Au cours de l’ovogenèse chez la mouche du vinaigre: Drosophila melanogaster, un groupe de cellules folliculaires appelées cellules de bord, migrent à travers les cellules nourricières pour atteindre l’ovocyte. Cet événement, nécessitant la transition épithélio- mésenchymateuse (TEM), la réorientation, puis l’arrêt, ressemble à la formation de métastases. L’endocytose est un régulateur clé de plusieurs événements polarisés, y compris la migration cellulaire. En effet, différentes protéines impliquées dans la migration, comme les intégrines et les E-cadhérines (cadhérines épithéliales), sont régulées par transport à travers les endosomes. De même, l’endocytose restreint au front de migration l’activité des récepteurs tyrosine kinases (RTKs) qui guident les cellules de bord dans leur mouvement. Cependant les mécanismes moléculaires de cette restriction spatiale de l’activité des RTKs demeurent largement inconnus. Nous avons testé l’implication du trafic vésiculaire à travers la machinerie d’endocytose, dans la migration dirigée des cellules de bord, car ce système est facilement accessible pour l’expression de protéines et l’analyse de mutants. Nous avons commencé par confirmer une observation précédente du rôle de l’endosome précoce dans la migration des cellules de bord. Ensuite, nous avons identifié l’endosome de recyclage (ER) comme un régulateur clé de cette migration. En effet, nous avons démontré que l’expression dans les cellules de bord d’une forme dominante négative de Rab11, la petite GTPase régulant le transport vésiculaire à travers l’ER, bloque la migration ou entraîne de sévères défauts de migration dans environ 80% des chambres d’œufs examinées. De plus, nous observons par immunofluorescence une relocalisation de l’activité des RTKs alors que d’autres protéines de migration ne sont pas affectées par Rab11 dominant négatif. Ce résultat a été par la suite confirmé par une interaction génétique entre Rab11 et les RTKs. D’autre part, nous avons montré que le complexe exocyste, un effecteur de Rab11, est impliqué dans la migration des cellules de bord. Nous avons trouvé par microscopie confocale en tissu fixé et par microscopie en temps réel que Sec15, un composant de ce complexe, est polarisé, de façon Rab11- dépendante, dans des vésicules qui s’accumulent au front de migration tout au long du mouvement des cellules de bord. De plus, la perte de l’activité de Sec15 perturbe à son tour la migration. Ainsi, toutes ces données démontrent le rôle fondamental d’un cycle d’endo- exocytose dans le maintien des RTKs actifs au niveau du front de migration des cellules de bord le long de leur mouvement. / During Drosophila melanogaster’s oogenesis, a cluster of folllicle cells, called border cells, perform an invasive migration through the surrounding nurse cells to reach the oocyte. This event resembles metastasis formation since it requires epithelial- mesenchymal transition, reorientation and arrest. Endocytosis plays a fundamental role in many polarized processes, including cell migration, since different migration proteins, like integrins and E-cadherins traffic through the endocytic pathway. Furthermore, receptor tyrosine kinases (RTKs) that guide border cells during their migration are regulated by endocytosis, although the mechanisms involved are largely unknown. We tested the implication of vesicular trafficking through the endocytic machinery, in border cells’ directed migration, because this system is easily accessible for protein expression and mutant analysis. We first confirmed previous observation that trafficking through the early endosome is necessary for border cells migration, and then we identified the recycling endosome as a key compartment for this migration. Indeed, we showed that overexpression in border cells of a dominant negative form of Rab11, the small GTPase regulating vesicular trafficking through the recycling endosome, blocks migration or leads to severe migration defects in about 80% of examined egg chambers. Furthermore, using immunofluorescence, we observed a relocalization of RTKs activity, whereas other migration proteins were not redistributed upon dominant negative Rab11 expression. This result was further confirmed by a genetic interaction between Rab11 and RTKs. Moreover, we showed that the exocyst complex, an effector of Rab11, is also involved in border cells migration. We found by using confocal microscopy of fixed tissues and time-lapse microscopy of living egg chambers, that Sec15, a member of this complex, is distributed in vesicles which are polarized, in a Rab11- dependent manner, throughout border cells migration. In addition, loss of Sec15 also impairs migration. Together these data demonstrate a fundamental role for an endo- exocytic cycle in the maintenance of active RTKs at the leading edge of border cells during their migration.

