L’érythropoïèse est le processus aboutissant à la production des hématies à partir d’une cellule souche hématopoïétique. La différenciation érythroïde implique des changements morphologiques en partie liés à la perte d’expression membranaire du récepteur à activité tyrosine kinase de classe III, c-Kit. En réponse à son ligand, le SCF, c-Kit est activé puis internalisé et dégradé par la voie du protéasome, via l’ubiquitine E3-ligase c-Cbl, ou par la voie lysosomale suite à une endocytose. Dans la première partie de ce travail, nous avons pu mettre en évidence qu’en absence de SCF et en réponse à un inhibiteur de tyrosine kinase, l’imatinib, les érythroblastes cultivés ex vivo perdent l’expression membranaire de c-Kit et accélèrent leur entrée en différenciation terminale. Au vu de ces observations, nous avons cherché à comprendre les mécanismes impliqués. Sur un modèle de cellules érytholeucémiques dépendantes de l’érythropoïétine, mais exprimant de manière endogène c-Kit, nous avons montré que l’imatinib induit une internalisation du récepteur ainsi que sa dégradation par la voie lysosomale et de manière indépendant de c-Cbl. De plus, nous avons montré que cet effet est réversible et que l’imatinib ne bloque pas la réexpression de c-Kit après son internalisation en réponse au SCF. Des marquages métaboliques ont permis de montrer que l’imatinib ne modifie ni la synthèse ni la maturation de c-Kit et que le profil phospho-tyrosine des cellules traitées à l’imatinib est globalement inchangé. Enfin, nous avons montré que la fixation de l’imatinib à la poche catalytique de c-Kit est indispensable à son internalisation, et par conséquent à sa dégradation. Il apparait donc que l’imatinib lève l’auto-inhibition de c-Kit, qui semble nécessaire pour son maintien à la membrane. Dans la seconde partie de ce travail, nous nous sommes intéressés aux changements morphologiques subis par les nucléoles, lieu de la biogenèse des ribosomes, au cours de différenciation des érythroblastes. L’étude de la taille et du potentiel prolifératif des cellules, ainsi que l’analyse morphologique des nucléoles, nous a permis de confirmer que la réduction de taille des cellules est contemporaine d’un ralentissement de leur prolifération ainsi que de la réduction du volume et de la surface du composé granulaire (CG), « matrice » du nucléole. En microscopie électronique, nous montrons la persistance des CG en fin de maturation. Enfin, nous avons également étudié l’évolution des nucléoles dans un contexte pathologique de syndromes myélodysplasiques de faible risque, qui se caractérisent par une hématopoïèse inefficace. Nous observons que les cellules pathologiques immatures ont des CG plus volumineux que les cellules normales immatures, et qu’au cours de la différenciation, la morphologie des nucléoles est identique entre les cellules normales et pathologiques. En conclusion, ce travail a permis de décrire 1) le mécanisme d’internalisation d’un récepteur à activité tyrosine kinase de classe III, c-Kit par l’imatinib et 2) la morphologie du nucléole au cours de la différenciation érythroïde normale et pathologique des syndromes myélodysplasiques de faible risque. / Erythropoiesis is the process leading to the production of red blood cells from hematopoietic stem cell. The erythroid differentiation involves morphological cell changes, in part related to the loss of membrane expression of the type III receptor tyrosine kinase, c-Kit. In response to its ligand SCF, c-Kit is activated, then internalized and degraded by the proteasome pathway via the E3 ubiquitin ligase c-Cbl, or by the lysosomal pathway, after endocytosis. In the first part of this work, we demonstrated that in the absence of SCF and in response to tyrosine kinase inhibitor, imatinib, erythroblasts cultured ex vivo, lose membrane expression of c-Kit and accelerate their terminal differentiation. In view of these observations, we sought to understand the mechanisms involved. On an erythropoietin dependent cell line expressing c-Kit at the membrane, we showed that imatinib induces receptor internalization and degradation by the lysosomal pathway, independently of c -Cbl. Furthermore, we showed that this effect is reversible and that imatinib does not block the c-Kit re-expression after its internalization, in response to SCF. Metabolic labelling showed that imatinib does not alter synthesis or maturation of c -Kit and that the phospho-tyrosine profile of cells treated with imatinib is generally unchanged. Finally, we showed that the binding of imatinib to the catalytic pocket of c-Kit is essential for its internalization, and