Ljudski genom sadrži milijune regulatornih elemenata - enhancera - koji kvantitativno reguliraju ekspresiju gena. Unatoč ogromnom napretku u razumijevanju načina na koji enhanceri reguliraju ekspresiju gena, području još uvijek nedostaje pristup koji je sustavan, integrativan i dostupan za otkrivanje i dokumentiranje cis-regulatornih odnosa u cijelom genomu. Razvili smo novu računalnu metodu - reg2gene - koja modelira i integrira aktivnost enhancera~ekspresije gena. reg2gene sastoji se od tri glavna koraka: 1) kvantifikacija podataka, 2) modeliranje podataka i procjena značaja, i 3) integracija podataka prikupljenih u reg2gene R paketu. Kao rezultat toga, identificirali smo dva skupa enhancer-gen interakcija (EGA): fleksibilni skup od ~ 230K EGA (flexibleC) i strogi skup od ~ 60K EGA (stringentC). Utvrdili smo velike razlike u prethodno objavljenim računalnim modelima enhancer-gen interakcija; uglavnom u lokaciji, broju i svojstvima definiranih enhancera i EGA. Izveli smo detaljno mjerenje performansi sedam skupova računalno modeliranih EGA-a, ali smo pokazali da se niti jedan od trenutno dostupnih skupova referentnih podataka ne može koristiti kao referentni skup podataka "zlatnI standard". Definirali smo dodatni referentni skup pozitivnih i negativnih EGA -a pomoću kojih smo pokazali da stringentC ima najveću pozitivnu prediktivnu vrijednost (PPV). Pokazali smo potencijal EGA-a za identifikaciju genskih meta nekodirajucih SNP-ova. Proveli smo funkcionalnu analizu kako bismo otkrili nove genske mete, pleiotropiju enhancera i mehanizme aktivnosti enhancera. Ovaj rad poboljšava naše razumijevanje regulacije ekspresije gena posredovane enhancerima. / Das menschliche Genom enthält Millionen von regulatorischen Elementen - Enhancern -, die die Genexpression quantitativ regulieren. Trotz des enormen Fortschritts beim Verständnis, wie Enhancer die Genexpression steuern, fehlt es in diesem Bereich immer noch an einem systematischen, integrativen und zugänglichen Ansatz zur Entdeckung und Dokumentation von cis-regulatorischen Beziehungen im gesamten Genom. Wir haben eine neuartige Methode - reg2gene - entwickelt, die Genexpression~Enhancer-Aktivität modelliert und integriert. reg2gene besteht aus drei Hauptschritten: 1) Datenquantifizierung, 2) Datenmodellierung und Signifikanzbewertung und 3) Datenintegration, die in dem R-Paket reg2gene zusammengefasst sind. Als Ergebnis haben wir zwei Sätze von Enhancer-Gen-Assoziationen (EGAs) identifiziert: den flexiblen Satz von ~230K EGAs (flexibleC) und den stringenten Satz von ~60K EGAs (stringentC). Wir haben große Unterschiede zwischen den bisher veröffentlichten Berechnungsmodellen für Enhancer-Gene-Assoziationen festgestellt, vor allem in Bezug auf die Lage, die Anzahl und die Eigenschaften der definierten Enhancer-Regionen und EGAs. Wir führten ein detailliertes Benchmarking von sieben Sets von rechnerisch modellierten EGAs durch, zeigten jedoch, dass keiner der derzeit verfügbaren Benchmark-Datensätze als "goldener Standard" verwendet werden kann. Wir definierten einen zusätzlichen Benchmark-Datensatz mit positiven und negativen EGAs, mit dem wir zeigten, dass das stringentC-Modell den höchsten positiven Vorhersagewert (PPV) hatte. Wir haben das Potenzial von EGAs zur Identifizierung von Genzielen von nicht-kodierenden SNP-Gene-Assoziationen nachgewiesen. Schließlich führten wir eine funktionelle Analyse durch, um neue Genziele, Enhancer-Pleiotropie und Mechanismen der Enhancer-Aktivität zu ermitteln. Insgesamt bringt diese Arbeit unser Verständnis der durch Enhancer vermittelten Regulierung der Genexpression in Gesundheit und Krankheit voran. / Human genome contains millions of regulatory elements - enhancers - that quantitatively regulate gene expression. Multiple experimental and computational approaches were developed to associate enhancers with their gene targets. Despite the tremendous progress in understanding how enhancers tune gene expression, the field still lacks an approach that is systematic, integrative and accessible for discovering and documenting cis-regulatory relationships across the genome. We developed a novel computational approach - reg2gene- that models and integrates gene expression ~ enhancer activity. reg2gene consists of three main steps: 1) data quantification, 2) data modelling and significance assessment, and 3) data integration gathered in the reg2gene R package. As a result we identified two sets of enhancer-gene associations (EGAs): the flexible set of ~230K EGAs (flexibleC), and the stringent set of ~60K EGAs (stringentC). We identified major differences across previously published computational models of enhancer-gene associations; mostly in the location, number and properties of defined enhancer regions and EGAs. We performed detailed benchmarking of seven sets of computationally modelled EGAs, but showed that none of the currently available benchmark datasets could be used as a “golden-standard” benchmark dataset. To account for that observation, we defined an additional benchmark set of positive and negative EGAs with which we showed that the stringentC model had the highest positive predictive value (PPV) across all analyzed computational models. We reviewed the influence of EGA sets on the functional analysis of risk SNPs and demonstrated the potential of EGAs to identify gene targets of non-coding SNP-gene associations. Lastly, we performed a functional analysis to detect novel gene targets, enhancer pleiotropy, and mechanisms of enhancer activity. Altogether, this work advances our understanding of enhancer-mediated gene expression regulation in health and disease.