Au laboratoire, une famille de tyrosine kinases propres aux bactéries et ne présentant aucune ressemblance structurale avec les protéine-kinases d’origine eucaryote a été identifiée. Ces enzymes, appelées BY-kinases, sont notamment impliquées dans la biosynthèse des polysaccharides extracellulaires, mais leurs rôles précis ainsi que leurs mécanismes catalytiques sont encore peu compris.Dans la première partie de ce travail, nous avons caractérisé le rôle physiologique de la phosphorylation sur la tyrosine de la protéine Ugd, une UDP-glucose déshydrogénase, par les BY-kinases Wzc et Etk d’E. coli. Nous avons démontré que la phosphorylation d’Ugd sur un site commun à Wzc et Etk augmente son activité. Nous avons également établi que la phosphorylation d’Ugd par Wzc participe à la régulation de la quantité d’acide colanique produit, tandis que la phosphorylation d’Ugd par Etk influence la résistance de la bactérie à la polymyxine.Nous avons également effectué une analyse structure-fonction du domaine cytoplasmique de deux BY-kinases, CapA1/CapB2 de S. aureus et Wzc d’E. coli. Nous avons montré que ces deux protéines s’associent en octamère, grâce au motif EX2RX2R et qu’elle s’autophosphoryle selon un mécanisme intermoléculaire. Nous avons, de plus, identifié le mécanisme d’activation de ces protéines et révélé l’importance d’un domaine particulier dans l’autophosphorylation de Wzc et la biosynthèse de l’acide colanique.La caractérisation structurale et fonctionnelle des BY-kinases représente une approche prometteuse et originale en vue de l’élaboration de molécules inhibant spécifiquement leur activité et pouvant affecter le pouvoir virulent des bactéries pathogènes. / A new class of bacterial enzymes, named BY-kinases, has been shown to catalyze protein-tyrosine phosphorylation. These enzymes share no structural and functional similarities with their eukaryotic counterparts. Evidence of their involvement in extracellular polysaccharide biosynthesis has been provided, but their accurate functions and their catalytic mechanism remain largely unknown.First, we characterized the physiological role of tyrosine phosphorylation of Ugd, a UDP-glucose dehydrogenase, by the BY-kinases Wzc and Etk of E. coli. We demonstrated that Ugd phosphorylation by Wzc or Etk occurs on the same site and increases its activity. We also established that Wzc-mediated phosphorylation of Ugd participates in the regulation of colanic acid production whereas Ugd phosphorylation by Etk influences resistance to polymyxin.In addition, we performed a structure-function analysis of the cytoplasmic domain of two BY-kinases, namely CapA1/CapB2 from S. aureus and Wzc from E. coli. We showed that these two proteins associate in a ring-shaped octamer in which the motif EX2RX2R plays a crucial role. In addition, we showed that BY-kinases autophosphorylate using an intermolecular mechanism. We also identified the activation mechanism of BY-kinases and we revealed the role of a particular domain, found specifically in BY-kinases from proteobacteria, in Wzc autophosphorylation and colanic acid biosynthesis.Structural and functional characterization of BY-kinases represents an original and promising approach in order to develop new molecules inhibiting specifically these enzymes and to affect the virulence of bacterial pathogens.

