Η φυλοσύνδετη θερμοευαίσθητη μετάλλαξη wisertsl (wings scalloped, eyes rough) οφείλεται σε ένθεση ενός Ρ μεταθέσιμου γενετικού στοιχείου στη 5΄ ρυθμιστική περιοχή του γονιδίου lawc (CG32711). Τα άτομα wisertsl πεθαίνουν στους 29οC, γεγονός που οφείλεται στην διαφορετική έκφραση της πρωτεΐνης Wiser/Lawc κατά τη διάρκεια της ανάπτυξης. Το γονίδιο lawc/wiser κωδικοποιεί μια μικρή πρωτεΐνη 73 αμινοξέων, η οποία εκφράζεται στο στάδιο του εμβρύου στα κύτταρα της κοιλιακής χορδής του κεντρικού νευρικού συστήματος, στα πολικά κύτταρα, στο έντερο, στην τραχεία, στα αιμοκύτταρα και στο περιφερικό νευρικό σύστημα. Στα ακμαία θηλυκά άτομα εκφράζεται στα τροφοκύτταρα των ωοθηκών. Επίσης εμπλέκεται στη μορφογένεση των φτερών, των ποδιών και των ματιών. Αλληλεπιδρά με τα γονίδια Notch και Beadex και Serrate και εμπλέκεται στον κυτταρικό πολλαπλασιασμό. Τέλος, η πρωτεΐνη Lawc είναι απαραίτητη για την κατάλληλη μεταγραφή από την RNA polymerase II σε μια διαδικασία που περιλαμβάνει το πυρηνικό πρωτεάσωμα, το οποίο είναι υπεύθυνο για την αποικοδόμηση άχρηστων ή καταστραμμένων πρωτεΐνών. Τα αποτελέσματα της παρούσας εργασίας αποκάλυψαν ότι οι μεταλλάξεις ap-Gal4 και fng-lacZ των γονιδίων apterous (ap) και fringe, αντίστοιχα αυξάνουν σημαντικά το φαινότυπο wisertsl τσιμπημένα/φαγωμένα φτερά, στα θηλυκά και αρσενικά άτομα των γενοτύπων wisertsl; ap-Gal4/+(CyO) και wisertsl;fng-lacZ/+(Sb). Το μεγαλύτερο μέρος της παρυφής των φτερών λείπει όπως και μέρος του ιστού τους. Η αύξηση του φαινοτύπου wisertsl στα άτομα των παραπάνω γενοτύπων συνοδεύεται και από σημαντικές αλλαγές στo πρότυπο έκφρασης των αντίστοιχων γονιδίων ap κα fng. Το γονίδιο ap, ενώ κανονικά εκφράζεται στο ραχιαίο διαμέρισμα του εμβρυικού δίσκου του φτερού και ορίζει το ραχιοκοιλιακό άξονα, στα άτομα wisertsl; ap-Gal4/+(CyO) η έκφρασή του επεκτείνεται και στο κοιλιακό διαμέρισμα. Το γονίδιο fng κανονικά εκφράζεται στο θύλακα των εμβρυικών δίσκων του φτερού, περιοχή που θα δώσει την πτέρυγα του ακμαίου ατόμου και στο κοιλιακό και ραχιαίο τμήμα του δίσκου που θα σχηματίσει τις αρθρώσεις των φτερών. Στα άτομα wisertsl;fng-lacZ/+(Sb) η έκφραση του γονιδίου fng περιορίζεται μόνο σε μέρος του ραχιαίου και κοιλιακού τμήματος του δίσκου των φτερών. Αύξηση του φαινοτύπου wisertsl προκαλεί και η μετάλλαξη Serrate1 στα άτομα wisertsl/Υ;Ser1. Υπερέκφραση των γονιδίων ap, fng, Ser και Chip, με τη χρησιμοποίηση των αντίστοιχων UAS διαγονιδίων τους, στην περιοχή έκφρασης του γονιδίου ap, με οδηγό τη μετάλλαξη ap-Gal4, διασώζουν πλήρως το φαινότυπο wisertsl στα άτομα wisertsl/Y;ap-Gal4/UAS-ap, wisertsl/Y;ap-Gal4/UAS-fng, wisertsl/Y;ap-Gal4/UAS-Ser και wisertsl/Y;ap-Gal4/UAS-chip τα οποία έχουν κανονικά φτερά (με κανονικές παρυφές). Τα γονίδια fng και Ser αποτελούν στόχους του γονιδίου ap που μαζί με το γονίδιο Delta ενεργοποιούν το γονίδιο Notch, γονίδιο κλειδί για την ανάπτυξη του φτερού. Το γονίδιο Chip κωδικοποιεί την πρωτεΐνη Chip που είναι συμπαράγοντας της πρωτεΐνης Αp. Τα αποτελέσματα αυτά δείχνουν ότι έντονος φαινότυπος φαγωμένα φτερά της μετάλλαξης wisertsl στα άτομα των γενοτύπων wisertsl; ap–gal4/+ ή CyO και wisertsl; fng-lacΖ/+ ή Sb1 οφείλεται σε μειωμένη δράση της πρωτεΐνης Ap για την οποία υπεύθυνη πρέπει να είναι η μετάλλαξη wisertsl. Η παρατήρηση ότι η μετάλλαξη wisertsl στις παραπάνω διασταυρώσεις δίνει αποτελέσματα παρόμοια με εκείνα που δίνει η υπερέχουσα μετάλλαξη Bx1 του γονιδίου Beadex στις αντίστοιχες διασταυρώσεις υποστηρίζει την άποψη ότι το γονίδιο lawc πρέπει να εμπλέκεται στην ανάπτυξη των φτερών μέσω της ρυθμίσεις της έκφρασης του γονιδίου Beadex. Αύξηση της πρωτεΐνης dLMO (Bx) μειώνει τη δράση του παράγοντα Ap μέσω της μείωσης των επιπέδων του τετραμερούς Ap-Chip-Chip-Ap. / The X-linked temperature sensitive mutation wisertsl (wings scalloped, eyes rough) is the result of an insertion of the P transposable element in