A tuberculose (TB) é uma doença infecciosa bacteriana causada por Mycobacterium tuberculosis (Mtb), acomete seres humanos em todo mundo, e é considerada uma das principais causas de morbidade e mortalidade dentre as doenças infecciosas. Nesse cenário, a TB ainda é vista como um grande desafio para a ciência e para medicina. Com o estudo de novos alvos moleculares para terapia e diagnóstico, por exemplo, a sinalização Notch tem sido descrita como uma via importante para a manutenção da resposta imunológica durante a infecção por Mtb. Assim, o objetivo deste trabalho foi avaliar a participação da via Notch na modulação da resposta imune durante a infecção por Mtb em humanos. Neste trabalho foram incluidos 13 pacientes com tuberculose ativa e 13 indivíduos saudáveis. Células mononucleares de sangue periférico (PBMCs) foram isoladas e avaliadas quanto à expressão de receptores Notch 1 - 2 em células T e ligantes Notch, DLL1 - 4 em subpopulações de monócitos por citometria de fluxo e expressão de genes relacionados à via Notch e resposta imune. Além disso, o plasma foi utilizado para avaliar níveis de citocinas, CD14s e CD163s. Ainda, avaliamos a função celular dependente da ativação da via Notch durante a infecção de células mononucleares de sangue periférico de indivíduos saudáveis com Mtb H37Rv in vitro. Observamos que pacientes com TB ativa apresentaram aumento na expressão de DLL4 em monócitos intermediários e não-clássicos e diminuição na expressão de Notch 2 em células T CD8+. In vitro, observamos nas culturas de PBMCs, aumento da expressão de DLL4 em monócitos intermediários e Notch 2 em células T CD4+. A expressão do ligante DLL4 em monócitos intermediários e a expressão do Notch 1 em células T CD4+ em pacientes com TB ativa se correlacionaram positivamente com o grau de lesão pulmonar. Além disso, pacientes com quadros de lesão pulmonar moderado e avançado têm maior expressão de Notch 1 em células T CD4+ quando comparados a pacientes com grau mínimo de lesão pulmonar. Pacientes com TB ativa apresentam aumento significativo nos níveis plasmáticos de IL-6, IP- 10, CD14s e CD163s e diminuição nos níveis de IFN-?, IL-17A, IL-4, IL-2, IL-1? e RANTES. Os níveis plasmáticos de IFN-?, TNF-?, IL-17A, IL-4, IL-2, IL-12, IL-1?, RANTES e IL-10 se correlacionaram positivamente com o maior grau de lesão pulmonar.Além disso, demonstramos que os níveis de IFN-?, IL-17A, IL-1?, IL-4 e IL-2 se relacionam com maior expressão do receptor Notch 1 em células T CD4+ , enquanto níveis plasmáticos de IP-10, CD163s e de procalcitonina se correlacionam com maior expressão de Notch 1 em células T CD8+. Evidenciamos maior tendência na expressão de HES1 em pacientes com TB ativa e células infectadas in vitro, indicando que a via Notch está sendo ativada durante a infecção por Mtb. De forma interessante, in vitro as culturas de PBMCs tratadas com inibidor farmacológico da via Notch (GSI), reduziram a produção de IL-17A, IL-2 e IL-10 e aumentaram IL-8, enquanto o tratamento com anti-hDLL4 promoveu o aumento significativo nos níveis de TNF-? e atividade fagocítica de monócitos. Em conclusão, a expressão de receptores e ligantes Notch é alterada durante a TB, em especial o ligante DLL4 em subpopulações de monócitos. Além disso, a via Notch parece ser importante na modulação de respostas pró e anti-inflamatórias. Nesse trabalho, sugerimos que a expressão de Notch 1 em células T e DLL4 em subpopulações de monócitos estão associados com a gravidade da TB. Assim, a detecção desses constituintes da via Notch em PBMCs de pacientes com TB ativa são potenciais biomarcadores para avaliar a progressão da doença. / Tuberculosis (TB) is a bacterial infectious disease caused by Mycobacterium tuberculosis (Mtb), affects humans worldwide, and is considered a major cause of morbidity and mortality among infectious diseases. In this scenario, TB is still seen as a major challenge for science and medicine. With the study of new molecular targets for therapy and diagnosis, for example, Notch signaling has been described as an important pathway for the maintenance of immune response during Mtb infection. Thus, the objective of this work was to evaluate the participation of the Notch pathway in the modulation of the immune response during Mtb infection in humans. In this study, 13 patients with active tuberculosis and 13 healthy individuals were included. Peripheral blood mononuclear cells (PBMCs) were isolated and evaluated for Notch 1 - 2 receptor expression in T cells and Notch, DLL1 - 4 ligands in monocyte subpopulations by flow cytometry and expression of Notch - pathway related genes and immune response. In addition, plasma was used to assess levels of cytokines, CD14s and CD163s. Furthermore, we evaluated the cellular function dependent on the activation of the Notch pathway during the infection of peripheral blood mononuclear cells of healthy subjects with Mtb H37Rv in vitro. We observed that patients with active TB had increased DLL4 expression in intermediate and nonclassic monocytes and decreased expression of Notch 2 in CD8+ T cells. In vitro, we observed in the cultures of PBMCs, increased expression of DLL4 in intermediate monocytes and Notch 2 in CD4+ T cells. The expression of the DLL4 linker in intermediate monocytes and the expression of Notch 1 in CD4+ T cells in patients with active TB correlated positively with the degree of lung injury. In addition, patients