Candida albicans, a commensal of gastrointestinal and uro-vaginal tract can cause superficial as well as life threatening disseminated infections under conditions of lowered immunity of the host such as HIV infection, drug induced immune suppression [given during organ transplantation to prevent rejection] and radiation therapy [head and neck cancer patients] (Odds, 1988; Fidel and Sobel, 1996). Candida albicans shows a range of morphologies, it can switch from budding yeast morphology to pseudohyphae (chains of elongated cells with visible constrictions at the sites of septa) and hyphae (linear filaments without visible constrictions at the septa) (Mitchell, 1998). The various factors that contribute to its virulence include its ability to undergo yeast to hyphal transition, formation of biofilms, adhesion and secretion of aspartyl proteinases. Hyphae are considered to be involved in invasive growth as they are frequently identified in infected tissues and strains defective in morphological transition (yeast to hyphal) are avirulent (Leberer et al., 1996; Lo et al., 1997; Stoldt et al., 1997). Morphological switching is not only necessary for successful establishment of infection but important for evading components host defense system like macrophages or dendritic cells. A network of signaling pathways that operate in C. albicans continuously assess the nutrient availability, cell density and other environmental conditions. The integrated output of these pathways determine the response of C. albicans under given set of environmental/media conditions and eventually determines the gene expression and morphogenic transition (Liu., 2001). C. albicans utilizes at least two major signaling pathways besides others for regulating the morphological transition. One of these two pathways uses Cph1 as transcription factor and is the homolog of Ste12 in S. cerevisiae which is shown to be involved in Pseudohyphal growth and mating. The other pathway includes Efg1 (homolog of Phd1 in S. cerevisiae) as transcription factor. Biofilm formation by Candida species is an important virulence factor and has gained considerable interest recently as these specialized survival structures are found in implanted devices such as indwelling catheters and prosthetic heart valves (Hawser and Douglas, 1994; Douglas, 2003). These biofilms lead to the failure of implants besides providing multiple drug resistance (Baillie and Douglas, 1999). A better understanding of the C. albicans interaction with the host at the site of infection and with the components of immune system will help in identifying new potential drug targets. (a) Genome wide expression profile of Candida albicans from patient samples and characterization of CaRPB4/7: To get a better insight in C. albicans response at the site of infection we were interested in mapping the expression profile of Candida albicans in active state of human infections. Patients suffering from head and neck cancer undergoing radiation therapy have high risk of C. albicans infection. We identified five such patients with heavy oral thrush infections and C. albicans samples were collected from them. Candida albicans was confirmed in these samples by various microbiological tests following which the samples were used for RNA isolation. The whole genome expression analysis leads to the identification of 188 up regulated and 88 down regulated genes in patient samples. Our data analysis revealed that Protein Kinase A pathway and many downstream genes of the same were differentially expressed. Analysis of saliva (saliva is known for antifungal and antibacterial activity) from these patients showed that unlike healthy individuals, the patient saliva favours yeast to hyphal transition of C. albicans cells. This might be a reason for high risk of infection. A major class of upregulated genes is found to be functionally involved in transcription which includes some RNA polymeraseII and III subunits. CaRPB4, the forth largest subunit of RNA polymeraseII, was found to be upregulated in patient samples. RPB4 has been shown to form sub complex with RPB7, the seventh largest subunit of RNA polymeraseII, and both subunits are known to play a role in a variety of stress conditions and pseudohyphal development in Saccharomyces cerevisiae. We characterized the CaRPB4 and CaRPB7 (homolog in Candida albicans) for their ability to complement their S. cerevisiae counterparts. CaRPB4 and CaRPB7 were able to complement majority of the phenotypes associated with these subunits in S. cerevisiae. Overexpression of CaRPB7 in S. cerevisiae enhances pseudohyphal growth. Considering the high degree of conservation of signaling pathways between S. cerevisiae and C. albicans it can be speculated that CaRPB7 might be involved in pseudohyphal development in C. albicans. We found that over expression of CaRPB4 in Candida albicans shows enhanced agar invasive growth which can be thought analogous to tissue invasion in host and hence might contribute for establishment of infection. This suggests that both the RNA polII subunits have a role to play in the virulence of C. albicans. (b) Characterization of UDP-Galactose 4-Epimerase (GAL10) from Candida albicans and their role in virulence. Enzyme UDP-Galactose-4-Epimerase [GAL10] is responsible for conversion of UDP-galactose to UDP-glucose which then gets metabolized by the cells through glycolysis and TCA cycle. The enzyme catalyzes a reversible reaction and can convert glucose to galactose in the absence of galactose as shown in Trypanosoma brucei and also involved in its virulence. In this study, we have identified the functional homolog of GAL10 in Candida albicans. S. cerevisiae and C. albicans GAL10 homologs are similar in their domainal organization as the proteins have a mutarotase and an epimerase domain. The former is responsible for conversion of ﾟ-D-galactose to a-D-galactose and the latter for epimerization of UDP-galactose to UDP-glucose. The synteny of galactose