the 5΄ regulatory region of the gene lawc (CG32711). wisertsl flies do not survive in the restrictive temperature of 29οC, because of the differential expression of the Lawc protein during embryonic development. The lawc gene encodes a small protein of 73 a.a. which is mainly expressed during the stage of third instar larva in the ventral mid-line cells of the central nervous system, in pole cells, in the intestine, in the trachea, in red blood cells and in the peripheral nervous system. In adult female flies Lawc is expressed in the nurse cells of the ovaries. Lawc is involved in development of wings, legs and eyes. The lawc gene also interacts with the genes Notch, Beadex (dLMO) and Serrate and it plays a role in the cell proliferation. Finally Lawc is required for proper transcription by RNA polymerase II in aprocess that involves the nuclear proteasome, which is responsible for degradation of damaged or needless proteins. The results of the present study revealed that the mutations ap-Gal4 and fng-lacZ of the genes apterous (ap) and fringe (fng) respectively, increase the wisertsl phenotype of notched wings in female and male flies of the genotype wisertsl; ap-Gal4/+(CyO) and wisertsl; fng-lacZ/+(Sb). Wings of these genotypes lack most of the wing margin and part of the blade. The increased phenotypes are accompanied by significant changes in the expression pattern of the genes ap and fng. Specifically while ap is normally expressed in the dorsal compartment of wing imaginal disc in flies of the genotype wisertsl; ap-Gal4/+(CyO) its expression expands into the ventral compartment. The gene fng is normally expressed in the pouch of wing imaginal disc which will give rise to wing blade and dorsal and ventral wing hinge. In the flies of the genotype wisertsl; fng-lacZ/+(Sb), fng expression is restricted only into a portion of dorsal and ventral hinge of wing imaginal disc. Mutation Serl also causes increased wisertsl phenotypes in flies of the genotype wisertsl/Υ; Ser1. Overexpression of the genes ap, fng, Ser and Chipin in the dorsal compartment wing imaginal discs by using their corresponding UAS transgenes and driver the mutation ap-Gal4, completely rescued the wisertsl phenotype. Flies of the genotypes wisertsl/Y; ap-Gal4/UAS-ap, wisertsl/Y; ap-Gal4/ UAS-fng, wisertsl/Y; ap-Gal4/UAS-Ser and wisertsl/Y; ap-Gal4/UAS-Chip have complete restoration of the wing margin. The genes fng and Ser, which are regulated by ap, with the Delta are responsible for the activation of the Notch gene that is key for the proper development of the wing. The gene Chip encodes the Chip protein that is co-factor of Ap. Taking into account all these data, we can conclude that the strong wisertsl phenotype in the flies of the genotype wisertsl; ap-gal4/+ or CyO and wisertsl; fng-lacΖ/+ or Sb1 is the result of reduced activity of the transcription factor Ap which might due to the wisertsl. Overexpressed the genes ap, fng, Ser and Chip, in the dorsal compartment wing imaginal discs in wisertsl genetic background. Flies of the genotype wisertsl/Y; ap-Gal4/UAS-ap, wisertsl/Y; ap-Gal4/ UAS-fng, wisertsl/Y; ap-Gal4/UAS-Ser and wisertsl/Y; ap-Gal4/UAS-Chip have complete restoration of the wing margin. Genes fng and Ser, which are regulated by ap, with the participation of Delta are responsible for the activation of Notch receptor that is a key component for the proper development of the wing. Gene Chip encodes Chip protein that is a co-factor of Ap. Taking into account all these data, we can conclude that the strong wisertsl phenotype in the flies of the genotype wisertsl; ap-gal4/+ or CyO and wisertsl; fng-lacΖ/+ or Sb1 is the result of reduced activity of the transcription factor Ap. The observation that the mutation wisertsl in the aforementioned crossing schemes, causes phenotypes similar to those caused by the dominant mutation Bx1 (hypermorphic allele) in the corresponding crosses, support the view that the gene lawc must implicated in wing development by regulating the expression levels of Beadex (dLMO) protein. Increased levels of dLMO protein reduce the activity of Ap by decreasing the levels of the active tetramers of Ap-Chip-Chip-Ap.

