O câncer de mama é importante problema de saúde pública. O microambiente onde as células cancerígenas se encontram possui moléculas como a laminina e seus peptídeos bioativos, que influenciam a biologia tumoral. Estudo anterior realizado no laboratório demonstrou que o peptídeo C16, derivado da laminina, aumenta a expressão gênica de GPNMB e SPOCK1. Nesse trabalho, demonstramos que o peptídeo C16 aumentou níveis moleculares de GPNMB e SPOCK em células malignas MDA-MB-231e MCF-7, comparado com células normais MCF-10A. C16 estimulou significantemente a invasão de células MDA-MB-231. Silenciamento de GPNMB diminuiu a invasão celular desencadeada por C16. Contextualizando in vivo nossos resultados in vitro, imunohistoquímica em tissue microarrays mostrou que a presença de GPNMB e SPOCK é significantemente maior em câncer de mama. Assim, C16 regula os níveis de GPNMB e SPOCK em células mamárias malignas. C16 e GPNMB cooperam regulando a invasão de células MDA-MB-231. GPNMB e SPOCK foram mais detectados em câncer de mama comparado com mama normal. / Breast cancer is an important public health. The microenvironment in which cancer cells are found contains molecules such as laminin and its bioactive peptides that influence tumor biology. Previous study conducted in the laboratory showed that the C16 peptide derived from laminin, increases the gene expression of GPNMB and SPOCK1. In this work, we demonstrate that the C16 peptide increased molecular levels of GPNMB and SPOCK in malignant cells MDA-MB-231e MCF-7 cells compared with normal cells MCF-10A. C16 significantly stimulated invasion of MDA-MB-231 cells. GPNMB silencing decreased cell invasion triggered by C16. Contextualizing in vivo our in vitro results, tissue microarrays immunohistochemistry showed that the presence of GPNMB and SPOCK are significantly higher in breast cancer. Thus, C16 regulates the levels of GPNMB and SPOCK in malignant breast cells. C16 and cooperate GPNMB regulating the invasion of MDA-MB-231 cells. SPOCK and GPNMB were more detected in breast cancer compared to normal breast.

O câncer de próstata é o segundo câncer mais freqüentemente diagnosticado em homens. Durante o crescimento tumoral, as células neoplásicas interagem com a matriz extracelular (MEC). Analisamos o efeito de C16, peptídeo derivado da clivagem da MEC, sobre a migração, invasão e regulação dos invadopódios em células de câncer de próstata (DU145). Ensaios de migração e invasão demonstraram que C16 promoveu um aumento da atividade migratória e invasiva de células DU145 de maneira dose dependente. Demonstramos que o peptídeo estimula a fosforilação de Src. Ensaios de degradação em substrato fluorescente mostraram que C16 promoveu a formação de invadopódios de células DU145. O immunoblot nos revelou que este peptídeo também estimula a expressão de Tks4, Tks5, cortactina e MT1-MMP. C16 estimulou a produção de espécies reativas de oxigênio, importantes para o fenótipo invasivo das células tumorais. Nossos resultados sugerem que o peptídeo C16 regula migração, invasão, invadopódios e suas moléculas-chave e a geração de espécies reativas de oxigênio em células DU145. / Prostate cancer is the second most frequently diagnosed cancer in males. During tumor growth, neoplastic cells interact with the extracellular matrix (ECM) Our Laboratory has demonstrated that peptides derived from ECM cleavage are involved in migration, invasion and invadopodia formation in different tumor cell lines. Invadopodia activity depends on expression of the proteins Tks4, Tks5, cortactin, MT1-MMP, as well as reactive oxygen species (ROS) generation. Migration and invasion assays in chemotaxis chambers demonstrated that C16 increased migration and invasion activities of DU145 cells in a dose-dependent manner. We observed that the peptide stimulated phosphorylation of Src. Fluorescent substrate degradation assay showed that C16 increased invadopodia activity of DU145 cells. Immunoblot revealed that this peptide stimulated Tks4, Tks5, cortactin and MT1-MMP expression. Furthermore, C16 increased ROS production. Our results strongly suggested that C16 regulates migration, invasion, invadopodia key-molecules, and ROS generation in DU145 cells.

