O linfoma canino é uma das neoplasias mais prevalentes em cães, diagnosticado frequentemente através de exame citológico de aspirados. A imunofenotipagem para diferenciação entre linfomas de células B e T é importante para definição prognóstica e tratamento, entretanto, dificilmente é realizada em materiais provenientes de aspirados citológicos. A citologia em meio líquido (CML) é uma metodologia automatizada de produção de esfregaços citológicos, que permite a conservação de todo o material da aspiração, e utilização do material residual para, por exemplo, produção de emblocado celular (EC) e imunocitoquímica. Este trabalho teve como objetivo aplicar a CML e EC com imunocitoquímica em amostras de linfonodos caninos suspeitos para linfoma. Além disso, pretendeu caracterizar morfologicamente as células linfoides neste tipo de preparado, comparando ao esfregaço convencional. Para isto, foram selecionados 54 cães com linfadenomegalia, 10 deles sorologicamente positivos para leishmaniose canina. Comparou-se a CML com a citologia convencional, quanto às características técnicas e testou-se dois métodos diferentes de EC: Bouin e agarose com formalina. Aplicou-se painel imunocitoquímico de anticorpos anti-CD3, anti-CD79a, anti-Pax-5 e anti-Ki67, para imunofenotipagem e determinação da proliferação celular. Adicionalmente, realizaram-se análises estatísticas para avaliação do desempenho e concordância interobservador comparada entre citologia convencional, CML e o uso conjunto de CML e imunocitoquímica do emblocado celular. Como resultado, as células linfoides apresentaram-se preservadas, com melhor definição de cromatina e nucléolos, porém com tamanho reduzido e pior definição citoplasmática, na CML. O EC de Bouin revelou grupos densos e bem definidos, que permitiram a aplicação da imunocitoquímica. Por outro lado, os emblocados de agarose, apresentaram-se frouxos, com células marcadamente dispersas, revelando-se inadequados para preparados de células linfoides. O anticorpo anti-Pax-5 mostrou-se mais adequado do que o anti-CD79a para marcação de células B em emblocados, por produzir menos fundo e pela marcação nuclear ser mais distinguível do que a citoplasmática. A concordância interobservadores da CML foi moderada (k= 0,434), enquanto a da citologia convencional foi boa (k=0,762). Ambas as técnicas exibiram a mesma acurácia e, com aplicação da CML, houve redução do percentual de amostras insatisfatórias. A CML em conjunto com EC e imunocitoquímica apresentou maior sensibilidade (75%), especificidade (99%) e acurácia (89,47%). Como conclusão, a CML em conjunto com EC e imunocitoquímica pode ser aplicada para diagnóstico de linfoma canino, com maior sensibilidade, especificidade e acurácia / Canine lymphoma is one of the most commonly treated neoplasia in dogs, frequently diagnosed through cytological smears. Differentiating B lymphomas from T lymphomas is important for prognosis and treatment, however, immunophenotyping is rarely applied to cytological preparations. Liquid-based cytology (LBC) is an automated method for producing cytological smears, allowing preservation of the residual material for other analysis, as cell block (CB) immunocytochemistry. The aim of this study was to apply LBC and cell block immunocytochemistry in the diagnosis of lymphoid samples from dogs suspected of lymphoma. Besides, we intended to characterize the lymphoid cells regarding to its morphology, comparing to conventional cytology. To accomplish these goals, 54 dogs were selected, being 10 of them with canine leishmaniasis. LBC smears were compared to conventional smears, with production of two types of CB: bouin and formalin with agarose. Also, an immunocytochemistry panel with anti-CD3, anti-CD79a, anti-Pax-5 and anti-Ki-67 was applied, for immunophenotyping and cellular proliferation determination. As results, lymphoid cells were well preserved, with better nuclear definition, but smaller size and worst cytoplasmic definition, in LBC. Bouin CBs revealed cohesive groups, which allowed immunocytochemistry. In the other hand, agarose CBs were loose, with disperse cells, inadequate for lymphoid aspirates. Anti-Pax-5 antibody was more adequate than anti-CD79a, once it produced less background and the nuclear marking was more distinct than cytoplasmic. CML interrater agreement was moderate (k=0,434), while conventional cytology agreement was good (k=0,762). Both techniques exhibited the same accuracy, and, with CML, there was reduction of unsatisfactory results. LBC with cell block immunocytochemistry presented the higher sensitivity (75,00%), specificity (99,99%) and accuracy (89,47%). As conclusion, LBC with immunocytochemistry CB can be applied for canine lymphoma diagnosis, with higher sensitivity, specificity and accuracy