O desenvolvimento embrionário requer um rigoroso controle da expressão de inúmeros genes que devem ser ativados no momento e local apropriados para o correto estabelecimento das estruturas e órgãos do organismo. Com a análise das seqüências expressas (Expressed Sequence Tags, EST) é possível identificar os genes expressos em tecidos específicos em diferentes estádios do desenvolvimento. A fim de identificar genes expressos durante o desenvolvimento embrionário de Gallus gallus, EST foram obtidas a partir da extremidade 5' dos clones de três bibliotecas de cDNA construídas a partir de (1) embrião inteiro, (2) membros anteriores e posteriores e (3) somitos e tubo neural. As EST foram analisadas pelos programas Phred, Cap3 e Consed para avaliação de qualidade das bases e clusterização. A análise dos dados resultou em 4998 EST válidas segundo parâmetros estabelecidos para este estudo. Todas as seqüências consenso dos clusters e singletons foram comparadas com as seqüências disponíveis no GenBank (http://www.ncbi.nlm.nih.gov) e classificadas em doze categorias segundo sua função. A categorização revelou que 25% dos genes não apresentaram homólogos no banco utilizado (Low e No hit). Aproximadamente 15% dos genes não apresentaram função definida (hipotéticos conservados). Os 60% restantes permitiram identificar genes envolvidos com a somitogênese, com a formação da musculatura esquelética e com a formação dos brotos de membros em vertebrados, além de genes de manutenção e estrutura celular. O conjunto das EST identificadas neste projeto possibilitou a construção de um banco com cerca de 5.000 EST de estádios embrionários de Gallus gallus, fonte para a identificação de novos genes, para a elucidação dos processos reguladores do desenvolvimento embrionário e para a identificação de SNPs (Single Nucleotide Polymorphism). / Embryo development requires rigorous control of expression of various genes, which may become active at an adequate time and site for the correct establishment of organ and structure of the organism. Identification of Expressed Sequence Tags (EST) allows the determination of genes expressed in specific tissues at various stages of development. To identify genes expressed during embryonic development of Gallus gallus, EST were obtained from 5' end of clones from three cDNA libraries, derived from (1) whole embryos, (2) limb buds (hindlimb and forelimb), and (3) somites and neural tube. EST sequences were analyzed using the softwares Phred, Cap3 and Consed to evaluate base quality and clustering, resulting in 4998 valid EST, according to the parameters established. All consensus sequences of clusters and singletons obtained were compared with sequences available at GenBank (http://www.ncbi.nlm.nih.gov) and classified into twelve categories according to function. Categorization revealed that about 25% of the genes were not described in the database consulted (Low and No hit). About 15% of the genes had no defined function (conserved hypothetical). The remaining sixty percent permitted identification of genes involved with somitogenesis, skeletal muscle and limb bud development in vertebrates and cellular maintenance and structure. The set of EST identified in this project resulted in a database with around 5000 EST of Gallus gallus embryonic stages, which is a new source of gene identification to facilitate investigation of regulatory processes in embryo development and identification of Single Nucleotide Polymorphisms (SNPs).