with moderate and advanced lung injury have higher Notch 1 expression in CD4+ T cells when compared to patients with a minimal degree of lung injury. Patients with active TB showed a significant increase in plasma levels of IL-6, IP-10, CD14s and CD163s, and decreased levels of IFN-?, IL-17A, IL-4, IL-2, IL-1? and RANTES. The plasma levels of IFN-?, TNF-?, IL-17A, IL-4, IL-2, IL-12, IL-1?, RANTES and IL-10 correlated positively with the highest degree of lung injury. In addition, we demonstrated that levels of IFN-?, IL-17A, IL-1?, IL-4 and IL-2 are related to higher Notch 1 receptor expression in CD4+ T cells, whereas plasma levels of IP-10,CD163s and procalcitonin correlate with increased Notch 1 expression in CD8+ T cells. We have shown a greater trend in HES1 expression in patients with active TB and in vitro infected cells, indicating that the Notch pathway is being activated during Mtb infection. Interestingly, in vitro cultures of PBMCs treated with the Notch pharmacological inhibitor (GSI), reduced IL-17A, IL-2 and IL-10 production and increased IL-8, while anti-hDLL4 treatment promoted the significant increase in TNF-? levels and monocyte phagocytic activity. In conclusion, the expression of Notch receptors and ligands is altered during TB, especially the DLL4 linker in monocyte subpopulations. In addition, the Notch pathway appears to be important in modulating pro and anti-inflammatory responses. In this work, we suggest that Notch 1 expression in T cells and DLL4 in monocyte subpopulations is associated with TB severity. Thus, detection of these constituents of the Notch pathway in PBMCs of patients with active TB are potential biomarkers to assess disease progression.

La conversion de la protéine prion cellulaire PrPC en une isoforme pathologique, la protéine prion scrapie PrPSc, est à l'origine d'un groupe de maladies neurodégénératives, les Encéphalopathies Spongiformes Transmissibles (EST). De nombreux travaux indiquent que la toxicité de la PrPSc implique une déviation de la fonction normale de la PrPC, cependant le rôle physiologique de la protéine prion n’est que partiellement compris. Dans ce travail, nous nous sommes attachés à identifier des voies de signalisation mobilisées par la PrPC qui pourraient à la fois rendre compte du rôle de cette protéine dans le développement du système nerveux et être impliquées dans la pathogénèse des EST. Nous montrons que la protéine prion contrôle l’activité de la voie Notch, une voie de signalisation qui joue un rôle majeur dans le développement mais également dans l’homéostasie du système nerveux central et la plasticité synaptique. Dans des modèles ex vivo et in vivo d’EST, nous mettons en évidence une diminution de l’activité de la voie Notch, ainsi que de l’expression des récepteurs de la famille Eph - connus pour leur implication dans l’activité synaptique. Cette diminution des Eph est retrouvée dans des cellules dépourvues de PrPC. Ainsi, l’observation d’un profil similaire entre la perte d’expression de la PrPC et l’infection par les prions renforce l’idée d’une déviation de la fonction normale de la PrPC par la PrPSc. Des inhibiteurs de l’activité histone désacétylase (HDAC) permettent de rétablir l’expression des acteurs de la voie Notch et des récepteurs Eph aussi bien dans les cellules déplétées en PrPC que dans celles infectées par les prions, suggérant que des mécanismes épigénétiques sont impliqués dans le contrôle transcriptionnel de ces gènes par la protéine prion. Ce travail fournit les bases pour évaluer un effet bénéfique des inhibiteurs de HDAC dans un modèle de souris infectées par les prions et ainsi déterminer si les HDAC pourraient constituer de nouvelles cibles thérapeutiques pour combattre les EST. / The conversion of the cellular prion protein PrPC into a pathogenic isoform, the scrapie prion protein PrPSc, lies at the root of a group of neurodegenerative disorders known as Transmissible Spongiform Encephalopathies (TSEs). Several lines of evidence indicate that PrPSc-mediated toxicity involves a subversion of PrPC normal function, however, our knowledge of PrPC physiological role is still far from complete. In this work, we sought to identify signalling pathways mobilized by PrPC that could accommodate both its role in central nervous system development and its implication in TSE pathogenesis. We show that the prion protein controls the activity of the Notch pathway, which plays an overriding role during embryonic development as well as central nervous system homeostasis and synaptic plasticity. In both ex vivo and in vivo models of TSE, we monitored a decrease in Notch activity, together with reduced expression of Eph receptors, which are key players in synaptic activity. The reduction in Eph is also found in PrPC-depleted cells. Hence, our observation of a similar signature of PrPC depletion and prion infection strengthens the view that PrPSc diverts PrPC function. We found a restoration of Notch and Eph effectors expression in response to histone deacetylase (HDAC) inhibitors, both in PrPC-depleted and prion-infected cells, suggesting that epigenetic mechanisms are involved in the PrP-dependent transcriptional control of these genes. This work provides a foundation for assessing a beneficial effect of HDAC inhibition in prion-infected mice and thereby defining whether HDAC could represent novel therapeutic targets to combat TSEs.

