Para elucidar as vias genéticas controlando a formação das câmaras cardíacas, foi analisada a regulação do promotor atrial-específico do gene de codorna que codifica a isoforma lenta da cadeia pesada de miosina (slow myosin heavy chain 3 -SMyHC3). Em camundongos transgênicos, a expressão atrial-específica dos 840 pb do promotor SMyHC3 fundido ao gene repórter da fosfatase alcalina (HAP), SMyHC3-HAP, é controlada pelos 72 pb mais distais deste promotor. Este fragmento contém sítios putativos para ligação a receptores nucleares os quais foram denominados ECRRN (elemento complexo de resposta a receptores nucleares), definidos por modelagem molecular. Tratamento de embriões SMyHC3-HAP com ácido retinoico (AR) expande o domínio atrial de expressão do transgene, enquanto que a inibição da via de sinalização por AR reduz o domínio de expressão do transgene. Ensaios de gel shift revelam que os receptores de AR, RAR/RXR se ligam fracamente a esse promotor. Também, esses receptores não ativam o promotor SMyHC3 em experimentos de transfecção transiente, mesmo na presença de coativadores, tais como p300, CBP e GRIP1. Isso sugere a participação indireta de AR na regulação deste marcador atrial. Ensaios de transfecção celular demonstram que COUPTF2 é capaz de ativar o promotor SMyHC3 e, siRNA contra COUPTF2 inibe esta transativação. Embriões tratados com AR apresentam um aumento geral da expressão de COUPTF2, reforçando a idéia de que AR age indiretamente ativando este gene. Estudos de bioinformática revelam que o ECRRN está presente na região 3 do gene AMHC1 de galinha e alinhamentos indicam que pode se tratar de um elemento móvel que pode ter sido adquirido por um evento de exaptação. Em resumo, COUPTF2 e AR controlam a expressão do promotor SMyHC3, porém a natureza da relação entre os 2 elementos necessita ser elucidada. / To elucidate the genetic pathways controlling cardiac chamber formation, the atrial specific gene promoter SMyHC3 was analyzed. In transgenic mice, the atrial specific expression an 840 bp of the SMyHC3 promoter was linked to reporter gene human alkaline phosphatase (HAP), SMyHC3-HAP. By directed mutagenesis and deletion analysis we identified the more distal 72 bp of the promoter as responsible of the atrial expression. This fragment contains putative binding sites to nuclear receptors, the CNRRE (complex nuclear receptor response element), defined by molecular modeling. RA (retinoic acid) treatment in SMyHC3-HAP embryos expands the atrial domain. However, the RA effectors, RXR/RAR bind with low affinity to the SMyHC3 promoter. These receptors are not able to activate the promoter even in the presence of coactivators such as p300, CBP and GRIP1. These results suggest that RA acts indirectly to regulate this atrial marker. Cell transient transfection shows that COUPTF2 activate the SMyHC3 promoter, and COUPTF2 siRNA inhibits the transactivation of the SMyHC3 promoter. Treatment of embryos with RA increases the COUPTF2 expression reinforcing the idea that this receptor could be regulatedindirectly by RA. Bioinformatic studies revealed that CNRRE is present in the 3 region of the ortholog chicken AMHC1 gene. Alignments indicate that this CNRRE could have been acquired by an exaptation event. In conclusion, the SMyHC3 promoter seems to be controlled by RA and COUPTF2 governing atrial expression. However the nature of relationships between RA and COUPTF2 need to be elucidated.

