This work presents the building up of basic biological units DNA, RNA and proteins as well as their function. Provided data are kept in biological databases which are connected worldwide to supply preferable communication along with all kinds of available information to be used in the scientific research. The secret of alive is hidden in genes coded in sequences of nucleotides. Genes enable the creation of proteins which are made of sequences of amino-acids. The wide-spread methods of comparing these sequences are FASTA and BLAST algorithms. Their base is used for the PSProt program which is described in this work. PSProt program is the tool for comparing the sequences of proteins. First it is necessary to synthesise the protein from the DNA oligonucleotide because it codes the surveyed protein. The most similar proteins are searched out by heuristic of hitpoints, then their final score that is essential for aligning is modified by semiglobal alignment algorithm.

No description available.

Les plantes présentent une incroyable diversité de tailles, formes et couleurs, étroitement liée à certaines de leurs fonctions biologiques telles que la photosynthèse, la reproduction, etc. De ce fait, la façon dont ces organismes multicellulaires acquièrent des formes complexes est une question clé en biologie du développement. La morphologie des organes végétaux résulte en effet de la modulation, à l’échelle cellulaire, de patrons d’expression génétique, de croissance et de différenciation. Bien que la morphogénèse ait été largement étudiée d’un point de vue moléculaire, nous ne savons toujours pas comment ces réseaux génétiques sont traduits en formes biologiques. Le but de ce projet de recherche est donc d’étudier le développement des organes latéraux (feuilles juvéniles, feuilles caulinaires et organes floraux, id sépales, pétales et anthères) chez l’espèce modèle Arabidopsis thaliana. Afin d’approcher la question du rôle des interactions complexes entre cellules et organes lors du développement, nous nous intéressons à la variabilité entre les organes, mais aussi à la variabilité cellulaire intrinsèque de chaque organe. Nous avons donc testé (1) si la diversité de formes observées chez les organes latéraux résulte de modulations d’un programme développemental commun; (2) si la croissance et le développement des organes latéraux est un phénomène stochastique ou dépend de mécanismes sous-jacents spécifiques. Pour ce faire, nous utilisons une approche multidisciplinaire basée sur la génétique, la microscopie confocale et l’analyse d’images 3D pour extraire les patrons de croissance inhérents aux différents organes. Les résultats de la première étude (Chapitre 2) montrent que la forme des organes dépend de l’équilibre entre croissance et différentiation, dont la régulation précise permet l'acquisition de fonctions hautement spécialisées. La feuille caulinaire, par exemple, présente un retard de différenciation qui permet une activité morphogénétique prolongée et une redistribution de la croissance. À travers la suppression transitoire de la croissance lors des premiers stades de développement, la trajectoire développementale de la feuille caulinaire permet sa double fonction, à la fois protectrice et photosynthétique.\par La deuxième étude (Chapitre 3), quant-à-elle, s’intéresse aux comportements des cellules individuelles, dont la croissance, bien que contrôlée par des informations positionnelles, est souvent hétérogène. Cette variabilité résulte de la différenciation de cellules spécialisés, les stomates, qui suivent un programme de développement spécifique. Le comportement autonome de ces cellules, asynchrone, est la principale source de variabilité dans des tissus dont la croissance est autrement homogènes. Dans l’ensemble, cette thèse a permis de mettre en lumière l’importance de la temporalité lors du développement des organes végétaux. Que ce soit à l’échelle de l’organe, du tissu ou de la cellule, la modulation et la synchronisation de la croissance et de la différentiation sont nécessaires à l’acquisition des formes stéréotypiques des organes et à leur complexité fonctionnelle. / Plants display an incredible diversity of sizes, shapes, and colors, closely linked to some of their biological functions, such as photosynthesis, reproduction, etc. How these multicellular organisms acquire complex shapes is, therefore, a key question in developmental biology. The morphology of plant organs results from cell-level modulation of patterns of gene expression, growth, and differentiation. Although morphogenesis has been extensively studied from a molecular point of view, how genetic networks are translated into biological forms is still unclear. Thus, the aim of this research project is to study the development of lateral organs (rosette leaves, cauline leaves, and floral organs, i.e. sepals, petals, and anthers) in the model species Arabidopsis thaliana. To address the question of the role of complex cell-organ interactions during development, we are interested not only in variability between organs but also in the intrinsic cellular variability of each organ. We, therefore, tested (1) whether the diversity of shapes observed in lateral organs results from modulations of a common developmental program; (2) whether the growth and development of lateral organs is a stochastic phenomenon or depends on specific underlying mechanisms. To this end, we are using a multidisciplinary approach based on genetics, confocal microscopy, and 3D image analysis to extract the growth patterns inherent in the different organs. The results of the first study (Chapter 2) show that organ shape depends on the balance between growth and differentiation, which fine regulation enables the acquisition of highly specialized functions. The cauline leaf, for example, shows a delay in differentiation that allows for prolonged morphogenetic activity and growth redistribution. Through the transient growth suppression at early stages, the cauline leaf developmental trajectory allows for its dual function, from protection to photosynthesis. The second study (Chapter 3) focuses on the behavior of individual cells, whose growth, although controlled by positional information, is often heterogeneous. This variability results from the differentiation of specialized cells, the stomata, which follow a specific developmental program. The autonomous, asynchronous behavior of these cells is the main source of variability in tissues whose growth is otherwise homogeneous. Overall, this thesis has shed light on the importance of timing in plant organ development. Whether at the organ, tissue, or cell level, modulation and synchronization of growth and differentiation are necessary for the acquisition of stereotypic organ shapes and functional complexity.

