Dissertations / Theses on the topic 'Ascidie'

Les embryons d'ascidies se développent avec un lignage cellulaire stéréotypé et évolutionairement conservé pour produire en quelques heures ou jours un têtard comportant un petit nombre de cellules. De ce fait, ils fournissent un cadre intéressant pour décrire avec une résolution cellulaire le programme de développement d’un organisme complet. Pendant mon doctorat, j’ai développé une approche quantitative pour décrire l’évolution morphologique embryonnaire pendant le développement de Phallusia mammillata. J’ai ensuite utilisé cette approche pour systématiquement caractériser en détail les logiques des événements de spécifications de destin cellulaire.Pour caractériser quantitativement les comportements cellulaires pendant l’embryogenèse, nous avons utilisé de la microscopie à feuille de lumière multi-angles pour imager des embryons entiers à haute résolution spatio-temporelle. Les membranes plasmiques étaient marquées pour permettre l’identification des cellules. Pour extraire les informations biologiques de ce jeu de donnés, j’ai développé une nouvelle méthode pour segmenter les cellules en 4D, ASTEC. Une fois appliquée aux embryons de Phallusia mammillata imagés pendant 6 heures entre le stade 64 cellules et le début des stades bourgeon caudal, cette méthode a permis de récupérer la forme et de suivre 1030 cellules pendant 640 divisions. L’embryon digital 4D résultant peut être formalisé par un graphe dynamique, dans lequel les cellules sont représentées par des sommets reliés par des arrêtes représentant au sein d’un point de temps leur voisinage spatial, et entre différents points de temps leur lignage cellulaire.Basé sur cette représentation digitale et quantitative, nous avons systématiquement identifié les événements de spécification cellulaire jusqu’au dernier stade de la gastrulation. Des simulations informatiques ont révélé que des règles remarquablement simples intégrant les aires de contacts cellulaires et les expressions spatio-temporelles booléennes de signaux moléculaires extracellulaires sont suffisantes pour expliquer les inductions cellulaires au cours du développement précoce. Ce travail suggère que pour les embryons établissant des contacts stéréotypés et précis entre cellules voisines, les contraintes génomiques sont relâchées, ce qui permet une évolution plus rapide du génome<br>Ascidian embryos develop with stereotyped and evolutionarily conserved invariant cell lineages to produce in a few hours or days tadpole larvae with a small number of cells. They thus provide an attractive framework to describe with cellular resolution the developmental program of a whole organism. During my PhD, I developed a quantitative approach to describe the evolution of embryonic morphologies during the development of the ascidian Phallusia mammillata. I then used this approach to systematically characterize in detail the logic of cell fate induction events. To quantitatively characterize cell behaviors during embryogenesis, we used multi-angle light-sheet microscopy to image with high spatio-temporal resolution entire live embryos with fluorescently labeled plasma membranes. To extract biological information from this imaging dataset, I then developed a conceptually novel automated method for 4D cell segmentation, ASTEC. Applied to a Phallusia mammillata embryo imaged for 6 hours between the 64-cell and the initial tailbud stages, this method allows the accurate tracking and shape analysis of 1030 cells across 640 cell divisions. The resulting 4D digital embryo can be formalized as a dynamic graph, in which cells are represented by nodes, linked within a time point by edges that represent their spatial neighborhood, and between time points by temporal edges describing cell lineages.Based on this quantitative digital representation, we systematically identified cell fate specification events up to the late gastrula stage. Computational simulations revealed that remarkably simple rules integrating measured cell-cell contact areas with boolean spatio-temporal expression data for extracellular signalling molecules are sufficient to explain most early cell inductions. This work suggests that in embryos establishing precise stereotyped contacts between neighboring cells, the genomic constraints for precise gene expression levels are relaxed, thereby allowing rapid genome evolution

Comment la morphogenèse embryonnaire peut-elle être conservée malgré une divergence importante des séquences codantes et non-codantes ? Pour répondre à cette question, nous avons travaillé sur le développement précoce d’ascidies divergentes, Phallusia mammillata et Ciona intestinalis. Ces espèces partagent une morphogénèse pratiquement identique et des lignages cellulaires stéréotypés. Or, leurs génomes sont tellement divergents que leurs séquences ne peuvent pas être alignées.Nous avons choisi d’étudier les cellules précurseuses de l’endoderme au cours de deux processus développementaux conservés : spécification du destin et la gastrulation. Nous avons comparé par hybridation in situ l’expression transcriptionelle des gènes régulateurs orthologues dans Phallusia et Ciona. Nous avons trouvé que l’expression dans l’endoderme de 8 gènes régulateurs impliqués dans ces processus développementaux est qualitativement conservée entre les deux espèces.Pour étudier comment ces gènes ont conservé leur régulation malgré une divergence non-codante importante, nous avons collaboré avec l’équipe Gomez-Skarmeta pour cartographier, par ATAC-seq, la chromatine ouverte dans les deux espèces pour identifier les régions régulatrices actives à l’échelle du génome. 35 sur les 39 séquences ouvertes avoisinant les gènes de l’endoderme ont été trouvé active avant le stade larval, par éléctroporation. La plupart des séquences testées ont conservé leur activité dans les deux espèces malgré la divergence de séquence. Nous avons alors identifié des sites de fixations pour facteurs de transcription potentiels se trouvant dans les enhancers pour l’endoderme pour identifier les régulateurs dans Phallusia et Ciona.Nos résultats suggèrent des changements assez importants de l’ordre des sites de fixations sans pour autant avoir de changement dans l’architecture dans les réseaux de gènes régulateurs ; ceci explique la conservation qualitative de l’expression des gènes entre ces ascidies divergentes. En outre, nous avons trouvé que les shadow enhancers sont plus répandus qu’anticipé<br>How can embryonic morphogenesis be evolutionarily conserved in spite of extensive divergence in coding and non-coding genome sequences? To address this question, we worked on the early development of two very divergent ascidians, Phallusia mammillata and Ciona intestinalis. These species share an almost identical early morphogenesis and stereotyped cell lineages. Remarkably, however, their genomes are divergent to the extent that their non-coding sequences cannot be aligned and gene order has not been conserved.We focus our attention on the behaviour of endoderm precursors throughout two important evolutionarily conserved developmental processes: initial fate specification and early gastrulation. We first compared by in situ hybridisation the transcriptional expression of orthologous regulatory genes in Phallusia and in Ciona. We found that the endodermal expression of 8 regulatory genes known to be involved in these developmental processes is qualitatively conserved between the two species.To study how these genes conserved their regulation in spite of extensive non-coding sequence divergence, we collaborated with the Gomez-Skarmeta lab to map, by ATAC-seq, open chromatin regions in both species to identify active regulatory regions genomewide. Three quarters of the 39 open chromatin regions for endodermal genes behaved as active regulatory sequences by the larval stage, when tested by electroporation in embryos. Many of the tested sequences had conserved cis-regulatory activity in both species in spite of sequence divergence. We have identifed putative transcription factor binding sites in endodermal enhancers in both species to identify conserved upstream regulators shared between Phallusia and Ciona.Taken together our results suggest that extensive transcription factor binding site turn over, without radical change in GRNs architecture, may explain the qualitative conservation of gene expression patterns between highly divergent ascidian genomes. Furthermore, we found that shadow enhancers are much more prevalent than initially anticipated.Taken together our results suggest that extensive transcription factor binding site turn over, without radical change in GRNs architecture, may explain the qualitative conservation of gene expression patterns between highly divergent ascidian genomes. Furthermore, we found that shadow enhancers are much more prevalent than initially anticipated

Durant ma thèse, j'ai participé à une étude fonctionnelle qui a démontré que p120-RasGAP, une protéine appartenant à la famille GAP (GTPase-activating protein), est le médiateur cytoplasmique de l'éphrine lors de l'atténuation d'ERK1/2. Pour confirmer cela, j'ai réalisé une expérience de co-immunoprécipitation et j'ai démontré que p120-RasGAP s'associe au récepteur de l'éphrine, Eph3, quand celui-ci est activé par un ligand éphrine. Ce résultat indique fortement que les signaux FGF et éphrine convergent au niveau de Ras et qu'ils contrôlent de manière antagoniste son activité. Dès lors, j'ai analysé les autres événements de spécification cellulaire impliquant l'antagonisme FGF/éphrine. Chez l'embryon d'ascidie, le signal FGF est décrit comme inducteur du destin neural dans les cellules ectodermiques qui, en absence du signal FGF, adoptent le destin épidermique. L'induction neurale des ascidies a lieu au stade 32 cellules et se traduit par la spécification de quatre précurseurs neuraux (ERK+) parmi les 16 cellules ectodermiques. J'ai démontré que le signal éphrine/Eph/RasGAP antagonise le signal FGF pour générer une activation d'ERK1/2 de type tout ou rien parmi les cellules ectodermiques. Enfin, en collaboration avec Philip Abitua, doctorant dans le laboratoire du Dr. Mike Levine (UC Berkeley), nous démontrons que l'antagonisme entre les signaux éphrine et FGF est impliqué dans la régionalisation antéro-postérieure de la plaque neurale

Le système nerveux périphérique des vertébrés se forme à partir de structures dorsales qui leurs sont propres : la crête neurale et les placodes ectodermiques. À l’inverse, chez les invertébrés chordés, les amphioxus et les ascidies, une partie du SNP provient d’une région neurogénique ventrale. Chez les deux groupes, il a été montré qu’un fort signal BMP induit l’ectoderme ventral à devenir neurogénique au sein de laquelle la formation de neurones épidermiques sensorielles est contrôlée par la voie Notch. Ainsi, il est probable que le système nerveux périphérique ventral soit un caractère ancestral chez les chordés, qui a été perdu chez les vertébrés. Afin de tester cette hypothèse, mon projet vise à étudier l’origine et l’évolution du SNPv des invertébrés chordés. Pour cela, j’ai réalisé une analyse comparative du modèle ascidie Phallusia mammillata avec le modèle amphioxus Branchiostoma lanceolatum. Des analyses transcriptomiques m’ont permis d’identifier de nouveaux marqueurs du SNPv chez les deux espèces et de proposer deux réseaux de régulation de gènes hypothétiques. Malgré une faible conservation au sein des invertébrés chordés, plusieurs similitudes ont été observées avec le SNP des vertébrés, suggérant un recyclage de certains modules de ces réseaux au niveau du SNP dorsal des vertébrés. Ensuite, j’ai mis en place une nouvelle méthode d’injection et validé l’inactivation génique grâce au système CRISPR/Cas9 chez l’amphioxus. Enfin, je me suis intéressé à la formation des palpes des ascidies. La fonction des voies de signalisation BMP et Wnt dans la formation des palpes suggère des similarités avec la mise en place du télencéphale antérieur des vertébrés<br>Vertebrates develop their peripheral nervous system from unique dorsal structures, the neural crest and the ectodermal placodes. By contrast, in the invertebrate chordates, amphioxus and ascidians, a part of the PNS originates from a ventral neurogenic field. In both groups, a biphasic mechanism regulates ventral PNS formation: high BMP levels specify the ventral ectoderm as a neurogenic territory within which epidermal sensory neurons formation is controlled by the Notch pathway. Thus, it is likely that ventral PNS is an ancestral feature in chordates and that it has been lost in vertebrates or redeployed dorsally to form the neural crest and placodes. In order to test this hypothesis, my project aims at studying the origin and evolution of the vPNS in invertebrate chordates. To this end, a comparative analysis of the ascidian Phallusia mammillata and the amphioxus Branchiostoma lanceolatum was performed. Transcriptomic analyses allowed me to identify novel vPNS markers in both species and to propose two hypothetical gene regulatory networks. Despite low conservation in invertebrate chordates, similarities were observed with vertebrate PNS, suggesting that ancestral vPNS gene networks have been redeployed in vertebrates. Then, in order to validate these networks, I set up a new injection method and established gene inactivation using the CRISPR/Cas9 system in B. lanceolatum. Finally, transcriptomic data mining led me to study the formation of the ascidian palps. Functional studies revealed essential roles for BMP and Wnt signalling pathways in the formation of the palps and suggest similarities with the formation of the anterior telencephalon of vertebrates