therefore its degradation. So, it appears that imatinib removes c-Kit self-inhibition, which seems necessary to its retention at the membrane. In the second part of this work, we studied the morphological changes of nucleoli, the site of ribosome biogenesis, during erythroid differentiation. We showed that the reduction of cell size takes place at the same time than reduction of cell proliferation and reduction of surface and volume of the Granular Compound (GC), the “matrix” of the nucleolus. Moreover, we showed by electronic microscopy, the persistence of GC at the end of maturation. Finally, we also studied the evolution of nucleoli in a pathological context of low risk myelodysplastic syndromes, which are characterized by ineffective hematopoiesis. We observed that immature pathological cells have larger GC than immature normal cells, but that during differentiation, the morphology of nucleoli is identical between normal and pathological cells. In conclusion, this work has allowed us to describe 1) the mechanisms of internalization of a class III receptor tyrosine kinase, c-Kit by imatinib and 2) the morphology of the nucleolus during normal and pathological low risk myelodysplastic syndromes of erythroid differentiation.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-06-12T18:19:02Z No. of bitstreams: 1 BismarckAscarSauaia.pdf: 1915965 bytes, checksum: 720a005798d94ec0d82a2ba0cf2b225e (MD5) / Made available in DSpace on 2017-06-12T18:19:02Z (GMT). No. of bitstreams: 1 BismarckAscarSauaia.pdf: 1915965 bytes, checksum: 720a005798d94ec0d82a2ba0cf2b225e (MD5) Previous issue date: 2016-03-31 / Estimation of blood glucose levels using invasive methods currently is a routine in emergency and ambulatory attendance of public health services, as well as in residential glucose monitoring, in order to prevent glucose levels variation, which may evolve to ophthalmic, renal, vascular and neurological complications. In order to develop a low-cost, socially accessible and non-invasive method to establish blood glucose levels, it was created a linear conversion mathematical model from salivary hidrogenionic potential (pH) values to blood glucose level. After signing the informed consent form and filling out the survey questionnaire to gather personal, clinical and family history data, it was collected a drop of blood from the tip of the right index finger, which was submitted to the glucometer for blood glucose level determination and after that, it was also collected 2 mL of saliva in a 5ml glass Beacker to salivary pH definition with a digital pH meter. Data collection was performed between May and November, 2014, with a sample of 333 cappilary blood glucose (CG) and salivary ph tests from non - diabetic adult volunteers, in fasting state from 2 to 4 hours after last meal, who were randomly selected from medicine and physiotherapy clinics of UNICEUMA. After entering data in EPINFO 2000 program, results were analyzed with Pearson’s correlation coefficient (r) in Bioestat 5.3 Software and presented a value of – 0,7522, showing the existence of a correlation between capillary blood glucose and salivary pH. Then, we defined that variables were related in a mathematical form of a straight line. Thus, we found the coefficient of determination between capillary blood glucose and salivary pH using simple linear regression test: R2 = 0,5658. Regarding alpha level = 0.05, we established a mathematical model: y = a + bx, where a = 9.3286 and b = - 0.0278. So, after calculating blood glucose level from pH (pH = F (Glycemia); we have: CG = (9.3286 - pH)/ 0.0278. The results demonstrated the relation among CG values (mg/dl), corresponding salivary ph and the diagnosis respectively: CG < 70  pH > 7,94 with (Variation (Δ) = 7,784 to 8,096) = Hypoglycemic Risk; CG between 70 and 100  pH 7,94 to 6,69 = Security Gap or normality; CG > 100  pH < 6,69 with (Δ = 6,536 to 6,848) = Hyperglycemic Risk. The proposed mathematical model may allow the definition of a diagnostic kit to estimate blood glucose levels from salivary pH through a non-invasive, low-cost, socially accessible and less traumatic method either in healthy patients or at risk for developing diabetes. / A estimativa dos níveis de açúcar no sangue, por método invasivo, atualmente é rotina no atendimento de emergência e ambulatorial das unidades públicas de saúde, assim como, no controle e monitoramento glicêmico domiciliar, para prevenir variações, que podem evoluir em complicações oftalmológicas, renais, vasculares, neurológicas, entre outras. Com a finalidade de minimizar gastos e disponibilizar um kit de baixo