Dentre os vários tipos de leucemia, destaca-se a Leucemia Mielóide Crônica (LMC), um distúrbio mieloproliferativo em que ocorre a translocação entre o gene BCR no cromossomo 22 e o gene ABL1 no cromossomo 9. Essa translocação cria um cromossomo conhecido como Philadelphia (t 9,22)(q34;q11), ou Ph+, e a consequente formação de um produto único de proteínas BCR-ABL1. Essa proteína tem atividade de quinase constitutiva e impulsiona a proliferação descontrolada de células tronco hematopoiéticas. O surgimento de uma nova classe farmacológica no início dos anos 2000 - os inibidores de tirosina quinases, revolucionou o tratamento e o prognóstico da LMC, permitindo que esse câncer fosse tratado praticamente como uma doença crônica, com farmacoterapia oral. A droga de estréia dessa classe, o Mesilato de Imatinib, foi desenvolvida através de modelagem molecular para ser alvo-específica, mas apesar do desenvolvimento exitoso, após o início da comercialização, foram observadas falhas na ação em determinados pacientes. Há evidências de que a avaliação da relação entre a dose de imatinibe (e seus níveis sanguíneos) e a eficácia do tratamento medida através das respostas Hematológica, Citogenética e Molecular, seja uma forma de realizar o ajuste da dose reduzindo efeitos colaterais e custo do tratamento. No presente estudo foram avaliadas as concentrações séricas de imatinib, Cmin e Cmax, em 51 pacientes com Leucemia Mielóide Crônica, dos quais 33 atingiram Resposta Molecular Maior em até 12 meses de tratamento, 11 levaram mais que 12 meses para antingir, e 7 não atingiram. As concentrações séricas obtidas desses pacientes indicaram que no grupo que atingiu RMM em até 12 meses, os valores de vale (Cmin) se apresentaram com mediana de 889.2 ng/mL (721.9 e 1202.4 para primeiro e terceiro quartis respectivamente), sendo que o grupo que levou mais de 12 meses para atingir RMM, a concentração mediana observada foi de 611.0 ng/mL (493.0 e 816.0 para primeiro e terceiro quartis respectivamente), sendo essa diferença estatisticamente significativa (p < 0.05). Dessa forma demonstrou-se a importância do monitoramento das concentrações séricas de imatinib para o ajuste da dose e para a gestão do tratamento na mudança para segunda geração de inibidores de tirosina quinase. Através da análise comparativa dos dados populacionais estudados, observou-se não haver correlação significativa entre as concentrações séricas e índice de massa corpórea (IMC), peso, idade ou sexo / Among the various types of leukemia, Chronic Myeloid Leukemia (CML) stands out as a myeloproliferative disorder in which translocation occurs between the BCR gene on chromosome 22 and the ABL1 gene on chromosome 9. This translocation creates a chromosome known as Philadelphia (t 9,22) (q34; q11), or Ph +, and the consequent formation of a unique BCR-ABL1 protein product. This protein has constitutive kinase activity and drives the uncontrolled proliferation of hematopoietic stem cells. The launch of a new pharmacological class in the early 2000s - the tyrosine kinases inhibitors, revolutionized the treatment and prognosis of CML, allowing that cancer to be treated virtually as a chronic disease with oral pharmacotherapy. The newbie drug of this class, Imatinib Mesylate, was developed through molecular modeling to be target-specific, but despite the successful development, after the beginning of marketing, certain patients presented some failures in the response. There is an evidence that an assessment of the relationship between a dose of Imatinib (and its blood levels) and the efficacy of treatment from its Hematologic, Cytogenetic and Molecular Responses, is a really effective way to perform dose adjustment reducing side effects and cost of treatment. In the present study, the serum concentrations of Imatinib, Cmin and Cmax were evaluated in 51 patients with chronic myeloid leukemia, of which 33 reached major molecular response in up to 12 months of treatment, 10 took more than 12 months to achieve it, and 7 did not reach that. The serum concentrations obtained from those patients indicated that in the group that reached Major Molecular Response (MMR) within 12 months, the trough level (Cmin) presented a median of 889.2 ng / mL (721.9 and 1202.4 for first and third quartiles, respectively), and the group which took more than 12 months to reach MMR, the median concentration observed was 611.0 ng / mL (493.0 and 816.0 for the first and third quartiles respectively), and this difference was statistically significant (p < 0.05). Thus, the importance of monitoring serum imatinib concentrations for dose adjustment and treatment management in switching to second-generation tyrosine kinase inhibitors has been demonstrated. Through the comparative analysis of the population data, there was no significant correlation between serum concentrations and body mass index (BMI), weight, age or gender