O linfoma folicular é entidade clinicamente heterogênea, com carência de marcadores prognósticos que estratifiquem grupos de risco para otimização do manejo. É relativamente raro em pacientes abaixo de 40 anos. Os aspectos clínicos e patológicos desse tipo de linfoma, nessa faixa etária, assim como o comportamento biológico, são pouco conhecidos. No presente estudo, uma série de 208 pacientes entre 19-40 anos de idade foi, retrospectivamente, avaliada quanto aos achados anatomoclínicos e moleculares. Essas variáveis foram, então, correlacionadas com seguimento e sobrevida. A mediana de idade na apresentação foi de 35 anos, com leve predomínio no sexo feminino (56%). A maioria dos casos se manifestou como doença nodal (87%). Concomitância de linfoma folicular com linfoma difuso de grandes células B foi encontrada em 7 (3%) pacientes. Estudos imuno-histoquímicos revelaram expressão de CD10 (91%), BCL6 (97%), BCL2 (95%), MUM1/IRF4 (17%) e CD23 (25%). Rearranjos envolvendo os genes BCL2 e BCL6 foram encontrados em 74% e 20%, respectivamente. A sobrevida média geral estimada dos pacientes, em que foi possível o seguimento, foi de 13 anos. Presença de anemia, elevação da desidrogenase lática, acometimento de medula óssea, índice prognóstico internacional do linfoma folicular na faixa de alto risco, padrão de \"céu estrelado\", Ki-67 >= 50% e ausência do rearranjo do gene BCL2 e presença do BCL6 relacionaram-se diretamente com pior sobrevida geral. O estadiamento de Ann Arbor III/IV e MDM2 >=20% têm fortes indícios desta associação negativa com sobrevida geral. A combinação BCL2+ e BCL6- correlacionou-se com maior sobrevida média. O grau histológico determinou redução gradual da sobrevida, com semelhanças nas curvas de sobrevida entre os graus 1, 2 e 3A. Não houve diferença significativa para as curvas de sobrevida relacionando faixa etária, persistência da zona do manto, presença de áreas difusas, fibrose, expressão de CD10, BCL6 ou CD23, padrão de trama de células foliculares dendríticas ou clonalidade para cadeia leve de imunoglobulina. Esses achados revelaram que o linfoma folicular em adultos jovens apresenta similaridades com o linfoma folicular que ocorre em adultos mais velhos, incluindo a frequência de apresentação nos diversos sítios anatômicos, grau histológico e fatores prognósticos adversos / Follicular lymphoma is a clinically heterogeneous group of disease and therefore with a need of characterization of prognostic markers to stratify risk groups and to optimize clinical management. It is relatively rare in patients younger than 40 years, and the clinicopathologic characteristics and biological behavior in this age group are poorly understood. In the current study, samples from a cohort of 208 patients between 19-40 years of age were evaluated retrospectively with respect to clinical, histologic and molecular characteristics. These findings were then correlated with the follow up and the clinical outcome. The median age at presentation was 35 years with a slight female preponderance (56%). Most of the cases presented with nodal disease (87%). Concomitant follicular lymphoma and diffuse large B-cell lymphoma was observed in 7 (3%) patients. Immunohistological studies showed the expression in the following frequency: CD10 (91%), BCL6 (97%), BCL2 (95%), MUM1/IRF4 (12%), MDM2 (17%) and CD23 (25%). BCL2 and BCL6 rearrangements were present in 74%, and 20%, respectively. The estimated overall survival of patients was 13 years (mean). The presence of anemia, elevated lactose dehydrogenase, bone marrow involvement, high-risk follicular lymphoma international prognostic index, \"starry sky\" appearance, proliferative index >= 