Orientador: Claudio Antonio Barbosa de Toledo / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T19:38:46Z (GMT). No. of bitstreams: 1 Santana_RenatoFigueiredode_M.pdf: 3137636 bytes, checksum: bfb0cb5b3693de7bf1e46cd7ce5de3db (MD5) Previous issue date: 2007 / Resumo: Glutamato, um dos principais neurotransmissores excitatórios do sistema nervoso central participa do tráfego de informação neural nos núcleos de base de aves e mamíferos através de receptores ionotrópicos do tipo AMPA. Investigamos a variação da expressão das subunidades que compõem esse receptor em áreas motoras telencefálicas do encéfalo de pombos (C. lívia) em diferentes idades: do nascimento até 10 dias de vida e na idade adulta. As aves foram perfundidas e seus cérebros foram processados por imunohistoquímica usando anticorpos contras as subunidades GluR1 e GluR4 e contra um epítopo comum às subunidades GluR2 e GluR3 (GluR2/3); além disso, anticorpos contra proteínas ligantes de cálcio foram utilizados como marcadores. O globo pálido (GP) apresentou marcação crescente de neurônios GluR1+ durante os primeiros dias de vida, atingindo pico em P3, decaindo em seguida e sendo novamente expresso na fase adulta. A expressão de GluR2/3 mostrou-se relativamente estável no GP durante os primeiros dias de vida, mas decaiu após P3. Células expressando GluR4 foram principalmente evidentes entre P3 e P7 no GP. Com relação ao corpo estriado, a neurópila estriatal foi intensamente marcada para GluR1 e GluR2/3, contrastando com a pobre marcação de neurópila apresentada pelo globo pálido. Nos diferentes estágios de desenvolvimento a neurópila estriatal foi pobre em GluR4. O número de células GluR1+ no corpo estriado mostrou pico máximo em P5 e o de GluR2/3+ foi crescente ao longo das idades. A região lateral do estriado lateral indicou marcação semelhante à região central em todas as idades, com padrão estável de crescimento para GluR2/3, um pico em P5 para GluR1 e escassa presença de marcação GluR4+ em todas as idades. O número de células PV+ decai durante o desenvolvimento e CB foi expressa nos gânglios da base somente na fase adulta. Nossos resultados indicam que subunidades do receptor de glutamato do tipo AMPA são diferencialmente expressas durante o crescimento pós-natal. Estas alterações podem estar relacionadas com mudanças funcionais que conduzem à estruturação final da circuitaria sináptica envolvida nas respostas motoras desta espécie, cujo desenvolvimento até o estágio de auto-suficiência depende dessa habilidade / Abstract: AMPA-type glutamate receptors mediate the responses of neurons of the striatal part of the basal ganglia to excitatory cortical input in both birds and mammals. We have investigated the maturation of the expression of the four AMPA receptor subunits in 42 pigeons (C. livia) at different stages, from hatching (P0) to 1, 3, 5, 7, or 10 days posthatch (P1-10), to adulthood. Pigeons are altricial, become relatively mobile by 12 days, and independent by one month. The birds were perfused and their brains processed by standard immunolabeling using antibodies against the GluR1 or GluR4 subunits, or against an epitope common to both GluR2 and GluR3 (GluR2/3) subunits. Antibodies against calcium binding proteins parvalbumin (PV) and calbindin (CB) were used as neuronal marker. Globus pallidus at P0 showed labeling in neurons for GluR1 and GluR2/3, which reached its peak at P3. The GluR1 perikaryal labeling in globus pallidus decreased after P5, but was labeling another time at adulthood. GluR2/3 remained stable into early age, but decrease after P3. The perikarya of globus pallidus and their processes were also labeled for GluR4 at P0, and GluR4 cells GluR4+ was peak between P3 and P7. PV was decrease during