A paracoccidioidomicose é uma micose sistêmica de natureza profunda que afeta preferencialmente o tecido pulmonar podendo disseminar via linfo-hematogênica para outros órgãos e tecidos, sendo causada principalmente pelo Paracoccidioides brasiliensis, fungo que apresenta dimorfismo térmico. O sistema imune inato mediado por macrófagos é extremamente importante para o controle de infecções e está envolvido na indução e regulação da resposta imune/inflamatória. Estas células são capazes de reconhecer patógenos por meio de receptores de reconhecimento de padrões (PRRs), tais como receptores Toll-like (TLR). Além desses PRRs, recentemente, demonstrou-se a importância da via de sinalização Notch no sistema imune inato e na regulação da atividade dos macrófagos. Nossos dados demonstram que a cepa Pb18 do P. brasiliensis é capaz de ativar o receptor Notch1 em macrófagos J774. A ativação desse receptor concomitante com a ativação de TLR 4 (via LPS) induz a produção de IL-6, e apresenta elevada carga fúngica e menor fagocitose, o que favorece a patogenia. Ao utilizarmos um inibidor farmacológico da γ-secretase (DAPT) para inibir a ativação do receptor Notch1 em macrófagos, é possível observar diminuição da carga fúngica, diminuição de IL-6, aumento de TNF-α e aumento da fagocitose. Entretanto, a ausência do receptor TLR 4 em macrófagos derivados de medula óssea de camundongos TLR 4-/-, na presença de DAPT, percebe-se diminuição da capacidade fagocítica desses macrófagos e também diminuição da carga fúngica, evidenciando a relação entre TLR 4 e Notch1. Em adição, realizamos um tratamento em camundongos BALB/c com DAPT previamente à infecção com Pb18. Nossos resultados evidenciaram que animais com este tratamento apresentaram diminuição da carga fúngica dos pulmões, diminuição de IL-6, ativação de macrófagos e aumento de IgG, após 45 dias de infecção, indicando um perfil de cura desses animais. O mesmo tratamento foi realizado em camundongos BALB/c NUDE, seguido da infecção com Pb18. Nestes animais, verificamos que há maior produção de citocinas pró-inflamatórias no pulmão, aumento de células CD19+ e a carga fúngica dos animais tratados manteve-se similar ao dos animais não tratados, indicando que o perfil protetor observado em animais com DAPT é dependente da resposta das células T. Juntos, esses resultados evidenciam que o Pb18 é capaz de ativar o receptor Notch1 em macrófagos e utiliza a via de sinalização Notch-TLR 4 como um possível mecanismo de escape, podendo fornecer uma nova abordagem de estudo da imunidade envolvida na paracoccidioidomicose experimental. / Paracoccidioidomycosis is a systemic mycosis of deep nature that primarily affects the lung and can spread via lymphatic and hematogenous to other organs and tissues. It is mainly caused by Paracoccidioides brasiliensis fungus which exhibit thermal dimorphism. The innate immune system mediated by macrophages is extremely important for the control of infection and is involved in the induction and regulation of immune/inflammatory response. These cells are able to recognize pathogens through pattern recognition receptors (PRRs) such as Toll-like receptors (TLR). Beyond these PRRs, the importance of Notch signaling has recently been demonstrated in the innate immune system and the regulation of macrophage activity. Our data demonstrate that the Pb18 strain of P. brasiliensis is able to activate the Notch1 receptor in J774 macrophages. Activation of this receptor with also activation of TLR 4 (via LPS) induces IL-6 production, induces phagocytosis and decreases fungal burden, which favors the pathogenesis. By using a γ-secretase pharmacological inhibitor (DAPT) for inhibiting the activation of Notch1 receptor on macrophages, it is possible to observe decreased fungal burden, less production of IL-6, and increased TNF-α and phagocytosis. However, due to the absence of TLR 4 receptor in bone marrow derived macrophages from TLR 4-/- mice, these macrophages showed decreased phagocytic ability and also reduced fungal burden in the presence of DAPT, showing a relationship between TLR 4 and Notch1. In addition, we made a treatment with DAPT in BALB/c mice prior to infection with Pb18. And our results showed that DAPT-treated animals exhibited a decrease of fungal burden in the lungs, and a decrease of IL-6. Furthermore, we observed an increase of IgG after 45 days of infection, indicating probably a healing of these animals. Same treatment was made in BALB/c NUDE mice, followed by infection with Pb18. In these animals, we observed an increased production of proinflammatory cytokines in the lung and increased CD19+ cells, but fungal burden was similar in both group (treated and untreated), which indicates that treatment with DAPT is dependent on T cell response. Taken together, these results showed that Pb18 is able to activate the Notch 1 receptor on macrophages and uses the Notch-TLR 4 signaling pathway as a possible escape mechanism, and may provide a new immunity study approach in experimental paracoccidioidomycosis.

