No description available.

No description available.

Vitronectin (VTN) is a blood protein produced mainly by the liver. We show that VTN leaks from the bloodstream into the injury site and neighboring subventricular zone (SVZ) following ischemic stroke (middle cerebral artery occlusion, MCAO) in adult mice. MCAO is known to increase neurogenesis after stroke. VTN inhibits this response in females, but not in males, as shown by ~70% more stroke-induced SVZ neurogenesis in female VTN−/− mice at 14 d. In female VTN−/− mice, stroke-induced expression of interleukin-6 (IL-6) at 24 h was reduced in the SVZ. The closely related leukemia inhibitory factor (LIF) or pro-neurogenic ciliary neurotrophic factor (CNTF) were not affected. The female-specific effect of VTN on IL-6 expression was not due to sex hormones, as shown by ovariectomy and castration. IL-6 injection next to the SVZ reversed the MCAO-induced increase in neurogenesis seen in VTN−/− mice. Our in vitro and vivo data suggest that plasma VTN activates focal adhesion kinase (FAK) in the SVZ following MCAO, which reduces IL-6 expression in astrocytes but increases it in other cells such as microglia/macrophages. Inducible conditional astrocytic FAK deletion increased MCAO-induced IL-6 expression in females at 24 h and blocked MCAO-induced neurogenesis at 14 d, confirming a key detrimental role of IL-6. Collectively, these data suggest that leakage of VTN into the SVZ reduces the neurogenic response to stroke in female mice by promoting IL-6 expression. Reducing VTN or VTN signaling may be an approach to promote neurogenesis for neuroprotection and cell replacement after stroke in females.

La FAK est une tyrosine kinase cytoplasmique qui régule divers processus cellulaires, dont la survie, la prolifération, la différenciation et la motilité. Bien que diverses études aient démontré l'importance du FAK dans la pathogenèse du SMD et de la LAM, le rôle de cette molécule dans le microenvironnement des tumeurs du SMD et de la LAM reste à déterminer davantage. En examinant les CSM de la moelle osseuse qui dérivent de patients atteints de SMD et de LAM, nous avons observé une augmentation continue de l'expression et de l'activation de la FAK pendant la progression du SMD vers de la LAM, semblable à celle observée chez les patients hémopoïétiques. Dans le SMD à faible risque, on a constaté que les CSM se caractérisaient par une faible expression et une faible activation du FAK. Ils présentaient une morphologie modifiée, un immunophénotype, une différenciation et l'expression de facteurs favorables à l'hématopoïèse. Il convient de noter que ces caractéristiques pourraient être largement reproduites dans les CSM saines par inhibition FAK. De plus, l'appauvrissement en FAK dans la lignée cellulaire stromale pourrait induire une expansion massive et l'apoptose des CSH normaux. Nos résultats mettent en évidence le rôle crucial du FAK dans le maintien des fonctions des CSM et fournissent la preuve que la dysrégulation du FAK dans les CSM contribue à la perturbation de l'hématopoïèse et éventuellement à la progression des tumeurs malignes myéloïdes. Une meilleure compréhension du rôle que joue le microenvironnement du SMD et de la LAM permettra de mieux reconnaître les patients à faible risque et de mettre au point des traitements ciblant les CSM défectueuses, améliorant ainsi le résultat clinique / FAK is a cytoplasmic tyrosine kinase that regulates diverse cellular processes, including survival, proliferation, differentiation, and motility. Though various studies have demonstrated the importance of FAK in MDS and AML pathogenesis, the role of this molecule in MDS and AML tumor microenvironment remained to be further determined. By examining BM MSCs derived from MDS and AML patients, we have observed a continues increase of FAK expression and activation during MDS progression to AML, similar to those detected in hemopoietic counterparts. In LR-MDS, MSCs were found to be characterized by low FAK expression and activation. They exhibited altered morphology, immunophenotype, differentiation, and expression of hematopoiesis-supporting factors. Of note, these features could be largely reproduced in normal MSCs by FAK inhibition. Furthermore, FAK depletion in BM stromal cell line could induce massive expansion and apoptosis of normal HSPCs. Our results highlight a critical role of FAK in maintaining the functions of BM MSCs and provide evidence that dysregulation of FAK in MSCs contribute to the disturbed hematopoiesis and possibly the progression of myeloid malignancies. A greater understanding of the role that BM microenvironment plays in MDS and AML will enable an increased recognition of poor-risk patients and the development of therapies that target the defected MSCs, thereby improving the clinical outcome

No description available.