A laminina contém peptídeos que podem ser liberados por proteólise. Nosso laboratório estuda os efeitos de peptídeos da laminina em biologia tumoral. Neste trabalho, verificamos se C16 (cadeia g1) estimularia invasão e atividade de invadopódios em células de carcinoma epidermóide (CAL27) e fibrossarcoma (HT1080). C16 promoveu aumento na taxa de invasão e atividade de invadopódios em ambas às linhagens celulares, comparado ao peptídeo controle C16SX. Microscopia em time-lapse demonstrou que C16 induz aumento na atividade de invadopódios em função do tempo. C16 estimula fosforilação de Src e ERK 1/2, e inibição da via ERK reduz invasão e atividade de invadopódios relacionados ao peptídeo. C16 conjugado à rodamina foi encontrando decorando a membrana de células CAL27, sugerindo possível interação com receptores. Diminuição dos níveis de integrina b1 reduzem atividade de invadopódios em amostras tratadas com C16. Nossos dados sugerem que C16 regula invasão e atividade de invadopódios em células CAL27 e HT1080, provavelmente por meio de Src, ERK e integrina b1. / Laminin harbors bioactive peptides released upon tumor-induced proteolysis. Our Laboratory has been studying laminin peptides effects in tumor biology. Here we addressed whether C16 (g1 chain) would regulate invasion and invadopodia activity in cell lines from squamous cell carcinoma (CAL27) and fibrosarcoma (HT1080). C16 increased invasion rate and invadopodia activity compared to control peptide (C16SX). Through time-lapse microscopy, we observed that C16 stimulated invadopodia activity overtime. We searched for signaling pathways related to peptide effects. C16 stimulated Src and ERK 1/2 phosphorylation, and ERK signaling cascade inhibition decreased C16-induced invasion and invadopodia. Next, we addressed how C16 would interact with tumor cells. Rhodamine-conjugated C16 was found decorating CAL27 cell membrane, suggesting an interaction with receptors. Knockdown of b1 integrin reduced invadopodia activity of C16-treated cells. We propose that C16 regulates invasion and invadopodia activity of CAL27 and HT1080 cells through Src, ERK and b1 integrin.

O Lopap é um ativador de protrombina da lagarta L. obliqua, pertence à família das lipocalinas e apresenta atividade antiapoptótica. O Lopap foi obtido na forma recombinante (rLopap), na levedura P. pastoris, por metodologia escalonável, e sua atividade foi avaliada in vitro e in vivo. O tratamento com rLopap reduziu o tempo de sangramento em animais anticoagulados com enoxaparina. Por outro lado, um peptídeo derivado do Lopap, designado antiapoptotic peptide (AP), foi capaz de induzir a síntese de colágeno em cultura de fibroblastos e na derme de animais. A região correspondente a AP apresentou propriedades físicas e estruturais semelhantes a seqüências relacionadas em outras lipocalinas com atividade antiapoptótica. Estes resultados abrem perspectivas para aplicações do Lopap, como uma molécula procoagulante, e de AP, através de sua ação na modulação celular, como um componente cosmético, no reparo e remodelamento tecidual e em disfunções que envolvem morte celular e perda de colágeno. / Lopap is a prothrombin activator from the L. obliqua caterpillar, belongs to the lipocalin family, and displays antiapoptotic activity. Lopap was obtained in the recombinant form (rLopap) in the P. pastoris yeast, by a scaled up methodology, and its activity was evaluated in vitro and in vivo. Treatment with rLopap reduced the bleeding time in animals anticoagulated with enoxaparin. On the other hand, a Lopap-derived peptide, designated antiapoptotic peptide (AP), was able to induce collagen synthesis in fibroblast culture and in the animal dermis. The region corresponding to AP had similar physical and structural properties when compared with other antiapoptotic lipocalins. These results open perspectives for the use of Lopap, as a procoagulante molecule, and the use of AP, based on its cell modulation effects, as a cosmetic component, aiding tissue repair and in dysfunctions involving cell death and loss of collagen.