Au cours de l’ovogenèse chez la mouche du vinaigre: Drosophila melanogaster, un groupe de cellules folliculaires appelées cellules de bord, migrent à travers les cellules nourricières pour atteindre l’ovocyte. Cet événement, nécessitant la transition épithélio- mésenchymateuse (TEM), la réorientation, puis l’arrêt, ressemble à la formation de métastases. L’endocytose est un régulateur clé de plusieurs événements polarisés, y compris la migration cellulaire. En effet, différentes protéines impliquées dans la migration, comme les intégrines et les E-cadhérines (cadhérines épithéliales), sont régulées par transport à travers les endosomes. De même, l’endocytose restreint au front de migration l’activité des récepteurs tyrosine kinases (RTKs) qui guident les cellules de bord dans leur mouvement. Cependant les mécanismes moléculaires de cette restriction spatiale de l’activité des RTKs demeurent largement inconnus. Nous avons testé l’implication du trafic vésiculaire à travers la machinerie d’endocytose, dans la migration dirigée des cellules de bord, car ce système est facilement accessible pour l’expression de protéines et l’analyse de mutants. Nous avons commencé par confirmer une observation précédente du rôle de l’endosome précoce dans la migration des cellules de bord. Ensuite, nous avons identifié l’endosome de recyclage (ER) comme un régulateur clé de cette migration. En effet, nous avons démontré que l’expression dans les cellules de bord d’une forme dominante négative de Rab11, la petite GTPase régulant le transport vésiculaire à travers l’ER, bloque la migration ou entraîne de sévères défauts de migration dans environ 80% des chambres d’œufs examinées. De plus, nous observons par immunofluorescence une relocalisation de l’activité des RTKs alors que d’autres protéines de migration ne sont pas affectées par Rab11 dominant négatif. Ce résultat a été par la suite confirmé par une interaction génétique entre Rab11 et les RTKs. D’autre part, nous avons montré que le complexe exocyste, un effecteur de Rab11, est impliqué dans la migration des cellules de bord. Nous avons trouvé par microscopie confocale en tissu fixé et par microscopie en temps réel que Sec15, un composant de ce complexe, est polarisé, de façon Rab11- dépendante, dans des vésicules qui s’accumulent au front de migration tout au long du mouvement des cellules de bord. De plus, la perte de l’activité de Sec15 perturbe à son tour la migration. Ainsi, toutes ces données démontrent le rôle fondamental d’un cycle d’endo- exocytose dans le maintien des RTKs actifs au niveau du front de migration des cellules de bord le long de leur mouvement. / During Drosophila melanogaster’s oogenesis, a cluster of folllicle cells, called border cells, perform an invasive migration through the surrounding nurse cells to reach the oocyte. This event resembles metastasis formation since it requires epithelial- mesenchymal transition, reorientation and arrest. Endocytosis plays a fundamental role in many polarized processes, including cell migration, since different migration proteins, like integrins and E-cadherins traffic through the endocytic pathway. Furthermore, receptor tyrosine kinases (RTKs) that guide border cells during their migration are regulated by endocytosis, although the mechanisms involved are largely unknown. We tested the implication of vesicular trafficking through the endocytic machinery, in border cells’ directed migration, because this system is easily accessible for protein expression and mutant analysis. We first confirmed previous observation that trafficking through the early endosome is necessary for border cells migration, and then we identified the recycling endosome as a key compartment for this migration. Indeed, we showed that overexpression in border cells of a dominant negative form of Rab11, the small GTPase regulating vesicular trafficking through the recycling endosome, blocks migration or leads to severe migration defects in about 80% of examined egg chambers. Furthermore, using immunofluorescence, we observed a relocalization of RTKs activity, whereas other migration proteins were not redistributed upon dominant negative Rab11 expression. This result was further confirmed by a genetic interaction between Rab11 and RTKs. Moreover, we showed that the exocyst complex, an effector of Rab11, is also involved in border cells migration. We found by using confocal microscopy of fixed tissues and time-lapse microscopy of living egg chambers, that Sec15, a member of this complex, is distributed in vesicles which are polarized, in a Rab11- dependent manner, throughout border cells migration. In addition, loss of Sec15 also impairs migration. Together these data demonstrate a fundamental role for an endo- exocytic cycle in the maintenance of active RTKs at the leading edge of border cells during their migration.

The Tec family kinases Itk and Rlk have been previously shown to have an important role in signaling downstream of the T cell receptor [TCR]. Almost all of the work done in the past on these two kinases looked at their role in conventional αβ T cells, specifically CD4+ T cells. These studies demonstrated functions for Itk [primarily] and Rlk in T cell development, activation, and differentiation. However, despite the wealth of knowledge on conventional CD4+ T cells, prior to the work presented here little to no studies addressed the role of Tec family kinases on CD8+ or innate T cell development. My studies show a clear role for Itk [and in some cases Rlk] in innate T cell development; whether it be deprecating, in the case of innate CD8+ T cells or some subsets of γδ T cells, or beneficial, in the case of NKT cells. I show that Itk has a crucial role in conventional CD8+ T cell development, as absence of Itk [or Itk and Rlk] causes strongly reduced numbers of conventional CD8+ T cells and a vigorous enhancement of an innate-like CD8+ T cell population. In NKT cells, my work demonstrates that Itk [and to a lesser extent Rlk] is required for terminal maturation, survival, and cytokine secretion. Finally, on γδ T cells Itk is important in maintaining the Th1 cytokine secretion profile usually associated with these cells, and regulating the development of CD4+ or NK1.1+ γδ T cells. Taken together, this work clearly illustrates an important role for Tec family kinases in innate T cell development and function.