The role that signaling molecules play during morphogenesis and their interactions is a field of intense study and the sea urchin represents a facile system to study these aspects of development in the early embryo. In many instances, the S. purpuratus genome contains relatively simple receptor-ligand signaling systems compared to vertebrate counterparts and this provides interesting opportunities to study their diversity of function during the morphogenetic events that shape the embryo. The Eph-Ephrin signaling components are an excellent example of this and they are represented by dozens of members in the vertebrate system with developmental functions that include axon guidance, cell migration and tissue segregation. In contrast, the sea urchin genome contains a single Eph receptor and a single Ephrin ligand and by interacting with different effectors of signal transduction, this simple, bipartite system can fulfill a variety of functional roles during morphogenesis. Studying the function of Eph-Ephrin signaling in the sea urchin embryo, I have revealed two distinct morphogenetic movements in which Eph-Ephrin signaling is necessary; apical constriction of ciliary band cells and pigment cell migration. In both examples, a functionally relevant Ephrin gradient establishes spatial information in the developing tissues, producing a reaction from cells expressing the Eph receptor. In the case of pigment cells, the distribution of migrating cells is affected and in the case of ciliary band cells, apical constriction occurs. The different outcomes of Eph-Ephrin signaling in these two tissues exemplifies signaling components communicating spatial information and initiating morphogenetic programs with outcomes dependent on cellular context. Furthermore, I have identified downstream components of Eph-Ephrin signaling that have necessary functions in both models, illustrating how different cellular programs can be induced by the same signaling iii iv components. My research contributes to understanding fundamental aspects of how complex 3 dimensional tissues arise from the genes and regulatory elements encoded in metazoan genomes. / Graduate

The aim of biomaterials design is to create an artificial environment that mimics the in vivo extracellular matrix for optimized cell interactions. A precise synergy between the scaffolding material, bioactivity, and cell type must be maintained in an effective biomaterial. In this work, we present a technique of nanofabrication that creates chemically nanopatterned bioactive silicon surfaces for cell studies. Using nanoimprint lithography, RGD and mimetic BMP-2 peptides were covalently grafted onto silicon as nanodots of various dimensions, resulting in a nanodistribution of bioactivity. To study the effects of spatially distributed bioactivity on cell behavior, mesenchymal stem cells (MSCs) were cultured on these chemically modified surfaces, and their adhesion and differentiation were studied. MSCs are used in regenerative medicine due to their multipotent properties, and well-controlled biomaterial surface chemistries can be used to influence their fate. We observe that peptide nanodots induce differences in MSC behavior in terms of cytoskeletal organization, actin stress fiber arrangement, focal adhesion (FA) maturation, and MSC commitment in comparison with homogeneous control surfaces. In particular, FA area, distribution, and conformation were highly affected by the presence of peptide nanopatterns. Additionally, RGD and mimetic BMP-2 peptides influenced cellular behavior through different mechanisms that resulted in changes in cell spreading and FA maturation. These findings have remarkable implications that contribute to the understanding of cell-extracellular matrix interactions for clinical biomaterials applications.

