deux techniques de nanoscopie de fluorescence basées sur la microscopie TIRF (Total Internal Reflection Fluorescence). Cette technique repose sur création d’une onde évanescente à proximité d’une interface. J’ai développé pour cela un montage optique, qui permet de contrôler finement les caractéristiques de l’onde évanescente (longueur d’atténuation, état de polarisation, etc.). L’adhésion des vésicules a été étudiée par nTIRF (TIRF normalisé) : les images TIRF sont normalisées par des images en épi-fluorescence. J’ai pu ainsi caractériser l’adhésion non spécifique (interaction électrostatique) et spécifique (interaction biotine-streptavidine) de vésicules sur différentes surfaces fonctionnalisées. Pour quantifier l’adhésion des cellules, j’ai utilisé l’approche VA-TIRF (TIRF à angle variable). Cette dernière consiste à enregistrer une série d’images en régime évanescent à différents angles d’incidence. Ceci nous a permis d’établir une cartographie des distances entre la membrane ventrale d’une cellule et la surface pour différents comportements d’adhésion initiés sur divers substrats : chimiques ou protéiques. Ces deux techniques permettent de mesurer la distance membrane-surface avec une précision nanométrique, ≈20nm, ce qui est particulièrement adapté à l’étude du processus d’adhésion / The aim of my thesis was to characterize the adhesion of Giant Unilamellar Vesicles and living cells. In order to obtain a quantitative information about the state of the adhesion, I developed two fluorescence nanoscopy techniques based on microscopy TIRF (Total Internal Reflection Fluorescence). This technique consist of creating an evanescent wave in the vicinity of an interface. I developed the experimental setup, which allows an accurate control of characteristics of the evanescent wave (penetration depth, polarization state, etc.). The vesicles adhesion was studied by nTIRF (normalized TIRF). TIRF images are normalized by epi-fluorescence images. I was able to characterize the nonspecific adhesion (electrostatic interaction) and specific adhesion (biotin-streptavidin interaction) of vesicles on different functionalized surfaces. To quantify the adhesion of cells, I used the VA-TIRF approach (variable angle TIRF). The latter is to record a series of images at different angles of incidence in the evanescent regime. This allowed us to map the distances between the ventral membrane of a cell and the surface for different adhesion behaviors initiated on various substrates: chemical or protein. These two techniques permit to measure the membrane surface-distance with the nanometer precision ≈20nm, which is particularly suitable for the study of the adhesion process

SHIP2 is a phosphatase that belongs to the family of the phosphoinositide 5-phosphatases. It is known to dephosphorylate PI(3,4,5)P3 to PI(3,4)P2 imparting a tight control of the PI 3-kinase pathway. Over the last decade, SHIP2 has been described as a tumor promotor or tumor suppressor in several cancer types such as glioblastoma, colorectal cancer or breast cancer cells. Several studies have proposed a role of SHIP2 in breast cancer cells, but its tumor promoting function was unclear at the beginning of this thesis especially in terms of its mode of regulation. In 2013, the INPPL1 gene that encodes SHIP2 has been found to be mutated in opsismodysplasia (OPS), a rare autosomal recessive disease characterized by delayed bone maturation but no molecular mechanism was provided to explain the mechanism. In this thesis, we first contributed to establish a negative regulation of SHIP2 on cell migration in 1321 N1 glioblastoma (GBM) cells. Our studies revealed a dephosphorylation activity of SHIP2 on PI(4,5)P2 at the plasma membrane to control cell migration. This study was done in collaboration with Dr. Elong Edimo in the lab. We have also shown that the regulation of cell motility cannot be generalized to all the GBM cells. In LN229 and U-251 GBM cells we observed a positive regulation of cell migration by SHIP2. We next took advantage of a unique model comparing fibroblasts derived from non-affected and OPS patients (in collaboration with Dr. Valérie Cormier-Daire). We have shown that the fibroblasts from the OPS patients are SHIP2 deficient and migrate slower as compared to fibroblasts from non-affected individuals. Finally, the major part of the thesis was the study of breast cancer cells: in the model MDA-MB-231 cells, we established a positive regulation of SHIP2 on cell migration. We extended this regulation on cell migration to different breast cancer cell models using a SHIP2 inhibitor AS1949490. We confirmed that this inhibitor blocks the phosphatase activity of SHIP2 and showed its selectivity towards SHIP2 in cell migration assay. In MDA-MB-231 cells we deciphered a second messenger role of PI(3,4)P2 to control cell migration. Our data in this model rely on the use of SHIP2 depleted cells obtained by lentiviral infection and shRNA. We confirmed the positive role of SHIP2 on cell migration in the model of rat chondrosarcoma SHIP2CRISPR cells (in collaboration with Dr. Pavel Krejci).A major goal of this thesis was achieved thanks to in-vivo studies: using MDA-MB-231 cells injected in SCID mice, we found a tumor promoting role of SHIP2 by determining the tumor weight. We also observed less lung metastasis of SHIP2 depleted injected cells as compared to control cells suggesting SHIP2 to be important for invasiveness of triple negative breast cancers. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished