Dissertations / Theses on the topic 'Notch1 Receptor'

Gain of function NOTCH1 mutations are common in both patients with T-ALL and in mouse models of the disease. Inhibiting the Notch pathway in T-ALL cell lines results in growth arrest and/or apoptosis in vitro, suggesting a requirement for Notch signaling in T-ALL. Therefore, we sought to examine the role of Notch1 signaling in both premalignancy and in the maintenance of leukemic growth. Using a murine model of T-ALL, in which expression of the Tal1 and Lmo2 oncogenes arrests thymocyte development, our preleukemic studies reveal that Notch1 mutations are early events that contribute to the clonal expansion of DN3 and DN4 progenitors. We also demonstrate that progenitors are maintained within the tumor and are enriched in leukemia-initiating cell (L-IC) activity, suggesting Notch1 may contribute to L-IC self-renewal. By studying the effects of Notch signaling in murine T-ALL cell lines, we also demonstrate that Notch1 promotes the proliferation and survival of leukemic blasts through regulation of Lef1 and the Akt/mTOR pathways. Given that T-ALL cell lines are dependent on Notch signaling in vitro, we investigated the effects of Notch inhibition in vivo. We provide evidence that Notch1 can be successfully targeted in vivo and that Notch inhibition, with γ-secretase inhibitors (GSIs), significantly extends the survival of leukemic mice. We also demonstrate that administration of GSIs in combination with rapamycin inhibits human T-ALL growth and extends survival in a mouse xenograft model. Given that NOTCH1 may be required to maintain both L-IC and bulk leukemic growth, targeting NOTCH1 may prove to be an efficacious targeted therapy for T-ALL patients with aberrant NOTCH1 activation.

The cardiac conduction system is responsible for maintaining and orchestrating the rhythmic contractions of the heart. Results from lineage tracing studies indicate that precursor cells in the ventricles give rise to both cardiac muscle and conduction cells. Using chick embryonic hearts, we have found that Notch signaling plays an important role in the differentiation of cardiac muscle and conduction cell lineages in the ventricles. Notch1 expression coincides with a conduction marker at early stages of conduction system development. Mis-expression of constitutively active Notch1 (NIC) in early heart tubes exhibited multiple effects on cardiac cell differentiation. Cells expressing NIC had a significant decrease in the expression of cardiac muscle markers, but an increase in the expression of conduction cell markers. Loss-of-function studies further support that Notch1 signaling is important for the differentiation of these cardiac cell types. Functional electrophysiology studies show that the expression of constitutively active Notch1 resulted in abnormalities in ventricular conduction pathway patterns. During cardiogenesis, groups of myocardial cells become separated from each other, and migrate to form the trabeculae. These finger-like projections found within the ventricular chamber coalesce to generate the muscular portions of the interventricular septum, the thickened myocardium, and future sites of the conduction system. We have found that Notch signaling regulates the migration of cardiac cells during cardiogenesis. Over-expression of constitutively active Notch causes cells to localize more centrally within the heart, while loss-of-Notch function results in cells distributed within the periphery of the heart. Staining of heart sections shows that Notch signaling regulates the expression of N-cadherin, the predominant adhesion molecule in cardiomyocytes. We find that the effects of Notch on cell migration are two-fold: delamination and cell motility. Time-lapse studies demonstrate that Notch signaling increases cell motility, but does not affect speed or directionality of migration. Furthermore, we find that the effects of Notch on cell migration is independent of its effects on differentiation.

Although cure rates have significantly improved for children with T-cell acute lymphoblastic leukemia (T-ALL), 20-30% undergo induction failure or relapse with most succumbing to disease. Leukemia-initiating cells (L-ICs) are hypothesized to be resistant to conventional chemotherapy and radiation and are thereby responsible for disease recurrence. Using an in vivo limiting dilution assay, we previously showed that the murine T-ALL L-IC is quite rare, with only 0.003-0.05% of cells capable of initiating disease, and demonstrated that the L-IC is a subset of the leukemic DN3 thymic progenitor population. Work described in this thesis validates the L-IC assay using two transplantation methods to rule out effects of homing and/or microenvironment on T-ALL L-IC survival and maintenance. Using this assay, we demonstrate that sustained Notch1 signaling is required for T-ALL initiation in vivo and show that treatment with a Notch1 inhibitor reduces or in some cases eliminates the L-IC population. We further analyze the effects of inhibiting c-Myc, a Notch1-regulated gene, on L-IC frequency and uncover an essential role for c-Myc in L-IC survival and expansion. Suppressing c-Myc by using specific shRNAs or a c-Myc inhibitor reduces the L-IC population and interferes with leukemia initiation. Together, these findings reveal a critical role of the Notch1-c-Myc pathway in T-ALL initiation and suggest that therapeutics targeted at this pathway could be used to treat and/or prevent disease relapse in patients.

Flotillin-1 és una proteïna associada a membrana plasmàtica implicada en processos de trànsit de vesícules, reordenació del citoesquelet i transducció de senyals. Estudis previs en el laboratori han demostrat que Flotillin-1 és capaç de translocar-se a nucli en resposta a un estímul mitogènic i afavorir la proliferació de diverses línies cel·lulars. Els mecanismes mitjançant els quals provoca aquests efectes són desconeguts i objecte del present estudi.
D'una banda demostrem que Flotillin-1 és un factor regulador de la cinasa Aurora B, una proteïna que intervé en el control de la mitosi i més concretament en el anaphase checkpoint. El knock-down de Flotillin-1 provoca events mitòtics aberrants, acompanyats del descens tant en l'expressió d'Aurora B com de la seva activitat mesurada com els nivells de fosforilació de la histona H3. Flotillin-1 interacciona amb Aurora B i evita la seva degradació per la via del proteasoma.
D'altra banda, Flotillin-1 interacciona amb el receptor transmembrana Notch1, implicat en nombrosos processos de regulació de proliferació, diferenciació, apoptosi, etc. Flotillin-1 regula la localització subcel·lular de Notch1 així com la seva capacitat com activador transcripcional. La depleció o mutació de Flotillin-1 dificulta l'entrada de Notch1 a nucli i l'expressió dels gens diana de les famílies Hes/Hrt.
En conjunt, es presenta a Flotillin-1 com una proteïna capaç d'actuar a diferents nivells i regular processos i vies de senyalització cel·lular que li confereixen un paper com a regulador de la proliferació cel·lular.
Flotillin-1 is a protein associated to plasma membrane involved in vesicle trafficking, cyotskeleton reorganization and signal transduction. Previous findings in our laboratory has shown that Flotillin-1 is able to translocate the nucleus under mitogenic stimulus and increase proliferation rates of several cell lines. The mechanisms of action are unknown and object of the present study.
First, we show that Flotillin-1 is a regulator factor of the mitotic kinase Aurora B, a protein involved in control of mitosis and, specifically, in the anaphase checkpoint. The knock-down of Flotillin-1 causes aberrant mitotic events, decrease in Aurora B levels and its activity, measured as protein levels of phosporilated histone H3. Flotillin-1 interacts with Aurora B and avoid its degradation by the proteasome pathway.
In addition, Flotillin-1 interacts with the transmembrane receptor Notch1, involved in many regulatory processes of proliferation, differentiation, apoptosis, etc. Flotillin-1 regulates the subcellular localization of Notch1 and its activity as transcriptional activator. The mutation or depletion of Flotillin-1 difficult the entry of Notch1 in the nucleus and the expression of its target genes Hes/ HRT.
Overall, Flotillin-1 is a protein capable of acting at different levels, processes and signaling pathways in order to be a regulator of cell proliferation.

Orientador: José Andres Yunes
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A leucemia linfóide aguda (LLA) é o câncer mais frequente na infância, e destas, 15% são do tipo T (LLA-T). A hiperativação da via PI3K/Akt tem sido amplamente descrita em tumores e em linhagens celulares de LLA-T. PTEN é o principal regulador negativo dessa via e frequentemente encontra-se inativado em cânceres humanos. Com frequência, pacientes com LLA-T apresentam mutações ativadoras de NOTCH1. NOTCH1 pode regular transcricionalmente PTEN, contudo ainda não está claro como as mutações ativadoras de NOTCH1 influenciariam a expressão de PTEN nas LLA-T. Nós encontramos uma ocorrência de 11 (17,7%) mutações no éxon 7 do PTEN em 62 casos de LLA-T estudados consecutivamente. Contudo, nenhuma mutação foi encontrada na análise de 71 casos de LLA-B derivada. A maioria das mutações de PTEN apresentavam inserções/deleções de mais de 3 nucleotídeos. Não encontramos associação entre mutações em PTEN e o gênero, a idade e a contagem de glóbulos brancos ao diagnóstico. Pacientes com alterações no PTEN apresentaram uma tendência a pior sobrevida global (OS, p=0.07). Dentre os pacientes de LLA-T classificados como alto risco (n=56), aqueles possuindo anormalidades no PTEN mostraram-se associados significativamente a menor OS (p=0.019) e sobrevida livre de leucemia (LFS 47% vs 76%; p=0.045). As curvas de LFS foram significativamente diferentes (p=0.003), mesmo considerando apenas pacientes que atingiram a remissão no dia 28 do tratamento para a análise. Nosso estudo também mostrou que pacientes com mutações em NOTCH1 apresentavam aumento na transcrição de MYC e menor expressão de PTEN mRNA comparados a pacientes com NOTCH1 selvagem. Nós recentemente demonstramos que células de LLA-T apresentavam fosforilação de PTEN mediada por CK2, resultando na estabilização e consequentemente inativação da proteína PTEN. Corroborando ao estudo anterior, os casos de LLA-T analisados, independente do status de mutação em NOTCH1, expressam níveis significativamente mais altos de proteína PTEN do que controles normais. Para avaliar o impacto da regulação transcricional de NOTCH e a inativação postranscricional por CK2 de PTEN, nós tratamos as células de LLA-T com inibidores de gamma-secretase (DAPT e de CK2 (DRB/TBB). Nosso estudo enfatiza a relevância biológica e clínica da regulação do PTEN em LLA-T. E sugerimos o uso combinado de inibidores de gamma-secretase e CK2 devem possuir potencial terapêutico nas LLA-T
Abstract: T-cell acute lymphoblastic leukemia (T-ALL) accounts for approximately 15% of pediatric ALL. Patients with T-ALL are at increased risk of relapse compared with children treated for B-cell precursor ALL. Mutations in the phosphatase and tensin homolog (PTEN) gene leading to PTEN protein deletion and subsequent activation of the PI3K/Akt signaling pathway are common in cancer. PTEN is the main negative regulator of the PI3K/Akt survival pathway. T-ALL patients frequently display NOTCH1 activating mutations and Notch can transcriptionally down-regulate the tumor suppressor PTEN. However, it is not clear whether NOTCH1 mutations associate with decreased PTEN expression in primary T-ALL. We report that PTEN exon 7 mutations occurred in 11 (17.7%) out of 62 consecutive pediatric T-cell acute lymphoblastic leukemia (T-ALL) but in none of 71 precursor B-ALL patients. Most PTEN mutations were insertions/deletions of more than 3 nucleotides. No associations were found between PTEN mutation and age, gender, WBC at diagnosis, early response to therapy and remission rate. Patients with PTEN mutation (n=11) had a tendency toward worse overall survival (OS, p=0.07). Remarkably, PTEN mutations were significantly associated with lower OS (p=0.019) and leukemia-free survival (LFS 47% vs 76%, p=0.045) within patients classified in the high risk group (n=56). LFS curves were significantly different (p=0.003) even if only patients who reached remission on day 28 were considered for analysis. We compared patients with or without NOTCH1mutations and report that the former presented higher MYC transcript levels and decreased PTEN mRNA expression. We recently showed that T-ALL cells frequently display CK2-mediated PTEN phosphorylation, resulting in PTEN protein stabilization and concomitant functional inactivation. Accordingly, the T-ALL samples analyzed, irrespectively of their NOTCH1 mutational status, expressed significantly higher PTEN protein levels than normal controls. To evaluate the integrated functional impact of NOTCH transcriptional and CK2 post-translational inactivation of PTEN, we treated TALL cells with both the gamma-secretase inhibitor DAPT and the CK2 inhibitors DRB/TBB. Our data suggest that combined use of gamma-secretase and CK2 inhibitors may have therapeutic potential in T-ALL. And emphasize the biological and clinical relevance of PTEN regulation in pediatric T-ALL
Doutorado
Genetica Animal e Evolução
Doutor em Genetica e Biologia Molecular