No description available.

No description available.

La réparation des cassures double brin (CDB) de l'ADN par recombinaison est essentielle au maintien de l'intégrité du génome de tous les être vivants. Ce processus doit cependant être finement régulé puisque la recombinaison peut générer des mutations ou des réarrangements chromosomiques, parfois extrêmement délétères pour la cellule. Les CDB peuvent être réparées par deux mécanismes : la recombinaison non homologue (ou jonction des extrémités d'ADN) ou la recombinaison homologue (impliquant une homologie de séquence entre les molécules recombinantes). Dans les cellules somatiques, les deux voies principales de recombinaison homologue (RH) sont la voie Synthesis Dependent Strand Annealing (SDSA) dépendante de la recombinase RAD51 et la voie Single Strand Annealing (SSA) indépendante de RAD51. Nos résultats ont d'abord mis en évidence un rôle inattendu de XRCC2, RAD51B et RAD51D - trois paralogues de RAD51 - dans la voie SSA. Nous avons confirmé que la fonction de la protéine XRCC2 dans la voie SSA ne dépend pas de RAD51, ce qui démontre que certains paralogues de RAD51 ont acquis des fonctions indépendantes de la recombinase. La différence de sévérité des phénotypes des mutants individuels ainsi que les analyses d'épistasie menées sur le double et le triple mutant suggèrent des fonctions individuelles de ces protéines au cours du SSA. Nous proposons qu'elles facilitent l'étape d'hybridation des deux séquences complémentaires situées de part et d'autre de la cassure, bien que ceci reste à confirmer par des études in vitro. L'étude des fonctions de l'hétérodimère XPF-ERCC1 - un complexe impliqué dans le clivage des extrémités d'ADN non homologues au cours des voies de RH - a révélé un rôle inhibiteur de ce complexe sur la voie SDSA. Cette action est dépendante de son activité endonucléasique et serait liée au clivage des longues extrémités 3' sortantes réalisant l'invasion d'un duplex d'ADN homologue, l'étape initiale de la voie SDSA. Notre étude a de plus confirmé que le rôle du complexe dépend de la longueur des extrémités non homologues chez Arabidopsis, comme chez les mammifères et la levure. Bien que le complexe XPF-ERCC1 soit essentiel au clivage des longues extrémités d'ADN non homologue, il n'est pas requis à l'élimination des courtes extrémités au cours de la RH. / The repair of DNA double-strand breaks (DSB) by recombination is essential for the maintenance of genome integrity of all living organisms. However, recombination must be finely regulated as it can generate mutations or chromosomal rearrangements, sometimes extremely deleterious to the cell. DSB can be repaired by two classes of recombination mechanism: non-homologous recombination (or DNA End Joining) or homologous recombination (implicating DNA sequence homology between the recombining molecules). In somatic cells, the two main pathways of homologous recombination (HR) are RAD51-dependent Synthesis Dependent Strand Annealing (SDSA) and RAD51-independent Single Strand Annealing (SSA). Our results have demonstrated an unexpected role of XRCC2, RAD51B and RAD51D - three RAD51 paralogues – in the SSA pathway. We confirmed that the function of XRCC2 in SSA does not depend upon RAD51, thus demonstrating that some RAD51 paralogues have acquired RAD51 recombinase-independent functions. The different severities of individual mutant phenotypes and epistasis analyses carried out on the double and triple mutants suggest individual functions of these proteins in SSA recombination. We propose that they facilitate hybridization of the two complementary sequences located on both sides of the break, although this remains to be confirmed by in vitro experiments. Study of the roles of XPF-ERCC1 - a complex involved in the cleavage of non-homologous DNA ends during HR - revealed an inhibitory role of this complex on the SDSA pathway. This is dependent on its endonuclease activity and is probably due to the cleavage of long 3' ends performing the homologous DNA duplex invasion, the initial step of the SDSA pathway. Our analyses also confirmed that the role of the complex depends on the length of the nonhomologous ends, as seen in mammals and yeasts. Although XPF-ERCC1 is essential for the cleavage of long nonhomologous DNA ends, it is not required for the elimination of short ends during HR.