In order to study the evolution of metallothioneins (MTs) in deuterostomes I have analysed these proteins in Tunicates, which are invertebrate chordates, sister-group of vertebrates. I have isolated the complete cDNA sequences of Ciona intestinalis, Molgula manhattensis and Ascidiella aspersa MTs. Their deduced amino acid sequences are similar in length (39 to 41 amino acids) and each protein has about 30% of cysteine residues which are organised in the typical clusters of vertebrate MTs. Afterword, my investigation was mainly focused on C. intestinalis, a model tunicate of which the whole genome is available. Nevertheless, no MTs had been previously annotated. After the identification of MT, the research has followed two pathways. The first one was aimed at identifying the tissues involved in the transcription and the gene expression profiles in response to heavy metals such as Zn, Cd and Cu. As regards this last point, in addition to MT, I also considered genes for phytochelatin synthase (PCS), Cu/Zn superoxide dismutase (Cu/Zn SOD), glutamate-cysteine ligase (GCLC) catalytic subunit, glutathione synthetase (GS), glutathione peroxidase 7 (GPX7) and proliferating cell nuclear antigen (PCNA), looking for relationships between them. In order to define their possible evolution, the second approach involved bioinformatic analyses, aimed at understanding the organisation of the gene structure, promoter regions and sequence identity of MTs and of all the other considered proteins. The deduced protein sequence of C. intestinalis MT (CiMT-1) is quite different from the other deuterostome MTs: it is the shortest MT so far identified in this group, with only 39 amino acids in comparison with 61-68 amino acids of the other taxa. In addition, the KKS motif, that links the two domains (α and β) of the vertebrate MTs, is completely missing. The identity between CiMT-1 and other deuterostome MTs is quite low, ranging from 18.8 and 38.3%. Vertebrate MT genes show a tripartite structure, with three exons and two introns. Introns differ in length, but their relative position is conserved: the first intron interrupts the sequence at amino acid 9, while the second intron at amino acid 31 or 32, at the junction of the α and β domains. CIMT-1 shows the same tripartite structure, with the first intron in the typical vertebrate position, while the second intron is located in the 3’UTR, as in echinoderm MTs. The promoter region of CIMT-1 shows features present in both vertebrates and in echinoderms: it contains three half-AREs (antioxidant responsive element) as in echinoderms, and one typical vertebrate ARE. Four MREs (metal responsive elements) are also present. Cd exposure increases the transcription of CIMT-1 in a non linear way: an initial increase, at 6 h, is followed by a decrease to the control levels at 24 h, then another increase is present after 96 h. Treatments with Zn and Cu, however, induce the gene transcription progressively up to the end of treatment (120 h) when the messenger levels are fourfold the control values. CiMT-1 mRNA is present only in haemocytes, specifically in granulocytes. These cells, after 24 h of treatment with Cd, are present in tunic and their amount is twice that of controls. Therefore, the decrease of transcript at 24 h may be a result of this cell migration. The subsequent increase is presumably due to specific cellular proliferation of granulocytes in the blood lacunae. This scenario is confirmed by the high rate of cell death in tunic, where haemocytes accumulate, and also by the massive transcription of PCNA in treated samples. Indeed, both CiMT-1 and PCNA show a peak of expression at 96 h. The results obtained with phytochelatin synthase fit with this hypothesis. In C. intestinalis, PCS is expressed in granulocytes and the mRNA levels remain comparable to those of controls up to 72 h, but then they rise up to 96 h. From literature data it is known that this gene is not inducible, but it is activated in presence of divalent metal ions. Again, it is very likely that the observed messenger peak is not a consequence of an induction phenomenon, but of a strong proliferation of granulocytes. This is the first data on the transcription of this gene, on the location at granulocyte level and on its involvement in metal detoxification processes in deuterostomes. The study of the behaviour of the genes involved in detoxification suggests that the enzymes (SOD, GPX) may have a marginal role, whereas the cystein-rich molecules (PCS, GS, GCL) play an important role in presence of Cd, Cu and Zn. Moreover, the localisation of these transcripts, by in situ hybridisation, suggests that circulating haemocytes are the cells mostly involved in detoxification processes.<br>Lo scopo di questa ricerca è quello di analizzare le metallotioneine (MT) nei Tunicati, taxon il sister-group dei Vertebrati, allo scopo di studiare l’evoluzione delle metallotioneine nei deuterostomi invertebrati. Ho quindi isolato sequenze complete di cDNA per MT nei Tunicati Ciona intestinalis, Molgula manhattensis e Ascidiella aspersa. In questi organismi le MT presentano sequenze aminoacidiche dedotte simili, di lunghezza compresa tra 39 e 41 aminoacidi e con circa il 30% di residui cisteinici organizzati nei tipici cluster conservati delle MT dei Vertebrati. La mia indagine si è quindi concentrata su C. intestinalis, organismo modello dei Tunicati per il quale è disponibile la sequenza dell’intero genoma. Nonostante questo, nessuna MT era stata annotata. Dopo aver identificato la MT, la ricerca si è orientata su due percorsi. Il primo percorso era volto a comprendere quali fossero i tessuti coinvolti nella trascrizione di questo gene e quali ne fossero i profili di espressione in risposta a metalli pesanti quali Zn, Cd e Cu. In quest’ambito ho anche considerato la fitochelatino sitetasi (PCS), la Cu/Zn superossido dismutasi (Cu-Zn SOD), la glutammato cistein-ligasi (GCLC) subunità catalitica, la glutatione sintetasi (GS), la glutatione perossidasi 7 (GPX7) e l’antigene nucleare di proliferazione cellulare (PCNA), al fine di valutarne le relazioni. Il secondo approccio, di tipo bioinformatico, era volto a chiarire l’organizzazione genica, la struttura delle regioni promotrici e le identità di sequenza delle metallotioneine e delle altre proteine studiate, al fine di far luce sulla possibile evoluzione delle sequenze prese in esame. Le sequenza proteica dedotta da C. intestinalis (CiMT-1) è decisamente diversa dalle MT degli altri deuterostomi: essa risulta essere la più corta MT fino ad ora individuata in questo gruppo, presentando 39 aminoacidi contro 61-68 degli altri taxa. Inoltre, manca completamente il motivo KKS che connette i due domini (alpha e beta) delle MT dei vertebrati. Si evidenziano quindi bassi livelli di identità aminoacidica, tra 18,8% e 38,3%, con le MT degli altri deuterostomi. Tutte le MT dei vertebrati hanno struttura genica tripartita, con tre esoni e due introni. Gli introni differiscono in lunghezza, ma la loro posizione relativa è conservata: il primo introne interrompe la sequenza all’aminoacido 9, mentre il secondo introne interrompe la sequenza all’aminoacido 31 o 32, alla giunzione dei domini alpha e beta. CiMT-1 mostra la stessa struttura tripartita, con il primo introne nella posizione tipica dei vertebrati, mentre il secondo introne è posto nella regione 3‘UTR, come nelle MT degli echinodermi. Anche la regione promotrice di CiMT-1mostra caratteristiche presenti sia negli echinodermi che nei vertebrati: possiede tre half-ARE (antioxidant responsive element) come negli echinodermi ed una sequenza ARE tipica dei vertebrati. Esso presenta inoltre quattro MRE (metal responsive elements) ben distanziati. Il trattamento con Cd induce aumenti del trascritto di CiMT-1 con andamento irregolare: un primo aumento è presente a 6 h, seguito da una diminuzione fino a quasi i livelli di controllo a 24 h per poi ricrescere fino ad un picco massimo dopo 96 h. I trattamenti con Zn e Cu, invece, inducono valori di trascrizione quattro volte superiori rispetto ai livelli dei controlli solo a fine trattamento (120 h) con un andamento di crescita pressoché lineare nel tempo. Il trascritto per CiMT-1 è presente a livello dei soli granulociti. Tali cellule a 24 h di trattamento con Cd si accumulano nella tunica in numero circa doppio rispetto ai controlli. Pertanto la diminuzione di trascritto riscontrato a 24 h può essere conseguenza di questa migrazione cellulare, mentre il successivo aumento è presumibilmente da imputare a proliferazione cellulare specifica dei granulociti in circolo. Questo scenario è confermato dall’alto tasso di morte cellulare nella tunica, dove gli emociti si accumulano, ed inoltre dalla massiccia trascrizione di PCNA nei campioni trattati. Infatti sia CiMT-1 che PCNA presentano un picco di espressione a 96 h.. A confermare tale ipotesi sono i risultati relativi alla fitochelatino-sintetasi. In C. intestinalis il gene viene espresso dai granulociti ed i livelli di trascritto rimangono simili a quelli dicontrollo fino a 72 h, per poi crescere a 96 h. Da dati di letteratura è noto che questo gene non è inducibile, ma viene attivato in presenza di ioni metallici bivalenti. E’ molto probabile quindi, che questo picco non sia una conseguenza di un fenomeno di induzione, ma della forte proliferazione cellulare dei granulociti. E’ da notare che questo è il primo dato che dimostra la trascrizione di questo gene in organismi deuterostomi, la sua localizzazione esclusiva a livello dei granulociti e il suo coinvolgimento in processi di detossificazione da metalli. Lo studio degli andamenti degli altri geni coinvolti nella detossificazione suggerisce che la componente enzimatica (SOD, GPX) possa avere un ruolo marginale, mentre la componente tiolica (PCS, GS, GCL) svolga un ruolo primario in presenza sia di Cd, che di Cu e Zn. Inoltre la localizzazione tramite ibridazione in situ di questi trascritti evidenzia che gli emociti circolanti sono le cellule maggiormente coinvolte nei processi di detossificazione.

Chez l’ascidie coloniale Botryllus schlosseri, en plus de l’embryogénèse existent deux voies de développement aboutissant à la production de la même structure : l’organisme adulte ou zooide. Ces développements alternatifs ont lieu lors de processus biologiques distincts : le bourgeonnement palléal (BP) et le bourgeonnement vasculaire (BV). Le BP est un processus de multiplication asexuée présentant une ontogénèse stéréotypée. En revanche, le BV est un phénomène régénératif, induit dans les vaisseaux sanguins de la colonie par l’ablation de tous les zooides et bourgeons palléaux. Mes travaux de recherche ont eu pour objectif de caractériser les bases moléculaires et cellulaires régissant le BP et le BV chez B. schlosseri. L’étude de gènes marqueurs des lignées méso-, endo- et ectodermiques a révélé l’existence de territoires présomptifs pour chacune de ces lignées, dès les premiers stades du BV et du BP, et suggéré l’existence d’un programme unique aux deux processus. Les lignées neurales et musculaires ont été étudiées plus en détail lors du BP, indiquant un double rôle potentiel, neuro- et myo-génétique, au tube dorsal, une structure jusqu’à présent uniquement associée au système nerveux. Une caractérisation morphologique poussée a mené à l’identification de stades précoces stéréotypés du BV lors de la régénération. Enfin, l’analyse de transcriptomes de différents stades du BP et de la régénération ont initié l’étude non biaisée des bases moléculaires du bourgeonnement chez Botryllus. L’objectif à long terme de ces travaux est de décrypter les bases moléculaires et génétiques facilitant, chez les métazoaires, l’évolution de voies de développement alternatives<br>In addition to embryogenesis, the colonial ascidians Botryllus schlosseri evolved two alternative developmental pathways leading to the same final structure: the adult body, or zooid. These non-embryonic ontogenesis occur during distinct biological processes: palleal budding (PB) and vascular budding (VB). PB is a process of asexual propagation, with a very stereotyped morphogenesis. Conversely, VB is a purely regenerative phenomenon, induced in the vascular system of the colony by the ablation of all zooids and palleal buds. My research work followed the objective to characterize the molecular and cellular basis of both PB and VB in B. schlosseri. The study of meso-, endo- and ectodermal lineage marker genes revealed the existence of presumptive territories of these lineages in the early palleal and vascular buds and that a single developmental program was launched in both VB and PB. Neural and muscle fates were studied in more detail for PB, indicating a potential double function, both neuro- and myo-genic for the dorsal tube, a structure so far associated with the nervous system only. A detailed morphological description of VB allowed to identify stereotyped stages during early regeneration. Eventually, a transcriptomic characterization of early VB and PB processes initiated an unbiased study of the molecular basis underlying the budding phenomenon in Botryllus. The overall goal of these research works is to unravel the molecular and genetic basis that facilitated, in Botryllus and globally in metazoan, the evolution of alternative developmental pathways

En biologie développementale, l'étude d'organismes modèles vise à comprendreles mécanismes génétiques responsables de la morphogenèse chez le vivant. Lamicroscopie confocale à fluorescence permet aujourd'hui d'observer in vivo àl'échelle de la cellule et avec une haute fréquence temporelle le développementd'organismes. Les séquences d'images 3D+t ainsi obtenues nécessitent d'avoirdes outils de traitement d'images adaptés.Dans cette thèse, nous construisons des outils dédiés à l'étude dudéveloppement de deux organismes, l'embryon de l'ascidie Phallusiamammillata et le bouton floral d'Arabidopsis thaliana.Nous développons d'abord une méthode de comparaison de segmentationsadaptée aux images de tissus épithéliaux d'organismes en développement.Nous nous appuyons sur cet outil pour valider notre seconde contribution quiporte sur la mise en place d'un outil de détection et de reconstruction demembranes cellulaires conçu afin de procéder à la segmentation de cellulesdans les images d'ascidies et d'arabidopsis.Nous utilisons ensuite l'outil de segmentation de membranes précédemmentintroduit pour construire une stratégie de recalage spatial inter-individusappliquée aux embryons d'ascidies. Enfin, nous élaborons une stratégie derecalage spatio-temporel inter-individus appliquée à des séquences d'images 3Dde méristèmes floraux<br>In developmental biology, the study of model organisms aims for theunderstanding of genetic mechanisms responsible of morphogenesis. Today,fluorescent confocal microscopy is a means for in vivo imaging of developingorganisms at cell level with a high spatio-temporal resolution. To handle such3D+t image sequences, adapted computer-assisted methods are highlydesirable.In this thesis, we build dedicated tools for the study of two developingorganisms, the ascidian Phallusia mammillata's embryo and the Arabidopsisthaliana's floral meristem.We first develop a method for segmentation comparison adapted to developingorganism epithelial tissue images. This tool is then used to validate our secondcontribution that is about the development of a cell membranes detection andreconstruction tool for cell shape segmentation process applied to ascidian andarabidopsis images.We then use the previously introduced membrane detection tool to build aninter-individual spatial registration strategy applied to ascidian embryo images.Finally, we develop an inter-individual spatio-temporal registration strategyapplied to 3D image sequences of arabidopsis floral meristems