The 21st century brought about a dramatic increase in knowledge about genetic and molecular profiles of cancer. This information has validated the complexity of tumor cells and increased awareness of “nodal proteins”, but has yet to advance the development of rational targeted cancer therapeutics. Nodal proteins are critical cellular proteins that collect biological inputs and distribute the information across diverse biological processes. Survivin acts as a nodal protein by interfacing the multiple signals involved in mitosis and apoptosis and functionally integrate proliferation, cell death, and cellular homeostasis. By characterizing survivin as a target of both Type 1 Insulin-like Growth Factor (IGF-1) and Notch developmental signaling, we contribute to the paradigm of survivin as a nodal protein. The two signaling systems, Notch and IGF-1, regulate survivin by two independent mechanisms. Notch activation induces survivin transcription preferentially in basal breast cancer, a breast cancer subtype with poor prognosis and lack of molecular therapies. Activated Notch binds the transcription factor RBP-Jк and drives transcription from the survivin promoter. Notch mediated survivin expression increases cell cycle kinetics promoting tumor proliferation. Inhibition of Notch in a breast xenograft model reduced tumor growth and systemic metastasis. On the other hand, IGF-1 signaling drives survivin protein translation in prostate cancer cells. Binding of IGF-1 to its receptor activates downstream kinases, mammalian target of rapamycin (mTOR) and p70 S6 protein kinase (p70S6K), which modulates survivin mRNA translation to increase the apoptotic threshold. The multiple roles of survivin in tumorigenesis implicate survivin as a rational target for the “next generation” of cancer therapeutics.

A intensa exposição ao Sol sujeita o lábio, principalmente o inferior, aos danos provocados pela absorção de radiação ultravioleta (UV). O carcinoma epidermoide é a neoplasia maligna que se desenvolve nos lábios após exposição crônica prolongada aos raios UV e acredita-se que todos os casos sejam precedidos pela desordem potencialmente maligna denominada queilite actínica. Ambas as lesões são causados pelos efeitos nocivos da radiação UV agindo diretamente sobre o DNA, por meio do fenômeno da fotocarcinogênese. Nesse processo, a radiação provoca mutações que são capazes de causar a iniciação, progressão e a promoção de neoplasias. No entanto, é também importante considerar que outros eventos moleculares, além das mutações, estão envolvidos na iniciação e progressão do câncer. Alterações moleculares com ganho ou perda de função de componentes da via de sinalização Notch estão envolvidas em diferentes tipos de cânceres hematológicos e sólidos. Entretanto, a participação da sinalização Notch em câncer de lábio ainda é desconhecida. Assim, o objetivo desse trabalho foi investigar se a via Notch está relacionada às lesões de queilite actínica e de carcinoma epidermoide de lábio e sua participação na fotocarcinogênese bucal. Para isso, foram utilizados 45 casos de queilite actínica, 15 casos de carcinoma epidermoide de lábio e 05 casos de lábio com aspecto de normalidade, nos quais foi analisada a expressão de Notch1 e Jagged1 por meio da técnica de imuno-histoquímica. Os resultados demonstraram que houve perda da expressão de Notch1 em 40% dos carcinomas epidermoides de lábio, sugerindo que a expressão reduzida de Notch1 pode converter os queratinócitos a um estado ativado e imaturo. Observou-se ainda, diferença nos padrões de marcação de Nocth1 e Jagged1 nas células epiteliais sugerindo que o sinal da via Notch seja transmitido a partir de uma célula apical para uma célula basal devido a localização das células que expressam o receptor e das que expressam o ligante. Concluiu-se, assim, que os resultados imuno-histoquímicos não apontam a uma regulação diferencial da expressão da proteína Notch1 e Jagged1 em lesões UV induzidas.
The intense exposure to the sun subject the lips, particularly the lower, the damage caused by the absorption of ultraviolet (UV) radiation. The squamous cell carcinoma is a malignant tumor that develops on the lips after prolonged chronic exposure to UV rays and it is believed that all cases are preceded by potentially malignant disorder called actinic cheilitis. Both lesions are caused by the harmful effects of UV radiation acting directly on the DNA, through the phenomenon of photocarcinogenesis. In this process, the radiation causes mutations that are capable of causing the initiation, progression and promotion of cancer. However, it is also important to consider that other molecular events, apart from the mutations are involved in the initiation and progression of cancer. Molecular abnormalities with gain or loss of Notch pathway components function are involved in several types of hematological and solid cancer. However, the participation of Notch signaling in lip cancer is still unknown. The objective of this study was to investigate whether the Notch pathway is related to injuries actinic cheilitis and squamous cell carcinoma of the lip and participation in oral photocarcinogenesis. For this, were used 45 cases of actinic cheilitis, 15 cases of squamous cell carcinoma of the lip and lip 05 cases with normal aspect in which we analyzed the expression of Notch1 and Jagged1 by immunohistochemistry. The results showed loss of Notch1 expression in 40% of squamous cell carcinomas of the lip, suggesting that reduced expression of Notch1 can convert to an activated keratinocytes and immature state. There was also a difference in labeling patterns of Notch 1 and Jagged1 epithelial cells suggesting that the Notch pathway signal is transmitted from an apical cell to a basal cell due to localization of cells expressing the receptor and expressing the ligand. In summary, the immunohistochemical results do not show a differential regulation of Notch 1 and Jagged1 expression in UV induced lesions.

The Notch receptor is part of a highly conserved signaling pathway shared in Drosophila, C. elegans and mammals. Extensive studies of Notch signaling have revealed its participation in the development of diverse organ systems including brain, blood cells, blood vessels, gut, and skin. Many genetic modifiers of the Notch signaling pathway have been identified, including some which act at the membrane and others in the nucleus. One such member is Deltex, an E3 ubiquitin ligase, which was originally identified as a modifier of Notch in a Drosophila genetic screen. In early lymphoid development, Deltex has been demonstrated functionally to antagonize Notch signaling but the precise molecular mechanism for this functional antagonism between Notch and Deltex is not understood. However, in Drosophila, recent data supports the formation of a trimeric complex between Deltex, Kurtz and Notch that promotes Notch ubiquitin-mediated proteosomal degradation. Beta-arrestin1 is one of the closest mammalian homologues of Kurtz and functions as an adaptor protein in a variety of cellular processes such as endocytosis, ubiquitination and nuclear shuttling. We hypothesize that a similar interaction occurs in mammalian cells between Notch, beta-arrestin1 and Deltex to negatively modulate the Notch signaling pathway. Our data reveal a physical interaction between beta-arrestin1 and the Notch receptor. We could not, however, detect an interaction between Deltex and beta-arrestin1 by co-immunoprecipitation. We also demonstrate that Notch and beta-arrestin1 physically associate with both a membrane-bound form of activated Notch, as well as the intracellular form of Notch after membrane cleavage. Using RNA interference, as well as overexpression of beta-arrestin1, we demonstrate that beta-arrestin1 negatively regulates a Notch/CSL dependant reporter assay. We also show that the presence of Deltex enhances the negative modulation of the Notch signaling pathway mediated by beta-arrestin1. Therefore, we reveal a new Notch interacting protein and a novel role for beta-arrestin1 in the Notch signaling pathway.

The Notch receptor family plays a key role in development and disease. In cancer, Notch can act either as an oncogene or as a tumour suppressor, and possibly as a cancer stem-cell factor. Whereas most research has focused on downstream signalling events, little is known about the cell surface organisation of Notch and its ligands. The extracellular part of Notch consists mainly of 36 epidermal growth factor-like domains (EGF-domains), many of which bind calcium. Studies have shown that tandem repeats of calcium-binding EGF domains form a rigid linear arrangement; however, the lack of calcium binding in EGF6, EGF10 and EGF22 led to the hypothesis that these might be sites of flexibility. This thesis addresses the effect of these domains on the organisation of the extracellular region of Notch and provides further insight into the calcium-binding properties of Notch. NMR residual dipolar coupling (RDC) measurements of these regions are presented, together with the X-ray crystallographic data obtained in collaboration. The crystal structure of the human Notch-1 construct EGF4-7 shows a tilt angle of 90° at the EGF5-6 interface which is much larger than the tilt angles of 10-20° observed for the EGF11-13 crystal structure. RDC measurements demonstrated an angle of ~70° in solution. The crystal structures of EGF21-23 and EGF20-23 showed a rod-shaped interface for the EGF21-22 domain, in which a cis-proline forms the packing interaction to a tyrosine at the β-turn in the major β-sheet of EGF22. These two interfaces are novel and demonstrate the possibility of interface formation without Ca²⁺. Crystallisation was unsuccessful for the EGF8-11 construct. However, RDC measurements indicate interdomain motion between EGF9 and EGF10 demonstrating a flexible interface. These data establish new information on the structural organisation and calcium-binding properties of the extracellular region of Notch and identify flexible and rigid interfaces within multiple tandem repeats of EGF domains. This information will be invaluable in constructing models of Notch-ligand complexes for testing in future functional experiments.