Le métabolisme des phénylpropanoïdes est un métabolisme secondaire spécifique au règne végétal. Il conduit, à partir de la phénylalanine, à la synthèse d'une grande variété de substances telles que les anthocyanes, les isoflavonoïdes, les stilbènes, des esters d'acides hydroxycinnamiques, ou encore à la lignine. Ces métabolites secondaires interviennent dans la pigmentation florale ou encore la protection des tissus végétaux contre divers stress biotiques et abiotiques. Quant à la lignine, elle assure rigidité aux parois cellulaires végétales et imperméabilité aux tissus conducteurs. La lignine est un polymère tridimensionnel constitué de trois unités monomériques qui possèdent le même squelette carboné phénylpropane mais diffèrent par leur degré de méthoxylation et d'hydroxylation. Une partie de mon travail de thèse a consisté à étudier la relation structure/fonction de la caféoyl-coenzyme A O-méthyltransférase (CCoAOMT) de N. tabacum, responsable de l'introduction de la première des deux fonctions méthyles. Des études bioinformatiques couplées à des approches de biochimie et de mutagenèse dirigée, nous ont permis de modéliser l'interaction de la CCoAOMT avec son substrat, le caféoyl-CoA. Trois acides aminés du site actif ont notamment été identifiés comme intervenant dans la reconnaissance spécifique de la chaîne latérale de CoA. J'ai également caractérisé, chez N. tabacum, une nouvelle acyltransférase à activité HydroxyCinnamoyl-CoA : shikimate/quinate hydroxycinnamoyl Transférase (HCT) impliquée dans le métabolisme des phénylpropanoïdes. Nous avons montré que l'enzyme HCT recombinante synthétisait, in vitro, les substrats de l'hydroxylation en position 3 du noyau aromatique. De plus, la répression de l'expression du gène HCT par le «VIGS» conduit à un ralentissement de la croissance des plantes, à une perturbation importante du pool d'acide chlorogénique, ainsi qu'à une diminution de la quantité et à une modification de la composition de la lignine synthétisée.

ABSTRACT The xyloglucan endo-transglycosylase/hydrolase (XTH) genes are found in all vascular and some nonvascular plants. The XTH genes encode proteins which comprise a subfamily of glycoside hydrolase (GH) family 16 in the Carbohydrate-Active enZYmes (CAZY) classification. The XTH gene products are believed to play intrinsic role in cell wall modification during growth and development throughout the lifetime of the plant. In the present investigation, biochemical and reverse genetic approaches were used to better understand the functions of individual members of the XTH gene family of two important plants: the model organism Arabidopsis thaliana and the grain crop barley (Hordeum vulgare). A phylogenetic tree of the xyloglucan-active enzymes of GH16 has previously been constructed, where enzymes with similar activities have been shown to cluster together. Several members of phylogenetic Group I/II and III-B, predicted to exhibit xyloglucan endo-transglycosylase activity (EC 2.4.1.207) and members of Group III-A, predicted to exhibit xyloglucan endo-hydrolase activity (EC 3.2.1.151), were included to analyze the functional diversity of XTH gene products. A heterologous expression system using the yeast Pichia pastoris was found to be effective for recombinant protein production with a success rate of ca. 50%. XTH gene products were obtained in soluble and active forms for subsequent biochemical characterization. In order to be able to screen larger numbers of protein producing clones, a fast and easy method is required to identify clones expressing active protein in high enough amounts. Thus, a miniaturized XET/XEH assay for high-throughput analysis was developed, which was able to identify activities with good precision and with a reduced time and materials consumption and a reduced work load. Enzyme kinetic analysis indicated that the XET or XEH activity of all XTH gene products characterized in the present study corresponded to predictions based on the previously revised phylogenetic clustering. To gain insight into the biological function of the predominant XEHs AtXTH31 and AtXTH32, which are highly expressed in rapidly developing tissues, a reverse genetic approach was employed using T-DNA insertion lines of the A. thaliana Columbia ecotype. Genotypic and phenotypic characterization, together with in situ assays of XET and XEH activities, in single- and double-knock-out mutants indicated that these Group III-A enzymes are active in expanding tissues of the A. thaliana roots and hypocotyl.  Although suppression of in muro XEH activity was clearly observed in the double-knock-out, no significant growth phenotype was observed, with the exception that radicle emergence appeared to be faster than in the wild type plants. Keywords: Arabidopis thaliana, Hordeum vulgare, plant cell wall, xyloglucan, glycoside hydrolase family 16, xyloglucan endo-transglycosylase/hydrolase gene family, xyloglucan endo-transglycosylase, xyloglucan endo-hydrolase, heterologous protein expression, Pichia pastoris, T-DNA insertion, in situ XET/XEH assay, high-throughput screening / QC 20110114