custo, acessível e não invasivo para a determinação de níveis glicêmicos capilares, foi criado um modelo matemático linear de conversão dos valores do potencial Hidrogeniônico (pH) salivar em glicêmico capilar. Após assinatura do Termo de Consentimento livre e Esclarecido (TCLE) e preenchimento do questionário de avaliação contendo dados pessoais, clínico e de histórico familiar do paciente, coletou-se uma gota de sangue da extremidade do indicador direito, a qual foi submetida à glicosímetro para a definição do nível de glicemia, e a seguir, em um recipiente de 5 ml, coletou-se 2 ml de saliva para leitura do pH em phgâmetro digital. A coleta foi realizada entre os meses de maio a novembro de 2014, sendo a amostragem constituída por 333 exames de Glicemia Capilar (GC) e pH do fluido, de voluntários, adultos, não diabéticos, em jejum alimentar de 2 a 4 horas, os quais foram selecionados aleatoriamente, provenientes da demanda da clínica médica e de fisioterapia do UNICEUMA. Os resultados após tabulados no banco do programa EPINFO 2000 e, analisados com auxílio do Software Bioestat 5.3 a partir da aplicação do teste de correlação de Pearson apresentaram o valor de r (coeficiente de correlação) igual a – 0,7522, admitindo-se a existência de correlação entre GC e o pH salivar. Em seguida, definiu-se a forma matemática em que as variáveis estão relacionadas, nesse caso, uma reta. Desta forma, a partir da aplicação do teste de regressão linear simples obteve-se o coeficiente de determinação ou explicação da correlação entre GC e pH da saliva: R2 = 0,5658. Considerando-se o nível de decisão alfa = 0.05, estabeleceu-se um modelo matemático: y = a + bx, sendo a = 9,3286 e b = - 0,0278. Logo, calculando glicemia a partir do pH (pH = F(Glicemia); temos: GC = (pH - 9,3286)/ - 0,0278. Os resultados demonstraram a relação entre valores de GC (mg/dl), seu correspondente no pH da saliva e o diagnóstico, respectivamente: GC < 70  pH > 7,94 com (Variação (Δ) = 7,784 a 8,096) = Risco Hipoglicêmico; GC Entre 70 e 100  pH 7,94 a 6,69 = Faixa de Segurança ou normalidade; GC > 100  pH < 6,69 com (Δ = 6,536 a 6,848) = Risco Hiperglicêmico. O modelo matemático proposto pode ser utilizado para a definição de um kit de diagnóstico na estimativa dos níveis glicêmicos, a partir do pH salivar por método não invasivo, de baixo custo, socialmente acessível, com menos trauma em pacientes adultos hígidos ou com risco de desenvolver a diabetes

La mastocytose est une maladie hétérogène, caractérisée par une accumulation de mastocytes dans l’organisme. Les enfants et les adultes ont des mutations différentes de c-Kit. Dans un premier travail, nous avons montré que seules les formes adultes sont associées à la réactivation de la télomérase, alors que les formes pédiatriques ne sont pas. Cela semble être lié aux différences de mutations de c-Kit observées entre adultes et enfants et pourrait expliquer pourquoi seules les formes pédiatriques de mastocytose régressent spontanément et non les formes adultes. Ces résultats aident à mieux comprendre la physiopathologie de la mastocytose. Dans un second travail, nous a étudié le lien entre la longueur des télomères et les troubles psychologiques des adultes atteints de mastocytose. Nous avons montré que réactions émotionnelles négatives sont corrélées au raccourcissement de la longueur des télomères des leucocytes et que l’érosion télomérique est fortement prédite par les défauts de régulation des émotions. Nous émettons l'hypothèse qu’au cours des troubles neuropsychologiques, le mastocyte pourrait être impliqué dans le raccourcissement de la longueur des télomères en périphérie. / Mastocytosis is a heterogeneous disease characterized by an accumulation of mast cells. Children and adults hold different c-Kit mutations. In a first work, we showed that only adult forms are associated with reactivation of telomerase whereas pediatric forms are not. This seems to be linked to the differential c-Kit mutations observed between adults and children and could explain why only pediatric mastocytosis spontaneously regress in comparison with adult forms. These results help to better elucidate the pathophysiology of mastocytosis. In a second work, we studied the link between the telomere length and the psychological features of adults with mastocytosis and showed that negative emotionality correlated negatively to telomere length and that telomere shortening was strongly predicted by emotion regulation deficits. We hypothesize that in psychological disorders, mast cell may represent the link between brain and periphery and induce telomere shortening.