metabolizing structural genes is conserved among some fungi. To study the importance of CaGAL10 we generated deletion mutant of the gene in C. albicans. Our studies show that CaGAL10 [C. albicans GAL10] is involved in cell wall organization and in oxidative stress response. The mutant strain of GAL10 is hyperfilamentous in Lee’s and spider medium and the biofilm formed is morphologically different from the wild type strain. These set of results suggests that CaGAL10 plays an important role in organization/integrity of cell wall in C. albicans and speculate that it might be involved in virulence. (c) Study of Candida albicans-macrophage interaction and identification of transcriptional regulator of genes encoding proteins of translation machinery: Macrophages serve as the effector cells of cell mediated immunity in the control of infections. They are considered to be important for resistance to muco-cutaneous and systemic candidiasis. Our studies were aimed at understanding the response of Candida albicans cells to the presence of macrophages for extended period of time. The response was monitored using microarrays. Specifically genes involved in galactose, protein and lipid metabolism and stress response undergo concerted changes in their transcript levels. We analyzed the promoters of coregulated genes to identify common DNA elements present in them which might be involved in their transcriptional regulation. Promoter analysis of differentially expressed genes revealed presence of CPH1 and EFG1 transcription factor binding sites. Besides identifying CPH1 and EFG1 Binding sites, we identified two novel DNA elements in promoters of coregulated gene. A conserved motif TGAAAAGGAAG was identified in the promoters of genes involved in energy generation. Another 18 mer consensus palindromic sequence TAGGGCTNTAGCCCTAAT was identified in the promoters of about 48 genes. Majority of these genes encode ribosomal proteins. With the help of techniques like EMSA (Electophoretic Mobility Shift Assay) and south-western we had shown the presence of a protein of ~66 KDa molecular weight binding to the sequence with high specificity.

Decapentaplegic (Dpp), a member of the TGF-[Beta] superfamily, acts as a morphogen to direct cell differentiation, determine cell fate and promote cell survival and proliferation in Drosophila wing development. To investigate the role of Dpp in Drosophila wing development, three aspects of the patterning role of Dpp have been analyzed. First, I investigated the cellular responses to Dpp signaling by a loss of function strategy. The consequences of lacking Dpp signal transduction on cell morphology and tissue integrity were analyzed. Second, I investigated whether Dpp signaling is down-stream of Hh signaling to maintain the normal cell segregation at the A/P boundary by clonal analysis. Third, I investigated whether cross talk among the Hh, Dpp and Wg signaling pathways exists and what its relevance for wing patterning is. To investigate the role of Dpp in Drosophila wing development, the general strategies are to look at the phenotypes of loss-of-function and gain-of-function. Mutant clones lacking Dpp signal transduction by knock down Dpp receptor Thick veins (Tkv) do not survive in wing blade due to JNK dependent apoptosis. To get larger mutant clones for analysis, JNK pathway was inhibited by knock down bsk (encodes JNK) in mutant clones lacking Dpp signaling using FLP-FRT system. Clones double mutant for tkv and bsk did not undergo apoptosis, but recovered at very low frequencies compared to sibling clones. Here, I showed that the low recovery of tkv bsk double mutant clones are due to the extrusion of mutant cells. The extrusion of tkv bsk double mutant cells correlated with changes in the actin cytoskeleton and a dramatic loss of the apical microtubule web normally present in these cells. These results suggest that Dpp signaling is required for cell morphogenesis in Drosophila wing development. We propose that Dpp acts as a survival factor in the wing disc epithelium by orchestrating proper cytoskeletal organization and maintaining normal cell-cell contact. Drosophila wing is subdivided into anterior (A) and posterior (P) compartments. This developing into adjacent compartments is crucial for the patterning of Drosophila wing. Previous study has shown that Hedgehog (Hh) signaling is required in A cells to maintain the A/P boundary and is sufficient to specify A type cell sorting. A previous study has in addition implicated the signaling molecule Decapentaplegic (Dpp) in maintaining the A/P boundary. However, this study did not address whether and in which cells, A and/or P, Dpp signal transduction was required to maintain this boundary. Here, I have analyzed the role of components of the Dpp signal transduction pathway and the relation of Dpp and Hh signaling in maintaining the A/P boundary by clonal analysis. I showed that Dpp signaling mediated by the Dpp target gene, T-box protein Optomotor-blind (Omb), is required in A cells, but not in P cells, to maintain the normal position of the A/P boundary. During patterning formation, it is essential for cells to receive precise positional information to pattern the tissue. It has been proposed for a long time that different signaling pathways such as Hedgehog (Hh), Dpp and Wingless (Wg) signaling pathways provide positional information for tissue patterning in an integrated manner. Recently, evidence of interactions between Hh and Dpp as well as Wg and Hh signaling pathways has been reported in Drosophila wing. Here, I have identified additional interactions among Hh, Dpp and Notch/Wg signaling. We propose that the selector gene engrailed, Hh and Dpp signaling interact with each other to regulate target genes expression and thus to pattern the wing along the A/P axis. Further more, I showed that Dpp signaling is also participating in the patterning along the D/V axis by interaction with the selector gene apterous and Notch/Wg signaling.