Chez l’ascidie coloniale Botryllus schlosseri, en plus de l’embryogénèse existent deux voies de développement aboutissant à la production de la même structure : l’organisme adulte ou zooide. Ces développements alternatifs ont lieu lors de processus biologiques distincts : le bourgeonnement palléal (BP) et le bourgeonnement vasculaire (BV). Le BP est un processus de multiplication asexuée présentant une ontogénèse stéréotypée. En revanche, le BV est un phénomène régénératif, induit dans les vaisseaux sanguins de la colonie par l’ablation de tous les zooides et bourgeons palléaux. Mes travaux de recherche ont eu pour objectif de caractériser les bases moléculaires et cellulaires régissant le BP et le BV chez B. schlosseri. L’étude de gènes marqueurs des lignées méso-, endo- et ectodermiques a révélé l’existence de territoires présomptifs pour chacune de ces lignées, dès les premiers stades du BV et du BP, et suggéré l’existence d’un programme unique aux deux processus. Les lignées neurales et musculaires ont été étudiées plus en détail lors du BP, indiquant un double rôle potentiel, neuro- et myo-génétique, au tube dorsal, une structure jusqu’à présent uniquement associée au système nerveux. Une caractérisation morphologique poussée a mené à l’identification de stades précoces stéréotypés du BV lors de la régénération. Enfin, l’analyse de transcriptomes de différents stades du BP et de la régénération ont initié l’étude non biaisée des bases moléculaires du bourgeonnement chez Botryllus. L’objectif à long terme de ces travaux est de décrypter les bases moléculaires et génétiques facilitant, chez les métazoaires, l’évolution de voies de développement alternatives<br>In addition to embryogenesis, the colonial ascidians Botryllus schlosseri evolved two alternative developmental pathways leading to the same final structure: the adult body, or zooid. These non-embryonic ontogenesis occur during distinct biological processes: palleal budding (PB) and vascular budding (VB). PB is a process of asexual propagation, with a very stereotyped morphogenesis. Conversely, VB is a purely regenerative phenomenon, induced in the vascular system of the colony by the ablation of all zooids and palleal buds. My research work followed the objective to characterize the molecular and cellular basis of both PB and VB in B. schlosseri. The study of meso-, endo- and ectodermal lineage marker genes revealed the existence of presumptive territories of these lineages in the early palleal and vascular buds and that a single developmental program was launched in both VB and PB. Neural and muscle fates were studied in more detail for PB, indicating a potential double function, both neuro- and myo-genic for the dorsal tube, a structure so far associated with the nervous system only. A detailed morphological description of VB allowed to identify stereotyped stages during early regeneration. Eventually, a transcriptomic characterization of early VB and PB processes initiated an unbiased study of the molecular basis underlying the budding phenomenon in Botryllus. The overall goal of these research works is to unravel the molecular and genetic basis that facilitated, in Botryllus and globally in metazoan, the evolution of alternative developmental pathways

Après la fécondation d’un ovocyte, un aster de microtubules se forme autour de l’ADN mâle. Cet aster spermatique permet d’amener le pro-noyau femelle jusqu’au pro-noyau mâle pour qu’ils puissent fusionner. Il permet aussi de déplacer l’ADN fusionné jusqu’au centre de la cellule pour assurer une division cellulaire équitable. Les mécanismes de centration d’un aster ou d’un fuseau ont donné lieu à de nombreuses recherches, que ce soit par modélisation, expérimentalement chez des espèces telles C. elegans, P. lividus, M.musculus ou in vitro sur des extraits de Xenopus laevis. Trois mécanismes principaux se dégagent : le pushing, le cortical pulling et le cytoplasmic pulling (ou bulk pulling). En étudiant le déplacement de l’aster et du fuseau mitotique chez le zygote de l’ascidie P. mammillata j’ai découvert un système qui combine ces trois mécanismes en s’appuyant sur l’alternance des étapes du cycle cellulaire. En méiose, l’aster utilise la polymérisation des microtubules qui le composent pour pousser contre le cortex d’actine et s’en décoller (pushing). Arrivé en interphase, l’aster retourne contre le cortex grâce à une traction qu’exerce la membrane sur les microtubules (cortical pulling). Enfin à l’entrée en mitose, la traction membranaire cesse et libère les asters du fuseau mitotique, qui cèdent donc aux forces exercées par le transport d’organelles vers le centre de l’aster (cytoplasmic pulling) qui semblent constantes durant le cycle cellulaire. Cela permet de centrer le fuseau. En même temps que l’aster se forme et se déplace, une réorganisation des compartiments intracellulaires se met en place. Pour comprendre de quelle manière l’organisation intracellulaire peut être perturbée par la formation de l’aster, j’ai étudié le cas du vitellus. En effet, le vitellus, qui est présent sous forme de vésicules, est initialement abondant et homogène dans l’ovocyte non fécondé. Cependant, dès que l’aster apparaît, sa répartition change et les vésicules de vitellus sont exclues de la zone contenant l’aster. Cette exclusion générée à la formation de l’aster chez le zygote, est maintenue au cours du développement. Dans mes travaux, j’ai pu observer qu’elle est majoritairement due à l’accumulation à l’aster d’autres organelles comme le réticulum endoplasmique. La fonction de transport des microtubules de l’aster suffit donc à réorganiser complètement la cellule en excluant certaines organelles et en en accumulant d’autres. Les déplacements de l’aster et du fuseau mitotique, leur régulation par le cycle cellulaire, et la réorganisation intracellulaire, identifiés ici chez le zygote d’ascidie, s’appuient sur le fonctionnement d’éléments fondamentaux d’une cellule, à savoir : les microtubules, le cortex d’actine, le réticulum endoplasmique, les protéines du cycle cellulaire, etc. Les découvertes présentées revêtent ainsi une portée universelle, adaptable aux spécificités de différents types cellulaires<br>After oocyte fertilization, a microtubule aster forms around the male DNA. The sperm aster brings the female pro-nucleus to the male pro-nucleus so they can fuse, but it also moves the fused nuclei to the cell center to ensure an equitable cell division. Numerous studies performed in vitro, by modeling or experimentally in species such as C. elegans, P. lividus, and M. musculus, addressed the aster and spindle centration mechanisms. Three main mechanisms emerged; pushing, cortical pulling, and cytoplasmic pulling. By studying aster centration in the zygote of the ascidian P. mammillata, I discovered a system that combines these three mechanisms based on the cell cycle stages. In meiosis, the aster uses the polymerization of its microtubules to push against the actin cortex and move away from it (pushing). Once in interphase, the aster returns to the cortex by a pull exerted by the membrane on the microtubules (cortical pulling). At mitosis entry, cortical pulling stops, and releases the mitotic spindle's asters. In consequence, the asters give in to the forces exerted by the transport of organelles to the aster center (cytoplasmic pulling), that appeared constant during the cell cycle. Cytoplasmic pulling hence participate in centering the spindle While the aster forms and moves, the intracellular compartments reorganize. To understand how intracellular organization can be disrupted by aster formation, I studied the case of yolk. The yolk, in the form of vesicles (called granules or platelets), is initially abundant and homogeneous in the unfertilized oocyte. However, as soon as the aster appears, its distribution changes and the yolk platelets are excluded from the region containing the aster. This exclusion generated by the aster formation in the zygote is maintained during development. I observed that yolk exclusion is mainly due to the accumulation at the aster of other organelles such as the endoplasmic reticulum. The transport function of the aster microtubules is therefore sufficient to completely reorganize the cell by excluding some organelles and accumulating others. The movements of the aster and the spindle, their regulation by cell cycle, and the intracellular reorganization, identified here in the ascidian zygote, rely on basic elements of a cell, namely: the microtubules, the actin cortex, the endoplasmic reticulum, the proteins of the cell cycle, etc. Thus, the discoveries presented here cover a broad scope, and seem adaptable to the specificities of different cell types

Durant ma thèse, j’ai participé à une étude fonctionnelle qui a démontré que p120-RasGAP, une protéine appartenant à la famille GAP (GTPase-activating protein), est le médiateur cytoplasmique de l’éphrine lors de l’atténuation d’ERK1/2. Pour confirmer cela, j’ai réalisé une expérience de co-immunoprécipitation et j’ai démontré que p120-RasGAP s’associe au récepteur de l’éphrine, Eph3, quand celui-ci est activé par un ligand éphrine. Ce résultat indique fortement que les signaux FGF et éphrine convergent au niveau de Ras et qu’ils contrôlent de manière antagoniste son activité. Dès lors, j’ai analysé les autres événements de spécification cellulaire impliquant l’antagonisme FGF/éphrine. Chez l’embryon d’ascidie, le signal FGF est décrit comme inducteur du destin neural dans les cellules ectodermiques qui, en absence du signal FGF, adoptent le destin épidermique. L’induction neurale des ascidies a lieu au stade 32 cellules et se traduit par la spécification de quatre précurseurs neuraux (ERK+) parmi les 16 cellules ectodermiques. J’ai démontré que le signal éphrine/Eph/RasGAP antagonise le signal FGF pour générer une activation d’ERK1/2 de type tout ou rien parmi les cellules ectodermiques. Enfin, en collaboration avec Philip Abitua, doctorant dans le laboratoire du Dr. Mike Levine (UC Berkeley), nous démontrons que l’antagonisme entre les signaux éphrine et FGF est impliqué dans la régionalisation antéro-postérieure de la plaque neurale<br>During my thesis study, I was involved in functional studies to demonstrate that p120-RasGAP, a GTPase-activating-protein (GAP), is a cytoplasmic mediator of the ephrin-mediated ERK attenuation. To confirm this notion, I conducted a co-immunoprecipitation experiment and demonstrated that p120-RasGAP associates with an ephrin receptor, Eph3, when the latter is activated by an ephrin ligand in ascidian embryos. These results strongly indicate that FGF and ephrin signals converge at the level of Ras and control its activity antagonistically. Following this finding, I looked for other cell fate specification events controlled by the antagonism between ephrin and FGF signals. In ascidian embryos, FGF signals are known to induce neural fates in ectodermal cells which otherwise adopt epidermal fates. Ascidian neural induction takes place at the 32-cell stage, resulting in specification of specific four cells as ERK1/2-active neural precursors among 16 ectodermal cells. I was able to demonstrate that ephrin/Eph/RasGAP signals counterbalance FGF neural inducing signals to generate the ON-OFF response of ERK activation among the ectodermal cells. Finally, in collaboration with a PhD student in Dr. Mike Levine’s lab (UC Berkeley), the antagonism between ephrin and FGF signals plays a role in regionalisation of the neural plate along the anterior-posterior axis

Le but de mon projet était de décrire les molécules impliquées dans le développement asexué de l'ascidie coloniale Botryllus schlosseri et d'en déduire les mécanismes associés. Les ascidies appartiennent au sous embranchement des Tuniciers, groupe frère des Vertébrés. Elles sont les organismes les plus proches de l'humain capable de se reproduire de manière asexuée par bourgeonnement. Le travail a été réalisé sur l'ascidie coloniale Botryllus qui est composée de plusieurs individus (zoïde) réunis dans une tunique commune. Lors du développement asexué de Botryllus le zoïde peut sauter les stades embryonnaires et larvaires pour directement former un nouveau corps adulte incluant toute la musculature et le système nerveux en bourgeonnant. Dans le but d'étudier l'origine cellulaire et les mécanismes sous-jacents potentiels à la myogenèse non embryonnaire, j'ai suivi l'expression de ces gènes myogéniques lors du bourgeonnement de Botryllus et j'ai reconstruit la dynamique des précurseurs des muscles. Les orthologues de ces molécules ne sont pas exprimés seulement chez les ascidies mais également lors du développement du muscle cardio-pharyngé chez les Vertébrés et l'origine commune du cœur et des muscles pharyngés dans le champ cardio-pharyngé était déjà présente chez l'ancêtre commun des Tuniciers et des Vertébrés. En étudiant l'origine cellulaire lors de la neurogenèse j'ai pu observer que des gènes larvaires qui déterminent l'axe antérieur postérieur (AP) ont été cooptés dans une structure transitoire neuronale qui donne le ganglion cérébroïde. Ils divisent la structure en trois parties, évoquant ainsi de la tripartition de système nerveux des Vertébrés<br>The aim of this work is to describe via molecular-biological methods the asexual form of development of the colonial ascidian Botryllus schlosseri focusing on the formation of different tissues, namely muscles and nervous system, as well as exploring the potential presence of structures or cells homologous to the neural crest. Ascidians belong to the subphylum tunicates, sister group of vertebrates and are the closest relatives to man that can reproduce asexually, by budding. As colonial ascidian, the metamorphosis of Botryllus schlosseri specimen is followed by a lifelong, recurring, highly coordinated budding process, where multiple individuals (zooids) are connected and embedded in a common tunic. During asexual development, zooids can develop in a direct manner without embryonic and larval stages. To study the cellular origin and mechanisms of non-embryonic myogenesis I followed the expression pattern and dynamics of myogenic genes during asexual development and reconstructed muscle precursors. Orthologs of these genes are not only expressed during muscle formation via larval development but also during the formation of cardio-pharyngeal muscles in the vertebrate embryogenesis. I further drew a comparison of the regionalization of a transitory neurogenic structure, the dorsal tube, along the anteroposterior axis during budding with its larval counterpart, the neural tube, thus adding a RNA-expression profile of neural genes hereby proposing a scenario of cerebral ganglion formation by delamination. To better understand the nature of the dorsal tube and gangliogenesis I investigated potential involvement gene orthologs implemented in vertebrate neural crest formation