Na primeira parte desta tese, estudamos os princípios operacionais das decisões celulares mediadas pelo sistema de sinalização Notch. Este sistema tem papel importante nas decisões celulares que ocorrem durante o desenvolvimento embrionário, cicatrização de feridas e na formação de tumores. O circuito de sinalização é ativado quando o receptor Notch de uma célula interage com um dos ligantes - Delta ou Jagged - de uma célula vizinha. O circuito Notch-Delta forma um comutador intercelular, e duas células vizinhas tendem a adotar estados diferentes - Emissor (muito ligante e pouco receptor) e Recebedor (pouco ligante e muito receptor). Neste manuscrito, apresentamos uma nova abordagem teórica que integra ambos Delta e Jagged no circuito Notch. Mostramos que o circuito Notch-Delta-Jagged permite um novo estado - um híbrido Emissor/Recebedor (E/R) com concentrações intermediárias de receptores e ligantes, e portanto o circuito é age como uma chave de três vias. Em seguida, observamos que a taxa de produção de ambos os ligantes, assim como a modulação assimétrica da afinidade de ligação do Notch com seus ligantes mediada pela glicosiltransferase Fringe, afeta severamente o intervalo de existência dos estados e sua relativa estabilidade - altos níveis de Jagged, mas não de Fringe ou Delta, promovem o estado híbrido E/R e o processo de indução lateral. Nós elucidamos o papel de Jagged na determinação dos estados celulares e discutimos sua possível implicação no entendimento da comunicação entre tumor e estroma, que frequentemente envolve comunicação via interações Notch-Jagged. Posteriormente, avaliamos a interação entre Notch, inflamação e a população de Células Cancerígenas Estaminais (CCE). Mostramos que inflamação pode expandir a população de CCE por meio do aumento dos níveis de produção de Jagged que posteriormente ativa o sistema de sinalização Notch em células vizinhas não-CCE. Nossos resultados sugerem que a inibição da produção de Jagged atenua o efeito da expansão de CCE devido a inflamação, indicando que inflamação cresce a população de CCE via interações Notch-Jagged. Nossos resultados são consistentes com observações em câncer de mama do subtipo basal (triplo negativo), onde a perda de Fringe e a ativação constitutiva do eixo NF-kB - Jag1 promove a expansão da população de CCE. Nossa abordagem computacional pode ser adaptada para incluir circuitos adicionais tais como p53 e hipóxia, que afetam a plasticidade celular, proporcionando assim uma plataforma útil para a projeção de novas terapias. Na segunda parte desta tese, introduzimos um novo método para seleção de características: Suvrel. Este é um método variacional, inspirado em aprendizado de relevância, para determinar tensores métricos para definição de distâncias baseadas em similaridades, para utilização em métodos de classificação. Nós introduzimos uma nova metodologia na qual o tensor métrico pode ser calculado analiticamente. O preprocessamento das características por uma transformação linear utilizando o tensor métrico calculado via Suvrel melhora a eficiência dos classificadores. Testamos nosso método para conjuntos de dados públicos, utilizando os classificadores mais comumente utilizados. Nós também aplicamos esta metodologia no estudo da relação entre parâmetros estruturais globais e o sistema de classificação de função enzimática. Por último, introduzimos uma nova metodologia para a identificação de genes diferencialmente expressos utilizando a tecnologia de microarranjos de DNA. Diferentemente das abordagens tradicionais, nossa metodologia evita passos intermediários de preprocessamento que são desnecessários e devido a isto não acumula erros destas análises, o que resulta em um método mais sensível e robusto. / In the first part of this thesis, we studied the operating principles of cell fate decisions mediated by Notch signaling pathway. This pathway have important role in cell fate determination during embryonic development, wound healing and tumorigenesis. Notch signaling is activated by binding of Notch receptor of one cell to either of its ligand- Delta or Jagged- of another cell. Notch-Delta circuit forms an intercellular toggle switch, and two neighboring cells tend to adopt different fates - Sender (high ligand, low receptor) and Receiver (low ligand, high receptor). Here, we present a new tractable theoretical framework that incorporates both Delta and Jagged in Notch signaling, and show that Notch-Delta-Jagged circuit enables an additional fate - hybrid Sender/Receiver (S/R) (medium ligand, medium receptor) and behaves as a three-way switch. Further, we found that production rates of both the ligands and the asymmetric modulation of binding affinity of Notch to its ligands by glycosyltransferase Fringe severely affects the parameter range of the existence of these states and their relative stability - high levels of Jagged, but not that of Fringe or Delta, promote hybrid S/R state and lateral induction. We elucidate the role of Jagged in cell fate determination and discuss its possible implications in understanding tumor-stroma crosstalk, which frequently entails Notch-Jagged communication. We further evaluate the interplay among Notch signaling, inflamation and Cancer Stem Cell population. We show that inflammation can expand the population of Cancer Stem Cells (CSCs) by increasing the levels of Jagged in cells that can further activate Notch signaling pathway in neighboring non-CSCs. Our results suggest that, inhibiting the production of Jagged dampens the effect of inflammation in expanding the CSC population, indicating that inflammatory signal function through Notch-Jagged signaling to increase CSCs. Our results are consistent with observations in basal-like breast cancer, where loss of Fringe and constitutive activation of NF-kB-Jag1 axis promotes CSC population. Our computational framework can be tailored to include additional signals such as p53 and hypoxia that affect this plasticity to gain stemness, thus providing a platform that can be useful in designing novel therapies. In the second part of the thesis, we introduce a new method for feature selection: Supervised Variational Relevance Learning (Suvrel). This is a variational method to determine metric tensors to define distance based similarity in pattern classification, inspired in relevance learning. We propose a new methodology where the metric tensor can be calculated analytically. Preprocessing the patterns by doing linear transformations using the metric tensor yields a dataset which can be more efficiently classified. We test our methods using publicly available datasets, for some standard classifiers. We also applied this methodology to study the relationship between global structural parameters and the Enzyme Commission hierarchy. Lastly, we propose a new methodology for identifying the differentially expressed genes using DNA microarray technology. Unlike traditional approaches, our methodology skips intermediate unnecessary preprocessing steps and therefore does not accumulate errors due to these analysis, resulting in a more sensitive and robust method.