As vias de sinalização celular por meio de moléculas são um dos principais meios de controle funcional de um organismo. O entendimento das funções de moléculas sinalizadoras facilita a compreensão das vias metabólicas de um organismo, assim possibilitando uma melhor compreensão de vários eventos biológicos e também de várias doenças. A sinalização pelo ácido retinóico (AR), e seus derivados, é responsável pelo controle de várias funções, por exemplo: crescimento celular, diferenciação celular, formação da retina, desenvolvimento cardíaco e também relacionado a várias patologias como diabetes, obesidades, cânceres, e doenças cardiovasculares. A ação do ácido retinóico é controlada em dois níveis: no metabolismo de síntese/degradação e na sua utilização na sinalização para a expressão gênica. A maquinaria que controla o metabolismo inclui as enzimas de síntese do AR (aldeído desidrogenase ALDH) e as enzimas de degradação do AR (Cyp26), que controlam a distribuição espaço-temporal do AR durante a embriogênese. As ALDHs são enzimas NAD(P)+ dependentes, que oxidam uma ampla gama de aldeídos para os seus correspondentes ácidos carboxílicos, sendo ALDH1A2 a principal enzima na transformação de retinal em ácido retinóico. A maquinaria da sinalização celular por AR contém os receptores nucleares controlados por AR (RARs) que estão envolvidos com o controle da transcrição gênica. Os mecanismos de controle de expressão mais comuns são os que ocorrem na fase transcricional. Um desses mecanismos envolve proteínas que se ligam às regiões promotoras de transcrição, representadas por trechos de DNA que geralmente estão localizados próximo à região de início da transcrição, mas que também podem estar a centenas ou até milhares de pares de bases desse início. Essas proteínas modulam a maquinaria transcricional, podendo ativá-la ou inibi-la. A associação de várias técnicas como a biologia molecular, bioinformática, filogenia, análises estruturais de biomoléculas, mecânica molecular e métodos termodinâmicos tem se mostrado uma poderosa abordagem para compreensão de sistemas biológicos simplificando e agilizando o desenvolvimento do conhecimento científico. Nessa direção, esse estudo desenvolveu duas análises: a primeira estudando a evolução das funções das enzimas ALDH, utilizando-se de técnicas de genômica combinatória, filogenia, bioinformática, estrutura de biomoléculas e de biologia do desenvolvimento, tentando compreender o modo como as ALDHs, que apresentam as seqüências de aminoácidos bastante similares, puderam divergir para gerar funções diversas como a destoxificação e a sinalização. Para este estudo foram analisados os genomas de 487 organismos em busca de seqüências de ALDHs e também o genoma do organismo modelo Branchiostoma floridae. Foram obtidas 190 seqüências que foram utilizadas em uma análise filogenética para tentar compreender a função primordial e também para definir grupos de aminoácidos candidatos a marcadores das diferentes famílias de ALDHs. Essas 190 seqüências também foram modeladas estruturalmente e analisada a forma e o volume do canal onde se aloja o aldeído a ser oxidado. A partir dessas informações foi possível prever que as ALDHs passaram das funções ancestrais de controle do padrão corporal para algo mais abrangente como funções protetoras. A segunda análise, utilizando-se das estruturas tridimensionais dos fatores de transcrição ligados ao DNA em diferentes posições e submetendo esses complexos a processos de mecânica molecular, cálculos termodinâmicos e análises das ligações de hidrogênio para tentar prever os mais prováveis sítios de interação entre os receptores e o DNA. O modelo escolhido para essa análise foram os fatores de transcrição regulados por ácido retinóico o RAR e RXR utilizando a região promotora do gene RARE-2 para avaliar as mais prováveis regiões de ligação desses fatores. Para esse estudo foram construídos 71 complexos proteína-DNA que foram submetidos a processos de mecânica molecular e cálculos termodinâmicos. A partir dessas informações foi possível prever uma região de maior afinidade entre o fator de transcrição e o DNA. As análises de ligações de hidrogênio possibilitaram definir exatamente a região de interação entre os fatores de transcrição e o DNA, e também descrever as interações moleculares responsáveis pela especificidade da