Cyclic nucleotide-gated channels (CNGCs) are non-selective cation channels that were first identified in vertebrate photosensory and olfactory neurons. Although the physiological roles and biophysical properties of animal CNGCs have been well studied, much less is known about these channels in plants. The Arabidopsis genome encodes twenty putative CNGC subunits that are postulated to form channel complexes that mediate various physiological processes involving abiotic and biotic stress responses, ion homeostasis and development. The identification of Arabidopsis autoimmune CNGC mutants, such as defense no death class (dnd1 and dnd2), and the constitutive expressor of pathogenesis related genes 22 (cpr22) implicate AtCNGC2, 4, 11 and 12 in plant immunity. Here, I present a comprehensive study of the molecular mechanisms involved in CNGC-mediated signaling pathways with emphasis on pathogen defense. Previously, a forward genetics approach aimed to identify suppressor mutants of the rare gain-of-function autoimmune mutant, cpr22, identified key residues that are important for CNGC subunit interactions and channel function. First, I present a structure-function analysis of one of these suppressor mutants (S58) that revealed a key residue in the cyclic nucleotide binding domain involved in the stable regulation of CNGCs. Second, I present a new suppressor screen using AtCNGC2 T-DNA knockout mutants that specifically aimed to identify novel downstream components of CNGC-mediated pathogen defense signaling. In this screen, I successfully isolated and characterized the novel Arabidopsis mutant, repressor of defense no death 1 (rdd1), and expanded this study to demonstrate its involvement in AtCNGC2 and AtCNGC4-mediated signal transduction. Additionally, I demonstrated for the first time, the physical interaction of AtCNGC2 and AtCNGC4 subunits in planta. The findings presented in this thesis broaden our current knowledge of CNGCs in plants, and provide a new foundation for future elucidation of the structure-function relationships and signal transduction mediated by these channels.

Cyclic nucleotide-gated channels (CNGCs) are non-selective cation channels that were first identified in vertebrate photosensory and olfactory neurons. Although the physiological roles and biophysical properties of animal CNGCs have been well studied, much less is known about these channels in plants. The Arabidopsis genome encodes twenty putative CNGC subunits that are postulated to form channel complexes that mediate various physiological processes involving abiotic and biotic stress responses, ion homeostasis and development. The identification of Arabidopsis autoimmune CNGC mutants, such as defense no death class (dnd1 and dnd2), and the constitutive expressor of pathogenesis related genes 22 (cpr22) implicate AtCNGC2, 4, 11 and 12 in plant immunity. Here, I present a comprehensive study of the molecular mechanisms involved in CNGC-mediated signaling pathways with emphasis on pathogen defense. Previously, a forward genetics approach aimed to identify suppressor mutants of the rare gain-of-function autoimmune mutant, cpr22, identified key residues that are important for CNGC subunit interactions and channel function. First, I present a structure-function analysis of one of these suppressor mutants (S58) that revealed a key residue in the cyclic nucleotide binding domain involved in the stable regulation of CNGCs. Second, I present a new suppressor screen using AtCNGC2 T-DNA knockout mutants that specifically aimed to identify novel downstream components of CNGC-mediated pathogen defense signaling. In this screen, I successfully isolated and characterized the novel Arabidopsis mutant, repressor of defense no death 1 (rdd1), and expanded this study to demonstrate its involvement in AtCNGC2 and AtCNGC4-mediated signal transduction. Additionally, I demonstrated for the first time, the physical interaction of AtCNGC2 and AtCNGC4 subunits in planta. The findings presented in this thesis broaden our current knowledge of CNGCs in plants, and provide a new foundation for future elucidation of the structure-function relationships and signal transduction mediated by these channels.