50%, absence of BCL2 rearrangement and presence of BCL6 rearrangement correlated with adverse overall outcome. Ann Arbor stage III/IV and MDM2 >= 20% correlated with high trend toward worse overall survival. The combination BCL2+ and BCL6- was associated with better overall survival. No impact on overall survival was observed related to age, persistence of mantle zone, presence of diffuse areas, fibrosis, expression. of CD10, BCL6 or CD23, follicular dendritic cells meshwork or clonality. These findings revealed that follicular lymphoma in young adults demonstrate similarities with that of older adults, including the frequency of presentation at various anatomic sites, grade, and adverse prognostic factors

Aproximadamente 40% dos casos de Linfoma não Hodgkin (LNH) se originam fora dos linfonodos, sendo então denominados linfomas extralinfonodais. No trato gastrointestinal (TGI), o estômago é o local mais freqüentemente envolvido, representado pelo linfoma MALT e pelo linfoma difuso de grandes células B (LDGCB). No Brasil, apesar da sua freqüência e importância, existem poucos dados epidemiológicos em relação aos linfomas, especialmente no que se refere aos linfomas de origem extralinfonodal. Para avaliar as características dos linfomas primários gástricos em uma população brasileira, 60 casos foram avaliados retrospectivamente. Trinta e oito (63,3%) foram classificados como LDGCB e 22 (36,6%) como MALT. Entre os dois grupos, não houve diferenças significativas em termos de sexo, idade, sintomas dispépticos, sintomas B, presença de massa Bulky, infiltração de medula óssea, estádio, infecção por H. pylori, achados laboratoriais e endoscópicos. Foram adotados diferentes protocolos de tratamento. A taxa de remissão completa foi de 73,1% no LDGCB e de 95% no linfoma MALT. A taxa de sobrevida livre de doença em 7 anos foi de 84,8% no LDGCB e de 94,1% no linfoma MALT. A taxa de sobrevida global em 7 anos foi de 65,7% no LDGCB e de 92,9% em 5 anos no linfoma MALT. Como não conseguimos demonstrar diferenças entre os dois tipos histológicos, concluímos que o diagnóstico histológico correto é essencial para a terapêutica mais adequada. / Approximately 40% of the non-Hodgkins Lymphoma arises outside lymph node tissue, being then termed extranodal lymphoma. In the gastrointestinal tract, gastric is the commonest localization represented by MALT and diffuse large B-cell lymphoma (DLBCL). In Brazil, despite its importance and frequency there are very few epidemiological data concerning lymphomas, specially the extranodal ones. In order to study the primary gastric lymphoma features in a Brazilian population, 60 patients were retrospectively evaluated. Thirty eight cases (63.3%) were classified as DLBCL and 22 (36.6%) as MALT lymphoma. There were no significant differences between the 2 groups in terms of sex, age, gastric symptoms, B symptoms, Bulky disease, bone marrow infiltration, stage, H. pylori infection, laboratory and endoscopic findings. Different treatment methods were adopted. The complete remission rate was 73.1% for DLBCL and 95% for MALT lymphoma. The disease free survival in 7 years was 84.8 for DLBCL and 94.1% for MALT lymphoma. The 7 year overall survival (OS) rate for DLBCL was 65.7% and 5 year OS for MALT was 92.9%. Because we could not demonstrate differences between the two histological groups, we conclude that the correct histological diagnosis is essential for choosing the best therapeutic approach.

Malt lymphoma is a malignant disease that can arise in a variety of extra nodal sites. Previous studies indicate that tumour arise from more mature B-cells. Our purpose was to examine the presence of clonality and somatic hypermutation of immunoglobulin (IgVн) of MALT lymphomas. Paraffin-embedded tumour samples from13 MALT lymphoma were subjected to rearrangement analysis, by using PCR, heteroduplex gels and sequence analysis. Successful amplification was seen in 10/13 cases and sequences of IgVн genes were obtained in 6/13, all of them were mutated. The percentage of mutation compared to germline sequences was 1,1% to 8,6% monoclonal rearrangemang. It was demonstrated that 5 of 7 clones were derived from the Vн3 family, 2 from Vн1 and 1 from the Vн 4 family.