Οι εξωκυττάριες ουσίες παράγονται από τα κύτταρα, εκκρίνονται από αυτά και αλληλεπιδρώντας τόσο μεταξύ τους όσο και με τα κύτταρα ρυθμίζουν πολλές αναπτυξιακές πορείες. Μελετήσαμε τη χρονική και τοπική εμφάνιση των mRNAs της εντακτίνης και της λαμινίνης, γλυκοπρωτεϊνών των εξωκυττάριων ουσιών, στο πρώϊμο έμβρυο όρνιθας. Χρησιμοποιήσαμε την τεχνική της in situ υβριδοποίησης, που κατέδειξε διαφορική έκφραση της εντακτίνης και των αλυσίδων της λαμινίνης σε ξεχωριστούς κυτταρικούς πληθυσμούς και όργανα σε διαφορετικά στάδια ανάπτυξης του εμβρύου. Επίσης μελετήσαμε την χρονική και τοπική κατανομή του πολυπεπτι- δίου της εντακτίνης από το στάδιο του μοριδίου ως την αρχή της οργανογένεσης με τις τεχνικές του ανοσοφθορισμού και της ανοσοκατακρήμνισης. Ανιχνεύσαμε την παρουσία της εντα-κτίνης ήδη από το στάδιο του όψιμου μοριδίου. Με τη χρήση αντισωμάτων για την εντακτίνη μελετήσαμε το ρόλο της κατά την ανάπτυξη του πρώϊμου εμβρύου. Στα αποτελέσματά μας φάνη-κε οτι η εντακτίνη είναι απαραίτητη για το σωστό προσανατο-λισμό των κυττάρων καθώς μεταναστεύουν κατά την γαστριδίω-ση. Αυτό έχει ως αποτέλεσμα την αναστολή του σχηματισμού του εμβρυϊκού άξονα. / The extracelular matrix is a complex cell product which regulates many developmental processes. We used specific antisense RNA probes for the laminin α1, β1 and γ1 chains and for entactin, two extracellular matrix glycoproteins, to study the tissue specific and temporal patterns of their mRNAs in the early chick embryo. Our work employing in situ hybridization, showed strong signals of the laminin and of entactin mRNAs at the morula stage and differential express-ion of these mRNAs in the forming embryonic tissues and organs in the developing chick embryo. We also studied the time of appearance and subsequent distribution of the entactin polypeptide using immunofluorescence and immunopre-cipitation from the morula stage up to the early organogene-sis in the chick embryo. The first presence of entactin was detected at the late morula stage and showed differential expression in the various cell populations in the develop- ing embryo. To study the role of entactin in the major cell-ular migrations during gastrulation we used blocking anti- bodies in set of functional studies. Entactin seemed to be essential for the directional migrations of cells during gastulation and the embryonic axis was not formed in the embryos treated with the anti-entactin antibodies.

Die Zellteilung/Zytokinese ist ein grundlegender zellulärer Prozess und essentiell für das Wachstum von einzelligen und mehrzelligen Organismen. Reguliert wird dieser Prozess durch komplexe molekulare Mechanismen sowie einer Vielzahl von interaktiven Netzwerken. In Pilzen koordiniert eine Kinase-Kaskade, das Septierungs-Initiierungs Netzwerk (SIN) das Fortschreiten des Zellzyklus mit dem Beginn der Zellteilung und kontrolliert die Septenbildung. Fehlregulation des homologen Hippo Netzwerks in Tieren führt zu Gewebewucherungen und Tumorbildung, was die konservierte Bedeutung dieser Regulationsnetzwerke in verschiedenen Organismen unterstreicht. Obwohl die Septenbildung essentiell für das Wachstum und die Differenzierung von Schimmelpilzen ist, bleibt die Frage wie die Septierung reguliert wird und aus welchen Komponenten sich das SIN Netzwerk in filamentösen Pilzen zusammensetzt bisher noch unbeantwortet. Mit Hilfe von in silico Analysen konnten homologe Proteine für fast alle SIN Netzwerk Komponenten im Modellorganismus Neurospora crassa identifiziert werden. Die Analyse dieser vorhergesagten SIN Komponenten ermöglichte die Charakterisierung der SIN-Kinase-Kaskade, bestehend aus CDC-7, SID-1 und DBF-2 sowie den entsprechenden, regulatorischen Untereinheiten CDC-14 und MOB-1. Es konnte gezeigt werden, dass DBF-2 durch SID-1 am hydrophoben Motiv phosphoryliert und aktiviert wird und dass eine SID-1 abhängige Stimulation von DBF-2 durch Zugabe von CDC-7 weiter gesteigert wird. Diese Daten liefern den ersten biochemischen Nachweis für die schrittweise Aktivierung einer dreistufigen SIN-Kinase-Kaskade in Pilzen. Es wurde weiterhin gezeigt, dass die gesamte SIN Kaskade konstitutiv und Zellzyklus unabhängig an den Spindelpolkörpern akkumuliert und dass alle SIN Proteine an kontrahierenden Septen lokalisieren. Demzufolge ist im Gegensatz zu den einzelligen Pilzen die Lokalisation und Aktivität der SIN Komponenten in Synzytium-bildenden Ascomyzeten Zellzyklus unabhängig. Darüber hinaus deutet die Charakterisierung von DBF-2 Mutanten, in denen die beiden regulatorischen Aminosäuren (Ser499 and Thr671) mutiert sind, darauf hin, dass ein dynamischer Phosphorylierungs-/Dephosphorylierungszyklus des Ser499 entscheidend für die Aktivität und Funktion von DBF-2 in N. crassa ist. Diese Daten haben Einfluss auf das allgemeine Verständnis der Aktivierung von NDR Kinasen, denn bisher wurde für NDR Kinasen höherer Eukaryonten eine folgegebundene