Orientador: Kleber Gomes Franchini / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-17T19:53:30Z (GMT). No. of bitstreams: 1 Santos_AlineMarados_D.pdf: 7023687 bytes, checksum: ccd063ea57be631b6b555f86e24af597 (MD5) Previous issue date: 2011 / Resumo: A tirosino-quinase de adesão focal (FAK) tem papel crítico na mediação da migração, sobrevivência e proliferação celular. Estudos anteriores de nosso laboratório demonstraram que a FAK é ativada pelo estresse mecânico em miócitos cardíacos e que ela se coimunoprecipita com a miosina sarcomérica. No presente trabalho foi demonstrado que o domínio FERM da FAK medeia à interação com a miosina sarcomérica, sendo que esta interação leva a inibição da autofosforilação da FAK, enquanto que a ativação prévia da FAK reduz sua interação com a miosina in vitro. Ensaios de cross linking acoplado a espectrometria de massas e espalhamento de raios X a baixos ângulos demonstraram que a miosina interage em uma fenda localizada entre os subdomínios do domínio FERM. Experimentos de microscopia confocal demonstraram que estas proteínas estão colocalizadas em miócitos cardíacos de ratos neonatos e adultos. Ensaios de imunoprecipitação revelaram que aproximadamente 40% da FAK está basalmente associada à miosina sarcomérica enquanto que, após o estiramento celular esta associação reduziu paralelamente à ativação da FAK. A porcentagem de FAK associada à miosina não se alterou com a ativação da FAK após tratamento com fenilefrina, diferente da ativação pelo estresse mecânico. A interferência na interação FAK/miosina pelo silenciamento gênico da miosina culminou com a ativação da FAK e o tratamento dos miócitos cardíacos com o peptídeo FP-1, derivado do subdomínio F2 do domínio FERM, levou a uma diminuição na interação FAK/miosina e ao aumento na fosforilação/ativação da FAK. O tratamento prolongado com FP-1 resultou em hipertrofia dos miócitos cardíacos de ratos neonatos, efeito concomitante à ativação da via de sinalização Akt, TSC2 e S6Kinase. Tanto o silenciamento da FAK quanto o tratamento com rapamicina bloquearam a hipertrofia decorrente do tratamento com FP-1. Os dados deste trabalho indicam que a interação da FAK com a miosina sarcomérica é sensível ao estresse mecânico e que possui papel regulatório na manutenção da quiescência basal da FAK e no controle das vias de sinalização mediadas por esta quinase, como a via de crescimento celular AKT/mTOR/S6Kinase, em miócitos cardíacos em cultura / Abstract: The Focal Adhesion Kinase (FAK) plays a critical role in mediating the migration, survival and cell proliferation. Previous studies from our laboratory demonstrated that FAK is activated by mechanical stress in cardiac myocytes and it co-immunoprecipitate with sarcomeric myosin. Here, we demonstrated that the FAK FERM domain mediates the interaction with sarcomeric myosin, and that this interaction leads to inhibition of FAK autophosphorylation in vitro, whereas the previous activation of FAK reduces its affinity to myosin. A model based on small angle X-ray scattering analyses and crosslinking technology coupled with mass spectrometry indicated that a cleft in FERM domain is critical to the interaction of FAK to myosin. Confocal microscopy experiments showed that these proteins are colocalized in cardiomyocytes of neonatal and adult rats. Immunoprecipitation assays revealed that approximately 40% of FAK is basally associated with sarcomeric myosin while cardiomyocyte stretching reduced this association in parallel with FAK activation. The percentage of FAK associated with myosin was not change in response to FAK activation by treatment with phenylephrine, unlike in response to FAK by mechanical stress. The interference in the FAK/Myosin interaction by myosin silencing approach culminated with the activation of FAK. The treatment of cells with the FP-1 peptide, derived from the FAK FERM domain, lead to a decrease in the interaction with sarcomeric myosin and an increase in FAK activation. Prolonged treatment with FP-1 resulted in morphological hypertrophy of neonatal rat cardiomyocytes which was an effect concomitant with activation of the Akt, TSC2 and S6Kinase signaling pathway. Both FAK silencing and the rapamycin treatment blocked the morphological hypertrophy resulting of the treatment with FP-1. This study indicated that the interaction of FAK with sarcomeric myosin is sensitive to mechanical stress and it has regulatory role in maintaining of FAK quiescence and control of signaling pathways mediated by this kinase, such as cell growth via AKT/mTOR/S6Kinase in cardiac myocytes in culture / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Ciências