Peripheral T-cell non-Hodgkin lymphoma (PTCL) are heterogeneous, rare, and aggressive diseases mostly incurable with current cell cycle therapies. Aurora kinases (AKs) are key regulators of mitosis that drive PTCL proliferation. Alisertib (AK inhibitor) has a response rate similar to 30% in relapsed and refractory PTCL (SWOG1108). Since PTCL are derived from CD4(+)/CD8(+) cells, we hypothesized that Program Death Ligand-1 (PDL1) expression is essential for uncontrolled proliferation. Combination of alisertib with PI3K alpha (MLN1117) or pan-PI3K inhibition (PF-04691502) or vincristine (VCR) was highly synergistic in PTCL cells. Expression of PD-L1 relative to PD-1 is high in PTCL biopsies (similar to 9-fold higher) and cell lines. Combination of alisertib with pan-PI3K inhibition or VCR significantly reduced PD-L1, NF-kappa B expression and inhibited phosphorylation of AKT, ERK1/2 and AK with enhanced apoptosis. In a SCID PTCL xenograft mouse model, alisertib displayed high synergism with MLN1117. In a syngeneic PTCL mouse xenograft model alisertib demonstrated tumor growth inhibition (TGI) similar to 30%, whilst anti-PD-L1 therapy alone was ineffective. Alisertib + anti-PD-L1 resulted in TGI > 90% indicative of a synthetic lethal interaction. PF-04691502 + alisertib + anti-PD-L1 + VCR resulted in TGI 100%. Overall, mice tolerated the treatments well. Co-targeting AK, PI3K and PD-L1 is a rational and novel therapeutic strategy for PTCL.

Ce travail a eu pour objectif de proposer des modèles conjoints d'intensités de processus d'événements récurrents et d'un événement terminal dépendant. Nous montrons que l'analyse séparée de ces événements conduit à des biais d'estimation importants. C'est pourquoi il est nécessaire de prendre en compte les dépendances entre les différents événements d'intérêt. Nous avons choisi de modéliser ces dépendances en introduisant des effets aléatoires (ou fragilités) et de travailler sur la structure de dépendance. Ces effets aléatoires prennent en compte les dépendances entre événements, les dépendances inter-récurrences et l'hétérogénéité non-observée. Nous avons, en premier lieu, développé un modèle conjoint à fragilités pour un type d'événement récurrent et un événement terminal dépendant en introduisant deux effets aléatoires indépendants pour prendre en compte et distinguer la dépendance inter-récurrences et celle entre les risques d'événements récurrents et terminal. Ce modèle a été ajusté pour des données de patients atteints de lymphome folliculaire où les événements d'intérêt sont les rechutes et le décès. Le second modèle développé permet de modéliser conjointement deux types d'événements récurrents et un événement terminal dépendant en introduisant deux effets aléatoires corrélés et deux paramètres de flexibilités. Ce modèle s'avère adapté pour l'analyse des risques de récidives locorégionales, de récidives métastatiques et de décès chez des patientes atteintes de cancer du sein. Nous confirmons ainsi que le décès est lié aux récidives métastatiques mais pas aux récidives locorégionales tandis que les deux types de récidives sont liés. Cependant ces approches font l'hypothèse de proportionnalité des intensités conditionnellement aux fragilités, que nous allons tenter d'assouplir. Dans un troisième travail, nous proposons de modéliser un effet potentiellement dépendant du temps des covariables en utilisant des fonctions B-Splines. / This work aimed to propose joint models for recurrent events and a dependent terminal event. We show how separate analyses of these events could lead to important biases. That is why it seems necessary to take into account the dependencies between events of interest. We choose to model these dependencies through random effects (or frailties) and work on the dependence structure. These random effects account for dependencies between events, inter-dependence recurrences and unobserved heterogeneity. We first have developed a joint frailty model for one type of recurrent events and a dependent terminal event with two independent random effects to take into account and distinguish the inter-recurrence dependence and between recurrent events and terminal event. This model was applied to follicular lymphoma patient’s data where events of interest are relapses and death. The second proposed model is used to model jointly two types of recurrent events and a dependent terminal event by introducing two correlated random effects and two flexible parameters. This model is suitable for analysis of locoregional recurrences, metastatic recurrences and death for breast cancer patients. It confirms that the death is related to metastatic recurrence but not locoregional recurrence while both types of recurrences are related. However, these approaches do the assumption of proportional intensities conditionally on frailties, which we want to relax. In a third study, we propose to model potentially time-dependent regression coefficient using B-splines functions.