Phosphorylierung beider regulatorischer Reste angenommen. Der Ste20-verwandten Kinase MST-1 konnte eine Funktion als SIN-assoziierte Kinase, die parallel zu SID-1 agiert, zugeordnet werden. SID-1 und MST-1 werden auf entgegengesetzte Weise von der oberhalb agierenden SIN Kinase CDC-7 reguliert, was nahelegt, dass MST-1 für die Feinabstimmung des SIN erforderlich ist. Lifeact- und Formin-GFP Reporter Konstrukte zeigten, dass in der Δmst-1 Mutante abnormale, kortikale Actomyosin-Ringe gebildet werden, was eine Fehlpositionierung der Septen und die Bildung von unregelmäßigen Spiralen zur Folge hat. Diese Defekte entsprechen partiell jenen der MOR Mutanten. Diese Mutanten weisen ein defektes NDR Kinase Netzwerk auf, welches für das polare Wachstum verantwortlich ist (MOR). Es stellte sich heraus, dass MST-1 mit den zentralen MOR Kinasen POD-6 und COT-1 interagiert und sowohl die SIN Effektor Kinase DBF-2 als auch die MOR Effektor Kinase COT-1 aktiviert. Somit fungiert MST-1 als dual-spezifisches Enzym. Eine weitere Vernetzung beider Signalwege ist durch die Bildung von Heterodimeren gegeben. Die in dieser Studie identifizierten verschiedenen Ebenen der Vernetzung des SIN und MOR, sowie entsprechende Daten aus anderen Modellorganismen wie S. pombe und D. melanogaster, lassen vermuten, dass antagonistische Interaktionen zwischen homologen NDR Kinase Netzwerken ein genereller Mechanismus zur Koordination beider Signalwege darstellt und auch in höheren Organismen konserviert ist. Durch die Annotierung mehrerer Pilzgenome wurden zahlreiche Gene mit einer Homologie zu den S. cerevisiae BUD Genen auch in filamentösen Pilzen identifiziert. Epistatische und biochemische Analysen ergaben, dass das MOR Netzwerk als negativer Regulator der Septenbildung oberhalb des BUD komplex fungiert und dass COT-1 im Gegensatz zu DBF-2, die beiden Septierungsmarkerproteine BUD-3/BUD-4 phosphoryliert. Folglich könnte die Regulation von BUD-3 (und eventuell auch BUD-4) durch COT-1 ein Mechanismus des MOR Netzwerks sein, um die Septenbildung in N. crassa zu inhibieren.

Les urodèles amphibiens, dont fait partie l’axolotl (Ambystoma mexicanum), ont la capacité de régénérer leurs organes et membres suite à une amputation, tout au long de leur vie. La patte est l’organe dont le processus de régénération est le mieux caractérisé et ce dernier est divisé en deux phases principales. La première est la phase de préparation et commence immédiatement suite à l’amputation. Elle renferme des étapes essentielles au processus de régénération comme la guérison de la plaie et la formation d’une coiffe apicale ectodermique. Par la suite, les fibroblastes du derme et certaines cellules musculaires vont revenir à un état pluripotent via un processus appelé dédifférenciation cellulaire. Une fois dédifférenciées, ces cellules migrent et s’accumulent sous la coiffe apicale pour former le blastème. Lors de la phase de redéveloppement, les cellules du blastème se divisent puis se redifférencient pour régénérer la partie amputée. Fait intéressant, la régénération d’un membre ou la guérison d’une plaie chez l’axolotl ne mène jamais à la formation d’une cicatrice. Afin d’en apprendre plus sur le contrôle moléculaire de la régénération, les gènes Heat-shock protein-70 (Hsp-70) et Transforming growth factor-β1 (Tgf-β1) ont été sélectionnés. Ces gènes jouent un rôle important dans la réponse au stress et lors de la guérison des plaies chez les mammifères. HSP-70 est une chaperonne moléculaire qui est produite pour maintenir l’intégrité des protéines cellulaires lorsqu’un stress se présente. TGF-β1 est une cytokine produite suite à une blessure qui active la réponse inflammatoire et qui stimule la fermeture de la plaie chez les amniotes. Les résultats présentés dans cette thèse démontrent que Hsp-70 est exprimé et régulé lors du développement et de la régénération du membre chez l’axolotl. D’autre part, nos expériences ont mené à l’isolation de la séquence codante pour Tgf-β1 chez l’axolotl. Nos résultats montrent que Tgf-β1 est exprimé spécifiquement lors de la phase de préparation dans le membre en régénération. De plus, le blocage de la voie des Tgf-β avec l’inhibiteur pharmacologique SB-431542, lors de la régénération, mène à l’inhibition du processus. Ceci démontre que la signalisation via la voie des Tgf-β est essentielle à la régénération du membre chez l’axolotl. / Urodele amphibians, such as the axolotl (Ambystoma mexicanum), have the unique ability, among vertebrates, to perfectly regenerate many parts of their body throughout their life. Among the complex structures that can be regenerated, the limb is the most widely studied. Limb regeneration is divided in two main phases. The preparation phase, which begins right after amputation, includes wound healing and the formation of an apical ectodermal cap. During this phase, dermal fibroblasts and muscle cells will