La perception de la viande bovine par le consommateur est un enjeu crucial pour l’industrie de la viande. La tendreté reste l’une des qualités organoleptiques essentielle aux yeux du consommateur, avec la jutosité et la flaveur. L’objectif de ma thèse a été d’étudier les propriétés structurales et biochimiques du tissu conjonctif intramusculaire responsables de la dureté de base et de faire le lien avec la tendreté et plus largement avec les qualités organoleptiques de la viande. Le développement et la mise au point d’une méthode d’analyse d’images a permis d’étudier objectivement les caractéristiques des composantes périmysiale et endomysiale du tissu conjonctif intramusculaire et de montrer que la surface occupée par ces deux structures influençait la tendreté de la viande. Préalablement à l’étude biochimique et moléculaire du tissu conjonctif intramusculaire le dosage des protéoglycanes totaux a été développé et mis en place au laboratoire. Les résultats de cette deuxième partie ont permis de mettre en avant la complémentarité entre protéoglycanes, collagène total et liaisons de réticulation au sein du muscle et leur rôle sur la jutosité. Par ailleurs, nous avons pu mettre en évidence le rôle positif des protéoglycanes de haut poids moléculaire sur la jutosité, alors que les protéoglycanes de faible poids moléculaire, telle que la décorine,joueraient plutôt un rôle sur la dureté de la viande. L’ensemble des résultats obtenus confirme nos hypothèses, à savoir que la composante structurale ainsi que les molécules minoritairement présentes dans le tissu conjonctif peuvent influencer les qualités organoleptiques de la viande bovine. / The perception of beef by consumers is a crucial issue for the meat industry. Tenderness remains one of the sensory qualities essential to the consumer, with the juiciness and flavour. The aim of my thesis was to study the structural and biochemical properties of intramuscular connective tissue responsible for the background toughness and study relationships with tenderness and more widely with the sensory qualities of beef. The development of a method of image analysis was used to study objectively the characteristics of the perimysial and endomysial component of the intramuscular connective tissue. The results showed that the area occupied by these two structures influenced the beef tenderness. An assay for total proteoglycan measurement has been developed previously to the study of biochemical and molecular characteristics intramuscular connective tissue. The results obtained in this second part have highlighted the complementarities between proteoglycans, total collagen and cross-links within the muscle and their role on the juiciness. Furthermore, we were able to highlight the positive role of high molecular weight proteoglycans on juiciness, while low molecular weight proteoglycans, such as decorin, rather played a role in beef toughness. The overall results confirm our hypothesis that the structural components of the connective tissue and the small quantity of extracellular matrix molecules could influence the sensory qualities of beef.

There is a basic rule to mammalian neocortical expansion: as it expands, so does it fold. The degree to which it folds, however, cannot strictly be attributed to its expansion. Across species, cortical volume does not keep pace with cortical surface area, but rather folds appear more rapidly than expected. As a result, larger brains quickly become disproportionately more convoluted than smaller brains. Both the absence (lissencephaly) and presence (gyrencephaly) of cortical folds is observed in all mammalian orders and, while there is likely some phylogenetic signature to the evolutionary appearance of gyri and sulci, there are undoubtedly universal trends to the acquisition of folds in an expanding neocortex. Whether these trends are governed by conical expansion of neocortical germinal zones, the distribution of cortical connectivity, or a combination of growth- and connectivity-driven forces remains an open question. But the importance of cortical folding for evolution of the uniquely mammalian neocortex, as well as for the incidence of neuropathologies in humans, is undisputed. In this hypothesis and theory article, we will summarize the development of cortical folds in the neocortex, consider the relative influence of growth- vs. connectivity-driven forces for the acquisition of cortical folds between and within species, assess the genetic, cell-biological, and mechanistic implications for neocortical expansion, and discuss the significance of these implications for human evolution, development, and disease. We will argue that evolutionary increases in the density of neuron production, achieved via maintenance of a basal proliferative niche in the neocortical germinal zones, drive the conical migration of neurons toward the cortical surface and ultimately lead to the establishment of cortical folds in large-brained mammal species.