interação. / Cellular signaling paths through molecules are one of the main processes of functional control of an organism. The comprehension of signaling molecules functions enables one to understand the metabolic pathways of an organism, along with related biological events and several diseases. The signaling through retinoic acid (RA) and its secondary products is responsible for controlling several functions, such as cellular growth and differentiation, retinas formation and cardio development, and is also related to several pathologies such as diabetes, obesity, cancers and cardiovascular disorders. There are two levels of control of retinoic acid activity: synthesis/degradation metabolism and its use in gene expression signaling. The machinery that controls the metabolism includes RAs synthesis (aldehyde dehydrogenase ALDH) and degradation (Cyp26) enzymes, which control the space-temporal distribution of RA during the embryogenesis. The ALDHs are NAD(P)+ dependent enzymes that oxidize many types of aldehydes into the related carboxylic acids, being the ALDH1A2 the main enzyme involved in the process of transformation of retinal into retinoic acid. The machinery of cellular signaling through RA contains the nuclear receptors controlled by RA (RARs) that are involved in the control of gene transcription. The most common mechanisms of expression control are the ones that occur during the transcriptional phase. One of these mechanisms involves proteins that bind to the transcription promoter regions, represented by DNA sequences that are usually located close to the region where the transcription starts, but can also be hundreds or thousands of base pairs apart from the starting point. These proteins modulate the transcriptional machinery, being responsible for both its activation and inhibition. The association of several techniques as molecular biology, bioinformatics, phylogeny, structural analysis of biomolecules, molecular mechanics and thermodynamic methods has been shown as a powerful tool for the understanding of biological systems, simplifying and speeding up the production of related scientific knowledge. Facing this direction, the present study developed two analyses. The first one studied the evolution of ALDH enzymes functions, using the techniques of combinatory genomic, phylogeny, bioinformatics, structure of biomolecules and developmental biology, in the attempt of understanding how the ALDHs could diverge and acquire different functions as detoxification and signaling, despite the fact that they have very similar aminoacid sequences. For this study, ALDHs sequences were searched for in the genome of 487 organisms plus the model organisms, Branchiostoma floridae. All 190 sequences obtained were used in a phylogenetic analysis, in the attempt of understanding the primordial function of the enzyme and defining possible groups of conserved aminoacids in the different families of ADLHs. These 190 sequences were also structurally modeled and the shape and volume of the channel where the aldehyde is placed to be oxidized were analyzed. Based on this information, it became possible to predict that the ALDHs moved from ancestral functions of corporal pattern control to a wider spectrum of protection functions. For the second analysis we submitted the complex formed by tridimensional structures of the transcriptional factors bond to DNA in different positions to processes of molecular mechanics, thermodynamic calculi and analysis of the hydrogen bonds, in order to predict the most probable sites of interaction between the receptors and the DNA. The model chosen for this analysis were the transcription factors regulated by retinoic acid, RAR and RXR, using the promoter region of the gene RARE-2 to assay the most probable binding regions of these factors. For this study, 71 protein-DNA complexes were built and submitted to processes of molecular mechanics and thermodynamic calculi. Based on the resulting data, it became possible to predict a region of greater affinity between the transcription factor and the DNA. The analyses of hydrogen bonds enabled us to define the exact region where the interaction between the transcription factor and the DNA takes place and also enabled us to describe the molecular interactions responsible for the specificity of this interaction.