Plus de 80% des cancers colorectaux sont initiés par la perte de fonction du gène Apc. Afin d’identifier de nouvelles cibles thérapeutiques, nous avons utilisé des modèles murins présentant des mutations du gène Apc et recherché par des analyses de puces à ADN de nouveaux événements moléculaires impliqués au cours de la carcinogenèse intestinale.Cette approche nous a permis d’identifier une activation de la signalisation Notch tout au long du processus tumoral. Toutefois, cette activation n’est pas un élément clé de la progression tumorale puisque son inhibition n’empêche pas le phénotype tumoral induit par la perte du gène Apc. En parallèle, nos travaux ont permis d’identifier une induction de l’autophagie tout au long de la carcinogenèse intestinale. L’activation de ce processus biologique ouvre, quant à lui, de nouvelles perspectives thérapeutiques dans le traitement du CCR. / Over 80% of colorectal cancers are linked to an Apc mutation. To identify new therapeutic targets, we used mouse models with Apc mutations and performed microarray experiments to identify key molecular events involved in intestinal carcinogenesis. This approach allowed usto identify an activation of the Notch signaling all along tumor progression. However, this induction is dispensable for tumor development since its inhibition did not prevent the Apc phenotype. In addition, we have identified an induction of autophagy throughout intestinal carcinogenesis which appears to be an attractive therapeutic target in the treatment of CRC patients.

L’hématopoïèse est le processus physiologique qui permet le développement de l’ensemble des cellules sanguines matures, leur renouvellement et leur homéostasie tout au long de la vie. L’hématopoïèse est généralement décrite de façon hiérarchique avec, au sommet, les cellules souches hématopoïétiques qui s’autorenouvellent et se différencient en progéniteurs puis en cellules matures. La voie de signalisation NOTCH canonique, contrôle l’activité du facteur de transcription RBPJ. Elle joue un rôle dans le développement des lymphocytes T et la spécification de la différenciation des cellules souches hématopoïétiques normales vers la lignée mégacaryocytaire. Les protéines de la famille OTT1 (OTT1, OTT3 et SHARP) s’expriment de façon ubiquitaire et sont impliquées dans le contrôle de l’activité de RBPJ. Les modalités de régulation de ces activités et l’intégration de signaux provenant d’autres voies de signalisation sont mal caractérisées. L’utilisation d’un modèle de différenciation in vitro de cellules souches hématopoïétiques sur des cellules stromales (OP9) exprimant le ligand NOTCH Delta-like 1 (DL1) ainsi que l’utilisation de modèles murins, nous a permis de montrer un lien entre la voie NOTCH et la voie PI3K/AKT dans le développement mégacaryocytaire. Nos résultats indiquent que la différenciation mégacaryocytaire peut être engagée à partir de progéniteurs myéloïdes engagés dépendant principalement de la voie PI3K/AKT, mais également directement à partir de cellules souches hématopoïétiques pour lesquelles une activation de la voie PI3K/AKT conduit à une synergie avec la voie NOTCH, mais n’est pas essentielle à la spécification mégacaryocytaire. D’autre part, pour comprendre le mécanisme de régulation de la protéine OTT1, j’ai recherché ses partenaires protéiques par crible double hybride chez la levure, et identifié des interactions avec, entre autres, des protéines à activité tyrosine kinase de la famille SRC (dont LYN) et SHARP. La spécificité d’interaction entre OTT1 et LYN a été validée dans un modèle de surexpression ainsi que dans une lignée modélisant la leucémie aigüe mégacaryocytaire. Dans nos modèles, l’interaction avec LYN conduit à la phosphorylation d’OTT1. Les analyses fonctionnelles préliminaires n’ont pas permis à ce jour de mettre en évidence un rôle essentiel de cette interaction dans le développement mégacaryocytaire. / Hematopoiesis is generally described as a hierarchical system, with at the top hematopoietic stem cells which self-renew and differentiate in progenitors, then in mature cells. Canonical Notch signaling controls RBPJ transcriptional activity. It plays a role in T lymphocyte development and stem cell fate. OTT1 family proteins (OTT1, OTT3 and SHARP) are expressed ubiquitously and are implied in control of RBPJ activity. The regulation of these activities and signal integration are all not well characterised. The use of an in vitro model of differentiation for hematopoietic stem cells on OP9 stroma cells expressing the NOTCH Delta-like-1 (DL1) ligand and the use of murine models, allowed us to show a link between NOTCH and PI3K/AKT in megakaryocytic development. Our results indicate that megakaryocytic differentiation can be engaged from myeloid progenitors depending mostly on the PI3K pathway but also from hematopoietic stem cells for which, an activation of PI3K/AKT lead to a synergy with NOTCH, but is not essential for megakaryocytic specification. On the other hand, to understand OTT1’s mechanisms of regulation, I looked for proteic binding partners by the double hybrid screen technique. Among the candidates I identified SHARP and SRC family kinases as LYN. The specific interaction between OTT1 and LYN was validated in a overexpression model and in a cell line modeling acute megakaryoblastic leukemia. In our models, the interaction with LYN lead to the phosphorylation of OTT1. However, the first analysis did not point out an essential role of this interaction in megakaryocytic development.