Les lymphomes sont constitués de sous-groupes tumoraux définis par différents critères cliniques, morphologiques, immunophénotypiques et moléculaires. Néanmoins, même au sein d’un sous-groupe, il existe une grande hétérogénéité dans l’évolution clinique des patients, ce qui rend leur prise en charge difficile. Ceci souligne l’importance de découvrir de nouveaux biomarqueurs pronostiques qui amélioreraient l’approche thérapeutique personnalisée. <p>La revue de la littérature fait apparaitre les galectines-1 et -3 comme des protéines impliquées dans le développement des cancers ainsi que dans la biologie des lymphocytes. Pourtant, au moment où nous avons débuté ce travail, peu de chercheurs s’étaient intéressés à l’implication de ces galectines dans les lymphomes.<p>Nous avons donc décidé d’évaluer le rôle diagnostique ou pronostique de la galectine-1 et de la galectine-3 dans les lymphomes.<p><p>La première étude a consisté en la caractérisation de l’expression immunohistochimique de ces deux galectines au sein d’une série clinique de lymphomes systémiques humains en comparaison au tissu lymphoïde normal. <p>En ce qui concerne la galectine-1, les cellules lymphomateuses l’expriment peu, de façon similaire à ce qui est observé pour les cellules lymphoïdes normales. Au contraire, les cellules endothéliales associées aux lymphomes sont caractérisées par une augmentation de l’expression de galectine-1 en comparaison aux cellules endothéliales normales. Cette expression endothéliale ne montre pas de relation avec les variables cliniques de notre étude mais est corrélée à la densité microvasculaire des lymphomes. <p>En ce qui concerne la galectine-3, les cellules lymphomateuses des lymphomes B diffus à grandes cellules se caractérisent par une expression plus élevée comparativement aux autres sous-groupes de lymphomes et aux cellules lymphoïdes normales. Cette expression de galectine-3 n’est pas associée aux variables cliniques dans notre étude. Contrairement à la galectine-1, la galectine-3 est exprimée de façon similaire par les cellules endothéliales des vaisseaux des tissus lymphoïdes normaux et des lymphomes.<p>Ces données suggèrent un rôle angiogénique de la galectine-1 dans les lymphomes systémiques.<p><p>Dans la deuxième étude, nous avons caractérisé l’expression des galectines-1 et -3 au sein d’une série clinique de lymphomes primitifs du système nerveux central (LPSNC). <p>Nous avons observé que les profils d’expression des cellules lymphomateuses sont similaires pour les lymphomes systémiques et les LPSNC, caractérisés par une absence d’expression de galectine-1 et une expression variable de galectine-3. <p>A l’opposé, nous avons observé une expression endothéliale de ces galectines différente entre les lymphomes systémiques et les LPSNC. La galectine-1 n’est pas exprimée par les cellules endothéliales des vaisseaux des LPSNC. Par contre, la galectine-3 est exprimée par les cellules endothéliales de certains LPSNC. Cette expression est associée à un mauvais pronostic. <p>Nous avons également démontré que l’hyperplasie endothéliocapillaire est associée à une survie diminuée. <p>Cette hyperplasie endothéliocapillaire et/ou l’expression endothéliale de galectine-3 sont des facteurs pronostiques indépendants au sein de notre série de patients immunocompétents atteints de LPSNC et traités par chimiothérapie.<p><p>Au vu des résultats des deux premières études, nous pensons que les galectines-1 et -3 jouent un rôle dans l’angiogenèse des lymphomes. Ce rôle est très certainement complexe comme l’illustre la différence d’expression endothéliale de ces galectines entre les lymphomes systémiques et les LPSNC. Ces variations d’expressions selon l’organe hôte sont à intégrer avec la notion générale d’hétérogénéité des cellules endothéliales. Cette hétérogénéité s’observe également entre les cellules endothéliales des vaisseaux normaux et les cellules endothéliales des vaisseaux dans un contexte tumoral (TAECs - tumor-associated endothelial cells). <p>Suite à ces observations, nous avons décidé d’étudier l’implication des galectines-1 et -3 