the development. CB was expressed in basal ganglia only at adulthood. At P0, the striatal neuropil was intensely (though diffusely) labeled for GluR1 and GluR2/3, which contrasted distinctly with the poor neuropil labeling for both in globus pallidus. GluR1+ was peak at P5 and GluR2/3 increase during the time. At all stages, the striatal neuropil immunolabeling was poor for GluR4, although neurons were labeled for GluR4. GluR4 was peak at P7. PV was peak at adult age. The labeling of GluR2/3 in lateral region of lateral striatun was similar to central region, but GluR1+ was increase by ages. Our results indicate that AMPA receptor subunits was expressed differentially at the development post-hatching. This may support maturational changes that facilitate the motor development of this altricial species / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Au cours du développement, les cellules sont progressivement séparées dans des territoires distincts délimités par des frontières embryonnaires. La première ségrégation a lieu pendant la gastrulation, quand l’embryon s’organise en trois feuillets embryonnaires, l’ectoderme, le mésoderme et l’endoderme. Les mécanismes moléculaires et cellulaires assurant cette ségrégation n’ont pas encore été élucidés. Au cours de ma thèse, je me suis focalisée sur l’internalisation de l’endoderme chez le poisson-zèbre. À partir de résultats in vitro, il a été suggéré que les progéniteurs de feuillets embryonnaires soient ségrégés par un tri cellulaire passif. En combinant des expériences de transplantation de cellules, une imagerie confocale en temps réel et des analyses fonctionnelles, j’ai montré que l’internalisation des cellules endodermiques est due en réalité à un processus de migration active dépendante de Rac1 et de son effecteur Arp2/3, un régulateur direct de l’actine. De manière surprenante, les cellules endodermiques ne sont pas attirées par leur destination interne, mais semblent plutôt migrer hors de leurs voisines. Ce processus est dépendant de la voie Wnt/PCP et de la N-cadhérine. De plus, la N-cadhérine est suffisante pour induire l’internalisation de cellules ectodermiques, sans modifier leur identité. Dans leur ensemble, ces résultats conduisent à un nouveau modèle de formation des feuillets embryonnaires dans lequel les cellules endodermiques migrent activement hors de l’épiblaste pour atteindre leur position interne dans l’embryon. / During development, cells are progressively separated into distinct territories, delimited by embryonic boundaries. The first segregation event occurs during gastrulation, when the embryo is organised in three germ-layers, the ectoderm, the mesoderm and the endoderm. The molecular and cellular mechanisms ensuring this segregation have not yet been elucidated. During my PhD thesis, I have focused on the endoderm internalisation in the zebrafish embryo. Based on in vitro results, it has been suggested that germ-layer progenitors would be segregated by a passive cell sorting. Combining cell transplantation, live confocal microscopy and functional analyses, I have shown that endodermal cell internalisation actually results from an active migration process dependent on Rac1 and its effector Arp2/3, a direct regulator of actin. Strikingly, endodermal cells are not attracted to their internal destination but rather appear to migrate out of their neighbouring cells. This process is dependent on the Wnt/PCP pathway and N-cadherin. Furthermore, N-cadherin is sufficient to trigger the internalisation of ectodermal cells, without affecting their fate. Overall, these results lead to a new model of germ-layer formation, in which endodermal cells actively migrate out of the epiblast to reach their internal position.