Dentre os vários tipos de leucemia, destaca-se a Leucemia Mielóide Crônica (LMC), um distúrbio mieloproliferativo em que ocorre a translocação entre o gene BCR no cromossomo 22 e o gene ABL1 no cromossomo 9. Essa translocação cria um cromossomo conhecido como Philadelphia (t 9,22)(q34;q11), ou Ph+, e a consequente formação de um produto único de proteínas BCR-ABL1. Essa proteína tem atividade de quinase constitutiva e impulsiona a proliferação descontrolada de células tronco hematopoiéticas. O surgimento de uma nova classe farmacológica no início dos anos 2000 - os inibidores de tirosina quinases, revolucionou o tratamento e o prognóstico da LMC, permitindo que esse câncer fosse tratado praticamente como uma doença crônica, com farmacoterapia oral. A droga de estréia dessa classe, o Mesilato de Imatinib, foi desenvolvida através de modelagem molecular para ser alvo-específica, mas apesar do desenvolvimento exitoso, após o início da comercialização, foram observadas falhas na ação em determinados pacientes. Há evidências de que a avaliação da relação entre a dose de imatinibe (e seus níveis sanguíneos) e a eficácia do tratamento medida através das respostas Hematológica, Citogenética e Molecular, seja uma forma de realizar o ajuste da dose reduzindo efeitos colaterais e custo do tratamento. No presente estudo foram avaliadas as concentrações séricas de imatinib, Cmin e Cmax, em 51 pacientes com Leucemia Mielóide Crônica, dos quais 33 atingiram Resposta Molecular Maior em até 12 meses de tratamento, 11 levaram mais que 12 meses para antingir, e 7 não atingiram. As concentrações séricas obtidas desses pacientes indicaram que no grupo que atingiu RMM em até 12 meses, os valores de vale (Cmin) se apresentaram com mediana de 889.2 ng/mL (721.9 e 1202.4 para primeiro e terceiro quartis respectivamente), sendo que o grupo que levou mais de 12 meses para atingir RMM, a concentração mediana observada foi de 611.0 ng/mL (493.0 e 816.0 para primeiro e terceiro quartis respectivamente), sendo essa diferença estatisticamente significativa (p < 0.05). Dessa forma demonstrou-se a importância do monitoramento das concentrações séricas de imatinib para o ajuste da dose e para a gestão do tratamento na mudança para segunda geração de inibidores de tirosina quinase. Através da análise comparativa dos dados populacionais estudados, observou-se não haver correlação significativa entre as concentrações séricas e índice de massa corpórea (IMC), peso, idade ou sexo / Among the various types of leukemia, Chronic Myeloid Leukemia (CML) stands out as a myeloproliferative disorder in which translocation occurs between the BCR gene on chromosome 22 and the ABL1 gene on chromosome 9. This translocation creates a chromosome known as Philadelphia (t 9,22) (q34; q11), or Ph +, and the consequent formation of a unique BCR-ABL1 protein product. This protein has constitutive kinase activity and drives the uncontrolled proliferation of hematopoietic stem cells. The launch of a new pharmacological class in the early 2000s - the tyrosine kinases inhibitors, revolutionized the treatment and prognosis of CML, allowing that cancer to be treated virtually as a chronic disease with oral pharmacotherapy. The newbie drug of this class, Imatinib Mesylate, was developed through molecular modeling to be target-specific, but despite the successful development, after the beginning of marketing, certain patients presented some failures in the response. There is an evidence that an assessment of the relationship between a dose of Imatinib (and its blood levels) and the efficacy of treatment from its Hematologic, Cytogenetic and Molecular Responses, is a really effective way to perform dose adjustment reducing side effects and cost of treatment. In the present study, the serum concentrations of Imatinib, Cmin and Cmax were evaluated in 51 patients with chronic myeloid leukemia, of which 33 reached major molecular response in up to 12 months of treatment, 10 took more than 12 months to achieve it, and 7 did not reach that. The serum concentrations obtained from those patients indicated that in the group that reached Major Molecular Response (MMR) within 12 months, the trough level (Cmin) presented a median of 889.2 ng / mL (721.9 and 1202.4 for first and third quartiles, respectively), and the group which took more than 12 months to reach MMR, the median concentration observed was 611.0 ng / mL (493.0 and 816.0 for the first and third quartiles respectively), and this difference was statistically significant (p < 0.05). Thus, the importance of monitoring serum imatinib concentrations for dose adjustment and treatment management in switching to second-generation tyrosine kinase inhibitors has been demonstrated. Through the comparative analysis of the population data, there was no significant correlation between serum concentrations and body mass index (BMI), weight, age or gender