Thesis (M.S.)--Montana State University--Bozeman, 2005. / Title from PDF t.p. (viewed on June 12, 2006). Chairperson, Graduate Committee: Scott Creel. Includes bibliographical references (p. 71-80).

Esta dissertação é parte integrante da linha de pesquisa Currículo: sujeitos, conhecimentos e cultura e do grupo de pesquisa Currículo, cultura e diferença coordenado pela Prof Dr Elizabeth Fernandes de Macedo. Neste trabalho analisei os embates através das articulações discursivas em torno do kit anti-homofobia do Projeto Escola Sem Homofobia do Ministério da Educação. O objetivo deste trabalho era responder o que pode ter contribuído para que o discurso contrário à implementação deste projeto tenha saído hegemônico nesta disputa, e se seria este material um desestabilizador na discussão binária que envolve esta temática ou se reforçaria este binarismo. Para isso, utilizei como aporte teórico estudos pós-coloniais, estudos pós-estruturais, teoria do discurso, discussões do campo do currículo e teoria queer. Metodologicamente, acompanhei os argumentos dxs atorxs políticos que posicionaram-se favoráveis à utilização deste kit nas escolas e os argumentos dos que posicionaram-se contrariamente em alguns veículos de imprensa de relevância nacional e nos pronunciamentos nos plenários da Câmara e no Senado no período de novembro de 2010 a outubro de 2012. A análise evidenciou que os embates discursivos provocados por tais ações governamentais podem ser um convite às resignificações da tradição cultural brasileira tão marcada pelo sexismo e a homofobia. / This dissertation is an integral part of the research line Currículo: sujeitos, conhecimentos e cultura which is coordinated by Prof. Dr. Elizabeth Fernandes de Macedo. In this work I have analysed the conflicts that took place through discursive articulations on the anti-homophobia kit of the project Escola Sem Homofobia from the Ministry of Education. The aim of this work was to answer about what might have contributed for the discourse against the implementation of such project to have come out of these conflicts as a hegemonic one. Moreover, the work also aims at answering if this material would be a destabilizer in the binary discussion that is involved in this set of themes or if it world reinforce such binary frame. To that end, I used the following as theoretical resources: post-colonial studies, post-structural studies, discourse theory, discussions on the field of curriculum, and queer theory. Methodologically I followed both the arguments of the political actors who positioned themselves favourably to the utilization of this kit in schools and the arguments of those who positioned themselves against it in some nationally relevant media outlets, as well as in the pronouncements issued in the House of Representatives and the Senate plenary sessions, from November 2010 to October 2012. The analysis evidenced that the discursive conflicts provoked by such governmental actions may be an invitation to resignifying Brazilian cultural tradition, which is so marked by sexism and homophobia.