La voie de signalisation Notch est une signalisation fortement conservée chez les metazoans. Elle régule nombreux processus biologiques, y compris le développement embryonnaire, l'homéostasie tissulaire en particulier dans la décision du destin cellulaire. Largement décrit dans la tumeurigénèse, la voie Notch est aussi un régulateur clé dans l'angiogenèse. De plus en plus d'études montrent que la voie Notch joue un rôle important dans régulation d'angiogenèse tumorale. Par conséquent, divers d'efforts ont été faits pour inhiber la voie canonique de Notch. Jusqu'à aujourd'hui, appart Notch1, d'autre récepteurs Notch ont été très peu étudiés dans l'angiogenèse tumorale. Notch3 exprimé restreint dans le systèm vasculaire, n'a jamais été étudié dans le contexte de l'angiogenès tumorale. Par consequent, l'objectif de ma thèse est d'étudier le rôle de Notch3 dans l'angiogenès tumorale. Ici, nous avons montré une expression abérrant de Notch3 dans les cellules endotheliales tumorales où il présente un effect inattendu. En inhibant Noth3 dans le microenvironnement de la tumeur, nous avons montré que Notch3 limite l'angiogenèse tumorale par sa fonction pro-apoptotique dans les cellules endothéliales tumorales, ce qui entraîne une inhibition de la croissance tumorale. De plus, nous avons constaté que l'expression de JAG1 ont été argumenté dans une fraction des cancers humains. En outre, JAG1 dérivé de la tumeur favorise la survie des cellules endothéliales tumorales en inhibant l'effet pro-apoptotique de Notch3. Nous présentons ici pour la première fois que Notch3 est un récepteur de dépendance induisant une apoptose dans les cellules endothéliales tumorales qui est bloqué par JAG1. Par conséquent, nous montrons que l'effet antitumoral bien documenté médié par l'inhibition de la ?-sécrétase dépend au moins en partie de l'apoptose induite par Notch3 dans les cellules endothéliales
The Notch signalling is a highly conserved signalling which mediates numerous of biological processes, including embryonic development, tissue homeostasis especially in the cell fate decision. Widely implicating in tumorigenesis, the Notch signalling is also a key regulator of angiogenesis. Increasing number of studies showed that, the Notch signalling plays an important role in mediating tumour angiogenesis. Therefore, various efforts have been made to inhibit the canonical Notch pathway. To date, besides Notch1, other Notch receptors were few studied in tumour angiogenesis. Notch3, expressed restrictedly in vascular system, has never been studied in the tumour angiogenesis context. Therefore, the objective of my thesis is to study the role of Notch3 during tumour angiogenesis. Here, we showed that Notch3 is aberrantly expressed in tumour endothelial cells where it presents an unexpected pro-apoptotic effect. By silencing Noth3 in the tumour microenvironment, we showed that Notch3 limits tumour angiogenesis via its pro-apoptotic function in tumour endothelial cells which results in inhibition of tumour growth. In addition, we found that JAG1 is up-regulated in a fraction of human cancers. Furthermore, tumour derived JAG1 favourite the survival of tumour endothelial cells by inhibiting the pro-apoptotic effect of Notch3. We thus present here for the first time that Notch3 as a dependence receptor inducing apoptosis in tumour endothelial cells which is blocked by JAG1. Consequently, we show that the well-documented anti-tumour effect mediated by g-secretase inhibition is at least in part dependent on the apoptosis triggered by Notch3 in endothelial cells

The Notch family of receptors play important roles in cell fate assignation and pattern formation during development. Notch ligands, encoded by multiple Delta and Serrate/Jagged genes, have functionally significant differences in the way they interact with Notch, and the relative gene dosages of Notch and its ligands are crucial for normal development, reflected in phenotypes that correlate with copy number. Serrate and Delta have structural and functional similarities, but exhibit different expression patterns and mutant phenotypes, indicating there might be differences in the way Delta and Serrate bind to and elicit a signal from Notch. Genetic analyses have suggested the associations of Notch with its ligand in cultured cells are analogous to those in vivo. The ability of stably transfected Notch-expressing S2 cells to aggregate with Delta- or Serrate-expressing cells was examined, and unlike the binding of Delta to Notch, which is primarily dependent on the relative amounts of each protein, an additional factor influencing Notch:Serrate binding. In Drosophila, the activity of Serrate, and the way it interacts with Notch, is modulated by relative concentrations of Serrate, Notch and Wingless. Addition of Wingless to Notch- and Serrate-expressing cells promotes their aggregation, indicating Wingless facilities binding of Serrate to Notch, which would activate Notch signalling. In addition, preincubation of Notch with Wingless blocked its ability to subsequently bind Delta, indicating Wingless may change the conformation of the Notch protein. In vivo, this could profoundingly affect signalling if Wingless both blocked Delta and promoted Serrate interactions with Notch. I also present evidence that these interactions are mediated by different EGF-like repeats of Notch. Retinal progenitor cells can differentiate into any one of the neuronal or glial cell types of the mature retina and require temporal and positional information cues to adopt appropriate fates.

The adult zebrash brain has a remarkable constitutive neurogenic capacity. The regulation and maintenance of its adult neurogenic niches are poorly understood. In mammals, Notch signaling is involved in stem cell maintenance both in embryonic and adult CNS. To better understand how Notch signaling is involved in stem cell maintenance during adult neurogenesis in zebrafish we analysed Notch receptor expression in five neurogenic zones of the adult zebrafish brain. Combining proliferation and glial markers we identified several subsets of Notch receptor expressing cells. We found that 90 [Formula: see text] of proliferating radial glia express notch1a, notch1b and notch3. In contrast, the proliferating non-glial populations of the dorsal telencephalon and hypothalamus rarely express notch3 and about half express notch1a/1b. In the non-proliferating radial glia notch3 is the predominant receptor throughout the brain. In the ventral telencephalon and in the mitotic area of the optic tectum, where cells have neuroepithelial properties, notch1a/1b/3 are expressed in most proliferating cells. However, in the cerebellar niche, although progenitors also have neuroepithelial properties, only notch1a/1b are expressed in a high number of PCNA [Formula: see text] cells. In this region notch3 expression is mostly in Bergmann glia and at low levels in few PCNA [Formula: see text] cells. Additionally, we found that in the proliferation zone of the ventral telencephalon, Notch receptors display an apical high to basal low gradient of expression. Notch receptors are also expressed in subpopulations of oligodendrocytes, neurons and endothelial cells. We suggest that the partial regional heterogeneity observed for Notch expression in progenitor cells might be related to the cellular diversity present in each of these neurogenic niches.

The adult zebrash brain has a remarkable constitutive neurogenic capacity. The regulation and maintenance of its adult neurogenic niches are poorly understood. In mammals, Notch signaling is involved in stem cell maintenance both in embryonic and adult CNS. To better understand how Notch signaling is involved in stem cell maintenance during adult neurogenesis in zebrafish we analysed Notch receptor expression in five neurogenic zones of the adult zebrafish brain. Combining proliferation and glial markers we identified several subsets of Notch receptor expressing cells. We found that 90 [Formula: see text] of proliferating radial glia express notch1a, notch1b and notch3. In contrast, the proliferating non-glial populations of the dorsal telencephalon and hypothalamus rarely express notch3 and about half express notch1a/1b. In the non-proliferating radial glia notch3 is the predominant receptor throughout the brain. In the ventral telencephalon and in the mitotic area of the optic tectum, where cells have neuroepithelial properties, notch1a/1b/3 are expressed in most proliferating cells. However, in the cerebellar niche, although progenitors also have neuroepithelial properties, only notch1a/1b are expressed in a high number of PCNA [Formula: see text] cells. In this region notch3 expression is mostly in Bergmann glia and at low levels in few PCNA [Formula: see text] cells. Additionally, we found that in the proliferation zone of the ventral telencephalon, Notch receptors display an apical high to basal low gradient of expression. Notch receptors are also expressed in subpopulations of oligodendrocytes, neurons and endothelial cells. We suggest that the partial regional heterogeneity observed for Notch expression in progenitor cells might be related to the cellular diversity present in each of these neurogenic niches.

The core aim of this project was to use the nematode worm C. elegans to probe the function of the N-terminus of Notch ligands using com- plementary molecular and in vivo approaches. Sequence analysis and structural predictions suggested that the N-termini of C. elegans Notch ligands LAG-2 and APX-1 may comprise a C2 domain, although it was concluded structural information would be necessary to confirm this. Eu- karyotic expression systems were used to produce recombinant protein fragments, and ultimately yielded a large quantity of the fragment APX- 1NE1FcHis that was purified and used for crystallography studies, although no structural information has yet been obtained from this. In parallel, assays were established to assess rescue of the mutant phenotypes associ- ated with the lag-2(q420) allele. Extrachromosomal expression of LAG-2 and APX-1 partially rescued the Lag phenotype, although overexpres- sion of ligand appeared to cause an increase in embryonic lethality. An alternative assay was established, to assess rescue of the anchor cell du- plication phenotype. Using this assay, it was demonstrated that deletion of the N-terminal MNNL or DSL domains of APX-1 abolished rescue of the phenotype observed for WT APX-1 confirming the importance of these domains in Notch signalling. Rescue was still achieved after dele- tion of the EGF-like 1 domain of APX-1, and substitutions targeting loop regions in the DSL and MNNL (DSL(ETSD), I80-K86/SS, respec- tively), but the extent of this rescue appeared to be reduced in all three constructs, suggesting these regions are required for optimised Notch signalling.