Orientador: Kleber Gomes Franchini / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T14:22:48Z (GMT). No. of bitstreams: 1 Cordeiro_IsabelleBezerra_D.pdf: 17466566 bytes, checksum: de71f83d2b4d3c8e0f0269f5d61d332d (MD5) Previous issue date: 2014 / Resumo: A proteína Focal Adhesion Kinase (FAK; PTK2) participa de vários processos celulares. A identificação das proteínas parceiras de FAK tem contribuído para o entendimento de suas múltiplas funções celulares. Utilizando um sistema de indução de FAK fusionada a uma cauda de FLAG, combinada com análises por espectrometria de massas (MS), identificamos proteínas associadas à FAK em células HEK293. Um total de 153 proteínas foram repetidamente identificadas com alta resolução por experimentos de MS. Além de confirmar interações já previamente descritas como Paxillin (PXN), Heat shock protein Hsp90 (HSP90) e Transforming growth factor beta-1-induced transcript 1 protein (TGF?1I1), as análises por MS revelaram um novo conjunto de proteínas associadas à FAK, incluindo proteínas envolvidas nas vias de síntese de proteínas, expressão gênica, crescimento celular, proliferação, morte e sobrevivência celular. Além disso, análises de bioinformática das vias indicaram que a FAK se associa com um conjunto de 30 proteínas envolvidas com a via de modificação pós-transcricional do RNA, incluindo a proteína Serine/arginine-rich splicing factor 2 (SC-35; SRSF2), que é um marcador de speckles nucleares. Esse conjunto de proteínas está associado ao spliceossomo e um conjunto similar de proteínas também foi identificado com os experimentos que utilizaram somente o domínio FERM da FAK. Validações por Western Blotting e imunocitoquímica demonstraram que FAK se associa e co-localiza com a proteína SC-35 no núcleo celular. Ainda identificamos um papel funcional da FAK no splicing do mRNA. Demonstramos que FAK pode modificar o padrão de splicing de um gene repórter E1A ao ser superexpressa em células HEK. Nosso trabalho propõe novas funções da FAK no núcleo, indicando que esta pode estar envolvida em eventos relacionados à função do RNA, como a regulação do splicing do mRNA / Abstract: Focal adhesion kinase (FAK; PTK2) has roles in many cellular processes. The identification of protein partners for FAK has greatly contributed to our understanding of its multiple function. Using inducible FLAG-tagged FAK combined with affinity/purification mass spectrometry (MS) approach, we identified proteins associated with FAK in HEK293 cells. A total of 153 proteins were repeatedly detected in high-resolution MS measurements. Beyond the well characterized partnering with Paxillin (PXN), Heat Shock protein 90 (HSP90) and Transforming growth factor beta-1-induced transcript 1 protein (TGFB1I1), analysis of MS data uncovered novel sets of proteins that associate with FAK, playing a role in protein synthesis, gene expression, cellular growth, proliferation, death and survival. In addition, the network analysis established the unexpected finding that a module of 30 proteins linked to RNA post-transcriptional modifications are recruited by FAK, including Serine/arginine-rich splicing factor 2 (SC-35; SRSF2), which is a marker of the nuclear speckles. Indeed, this module is found to be enriched by proteins associated with spliceosome. Remarkably, a similar set of proteins was also recruited by FAK N-terminal FERM domain. Biochemical and imunofluorescence validations established that FAK associates and co-localizes with SC-35 protein at the cell nuclei. We further pinpoint a functional role of FAK in mRNA splicing. We showed that FAK can modify the splicing site selection of the adenoviral E1A minigene in a dose-dependent manner. Our work provides new insights into the molecular function of FAK in the nucleus, indicating that it may be involved in events related to RNA function, such as pre-mRNA splicing / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