C-Kit est généralement considéré comme un récepteur à tyrosine kinase et comme un proto-oncogène, dont la surexpression et la mutation conduisent à une progression tumorale médiée par son activité kinase. En clinique, les traitements ciblant l’activité kinase de c-Kit, comme l’Imatinib (Gleevec), ont été largement utilisés pour traiter les patients atteints de maladies liées à c-Kit. Alors que le rôle de c-Kit comme proto-oncogène ne fait aucun doute, certaines études et analyses de bases de données diffèrent avec l’idée d’un rôle pro-tumoral de c-Kit, laissant penser à un rôle différent de c-Kit dans le cancer. Ici, nous montrons que c-Kit appartient à la famille des récepteurs à dépendance, de la même façon que d’autres récepteurs de la famille des tyrosine kinases tel que MET, RET et TrkC. En absence de son ligand Stem Cell Factor (SCF), au lieu de rester inactif, c-Kit déclenche l’apoptose, qui peut être renforcée par l’invalidation de son activité kinase. En parallèle, nous montrons que c-Kit est capable de se lier à la caspase-9 et de l’activer. De plus, à la manière d’autres récepteurs à dépendance, c-Kit est aussi clivé par des protéases de type caspase sur son résidu acide aspartique D816, qui est nécessaire à son activité pro-apoptotique. La mutation du site D816 inhibe l’interaction entre c-kit et la caspase-9 et invalide l’activité pro-apoptotique de c-Kit. De façon intéressante, la mutation D816 est l’une des mutations les plus communes de ce récepteur dans la plupart des cancers liés à c-Kit, et cette mutation favorise la résistance au traitement Gleevec. Nous montrons aussi que la surexpression de c-Kit invalidé pour son activité kinase est capable d’inhiber la croissance tumorale dans des modèles animaux, alors que la mutation du site D816 empêche son effet suppresseur de tumeur. En outre, nous avons développé un outil permettant de bloquer l’interaction entre SCF et c-Kit, déclenchant l’activité pro-apoptotique de c-Kit dans les cancers positifs pour ce récepteur. En utilisant l’activité pro-apoptotique de c-Kit, en combinaison avec des inhibiteurs de kinases comme le Gleevec, nous proposons une nouvelle stratégie thérapeutique. En conclusion, nous démontrons que c-Kit est un membre de la famille des récepteurs à dépendance, présentant une activité pro-apoptotique, et pouvant être utilisé comme un outil alternatif dans le cadre d’un traitement contre le cancer / C-Kit has been generally considered as a receptor tyrosine kinase and a proto-oncogene, whose upregulation and mutation lead to tumor progression through its kinase activity. Clinically, drugs targeting the kinase activity of c-Kit, such as Imatinib (Gleevec), have been wildly used to treat patients with c-Kit related diseases. While the role of c-Kit as a proto-oncogene is of no doubt, some research reports and database analysis do not fit well the tumor promoting role of c-Kit, indicating a possible different role of c-Kit in cancer. Here, we show that c-Kit belongs to the dependence receptor family, similarly to other receptor tyrosine kinases such as MET, RET and TrkC. In the absent of its ligand SCF (stem cell factor), instead of staying inactive, c-Kit triggers apoptosis, which can be enhanced by silencing its kinase activity. Besides, we have shown that c-Kit is able to bind and activate caspase-9. Moreover, similarly to other dependence receptors, c-Kit is also cleaved by caspases-like protease at aspartic acid residue D816, which is crucial for its pro-apoptotic activity. The mutation of D816 site inhibits the c-Kit/caspase-9 binding and silences the pro-apoptotic activity of c-Kit. Of interest, c-Kit D816 mutation is one of the most common mutation of this receptor in many c-Kit related cancers and it promotes resistance against Gleevec treatment. We also show that overexpression of kinase mutated c-Kit is able to inhibit tumor growth in animal models, while the mutation of D816 site impairs the tumor suppressing activity. Furthermore, we develop a tool to block the SCF/c-Kit interaction, which unleashes the pro-apoptotic activity of c-Kit in cancers expressing this receptor. By using the pro-apoptotic activity of c-Kit, in combination with kinase inhibitors like Gleevec, we propose a novel therapeutic strategy. In conclusion, we demonstrate that c-Kit is a member of dependence receptor family, harboring intrinsic pro-apoptotic activity, which can be used as an alternative tool in cancer treatment