Les récepteurs membranaires sont des acteurs majeurs des interactions entre la cellule et son environnement. Ils peuvent être à l'origine des signaux de survie, de différentiation, de migration ou bien de mort cellulaire. Les travaux de ce manuscrit ont été faits sur une famille de récepteurs nommés "récepteurs à dépendance". Ils sont caractérisés par leur fonctionnement dans la cellule plutôt que par leur structure: en présence de leurs ligands ces récepteurs induisent un signal de survie et en l'absence de ces mêmes ligands ils induisent un signal actif de mort cellulaire. Deux nouveaux récepteurs à dépendance ont été étudiés: Notch3 et Kremen1 dans le contexte du contrôle de l'homéostasie et plus particulièrement dans le contrôle de la tumorigenèse du cancer du sein. Nous montrons que le récepteur à dépendance Notch3 est perdu dans le cancer du sein, dû à un gain significatif de méthylation, entre le tissu normal et le tissu tumoral dans les patients. Notch3 a également un rôle pro-apoptotique dans les cellules endothéliales dans le cancer du poumon. Des études effectuées sur des cohortes de cancer nous ont permis de voir que le ligand Dickkopf1 (Dkk1), qui lie le récepteur Kremen1, est sur-exprimé dans plusieurs cancers tandis que le récepteur il est perdu dans les cancers. Rétablir l'expression de Kremen1 ou invalider Dkk1 dans la lignée de cancer du sein de type basal MDA-MB 231 conduit à une forte mort cellulaire de type autophagique. En ce qui concerne les enjeux thérapeutiques de ces travaux, nous avons pu sélectionner plusieurs anticorps dirigés contre le domaine extracellulaire de Kremen1, qui induisent la mort des cellules cancéreuses
Membrane receptors are major actors of the interaction between a cell and its environment. They are able to trigger different types of signals such as survival, differentiation, migration or cell death. The work presented in this manuscript has been done on a particular family of receptors called dependence receptors. They are characterized by their function rather than by their structure. In the presence of their ligand they induce a survival signal whereas in the absence of the ligand they induce an active signal of cell death. Two new dependence receptors have been studied: Notch3 and Kremen1, in the context of homeostasis control, and more particularly in the control of breast cancer tumorigenesis. We show that Notch3 dependence receptor is lost in breast cancer, because of a significant gain of methylation observed between the normal tissu and the tumoral tissue within the same patient. Notch3 plays also a pro-apoptotic role in endothelial cells of lung cancer. Experiences carried on cancer cohorts have allowed us to notice that the Dickkopf (Dkk1) ligand, which links the Kremen1 receptor, is over-expressed in several cancers whereas the receptor is lost in different cancers. Restoring Kremen1 expression or disabling Dkk1 in breast cancer basal type MDA-MB 231 cells, leads to large autophagic cell death. Concerning therapeutic approaches, we selected several antibodies against Kremen1 extra-cellular domain, which induce the death of cancer cells

O timo é o órgão linfóide primário responsável pelo estabelecimento inicial de um repertório funcional de células T. A via de sinalização Notch é essencial para o desenvolvimento de células T a partir de células-tronco hematopoiéticas, e a distribuição de seus receptores e ligantes no timo humano ainda é desconhecida. A expressão de AIRE é crucial para a seleção de um repertório de receptores de linfócitos T (TCR) sem autorreatividade. Neste estudo, analisamos o padrão de expressão de AIRE e a distribuição de Notch em timos pacientes com cardiopatias congênitas, parte dos quais com síndrome de Down. Descrevemos a localização intratímica e os tipos celulares capazes de expressar os diferentes receptores e ligantes Notch. A expressão de AIRE em células epiteliais medulares foi significantemente reduzida em timos de crianças com síndrome de Down, deficiência esta que pode explicar a alta incidência de doenças autoimunes nesta cromossomopatia.
The thymus is a primary lymphoid organ which is essential for the initial establishment of a functional repertoire of T cells. Notch signaling is crucial for T-cell lineage development from hematopoietic stem cells; however, distribution of Notch ligands and receptors in human thymus is still unknown. AIRE is crucial for the selection of a T-cell-receptor (TCR) repertoire purged of self-reactive specificities. In this study, we analyzed the expression patterns of AIRE and Notch in human thymuses from children with congenital cardiopathies that undergo heart surgery, part of whom with Down syndrome. We described the intra-thymic localization and the cell types that express Notch receptors and ligands. AIRE expression in medullary epithelial cells is significantly decreased in Down syndrome patients. This deficiency could explain higher incidence of autoimmune disease in Down syndrome.

Delta-Notch signaling is crucial for development of nearly every tissue in metazoans. Signals received by the Notch receptor influence transcription of select target genes that ultimately restrict the developmental fate of the signal receiving cell with respect to its neighbors. The Notch pathway also functions in contexts of abnormal proliferation and differentiation, e.g. cancer and inflammation. Therefore, understanding the regulation of signaling through the Notch receptor protein at the cellular and molecular level is of great significance. In this dissertation, I investigated three ways in which Notch signaling is regulated, namely (1) proteolysis of the Delta ligand; (2) endocytosis of the Delta ligand; and (3) proteolysis of the Notch receptor.. The Delta protein has three functions. First, Delta is a ligand for Notch when bound to it from an adjacent cell. Second, Delta is an inhibitor of Notch when coexpressed with it in the same cell. Third, Delta is hypothesized to be a receptor and, upon binding to Notch, signals to nucleus. Delta undergoes proteolysis by ADAM proteases and there are two contradictory models for the role of Delta cleavage: (1) cleavage disables Delta function; and (2) cleavage activates Delta function. Overall, the results presented in this dissertation strengthen the first model and weaken the second one. Consistent with the first model, we showed that preventing Delta cleavage strengthens its ligand function. As well, when co-expressed in the same with Notch, Delta cleavage is upregulated therefore disabling Delta function as inhibitor of Notch. In contrast to the second model, we showed that Delta proteolysis does not follow a previously established pattern of cleavages typical of cell surface proteins that are activated by proteolysis. Delta also undergoes endocytosis. Two general models have emerged that are again contradictory: (1) endocytosis downregulates cell surface expression of Delta and therefore diminishes its ability to bind Notch; (2) endocytosis of Delta invokes activation of Notch signaling. Overall, our results strengthen the first model and weaken the second one. In support of the first model, we first demonstrated that Notch activation shows a linear relationship to the amount of Delta ligand present on the cell surface and that subsequent inhibition of cell surface expression of Delta leads to its loss of function. In contrast to the second model, we showed that endocytosis of Delta is not required to activate Notch. We also resolved that earlier evidence in support for this model stemmed from misinterpretations of the properties of a Delta mutant protein. Proteolysis of Notch activates the signaling cascade. Binding of Delta to Notch was previously regarded as a requisite regulatory step to invoke receptor proteolysis. We identified the ability of Kuzbanian and TACE, ADAM proteases that cleave Notch in response to Delta stimulation, to activate Notch in a ligand-independent manner. Altogether, our results demonstrate that proteolysis and endocytosis of Delta are independent mechanisms that act to downregulate Delta function and are therefore an important means of attenuating the Notch signal. Alternatively, we find a novel means of enhancing Notch signals in specific contexts, namely through ligand-independent Notch activation by the ADAMs Kuzbanian and TACE. With respect to the latter observation, Kuzbanian and TACE expression is known to be elevated in several human diseases, and thus predicts that engagement of Notch signaling is a contributing factor in these pathologies.

A paracoccidioidomicose (PCM) é uma micose sistêmica de natureza profunda e granulomatosa, que afeta preferencialmente o tecido pulmonar causada pelo Paracoccidioides brasiliensis, um fungo que exibe dimorfismo térmico. O P. brasiliensis interage com células apresentadoras de antígenos (APCs), alterando suas principais funções biológicas. Entre as APCs, os macrófagos são células que desempenham um papel importante na indução e regulação da resposta imune e/ou inflamatória. São células do sistema fagocítico mononuclear que podem discriminar entre o que é próprio do organismo e os patógenos, através da expressão de receptores de reconhecimento padrão (PRR) que reconhecem padrões moleculares associados à patógenos (PAMPs), sendo células importantes no processo de fagocitose controlando o crescimento destes patógenos. Recentemente tem sido demonstrada a importância dos receptores Notch na regulação da atividade de macrófagos e do sistema imune. Os ligantes de Notch estão envolvidos nas locais inflamatórios de infecção devido ao aumento da expressão dos mesmos na superfície de células envolvidas neste processo. Considerando que a sinalização Notch pode estar envolvida na modulação da função de macrófagos, nós avaliamos se P. brasiliensis tem a capacidade de modular a ativação desta via e interferir na produção de citocinas pró-inflamatórias. Para isso, macrófagos J774, pré-estimulados com LPS ou não, foram interagidos com leveduras do fungo, seguido por análise de PCR em tempo real e citometria de fluxo, dosagem de citocinas e índice de fagocitose. Nossos dados revelaram que na presença do fungo existe aumento dos níveis de transcrição do receptor Notch 1 e diminuição da transcrição do ligante Delta 4 em macrófagos pré-estimulados com LPS. Entretanto, verificamos que o fungo sozinho não é capaz de induzir a transcrição de NF-κB, nem na presença do LPS, mas quando os macrófagos são estimulados com LPS e sofrem inibição da via de Notch existe aumento dos níveis de transcritos após interação com o fungo, sugerindo que esse fator é ativado na ausência de Notch. Nesse contexto de inibição de Notch, evidenciamos que a fagocitose de leveduras do fungo por macrófagos tornou-se mais eficiente, visto que houve aumento do índice de fagocitose na ausência de Notch. Foi possível verificar que o fungo tem a capacidade de promover a produção de IL-6 via TLR-Notch, fazendo-nos supor que essa citocina seja importante para o estabelecimento da doença ao ser benéfica para o fungo e prejudicar o hospedeiro. E concomitantemente ao aumento de IL-6 existe diminuição da produção de TNF-α. Com base nesses resultados, podemos sugerir que o P. brasiliensis utiliza a via de sinalização Notch como um mecanismo de escape. A interação entre as leveduras do fungo e os macrófagos promove a ativação dessa via, através do receptor Notch 1, induzindo maior produção de IL-6, citocina importante para o crescimento do fungo no hospedeiro, conjuntamente com a diminuição de TNF-α prejudicando a atividade fungicida dos macrófagos.
Paracoccidioidomycosis (PCM) is a systemic mycosis and deep granulomatous in nature, which affects mainly the lung tissue caused by Paracoccidioides brasiliensis, a fungus that exhibits thermal dimorphism. The P. brasiliensis interacts with antigen presenting cells (APCs), changing its main biological functions. Among the APCs, macrophages are cells that play an important role in the induction and regulation of the immune response and/or inflammatory response. They are cells of the mononuclear phagocytic system that can discriminate between what is characteristic of the organisms and pathogens, by expression of pattern recognition receptors (PRR) that recognizes the pathogen-associated molecular pattern (PAMPs), and are considered cells important in phagocytosis for controlling the growth of these pathogens. It has been recently demonstrated the importance of the Notch receptor in regulating the activity of macrophages and of the immune system. The ligands of Notch are involved in inflammatory sites of infection because there are increased expression of these ligands on cell surface involved in this process. Whereas the Notch signaling may be involved in modulating macrophage function, we evaluated whether P. brasiliensis has the ability to modulate the activation of this pathway and interfere with the production of pro-inflammatory cytokines. For this, J774 macrophages, pre-stimulated with LPS or not, are interacted with yeast fungus, followed by Real Time PCR analysis and flow cytometry, cytokine and phagocytosis index. Our data showed that the presence of the fungus exists increased levels of transcription of the Notch 1 receptor, and a decrease in ligand Delta 4 transcription on macrophages pre-stimulated with LPS. However, we found that the fungus itself is not able to induce transcription NF-κB, even in the presence of LPS, but when macrophages are stimulated with LPS and suffer inhibition of the Notch signaling, exists increased levels of transcripts after interaction with the fungus, suggesting that this factor is activated in the absence of Notch. Within the context of inhibition of Notch, we found that phagocytosis of yeasts by macrophages become more efficient, since the increased rate of phagocytosis in the absence of Notch. It was verified that the fungus has the ability to promote the production of IL-6 via TLR-Notch, making us suppose that this cytokine is important for the establishment of the disease to be beneficial for the fungus and damage the host. And concurrently with increased IL-6 there is decreased production of TNF-α. Based on these results, we suggest that P. brasiliensis uses the Notch signaling pathway as an escape mechanism. The interaction between the yeasts with macrophages promotes the activation of this pathway, by means of a Notch 1 receptor, inducing increased production of IL-6 cytokine important for the growth of fungus on host, together with a reduction of TNF-α, contributing with a damaging fungicidal activity of macrophages.