extracellulaires sur deux modèles in vitro de cellules endothéliales (cellules HUVEC et EA.hy926). La revue de la littérature nous a permis de poser l’hypothèse que la lignée EA.hy926 pourrait s’apparenter aux TAECs. La première partie de cette troisième étude a pour but de valider cette hypothèse. La comparaison morphologique des lignées EA.hy926 et HUVEC cultivées sur un substrat de matrigel a permis de montrer que les tubes formés par les cellules EA.hy926 sont plus hétérogènes comme ce que l’on peut observer dans les vaisseaux tumoraux. La comparaison des profils d’expression des gènes de ces deux lignées a mis en évidence que différents gènes surexprimés dans les TAECs le sont également dans les cellules EA.hy926. Les cellules EA.hy926 présentent un profil d’expression des récepteurs au VEGF (VEGFR- vascular endothelial growth factor receptor) caractérisé par un taux élevé de VEGFR1 et un faible taux de VEGFR2 en comparaison aux cellules HUVEC. L’analyse in vivo des VEGFRs au sein des cellules endothéliales de tissus humains normaux et tumoraux nous a permis de confirmer la surexpression de VEGFR1 par les TAECs. L’ensemble de ces résultats supportent donc notre hypothèse que la lignée EA.hy926 présente des caractéristiques plus proches des TAECs en comparaison à la lignée HUVEC.<p>Nous avons ensuite, dans la deuxième partie de cette troisième étude, analysé les effets respectifs de la galectine-1, de la galectine-3 et de leur combinaison sur ces deux lignées. Nous avons observé que la galectine-1 extracellulaire dans notre modèle de cellules endothéliales normales (HUVEC) augmente la croissance cellulaire ainsi que la formation de tubes sans mise en évidence de phosphorylation significative de VEGFR1 ni de VEGFR2. Dans notre modèle de TAECs (EA.hy926), la galectine-1 extracellulaire ne favorise pas la croissance cellulaire et est faiblement angiogénique via la phosphorylation du VEGFR2, sans phosphorylation du VEGFR1. <p>En ce qui concerne la galectine-3, nous avons observé dans nos deux modèles des résultats similaires, à savoir une absence d’effet sur la croissance cellulaire et un effet positif sur la formation de tubes s’accompagnant d’une phosphorylation de VEGFR2 sans phosphorylation de VEGFR1. <p>Le résultat le plus original consiste en la mise en évidence d’un effet synergique des deux galectines sur la croissance et la formation de tubes des cellules EA.hy926. Cet effet synergique s’accompagne d’une phosphorylation de VEGFR1 et de VEGFR2. Il est important de noter que l’activation de VEGFR1 n’a été observée que pour la lignée EA.hy926 et uniquement en présence des deux galectines. <p>Dans ces conditions, nous avons démontré que les voies de signalisation activées suite à l’ajout de ces galectines impliquent la voie de l’extracellular signal regulated kinase1/2 et de l’heat shock protein 27. <p><p>En conclusion, ce travail nous a donc permis, à partir de la caractérisation de l’expression de galectine-1 et -3 dans une série de lymphomes systémiques et cérébraux, de proposer un rôle angiogénique pour ces galectines. Les études in vitro ont souligné les rôles différents que joueraient ces galectines extracellulaires dans l’angiogenèse non tumorale et tumorale et l’implication du VEGFR1 dans l’angiogenèse tumorale. <p> / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Les imidazoquinoxalines (imiqualines), molécules bioactives originales analogues chimiques de l’imiquimod et présentant un important potentiel anticancéreux, ont été explorées afin d’élucider leurs mécanismes d'action. Les molécules EAPB0203 et EAPB0503, identifiées lors d’études antérieures comme étant les « têtes de séries », ont montré un effet cytotoxique puissant in vitro sur des lignées cellulaires cancéreuses humaines de mélanome et de lymphomes T. Nous avons étudié l’effet cytotoxique de 13 imiqualines nouvellement synthétisées sur une lignée cellulaire de mélanome humain (A375). Tous les composés ont montré un effet cytotoxique important. L’effet cytotoxique a été démontré sur d’autres lignées cellulaires cancéreuses humaines (côlon, sein et lymphome