A tuberculose (TB) é uma doença infecciosa bacteriana causada por Mycobacterium tuberculosis (Mtb), acomete seres humanos em todo mundo, e é considerada uma das principais causas de morbidade e mortalidade dentre as doenças infecciosas. Nesse cenário, a TB ainda é vista como um grande desafio para a ciência e para medicina. Com o estudo de novos alvos moleculares para terapia e diagnóstico, por exemplo, a sinalização Notch tem sido descrita como uma via importante para a manutenção da resposta imunológica durante a infecção por Mtb. Assim, o objetivo deste trabalho foi avaliar a participação da via Notch na modulação da resposta imune durante a infecção por Mtb em humanos. Neste trabalho foram incluidos 13 pacientes com tuberculose ativa e 13 indivíduos saudáveis. Células mononucleares de sangue periférico (PBMCs) foram isoladas e avaliadas quanto à expressão de receptores Notch 1 - 2 em células T e ligantes Notch, DLL1 - 4 em subpopulações de monócitos por citometria de fluxo e expressão de genes relacionados à via Notch e resposta imune. Além disso, o plasma foi utilizado para avaliar níveis de citocinas, CD14s e CD163s. Ainda, avaliamos a função celular dependente da ativação da via Notch durante a infecção de células mononucleares de sangue periférico de indivíduos saudáveis com Mtb H37Rv in vitro. Observamos que pacientes com TB ativa apresentaram aumento na expressão de DLL4 em monócitos intermediários e não-clássicos e diminuição na expressão de Notch 2 em células T CD8+. In vitro, observamos nas culturas de PBMCs, aumento da expressão de DLL4 em monócitos intermediários e Notch 2 em células T CD4+. A expressão do ligante DLL4 em monócitos intermediários e a expressão do Notch 1 em células T CD4+ em pacientes com TB ativa se correlacionaram positivamente com o grau de lesão pulmonar. Além disso, pacientes com quadros de lesão pulmonar moderado e avançado têm maior expressão de Notch 1 em células T CD4+ quando comparados a pacientes com grau mínimo de lesão pulmonar. Pacientes com TB ativa apresentam aumento significativo nos níveis plasmáticos de IL-6, IP- 10, CD14s e CD163s e diminuição nos níveis de IFN-?, IL-17A, IL-4, IL-2, IL-1? e RANTES. Os níveis plasmáticos de IFN-?, TNF-?, IL-17A, IL-4, IL-2, IL-12, IL-1?, RANTES e IL-10 se correlacionaram positivamente com o maior grau de lesão pulmonar.Além disso, demonstramos que os níveis de IFN-?, IL-17A, IL-1?, IL-4 e IL-2 se relacionam com maior expressão do receptor Notch 1 em células T CD4+ , enquanto níveis plasmáticos de IP-10, CD163s e de procalcitonina se correlacionam com maior expressão de Notch 1 em células T CD8+. Evidenciamos maior tendência na expressão de HES1 em pacientes com TB ativa e células infectadas in vitro, indicando que a via Notch está sendo ativada durante a infecção por Mtb. De forma interessante, in vitro as culturas de PBMCs tratadas com inibidor farmacológico da via Notch (GSI), reduziram a produção de IL-17A, IL-2 e IL-10 e aumentaram IL-8, enquanto o tratamento com anti-hDLL4 promoveu o aumento significativo nos níveis de TNF-? e atividade fagocítica de monócitos. Em conclusão, a expressão de receptores e ligantes Notch é alterada durante a TB, em especial o ligante DLL4 em subpopulações de monócitos. Além disso, a via Notch parece ser importante na modulação de respostas pró e anti-inflamatórias. Nesse trabalho, sugerimos que a expressão de Notch 1 em células T e DLL4 em subpopulações de monócitos estão associados com a gravidade da TB. Assim, a detecção desses constituintes da via Notch em PBMCs de pacientes com TB ativa são potenciais biomarcadores para avaliar a progressão da doença. / Tuberculosis (TB) is a bacterial infectious disease caused by Mycobacterium tuberculosis (Mtb), affects humans worldwide, and is considered a major cause of morbidity and mortality among infectious diseases. In this scenario, TB is still seen as a major challenge for science and medicine. With the study of new molecular targets for therapy and diagnosis, for example, Notch signaling has been described as an important pathway for the maintenance of immune response during Mtb infection. Thus, the objective of this work was to evaluate the participation of the Notch pathway in the modulation of the immune response during Mtb infection in humans. In this study, 13 patients with active tuberculosis and 13 healthy individuals were included. Peripheral blood mononuclear cells (PBMCs) were isolated and evaluated for Notch 1 - 2 receptor expression in T cells and Notch, DLL1 - 4 ligands in monocyte subpopulations by flow cytometry and expression of Notch - pathway related genes and immune response. In addition, plasma was used to assess levels of cytokines, CD14s and CD163s. Furthermore, we evaluated the cellular function dependent on the activation of the Notch pathway during the infection of peripheral blood mononuclear cells of healthy subjects with Mtb H37Rv in vitro. We observed that patients with active TB had increased DLL4 expression in intermediate and nonclassic monocytes and decreased expression of Notch 2 in CD8+ T cells. In vitro, we observed in the cultures of PBMCs, increased expression of DLL4 in intermediate monocytes and Notch 2 in CD4+ T cells. The expression of the DLL4 linker in intermediate monocytes and the expression of Notch 1 in CD4+ T cells in patients with active TB correlated positively with the degree of lung injury. In addition, patients with moderate and advanced lung injury have higher Notch 1 expression in CD4+ T cells when compared to patients with a minimal degree of lung injury. Patients with active TB showed a significant increase in plasma levels of IL-6, IP-10, CD14s and CD163s, and decreased levels of IFN-?, IL-17A, IL-4, IL-2, IL-1? and RANTES. The plasma levels of IFN-?, TNF-?, IL-17A, IL-4, IL-2, IL-12, IL-1?, RANTES and IL-10 correlated positively with the highest degree of lung injury. In addition, we demonstrated that levels of IFN-?, IL-17A, IL-1?, IL-4 and IL-2 are related to higher Notch 1 receptor expression in CD4+ T cells, whereas plasma levels of IP-10,CD163s and procalcitonin correlate with increased Notch 1 expression in CD8+ T cells. We have shown a greater trend in HES1 expression in patients with active TB and in vitro infected cells, indicating that the Notch pathway is being activated during Mtb infection. Interestingly, in vitro cultures of PBMCs treated with the Notch pharmacological inhibitor (GSI), reduced IL-17A, IL-2 and IL-10 production and increased IL-8, while anti-hDLL4 treatment promoted the significant increase in TNF-? levels and monocyte phagocytic activity. In conclusion, the expression of Notch receptors and ligands is altered during TB, especially the DLL4 linker in monocyte subpopulations. In addition, the Notch pathway appears to be important in modulating pro and anti-inflammatory responses. In this work, we suggest that Notch 1 expression in T cells and DLL4 in monocyte subpopulations is associated with TB severity. Thus, detection of these constituents of the Notch pathway in PBMCs of patients with active TB are potential biomarkers to assess disease progression.