This master’s thesis contains a description of mesenchymal stem cells (MSC). The work includes knowledge about the process of migration and differentiation of MSCs. The theoretical part of this thesis also deals with the problem of cell cultivation in 2D and 3D environments. The most used materials for creating 3D scaffolds are described here. The practical part contains the description of the cell culture protocol for passaging and also describes the experiment in the cell laboratory. The results of the experiment are discussed using confocal microscope images. The proposed experiment was tested with a sufficient number of repetitions. The processing of microscopic images was performed in the MATLAB programming environment.

This thesis engages in a study of mesenchymal stem cell migration in extracellular matrix based on principles of chemotaxis. First, attention is focused on a theoretical part associated with a clarification of basic terms such as extracellular matrix, migration, confocal microscopy, mesenchymal stem cells or chemotaxis. There is also included a list and a description of some basic methods for monitoring cell migration and a more detailed description of a method called transwell assay, which has been chosen for an experiment in a practical part of this thesis. This part includes protocols of individual steps for the preparation of the experiment, the procedure of data processing obtained by scanning cells with a confocal microscope and a description of the resulting confluence values.

Les interactions cellule-cellule et cellule-matrice extracellulaire (MEC) sont cruciales pour entretenir la cohésion tissulaire. Ces deux types d'adhésions sont fonctionnellement interconnectés par un dialogue permanent qui met en jeu des voies de signalisation convergentes régulant notamment l'architecture et la contractilité du cytosquelette d'acto-myosine sous-jacent. Ce dialogue permet d'établir un équilibre de forces intracellulaires en réponse à la tension appliquée par le milieu extérieur. L'endothélium des vaisseaux sanguins est un tissu soumis à des conditions mécaniques particulières. En plus des compressions intercellulaires subies par tout épithélium, les cellules endothéliales (CEs) doivent également subir et résister aux forces hémodynamiques du flux sanguin et à la rigidité de la lame basale – deux signaux mécaniques agissant de part et d'autre de l'endothélium. Les Cerebral Cavernous Maformations (CCM) ou encore angiomes caverneux sont des lésions vasculaires hémorragiques d'origine génétique qui se développent au niveau des capillaires du système nerveux central et qui se caractérisent par des défauts dans l'environnement proche des CEs. La perte des jonctions intercellulaires et du recouvrement par les cellules murales, l'organisation aberrante de la membrane basale aussi que la stagnation du flux sanguin sont les caractéristiques des CCM. C'est pourquoi nous avons choisi cette pathologie comme modèle intéressant de mécanotransduction mettant en jeu le dialogue entre les intégrines et les cadhérines. En effet, les trois gènes indifféremment mutés dans cette pathologie codent pour des protéines, CCM1-3, qui s'associent en un complexe ternaire et qui sont reconnues comme des acteurs importants de la régulation des jonctions adhérentes. Des études moléculaires et protéomiques montrant que le complexe CCM interagit avec la protéine ICAP-1, un régulateur négatif de l'intégrine β1, nous ont conduit à formuler l'hypothèse selon laquelle ce complexe jouerait un rôle pivot dans la signalisation croisée entre ces intégrines et cadhérines. Les études effectuées pendant ma thèse ont démontré que les protéines CCM régulent l'homéostasie tensionnelle médiée par les structures d'adhérence intercellulaires et à la MEC par leur action inhibitrice sur l'intégrine β1 et en controlant une balance d'activité entre les deux isoformes de ROCK, ROCK1 et ROCK2. Nous avons montré que, suite à la perte des protéines CCMs, la suractivation de l'intégrine β1 augmente la sensibilité des CEs aux signaux mécaniques comme la rigidité de la MEC ou les forces hémodynamiques du flux sanguin. Il en résulte une suractivation de la contractilité cellulaire dépendante de ROCK1 déclenchant une boucle de rétrocontrôle mécanique conduisant à l'amplification des tensions