In the last years, several studies are addressed to the investigation of mechanisms that permitted the appearance and evolution of those structures considered as extremely important in the vertebrates radiation. The rise of vertebrates was accompanied by the acquisition of a great complexity in the structural plan of the organisms that is related to the evolution of features associated with the nervous system, such as neural crests, cranial placodes and an elaborated brain. The aim of the doctoral project is inserted in this line of research. Particularly, the attention is addressed to those characters, which in the non- vertebrate chordates can be interpreted as crucial for the subsequent evolution of the vertebrate body-plan. The starting point is represented by previous morphological studies that evidenced the presence, in the tunicate embryo, of transitory ectodermal and multipotential territories located at the neural plate border. Our research, carried out using different approaches, is focused on the characterisation and description of structures that differentiate from these domains. In this regard, it is investigated the organisation of the larval papillae and their formation from the rostral placode. These structures play a pivotal role in triggering the mechanisms and changes that characterise the metamorphosis, which in ascidians constitute the lost of the chordate body-plan of the larva and the begin of the sessile post-embryonic phase. The analysis of the sensory components in ascidians, finalised to the identification of homologies with the structures that derive form the placodes in vertebrates, is also extended to the coronal organ. The coronal organ is recently discovered and possesses morphological, positional and ultrastructural features that, together with the presence of hair cells, are comparable to the lateral line and inner ear of vertebrates, which components derive from the acoustic-lateral placodes. A substantial part of the work is dedicated to the investigation, with a molecular approach, of the presence of structures comparable to the neural placodes in ascidians. The attention is focused on the colonial ascidian Botryllus schlosseri that permits a comparative study on the mechanisms and genic networks involved in both embryogenetic and blastogenetic development. We characterised orthologues of placodal genes and constructed probes for in situ hybridisation experiments. During the stages of bud differentiation, we localised territories interested by expression of genes normally involved in the placodal induction and specification in vertebrates. These regions, for their position and differentiative potentialities, are comparable to other embryonic domains of B. schlosseri that are considered as homologues to the neural placodes. The larva of ascidians possesses a striated symmetrical musculature sharing some features with that of vertebrates and it flanks the dorsal tube and the notochord, representing a peculiar propriety of chordates. The acquisition of this new locomotory system probably required the parallel appearance of a sophisticated control of the coordination. The nervous system established new interactions and differentiated sensory structures that permitted the rapid perception of the environment by the mobile organism. At metamorphosis, the larval musculature is reabsorbed, while the cardiac and unstriated muscle fibres differentiate de novo from circulating mesenchymal cells. This plasticity constitutes the source of the evolutive potentiality of ascidians and thus we investigate its molecular and morphological bases. We isolated and characterised transcripts coding for muscle-specific genes, analysing the expression during the blastogenetic cycle of Botryllus, from the bud appearance to the adult regression. The use of different methods allowed the description of the organisation and differentiation of the unstriated muscle, confirming its unique proprieties. Taking together, our results contribute to the understanding of the origin and development of structures that represent an important starting point in the evolution and radiation of vertebrates.<br>Negli ultimi anni numerosi studi si sono rivolti all’approfondimento di quei meccanismi che hanno permesso la comparsa ed evoluzione di strutture ritenute di estrema importanza nella radiazione dei vertebrati. La comparsa dei vertebrati è stata accompagnata da un enorme balzo nella complessità del piano strutturale degli organismi, largamente ascrivibile all’evoluzione di strutture associate al sistema nervoso come le creste neurali, i placodi craniali ed un cervello elaborato. La tematica trattata durante lo svolgimento del progetto di dottorato si inserisce in questa attuale linea di ricerca. In particolare, l’attenzione è stata rivolta a quei caratteri che nei cordati non-vertebrati possono essere letti come cruciali per la successiva evoluzione del piano strutturale dei vertebrati. Il punto di partenza è rappresentato da precedenti studi morfologici che hanno evidenziato la presenza, nell’embrione dei tunicati, di territori ectodermici transitori e multipotenti localizzati al confine con la piastra neurale. Il nostro studio, svolto mediante l'utilizzo di vari approcci metodologici, si è rivolto alla caratterizzazione e descrizione delle strutture che queste aree sono in grado di differenziare. A questo proposito, è stata analizzata l’organizzazione delle papille larvali e la loro formazione a partire dal placode rostrale. Queste strutture giocano un ruolo primario nell’innescare i meccanismi e i cambiamenti che caratterizzano la metamorfosi, ovvero quel processo che nelle ascidie segna la perdita del piano corporeo da cordato della larva e il passaggio alla fase post-embrionale sessile. L’analisi riguardante strutture sensoriali presenti nelle ascidie, allo scopo di identificare eventuali omologie con le corrispondenti strutture derivanti dai placodi nei vertebrati, è stata poi estesa all’organo coronale. L’organo coronale è stato scoperto solo recentemente e presenta caratteristiche morfologiche generali, posizionali e ultrastrutturali tali, come la presenza di cellule capellute, che lo rendono comparabile alla linea laterale ed all’orecchio interno dei vertebrati, i cui componenti derivano dai placodi acustico-laterali. Una parte consistente del lavoro è stata dedicata all’indagine, da un punto di vista molecolare, della presenza di strutture accomunabile ai placodi neurali nelle ascidie. L’attenzione è stata rivolta all’ascidia coloniale Botryllus schlosseri, che permette di svolgere uno studio comparativo sui meccanismi e reti geniche che intervengono sia durante lo sviluppo embriogenetico che blastogenetico. Abbiamo caratterizzato specifici geni e prodotto sonde utilizzate in esperimenti di ibridazione in situ. Durante le fasi di differenziamento della gemma sono stati individuati specifici territori caratterizzati da espressione di alcuni geni normalmente coinvolti nell’induzione e specificazione placodale nei vertebrati. Grazie alla loro posizione e potenzialità differenziativa, queste stesse regioni sono apparse confrontabili con altri territori embrionali di B. schlosseri e di altre ascidie considerati omologhi a placodi neurali dei vertebrati. La larva delle ascidie presenta una muscolatura simmetrica striata, con caratteri comuni a quella dei vertebrati, la quale fiancheggia il tubo dorsale e la notocorda e che rappresenta una proprietà peculiare dei cordati. L’acquisizione di questo nuovo sistema locomotorio ha verosimilmente richiesto la comparsa parallela di un sofisticato sistema di controllo della coordinazione. Il sistema nervoso ha stabilito nuove interazioni e differenziato strutture sensoriali che hanno permesso all’organismo mobile la rapida percezione dell’ambiente circostante. Alla metamorfosi, la muscolatura larvale viene completamente riassorbita, mentre le fibre muscolari non striate della parete del corpo e quelle cardiache si differenziano de novo da cellule mesenchimali circolanti. Questa plasticità sta alla base della potenzialità evolutiva delle ascidie e quindi ne abbiamo indagato le basi molecolari e morfologiche. Abbiamo quindi isolato e caratterizzato trascritti e geni muscolo-specifici, studiandone l’espressione durante il ciclo blastogenetico di Botryllus, dalla comparsa della gemma alla regressione dell’adulto. L ’utilizzo di vari approcci ha permesso la descrizione dell’organizzazione e differenziamento della muscolatura non striata, confermandone le caratteristiche uniche. Nel complesso i diversi risultati rappresentano contributi significativi per la conoscenza delle origini e sviluppo quelle strutture che hanno rappresentato un punto di partenza importante nell’evoluzione e radiazione dei vertebrati.

Cette these est consacree a l'etude de l'ascidie mexicaine stomozoa murrayi (kott, 1957) des recifs de corail des caraibes, qui n'est pas colonisee par des macro-epibiontes. Cette espece est decrite pour la premiere fois en mer des caraibes et elle donne lieu a une investigation chimique et ecologique. L'extraction chimique de cet organisme a conduit a l'isolement de l'indole-3-carbaldehyde et du 6-bromoindole-3-carbaldehyde. Ces deux composes, obtenus auparavant d'autres organismes marins, ont ete detectes en cg/sm dans la culture de la bacterie acinetobacter lwoffii, isolee de la surface de s. Murrayi. Le compose brome montre une inhibition in vitro de la fixation des larves de balane, des proprietes antibacteriennes moderees, mais ne montre pas d'activites antialgales ni phagorepulsives vis a vis de poissons. La bacterie a. Lwoffii pourrait jouer un role important en la faible colonisation sur la surface de l'ascidie s. Murrayi comme nous l'avons observe in situ.

Au cours de mon travail de thèse, j'ai établi une procédure pour ordonner des motifs courts selon leur distribution dans le génome de C. Intestinalis autour d'un ensemble de gènes : plus une combinaison de motifs est regroupée autour des gènes exprimés spécifiquement dans un tissu, plus le score est élevé. Dans cette approche, j'ai intégré les résultats de la dissection d'un enhancer exprimé dans le neurectoderme antérieur qui indiquait une structure dupliquée. Ma procédure montre que les sites GATTA dupliqués sont une caractéristique générale des éléments cis-régulateurs du neurectoderme antérieur. Une recherche dans le génome entier pour ces séquences suivi par un test in-vivo montre une expression dans ce territoire pour la moitié des éléments. Par la suite, j'ai essayé d'améliorer l'annotation des séquences cis-régulatrices déjà publiées, par une extraction automatique à partir de la littérature. Grâce à l'augmentation du nombre de publications en accès libre et l'amélioration des procédures expérimentales de plus en plus de ce type de données est disponible. Finalement, j'ai montré que malgré l'absence d'alignements non-codants entre les génomes des vertébrés et C. Intestinalis, on peut cependant trouver quelques loci avec une conservation extrême de l'arrangement des gènes. Pour ces cas, la recherche des éléments cis-régulateurs peut être limitée aux introns du gène voisin, ce qui était confirmé dans la littérature pour certains. Ces trois approches montrent l'utilité de la bioinformatique pour une meilleure caractérisation des séquences cis-régulatrices et ouvrent la voie aux validations expérimentales supplémentaires<br>In this thesis, a procedure is presented to rank combinations of short sequence motifs by their distribution around a set of genes. The better a combination matches around genes expressed in a certain tissue, the higher is its score. I applied this to an already characterized enhancer of C. Intestinalis expressed in the anterior neurectoderm which had been found by systematic mutations to be composed of a duplicated structure. The results of my procedure indicated that duplicated GA TTA-sites are an essential feature of cis-regulatory elements active in the anterior neurectoderm. Searching the genome for matches to this signature resulted in putative enhancers that drive a reporter gene in 50% of the cases in the anterior neurectoderm. In addition, I tried to improve the curation of already published cis-regulatory elements by extracting them automatically from the full text of the biological research articles. Thanks to the thriving open access publishing model and the improvement in experimental assays, more and more of this data is becoming available. Finally, I showed that in the absence of non-coding sequence alignments between the genomes of vertebrate and C. Intestinalis, one can nevertheless find a handful of loci with a very unusually conserved gene order. In these cases, the cis-regulatory search space is reduced to a set of introns, some of which were recently shown to harbor enhancers. Many of these loci have not been analyzed yet. Together, these computational approaches should lead to a better characterization of cis-regulatory sequences and pave the way for further experimental validations

Par purification biodirigee, ce travail a conduit a l'isolement et a la caracterisation de metabolites secondaires a partir des ascidies trididemnum cereum et cystodytes della chiajei. Plusieurs dicetopiperazines, dont la maculosine, ont ete isolees de trididemnum cereum, caracterisees par etudes spectroscopiques (r. M. N. #1h et #1#3c/1 et 2d, s. M. Principalement) et leurs structures confirmees par synthese en phase liquide. A partir de cystodytes della chiajei, trois cyclotetrapeptides ont ete mis en evidence, caracterises et synthetises. L'etude conformationnelle de ces peptides a ete aussi effectue et permet d'avancer une hypothese sur la difference d'activite cytotoxique entre molecules naturelles et produits synthetises

Els animals estan agrupats en grans grups o files. Aquestes agrupacions representen organismes que presenten característiques que els hi són pròpies i diferents a les que presenten altres organismes. Aquest treball es centra en l'estudi dels cordats; format pels subfiles dels urocordats, els cefalocordats i els vertebrats. Els elements transposables són un conjunt molt heterogeni de seqüències que es caracteritzen per la seva capacitat de moure's al llarg d'un genoma i per la presència en múltiples còpies per genoma. Aquests elements, a conseqüència de les característiques que els hi són pròpies poden produir efectes deleteris en el genoma que els allotja tot i que si es troben regulats per mecanismes de control de l'hoste poden arribar a ser beneficiosos ja que n'augmenten el potencial evolutiu. Els estudis d'aquests elements en els cordats gnatostomats com l'home han evidenciat la seva massiva presència. Ja que aquests elements deuen haver condicionat l'evolució dels genomes dels gnatostomats, en aquest treball es va voler establir la participació dels elements transposables de tipus non-LTR en els organismes que es troben en la transició dels cordats pre-vertebrats cap als vertebrats. Així, utilitzant aproximacions in vitro i in silico s'ha determinat el tipus i nombre d'aquests elements en els genomes de l'ascidi <i>Ciona intestinalis</i>, el cefalocordat <i>Branchiostoma floridae</i> i l'àgnat <i>Myxine glutinosa</i>. Les diferents metodologies ens han permès determinar clarament l'estructura de 5 retrotransposons non-LTR en el genoma de l'ascidi, 6 en el de l'amfiox i 1 en el de la mixina. Els baixos números de còpies observats (<200 còpies/genoma haploid) en l'ascidi i l'amfiox mostren com la gran explosió d'aquests elements es va produir en els vertebrats ja que el vertebrat basal <i>M. glutinosa</i> ja presenta valors significativament elevats (~50000 còpies per genoma haploid). A més l'absència de metilació del elements de l'ascidi i l'amfiox però no en la mixina suggereix que la utilització d'aquest marcador epigenètic de l'expressió gènica hauria estat una coopció exclusiva dels vertebrats i no pas de tots els cordats. Per altra banda, evidències indirectes de l'activitat d'aquests elements suggeririen que aquests elements podrien ser actius en tots els genomes que s'han analitzat en aquest treball.<br>The animal kingdom is divided in huge groups of animals or phyla. These groups represent animals which share some particular traits that make them distinguishable from other animals. This work is based on the study of the chordates, which include the urochordate, cephalochordate and vertebrate subphyla. The transposable elements are an heterogeneous array of sequences characterized by their capacity of self mobilization along the host genome and to be present in multiple copies. These elements, as a consequence of their characteristics, could be deleterious to the host but the presence of control mechanisms by the host or the element itself might allow a beneficial relationship as these elements increase the evolbability of the genome. The studies of transposable elements in gnathostomate vertebrates, as humans, have revealed their massive presence on the host genome. As these elements should have participated in the evolution of gnathostomate subphylum, this work has tried to elucidate the role of non-LTR retrotransposons in the evolution of the organisms that appeared during the transition from the prevertebrate chordates to the vertebrates. Using in vitro and in silico approaches, we have determined the kind and number of these elements in the genome of an ascidian (Ciona intestinalis), the cephalochordate amphioxus (Branchiostoma floridae) and the agnathan hagfish (Myxine glutinosa). These approaches allowed us to clearly determine the structure of 5 non-LTR retrotransposon in the ascidian genome, 6 in amphioxus and 1 in hagfish. The low copy number observed for these elements in the genomes of the ascidian and amphioxus (less than 200 copies per haploid genome) clearly shows that the great increase of these elements occurred in the vertebrates, because the hagfish shows a number of copies similar to those in other fishes (~50000 copies per haploid genome). In addition, the absence of methylation in the non-LTR retrotransposons of the ascidian and amphioxus, but not in hagfish, suggests that the use of this epigenetic marker should be coopted in the vertebrate lineage. Even more, indirect results point to the presence of active elements in the host genome of the three studied subphyla.