T de l’adulte).Un blocage du cycle cellulaire en phase G2/M a été mis en évidence par cytométrie en flux sur des cellules A375 traitées par EAPB0203 et EAPB0503. Cet arrêt du cycle cellulaire semble être en relation avec un effet inhibiteur de la polymérisation de la tubuline. En effet, nos résultats ont montré que l’EAPB0503 et 3 autres imiqualines inhibent la polymérisation de la tubuline. L’étude de modélisation moléculaire de la liaison à de la tubuline a montré que ces composés se fixent sur le site de la colchicine.Une analyse transcriptomique sur EAPB0503 a été effectuée pour élucider le mécanisme d'action des imiqualines. Cette étude a été faite sur la lignée A375 en comparaison avec 13 anticancéreux de référence. L’étude transcriptomique a montré que EAPB0503 a une composante antitubuline tout en révélant un mécanisme d’action original. Deux hypothèses mécanistiques pour EAPB0503 ont ainsi été identifiées : 1/ altération de la voie de signalisation liée aux intégrines, 2/ altération de la voie de signalisation du récepteur TNFR et de FasL.Des analyses fonctionnelles in vitro nous ont permis d’explorer ces hypothèses. Ainsi, une altération uniquement des voies de signalisation PI3K/AKT et RAS/MAPK, toutes deux liées aux intégrines, a été observée. La première hypothèse semble donc validée. De plus, ce résultat a été confirmé par l’étude de l’expression et de la phosphorylation de ERK.Finalement, nous avons développé une formulation injectable par voie intraveineuse de EAPB0503 à base de nanocapsules. Cette formulation a d’abord été testée in vitro et in vivo sur un modèle de lymphome. Ces études ont pu mettre en évidence l’absence de toxicité des nanoparticules vides et le maintien de l’activité cytotoxique de EAPB0503 encapsulé in vitro, avec un effet immunomodulateur in vivo qui demande à être exploré. / The imidazoquinoxalines (imiqualines), original bioactive molecules and chemical analogues of imiquimod and with significant anti-cancer potential, were explored in order to elucidate their mechanisms of action. The molecules EAPB0203 and EAPB0503 identified in previous studies as leader, showed potent in vitro cytotoxic effect on human cancer cell lines of melanoma and T lymphoma. We studied the cytotoxic effect 13 newly synthesized imiqualines on a cell line of human melanoma (A375). All compounds showed a significant cytotoxic effect. The cytotoxic effect was shown on other human cancer cell lines (colon, breast and adult T lymphoma).A cell cycle block in G2 / M phase was demonstrated by flow cytometry on A375 cells treated with EAPB0203 and EAPB0503. This cell cycle arrest seems to be related with an inhibitory effect on the polymerization of tubulin. Indeed, our results showed that EAPB0503 and three other imiqualines inhibit tubulin polymerization. The molecular modeling study of binding to tubulin showed that these compounds bind at the colchicine site.A transcriptomic analysis on EAPB0503 was performed to elucidate the mechanism of action of imiqualines. This study was made on the A375 line compared with 13 approuved anticancer molecules. This transcriptomic study showed that EAPB0503 has an antitubulin effect while revealing a novel mechanism of action. Two mechanistic hypotheses for EAPB0503 were identified: 1/ alteration of the signaling pathway linked to integrin, 2/ alteration of the signaling pathway TNFR receptor and FasL.In vitro functional analyses have allowed us to explore these hypotheses. Thus, alteration only PI3K / AKT and RAS / MAPK signaling pathways, both related to integrins, was observed. The first hypothesis seems confirmed. Moreover, this result was confirmed by the study of the expression and phosphorylation of ERK.Finally, we developed an intravenously injectable formulation of EAPB0503 based on nanocapsules. This formulation was first tested in vitro and in vivo on lymphoma model. These studies could highlight the lack of toxicity of empty nanoparticles and retention of the cytotoxic activity of encapsulated EAPB0503 in vitro, with an in vivo immunomodulatory effect which needs to be explored.