intra- et extracellulaire et brisant ainsi l'homéostasie tensionnelle pour favoriser le phénotype malin. / Cell-cell or cell-matrix interactions have crucial roles in the maintenance of the physical cohesion of any tissue. In addition, growing body of evidence indicates that these two adhesion systems do not act independently, but rather are functionally interconnected by a permanent crosstalk. This dialog usually operates via common molecules that trigger convergent signaling as well as by actomyosin network which, by providing physical link, contributes to establishment of intracellular force counterbalancing tension applied by extracellular surrounding. Blood vessels endothelium is a particular tissue in term of mechanical conditions. Apart from intracellular compression, endothelial lining needs to resist hemodynamic forces as well as rigidity of the basal membrane - two mechanical inputs acting from opposite sides of the endothelial layer. Cerebral Cavernous Malformation (CCM) is a sporadically acquired or inherited disease of venous capillaries within neuro-vascular unit characterized by defects in all aspects of local microenvironment. Loss of intra-endothelial junctions and mural cell coverage, aberrant organization of basal lamina as well as stagnant blood flow are features of CCM lesions. Thereby, CCM became for us an interesting model to study mechanotrasduction process and in this context, the cross-talk between integrin and cadherin mediated adhesion structures. Indeed, CCM proteins are well recognized players involved in a control of VE-cadherin mediated intracellular junctions. In addition, CCM1 was found to interact with ICAP-1, a negative regulator of β1 integrin, raising the possibility that this complex most likely acts as molecular node regulating β1 integrin/ VE-cadherin convergent signaling pathways.Studies performed during this thesis have demonstrated that CCM complex coordinates cadherin- and integrin-mediated tensional homeostasis by repressing β1 integrin activation and maintaining a balance of activity between the two isoforms of RhoA-associated kinases ROCK1 and ROCK2. We have found that β1 integrin sustained over-activation upon CCM proteins loss contributes to increased ECs sensitivity to mechanical cues, such as ECM physical reorganization or hemodynamic force that in turn activates ROCK1-dependent contractility. This establishes a positive feedback mechanical loop that breaks tensional homeostasis and switches on the malignant phenotype.

In Alzheimer’s disease (AD), different types of neurons and different brain areas show differential patterns of vulnerability towards neurofibrillary degeneration, which provides the basis for a highly predictive profile of disease progression throughout the brain that now is widely accepted for neuropathological staging. In previous studies we could demonstrate that in AD cortical and subcortical neurons are constantly less frequently affected by neurofibrillary degeneration if they are enwrapped by a specialized form of the hyaluronan-based extracellular matrix (ECM), the so called ‘perineuronal net’ (PN). PNs are basically composed of large aggregating chondroitin sulphate proteoglycans connected to a hyaluronan backbone, stabilized by link proteins and cross-linked via tenascin-R (TN-R). Under experimental conditions in mice, PN-ensheathed neurons are better protected against iron-induced neurodegeneration than neurons without PN. Still, it remains unclear whether these neuroprotective effects are directly mediated by the PNs or are associated with some other mechanism in these neurons unrelated to PNs. To identify molecular components that essentially mediate the neuroprotective aspect on PN-ensheathed neurons, we comparatively analysed neuronal degeneration induced by a single injection of FeCl3 on four different mice knockout strains, each being deficient for a different component of PNs. Aggrecan, link protein and TN-R were identified to be essential for the neuroprotective properties of PN, whereas the contribution of brevican was negligible. Our findings indicate that the protection of PN-ensheathed neurons is directly mediated by the net structure and that both the high negative charge and the correct interaction of net components are essential for their neuroprotective function.