Cancers often resurrect embryonic molecular programs to promote disease progression. In melanomas, which are tumors of the neural crest (NC) lineage, a molecular signature of the embryonic NC is often reactivated. These NC factors have been implicated in promoting pro-tumorigenic features like proliferation, migration and therapy resistance. However, the molecular mechanisms that establish and maintain NC identities in melanomas are largely unknown. Additionally, whether the presence of a NC identity has any clinical relevance for patient melanomas is also unclear. Here, using comparative genomic approaches, I have a) identified a novel role for GDF6-activated BMP signaling in reawakening a NC identity in melanomas, and b) identified a NC signature as a clinical predictor of melanoma progression. Like the genomes of many solid cancers, melanoma genomes have widespread copy number variations (CNV) harboring thousands of genes. To identify disease-promoting drivers amongst such huge numbers of genes, I used a comparative oncogenomics approach with zebrafish and human melanomas. This approach led to the identification of a recurrently amplified oncogene, GDF6, that acts via BMP signaling to invoke NC identities in melanomas. In maintaining this identity, GDF6 represses the melanocyte differentiation gene MITF and the proapoptotic factor SOX9, allowing melanoma cells to remain undifferentiated and survive. Functional analysis in zebrafish embryos indicated a role of GDF6 in blocking melanocyte differentiation, suggesting that the developmental function of GDF6 is reiterated in melanomas. In clinical assessments, a major fraction of patient melanomas expressed high GDF6, and its expression correlated with poor patient survival. These studies provide novel insights into regulation of NC identities in melanomas and offer GDF6 and components of BMP pathway as targets for therapeutic intervention. In additional studies, I wanted to test whether a broader NC identity in melanomas had any clinical relevance. In these studies, I performed transcriptome analysis of zebrafish melanomas and derived a 15-gene NC signature. This NC gene signature positively correlated with the expression of SOX10, a known NC marker in human melanomas. Patients whose melanomas expressed this signature showed poor overall survival. These findings identify an important predictive signature in human melanomas and also illuminate the clinical importance of NC identity in this disease.

Pendant le développement d'un organisme, les tissus subissent, génèrent des changements morphologiques drastiques nécessaires à l'obtention d'une forme finale ou intermédiaire spécifique et fonctionnelle. On comprend cette acquisition de formes comme un phénomène émergent résultant de l'interaction mécanique entre toutes les cellules composant le tissu. On sait que les structures de protéines du cytosquelette sont capables aussi bien de générer des forces ou de changer les propriétés mécaniques des cellules i.e de changer la réaction de celles-ci à un stress mécanique. Ces phénomènes émergents font l'objet de nombreuses études et mesures aussi bien lors d'expériences in vitro (solution de protéines purifiées) que sur cellules de cultures. Le travail décrit dans cette thèse s'est attaché à mesurer quantitativement les forces et propriétés mécaniques in vivo durant le développement d'un organisme. À terme, ces mesures sont utiles à l'élaboration d'un modèle mécanique qui amènerait une meilleure compréhension des phénomènes morphogénétiques.Pour réaliser ces mesures, un banc de mesures optiques a été développé. Il permet de réaliser des mesures de microrhéologie passives et actives durant l'extension de la bandelette germinale de l'embryon de drosophile. Des mesures quantitatives de viscosité, de raideur et de force jonctionnelle ont été réalisées. / Pendant le développement d'un organisme, les tissus subissent, génèrent des changements morphologiques drastiques nécessaires à l'obtention d'une forme finale ou intermédiaire spécifique et fonctionnelle. On comprend cette acquisition de formes comme un phénomène émergent résultant de l'interaction mécanique entre toutes les cellules composant le tissu. On sait que les structures de protéines du cytosquelette sont capables aussi bien de générer des forces ou de changer les propriétés mécaniques des cellules i.e de changer la réaction de celles-ci à un stress mécanique. Ces phénomènes émergents font l'objet de nombreuses études et mesures aussi bien lors d'expériences in vitro (solution de protéines purifiées) que sur cellules de cultures. Le travail décrit dans cette thèse s'est attaché à mesurer quantitativement les forces et propriétés mécaniques in vivo durant le développement d'un organisme. À terme, ces mesures sont utiles à l'élaboration d'un modèle mécanique qui amènerait une meilleure compréhension des phénomènes morphogénétiques.Pour réaliser ces mesures, un banc de mesures optiques a été développé. Il permet de réaliser desmesures de microrhéologie passives et actives durant l'extension de la bandelette germinale de l'embryon de drosophile. Des mesures quantitatives de viscosité, de raideur et de force jonctionnelle ont été réalisées.