La voie de signalisation Notch joue un rôle essentiel dans le développement et l'homéostasie de l’épithélium intestinal et présente un potentiel oncogénique dans le cancer du côlon (CRC). L'un de ses gènes cibles, HES1, est un répresseur transcriptionnel de divers gènes, dont KLF4, facteur impliqué dans l'homéostasie intestinale et qui favorise la différenciation des cellules à mucus. De plus, afin d’éviter une activité aberrante de la voie Notch, HES1 exerce une boucle de rétrocontrôle négative sur son propre promoteur. Notre laboratoire s’intéresse à RIP140, un corégulateur transcriptionnel qui réprime l'activité de nombreux facteurs de transcription impliqués dans divers processus physiopathologiques. Dans l'épithélium intestinal, RIP140 inhibe la prolifération cellulaire et régule la différenciation en cellules de Paneth. L'objectif de ce travail a été d'étudier le dialogue entre RIP140 et la voie de signalisation Notch/HES1 ainsi que son impact sur différents paramètres cellulaires en utilisant différentes lignées cellulaires cancéreuses colorectales humaines ainsi que des modèles murins présentant une invalidation du gène Rip140. Pour cela, diverses expériences ont été mises en place en utilisant le gène HES1 comme principal marqueur d’activation de la voie Notch dans les lignées cellulaires de CRC SW620 et HT29. L’expression du gène HES1 a été analysée au niveau protéique, ARNm et transcriptionel en utilisant respectivement les techniques de Western-blot et d’immunofluorescence, de RT-QPCR et de gène rapporteur luciférase. L'activité de la voie Notch a été modulée par l'expression ectopique du domaine intracellulaire de récepteur Notch (NICD) ou en utilisant un inhibiteur de la γ-sécrétase. Le dialogue entre RIP140 et la voie de signalisation Notch est étroitement lié au niveau d'activation de la voie Notch et au niveau d’expression du facteur de transcription HES1. A de faibles niveaux d’activité de la voie Notch, RIP140 est une cible négative de NICD et exerce un effet positif sur la transcription du gène HES1 qui implique, au moins en partie, le complexe RBPJ/NICD. A de niveaux élevés d’activé de la voie Notch, RIP140 devient une cible positive de HES1 et exerce un effet négatif sur la transcription de ce gène en contribuant à la boucle de rétrocontrôle négative de HES1. De manière intéressante, comme le gène HES1, RIP140 a un impact important sur différents paramètres cellulaires. En effet, RIP140, est non seulement capable d’inhiber la prolifération des cellules intestinales, mais est également capable d’augmenter l'expression du gène KLF4 et de favoriser la différenciation en cellules à mucus. Conformément à ce dialogue entre RIP140 et la voie Notch/HES1, nous avons ensuite montré que HES1 et RIP140 inhibent mutuellement leurs effets sur la différenciation et la prolifération cellulaires. Nos données démontrent ainsi l'existence d'une boucle de rétrocontrôle impliquant RIP140 et la voie Notch/HES1 dans les lignées cellulaires de CRC. En effet, le gène RIP140 est à la fois une cible et un régulateur de la voie de signalisation Notch/HES1. Une activité aberrante de la voie Notch bascule la régulation de l'expression du gène HES1 par RIP140 d'un effet positif à un effet négatif via la boucle de rétrocontrôle négative de HES1. De plus, ce lien puissant entre RIP140 et HES1 a un impact sur la différenciation et la prolifération cellulaires. Il sera nécessaire d’analyser le recrutement de RIP140 et HES1 sur différents promoteurs cibles et de valider l'impact de ce dialogue in vivo, en utilisant un modèle de souris que j’ai développé au sein du laboratoire et qui présentent une invalidation conditionnelle du gène Rip140 dans l'épithélium intestinal. / The Notch signaling pathway plays an essential role in intestinal development and homeostasis and has an oncogenic potential in colon cancer (CRC). One of its target genes HES1 is a transcription repressor of a number of genes, including KLF4, which is implicated in intestinal homeostasis and promotes Goblet cell differentiation. In addition, to avoid aberrant activity of the Notch pathway, HES1 exerts a negative feedback loop on its own promoter. Our laboratory is studying RIP140, a transcriptional coregulator which represses the activity of many transcription factors involved in various pathophysiological processes. In the intestinal epithelium, RIP140 inhibits cell proliferation and regulates differentiation towards the Paneth cell lineage. The goal of this work was to investigate the crosstalk between RIP140 and the Notch/HES1 pathway and to study its cellular impacts in human CRC cells. Various experiments have been set up using the HES1 gene as the main output of the Notch pathway in two CRC cell lines (SW620 and HT29). HES1 gene expression has been assessed at the protein, mRNA and transcriptional levels using western-blot/immunofluorescence, RT-QPCR and luciferase reporter assays, respectively. The activity of the Notch pathway has been modulated through ectopic expression of the Notch intracellular domain (NICD) or using a γ-secretase inhibitor. RIP140 crosstalk with the Notch