O timo é o órgão linfóide primário responsável pela maturação dos linfócitos T onde se observam estruturas e células especializadas. A sinalização intra-tímica durante a maturação de linfócitos T não é bem esclarecida. Entre outras, a via de sinalização Notch, composta por receptores (Notch 1 a 4) e ligantes (DLL1, 3 e 4, Jagged 1 e 2), pode regular este processo. Neste trabalho temos como objetivo avaliar a expressão gênica e proteica de receptores e ligantes Notch nas diferentes fases de maturação de linfócitos T, linfócitos T reguladores naturais (nTreg) e células dendriticas tímicas (tDC). Para tanto, timos de 10 crianças submetidas à cirurgia cardíaca corretiva foram manipulados e as populações CD4-CD8-, CD4+CD8+, CD4+CD8-, CD4-CD8+, nTreg e tDC foram purificadas por citometria de fluxo. O RNA total foi isolado e os genes NOTCH1, 2 e 3, DLL1 e 4, JAG1 e 2, FOXP3 foram amplificados por RT-PCR. Alguns fragmentos de tecido foram avaliados por imunohistoquímica, quanto a expressão dos receptres Notch 1, 2, 3 e 4 e dos ligantes, DLL1 e 4, Jagged 1 e 2 em timócitos totais, células FOXP3+ e células S100+ em cada região tímica. Todos os genes de receptores e ligantes Notch foram expressos nas populações estudadas. Na população CD4-CD8- o gene NOTCH1 é mais expresso em comparação as outras populações de timócitos imaturos. Na população CD4+CD8+ o gene NOTCH2 é menos expresso, e o gene JAG2 mais expresso quando comparados à população CD4+CD8-. Os demais genes de receptores ligantes Notch são expressos de maneira similar entre as populações de timócitos. A avaliação relativa dos genes Notch nas populações de timócitos mostrou uma maior expressão do gene DLL1 na população CD4+CD8- e JAG1 na população CD4-CD8+ em comparação à CD4-CD8-. A expressão dos receptores e ligantes Notch no tecido mostrou ser homogênea entre as regiões. As nTreg expressam o gene do ligante JAG1 em maior nível entre os avaliados. A expressão gênica relativa das nTreg mostrou uma maior expressão de NOTCH1, NOTCH2, DLL4 e JAG1 e menor expressão de NOTCH3, DLL1 e JAG2 em relação as CD4-CD8-. Quando a relação utilizou a população CD4+CD8- observamos que as nTreg expressam mais os genes NOCTH1, NOCTH2, NOTCH3, DLL4 e JAG1, e em níveis similares DLL1 e JAG2. A avaliação histológica mostrou uma maior expressão de DLL4 em nTreg em comparação às demais proteínas avaliadas, e uma distribuição homogênea entre as regiões com exceção de Notch3 e Jagged2. As tDC mostraram uma maior expressão do gene JAG1 em comparação aos demais, e uma maior expressão da proteína DLL4. A expressão do receptor Notch2 em tDC difere entre as regiões tímicas. Em conjunto, nossos resultados mostraram que os receptores e ligantes Notch são expressos de forma gênica e proteica em todos os estágios de maturação de linfócitos T, nTreg e tDC do timo humano, com algumas variações quanto ao nível de expressão e distribuição no timo. Dados que se assemelham às avaliações murinas, porém com pontos a serem discutidos. Nossa inédita avaliação em nTreg propõem uma nova abordagem ao envolvimento da via Notch em sua maturação e a avaliação das tDCs sugere sua participação direta via Notch na maturação dos timócitos humanos
The thymus is a primary lymphoid organ responsible of maturing T lymphocytes, where specialized structures and cells are observed. The intrathymic signaling during lymphocytes maturation is unclear. Among them, Notch signaling pathway, which comprises in receptors (Notch1-4) and ligands (DLL1, 2 and 3, Jaaged1 and 2), may regulate this process. In this work our aim is to evaluate the expression of Notch receptors and ligands in different phases of T lymphocytes maturation, natural T regulatory cells (nTreg), and thymic dendritic cells (tDC). For this purpose, thymuses from 10 children who underwent corrective cardiac surgery were manipulated and populations CD4-CD8-, CD4+CD8+, CD4+CD8-, CD4- CD8+, nTreg and tDC were sorted by flow cytometry. Total RNA was purified and genes NOTCH1, 2 and 3, DLL1 and 4, JAG1 and 2, FOXP3 were amplified by RT-PCR. Some thymic fragments were evaluated by immunohistochemistry and screened for expression of Notch 1, 2, 3 and 4 receptors, DLL1 and 4, Jagged and e 2 ligands in total thymocytes, FOXP3+ cells and S100+ cells in each thymic region. All Notch receptors and ligands genes were expressed in studied populations. In CD4-CD8- subset NOTCH1 gene is more expressed in comparison to others immature thymocytes. In CD4+CD8+ subset NOTCH2 gene is less expressed, and JAG2 gene is more expressed when compared to CD4+CD8- population. The other receptors and ligands genes were expressed in a similar level among developing lymphocytes subsets. The relative Notch genes evaluation in developing lymphocytes populations showed a higher expression of DLL1 gene in CD4+CD8- population and JAG1 gene in CD4-CD8+ subset in comparison to CD4-CD8- thymocytes. The Notch receptors and ligands expression in thymic tissue showed to be homogeneous between thymic regions. The nTreg cells express JAG1 ligand gene in highest level among evaluated genes. The relative gene expression in nTreg presented higher expression of NOCTH1, NOTCH2, DLL4 and JAG1 genes, and low levels of NOTCH3, DLL1 and JAG2 related to CD4-CD8- subset. When relative gene expression were performed using CD4+CD8- subset, we observed that nTreg cells expressed more NOCTH1, NOCTH2, NOTCH3, DLL4 and JAG1, and in a similar level of expression DLL1 and JAG2 genes. The histological analysis showed that DLL4 was more expressed in nTreg cells in comparison to others proteins evaluated in this work, and a homogeneous distribution in thymic regions, in exception Notch3 and Jagged2. tDC cells presented higher expression of JAG1 gene among the others, and a higher expression of DLL4 protein. Notch2 expression in tDC was different between thymic regions. All together, our results showed that both genes and proteins of Notch receptors and ligands are expressed in distinct developmental stages of the maturation of T lymphocytes and nTreg cells and in the tDC cells in human thymus, with some variations in levels of expression and distribution in the thymus. These data are similar to the murine evaluations, but with some issues to be discussed. Our unpublished assessment in nTreg propose a new approach about the involvement of Notch pathway in its maturation and the evaluation of tDC suggests its direct participation of Notch signaling in the process of human thymocytes maturation

A galectina-3, um membro da família das proteínas de ligação a glicanas, tem sido objeto de intensa pesquisa nos últimos anos devido ao seu importante papel na biologia tumoral, como a proliferação, transformação, apoptose, angiogênese, adesão, invasão e metástase tumoral. As diferentes funções de galectina-3 nas células tumorais resultam das suas diversas localizações inter- e subcelulares que lhe permite interagir com diferentes proteínas. Esta tese teve como objetivo identificar um papel específico de galectina-3 na regulação da via de sinalização Notch, que cada vez mais tem sido associada com a progressão tumoral e angiogênese. Inicialmente, demonstramos que galectina-3 interage com o receptor Notch-1 e modula diferencialmente a ativação da via pelos ligantes DLL4 e Jagged1. A galectin-3 regulou a expressão dos ligantes de Notch assim como o receptor Notch-1 e extracelularmente recuperou a ativação de Notch na ausência de galectina-3 endógena. Em câncer gástrico humano, a galectina-3 encontrou-se positivamente correlacionada com a expressão de Jagged1, enquanto que a galectina-1, um outro membro da família das galectinas, foi positivamente correlacionado com DLL4. De seguida estudou-se o papel biológico da regulação da via Notch pela galectina-3 na angiogênese. Demonstramos que nas células endoteliais, galectina-3 liga e aumenta a meia vida de Jagged1 promovendo a ativação preferencial da Jagged1/Notch em vez de DLL4/Notch de uma forma independente de VEGF. Verificamos que condições de hipóxia alteraram a expressão de galectina-3 assim como o status de glicosilação das células endoteliais de forma a promover a ativação de Jagged1/Notch e o aumento de angiogênese. A superexpressão de Jagged1 num modelo de carcinoma de pulmão de Lewis, acelerou o crescimento tumoral in vivo que foi inibido em camundongos Lgals3-/-. Por fim, avaliou-se o papel de galectina-3 na resistência das células tumorais a quimioterápicos. Observamos que a expressão de sialil-Tn, um produto biossintético da ST6GalNAc-I, diminuiu in vitro como in vivo a presença e os sítios de ligação de galectina-3 na superfície da células levando à sua acumulação no meio intracelular. Extracelularmente, galectina-3 não levou à indução de morte celular, no entanto contribuiu para a morte induzida por quimioterápicos. As células expressando sialil-Tn encontraram-se protegidas. Em amostras de tumor gástrico, os sítios de ligação de galectina-3 encontraram-se negativamente correlacionados com a expressão de sialil-Tn. Este conhecimento possui implicações diretas no desenvolvimento de estratégias visando o controle do crescimento tumoral e angiogênese e abre novas perspectivas no combate à resistência tumoral à terapia
Galectin-3, a member of a family of glycan binding proteins has been the subject of an intense research over the past few years due to its important role in cancer biology, such as cancer cell growth, transformation, apoptosis, angiogenesis, adhesion, invasion and metastasis. The different roles of galectin-3 on cancer cells behavior appears to have originated from its diverse inter- and subcellular localizations where it interacts with several different binding partners. The aim of this thesis was to pinpoint a specific role for galectin-3 in regulating Notch signaling pathway in cancer. Notch signaling has emerged as an important pathway in carcinogenesis, and activated Notch-1 signaling has being associated with cancer progression and angiogenesis. Initially, we found that galectin-3 was able to interact with Notch-1 receptor and to differentially modulate Notch signaling activation by DLL4 and Jagged1 ligands. Galectin-3 was found to regulate the expression of the Notch ligands and Notch-1 receptor and its extracellular form was able to rescue Notch activation in the absence of endogenous galectin-3. In human gastric cancer, galectin-3 was positively correlated with the expression of Jagged1 whereas galectin1, another member of the galectin family, was positively correlated with DLL4. Furthermore, we studied the biological role of Notch regulation by galectin-3 in angiogenesis. We showed that, in endothelial cells, galectin-3 binds to and increases Jagged1 protein half-life promoting Jagged1/Notch over DLL4/Notch signaling in a VEGF independent way. Hypoxic conditions changed galectin-3 expression and the glycosylation status of endothelial cells, acting in concert to promote Jagged1/Notch activation and sprouting angiogenesis. Jagged1 overexpression in Lewis lung carcinoma accelerated tumor growth in vivo that was prevented in Lgals3-/- mice. Finally, we evaluated the role of galectin-3 in cancer cell resistance to therapy. We found that the expression of sialyl-Tn, a biosynthetic product of ST6GalNAc-I, was able to decrease cell surface galectin-3 and galectin-3-binding sites both in vitro and in vivo leading to an intracellular accumulation of this protein. Exogenously added galectin-3 was found to have no effect on cancer cell death but contributed to chemotherapy-induced apoptosis. Sialyl-Tn expressing cells were protected. In human gastric cancer samples, galectin-3 binding sites were negatively correlated with the expression of sialyl-Tn. This knowledge has direct implications for the development of strategies aimed at controlling tumor growth and angiogenesis and open novel perspectives to overcome tumor resistance to therapy