Esta dissertação é parte integrante da linha de pesquisa Currículo: sujeitos, conhecimentos e cultura e do grupo de pesquisa Currículo, cultura e diferença coordenado pela Prof Dr Elizabeth Fernandes de Macedo. Neste trabalho analisei os embates através das articulações discursivas em torno do kit anti-homofobia do Projeto Escola Sem Homofobia do Ministério da Educação. O objetivo deste trabalho era responder o que pode ter contribuído para que o discurso contrário à implementação deste projeto tenha saído hegemônico nesta disputa, e se seria este material um desestabilizador na discussão binária que envolve esta temática ou se reforçaria este binarismo. Para isso, utilizei como aporte teórico estudos pós-coloniais, estudos pós-estruturais, teoria do discurso, discussões do campo do currículo e teoria queer. Metodologicamente, acompanhei os argumentos dxs atorxs políticos que posicionaram-se favoráveis à utilização deste kit nas escolas e os argumentos dos que posicionaram-se contrariamente em alguns veículos de imprensa de relevância nacional e nos pronunciamentos nos plenários da Câmara e no Senado no período de novembro de 2010 a outubro de 2012. A análise evidenciou que os embates discursivos provocados por tais ações governamentais podem ser um convite às resignificações da tradição cultural brasileira tão marcada pelo sexismo e a homofobia. / This dissertation is an integral part of the research line Currículo: sujeitos, conhecimentos e cultura which is coordinated by Prof. Dr. Elizabeth Fernandes de Macedo. In this work I have analysed the conflicts that took place through discursive articulations on the anti-homophobia kit of the project Escola Sem Homofobia from the Ministry of Education. The aim of this work was to answer about what might have contributed for the discourse against the implementation of such project to have come out of these conflicts as a hegemonic one. Moreover, the work also aims at answering if this material would be a destabilizer in the binary discussion that is involved in this set of themes or if it world reinforce such binary frame. To that end, I used the following as theoretical resources: post-colonial studies, post-structural studies, discourse theory, discussions on the field of curriculum, and queer theory. Methodologically I followed both the arguments of the political actors who positioned themselves favourably to the utilization of this kit in schools and the arguments of those who positioned themselves against it in some nationally relevant media outlets, as well as in the pronouncements issued in the House of Representatives and the Senate plenary sessions, from November 2010 to October 2012. The analysis evidenced that the discursive conflicts provoked by such governmental actions may be an invitation to resignifying Brazilian cultural tradition, which is so marked by sexism and homophobia.

Made available in DSpace on 2014-06-11T19:24:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-04-06Bitstream added on 2014-06-13T20:07:59Z : No. of bitstreams: 1 batistela_rf_me_rcla.pdf: 1706264 bytes, checksum: 1528e2f49afe43e101299ea98ab97c44 (MD5) Bitstreams deleted on 2017-03-10T13:07:26Z: batistela_rf_me_rcla.pdf,. Added 1 bitstream(s) on 2017-03-10T13:08:13Z : No. of bitstreams: 1 000324142.pdf: 1614795 bytes, checksum: cd5cf3acaed355ef552c6ed6fe7b0fa0 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetico desta pesquisa foi construir um kit de instrumentos feitos com espelhos planos. Para atingir esse propósito uma pesquisa bibliográfica foi desenvolvida a respeito de instrumentos similares existentes para o ensino de geometria. A descrição de tais objetos é apresentada. Os dados obtidos na descrição realizada foram analisados com recursos da fenomenologia. Através das interpretações efetuadas foram articulados temas convergentes desses instrumentos, expressas na pesquisa como: o que é, como é feito e para que é utilizado. Essas convergências constituíram-se o núcleo da construção do kit apresentado nessa dissertação. A criação e cosntrução do kit foram minuciosamente explicitadas. Esse kit é um instrumento inovador e importante para o ensino de geometria e permite, através da visualização e da cosntrução de bases, a exploração de conceitos e propriedades geométricas abordando temas como: polígonos, poliedros de Platão e de Arquimedes, padrões simétricos, tesselações do plano e do espaço, construções geométricas, simetria, reflexão, translação, rotação, ângulos, entre outros. / The purpose of this research was to construct a kit of instruments made out of plane mirrors. To reach this purpose, a bibliographic search was developed about already existing similar instruments to teach geometry. A description of such tools is presented. The data found in the description was analyzed from phenomenological perspective. Though the interpretation of these instruments convergent themes were found: what is, how is constructed and for what is used. These convergences constituted the core for the construction of this new kit presented in this dissertation. The creation and cosntruction of is presented in details. This kit is an important and new instrument and allows the work of geometrics concepts and properties through of visualization and construction of basis, to explore geometric concepts and properties and the studying of themes, such that: polygons, Arquimedes and Plato's polyhedron, symmetric patterns, spherical and plane tessellations, geometric constructions, symmetry, reflexion, translation, rotation, and others.