L'utilisation de transgènes actifs dans le cortex surrénalien de souris a permis de dériver de nouvelles lignées cellulaires de la zone fasciculée de la corticosurrénale (lignées ATC) et de tester le pouvoir transformant de gènes candidats au développement de tumeurs corticosurrénaliennes. Les cellules ATC produisent de la corticostérone et expriment l'ensemble des gènes impliqués dans la stéroïdogenèse sous le contrôle de l'ACTH. Elles ont permis d'explorer les rôles antagonistes des facteurs de transcription SF1 et DAX1 dans le mécanisme d'action de l'ACTH. Les modèles cellulaires et murins surexprimant IGF2 ou ayant perdu en partie l'expression de p57kip2 ont conduit à rejeter leur implication individuelle dans le développement des corticosurrénalomes. Nous avons montré que l'expression dans la corticosurrénale de souris d'une sous unité régulatrice RIalpha tronquée de la PKA reproduit l'hyperactivité endocrine rencontrée dans les hyperplasies micronodulaires pigmentées des surrénales

Le plan d'organisation des vertébrés est caractérisé par la répétition de segments tels que les vertèbres. Les premiers signes de segmentation sont observés pendant l'embryogenèse précoce, lorsque les précurseurs des vertèbres, appelés " somites ", se forment de manière périodique à partir du mésoderme paraxial présomitique (PSM). Il a été proposé qu'une "horloge de segmentation" contrôlerait la périodicité de formation des somites en interagissant avec un "front de détermination" reculant caudalement dans le PSM. Afin de comprendre les mécanismes établissant le nombre total de somites chez les vertébrés, j'ai comparé la régulation de la somitogenèse dans une espèce en formant un grand nombre-le serpent des blés- avec la souris, le poulet et le poisson zèbre. J'ai premièrement cloné et analysé par hybridation in situ l'expression des gènes impliqués dans la formation de front de détermination et de l'horloge de segmentation. Le patron d'expression des gènes associés au front s'est révélé conservé, alors que celui de lunatic fringe, un gène associé à l'horloge, s'est révélé particulièrement atypique. Une étude comparative basée sur un modèle mathématique nous a conduit à l'hypothèse que ce patron d'expression traduisait un rythme accéléré de l'horloge par rapport à la vitesse d'élongation de l'axe chez le serpent, expliquant ainsi sa production accrue de somites. En conclusion, notre étude, conduite sur un modèle original, suggère que la relation entre horloge et croissance de l'axe est un facteur important pour expliquer la différence du nombre de somites entre vertébrés.

Várias evidências, incluindo as originadas de estudos anteriores do LBR&MEC, sugerem que condições adversas durante o desenvolvimento intrauterino promovam alterações moleculares e estruturais em órgãos e sistemas vitais podendo comprometer o seu funcionamento no indivíduo adulto. A hiperglicemia é um fator que influencia negativamente o desenvolvimento fetal, modificando processos biológicos importantes, como o padrão de síntese e deposição dos componentes da matriz extracelular (MEC). A MEC participa diretamente do processo de ramificação e morfogênese do pâncreas, e pouco é conhecido a respeito dos efeitos da hiperglicemia materna sobre a MEC desse órgão durante seu desenvolvimento. Investigamos por meio de imuno-histoquímica como a hiperglicemia materna severa modifica a distribuição de panlaminina, das cadeias &#945 1 ey1 das lamininas e da integrina &#945 3, moléculas da MEC que desempenham um papel chave na diferenciação do pâncreas endócrino. Avaliamos o perfil proliferativo das células presentes nas ilhotas e ainda, a distribuição das células &#945 e &#946 por meio da marcação de glucagon e insulina no pâncreas de fetos de 19 dias. Analisamos por RT-qPCRa expressão dos fatores de transcrição Pdx1 e Pax4 que controlam o desenvolvimento e diferenciação das células &#946 pancreáticas. O modelo utilizado foi o de gestação complicada por diabetes mellitus do tipo 1 (DM1), desenvolvido por nosso grupo, quimicamente induzido por aloxana sem tratamento de reposição insulínica, em camundongos. Observamos que a marcação de panlaminina e das cadeias &#945 1 e y1 das lamininas é mais fraca no pâncreas endócrino dos fetos de mães hiperglicêmicas, quando comparado ao grupo controle. Por outro lado, vimos um aumento na deposição da integrina &#945 3 na membrana basal das ilhotas pancreáticas dos fetos gerados sob condições de hiperglicemia materna. O índice proliferativo das células endócrinas, observado por imuno-histoquímica para PCNA, também é menor nesse grupo. Observamos um aumento da área de ilhotas fetais imunomarcadas para a insulina, indicando aumento na massa de células &#946 nessas ilhotas, enquanto que a área imunomarcada para glucagon estava com marcação menos intensa no grupo experimental comparado ao controle. Identificamos que a expressão relativa de Pdx1 é menor no pâncreas do grupo experimental comparado a expressão nos