The olfactory system exhibits remarkable regenerative ability in it’s neuronal population. The success of continuous neurogenesis is thought to be due, at least in part, to its unique glia – olfactory ensheathing cells (OECs). OECs bear characteristics of both peripheral and central glia, and serve to ensheath, guide and promote growth of olfactory receptor neurons (ORNs) throughout both development and adult life. Brain lipid binding protein (BLBP) is most highly expressed by radial glia during embryonic development. It is largely down-regulated in the adult CNS, but BLBP expression is retained in the adult by special subpopulations of glia, including OECs. BLBP expression is induced in radial glia via Notch signaling, but it is not known if these same mechanisms regulate BLBP expression in the adult CNS. Axonal-glial signaling is a dynamic process whereby closely apposed neuronal and glial cells regulate the growth, maintenance and plasticity of one another through direct cell-cell signaling. Delta/Notch-like EGF-related receptor (DNER) is a transmembrane protein expressed by Purkinje cells which has been implicated in the regulation of BLBP in Bergmann glia during cerebellum development through Notch1 deltex-dependent non-canonical signaling. We have found that DNER is expressed in more mature ORNs, and other exclusive subpopulations of cells within the CNS. OECs in close apposition with DNER-expressing ORNs in vivo appear to maintain the highest BLBP expression found in the nervous system through development and adulthood. Immunofluorescence shows that this close relationship between BLBP expressing cells and DNER expressing cells also appears to be retained in specialized areas such as the hippocampus, retina and spinal cord, throughout mouse CNS development as well as in the mature system. Removing DNER or axonal input in vivo decreases the robustness of OEC BLBP expression, and the number of cells in OEC culture expressing BLBP decreases rapidly with time. OEC co-culture with a DNER expressing monolayer increases the number of OECs in vitro which express BLBP, providing evidence for the regulation of BLBP expression in OECs by DNER expression in apposing ORNs.

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

Du nematode a l'homme, les recepteurs transmembranaires de la famille notch (n) agissent tout au long du developpement embryonnaire et post-embryonnaire pour controler l'acquisition et le maintien d'un etat differencie. Nous avons cherche a comprendre les mecanismes moleculaires sous-jacents a la transduction du signal notch chez drosophila melanogaster. Nous avons montre que le gene suppressor of hairless su(h) joue un role essentiel dans la voie de signalisation notch. En effet, in vivo su(h) active de maniere directe l'expression des genes du complexe enhancer of split-m5 et m8 en reponse a l'activation du recepteur n lors de la formation du systeme nerveux central et peripherique. Puis, nous avons teste l'hypothese selon laquelle le recepteur active par son ligand subit une coupure de n dans sa partie sous-membranaire, liberant ainsi la partie intracellulaire de n (n#i#n#t#r#a). N#i#n#t#r#a passerait dans le noyau ou il se comporterait comme un activateur de la transcription, une fois ancree a l'adn par une interaction avec une proteine su(h). Nous avons montre que le domaine intracellulaire du recepteur n est un co-facteur nucleaire de su(h), et agit comme un activateur de la transcription in vivo. Nous avons egalement observe in vivo un evenement de coupure proteolytique intracellulaire d'un recepteur transmembranaire n active. Puis, nous avons etudie le controle negatif de voie notch par la proteine hairless (h). Nous avons montre que la proteine h inhibe l'activite transcriptionnelle de su(h), en empechant cette derniere de reconnaitre son site de fixation a l'adn par des interactions proteine-proteine directes in vitro. Finalement, nous avons etudie la fonction de la proteine h au cours de l'embryogenese precoce. Nous avons montre que bien que le gene h soit fortement exprime dans l'embryon precoce, de maniere surprenante la proteine h n'est pas requis au cours de l'embryogenese. Cependant l'activite h est requise dans la transduction du signal notch chez l'adulte.

Proper central nervous system development is critical for survival and depends on complex intracellular and extracellular signaling to regulate neural progenitor cell growth and differentiation; however, the mechanisms that mediate molecular crosstalk between pathways during neurogenesis are not fully understood. Here, we explored the integration of the Hedgehog (Hh) signaling pathway with the two critical developmental pathways, Receptor Tyrosine Kinase (RTK) and Notch signaling, in the growth and maintenance of neural progenitors in the developing neuroretina. We found combined and sustained RTK and Hh signaling was sufficient to establish long-term retinal progenitor cell (RPC) cultures and these cells maintained neurogenic and gliogenic, but not retinogenic, competence in vitro and in vivo. In addition, we identified crosstalk between Notch and Hh signaling, where Notch is required for Hh-mediated proliferation and Gli protein accumulation, and gain-of-function of Notch is sufficient to extend the window of Hh responsiveness in a subset of Müller glia. Both Hh-RPC monolayer establishment and Notch mediated Hh-responsiveness required Gli2. Taken together, we identified molecular cross-communication between the Hh pathway and two major pathways, Notch and RTK, during retinogenesis, advancing our understanding of mechanisms that influence Hh to control neural progenitor growth.

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Fundação Oswaldo Cruz.Instituto Oswaldo Cruz. Rio de janeiro, RJ, Brasil
A Leishmaniose Tegumentar Americana (LTA) é uma doença que se caracteriza pela presença de lesões cultâneas (LCL) e, em um número pequeno de pacientes, pelo desenvolvimento de lesão mucosa (ML). A evolução clínica e a resposta ao tratamento dependem de múltiplos fatores, incluindo o equilíbrio entre as citocinas Th1 e Th2, influenciando no controle parasitário e na extensão da lesão. Nesse equilíbrio complexo é possível que outras citocinas inflamatórias como IL-17 e IL-18, bem como fatores que influenciem na diferenciação de queratinócitos e nos processos inflamatórios da pele, como o sistema Notch, também possam se relacionar ao prognóstico das lesões. IL-18 é uma potente citocina indutora de IFN-γ, já tendo sido observado efeito protetor em doenças por protozoários. IL-17 é produzida por uma linhagem de células T descritas mais recentemente, que são consideradas essenciais em processos inflamatórios de vários tecidos e em doenças auto-imunes. O sistema de sinalização Notch é composto por um grupo de proteínas transmembrana que regulam processos decisórios em diversos tecidos, tanto no período embrionário como na vida adulta, incluindo a diferenciação de queratinócitos e de linfócitos T, na dependência do ligante envolvido em sua ativação (Delta-like – DLL1/DLL3/DLL4 ou Jagged 1 e 2). Estudamos a expressão das proteínas do sistema Notch e das citocinas IL-17 e IL-18 em lesões e sangue de pacientes com LTA causada por Leishmania (Viannia) braz possa estar associado com reação inflamatória mais intensa e retardo na resolução das lesões. Nas lesões mucosas foi observada maior expressão do receptor Notch3 e dos ligantes Delta-like (DLL1 e DLL4). O sistema Notch está associado à homeostase da pele regulando a diferenciação de queratinócitos e a cicratização. Nas células do sistema imune, quando linfócitos T CD2+ expressando Notch são ativados por células apresentadoras de antígenos expressando ligantes Delta-like, uma resposta Th1 ou Th17 é gerada. Nesse sentido, o papel do sistema Notch nas lesões de LTA pode ser associado tanto à homeostase do tecido cutâneo quanto ao sistema imune ou a ambos, além de poder estar correlacionado ao aumento de IL-17 observado no mesmo grupo de pacientes. A alta expressão de receptores Notch, ligantes Delta-like e das citocinas inflamatórias IL-17 e IL-18 sugere que o aumento desses fatores pode indicar pacientes para os quais esteja indicado um esquema terapêutico mais agressivo, ou pelo menos, um acompanhamento mais rigoroso

Glioma therapy relies on induction of cytotoxicity; however, the current combination of surgery, irradiation (IR) and temozolomide (TMZ) treatment does not result in a long-term cure. Our lab previously demonstrated that a small population of glioma cells enters a transient cell cycle arrest in response to chemotherapy. Treatment with TMZ significantly decreases initial neurosphere formation; however, after a short recovery period, a small number of cells resume neurosphere formation and repopulate the culture. This recovery of neurosphere growth recapitulates the inevitable glioma recurrence in the clinic. The focus of our laboratory is to study direct-target therapies that can be combined with TMZ to inhibit neurosphere recovery. The Notch pathway is a promising target because it is involved in cell growth and survival. Here, we demonstrate that blocking the Notch pathway using gamma-secretase inhibitors (GSIs) enhances TMZ treatment. The combination of TMZ and GSI treatments targets the cells capable of recovery. TMZ + GSI treated cells do not recover and are no longer capable of self-renewal. Interestingly, recovery is inhibited when the GSI is administered 24 hrs after TMZ treatment, demonstrating a sequence-dependent mechanism. TMZ + GSI treatment also decreases tumorigenicity. When glioma cell lines were treated in vitro and implanted in NU/NU nude mice, TMZ + GSI treatment extended latency and greatly increased survival. In addition, in vivo TMZ + GSI treatment completely blocked tumor progression and resulted in the loss of a palpable tumor in 50% of mice, while none of the TMZ-only treated mice survived. TMZ + GSI treated cultures and xenografts display a senescent phenotype. Cultures treated with TMZ + GSI have decreased proliferation, but no increase in cell death. We observed an increase in the number of cells expressing senescence-associated β-galactosidase in vitro and in vivo. This demonstrates that inhibition of the Notch pathway shifts TMZ-treated cells from a transient cell cycle arrest into a permanent senescent state. Senescent cells can stimulate the innate immune system. Here we demonstrate that TMZ + GSI treatment increases phagocytosis in vitro. New therapy combinations, such as TMZ + GSI, are arising in the field of therapy-induced senescence (TIS). Overall, this data demonstrates the importance of the Notch pathway in chemoprotection and maintenance of TMZ-treated gliomas. The addition of GSIs to current treatments is a promising target-directed therapy to decrease the rate of brain tumor recurrence by inducing senescence and tumor clearance.