lose their characteristics and become pluripotent through a process called cellular dedifferentiation. The dedifferentiated cells migrate and accumulate under the apical ectodermal cap to form the blastema. During the redevelopment phase, the cells in the blastema proliferate and redifferentiate to regenerate the lost structures. It is interesting to highlight the fact that regeneration never leads to scar formation in the axolotl. In order to learn more about the molecular control of limb regeneration, the genes Heat-shock protein-70 (Hsp-70) and Transforming growth factor-β1 (Tgf- β1) were selected for their important roles in stress response and wound healing in mammals. HSP-70 is a molecular chaperone which is produced to protect cellular proteins when the cell faces a stress. TGF-β1 is a cytokine produced after wounding that activates the inflammatory response and stimulates wound closure in amniotes. Results presented in this thesis show that Hsp-70 is expressed and regulated during limb development and regeneration in the axolotl. We were also able to isolate the cDNA coding for axolotl Tgf-β1 and our results show that this gene is expressed specifically during the preparation phase of limb regeneration. Treatment of regenerating axolotls with a specific inhibitor of Tgf-β signalling, SB-431542, led to complete inhibition of regeneration. This directly implies that Tgf-β signalling is essential for limb regeneration in axolotl.

La division cellulaire est un événement fondamental, indispensable au développement embryonnaire animal et à l’homéostasie des organismes adultes. Il s’agit d’un processus complexe qui doit être précisément contrôlé dans le temps et l’espace pour permettre la formation de deux cellules filles, au contenu génétique identique à celui de la cellule mère. Ceci requiert une coordination entre la ségrégation des chromosomes, opérée par les microtubules, et le clivage de la cellule, engageant une réorganisation dynamique du cytosquelette d’Actine. La modification de la forme des cellules en cours de division est en effet due au remodelage du cortex cellulaire, incluant la membrane plasmique et le réseau de filaments d’Actine sous-jacent. Bien que cette série de modifications du cortex soit indispensable au déroulement correct de la division cellulaire, les mécanismes moléculaires du contrôle l’organisation corticale en mitose restent mal caractérisés. Le PI(4,5)P2 est un phosphoinositide constituant de la membrane plasmique, notamment nécessaire à la division cellulaire. Nos travaux chez la drosophile mettent en évidence que ce phospholipide présente une distribution dynamique, homogène sur l’ensemble du cortex à l’entrée en mitose, puis se concentrant à l’équateur des cellules après la séparation des deux lots de chromosomes. Nous montrons que le PI(4,5)P2 est nécessaire au contrôle de la stabilité corticale et du fuseau mitotique, au moins en partie par son rôle favorisant l’activation de la dMoésine. La dMoésine régule l’interaction entre les filaments d’Actine et la membrane plasmique, jouant un rôle clé dans l’organisation locale du cortex des cellules en mitose et ses propriétés mécaniques. Nous montrons que l’interaction PI(4,5)P2/dMoésine participe à la contraction cellulaire à l’entrée en mitose, puis à l’élongation cellulaire caractéristique des étapes plus tardives de la division. A la fin de la mitose, nous montrons que la phosphatase pP1-87B inhibe l’activation de la dMoésine, indispensable à la relaxation du cortex des cellules en interphase. Par un crible fonctionnel systématique, nous avons recherché l’ensemble des facteurs indispensables à la production et à l’enrichissement localisé du PI(4,5)P2 au cortex mitotique. Nous montrons le rôle majeur de deux voies de biosynthèse, qui collaborent pour produire localement le PI(4,5)P2 à la membrane plasmique au cours de la mitose. Leur absence prévient l’activation et le recrutement membranaire de la dMoésine, et conduit à une instabilité corticale associée à des défauts du fuseau mitotique. Une troisième voie, nécessitant l’activité de la protéine dOcrl, contribue à l’homéostasie de ce phosphoinositide, en dégradant le PI(4,5)P2 présent sur les membranes internes de la cellule. L’inactivation de dOcrl empêche la formation normale et l’ingression du sillon de clivage. Ensemble, ces résultats identifient donc des régulateurs importants de la membrane plasmique et de son interaction avec le cytosquelette, permettant de mieux comprendre les mécanismes de la réorganisation de la forme cellulaire au cours de la mitose. / Cell division must be accurately controlled in time and space to permit the formation of two daughter cells whose genetic content is identical to that of the mother cell. This process requires successive modifications of cell shape, induced by cortical remodelling. Molecular mechanisms controlling cortical reorganization