Orientador: William Dias Belangero / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-28T23:43:39Z (GMT). No. of bitstreams: 1 Faria_LidiaDornelasde_M.pdf: 3066212 bytes, checksum: 6cb5506682740e2fcb2de4b1694998a3 (MD5) Previous issue date: 2015 / Resumo: A Terapia por ondas de choque (TOC) é uma alternativa não invasiva utilizada como método de indução a formação óssea que consiste em pulsos sonoros de alta energia transmitidas de modo focal a um tecido específico. Artigos demonstram aumento de vascularização, que ativação de proteínas como BMP (bone morphogenic protein) e Erk (extracellular signal-regulated kinase) induzindo diferenciação osteogênica após sua utilização no tecido ósseo. O presente estudo visou avaliar os níveis das proteínas Erk e Akt (akutely transforming), envolvidas na cascata protéica responsiva a deformação celular gerada por estímulo mecânico e consequente transformação em estímulo bioquímico induzindo a osteogênese. Os animais selecionados para o estudo foram anestesiados e divididos em dois diferentes grupos, onde no dia 1, o primeiro grupo foi submetido a TOC em sessão única de 500 impulsos gerados por aparelho eletrohidráulico a 0,12mJ/mm²na tíbia intacta e o segundo não recebeu TOC. Na sequência, os animais foram divididos em 3 subgrupos para cada tempo de segmento de 7, 14 e 21 dias A determinação dos níveis das proteínas propostas foi realizada por meio de immunoblotting. A fosforilação das proteínas Erk e Akt dos tecidos ósseos das tíbias extraídas dos ratos aumentou nos grupos submetidos a TOC após 7, 14 e se manteve elevado até o 21° dia quando comparado ao controle / Abstract: Extracorporeal shock wave therapy (ESWT) is a non-invasive alternative used as a method for inducing bone formation that consists of high-energy acoustic pulses transmitted in a focal way to a specific tissue. Studies show increase in vascularization which activate proteins such as BMP and Erk inducing osteogenic differentiation after its use in the bone tissue. The present study aimed at evaluating the levels of Erk and AKT proteins involved in the protein cascade responsive to cell deformation in biochemical stimulus inducing osteogenesis. The animals selected for the study were under anesthesia and divided in two different groups where on day 1 the first group was submitted to ESWT in one 500 pulse-session generated by an electrohydraulic device at 0,12mJ/mm² in intact tibia and fibula and the second did not receive ESWT. Then the animals were divided into 3 sub-groups, one for each segment times of 7, 14 and 21 days. Immunoblotting analysis was performed to determine the levels of the proposed proteins. The Erk and Akt protein phosphorylation of the bone tissues of extracted tibia from the animals increased in the groups submitted to ESWT and kept elevated until the 21st day when compared to the control group / Mestrado / Fisiopatologia Cirúrgica / Mestra em Ciências

Les protéines kinases sont importantes dans de nombreux processus biologiques comme la prolifération, la différenciation, la migration, l’apoptose cellulaire et dans le maintien de l’intégrité de l’ADN. Dans de nombreuses pathologies comme le cancer, ces kinases sont anormalement suractivées. Pour ces raisons, plusieurs inhibiteurs de tyrosine kinase (ITK) ont été développés comme le dasatinib (Sprycel®), un ITK ciblant principalement le BCR-Abl et la famille SRC, qui montre une grande efficacité contre la leucémie myéloïde chronique. Cependant, le dasatinib présente aussi d’importants effets indésirables qui se manifestent au niveau pulmonaire, comme le développement d’hypertension artérielle pulmonaire (HTAP) et l’apparition d’épanchements pleuraux dont l’incidence est respectivement de 0,45% et de 15-35%. Ces effets secondaires représentent donc un problème majeur de santé publique. Les mécanismes impliqués dans ces effets indésirables ne sont pas connus.Mon projet de thèse entre donc dans l’étude des mécanismes par lesquels le dasatinib peut induire ces effets indésirables. Dans ce contexte, nous avons testé l’hypothèse que l’utilisation du dasatinib est associée à l’inhibition inappropriée d’une ou de plusieurs kinases qui jouent un rôle central dans l’intégrité de l’endothélium pulmonaire.Mon travail s’est articulé autour de trois grands axes : (1) Le premier axe a visé à déterminer le rôle des atteintes de la perméabilité endothéliale par le dasatinib dans l’apparition d’épanchement pleural ; (2) Le