With the dawn of metazoans, the ability of cells to communicate with each other became of paramount importance in maintaining tissue homeostasis. The transforming growth factor β (TGF-β) signaling pathway, which plays important roles during embryogenesis and in the adult organism, signals via a heterodimeric receptor complex consisting of two type II and two type I receptors. After receptor activation through ligand binding, Smads mediate the signal from the receptor complex to the nucleus, where they orchestrate transcription. Depending on the context of activation, TGF-β can mediate a plethora of cellular responses, including proliferation, growth arrest, apoptosis and differentiation. In cancer, TGF-β can act as both as a tumor suppressor and promoter. During early stages of tumorigenesis, TGF-β prevents proliferation. However, TGF-β is also known to promote tumor progression during later stages of the disease, where it can induce differentiation of cancer cells towards a migratory phenotype. The aim of this thesis was to investigate how cells can differentiate their response upon TGF-β pathway activation. The first paper describes the role of Notch signaling in TGF-β induced growth arrest, demonstrating that TGF-β promotes Notch activity and that Notch signaling is required for prolonged TGF-β induced cell cycle arrest. In the second and third paper, we investigate the role of SIK, a member of the AMPK family of kinases, mediating signaling strength of TGF-β through degradation of the TGF-β type I receptor ALK5. While the second paper focuses on the effect of SIK on ALK5 stability and subsequent alterations in TGF-β signaling, the third paper emphasizes cooperation between SIK, Smad7 and the E3 ligase Smurf in degradation of ALK5. Finally, the fourth paper explores a novel role of SIK during TGF-β induced epithelial to mesenchymal transition (EMT). SIK binds to and degrades the polarity protein Par3, leading to enhanced EMT.

Bien que le rôle du P1GF soit encore débattu, des études ont mis en évidence le rôle du P1GF et de son récepteur, le VEGFR-1 dans la croissance tumorale et l'angiogenèse tumorale. Une étude récente a démontré q'un anticorps monoclonal anti-P1GF (aP1GF) inhibe la croissance tumorale et l'angiogenèse tumorale, y compris dans les modèles résistants aux anti-VEGF. La néo-vascularisation tumorale étant caractérisée par des vaisseaux anormaux, nous avons émis l'hypothèse que l'axe P1GFNEGFR-1 pouvait jouer un rôle dans le remodelage vasculaire anormal et que la signalisation du VEGFR-1 pouvait induire in vitro D114 et la forme active de Notch4 dans les HUVECs Notre première étude a montré, dans un modèle de CHC murin, (i) que les taux de P1GF et de VEGFR-1 étaient significativement supérieurs à ceux du VEGF-A et du VEGFR-2, aux stades précoces du CHC coïncidant avec l'initiation des anomalies vasculaires, (ii) que seules les CEs sinusoïdales tumorales expriment le VEGFR-1 et Notch4 et (iii) que la signalisation du VEGFR-1 par le P1GF et le VEGF-A induit l'expression de D114 et de Notch4 activé dans les HUVECs. Dans notre seconde étude, en collaboration avec Peter Carmeliet, nous avons montré dans 2 modèles différents de CHC que le blocage du P1GF par siRNA ou aP1GF inhibant la croissance tumorale et les anomalies vasculaires décrits dans le CHC à savoir l'artérialisation et la capillarisation des sinusoïdes. Dans notre troisième étude, nous avons montré qu'une statine diminuait la croissance tumorale et prolongeait la survie des animaux en diminuant l'angiogenèse et les anomalies vasculaires du CHC. En conclusion, nous mettons en évidence le rôle prépondérant de l'axe P1GFNEGFR-1 dans l'initiation des anoamlies vasculaires du CHC et le rôle de la signalisation du VEGFR-1 dans l'induction de D114 et de Notch4 activé dans les HUVECs
Even if the role of P1GF is still debated, studies have highlighted the role of P1GF and its receptor, VEGFR-1, in tumor growth and angiogenesis. A recent study has shown that a monoclonal antibody, anti-P1GF, inhibits tumor growth and angiogenesis, including models resistant to anti-VEGF treatments. Since tumor neovascularization is characterized by abnormal blood vessels, we hypothesized that the P1GFNEGFR-1 axis could play a role in abnormal vascular remodelling, and that VEGFR-1 signaling could induce D114 and the active form of Notch4 in HUVECs in vitro. Chu- first study carried out in a mutine model of HCC showed that: (i) the level of P1GF and VEGFR-1 were significantly higher than those of VEGF-A and VEGFR-2 at early stages of HCC coincided with the initiation of vascular anomalies, (ii) only the sinusoidal tumor endothelial cells express VEGFR-1 and Notch4 and (iii) VEGFR-1 signaling by P1GF and VEGF-A induces the expression of D114 and the active form of Notch4 in HUVECs. In our second study performed in collaboration with Peter Carmeliet, we have shown in two different models of HCC, that P1GF blockade, using siRNA or anti-P1GF antibodies, inhibited tumor growth and vascular anomalies described in HCC namely arterialization and capillarization of sinusoids. In our third study, we showed that statins decreased tumor growth and prolonged survival of animais by decreasing angiogenesis and abnormal vasculature in HCC. In conclusion, our results evidence the role of P1GFNEGFR-1 axis in the initiation of vascular abnormalities in HCC and the role of VEGFR-1 signaling in the induction of D114 and activated Notch4 in HUVECs

Le stress oxydant joue un rôle majeur dans le déclenchement et le développement de la sclérodermie systémique (ScS). Nous avons mis au point un modèle murin où la maladie est déclenchée par divers types de stress oxydant, puis nous avons exploré les différentes voies d'activation des fibroblastes sous l'effet des formes réactives de l'oxygène, afin de déterminer d'éventuelles cibles thérapeutiques. Pour apprécier les effets d’un stress oxydant chronique, des solutions contenant différents oxydants ont été injectées dans la peau de souris BALB/c et BALB/c SCID. Les solutions contenant le radical hydroxyl OH° ou HOCl ont induit une maladie caractérisée, comme la ScS diffuse, par une fibrose cutanée et viscérale, et des auto-anticorps anti-ADN topoisomérase-1. Les sérums de ces souris contenaient de grandes quantités de dérivés oxydés des protéines et induisaient la prolifération des fibroblastes et la production de formes réactives de l’oxygène par les cellules endothéliales. Une fibrose pulmonaire de moindre importance était induite chez les souris BALB/c SCID. Grâce à ce nouveau modèle murin de SSc, nous avons démontré que le stress oxydant était directement responsable des anomalies observées dans les fibroblastes, les cellules endothéliales et le système immunitaire. Nous avons ensuite utilisé ce modèle pour analyser les voies d’activation fibroblastique dans la ScS. Dans les fibroblastes des souris exposées à HOCl, on observe une dérégulation des voies des récepteurs Notch, des récepteurs aux cannabinoïdes, et des récepteurs au PDGF. On observe les mêmes dérégulations ex vivo dans les fibroblastes de patients atteints de SSc diffuse. Nous avons ainsi observé une amélioration clinique significative chez les souris sclérodermiques traitées avec un inhibiteur de l’activation de Notch, avec un agoniste des récepteurs aux cannabinoïdes, et avec des inhibiteurs de tyrosine-kinase ciblant le récepteur au PDGF. Puisque les fibroblastes sclérodermiques ont un phénotype activé et produisent de forts taux de formes réactives de l’oxygène, nous avons enfin mis à profit cette particularité pour induire l’apoptose sélective de ces cellules dans le derme des souris. Le trioxyde d’arsenic, molécule cytotoxique utilisée en thérapeutique humaine, augmente la production cellulaire de formes réactives de l’oxygène au-delà d’un seuil létal et induit ainsi l’apoptose des fibroblastes sclérodermiques. L’utilisation in vivo de cette molécule dans notre modèle murin prévient la fibrose cutanée et viscérale, et les anomalies endothéliales. Le trioxyde d’arsenic a un effet comparable dans le modèle murin de ScS associée à la réaction du greffon contre l’hôte en détruisant les lymphocytes T CD4+ alloréactifs activés et les cellules dendritiques plasmacytoïdes responsables de l’activation du système immunitaire. Les formes réactives de l’oxygène sont donc impliquées dans l’induction des lésions observées au cours de la ScS. Dans notre modèle, le rôle du système immunitaire intervient dans l'auto-entretien et l’extension systémique de la maladie. Le stress oxydant contribue à la dérégulation de diverses voies de signalisation dont les voies des récepteurs Notch, des récepteurs aux cannabinoïdes et du PDGF dans les fibroblastes. La modulation de ces voies permet d’obtenir une amélioration clinique chez les souris sclérodermiques, tout comme l’utilisation du trioxyde d’arsenic qui entraîne la délétion spécifique des fibroblastes sclérodermiques surpoduisant des formes réactives de l’oxygène. Le trioxyde d’arsenic montre également une efficacité intéressante dans le modèle de sclérodermie associée à la maladie du greffon contre l’hôte via la délétion des lymphocytes T CD4+ alloréactifs
We defend the thesis that the oxidative stress plays a major role in the initiation and the development of systemic sclerosis. To demonstrate this thesis, we designed an original mouse model: BALB/c and BALB/SCID mice were injected intra-dermally with prooxidative agents, bleomycin or PBS for 6 weeks. Hypochlorite and hydroxyl radicals induced cutaneous and lung fibrosis in BALB/c mice, in association with anti-DNA topoisomerase-1 auto-antibodies that characterize human diffuse systemic sclerosis. Pulmonary fibrosis was less extensive in BALB/c SCID mice submitted to the same protocol. In this model of HOCl-induced systemic sclerosis, cutaneous fibroblasts display a hyperactivated phenotype that prompted us to investigate several pathways of cellular activation. The NOTCH pathway and the PGDF-receptor pathways were found upregulated in the skin of HOCl-mice. DAPT (a gamma secretase inhibitor that prevents NOTCH cleavage), Sunitinib (an inhibitor of PGDF-receptor phosphorylation), and WIN-55,212, an agonist of the cannabinoid receptors 1 and 2, dramatically improved the clinical, histological and biological signs of systemic sclerosis in the HOCl model.In our model as in patients with SSc, activated fibroblasts produce reactive oxygen species that exert an autocrine effect on their own proliferation and collagen synthesis. By analogy with tumor cells that undergo apoptosis upon cytotoxic treatment that triggers an oxidative stress beyond a lethal threshold, we showed that activated fibroblasts can be selectively killed by the cytotoxic molecule arsenic trioxide (As2O3) that generates intracellular ROS. In the mouse model of sclerodermatous-graft versus host disease (Scl-GVHD), daily intra-peritoneal injections of As2O3 abrogated the clinical symptoms (diarrhea, alopecia, vasculitis, fibrosis of the skin and visceral organs) and specifically induced the apoptosis of activated CD4+ T cells and plasmacytoid dendritic cells. Those data provide a rationale for the evaluation of As2O3 in the management of patients affected by systemic sclerosis or chronic GVHD