MHC Klasse II Moleküle präsentieren Peptidantigene für die Überwachung durch CD4+ T Zellen an der Zelloberfläche. Um Sicherzustellen, dass diese Peptidliganden möglichst genau die intrazelluläre Proteinzusammensetzung widerspiegeln, hat sich im Verlauf der Evolution ein komplexer Prozessierungsweg entwickelt, welcher möglichst stabile Peptid/MHC Komplexe an die Zelloberfläche liefert. MHC Moleküle, welche ihren Liganden verloren haben, konvertieren zudem spontan in einen ‚nichtrezeptiven’ Zustand, was als zusätzlicher Sicherheitsmechanismus dient. Diese Studie zeigt jedoch, dass Aminosäureseitenketten kurzer Peptide diesen Sicherheitsmechanismus umgehen können indem sie katalytisch einen reversiblen Ligandenaustausch auslösen. Die katalytische Aktivität von Dipeptiden, wie z.B. Tyr-Arg (YR), war dabei stereospezifisch und konnte durch zusätzliche Modifikationen verstärkt werden, welche das konservierte H-Brückennetzwerk der so genannten P1-Tasche des MHC Moleküls adressierten. Die Dipeptide verstärkten dabei sowohl die Antigenbeladung als auch den Ligandenaustausch, wobei deren relative Aktivität genau mit den bekanten Ankerpräferenzen der P1 Tasche korrelierte. Letzteres weist somit auf eine direkte Interaktion der katalytischen Seitenkette des Dipeptides mit dieser Tasche hin. Der Verstärkungseffekt war auch in CD4+ T Zellassays zu beobachten, bei denen der alleleselektive Einfluss der Dipeptide direkt in eine deutliche Erhöhung der Sensitivität der antigenspezifischen T Zellantwort führte. Durch weitere molekulardynamische Berechnungen konnte die Hypothese unterstützt werden, dass die Besetzung der P1 Tasche durch Aminosäureseitenketten einen Kollaps der leeren Bindungstasche zum ‚nichtrezeptiven’ Zustand verhindert. Während der Antigenpräsentation könnte P1 somit unmittelbar als ‚Sensor’ für die Beladung mit Peptiden dienen. Diese Annahme konnte experimentell durch spektroskopische Untersuchungen unter Verwendung des ANS-Farbstoffes (8-Anilino-1-Naphtalensulfonsäure) sowie durch Messung der intrinsischen Tryptophanfluoreszenz bestätigt werden. Darüber hinaus konnten konformationsspezifische Antikörper, welche bislang lediglich mit unbeladenen MHC Molekülen in Verbindung gebracht wurden, hier als spezifische Sonden für den nichtrezeptiven Zustand definiert werden. Als mögliche Risikofaktoren könnten katalytische kurze Peptide eine Rolle bei der Auslösung von Autoimmunerkrankungen spielen. In dieser Studie konnte gezeigt werden, dass sie die Beladung von Glutenantigenen auf das Zöliakie-assozierte HLA-DQ2 Molekül verstärken können. Zumindest in vitro konnte ihre Anwesenheit deshalb auch die antigenspezifische Antwort von CD4+ T Zellen verstärken, welche zuvor von Zöliakiepatienten isoliert worden waren. Auf der einen Seite könnten diese Peptide als ‚MHC-loading enhancer’ (MLE) deshalb als mögliche Risikofaktoren die Ausbildung entzündlicher (Auto-) Immunerkrankungen beschleunigen. Auf der anderen Seite könnten sie jedoch auch als ‚drug-like’ Vakzinadditiv zur Verbesserung von Immuntherapien führen. / MHC class II molecules present antigenic peptides on the cell surface for the surveillance by CD4+ T cells. To ensure that these ligands accurately reflect the content of the intracellular MHC loading compartment, a complex processing pathway has evolved that delivers only stable peptide/MHC complexes to the surface. As additional safeguard mechanism, MHC molecules quickly acquire a ‘non-receptive’ state once they have lost their ligand. This study shows that amino acid side chains of short peptides can bypass these safety mechanisms by triggering the reversible ligand-exchange. The catalytic activity of dipeptides such as Tyr-Arg (YR) is stereo-specific and could be enhanced by modifications addressing the conserved H-bond network near the P1 pocket of the MHC molecule. It enhanced both antigen-loading and ligand-release and strictly correlated with reported