deuxième axe a consisté à étudier les mécanismes impliqués dans l’HTAP induite par le dasatinib ; (3) Le troisième axe a permis d’identifier le rôle de l’inhibition de la protéine kinase c-Abl dans l’intégrité de la cellule endothéliale pulmonaire.L’ensemble de nos données montre que le dasatinib altère, à forte dose, les fonctions des cellules endothéliales pulmonaires et ainsi fragilise l’intégrité vasculaire. Tout d’abord, nous avons en effet pu mettre en évidence qu’un traitement quotidien à fortes doses de dasatinib chez le rat augmente la perméabilité endothéliale et conduit à l’apparition d’épanchements pleuraux. De manière intéressante, nous avons pu démontrer que cette augmentation de perméabilité endothéliale est liée à une génération de dérivés oxygénés capable de redistribuer les protéines de jonctions intercellulaires. Deuxièmement, nous avons pu mettre en évidence que le dasatinib, à forte dose, induit une apoptose des cellules endothéliales (CE) pulmonaires, un phénomène lié en partie à la génération d’un stress oxydant d’origine mitochondriale. De plus, nous avons noté que des prétraitements de rats avec du dasatinib conféraient une prédisposition à l’hypertension pulmonaire expérimentale par la mise en place d’une dysfonction endothéliale. Ces dernières observations n’ont pas été retrouvées avec d’autres ITK proches du dasatinib comme l’imatinib. Enfin, nous avons pu identifier que c-Abl est une protéine kinase clef de l’intégrité de la cellule endothéliale pulmonaire. En effet, son inhibition par certain ITK conduit à un défaut de réparation des cassures ADN et ainsi à une altération de certaines fonctions des cellules endothéliales.En conclusion, les effets indésirables pulmonaires associés au dasatinib semblent directement liés à une perte de l’homéostasie de l’endothélium pulmonaire par des mécanismes faisant appel à la génération de stress oxydatif mitochondrial, à l’induction d’apoptose et à la perte de perméabilité vasculaire. / By catalysing reversible phosphorylation of their substrates, protein kinases play central role in regulating a multitude of cellular processes, including division, proliferation, apoptosis, and differentiation as well as in the maintenance of DNA integrity. Since deregulation of different protein kinases contribute to several human disorders, a multitude of tyrosine kinase inhibitors (TKIs) inhibiting various kinases have been developed and some of them are currently approved for different indications, and many more are under development. Dasatinib (Sprycel®), an orally available short-acting dual ABL/SRC tyrosine kinase inhibitor (TKI), is an effective treatment for Philadelphia-positive chronic myelogenous leukemia (CML) and for all phases of Philadelphia-positive CML with resistance or intolerance to prior therapy, including imatinib. However, dasatinib treatment is associated with the development of pulmonary arterial hypertension (PAH; 0.45%) and pleural effusion (15-35%). These serious pulmonary adverse events represent a serious public health problem.Therefore, my PhD project is seeking to identify the underlying molecular mechanisms responsible for these pulmonary adverse events induced by dasatinib. My work has followed three different strategic axes seeking to: 1) Determine whether dasatinib alters endothelial integrity, resulting in increased pulmonary vascular endothelial permeability and pleural effusion; 2) Study the mechanisms involved in the long-term development of dasatinib-induced PAH; 3) Precise the role of c-Abl protein kinase inhibition in pulmonary endothelial cell DNA integrity.First, our data indicate that dasatinib can lead to pulmonary endothelial dysfunction and thus affect vascular integrity. Interestingly, we demonstrated that this increased endothelial permeability is a reactive oxygen species (ROS)-dependent mechanism in vitro and in vivo. Second, we also found that high doses of dasatinib induce pulmonary endothelial cell apoptosis by increasing mitochondrial oxidative stress and cause endothelial cell apoptosis. Consistent with these observations, we found that pretreatment of rats with dasatinib leads pulmonary endothelial dysfunction and confers increased susceptibility to experimental pulmonary hypertension (PH) in contrast to rats pretreated with imatinib or vehicle. Finally, we identified c-Abl as a key protein kinase in pulmonary endothelial cells, since its inhibition by dasatinib and ponatinib leads to impaired DNA repair in human pulmonary endothelial cells.Taken together, my PhD results demonstrate the importance played by damage to the pulmonary endothelium in the onset of dasatinib-induced PAH and pleural effusion.