signaling pathway is tightly related to the level of activation of the Notch pathway and to the level of HES1 expression. At low Notch activity, RIP140 is a negative target of NICD and exerts a positive effect on HES1 gene transcription which involves, at least partly, the RBPJ/NICD complex. When the Notch pathway is fully activated, RIP140 becomes a positive target of HES1 and exerts a negative effect on HES1 gene transcription by contributing to the HES1 negative feedback loop. Interestingly, as it is the case for HES1, RIP140 has a strong impact on different cellular parameters. Indeed, we found that RIP140, not only decreases intestinal cell proliferation, but also increases KLF4 gene expression and Goblet cell differentiation. In line with the strong crosstalk between RIP140 and the Notch/HES1 pathway, we then showed that HES1 and RIP140 mutually inhibit their effects on cell differentiation and proliferation. Altogether, our data demonstrated the existence of a feed-back loop involving RIP140 and the Notch/HES1 pathway in CRC cells. Indeed, the RIP140 gene is both a target and a regulator of the Notch/HES1 signaling pathway. A high level of Notch/HES1 activity switches the regulation of HES1 gene expression by RIP140 from a positive to a negative effect through the HES1 negative feedback loop. Moreover, this strong link between RIP140 and HES1 has an impact on cell differentiation and proliferation. It would be however interesting to demonstrate the recruitment of each factor on target promoters and to validate the impact of this strong crosstalk, in vivo, using the newly mouse model that I developed with a conditional knock-out of the Rip140 gene in the intestinal epithelium.

This thesis investigates the connection between the Sirtuin1 protein (Sirt1) and the Notch signaling pathway in Drosophila melanogaster. We used RNAi to downregulate the Sirt1 gene expression in a tissue specific manner in the muscles, brain, fat body, and wing disc and tested the sensitivity of Notch pathway to dietary amino acids. We identified tissue specific requirements for Sirt1 protein to mediate the sensitivity of Notch pathway to amino acids.

Le facteur de transcription Ikaros est un régulateur essentiel de la lymphopoïèse. Ikaros est nécessaire à la différenciation des lymphocytes B et joue aussi un rôle important dans la suppression des LAL-T. Contrairement aux souris mutantes nulles pour Ikaros, les souris mutantes hypomorphes IkL/L développent des lymphocytes B matures après la naissance. Avec l’âge, toutes les souris IkL/L développent des leucémies T Notch dépendantes avec des mutations similaires à celles trouvées chez les patients atteints de LAL-T. La souris IkL/L est donc un excellent modèle pour étudier l’activation des cellules B matures et la pathogenèsedes LAL-T. Nous avons montré que la délétion spécifique du promoteur et de l’exon 1 de Notch1 dans les cellules T conduit à l’activation de promoteurs cryptiques dans la région 3’ du gène, qui génèrent des transcrits codant pour des protéines Notch1 constitutivement actives qui accélèrent la leucémogenèse dans la souris IkL/L. De plus, nous mettrons en évidence l’existence de cellules initiatrices de leucémie dans les tumeurs IkL/L puisque nous avons trouvé que des cellules ayant la capacité de s’auto-renouveler représentent 1 sur 500. Enfin, nous avons montré que les cellules B IkL/L ont une activation excessive d’ERK et dep38 après la stimulation du BCR, ce qui résulte en une hyper-prolifération et une production d’autoanticorps liés au lupus systémique érythémateux. Nos résultats suggèrent qu’Ikaros est un régulateur négatif de l’activation des lymphocytes B. / The Ikaros transcription factor is a critical regulator of lymphopoeisis. Ikaros is needed for the differentiation of B cell and plays an important role in tumor suppression in T cells. Contrary to the other Ikaros targeted mutant mice, the IkL/L mouse develops mature B cells after birth. With age, it develops Notch dependent tumors with similar mutations as thosefound in human T cell acute lymphoblastic leukemia (T-ALL). Thus, the IkL/L mouse is an excellent model to study mature B cell activation and T-ALL tumor development. Here, we have shown that the deletion of the promoter and exon1 of the Notch1 gene in IkL/L T cells activates a cryptic promoter in the 3’ region which leads to the production of truncated, constitutively activated Notch1 proteins that accelerate tumorigenesis in the IkL/L mice. Moreover, we give evidence for the existence of LICs in the IkL/L tumors as we have found a specific subpopulation of cells with a frequency of 1 in 500 which show self-renewal capacity. In addition, we demonstrated that some tumor cells have the ability to efflux the Hoechst dye and that this side population is enriched in quiescent cells. Finally, we elucidate that IkL/L B cells display an enhanced activation of ERK and p38 after BCR stimulation that results in hyper-proliferation and the production of autoantibodies relatedto systemic lupus erythematosus (SLE). Our results suggest that Ikaros is a negative regulator of B cell activation.