Células pluripotentes são aquelas que possuem a capacidade de dar origem às células dos três folhetos embrionários (ectoderma, mesoderma e endoderma), bem como também às células germinativas. As células-tronco embrionárias (CTE) são as células pluripotentes mais conhecidas, as quais apresentam uma elevada capacidade de diferenciação celular e autorenovação. Estas propriedades tornam as CTE potenciais ferramentas para a medicina regenerativa, porém seu uso na prática clínica enfrenta várias barreiras. Neste sentido, o acúmulo de conhecimento a respeito dos mecanismos envolvidos na manutenção da pluripotência, levou ao desenvolvimento de técnicas capazes de induzir a pluripotência em células somáticas adultas. Na maioria das abordagens, isto se dá pela expressão ectópica de fatores de transcrição envolvidos na pluripotência (como Oct4 e Nanog). Com isto em vista, torna-se evidente que estudos que levem a um melhor entendimento destas propriedades biológicas, podem levar ao desenvolvimento desta importante área. Apesar destas inovações, os mecanismos responsáveis pela manutenção ou indução da pluripotência e da autorenovação, continuam largamente inexplorados. Neste sentido, o conjunto de técnicas referidas como High Content Screening (HCS) apresenta características fundamentais que permitiriam a interrogação sistemática e em larga-escala de fatores que possam estar influenciando nestes processos. A técnica de HCS se baseia no uso de microscopia de fluorescência em placas de 96 ou mais poços, permitindo a aquisição e a análise automatizada das imagens, de forma a quantificar alterações fenotípicas nas células. O presente trabalho teve como objetivo estabelecer um modelo experimental para a avaliação funcional e em larga escala de fatores que possam influenciar a diferenciação celular. Tendo em vista a facilidade de cultivo e manuseio, a linhagem humana de células pluripotentes de carcinoma embrionário (CCE) NTera-2, foi utilizada. Para a padronização do modelo, o processo de diferenciação foi avaliado ao longo do tempo (em 2, 4 e 8 dias) na presença ou ausência de ácido transretinóico (atRA), utilizado como indutor de diferenciação celular. Para isso, os níveis transcricionais de Oct4, Nanog (marcadores da pluripotência) e de N-Caderina foram avaliados por PCR em tempo real. Finalmente, a expressão e a distribuição celular de Oct4, Nanog e da alfa-actina foi avaliada por meio de microscopia de fluorescência automatizada, com o uso de anticorpos ou faloidina marcada, utilizando um sistema de HCS (Operetta, Perkin Elmer) para a análise dos resultados. A proliferação celular das células submetidas à diferenciação foi avaliada pelo ensaio do XTT. O atRA inibiu a proliferação e induziu a diferenciação; como demonstrado, respectivamente, pelos resultados do ensaio do XTT, decaimento dos níveis de Oct4 e Nanog e, concomitante aumento de N-Caderina, ao longo do tempo. Também foi observada a diferenciação espontânea da linhagem, na ausência de atRA, porém, de forma reduzida. Finalmente, as avaliações de HCS evidenciaram que, durante o processo de diferenciação, a perda da expressão nuclear de Oct4 e Nanog está associada à alteração do fenótipo celular, com a redistribuição da actina cortical e a formação das stress fibers, caracterizando o processo de transição epitélio-mesenquima (EMT), um importante mecanismo envolvido na diferenciação celular. Os resultados obtidos neste trabalho demonstram a viabilidade do uso da linhagem NTera-2 como modelo para estudos futuros de HCS visando a identificação de moléculas que atuem na modulação de propriedades fundamentais das células tronco pluripotentes. / Pluripotent stem cells are those that possess the ability to generate cells from the three germ layers (ectoderm, mesoderm and endoderm), as well as the germ cells. The embryonic stem cells (ESC) are the best known pluripotent cells that present a high capacity of cell differentiation and self renewal. These properties of the ESC make them potential tools for the regenerative medicine, but their use in clinical practice faces several barriers. In this sense, the accumulation of knowledge about the mechanisms involved in the maintenance of pluripotency led to the development of techniques capable of inducing pluripotency in adult somatic cells. In most approaches, this is achieved by the ectopic expression of transcription factors involved in pluripotency (such as Oct4 and Nanog). With this in mind, it becomes clear that studies that provide a better understanding of these biological properties can lead to the development of this important area. Despite these innovations, the mechanisms responsible for the maintenance or induction of pluripotency and self-renewal remain largely unexplored. In this sense, the set of techniques such as High Content Screening (HCS) has fundamental characteristics that allow systematic and large-scale interrogation of factors that may be influencing these processes. The HCS technique is based on the use of fluorescence