Orientador : Kleber Gomes Franchini / Tese (Doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T00:30:15Z (GMT). No. of bitstreams: 1 Antunes_JoaoEustaquio_D.pdf: 5472161 bytes, checksum: d84f6389e6baf3e2959126f6345e6a14 (MD5) Previous issue date: 2013 / Resumo: A compreensão de como a quinase de adesão focal (FAK) contribui para os processos de hipertrofia, insuficiência cardíaca e câncer são de grande interesse científico. Um dos nossos objetivos específicos é desenvolver inibidores desta tirosina quinase com vistas à sua aplicação terapêutica no tratamento de insuficiência cardíaca e câncer. Portanto, foi realizado planejamento racional e a síntese de inibidores farmacológicos para a FAK. Estudos computacionais de docking e farmacocinéticos permitiram selecionar 28 estruturas de quinazolina mais promissoras em relação à capacidade de inibir a FAK. Desta forma, economiza-se tempo e dinheiro para obter-se a síntese apenas de 6 das estruturas pré-selecionadas. Uma quinazolina denominada 4-BZLO foi sintetizada após o planejamento racional. Tal quinazolina foi capaz de inibir em 50% da atividade da FAK in vitro com aproximadamente 1nM. Os testes de pureza da síntese, absorção por via oral, melhor resultado em triagem em células que superexpressam a FAK e o melhor resultado para triagem em duas linhagens de célula leucêmicas e tumor sólido permitiram direcionar o 4- BZLO para ser o composto líder deste estudo. O composto 4-BZLO apresentou resultado promissor para experimentos com camundongos submetidos à coarctação da aorta, que induz hipertrofia cardíaca. Para avaliar se o tratamento preventivo seria eficiente para hipertrofia cardíaca, foi realizado experimento em animais tratados com 30mg/Kg/dia durante 30 dias. Após este período, foi realizada cirurgia de coarctação da aorta para induzir a hipertrofia cardíaca nos animais e os mesmos foram tratados por mais 30 dias. Parâmetros tais como: peso médio do ventrículo esquerdo sobre peso corpóreo de cada animal (LVW/BW), medida da espessura da parede do ventrículo esquerdo (LVWT) e parâmetros histológicos (diâmetro dos miócitos cardíacos) demonstrou que nos animais tratados houve regressão da hipertrofia comparada ao controle (animais sem tratamento). Outro estudo realizado no qual os animais foram tratados de maneira curativa, ou seja, o tratamento foi realizado somente após a coarctação da aorta demonstrou uma melhora no quadro de hipertrofia e função cardíaca. O modelo de fibrose cardíaca foi usado para avaliar se tratamento com 4-BZLO é capaz de reduzir a fibrose cardíaca nos animais. Os resultados obtidos demonstraram que os animais tratados com 4-BZLO por 30 dias apresentaram redução da acumulação de colágeno, que é um indicador de fibrose, em relação ao controle. Nosso laboratório desenvolveu camundongos transgênicos específicos para a FAK que desenvolve moderada hipertrofia cardíaca. Assim, tal modelo permitiu testar o tratamento com o 4-BZLO para hipertrofia cardíaca induzida pela FAK. Os animais transgênicos específicos para a FAK foram tratados com o composto líder e houve melhora dos parâmetros cardíacos. Os resultados obtidos permitiram concluir que a quinazolina denominada 4-BZLO é um bom candidato a fármaco como inibidor da FAK / Abstract: Understanding how the focal adhesion kinase (FAK) contributes to the processes of hypertrophy, heart failure and cancer are of great scientific interest. One of our goals is to develop specific inhibitors of this tyrosine kinase with potential therapeutic application in the treatment of heart failure and cancer. In this view, we used rational design to select a group of possible FAK inhibitors. Computational studies such as docking and pharmacokinetic studies allowed us to select 28 structures most likely to inhibit FAK. In this way, we saved up time and money in devising the synthesis of 6 pre-selected structures. Accordingly, quinazoline 4-BZLO was prepared and was able to inhibit the in vitro activity of FAK by 50% at a concentration of approximately 1nM. Several factors contributed to 4-BZLO being chosen as the lead compound in this study: 1) the degree of purity achieved during synthesis; 2) good oral bioavailability; 3) the best inhibition values in cells over expressing FAK in screening, 4) the best result against two leukemic cell lines and one solid tumor target. 4-BZLO showed promising results in experiments with mice subjected to aortic coarctation, which develops cardiac hypertrophy. To assess whether 4- BZLO would be effective for the preventive treatment of cardiac hypertrophy, an experiment was conducted in animals treated with 30 mg/kg/day for 30 days. After this period, an aortic coarctation was performed surgically in order to induce cardiac hypertrophy in animals, and these were further treated for 30 days. Parameters such as left ventricular weight per body weight ratio (LVW/BW), measurement of the left ventricle wall thickness (LVWT), and histological parameters, such as the diameter of cardiac myocytes in treated animals showed that there was a regression of hypertrophy, compared to untreated animals (control). A similar study, where treatment with 4-BZLO was performed only after aortic coarctation showed an improvement regarding hypertrophy and cardiac function. A cardiac fibrosis model was used and the results obtained demonstrated that animals treated with 4-BZLO for 30 days showed a reduction of collagen accumulation, which is an indicator of fibrosis, equal to the control group. Our laboratory has developed transgenic mice specific for FAK, which develop moderate cardiac hypertrophy. Consequently, this model allows us to test 4-BZLO for the treatment of FAK induced cardiac hypertrophy. The transgenic animals were treated with 4-BZLO, leading to an improvement of the cardiac parameters. These results showed that synthetic quinazoline 4-BZLO is a good drug candidate for the inhibition of the FAK enzyme / Doutorado / Farmacologia / Doutor em Farmacologia