Abstract Collagen XIII is a type II transmembrane protein, which has a short intracellular domain and a large, mainly collagenous ectodomain. It is located at many cell-matrix junctions and in focal adhesions in cultured cells and it has a function in cell adhesive processes. Overexpression of collagen XIII molecules with an 83 amino acid deletion in part of the ectodomain leads to fetal lethality in Col13a1del transgenic mice. Doppler ultrasonography was performed at 12.5 days of gestation on fetuses resulting from heterozygous matings and matings between heterozygous and wild-type mice. Some fetuses had atrioventricular valve regurgitation (AVVR) and all of them were transgene positive. In addition, fetuses had pathological changes in functional parameters. Histological analysis showed the trabeculation of the ventricles to be reduced and the myocardium to be thinner in the fetuses with AVVR. Based on in situ hybridization (ISH), collagen XIII mRNA are normal constituents of these structures. Overexpression of mutant collagen XIII results in mid-gestation cardiac dysfunction in fetuses, and these disturbances in cardiac function may lead to death in utero. The heterozygous mice that were initially of normal appearance had an increased susceptibility to develop B cell lymphomas, which originated in the mesenteric lymph node. Collagen XIII protein was not detected in normal lymph nodes or in the lymphomas. The incidence of lymphomas was higher in conventional conditions than in a specific pathogen-free facility. In addition, the expression of collagen XIII was localized in the intestine and the basement membrane was highly abnormal. These findings suggest that collagen XIII is a critical determinant of lymphanogenesis. Using ISH, antibody staining and RT-PCR techniques collagen XIII expression was analyzed during carcinogenesis in mice and in man. Collagen XIII expression increased during carcinogenesis in mice and in man. In the malignant process collagen XIII mRNA localized in the basal epithelium and in the invasive cells. According to antibody staining malignant invasive cells were positive. Results may reflect the disturbed adhesion of epithelial cells and ECM and that may affect the behaviour of the malignant cells, suggesting that collagen XIII has a significant role in the initiation of the invasion.

Abstract Collagen XIII belongs to the subgroup of collagenous transmembrane proteins. It has a wide tissue distribution and has been localized to many sites of cell-matrix and cell-cell interaction in tissues. Biochemical and in silico analyses of collagen XIII and other collagenous transmembrane proteins revealed that the biosynthesis of this structurally varied group is characterized by a coiled-coil motif following the transmembrane domain, and these trimerization domains appear to be associated with each of the collagenous domains. The collagen XIII trimer was shown to have an interchain disulfide bond at the junction of the NC1 and COL1 domains, and several other collagenous transmembrane proteins have a pair of cysteines in the same location. Furthermore, furin cleavage at the NC1 domain can be expected in most of the proteins. Mice heterozygous for the Col13a1del transgene, encoding a mutant collagen XIII, developed clonal mature B-cell lineage lymphomas originating in the mesenteric lymph node (MLN). The incidence of disease in conventionally reared mice was 2-fold higher than for mice raised in a specific pathogen-free facility. The lymphomas often associated with large populations of macrophages and T cells. Lymphomas expressed little if any collagen XIII, suggesting that the effect of the mutation was B-cell extrinsic and likely to be associated with collagen XIII-positive tissues drained by the MLN. Studies of the small intestines of transgenic mice showed highly abnormal subepithelial basement membranes (BM), associated with heightened expression of genes involved in immune responses. These findings suggest that collagen XIII-dependent maintenance of the intestinal BM is a critical determinant of cancer susceptibility. Collagen XIII exhibited a wide tissue distribution at the protein level, and the most intense expression was found in lung. Tissues contained 1-4 collagen XIII polypeptides, their size ranging between 78 and 102 kDa. Collagen XIII staining was detected in a restricted set of blood vessels in the liver, pancreas, adrenal gland, epididymis and brain. Moreover, Col13a1del transgene expression in the absence of endogenous collagen XIII proved to be deleterious for mouse embryonal development, leading to early fetal mortality.