during mitosis remain partially uncharacterized. Our work in Drosophila cells demonstrates that PI(4,5)P2, a phosophoinositide of the plasma membrane, is enriched at the equatorial region at the onset of anaphase. This PI(4,5)P2 is necessary for the cortical stability of mitotic cells, and requires dMoesin activation. The dMoesin, linking actin to the plasma membrane, plays a critical role in the cortical organization of mitotic cells and in the regulation of its mechanical properties. We show that the interaction PI(4,5)P2/dMoesin participates in cellular contraction at the beginning of mitosis, then in cell elongation characteristic of subsequent steps. At the end of mitosis, the Pp1-87B phosphatase inactivates the dMoesin. By a systematic functional screen, we characterize the key role of two pathways acting in synergy to locally produce PI(4,5)P2, Skittles- and Pten-dependent, and the role of a third pathway requiring dOcrl activity to control PI(4,5)P2 homeostasis. Altogether, these results allow us to better understand the mechanisms controlling cortical remodelling and modifications of cell shape that occur during mitosis. / Doctorat réalisé en cotutelle avec le laboratoire de François Payre au Centre de Biologie du Développement à Toulouse, France (Université de Toulouse III - Paul Sabatier)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Objective was to evaluate the effects of different population arrangements in forage sorghum grazing on forage production, morphogenesis, pasture structure, performance, and animal behavior. They were used 36 crossed heifers between Charolais and Nellore, with initial age of 15 months and 262 kg of body weight (BW). The experimental period lasted 84 days, divided into periods of 28 days. The animals received daily supplementation to the level of 1% of body weight. Treatments consisted of a combination of spacings of 22 or 44 cm in planting spacing with two densities of seeds per hectare, 12 or 24 kg, resulting in arrangements population E44D12; E44D24; E22D12; E22D24. The grazing method was continuous with variable number of regulators animals. The forage mass and the mass of leaf blades did not differ between population arrangements, with average values of 1279.11 and 232.99 kg DM / ha. Similarly the stocking rate, dry matter accumulation rate and weight gain per area, with average values of 1172.32 kg LW / ha, 58.47 kg DM / ha / day and 303.31 kg PV / ha, in that order. The performance and development of the animals were not affected by population arrangements grassland, with weight gain and average final weight between treatments of 0.891 kg / day and 330.36 kg of PV. The relation weight:height among the population arrangements was 2.67 kg / cm, indicating that cross heifers between Charolais and Nellore with 18 months of age have fat thickness and appropriate weight for slaughter. Productive parameters of pasture and animal performance were influenced by evaluation periods. The different population arrangements change the behavioral patterns of animals, E44D24 and E22D12 treatments increase the grazing time. The E44D24 treatment decreases the bit rate when compared to E22D24. The lower density of plants per square meter causes the increase in the number of feeding stations / minute and the displacement rate of the animals. The spacing between rows 22cm promote a larger number of bits/food station. The use of higher density of seeds and larger spacing between lines increases the density of leaf blades in the lower and upper stratum of the pasture, respectively. The E44D12 and E22D24 treatments have higher amounts of stems in the upper stratum of the pasture (over 60 cm). Morphogenic variables were not significantly influenced by the different arrangements of plants used and the evaluation periods, being obtained elongation rate and leaf senescence of 1.43 and 1.11 cm/day/tiller, respectively, leaf appearance rate and leaf appearance interval of 0.28 leaves/day/tiller and 3.81 days phillochron and leaf life span of 72.99 and 351.68 degree day, in that order. Have the structural characteristics of pasture were influenced by evaluation periods, reflecting the forage production decline with advancing the use of the pasture cycle. The first to the third evaluation period was verified a reduction of 65.53% in the number of sheets in elongation and 47.79% in the number of living leaves. / Objetivou-se avaliar os efeitos de diferentes arranjos populacionais em pastagem de sorgo forrageiro na produção forrageira, morfogênese, estrutura do pasto, desempenho e comportamento animal. Foram utilizadas 36 novilhas de corte cruzadas entre as Charolês e Nelore, com idade média inicial de 15 meses e 262 Kg de peso vivo (PV). O período experimental durou 84 dias, divididos em períodos de 28 dias. Os animais receberam suplementação diária à nível de 1% do peso vivo. Os tratamentos