J’étudie dans cette thèse les mécanismes moléculaires de l’asymétrie précoce de l’ectoderme d’un chordé, l’ascidie Ciona intestinalis. Rose (1939) a été la première à mettre en évidence que les ectodermes antérieur et postérieur possèdent une différence de compétence à répondre aux signaux més-endodermiques. Le lignage antérieur a semble plus compétent que le lignage postérieur b à former du tissu neural antérieur (tel que le cerveau). L’identification d’un marqueur spécifique de l’ectoderme antérieur, le gène Ci-SFRP1/5, a permis l’étude des événements en amont de son expression au stade 64-cellules. J’ai ainsi identifié un élément cis-régulateur exprimé spécifiquement dans l’ectoderme antérieur dès le stade 64-cellules. Cet élément contient 3 sites putatifs de fixation Fox, et est activé par la sur-expression de Ci-FoxA-a, le gène antérieur le plus précoce connu. Ci-FoxA-a, exprimé dans la moitié antérieure de l’embryon dès le stade 8-cellules mais absent de l’ectoderme postérieur, joue un rôle global dans l’ectoderme, activant les marqueurs antérieurs et réprimant activement le programme postérieur. L’expression du gène postérieur le plus précoce, Ci-Nodal, semble inhibée antérieurement par la présence d’un Fox. Une compétition stérique entre FoxA-a et les activateurs GATA et ETS semble l’hypothèse la plus probable pour expliquer cette inhibition. Dans l’ectoderme antérieur, la perte de fonction de Ci-SFRP1/5, connu chez d’autres organismes comme inhibiteur sécrété des Wnt, apparaît insuffisante pour supprimer la formation de l’ectoderme antérieur, révélant une possible redondance fonctionnelle. L’activation de la voie canonique Wnt/ß-caténine est néanmoins incompatible avec le développement du tissu neural antérieur. Je discute enfin, au regard des autres modèles de détermination antéro-postérieure de l’ectoderme chez les chordés, les mécanismes conservés et les stratégies nouvelles développées par l’embryon d’ascidie<br>In this thesis, I address the molecular mechanism of the early asymmetry in the ectoderm of a chordate, the ascidian Ciona intestinalis. Since Rose (1939) it was known that anterior and posterior ectodermal lineages have different competence to respond to endo-mesodermal signals. The anterior a-line appears more competent than the posterior b-line to form anterior neural tissue (brain). The identification of a specific marker for the anterior ectoderm, the Ci-SFRP1/5 gene, allowed addressing the events upstream of its expression at the 64-cell stage. I identified a small cis-regulatory element expressed from the 64-cell stage specifically in the anterior ectoderm, and activated by Ci-FoxAa-a, the earliest known anterior specific gene. Ci-FoxA-a, expressed in the anterior half of the embryo from the 8-cell stage but absent from the posterior ectoderm plays a global role in the ectoderm, activating the anterior ectodermal markers while actively repressing the posterior ectodermal programme. I finally discuss, in light of other model for anteroposterior patterning in chordates, the common conserved mechanisms and the possible new strategies develop by the ascidian embryo

Le cortex des œufs d’ascidies est hautement polarisé suivant l’axe animal-végétatif (a-v) à l’issue de l’ovogenèse, puis suivant les axes Dorso-Ventral (D-V) et Antero-Posterieur (A-P) à partir de la fécondation jusqu’au 1er clivage. Les ovocytes matures d’ascidie sont caractérisés par la distribution en gradient (a-v) de - 1) un domaine sous-cortical riche en mitochondries (appelé myoplasme) ; - 2) d’un domaine riche en Reticulum Endoplasmique cortical (REc) et d’une classe d’ARN messagers corticaux d’origine maternelle (appelée ARNm postplasmiques/PEM). Nous avons montré que la polarisation (a-v) de ces domaines s’effectue au cours de la maturation des ovocytes. Le cortex des œufs subit à l’issue de la fécondation 2 phases majeures de réorganisations. Le myoplasme, le REc ainsi que les ARNm postplasmiques/PEM se concentrent dans le pôle de contraction végétatif (futur pôle Dorsal) au cours d’une première phase majeure de réorganisation acto-myosine dépendante. Le myoplasme, le REc/ARNm corticaux sont ensuite déplacés au niveau du pôle postérieur lors d’une seconde phase majeure de réorganisation dépendante des microtubules. Ces domaines sont répartis de façon équivalente entre les blastomères au cours du premier clivage. Aux stades 2-4 cellules, le myoplasme, le REc et les ARNm postplasmiques/PEM s’accumulent dans les blastomères postérieurs. Au stade 8 cellules, le REc et les ARNm postplasmiques/PEM se concentrent au niveau d’une structure macroscopique corticale appelée CAB (pour Centrosome Attracting Body) localisée dans les blastomères végétatifs les plus postérieurs (B4. 1). Le CAB est impliqué dans la genèse de 3 divisions inégales successives et la ségrégation des ARNm postplasmiques/PEM. Nous avons caractérisé pour la première fois l’évolution et la dynamique de cette polarité corticale en utilisant des cortex isolés à partir d’ovocytes, de zygotes et d’embryons au stade 8 cellules. Nous avons montré que deux ARNm postplasmiques/PEM, PEM1 et macho1, respectivement impliqués dans la formation des axes et la différenciation des cellules musculaires primaires, sont ancrés à la surface d’un réseau polarisé de RE corticale rugueux déjà présent dans les ovocytes matures. Après fécondation, ces ARNm corticaux se concentrent dans le cortex végétatif avec le REc (formant un domaine REc/ ARNm). Ce domaine REc/ARNm se relocalise ensuite en position postérieure avant le 1er clivage et s’accumulent avec celui-ci dans le CAB au stade 8 cellules. Nous discutons 1) le rôle du cytosquelette dans la relocalisation du domaine polarisé riche en REc/ARNm après fécondation, et dans la formation du CAB ; 2) les mécanismes de ségrégation des ARNm postplasmiques/PEM dans les blastomères postérieurs de l’embryon ; 3) les conséquences de ces remaniements dans la différenciation de l’embryon d’ascidie et en particulier celle des cellules musculaires primaires<br>The ascidian egg cortex is highly polarized along the animal-vegetal (a-v) axis at the end of oogenesis, and along the Dorso-Ventral (D-V) axis and Antero-Posterior (A-P) axis between fertilization and first cleavage. Mature ascidian oocytes display (a-v) gradients of 1) a mitochondria-rich subcortical domain (called myoplasm), 2) a network of cortical Endoplasmic Reticulum (cER), and several cortical maternal mRNAs called postplasmic/PEM RNAs. We show that these domains and mRNAs acquire their polarized distribution during oocyte maturation. After fertilization the oocyte cortex undergoes 2 major phases of reorganization. The cortical (cER) and subcortical (myoplasm) domains are first concentrated in the vegetal contraction pole (future dorsal pole) during an acto-myosin dependant cortical contraction(first major phase of reorganization). The myoplasm, cER/mRNA domains are then translocated posteriorly by a microtubule-dependant movement of the sperm aster with respect to the cortex (second major phase of reorganization). The domains are distributed equally between blastomeres during the first cleavage. At the 2-4 cell stage, the myoplasm, cER and postplasmic/PEM RNAs accumulate in posterior blastomeres. At the 8 cell stage, cER and postplasmic/PEM RNAs are concentrated in a cortical macroscopic structure called Centrosome Attracting Body (CAB) located in the vegetal posterior-most blastomeres (B4. 1). The CAB is involved in the formation of three successive unequal cleavages and in mRNA segregation in small posterior blastomeres. We have characterized for the first time the evolution and dynamics of this cortical polarity using cortex isolation and characterization in oocytes, zygotes and early embryos (8 cell stage). We observe that two postplasmic/PEM RNAs, PEM1 and macho1 respectively involved in axes formation and primary muscle cell formation, are anchored to the surface of the polarized network of cortical rough ER. After fertilization these cortical RNAs are concentrated in the vegetal cortex with the cER (forming a cER/mRNA domain). The cER/mRNA domain moves posteriorly before the first cleavage and compacts into the CAB at the 8 cell stage. We discuss how the cytoskeleton relocates the cER/mRNA domain and how the CAB may form from the translocation and compaction of polarized cER/mRNA domain already present in the oocyte. We also discuss how the segregation of postplasmic/PEM RNAs into specific blastomeres directs development and differentiation of the posterior region of the embryo and particular primary muscle cell formation

Ce travail a été réalisé dans le cadre du programme de recherche ECIMAR. Nous avons évalué le potentiel que représente la biodiversité marine en Méditerranée en terme de chimiodiversité, et cherché à mieux comprendre comment s’exprime et varie cette diversité chimique, en nous focalisant sur les ascidies et sur une éponge. Les profils métaboliques d’une cinquantaine d’ascidies ont été enregistrés. Leur comparaison a clairement mis en évidence une plus grande diversité chimique pour les ascidies coloniales que pour les ascidies solitaires. Les cartes d’identité chimique de deux espèces d’ascidies du genre Cystodytes et de l’éponge Dysidea avara ont été établies. L’étude de C. Solitus, a permis l’isolement et la caractérisation de deux dérivés de la staurosporine, famille chimique nouvelle dans le genre. De C. Dellechiajei, nous avons isolé et caractérisé trois nouvelles pyridoacridines. Nous avons également mis en évidence la présence d’un composé quinolinonique dont la structure n’a pu être entièrement établie. L’éponge D. Avara contient des terpènes et a été choisie comme espèce modèle pour l’étude de la fluctuation de l’expression des métabolites secondaires en fonction de divers facteurs biotiques et abiotiques. Enfin, nous avons proposé une nouvelle hypothèse de biosynthèse pour les composés de la famille des pyridoacridines. C. Dellechiajei, ascidie riche en pyridoacridines, a été choisie comme modèle d’étude pour la mise en place d’un nouveau protocole afin de valider notre hypothèse de biosynthèse en extrait acellulaire<br>This work was performed within the ECIMAR research program. We have evaluated the potential of marine biodiversity in the Mediterranean Sea in terms of chemodiversity and tried to better understand how this chemical diversity is expressed and varies, by focusing on ascidians and a sponge. The metabolic profiles of fifty ascidians were recorded. Their comparison has clearly shown a greater chemical diversity for colonial ascidians than solitary ones. Chemical identity cards of two ascidian species of the genus Cystodytes and of the sponge Dysidea avara were established. The study of C. Solitus, allowed the isolation and characterization of two staurosporine derivatives belonging to a chemical family reported for the first time in this genus. From C. Dellechiajei, we have isolated and characterized three new pyridoacridines. We have also demonstrated the presence of a quinolinonic compound, not fully characterized for now. The sponge D. Avara contains terpenes and was selected as a model species for studying the fluctuation of the secondary metabolites expression according to various biotic and abiotic factors. Finally, we have proposed a new biosynthetic pathway for pyridoacridine alkaloids. In order to validate our hypothesis we have developed a new method based on cell-free extracts on the model study C. Dellechiajei, which is well-known for containing a whole variety of pyridoacridines

Les chimistes s’intéressent de plus en plus aux organismes marins dépourvus de protection mécanique (carapace, coquille…) dont la survie dépend de leur capacité à émettre des substances répulsives, voire des poisons. Les spongiaires, les ascidies et les coraux mous sont ainsi producteurs de molécules souvent de structure complexe et potentiellement intéressantes pour des usages biomédicaux. Ce manuscrit présente les travaux de recherche réalisés sur cinq éponges et une ascidie récoltées dans l’Océan Indien, près de Madagascar ainsi que sur un corail mou collecté à l’Ile de la Réunion. D’après la littérature parmi les sept organismes marins sélectionnés, six n’avaient jamais été étudiés auparavant. Nos recherches ont été essentiellement axées sur la séparation et la purification de métabolites secondaires à l’aide des méthodes chromatographiques usuelles ainsi que sur la caractérisation de ces métabolites à partir de différentes techniques spectroscopiques (RMN 1D, 2D, SM…). Cette étude a permis de mettre en évidence 20 composés dont 13 sont connus mais nouvellement caractérisés dans les organismes marins étudiés ainsi que 7 nouveaux composés dont la Ptilomycaline D, un épimère et un diastéréoisomère de la Plakortide N, les Flexusines A, B et C et un épimukulol<br>The Chemists are interested more and more in marine organisms deprived of mechanical protection (shell. . . ) whose survival depends on their capacity to emit repulsive substances, even poisons. Sponges, ascidians and soft corals have produced a wide variety of biologically active (so biomedical interest) and structurally unique metabolites (often with complex structures). Our work concerned essentially the isolation and the identification of secondary metabolites isolated from five marine sponges and one ascidia of Madagascar as well as a soft coral collected in Reunion Island. According to the literature, among the seven marine organisms selected, six had never been studied before. Thus, after extraction and fractionation using various chromatographic methods (exclusion, reverse phase. . . ), we have isolated 20 compounds which have been identified using spectroscopic techniques (NMR, MS…). Among these 20 compounds, 13 are known but first reported in our marine organisms and 7 are new compounds including Ptilomycalin D, an epimer and a diastereoisomer of the Plakortide N, the Flexusines A, B and C, and an epimukulol

Cells analogous to vertebrate endocrine cells have been described in the gut epithelium of the ascidian Styela clava. As well as some histochemical similarities, ultrastructural correlations have been demonstrated, particularly the presence of electron dense granules, clustered around and mainly below the nucleus, thinning out towards the apex. Like the endocrine cells of the vertebrate gut, the cells are often pyramidal, with a narrow apex which is occasionally observed to extend to the lumen of the gut. In addition, strong secretin immunofluorescence was observed in the endocrine-like (E-L) cells of the mucous cap of the gastric ridges. Because of these observations, acid extracts of Styela gut were assayed for secretin in the rat and in the turkey. The Styela extracts as prepared were inactive but it is possible that this reflected faults in the extraction technique. Development of a perfused Styela gut preparation, however, produced evidence to support the hypothesis that a CCK-like peptide is released into the circulation, presumably from the E-L cap cells, although CCK-like immunoreactivity is not demonstrable in these cells. The observation that in addition to CCK, bombesin and physalaemin also induce enzyme release suggests that the pre-pancreatic zymogen cells contain a rich complement of receptors, corresponding to all the classes of PI stimulating receptors which have been found on vertebrate acinar cells. There is therefore the implication that these hormones or analogues may be present in Styela. As secretin was found not to act as a secretagogue in this system, the significance of its production is unclear. By analogy with vertebrate systems it may exert some control over the secretion of mucus by the cap cells.