This thesis describes the design of construction of the converter USB joystick on the Ethernet interface. In the first part are theoretically analyzed both protocols used for the converter. An analysis of current microprocessor trade and their possible using for application in this project. Next part including the specification of used microprocessor and all options of programming and debugging of the microprocessor. Another part deals with the detailed design of the hardware interface. The last part describes the software equipment of microprocessor and the final appreaciation of work.

Les mastocytoses systémiques (SM) constituent un groupe hétérogène de maladies rares, caractérisées par l’accumulation anormale de mastocytes malins dans la moelle osseuse et dans d’autres organes extra-cutanés. La majorité des patients avec SM ont une mutation activatrice du gène KIT, le plus souvent la mutation KIT D816V, retrouvée chez plus de 90% de tous les patients. Cette mutation induit l’activation constitutive du récepteur KIT en déclenchant de façon aberrante une cascade de voies de signalisation, dont la voie PI3K/AKT et JAK/STAT5, aboutissant à l’inhibition de l’apoptose et à l’augmentation de la prolifération et de la survie des mastocytes malins. Cependant, l’efficacité des inhibiteurs de tyrosines kinases (ITKs) sur cette mutation est limitée à cause de la résistance et/ou de toxicité liée à un manque de spécificité. Il est donc nécessaire de trouver de nouvelles approches thérapeutiques afin de contourner cette résistance au cours des SM KIT D816V+ avancées. Nous avons utilisé une approche consistant à cibler de façon combinée des molécules activées en aval de KIT D816V, comme AKT et STAT5, par des inhibiteurs pharmacologiques. Ceci nous a permis d’identifier une combinaison synergique entre un inhibiteur d’AKT (GSK690693) et un inhibiteur de STAT5 (BP-1-102). Ces composés sont capables d’inhiber la prolifération des cellules KIT D816V+ seuls ou en combinaison, mais à de très fortes concentrations, malheureusement non utilisables en thérapeutique. Néanmoins, ces premiers résultats ont permis de valider AKT et STAT5 comme cibles potentielles dans le traitement des SM avancées. La seconde approche employée a été de cibler directement le récepteur KIT D816V par des inhibiteurs pharmacologiques. A l’issu d’un criblage, nous avons identifié trois composés - BLU2317, BLU2718 et DCC-2618 - capables d’inhiber sélectivement la phosphorylation de KIT D816V. Ces composés inhibent la prolifération des cellules ROSAKIT D816V et HMC-1.2, et induisent l’apoptose des cellules de façon dose-dépendante. Bien que les effets de ces trois composés soient similaires, DCC-2618 agit à des concentrations plus faibles par rapport aux composés BLU2317 et BLU2718. Afin d’apprécier l’efficacité in vivo de DCC-2618, nous avons d’abord établi un nouveau modèle de SM basé sur l’injection intraveineuse des cellules ROSAKIT D816V-Gluc exprimant la Gaussia luciferase (Gluc) dans des souris NSG. La présence de la Gluc sécrétée par les cellules ROSAKIT D816V-Gluc facilite la mise en évidence de prise de greffe et permet un contrôle précis de la progression de la maladie. Ce modèle reproduit, au bout de 4 semaines, chez toutes les souris greffées, une SM avancée similaire à celle retrouvée chez l’homme, avec atteinte de la moelle osseuse, du sang, de la rate et du foie, tandis que la dégradation de l’état général des souris n’est observée qu’à partir de 12 semaines. Ce nouveau modèle offre suffisamment de temps pour explorer la cinétique de la progression de la maladie et surtout pour effectuer des études pharmacologiques précliniques. L’évaluation de l’effet de DCC-2618 in vivo a été réalisée sur ce modèle. Etonnamment, DCC-2618 n’a pas été capable d’inhiber la progression de la maladie chez les souris traitées, bien qu’atteignant des concentrations élevées dans la moelle osseuse et le plasma des souris traitées. Néanmoins, DCC-2618 s’est montré capable d’inhiber la phosphorylation de KIT dans les cellules issues de la moelle osseuse des souris traitées. En revanche, contrairement aux effets observés in