microscopy in 96-well or larger plates, allowing the automated acquisition and analysis of images, so as to measure phenotypic changes in the cells. This study aimed to establish an experimental model for functional and large-scale assessment of factors that may influence cellular differentiation. Due its simple cultivation and handling characteristics, a human lineage of pluripotent embryonal carcinoma cell (ECC) NTERA-2 was used. To standardize the model, the process of differentiation was evaluated over time (at 2, 4 and 8 days) in the presence or absence of all-trans retinoic acid (atRA), used as an inducer of cellular differentiation. The transcriptional levels of Oct4, Nanog (pluripotency markers) and Ncadherin were assessed by real time PCR. Finally, the expression and cellular distribution of Oct4, Nanog and alpha-actin was assessed by fluorescence microscopy, using antibodies or labelled phalloidin, using a HCS platform (Operetta, Perkin Elmer) for the analysis of the results. The proliferation of cells undergoing differentiation was assessed by XTT assay. atRA inhibited proliferation and induced differentiation, as shown by the XTT assay results, and the decay of Oct4 and Nanog, and concomitant increase of N-cadherin levels over time, respectively. It was also observed spontaneous differentiation in the absence of atRA although in less extent. Finally, the HCS results showed that during the differentiation process, the loss of nuclear expression of Oct4 and Nanog is associated with alteration of cell phenotype, with redistribution of cortical actin and formation of stress fibers, characterizing the epithelialmesenchymal transition (EMT), an important mechanism involved in cell differentiation. The results of this study therefore demonstrate the feasibility of using the NTERA-2 cell line as a model for future HCS studies aiming identification of molecules that act in the modulation of fundamental properties of pluripotent stem cells.

Marine environments are under constant pressure from anthropogenic pollution. Chemical pollutants are introduced into the aquatic environment through waste disposal, sewage, land runoff and environmental exploitation (harbours, fisheries, tourism) leading to disastrous effects on the marine wildlife. Developmental malformations, reproduction failure including sex changes and high death rates are commonly observed in aquatic animal populations around the world. Unfortunately, the underlying molecular mechanisms of these pollution effects, in particular for marine invertebrate species, are often unknown. One proposed mechanism through which environmental pollution affects wildlife, is the disruption of nuclear receptors (NRs), ligand-binding transcription factors in animals. Environmental pollutants can directly interact with nuclear receptors, inducing incorrect signals for gene expression and subsequently disrupt developmental and physiological processes. Elucidation of the exact mechanism in invertebrates, however, is sparse due to limited understanding of invertebrate endocrinology and molecular regulatory mechanisms. Here, I have investigated the presence, expression and function of NRs in the Pacific oyster, Crassostrea gigas, and explored their interrelation with known environmental pollutants. Using a suite of molecular techniques and bioinformatics tools I demonstrate that the Pacific oyster possesses a large variety of NR homologs (43 NRs), which display individual expression profiles during embryo/larval development and supposedly fulfil distinct functions in developmental and physiological processes. Functional studies on a small subset of oyster NRs provided evidence for their ability to regulate gene expression, including interactions with DNA, other NRs or small molecules (ligand-binding). Oyster receptors also show a high likeliness to be disrupted by environmental pollutants. Computational docking showed that the retinoid X receptor ortholog, CgRXR, is able to bind and be activated by 9-cis retinoic acid and by the well-known environmental contaminant tributyltin. A potential interaction between tributyltin and the peroxisome proliferator-activated receptor ortholog CgPPAR has also been found. In addition, exposure of oyster embryos to retinoic acids and tributyltin resulted in shell deformations and developmental failure. In contrast, computer modelling of another putative target for pollutants, the retinoic acid receptor ortholog CgRAR, did not indicate interactions with common retinoic acids, supporting a recently developed theory of loss of retinoid binding in molluscan RARs. Sequence analyses revealed six residues in the receptor sequence, which prevent the successful interaction with retinoid ligands. In conclusion, this investigative work aids the understanding of fundamental processes in invertebrates, such as gene expression and endocrinology, as well as further understanding and prediction of effects of environmental pollutants on marine invertebrates.