The blood cells of the solitary ascidian, Ciona intestinalis, were examined for lymphocyte-like functions with a view to clarifying the phylogenetic origin of lymphocytes in invertebrates. It was found that cells, present in the circulating blood, mediate the haemolysis of sheep red blood cells, and that a different cell type mediates cytotoxic activity against a range of mammalian tumour cell lines in vitro. The blood cells, cytotoxic to mammalian cell lines, were enriched by continuous density gradient centrifugation, and their activity was ameliorated by heat-treatment. Parameters of cytotoxic activity against the target cell line, WEHI, a mouse myelomonocytic leukemic cell (strain 3B), were ascertained by fluorochromasia and the phenomenon was found to be rapid, temperature dependent and sensitive to osmotic conditions. Cytotoxicity was also found to be dependent upon the presence of magnesium and calcium ions, effector to target cell binding, and active metabolic, cytoskeletal and secretory processes in the effector cells. The cytotoxic cells were non-adherent to glass or nylon wool and transmission electron microscope studies of the target-binding cells showed that they were undifferentiated, with a high nucleus to cytoplasm ratio, containing a few large mitochondria, some profiles of rough endoplasmic reticulum and many free ribosomes. In addition, TEM studies revealed close inter-digitation of the effector and target cell membranes and evidence of secretory activity within the effector cell, in the area of target cell binding. The effector cell population was cultured in vitro and proliferation in response to concanavalin A, phytohaemagglutinin-B, lipopolysaccharide, or allogeneic leucocytes, measured by the uptake of tritiated thymidine, showed that these cells respond to both T and B cell mitogens and exhibit a mixed leucocyte reaction. In addition, the culture of pharyngeal explants and the measurement of cytotoxic activity by cells migrating from the explants indicates that the cytotoxic cells originate in a thymus-like haemopoietic tissue. Therefore, the undifferentiated blood cells of C. intestinalis, possess functional and morphological properties consistent with phylogenetic precursors of vertebrate lymphocytes.

L'ascidie coloniale Cystodytes est une espèce cosmopolite présente en Méditerranée sous plusieurs morphotypes. La variabilité de la production en métabolites secondaires chez Cystodytes, a été étudiée en fonction d'un certain nombre de paramètres biologiques et écologiques. Pour cela un ensemble de techniques de biologie moléculaire, phylogéographie, chimie et microscopie électronique a été utilisé. Les analyses chimiques ont permis de distinguer deux chemotypes. Le premier est constitué de pyridoacridines soufrées comme la shermilamine B, la kuanoniamine D et leurs formes déacétylées. Ces alcaloïdes sont présents dans les cellules pigmentaires des colonies de couleur violette. Le second est constitué de pyridoacridines non soufrées comme l'ascididemine et la 11-hydroxyascididemine, présentes uniquement dans les formes chromatiques bleue et verte. Les extraits, enrichis en pyridoacridines, des différents morphotypes sont toxiques et réduisent significativement la prédation par les poissons mais pas par les oursins. Les analyses génétiques montrent l'existence de plusieurs espèces en Méditerranée, ne pouvant être identifiés ni par leur couleur ni par leur contenu spiculaire. De plus, les pics saisonniers de reproduction et de croissance de la forme violette ont lieu deux mois après ceux de la forme bleue. D'autre part, le cycle de reproduction conditionne fortement la quantité d'énergie disponible pour d'autres fonctions biologiques (production de métabolites secondaires, croissance). L'approche pluridisciplinaire utilisée a permis d'obtenir une vision plus globale et sûrement plus réaliste de l'évolution de cet organisme marin dans son environnement<br>The colonial ascidian Cystodytes is widely distributed in both tropical and temperate waters. At least two chemotypes exist in the Mediterranean. The first presents the sulfur-containing pyridoacridines shermilamine B, kuanoniamine D, and their deacetylated forms, and was found in a purple morph. The second presents the C9-unsubstituted pyridoacridines ascididemin and 11-hydroxyascididemin, found in blue and green morphs. An ultra-structural study of the tunic revealed that the main cell types were bladder cells, pigment cells, amebocytes, phagocytes, and morula cells and that the sulfur-containing pyridoacridines were stored in the pigment cells. All crude extracts, as well as ascididemin, were toxic and significantly deterred fish but not sea urchin predation. In contrast, acidity by itself, and spicular shape and concentration did not deter feeding. Phylogenetic and population genetic analyses pointed towards the existence of several species in the Mediterranean. In addition, both the reproductive and growth periods of the purple and blue morphs, although partially overlapping, showed significant temporal lags, reinforcing previous genetic and chemical results indicating that these morphs are distinct species. Moreover, the results showed that when the ascidian invested in reproduction, the energy allocated to other life cycle parameters, such as growth, chemical and physical defenses, was significantly reduced. The multidisciplinary approach used in this work provided an interesting case study through which to develop a better understanding of the biology, ecology and secondary chemistry of marine invertebrates

Depuis quelques annees, la classe d'invertebres marins des ascidies a fourni de nombreux principes actifs originaux, susceptibles d'entrer en therapeutique humaine (cytotoxiques, antitumoraux, antiviraux. . . ). L'objet de cette these est la recherche de nouveaux alcaloides d'origine marine doues d'activites biologique et pharmacologique. Pour cela nous avons selectionne sur des criteres ecologiques deux ascidies coloniales: cystodytes della chiajei (polycitoridae) et lissoclinum fragile forme hospes (didemnidae). Pour isoler ces metabolites, des tests biologiques sont utilises permettant une biodirection de toutes les etapes de fractionnement et de purification chromatographique. Nous avons extrait de ces deux ascidies trois alcaloides qui ont montre une interaction affine avec l'adn et dont les etudes structurales ont ete menees par les methodes spectroscopiques usuelles des substances naturelles (uv, ir, ms et rmn). De cystodytes della chiajei, nous avons ainsi caracterise l'ascididemnine qui est un alcaloide aromatique pentacyclique condense. De lissoclinum fragile nous avons identifie l'eudistomine u et l'isoeudistomine u: alcaloide a noyau carboline pour la premiere; alcaloide a noyau carboline (rare dans la nature) pour la deuxieme molecule. Enfin, l'activite cytotoxique de ces molecules a ete evaluee sur des cellules cancereuses lymphoblastiques humaines

Les ascidies présentent des propriétés remarquables au sein des métazoaires qui en font un modèle particulièrement intéressant pour étudier le fonctionnement et l’évolution des éléments cis-régulateurs dans un contexte développemental. Ciona intestinalis et Phallusia mammillata, deux espèces d’ascidies qui ont divergé il y a environ 300 millions d’années, combinent une grande conservation de leurs processus développementaux avec une grande divergence de leur séquence génomique. Pour comprendre comment « fabriquer » des embryons similaires avec des génomes divergents, nous avons identifié les éléments cis-régulateurs actifs au cours du développement de Ciona intestinalis et Phallusia mammillata en développant et en appliquant la méthode de ChIP-Seq sur des modifications d’histones sur des jeunes gastrulae. La définition puis la validation fonctionnelle de différentes catégories d'éléments cis-régulateurs nous a permis de révéler quelques propriétés de la cis-régulation au sein de génomes compacts et intensément remaniés. En sus, les données que nous avons produites constituent une resource fonctionnelle unique pour la caractérisation des éléments cis-régulateurs chez les ascidies et l'étude de leur évolution au sein des Chordés<br>Ascidians display remarkable features within metazoans making them particularly suited for the study of function and evolution of cis-regulatory elements in the context of embryonic development. Ciona intestinalis and Phallusia mammillata, two ascidian species that diverged about 300M years ago, combine high conservation of their developmental processes with high divergence of their genome sequence. To understand how to “make” similar embryos with divergent genomes, we identified active cis-regulatory elements during Ciona intestinalis and Phallusia mammillata development by developing and applying the ChIP-Seq method on histone modifications in early-gastrula embryos. Definition then functional validation of different categories of cis-regulatory elements led us to reveal some features of cis-regulation within compact and highly dynamic genomes. Together, our data constitute a unique functional resource for characterizing cis-regulatory elements in ascidians and questioning their evolution within the Chordates

Las invasiones biológicas son un tema de especial importancia para los biólogos marinos. Estas invasiones pueden causar daños tanto ecológicos como económicos. El Mar Mediterráneo es uno de los ecosistemas marinos con más biodiversidad de todo el globo. No obstante, es uno de los mares más castigados y contaminados por la gran actividad humana costera y por la explotación de sus recursos. En consecuencia, es una región altamente vulnerable para la introducción de especies de diferentes orígenes. Siguiendo la introducción a una nueva área (dispersión pre-frontera), los procesos post-frontera determinan el éxito en el establecimiento de las especies no indígenas. Sin embargo, se conoce poco sobre cómo estos procesos post-frontera dan forma a la composición genética de las especies no indígenas a escalas regionales. Además, las introducciones recurrentes de especies no indígenas generan interacciones nuevas entre especies residentes que varían con las condiciones locales y la composición de la comunidad receptora. Por otro lado, el estudio de especies invasoras es a menudo confundido por cuestiones taxonómicas y por lo tanto estas especies pueden no ser reconocidas como introducidas en nuevas áreas colonizadas. De entre los organismos marinos invasores, las ascidias se han convertido en un problema global. Puertos, marinas, instalaciones de acuacultura y otras estructuras artificiales suelen ser los hábitats donde son introducidas y proliferan. Concretamente, las ascidias no indígenas son una especial preocupación para la industria de acuacultura y, paradójicamente, el stock de bivalvos representa una importante vía para transportar estas ascidias por todo el mundo. El objetivo principal de esta tesis doctoral es estudiar ciertos aspectos ecológicos y genéticos de la biología de cuatro ascidias invasoras en el Mediterráneo occidental. En el primer capítulo profundizamos en los procesos post-frontera de Microcosmus squamiger estudiando su estructura genética poblacional. Esta ascidia presenta una capacidad relativamente alta de dispersión post-frontera que le permite un flujo genético entre diferentes poblaciones a pequeña escala, además, presenta dos grupos genéticos relacionados con las dos fuentes originarias (Australia) de las poblaciones introducidas mundialmente. Estos acervos genéticos están mezclados a nivel poblacional pero no a nivel de individuo. En el segundo capítulo evaluamos los interacciones biológicas en los primeros estadios del desarrollo de Microcosmus squamiger y Styela plicata entre ellas y con mejillones, otros dominantes en las comunidades bentónicas. Parece que las dos ascidias no presentan interacciones a nivel espermático y tampoco a nivel de asentamiento larvario. En cambio, los mejillones juveniles presentan altos niveles de depredación de las larvas de estas dos ascidias, además de influenciar en su comportamiento larvario. Esto nos demuestra la importancia de la comunidad residente para el establecimiento de especies introducidas. En el tercer capítulo estudiamos el ciclo de vida y la estructura genética poblacional del invasor Didemnum. vexillum. Esta ascidia presenta un ciclo de crecimiento y reproductor claramente estacional, afectado negativamente por las temperaturas cálidas del verano. Además, las poblaciones introducidas en el Mediterráneo muestran una diversidad baja, sugiriendo un proceso de cuello de botella en el proceso de introducción. Finalmente, en el cuarto capítulo estudiamos el ciclo de vida de Clavelina oblonga. Veremos que esta ascidia es una especie introducida en el Mediterráneo desde hace más de 80 años pero que fue erróneamente identificada como C. phlegraea, nativa de la región. Su ciclo reproductor y de crecimiento es marcadamente estacional, favorecido por las temperaturas altas del verano, presentando hibernación en los meses más fríos.<br>Biological invasions are of special concern to marine biologists. The Mediterranean is one of the most harmed seas by human activities. Among invasive marine organisms, ascidians have become a global problem. The main goal of this PhD thesis is to study ecological and genetic aspects of the biology of four invasive ascidians in the western Mediterranean. In the first chapter, we delve into the post-border processes of the ascidian Microcosmus squamiger studying its population genetic structure. This ascidian has a relatively high capacity of post-border dispersion, which allows gene flow between populations on a small scale. Moreover there are two genetic clusters related to the two original sources of the introduced worldwide populations. These genetic pools are mixed at the population level but not at the individual level. In the second chapter, we evaluate biological interactions in early-life history stages of Microcosmus squamiger and Styelaplicata, among themselves and with mussels. No interactions between the two ascidians were found in fertilization or larval settlement and metamorphosis processes. In contrast, larvae of both ascidians were consumed by the juvenile mussels, and the ascidians also presented shifts in larval behavior in the presence of the mussels. This highlights the importance of the resident community for the establishment of introduced species. In the third chapter, we study the life cycle and population genetic structure of the invasive Didemnum vexillum. The ascidian showed a marked seasonal growth and reproductive cycle, negatively influenced by warm summer temperatures. Additionally, introduced populations in the Mediterranean showed low diversity, suggesting a bottleneck process in the introduction. Finally, in the fourth chapter, we study the life cycle of Clavelina oblonga. This ascidian is in fact an introduced species in the Mediterranean for more than 80 years ago but was erroneously identified as C. phlegraea, native to the region. Their reproductive and growth cycle was markedly seasonal, favored by warm summer temperatures, hibernating in colder months.