animais do grupo controle, enquanto a expressão de Pax4 está aumentada. Concluímos por meio de nossas abordagens histoquímicas que a hiperglicemia materna altera a morfogênese do pâncreas endócrino fetal modificando o padrão de deposição de moléculas da membrana basal peri-ilhotas, promovendo uma diminuição da atividade proliferativa das células endócrinas, associada a alterações na expressão de fatores de crescimento importantes para o estado diferenciado e proliferativo das células &#946. Essas células apresentam aumento da massa funcional identificada pelo aumento da deposição de insulina no tecido pancreático. / Previous studies from our lab and others have shown that adverse conditions during intrauterine development promotes molecular and structural changes in vital organs and systems which may alter on their function in the adult individuals. Hyperglycemia impacts on fetal development by modifying important biological processes, such as the pattern of synthesis and deposition of extracellular matrix (ECM) components. ECM cooperates in pancreatic branching and morphogenesis and little is known about the effect of maternal hyperglycemia on the pancreas ECM during development. We investigate through immunohistochemistry, how severe maternal hyperglycemia modifies the distribution of panlaminin, laminins &#945 1 and &#9781 chains and integrin &#945 3, ECM molecules that play a key role in the differentiation of the endocrine pancreas. We evaluate the proliferative index of islet cells and, &#945 and &#946 cells distribution, by insulin and glucagon fetal (E19.0) pancreas staining. We analyzed by RT-qPCR the expression of the Pdx1 and Pax4 transcription factors that control the development and differentiation of pancreatic &#946 cells. The model used was created by our group, a pregnancy model complicated by type 1 diabetes mellitus (T1D) chemically induced by alloxan without treatment of insulin replacement, in mice. We observed that the labeling of panlaminin and laminins &#945 1 and y1 chains is weaker in the endocrine pancreas of the fetuses from hyperglycemic mothers. On the other hand, integrin &#945 3 deposition increased in the basement membrane of the pancreatic islets of the fetuses generated under maternal hyperglycemia. Immunohistochemistry for PCNA showed lower proliferative index of endocrine cells. There was an increase in the area of immunolabeled fetal islets indicating an increase in &#946-cell mass in these islets; whereas the glucagon-immunolabeled area was smaller in the experimental group compared to the control group. The relative expression of Pdx1 was lower in the pancreas from the experimental group, and the Pax4 expression was increased. We conclude from our histochemical approaches that maternal hyperglycemia alters fetal endocrine pancreas morphogenesis by modifying the pattern of peri-islet basement membrane molecules, promoting a decrease in endocrine cell proliferative activity associated with changes in the expression of important growth factors for the differentiated and proliferative state of the &#946-cells. These cells have increased functional mass identified by increased insulin deposition in pancreatic tissue.

Born and educated for the most part in the United States, I have enjoyed the luxury of excellent mentorship during my career thus far as an independent scientist in France. All these mentors have taken it on trust that my training for a Ph.D. also included the necessary tools for directing original research responsibly, at all levels. However, the habilitation is an obligate rite of passage for researchers in France, Germany, Sweden and a number of other European countries. It ensures both that I am competent to not only continue to conduct original research, and that I have a directive seam in my research interests over time that is sufficiently rich to support myself and those trainees who will learn from my experience and contribute their efforts by my side to advancing science. To demonstrate that the faith of these esteemed colleagues has been well-placed since my Ph.D., I hereby present, to the best of my ability, my acquired credentials and my near- to mid-term projects. The over-arching theme of my work has been to identify molecular hallmarks and improve the physiopathological understanding of congenital and progressive conditions implicating a highly plastic embryological cell population known as the neural crest. These neural crest cells (NCC) participate directly or indirectly in the formation of a stunning array of tissues organs during embryogenesis. When the genes regulating the differentiation, proliferation, or migratory and appropriately invasive behavior of NCC are muted, this can lead to associations of pediatric congenital malformations or tumorigenesis. I make use of avian and, more recently, murine models, as well as careful observations effected on tissues derived from normal human embryos, to tease apart those mechanisms.