L’acide rétinoïque (AR), dérivé actif de la vitamine A (rétinol) circulante, est une petite molécule lipophile contrôlant divers aspects de la mise en place du système nerveux central des vertébrés. L'AR influence notamment le développement précoce du cerveau antérieur, où il contrôle la prolifération et la survie des cellules progénitrices dans l'épithélium neural prosencéphalique. Le développement neural est un processus qui s'articule en trois grandes étapes : la phase d'expansion latérale (E9,5-E10,5 chez la souris), la phase de neurogenèse (E11,5-stades périnataux) et la phase de gliogenèse (stades périnataux-adulte). Nous avons montré que l'AR produit par les méninges à partir de E13 influence la spécification et la migration neuronale au cours de la phase de neurogenèse. De plus, nos travaux suggèrent un rôle plus précoce de l'AR pour la formation et la prolifération des populations progénitrices et neuronales avant et au début de la phase de neurogenèse. Une combinaison de signaux intrinsèques et extrinsèques contrôle divers aspects du développement neural cortical. Nos travaux placent l'AR parmi ces facteurs modulateurs de la neurogenèse corticale. / Retinoic acid (RA), an active vitamin A (retinol) metabolite, is a small lipophilic molecule controlling numerous events during central nervous system development in vertebrates. RA is involved in early forebrain development by controlling cell proliferation and survival in the prosencephalic neuroepithelium. Neural development is a process progressing through three key steps: a phase of lateral expansion (E9.5-E10.5 in the mouse), a phase of neurogenesis (E11.5-perinatal stages) and a gliogenic phase (perinatal stages-adult). My work has shown that RA produced by the developing meninges from E13 influences neuronal specification and migration during the phase of neurogenesis. Moreover, our data suggest an earlier role of RA during the production and proliferation of progenitor and neuronal populations, before and at the onset of the neurogenic phase. A combination of extrinsic and intrinsic signals is required to orchestrate the various aspects of cortical development. RA is likely to be one of such extrinsic factors modulating cortical neurogenesis.

Les cellules souches cancéreuses (CSC) représentent une sous-population de cellules tumorales à l’origine de l’hétérogénéité et de la croissance tumorale. Les CSC sont plus résistantes aux traitements, et à l’origine de la rechute et des métastases. L’identification des CSC constitue actuellement un enjeu majeur dans le développement de nouvelles thérapies ciblées pour inhiber la croissance tumorale et éradiquer le cancer. Dans ce travail, nous avons cherché à identifier, caractériser, et cibler les CSC dans l’adénocarcinome gastrique. Des modèles murins de xénogreffe de tumeurs primaires de patients atteints d'adénocarcinome gastrique hors cardia de types intestinal et diffus ont été développés, ainsi qu’un modèle de tumorsphere in vitro afin d’évaluer les capacités tumorigéniques de sous-populations tumorales. Nous avons identifié CD44 et l'aldéhyde déshydrogénase (ALDH) comme marqueurs d’enrichissement des CSC dans les 2 types d’adénocarcinomes gastriques, l’ALDH représentant un marqueur plus spécifique que CD44. Nous avons ensuite étudié l'effet de l’acide rétinoïque tout trans (ATRA), et nous avons montré que l'ATRA inhibe la formation et la croissance des tumorspheres in vitro ainsi que la croissance tumorale in vivo. Cet effet de l’ATRA passe par l’inhibition de l’expression des marqueurs souches et des capacités d'auto-renouvèlement des CSC. En conclusion, CD44 et ALDH sont des marqueurs de CSC dans les adénocarcinomes gastriques hors cardia de types intestinal et diffus, et