A long-range research interest of this laboratory is the evolution of the transcriptional control mechanisms of the vertebrate troponin I (TnI) gene family. It is likely that the vertebrate TnI gene family arose from a single TnI gene present in early chordate ancestors. Analysis of transcriptional control mechanisms of the TnI gene of a primitive chordate, such as the ascidian Ciona intestinalis, may therefore provide insight into the regulatory mechanisms of the vertebrate ancestral TnI gene. As an initial step in such a study, I localized transcriptional control regions within 1.5 kb of 5'-flanking DNA of the Ciona TnI gene. I prepared a series of deletion constructs in which Ciona TnI 5'-flanking DNA segments were fused to a nuclear-targeted beta-galactosidase reporter gene. Constructs were introduced into fertilized Ciona eggs by electroporation, and following development up to the mid tailbud stage (12 h), reporter gene expression was assessed by whole-mount beta-galactosidase histochemistry. (Abstract shortened by UMI.)

The hermaphrodite, colonial ascidian <I>Diplosoma listerianum </I>(Chordata: Urochordata) mates by releasing sperm that disperse to neighbours, where they fertilise eggs that have been retained internally rather than spawned. The species is able to utilise highly dilute sperm: comparison with published information on a sea urchin, which released both eggs and sperm for external fertilisation, showed that <I>D. listerianum</I> maintained comparable levels of fertilisation at sperm concentrations two or three orders of magnitude lower than the echinoderm. Laboratory clones of <I>D. listerianum</I> displayed surprisingly high levels of sexual incompatibility. Fecundities of numerous pairwise crosses varied widely and suggested a continuous scale of computability. Although correlations of computability between reciprocal crosses appeared positive, considerable noise was present in the data and some crosses showed strongly asymmetrical compatibility. Patterns of sperm precedence with a five-day mating internal showed clear initial bias towards the first of two acting males. The proportion of second-male paternity (P<SUB>2</SUB>) subsequently increased with time. Estimated P<SUB>2</SUB> for entire progeny arrays was consistently greater than 0.5, but varied widely. When mating interval was reduced, mate order effects appeared to be moderated. In competition with an alternative sperm source, acting males fathered more progeny if previously mated to a particular female than if no mating history existed, an advantage probably derived from fertilisations by stored sperm. When virgin acting female colonies were given mixtures of sperm at widely divergent concentrations, offspring were shared between the two sperm sources in approximately the ratio of each mixture. However, there existed a small but statistically significant deviation from the fair raffle model, in that sperm at the lower concentration consistently achieved a greater than expected share of paternity. Environmentally-determined fixed female preferences could be responsible for this negative frequency dependence ('rare male effect').

Orientador : A. Cecilia Z. Amaral<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia<br>Made available in DSpace on 2018-07-15T04:17:22Z (GMT). No. of bitstreams: 1 Rocha_RosanaMoreirada_M.pdf: 9020561 bytes, checksum: c9d29580b7f177c090315e52aa7c1265 (MD5) Previous issue date: 1988<br>Resumo: Apesar dos Ascidiacea constituirem um grupo muito comum nos costões rochosos do litoral de São Paulo, foram muito pouco estudados, principalmente no que se refere aos aspectos da biologia e ecologia das espécies. No Canal de São Sebastião existe uma fauna diversificada de ascídias coloniais que recobre a face inferior de pequenos blocos de rocha, tocas e frestas. O interesse em estudar alguns aspectos da biologia das espécies, suas interações na comunidade e a influência dos mesmos na colonização e manutenção do espaço foi o que levou à execução deste trabalho. O estudo do período reprodutivo foi feito através da imersão mensal de placas de recrutamento de lajota natural. O estudo da forma e velocidade de crescimento e da sucessão de ascídias coloniais, incluindo observações sobre interações entre as espécies, foi feito através do acompanhamento de placas artificiais durante 9 meses, imersas em duas épocas diferentes (verão e inverno). As cinco espécies estudadas em maior detalhe foram: Didemnum speciosum, Diplosoma macdonaldi, Clavelina oblonga, Symplegma viride e Botrylloides nigrum, pois eram as mais frequente no substrato artificial. Estas espécies apresentaram período reprodutivo longo com interrupção nos meses mais frios, observada em C. oblonga e B. nigrum. ...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital<br>Abstract: Although ascidians are very abundant on rocky shores of the Southeastern Brazilian coast (Sao Paulo State), there is little information about the species biology and ecology. In the São Sebastião Channel occurs a diversified fauna of compound ascidians, which cover the underside surface of boulders, crevices and cracks. To fulfill this gap of knowledge, the study of some life-history features of the ascidian species their interrelations in the community and how these aspects influence their colonization and space maintenance ability was undertaken. The reproductive period was determined by observations of one-month submerged artificial plates throughout one year, and by dissecting colonies to look for larvae. The ascidian community succession starting on clean plates were monthly observed on 9 month submerged plates, .on which the shape and growth rate of the colonies was also analyzed. Five species were discussed: Didemnum speciosum, Diplosoma macdonaldi, Clavelina oblonga, Symplegma viride e Botrylloides nigrum. They showed a long reprodutive period with a short interruption in C. oblonga and B. nigrum during winter. ...Note: The complete abstract is available with the full electronic digital thesis or dissertations<br>Mestrado<br>Mestre em Ecologia

Invasive species constitute a major threat to global biodiversity and cause important economic losses and ecological impacts. In the marine realm, ascidians include several aggressive invasive species, some of which have worldwide colonisation ranges, such as Didemnum vexillum. In this thesis, some biological and ecological characteristics implicated in the invasiveness of the species are assessed. First, we performed a 20 month monitoring to determine settlement and growth preferences of invasive ascidian species in the Ebro Delta aquaculture facility, including D. vexillum. Our results indicated that D. vexillum has a preference for complex substrates. To minimise fouling on bivalves, spat immersion during fall and below 1 m depth is recommended. To detect new introduced species, a follow-up program based on occurrences would be sufficient. Second, a protocol for small DNA samples combining whole genome amplification (WGA) and genotyping-by-sequencing (GBS) was developed and applied to D. vexillum using a single zooid per colony to determine patterns of genomic diversity and differentiation, describe the colonisation history of the species, and study its capability to form chimeras. Our results confirmed that Japan is in the native area of the species and only one clade spread worldwide. We found that the two main mitochondrial clades are strongly differentiated at the genomic level suggesting reproductive isolation, we determined that three independent colonisation events shaped the global distribution of the species, and we found that populations are diverse and well differentiated indicating a high expansion potential of D. vexillum. Third we detected high prevalence of chimerism, and fusion was unlinked to global genetic relatedness. Finally, we analysed the microbiome of D. vexillum that showed markedly different composition than a congeneric species and water. The invasive clade had a small but abundant core and a highly diverse variable microbiome component with a strong capacity to enrich the symbionts from the environment. The microbiome structure correlated to host genetic distance, temperature and geographical distances, pointing to vertical and horizontal transmission. In conclusion, D. vexillum is an aggressive invasive species with a high adaptive capacity that may contribute to the invasiveness of this global pest.

Du fait de la complexité des processus de spéciation, l'étude des relations évolutives entre espèces nécessite une approche intégrative. Nous avons combiné croisements expérimentaux et études moléculaires (séquençage mitochondrial et RAD-seq) pour étudier les relations évolutives entre espèces indigènes et introduites du genre Ciona, en Europe. Nous avons examiné deux mécanismes post-zygotiques restant à explorer pour expliquer l'absence d'introgression de C. robusta et C. intestinalis dans leur zone de sympatrie en Manche : 1) la contre-sélection des hybrides par l'environnement (température et salinité) et 2) des incompatibilités de type Dobzhansky-Muller (DMIs). L'absence de dépression hybride (étudiée chez 725 hybrides F1) nous a conduit à rejeter le premier mécanisme. Le second a été exploré grâce à l'étude de sept familles double-hybrides ou issues de rétrocroisements, analysées par RAD-seq. Une stérilité sur la fonction mâle des hybrides F1 et d'importantes distorsions de ségrégation sont en faveur de la présence de DMIs. Nous avons étendu notre étude à d'autres espèces du genre Ciona. Les principaux résultats, notamment soutenus par une approche phylogénomique (7 taxons - 89 individus), sont : 1) la remise en cause du statut d'espèce de C. roulei par rapport à C. intestinalis (probablement deux lignées allopatriques), 2) les premières données montrant la forte divergence évolutive de C. edwardsi avec les autres Ciona spp. et 3) la présence de variabilité génétique et/ou d'introgression chez C. intestinalis type C et type D. Ces résultats ouvrent d'intéressantes perspectives de recherches dont des études populationnelles des espèces présentes en Méditerranée<br>Speciation is a complex process. Evolutionary relationships between close species thus required an integrative approach. We combined experimental crosses and molecular data (mitochondrial and RAD-tag sequencing) to investigate evolutionary relationships between native and non-indigenous species of the genus Ciona nowadays found in Europe. We studied two post-zygotic mechanisms, still to be examined, to explain the lack of introgression between C. robusta and C. intestinalis in their sympatric range –i.e. the English Channel: i) selection against hybrids, by the environment (here temperature and salinity) and ii) Dobzhansky-Muller incompatibilities (DMIs). The absence of outbreeding depression (over 725 F1-hybrids) makes unlikely the first mechanism. The second has been investigated with seven families of F2-hybrids and backcrosses, all studied with RAD-tag sequences. Male sterility on F1-hybrids and many segregation distortions are in favor of presence of DMIs. We then expanded our study to other Ciona species. The main outcomes, supported by a phylogenomic approach (7 taxa - 89 individuals) are: 1) a needed reappraisal of the species status of C. roulei and C. intestinalis, which are most likely two allopatric lineages of a same species (as defined by biological and phylogenetic species concepts), 2) the first data showing that C. edwardsi is strongly evolutionary divergent from the other Ciona species and 3) the presence of unexpected genetic variability and/or introgression in C. intestinalis type C and D. All together these results open new research perspectives, in particular based on population studies of the species nowadays found in the Mediterranean Sea