Topographical and mechanical properties of adhesive substrates provide important biological cues that affect cell spreading, migration, growth, and differentiation. The phenomenon has led to the increased use of topographically patterned and flexible substrates in studying cultured cells. However, these studies may be complicated by various limitations. For example, the effects of ligand distribution and porosity are affected by topographical features of 3D biological constructs. Similarly, many studies of mechanical cues are compounded with cellular deformation from external forces, or limited by comparative studies of separate cells on different substrates. Furthermore, understanding cell responses to mechanical input is dependent upon reliable measurements of mechanical properties. This work addresses each of these issues. To determine how substrate topography and focal adhesion kinase (FAK) affect cell shape and movement, I studied FAK-null (FAK -/-) and wild type mouse 3T3 fibroblasts on chemically identical polystyrene substrates with either flat surfaces or micron-sized pillars, I found that, compared to cells on flat surfaces, those on pillar substrates showed a more branched shape, an increased linear speed, and a decreased directional stability, which were dependent on both myosin-II and FAK. To study the dynamic responses to changes in substrate stiffness without other confounding effects, I developed a UV-modulatable substrate that softens upon UV irradiation. As atomic force microscopy (AFM) proved inadequate to detect microscale changes in stiffness, I first developed and validated a microsphere indentation method that is compatible with fluorescence microscopy. The results obtained with this method were comparable to those obtained with AFM. The UV-modulatable substrates softened by ~20-30% with an intensity of irradiation that has no detectable effect on 3T3 cells on control surfaces. Cells responded to global softening of the substrate with an initial retraction followed by a gradual reduction in spread area. Precise spatial control of softening is also possible - while there was little response to posterior softening, anterior softening elicited a pronounced retraction and either a reversal of cell polarity or a significant decrease in spread area if the cells move into the softened region. In conclusion, these techniques provide advances in gaining mechanistic insight into cellular responses to topographical and mechanical cues. Additionally, there are various other potential applications of the novel UV-softening substrate, particularly in regenerative medicine and tissue engineering.

Src family kinases (SFKs) are key regulators of cell proliferation, differentiation, and survival. The expression of these non-receptor tyrosine kinases is strongly correlated with cancer development and tumor progression. Thus, this family of proteins serves as an attractive drug target. The activation of SFKs can occur via multiple signaling pathways, yet many of them are poorly understood. Here, we summarize the current knowledge on G protein-coupled receptor (GPCR)- mediated regulation of SFKs, which is of considerable interest because GPCRs are among the most widely used pharmaceutical targets. This type of activation can occur through a direct interaction between the two proteins or be allosterically regulated by arrestins and G proteins. We postulate that a rearrangement of binding motifs within the active conformation of arrestin-3 mediates Src regulation by comparison of available crystal structures. Therefore, we hypothesize a potentially different activation mechanism compared to arrestin-2. Furthermore, we discuss the probable direct regulation of SFK by GPCRs and investigate the intracellular domains of exemplary GPCRs with conserved polyproline binding motifs that might serve as scaffolding domains to allow such a direct interaction. Large intracellular domains in GPCRs are often understudied and, in general, not much is known of their contribution to different signaling pathways. The suggested direct interaction between a GPCR and a SFK could allow for a potential immediate allosteric regulation of SFKs by GPCRs and thereby unravel a novel mechanism of SFK signaling. This overview will help to identify new GPCR–SFK interactions, which could serve to explain biological functions or be used to modulate downstream effectors.