le traitement par l’ATRA constituerait une stratégie commune de traitement pour cibler spécifiquement les CSC et inhiber la croissance tumorale dans ces deux types de cancer gastrique. / Cancer stem cells (CSCs) are a subpopulation of tumor cells at the origin of the heterogeneity and growth of tumors. CSCs are more resistant to treatment, and are responsible for relapse and metastasis. The identification of CSCs is a major challenge for the development of new targeted therapies to inhibit tumor growth and eradicate cancer. In this work, we aimed to identify, characterize, and target CSCs in gastric adenocarcinoma. Mouse models of primary tumor xenografts from intestinal and diffuse type non-cardia gastric adenocarcinomas from patients were developed, as well as an in vitro tumorsphere assay, to assess the tumorigenic capacity of subpopulations of tumor cells. We identified CD44 and aldehyde dehydrogenase (ALDH) as CSC enrichment markers in the two types of gastric adenocarcinoma, ALDH representing a more specific marker than CD44. We then studied the effect of All-trans retinoic acid (ATRA), and showed that it inhibited the formation and growth of tumorspheres in vitro and tumor growth in vivo. This effect of ATRA is due to the inhibition of stem marker expression and the self-renewal capacity of CSCs. In conclusion, CD44 and ALDH are effective CSC markers in intestinal and diffuse type non-cardia gastric adenocarcinomas, and treatment with ATRA provides a common treatment strategy to specifically target CSCs and inhibit tumor growth in both subtypes of this gastric cancer.

As coronárias derivam do proepicárdio, uma estrutura formada por precursores dos constituintes de vasos coronários, células endoteliais e musculares lisas (CoSMC). In vivo observa-se um marcante atraso entre a diferenciação endotelial e a integração de CoSMC à parede do vaso. O objetivo deste trabalho foi identificar os mecanismos que inibem a diferenciação a CoSMC in vivo. Baseados na perda progressiva da expressão de raldh2, a principal enzima de síntese de ácido retinóico (AR), nós exploramos a sinalização por AR como um possível inibidor da diferenciação a CoSMC. Através de um vetor adenoviral de expressão de raldh2 e da inibição in vivo da síntese de AR nós demonstramos que a sinalização por AR bloqueia a diferenciação a CoSMC dos precursores coronários. Nós também identificamos o VEGF como um fator chave no controle da diferenciação a CoSMC. Em conjunto, nossos dados suportam o modelo que a síntese de AR e VEGF durante o desenvolvimento cardíaco foi co-optada para o bloqueio da diferenciação a CoSMC até o estabelecimento de uma vasta malha vascular. / Coronary vessels derive from the proepicardium (PE), a structure formed by precursor of coronary vessels cells, endothelial and smooth muscle cells (CoSMC). In vivo there is a clear gap between the endothelial differentiation and the integration of CoSMC into the vascular tubes. The aim of this work was to understand the mechanisms controlling the delayed in vivo CoSMC differentiation. Based on the progressive loss of expression of raldh2, the main retinoic acid (RA) synthesizing enzyme, we explored the RA signaling as a possible candidate inhibitor of CoSMC differentiation. Using a adenoviral raldh2 expression system and in vivo inhibition of RA synthesis we showed that RA signaling act as a brake to slow CoSMC differentiation in PE-derived cells. We also identified VEGF as key factor acting on the control of CoSMC differentiation. Together our results support a model that AR and VEGF synthesis during cardiac development was co-opted to block the CoSMC differentiation of coronary precursors before an extensive endothelial network of tubes is established.