consistiram na combinação de espaçamentos de 22 ou 44 cm na entrelinha de plantio com duas densidades de sementes por hectare, 12 ou 24 Kg, resultando nos arranjos populacionais E44D12; E44D24; E22D12; E22D24. O método de pastoreio utilizado foi contínuo, com número variável de animais reguladores. A massa de forragem e a massa de lâminas foliares não diferiram entre os arranjos populacionais, apresentando valores médios de 1279,11 e 232,99 Kg de MS/ha. Da mesma forma a carga animal, taxa de acúmulo de matéria seca e o ganho de peso por área, apresentando valores médios de 1172,32 Kg de PV/ha, 58,47 Kg de MS/ha/dia e 303,31 Kg de PV/ha, nessa ordem. O desempenho e o desenvolvimento dos animais não foram influenciados pelos arranjos populacionais da pastagem, apresentando ganho de peso e peso final médio entre os tratamentos de 0,891 Kg/dia e 330,36 Kg de PV. Os parâmetros produtivos da pastagem e desempenho animal foram influenciados pelos períodos de avaliação. Os diferentes arranjos populacionais alteraram os padrões comportamentais dos animais, os tratamentos E44D24 e E22D12 aumentam o tempo de pastejo. O tratamento E44D24 diminui a taxa de bocado quando comparado ao E22D24. A menor densidade de plantas por metro quadrado ocasiona o aumento no número de estações alimentares/minuto e a taxa de deslocamento dos animais. O espaçamento 22 cm entrelinhas promove um maior número de bocados/estação alimentar. A utilização de maior densidade de sementes e maiores espaçamentos entrelinhas aumenta a densidade de lâminas foliares no estrato inferior e superior da pastagem, respectivamente. Os tratamentos E44D12 e E22D24 apresentaram maiores quantidades de colmos no estrato superior do pasto (acima de 60 cm). As variáveis morfogênicas não foram influenciadas significativamente pelos diferentes arranjos de plantas utilizados e pelos períodos de avaliação, sendo obtidos taxa de alongamento e senescência foliar de 1,43 e 1,11 cm/dia/afilho, respectivamente, taxa de aparecimento foliar e intervalo de surgimento de folhas de 0,28 folhas/dia/afilho e 3,81 dias, filocrono e duração de vida foliar de 72,99 e 351,68 graus dia, nessa ordem. Já as características estruturais da pastagem foram influenciadas pelos períodos de avaliação, refletindo a queda de produção forrageira com o avançar do ciclo de utilização da pastagem. Do primeiro para o terceiro período de avaliação foi verificado uma redução de 65,53% no número de folhas em alongamento e de 47,79% no número de folhas vivas.

No description available.

La division bactérienne résulte de la constriction de la membrane, menée par la protéine du cytosquelette FtsZ, et de l’expansion et du remodelage de la paroi, réalisés par des synthétases et des hydrolases de la paroi. La coordination de ces processus au sein d’un macrocomplexe protéique, le divisome, est nécessaire au maintien de la forme et de l’intégrité bactérienne. J’ai étudié deux aspects importants de ce mécanisme de coordination chez le pathogène humain Streptococcus pneumoniae. J’ai déterminé in vivo la nanostructure de la protéine FtsZ en développant l’utilisation du PALM (PhotoActivated Localization Microscopy)chez le pneumocoque. Cette technique, basée sur la détection de molécules uniques et permettant une résolution de 20-40 nm, a révélé des aspects inattendus (dimensions, amas, sous-structures) de l’architecture de l’anneau de FtsZ au cours du cycle cellulaire. En parallèle, j’ai étudié le rôle de l’hydrolase Pmp23 par génétique, biochimie et microscopie à fluorescence. Mon travail a montré que Pmp23 est requise pour la stabilité des macrostructures du divisome du pneumocoque, révélant une nouvelle connexion entre le métabolisme de la paroi et la division cellulaire. / Bacterial division results from the combination of membrane constriction, driven by the cytoskeletal protein FtsZ, with cell wall expansion and remodeling, performed by cell wall synthases and hydrolases. Coordination of these processes within a large protein complex known as the divisome ensures cell integrity and maintenance of cell shape. I have investigated two important aspects of this coordination mechanism in the human pathogen Streptococcus pneumoniae. I determined the in vivo nanostructure of the divisome scaffolding protein FtsZ by developing the use of PhotoActivated Localization Microscopy (PALM) in the pneumococcus. PALM, which is based on the detection of single fluorescent labels and allows 20-40 nm resolution, has revealed unexpected features (dimensions, clusters, new substructures) of the FtsZ-ring architecture along the cell cycle. In parallel, I studied the role of the cell wall hydrolase Pmp23 using genetics, biochemistry and fluorescence microscopy. My work has shown that Pmp23 is required for the stability of divisome macrostructures in the pneumococcal cell, revealing a new connection between cell wall metabolism and cell division.