vitro, DCC-2618 a induit une surexpression de phospho-ERK1/2 dans les cellules malignes des souris greffées. Ceci suggère qu’ERK1/2 joue un rôle important dans la résistance au composé DCC-2618 et éventuellement à d’autres ITKs, indépendamment du récepteur KIT. ERK1/2 pourrait donc être une nouvelle cible thérapeutique d’intérêt dans le traitement des SM résistantes aux ITKs / Systemic mastocytosis (SM) is a heterogeneous group of rare diseases characterized by abnormal accumulation of malignant mast cells (MCs) in the bone marrow (BM) and other extra-cutaneous organs. The majority of SM patients have an activating mutation in the KIT gene, usually the D816V point mutation, which is found in more than 90% of all patients. This mutation induces constitutive activation of the KIT receptor by triggering a cascade of signaling pathways, including the PI3K/AKT and the JAK/STAT5 pathways, resulting in the inhibition of apoptosis and increased survival and proliferation of malignant mast cells. However, the efficacy of the tyrosine kinase inhibitors (TKIs) on this mutation is limited due to resistance and/or toxicity associated with a lack of specificity. It is therefore critical to find new therapeutic approaches to overcome this resistance to TKIs, particularly for advanced KIT D816V+ SM. In the present thesis, we have used an approach consisting in targeting molecules activated downstream of KIT D816V, such as AKT and STAT5, using pharmacological inhibitors in combination. This allowed us to identify a synergistic combination of an AKT inhibitor (GSK690693) and an inhibitor of STAT5 (BP-1-102). These compounds are able to inhibit proliferation of KIT D816V+ cells, alone or in combination, but at very high concentrations, unfortunately not useful therapeutically. Nevertheless, these initial results have validated STAT5 and AKT as potential targets for the treatment of advanced SM. The second approach used was to target directly the KIT D816V receptor by pharmacological inhibitors. After a large screening, we identified three compounds - BLU2317, BLU2718 and DCC-2618 - which selectively inhibit the phosphorylation of KIT D816V. These compounds inhibit the proliferation of ROSAKIT D816V and HMC-1.2 cells, and induce apoptosis of these cells in a dose-dependent manner. Although the effects of these three compounds are similar, the DCC-2618 compound acts at lower concentrations relative to BLU2317 and BLU2718 compounds. In order to assess the in vivo efficacy of DCC-2618, we first established a new model of SM based on intravenous injection of cells expressing Gaussia luciferase (Gluc), ROSAKIT D816V-Gluc cells, in NSG mice. The presence of the secreted Gluc in ROSAKIT D816V-Gluc cells facilitates the detection of engraftment and allows precise monitoring of disease progression. This model reproduced within four weeks, in all grafted mice, an advanced SM similar to the one found in humans, with neoplastic MCs infiltration in BM, blood, spleen and liver, while the terminal deterioration of the clinical condition of the mice was observed after 12 weeks. Thus, this new in vivo model allows modulating the aggressiveness of the disease by varying the number of injected cells. It provides sufficient time to explore the kinetics of disease progression and especially to conduct preclinical pharmacological studies. We then evaluated the effect of DCC-2618 compound in vivo on this model. Surprisingly, DCC-2618 was not able to inhibit disease progression in treated mice, although it reached high concentrations in the BM and the plasma of treated mice. Nevertheless, we showed that the compound was able to inhibit the phosphorylation of the KIT receptor in cells derived from the BM of treated mice. In addition, contrasting to the effects observed in vitro, DCC-2618 induced an over-expression of phospho-ERK1/2 in the malignant cells of transplanted mice. This suggests that ERK1/2 may play a critical role in the resistance to DCC-2618, and possibly to other TKIs, independently of the KIT receptor. ERK1/2 could thus be a new interesting therapeutic target in the treatment of advanced SM resistant to TKIs