Plusieurs méthodes de capture antigénique ont été décrites dans l’intestin grêle, surtout en cas d’infection: échantillonnage direct par les cellules dendritiques (DC), capture par les macrophages qui délivrent ensuite l’antigène aux DC du stroma, passage des antigènes à travers les cellules caliciformes. Des travaux antérieurs in vitro dans le laboratoire ont montré l’importance de la myosine IIA dans la coordination de la migration des DC avec la capture et de l’apprêtement antigénique. L’objectif de ma thèse était de combiner plusieurs méthodes d’imagerie telle que la microscopie intravitale, la microscopie confocale ex vivo et l’immunofluorescence sur tissus à la cytométrie en flux pour déterminer l’impact de la myosine IIA sur la capture antigénique in vivo. Cette étude montre que les DC patrouillent en permanence dans l’épithélium de l’intestin grêle, y compris hors conditions infectieuses. Elles sont recrutées dans la lamina propria (LP) et pénètrent dans l’épithélium par transmigration à travers la membrane basale qui sépare ces deux compartiments. La myosine IIA est indispensable à la transmigration de CD103+CD11b+DC. Ces événements de transmigration surviennent plus fréquemment dans les parties proximales de l’intestin grêle, duodénum and jéjunum, que dans l’iléon. Chez les souris adultes, ces DC ne sont pas recrutées sous l’influence du microbiote mais sont sensibles au rétinal, un métabolite de la vitamine A qu’elles transforment en une molécule active l’acide trans-rétinoïque (AtRA). D’après notre analyse transcriptomique, les DC intra-épithéliales constituent une population homogène dont le profil est distinct de celui de leurs homologues de la LP. Elles sont enrichies en ARN des voies liées à l’apprêtement antigénique, l’autophagie et les lysosomes. Ces résultats suggèrent qu’elles ont une fonction différente des CD103+CD11b+DC de la LP: elles n’agissent pas sur la prolifération ni la différenciation des lymphocytes T mais contrôlent spécifiquement l’effectif des lymphocytes intra-épithéliaux CD8+αβ. Ces découvertes reflètent l’importance de l’épithélium comme première ligne de défense contre les pathogènes. Elles soulèvent également de nouvelles questions concernant la régulation de la réponse immune dans l’épithélium et les interactions mutuelles entre la lumière intestinale, l’épithélium et le stroma des villosités. / Several routes for antigen capture have been described in the small intestine, mainly upon pathogenic infection: direct sampling by Dendritic Cells (DCs), sampling by macrophages that deliver antigens to DCs in the stroma, antigenic passage through goblet cells. Previous in vitro work in the lab showed that myosin IIA is essential to coordinate antigen uptake and processing with DC migration. The objective of my thesis was to combine several imaging methods including intravital microscopy, ex vivo confocal microscopy and immunofluorescence on gut tissue to flow cytometry in order to unravel the impact of myosin IIA on DC physiology in vivo. My work shows that CD103+CD11b+ DCs, which are unique to the gut, constantly patrol the epithelium of the small intestine at steady state: they are recruited from the lamina propria (LP) and penetrate into the epithelium by transmigrating through the basal membrane that separates these two compartments. DC transmigration requires myosin IIA in vivo. Remarkably, we found that DC transmigration into the epithelium occurs mainly in the upper parts of the small intestine, the duodenum and the jejunum, but is not observed in the ileum. DC transmigration does not require the gut microbiota but relies on retinal, a vitamin A metabolite of that they convert into its active form all-trans retinoic acid (AtRA). Strikingly, single cell RNA-seq showed that intra-epithelial CD103+CD11b+ DCs constitute a homogenous cell population with a distinct transcriptomic signature from their LP counterpart. They are enriched with RNA related to antigen presentation, autophagy and lysosome pathways. Our results further suggest that these cells have a different function from LP CD103+CD11b+ DCs, as they do not significantly impact proliferation or differentiation of T helper lymphocytes but control the CD8+αβ intraepithelial lymphocytes (IELs) pool. These findings highlight the importance of the epithelial tissue as a first line of defense against pathogens in the upper parts of the small intestine. They also raise new questions about the regulation of the immune response in the epithelium and the mutual influences between lumen, epithelium and intestinal lamina propria.

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