Notch receptors and ligands are single-pass transmembrane proteins which play important roles in cell-cell communication. Notch in ‘harmony’ with other signaling pathways regulate the entire diversity of metazoan life (Artavanis-Tsakonas & Muskavitch, 2010). These signaling pathways also play key roles in regulatingseveral developmental processes. Given the importance of Notch signaling in various developmental decisions, it is not surprising that aberrant gain or loss-of-function of Notch pathway leads to several human diseases including cancer (Ranganathan et al, 2011). Notch signaling has also been implicated in various human cancers, most notably in T-cell acute lymphoblastic leukemia (T-ALL) (Weng et al, 2004). In view of the importance of Notch signaling in cancers, therapeutic molecules targeting this pathway are making their way into clinical trials (Rizzo et al, 2008). This underscores the importance of understanding the mechanism of Notch receptor activation in normal and patho-physiological conditions. In this thesis, antibodies against different domains of human Notch1 receptor have been used as tools to understand the mechanism of receptor activation. This work has provided insights into the role of Notch1 extracellular domain in ligand-dependent receptor activation. Further, the mechanism of ligand-independent receptor activation in T-ALL associated mutant Notch1 has also been investigated. This understanding of ligand-dependent and independent receptor activation facilitated development of mechanistic inhibitors of Notch signaling for therapeutic targeting of the cancer stem cells (CSCs) across the pectrum of cancers. The thesis is divived into two parts. Part-I focuses on understanding the role of Notch1 extracellular domain in receptor-ligand interactions using antibodies as a tool. In part-II, implications of these antibodies in therapeutic targeting of CSCs has been investigated. Part-I Unfolding the Mechanism of Notch1 Receptor Activation The extracellular domain of Notch1 receptor consists of 36 EGF-like repeats that contribute to ligand binding (Kopan & Ilagan, 2009). Despite extensive studies on the downstream consequences of Notch signaling, the initial events of ligandreceptor interactions have not been clearly elucidated. In the absence of structural insights into the receptor-ligand interactions, it was important to decipher the roles of various receptor domains in ligand-binding and consequent signaling. In this study, antibodies have been employed as tools for in-depth analyses of Notch receptorligand, interactions. Studies in Drosophila Notch receptor suggest that EGF-like repeats 11-12 are necessary and sufficient for ligand binding (Rebay et al, 1991). However, the role of these repeats in human Notch1 receptor-ligand interaction(s) was not clearly elucidated. Antibodies were generated against Notch1 EGF-like repeats 11-15. Further, these antibodies were characterized for their specificity for Notch1 receptor in various ligand-binding and signaling assays. The results suggest that the monoclonal antibodies (MAbs) against EGF-like repeats 11-12 were more potent inhibitors of ligand-binding compared to the antibodies against EGF-like repeats 13-15. As a part of these investigations, the Notch ligands Jagged1 and Jagged2, Delta-like1 and Delta-like4 were purified and characterized in various assays. Ability of these ligands to interact with Notch1 EGF-like repeat 11-15 was determined using Surface Plasmon Resonance. The Jagged family of ligands demonstrated higher affinity for this recept or fragment when compared to the Delta family of ligands. The relatively low affinities (μM) of all the ligands suggested possibile involvement of other EGF-like repeats in ligand-binding. This was further investigated using antibodies against other EGF-like repeats of Notch1. In Drosophila Notch EGF-like repeats 24-29 have been implicated in the ligand-dependent gain-of-function phenotype, suggesting a plausible involvement of this region in receptor activation (Pei & Baker, 2008). Therefore, role of human Notch1 EGF-like repeats 21-30 in ligand-binding and signaling was investigated. These EGF-like repeats demonstrated specific interaction with the ligand-binding domain (EGF-like repeats 11-15). This suggested that in the absence of the ligand, these inter-domain interactions keep the receptor in an auto-inhibited conformation. Further, ligand binding to EGF-like repeats 11-15 dissociated pre-formed interdomain interactions. These results suggested that, the binding of ligand to EGF-like repeat 11-12 overcomes the negative constraint imposed by the intra-domain interactions which might lead to receptor activation. Next, to understand the role of EGF-like repeats 21-30 in ligand binding, polyclonal antibodies were generated against the same and extensively characterized in various solid-phase and cell-based assays. These antibodies demonstrated partial inhibition of ligand-binding. Further, using immunoaffinity purified antibodies it was demonstrated that antibodies against EGF-like repeats 25-26 were most potent inhibitors of ligand-binding compared to antibodies against EGF-like repeats 21-24 and 27-30. These results provided novel insights into Notch1 receptor activation. The model proposed on the basis of these results suggested that ligand-binding to EGF-like repeats 11-12 competes with the inter-domain interaction, in turn dissociating EGF-like repeats 21-30 from the ligandbinding domain. It emerged that this altered conformation of the receptor creates a secondary ligand-binding site at EFG-like repeats 25-26. Overall these results provided novel insight into the mechanism of Notch receptor-ligand interaction(s). Part-II Implication in Cancer Stem Cell Targeting Recent studies have suggested existence of the CSC population in various cancers (Clevers, 2011). Notch signaling plays an important role in maintenance of these CSCs (Pannuti et al, 2010). Thus, targeting Notch signaling may provide a potential therapeutic tool for CSC targeting. Several studies have indicated that Notch1 receptor and ligands are overexpressed in breast cancer cells compared to the normal breast epithelium (Mittal et al, 2009; Reedijk et al, 2005; Reedijk et al, 2008). Moreover, it has been suggested that Notch1 signaling plays a key role in breast carcinogenesis (Stylianou et al, 2006). Monoclonal antibodies (MAbs) were used as mechanistic inhibitors of aberrant Notch1 signaling for therapeutic targeting of CSCs. One such antibody, MAb 602.101, against Notch1 ligand-binding domain (EGF-like repeat 11-12) inhibited proliferation and depleted breast CSCs. This MAb also modulated genes associated with stemness and epithelial to mesenchymal transition (EMT). Furthermore, MAb 602.101 irreversibly inhibited the sphere-forming potential of breast cancer cells by modulating long-term self renewing capacity of breast CSCs. Inhibition of Notch1 signaling by the MAb also depleted the chemoresistant CD44Hi/CD24Low sub-population in breast cancer cells. Interestingly, antibody treatment led to elevated expression of genes associated with myoepithelial lineage, which suggested that inhibition of Notch1 signaling might induce a differentiation program leading to reduction in the CSC population. This study demonstrated the importance of Notch1 signaling in CSCs and effectiveness of antibodies as a tool for specific targeting of individual Notch receptors in cancer therapeutics. While aberrant expression of receptors and ligands leads to breast cancer (Reedijk et al, 2005), gain-of-function mutations are associated with 40-50% of TALL\ patients (Weng et al, 2004). These mutations lead to ligand-independent receptor activation (Malecki et al, 2006). Despite several attempts of successful antibodymediated therapeutic targeting of Notch1 (Aste-Amézaga et al, 2010; Wu et al, 2010), specific antibodies recognizing T-ALL associated mutant Notch1 remains elusive. Using homology modeling, the mutation induced conformational change in T-ALL associated mutant Notch1 was predicted. These results suggested that mutation led to conformational changes in the Notch1 negative regulatory region (NRR) This conformation change might result in the constitutive activation of Notch1 signaling leading to pathogenesis. Next, MAbs were generated against the wild-type Notch1 NRR and characterized in flow-cytometry based assays for identification of conformation specific antibodies. These antibodies were classified as either wild-type specific, mutant specific or unbiased to receptor conformations. One such mutant specific MAb 604.107 demonstrated higher binding to mutant Notch1 in flowcytometer and SPR based experiments. This MAb also demonstrated specific inhibition of T-ALL associated mutant Notch1 signaling without affecting the wildtype signaling. Moreover, antibody treatment also inhibited proliferation and depleted leukemia initiating sub-population in patient derived T-ALL cells. Taken together, this study provides a novel tool for specific targeting of mutant Notch1 receptors in TALL. CSCs are inherently chemo-resistant and lead to tumor relapse (Chen et al, 2012). Recent studies have demonstrated a strong correlation between Notch1 signaling in lung CSCs and chemotherapy resistance (Hassan et al, 2013). In this study, Notch1 heterogeneity in solid tumors viz. breast and colon cancers was investigated. Using the antibodies generated previously in this study, Notch1High and Notch1Low sub-populations from MDA-MB-231 (breast cancer) and HCT-116 (colon cancer) cell lines were flow-sorted. It was demonstrated that the Notch1High subpopulation represented the sphere-forming CSCs in breast and colon cancer. The Notch1High sub-population also demonstrated chemo-resistant properties and expressed higher level of EMT and stemness markers. These results suggested explicit involvement of Notch1 signaling in EMT and maintenance of CSCs subpopulation in these cancers. The anti-Notch1 MAb also inhibited proliferation of the chemo-resistant Notch1High sub-population. Further, treatment with MAb inhibited expression of ABCC1 transporters in these drug-resistant cells leading to augmentation of chemotherapeutic response. Using mouse xenograft assays, it was demonstrated that Notch1 signaling plays an important role in the maintenacne of tumor-initiating sub-population in breast and colon cancer cells. Prior exposure of breast and colon cancer cells to MAb inhibited the tumor forming potential of these cells in xenotransplantation assays. Treatment with MAb alone or in combination with chemotherapy led to regression of pre-formed tumors in breast and colon xenograft models. These results demonstrated existence of Notch1 heterogeneity in breast and colon cancer cells and emphasised the importance of targeting Notch1 signaling to overcome drug-resistance in these cancers. The results described above have provided important insights into Notch1 receptor activation and this understanding was translated into therapeutic targeting of CSCs. This “proof-of-principle” demonstration has significant mechanistic and applied implications in Notch and cancer biology.