RESUMEN Introducción: Desde que en 1996 el Club Internacional de la Ascitis (CIA) publicó los criterios diagnósticos de la ascitis refractaria (AR) y del síndrome hepatorrenal (SHR) pocos estudios han evaluado la historia natural de la ascitis en series amplias de pacientes. Además, aunque el SHR es un tipo de insuficiencia renal (IR) bien caracterizado en pacientes cirróticos con ascitis, la información sobre otros tipos de IR funcional (IRF) en estos pacientes es escasa. Hipótesis: Un mejor conocimiento de la historia natural de la ascitis y de sus complicaciones podría mejorar el manejo de los pacientes cirróticos con ascitis. Objetivos: 1. Estudiar la historia natural de la ascitis y sus complicaciones en una serie amplia de pacientes cirróticos e identificar los factores pronósticos que determinan la aparición de hiponatremia dilucional (HD), AR, SHR y la supervivencia de los pacientes cirróticos con ascitis. 2. Evaluar la incidencia y pronóstico de los diferentes tipos de IRF que pueden complicar la historia natural de la ascitis, así como identificar los factores pronósticos que intervienen en su desarrollo. Métodos: Estudio prospectivo-retrospectivo realizado en una cohorte de 263 pacientes consecutivos con cirrosis, hospitalizados en dos Hospitales Universitarios del área de Barcelona y seguidos durante una media de 41 ± 3 meses a partir de su primera descompensación ascítica. Se consideraron 3 tipos de IRF: IR-prerenal cuando la IR se asoció a depleción del volumen intravascular, IR asociada a infección que no cumplía criterios de SHR y el SHR. Resultados: Durante el seguimiento 74 (28,1%) pacientes desarrollaron HD, 30 (11,4%) AR y 20 (7,6%) SHR (tipo1, 7; tipo 2, 13). La probabilidad a los 5 años de desarrollar HD, AR y SHR fue del 37,1, 16,7 y 11,4%, respectivamente. La probabilidad de supervivencia a 1 y 5 años fue del 85 y 56,5%, respectivamente. Los factores predictivos independientes de supervivencia fueron la edad y Child-Pugh basales y el desarrollo de HD. La probabilidad de supervivencia al año tras desarrollar HD, AR, SHR tipo 2 fue del 25,6, 31,6 y 38,5%, respectivamente. La supervivencia media tras desarrollar SHR tipo 1 fue de tan solo 7±2 días. En nuestra cohorte, 129 (49%) pacientes desarrollaron algún tipo de IRF, siendo la pre-renal la más frecuente (27,4%), seguida de la asociada a infección (14,1%) y el SHR (7,6%). La probabilidad al año de desarrollar cualquier tipo de IRF fue del 23,6%. Los factores predictivos independientes de desarrollo de IRF fueron la edad, el Child-Pugh y la creatinina sérica basales. La probabilidad de supervivencia al año fue del 91% en los pacientes sin IR y del 46,9% en los que desarrollaron cualquier tipo de IRF (p=0,0001). Conclusiones: 1. La supervivencia de los pacientes cirróticos con un primer episodio de ascitis es relativamente elevada y viene determinada por la edad y la puntuación de Child-Pugh en el momento de la descompensación ascítica y el desarrollo de HD. 2. La probabilidad de desarrollar AR y SHR es baja pero se asocia a un mal pronóstico.3. Aproximadamente el 50% de los pacientes cirróticos con ascitis desarrolla algún tipo de IRF durante el seguimineto, lo que se asocia a un peor pronóstico.4. Deberían tomarse medidas para prevenir la aparición de IRF en estos pacientes.<br>Background: Since the International Ascitis Club published the diagnostic criteria of refractory ascites (RA) and hepatorenal syndrome (HRS) in 1996, there have been few studies assessing the natural history of ascites. Moreover, although HRS is a well characterized type of terminal renal failure (RF) that occurs in patients wtih ascites, information about other types of functional renal failure (FRF) in these patients is scarce. Hypothesis: Best knowledge of natural history of ascites and their complications could improved management of cirrhotic patients with ascites. Aims: 1. To define the natural history of ascites and their complications in a large cohort of cirrhotic patients and to identify prognostic factors for dilutional hyponatremia (DH), RA and HRS development and survival. 2. To assess the incidence and prognosis of different types of FRF that could complicate natural history of ascites and to investigate prognostic factors for these disorders. Methods: A prospective-retrospective study was performed in a 263 consecutive cirrhotic patients cohort, hospitalized in two university hospitals in the area of Barcelona and followed for 41 ± 3 months after their first significant ascites. Three types of FRF were considered: pre-renal failure (when RF was associated with a depletion of intravascular volume), RF induced by infection that did not result in HRS, and HRS. Results: During follow-up 74 (28.1%) patients developed DH, 30 (11.4%) RA and 20 (7.6%) HRS (type 1, 7; type 2, 13). The 5-year probability of DH, RA and HRS was 37.1%, 16.7% and 11.4%, respectively. The probability of survival at 1 and 5 years was 85% and 56.5%, respectively. The independent predictors for survival were baseline age, baseline Child-Pugh score and DH development. The 1-year probability of survival after developing DH, RA, and type 2 HRS was 25.6%, 31.6% and 38.5%, respectively. In contrast, the mean survival was only 7±2 days in those patients developing type 1 HRS. In our cohort, 129 (49%) patients developed some type of FRF. The most frequent was pre-renal failure (27.4%), followed by RF induced by infection (14.1%), and then HRS (7.6%). The 1-year probability of developing the first episode of any FRF was 23.6%. the independent predictors of FRF development were baseline age, Child-Pugh score, and serum creatinine. Although the 1-year probability of survival was 91% in patients without RF, it decreased to 46.9% in those patients who developed any FRF. (P=.0001). Conclusions: 1.The survival of cirrhotic patients with ascites is relatively high, and is mainly influenced by age and Child-Pugh score at the time of ascites descompensation, as well as by DH development. 2. The probability of RA and HRS development is relatively low, but they are associated with a poor prognosis. 3. Approximately 50% of the cirrhotic patients with ascites developed some type of FRF during the follow-up period. RF was associated with worse prognosis.4. Efforts should be made to prevent RF in cirrhotic patients with ascites.

Ácaros da família Ascidae sensu Lindquist e Evans, 1965 são encontrados em vários habitats, principalmente em folhedo, produtos armazenados e flores. Eles são mais conhecidos por seu hábito predatório, alimentando-se de ácaros, outros pequenos artrópodes e nematóides, mas muitas espécies também são conhecidas por se alimentarem de fungos, pólen e néctar, sendo algumas ainda relatadas em associação com baratas e traças. Uma vez que essas espécies apresentam potencial no controle de pragas no solo, sobre plantas ou em produtos armazenados, considera-se conveniente o estudo detalhado de sua taxonomia, fundamental para o desenvolvimento subsequente de estudos biológicos e ecológicos que possibilitem a utilização desses ácaros no controle de pragas. Este trabalho objetivou listar as espécies de Ascidae sensu Lindquist e Evans, 1965 descritas em todo o mundo, elaborar uma chave para auxiliar na separação dos gêneros desta família, identificar espécies disponíveis na coleção de referência de ácaros da ESALQ além de descrever espécies novas encontradas, redescrever a espécie Lasioseius floridensis Berlese de importância no controle biológico de praga, bem como avaliar o potencial dessa espécie no controle do ácaro branco. Um total de 901 espécies válidas pertencentes a 40 gêneros foi mencionado na lista de espécies de Ascidae sensu Lindquist e Evans (1965) e uma chave dicotômica foi elaborada para a separação dos gêneros dessa família. Duas espécies novas, uma de Blattisocius Keegan e outra de Proctolaelaps Berlese, foram descritas. A primeira foi coletada em associação com Tyrophagus putrescentiae (Schrank) (Acaridae) em ração comercial para cães, em Charqueada, enquanto a segunda foi coletada de flores de Heliconia angusta Vellozo, em Registro, ambas no Estado de São Paulo. Blattisocius keegani Fox e Lasioseius floridensis Berlese foram redescritas com base em exemplares coletados de Gerbera sp. (Asteraceae) no Estado de São Paulo. Uma chave para a separação das espécies de Blattisocius de todo o mundo foi elaborada. A biologia de L. floridensis foi estudada, por sua importância potencial como agente de controle biológico. Neste estudo, observou-se a possível limitação do efeito deste predador sob condições de baixa umidade. A diversidade de espécies de Ascidae sensu Lindquist e Evans (1965) foi estudada em flores tropicais em diversos pontos do Brasil. No total, 23 espécies, pertencentes a Asca Heyden, Cheiroseius Berlese, Iphidozercon Berlese, Lasioseius, Proctolaelaps e Tropicoseius Baker e Yunker foram identificadas; os maiores números de exemplares examinados pertencem às espécies Proctolaelaps n.sp 1 e Tropicoseius venezuelensis Baker e Yunker. A dinâmica populacional de ácaros desta família foi estudada em Piracicaba, determinando-se que a espécie mais comum foi T. venezuelensis, e que os maiores níveis populacionais destes ácaros ocorreram no final de um ano e início do ano seguinte.<br>Mites of the family Ascidae sensu Lindquist and Evans (1965) are found in various habitats, especially in litter, stored products and flowers. They are best known for their predatory habits on mites, other small arthropods and nematodes, but many are also known to feed on fungi, pollen and nectar, and some have been reported in association with cockroaches and moths. Given the potential of this mite group in the control of pests in the soil, on plants or stored products, detailed studies on their taxonomy are warranted. These studies can help further development of biological and ecological studies to enable their use in control pest. The aim of the present study was to list the world species of Ascidae sensu Lindquist and Evans (1965), to develop a dichotomous key to assist in the separation of the genera of the family, identify species deposited in the mite reference collection of ESALQ and describe new species found in the study, redescribe a species of Lasioseius Berlese of possible importance in biological control of pests, and to evaluate the potential of this species in the control of the broad mite, Polyphagortarsonemus latus (Banks) (Tarsonemidae). A total of 901 valid species belonging to 40 genera were cited in the species list and a dichotomous key was developed for the separation of the genera of this family. Two new species, one of Blattisocius and another of Proctolaelaps, were described. The former was collected in association with Tyrophagus putrescentiae (Schrank) (Acaridae) on a commercial dog food, in Charqueada, while the latter was collected from flowers of Heliconia angusta Vellozo, in Registro, both in the State of Sao Paulo. Blattisocius keegani Fox and Lasioseius floridensis Berlese were redescribed based on specimens collected from Gerbera sp. (Asteraceae) in the State of Sao Paulo. A key to the separation of world species of Blattisocius was prepared. The biology of L. floridensis was studied for its potential importance as a biological control agent. The possible limitation of this predator under low humidity was determined in the study. The diversity of Ascidae sensu Lindquist and Evans (1965) species was studied in tropical flowers in various parts of Brazil. A total of 23 species belonging to Asca, Cheiroseius, Iphidozercon, Lasioseius, Proctolaelaps and Tropicoseius were identified, the largest numbers of specimens referring to Proctolaelaps n.sp and Tropicoseius venezuelensis Baker and Yunker. The population dynamics of mites of this family was studied in Piracicaba, determining that the prevalent species was T. venezuelensis, and that the highest population level of the mite species occurred at the end of one year and the beginning of the following year.

Submitted by JANDIR CRUZ SANTOS null (jandir_jc@hotmail.com) on 2017-05-15T18:13:46Z No. of bitstreams: 1 Tese_Jandir_Cruz Santos.pdf: 5587068 bytes, checksum: acb65ead4a03782c42c7fb8d0228224b (MD5)<br>Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-05-16T14:46:03Z (GMT) No. of bitstreams: 1 santos_jc_dr_jabo.pdf: 5587068 bytes, checksum: acb65ead4a03782c42c7fb8d0228224b (MD5)<br>Made available in DSpace on 2017-05-16T14:46:03Z (GMT). No. of bitstreams: 1 santos_jc_dr_jabo.pdf: 5587068 bytes, checksum: acb65ead4a03782c42c7fb8d0228224b (MD5) Previous issue date: 2017-04-20<br>Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)<br>Em diversos países, muitas empresas têm investido valores significativos na viabilização do uso do controle biológico de pragas, em função da demanda do mercado por produtos alimentícios saudáveis, livres de resíduos químicos. O uso de ácaros predadores tem crescido significativamente nos últimos anos principalmente com ácaros das famílias Phytoseiidae, Laelapidae e Macrochelidae, no entanto outras famílias como Ascidae, Blattisociidae e Melicharidae têm demonstrado bons resultados em laboratório no controle de pragas. Dentro da linha de trabalho priorizada pelas instituições em que o presente trabalho foi conduzido (FCAV/UNESP e ESALQ/USP), o objetivo deste trabalho foi conhecer a fauna de ácaros das famílias Ascidae, Blattisociidae e Melicharidae nas diferentes regiões do Brasil e estabelecer bases de dados sobre a ocorrência destes no Globo Terrestre. Espécimes disponíveis na Coleção de Referência de Ácaros da ESALQ/USP e coletados pelo autor deste documento foram analisados e identificados, descrevendo-se três espécies novas para a ciência e redescrevendose uma espécie (Leioseius basis Karg, 1994). Foram também criadas bases de dados sobre a distribuição de ácaros destas três famílias e sobre os substratos em que foram encontrados, para disponibilização “on-line”. No total, foram examinados 1657 exemplares, de 76 espécies de 19 gêneros. Vinte e seis espécies e três dos gêneros encontrados eram novos para a ciência e destes taxa, três espécies novas (uma de Leioseius e duas de Cheiroseius) foram descritas. Duas chaves dicotômicas taxonômicas foram elaboradas, uma para as espécies de Leioseius do Globo e uma para as espécies de Cheiroseius conhecidas no Brasil. As bases de dados estabelecidas correspondem a uma complementação das informações taxonômicas constantes do catálogo destas famílias publicado em 2016. Cerca de 820 publicações foram obtidas e analisadas, correspondendo a cerca de 1,3 vezes o número de publicações citadas naquele catálogo. As bases de dados permitem o rápido acesso às informações sobre a distribuição por país (por estado ou divisão política correspondente nos sete maiores países) e sobre os substratos em que estes ácaros foram relatados. As bases constam de aproximadamente 2200 registros, cada um correspondente ao número de pontos de constatação citados em cada publicação de dados primários, para cada uma das quase 1000 espécies distribuídas em 45 gêneros destas famílias. Os países com maior diversidade conhecida em cada família são: Ascidae – Rússia (56), Estados Unidos da América (42), China (37) e Polônia (36); Blattisociidae – China (47), Estados Unidos da América (41), Equador (38), Índia (33) e Polônia (32); Melicharidae – Estados Unidos da América (46), Brasil (23), Equador (20) e Polônia e Alemanha (15). No entanto, nenhuma espécie destas famílias é conhecida em cerca de 44% dos países.<br>In several countries, many companies have invested significant amounts in making viable the use of biological control of pest organisms, due to the demand for healthy food products, free of chemical residues. The use of predatory mites have grown significantly in recent years especially with mites Phytoseiidae families, and Laelapidae Macrochelidae, however other families like Ascidae, Blattisociidae Melicharidae and have shown good results in the laboratory to control pests. In line the research priorities of the institutions in which this work was conducted (FCAV/UNESP and ESALQ/USP), the objective of this work was to investigate the mite fauna of the families Ascidae, Blattisociidae and Melicharidae in different regions of Brazil and to establish databases on the world occurrence and substrates of these mites. Specimens available at ESALQ/USP Mite Reference Collection and specimens collected by the author of this document were analyzed and identified, describing three species new to science and redescribing a species (Leioseius basis Karg, 1994). The work also involved the establishment of databases on the distribution of mites of these three families and on substrates that have been found, for online availability. In total, 1657 specimens from 76 species of 19 genera were examined. Six species and three genera found were new to science and from these taxa, three new species (one Leioseius and two Cheiroseius) were described. Two dichotomous taxonomic keys were elaborated, one for the species of Leioseius of the world and one for the species of Cheiroseius known in Brazil. The established databases correspond to a complementation of the taxonomic information included in the catalog of these families published in 2016. About 820 publications were obtained and analyzed, corresponding to about 1.3 times the number of publications cited in that catalog. The databases allow rapid access to information on the distribution by country (by state or corresponding political division in the seven largest countries) and on the substrates in which these mites have been reported. The bases contain approximately 2200 records, each corresponding to the number of observation points in each publication of primary data, for each of the nearly 1000 species in 45 genera of these families. The countries with the highest known diversity in each family are: Ascidae - Russia (56), United States of America (42), China (37) and Poland (36); Blattisociidae - China (47), United States of America (41), Ecuador (38), India (33) and Poland (32); Melicharidae - United States of America (46), Brazil (23), Ecuador (20) and Poland and Germany (15). However, no species of these families is known in about 44% of the countries.