Plantas forrageiras se adaptam ao pastejo por meio de modificações em forma e função alterando seus padrões de expressão morfogênica e, consequentemente, de acúmulo e composição morfológica da forragem produzida. O objetivo deste estudo foi avaliar as respostas morfogênicas e as estruturais de perfilhos em pastos de capim-mulato submetidos a estratégias de pastejo rotativo de fevereiro de 2008 a abril de 2009. Os tratamentos corresponderam combinações entre duas condições pós-pastejo (alturas pós-pastejo de 15 e 20 cm - APP) e duas condições pré-pastejo (95% e máxima interceptação de luz pelo dossel forrageiro - IL), e foram alocados às unidades experimentais (piquetes de 1200 m2) segundo arranjo fatorial 2x2 e delineamento de blocos completos casualizados, com 4 repetições. Foram avaliadas as seguintes variáveis-resposta: taxa de aparecimento de folhas (TApF); filocrono (FIL); taxa de alongamento de folhas (TAlF); taxa de alongamento de colmos (TAlC); taxa de senescência de folhas (TSeF); encurtamento do colmo (EC); duração da vida da folha (DVF); duração do alongamento foliar (DAF); comprimento final da folha (CFF); número de folhas vivas (NFV), em expansão (NFEx), expandidas (NFE) e senescentes (NFS) por perfilho; comprimento do colmo (CC) e relação folha:colmo por perfilho (F:C). Tanto perfilhos basais como aéreos apresentaram sazonalidade de desenvolvimento caracterizada por ritmos morfogênicos mais lentos durante o outono/inverno/início de primavera e mais acelerados durante o final de primavera e verão. No caso de perfilhos basais, pastos manejados a 95% de IL apresentaram maiores valores de TApF no Verão 2. No Verão 1, o EC nesses pastos foi menor, sendo observado comportamento inverso no final da primavera. Menores valores de TAlC e TSeF foram registrados nos pastos manejados a 95% relativamente àqueles manejados com máxima IL (99%). Adicionalmente, pastos manejados com altura pós-pastejo 20 cm apresentaram maiores valores de TAlF, TSeF e DAF que pastos manejados a 15 cm, especialmente na condição pré-pastejo de 95% de IL. Nos perfilhos aéreos, maiores valores de TAlC e TSeF foram registrados nos pastos manejados com máxima IL (99%) relativamente àqueles manejados a 95% de IL. Com relação às características estruturais, a APP afetou apenas aquelas relacionadas com o porte da planta (CFF e CC). Apesar das diferenças estatísticas, o NFV foi relativamente constante para aéreos e basais (2,5 e 4,0 folhas por perfilho, respectivamente), com as diferenças entre categorias de perfilhos refletindo diferenças em NFS e NFE e não em NFEx. Perfilhos basais foram maiores que aéreos, porém com menor F:C. Para perfilhos basais, NFV, NFEx e F:C foram maiores em pastos manejados a 95% de IL e, para aéreos, naqueles manejados com máxima IL, padrão condizente com o fato de perfilhos aéreos serem provenientes de perfilhos basais reprodutivos decapitados. De forma geral, as características estruturais foram mais afetadas pela IL e época do ano do que pela APP, indicando, claramente, importância relativa maior da frequência comparativamente à severidade de desfolhação para controle da estrutura do dossel. Diante do exposto, a condição ideal para interrupção do processo de rebrotação dos pastos de capim-mulato é quando o dossel atinge 95% de IL com uma altura pós-pastejo de 20 cm. / Forage plants adapt to grazing through morphological and physiological changes that modify their morphogenesis and, in turn, herbage accumulation and morphological composition of the produced herbage. The objective of this study was to evaluate the morphogenetic responses and the structural characteristics of individual tillers on mulato grass swards subjected to strategies of rotational stocking management from February 2008 until April 2009. Treatments corresponded to combinations between two post-grazing (post-grazing heights of 15 and 20 cm) and two pre-grazing (95% and maximum light interception by sward canopy LI) conditions, and were allocated to experimental units (1200 m2 paddocks) according to a 2x2 factorial arrangement and a randomised complete block design, with four replications. The following response variables were evaluated: rates of leaf appearance (LAR), leaf elongation (LER), stem elongation (SER), leaf senescence (LSR), reduction in stem length (RSL), phyllochron (PHY), leaf lifespan (LLS), leaf elongation duration (LED), final leaf length (FLL), number of live (NLL), expanding (NExL), expanded (NEL) and senescing (NSL) leaves per tiller, stem length (SL) and the leaf:stem ratio per tiller (L:S). Both basal and aerial tillers showed a clear seasonal pattern of growth characterised by slow morphogenetic rhythms during autumn/winter/early spring and fast rhythms during late spring and summer. For basal tillers, swards managed at 95% LI showed highest values of LAR in summer 2. In Summer 1, RSL on those swards was lowest, the reverse happening in late spring. Lower values of SER and LSR were recorded on swards managed at 95% relative to those managed at maximum LI (99%). Further, swards managed with the postgrazing height of 20 cm showed larger values of LER, LSR and LED than those with 15 cm, particularly for the 95% LI pre-grazing condition. For aerial tillers, larger values of SER and LSR were recorded on swards managed at maximum LI (99%) relative to those managed at 95% LI. In relation to structural characteristics, postgrazing height only influenced those related to plant size (FLL and SL). In spite of the statistical differences, NLL was relatively stable for aerial and basal tillers (2.5 and 4.0 leaves per tiller, respectively), with differences between tiller categories mainly due to differences in NSL and NEL, not NExL. Basal tillers were bigger than aerial tillers, although had lower L:S. For basal tillers, NLL, NExL and L:S were larger on swards managed at 95% LI and, for aerial tillers, larger values were recorded on swards managed at maximum LI, a pattern in line with the fact that aerial tillers are originated from decapitated reproductive basal tillers. Overall, structural characteristics were more influenced by LI and season of the year than by postgrazing height, highlighting the larger importance of frequency relative to severity of defoliation for controlling sward structural characteristics. As a result, the ideal 14 condition for interrupting regrowth of rotationally stocked mulato grass correspond to a pre-grazing condition of 95% LI and a post-grazing height of 20 cm.