anchor preferences of P1, the specific target site for the catalytic side chain of the dipeptide. The effect was evident also in CD4+ T cell assays, where the allele-selective influence of the dipeptides translated into increased sensitivities of the antigen-specific immune response. The hypothesis that occupation of P1 prevents the ‘closure’ of the ‘empty’ peptide binding site into the ‘non-receptive’ state was further supported by molecular dynamic calculations. During antigen processing and presentation P1 may therefore function as important ‘sensor’ for peptide-load. Spectroscopic studies using ANS dye (8-aninilino-1-napthalenesulfonic acid) and intrinsic tryptophan fluorescence data, confirm the postulate by providing direct evidence for the conformational transitions. Moreover conformation specific antibodies previously described to be specific for ‘empty’ MHC could be shown to be a ‘probe’ for ‘receptive conformation’. As potent risk factors short peptides may be involved in the induction of autoimmune diseases. It could be shown here that they could enhance the loading of gluten derived antigen on celiac disease linked-HLA-DQ2 allele. At least in vitro the effect could enhance gluten specific CD4+ T cell response on T cell clones obtained from celiac disease patients. Thus, on one hand short peptides might work as ‘MHC loading enhancer’ (MLE) in the precipitation of inflammatory-‘autoimmune’ disorder, on the other hand they might be used as drug like vaccine ‘additive’ in various therapeutic settings.

Implications des complexes Polycomb et Trithorax au cours du développement précoce chez Ciona intestinalisLes protéines des groupes Polycomb (PcG) et Trithorax (TrxG) ont été initialement découvertes chez Drosophila melanogaster. Ces deux groupes sont classiquement connus pour leurs rôles respectifs de répresseurs et d'activateurs épigénétiques qui contrôlent et maintiennent les états chromatiniens au cours du temps. Ces facteurs régulent de nombreux gènes cibles dont les gènes homéotiques. Au cours de ma thèse, j'ai étudié trois composants de ces deux groupes : Enhancer of zeste (E(z)), appartenant au complexe PRC2 du PcG et responsable du dépôt de la marque de répression génique H3K27me3, Polyhomeotic (Ph), appartenant au complexe PRC1 du PcG et dont le rôle exact reste à déterminer, et Trithorax (Trx), appartenant au complexe TAC1 du TrxG et responsable du dépôt de la marque d'activation génique H3K4me3. Jusqu'à présent, aucune étude n'a abordé la régulation épigénétique via les PcG et TrxG chez l'ascidie solitaire Ciona intestinalis. Cette espèce présente un cluster des gènes Hox désorganisé et ne possède pas la protéine Polycomb (Pc) du PRC1, responsable de la reconnaissance de la marque de répression H3K27me3 déposée par la protéine E(z).Nos travaux montrent que la protéine E(z) est fonctionnelle et conserve son activité méthyltransférase sur le résidu H3K27 chez Ciona intestinalis. Nous avons ensuite observé, par des expériences de knockdown par micro-injection de morpholinos, que les inhibitions protéiques d'E(z), Ph et Trx ont des conséquences dramatiques sur la différenciation et la mise en place des différents tissus au cours du développement larvaire, notamment sur la mise en place de la notochorde puisque celle-ci est totalement absente chez les morphants E(z) et Ph. Les défauts de phénotype du morphant E(z) sont corrélés à la perte du dépôt d'H3K27me3 et nous avons mis en évidence, lors de l'inhibition d'E(z), une dérépression des gènes tissu-spécifiques impliqués dans le développement embryonnaire précoce alors que les gènes tardivement exprimés sont réprimés. De plus, l'expression des gènes Hox n'est pas significativement modifiée au cours du développement embryonnaire lorsque la protéine E(z) est inhibée, à l'exception du gène Hox12 qui est déréprimé, comme attendu.L'ensemble de ces résultats permet d'émettre l'idée innovante selon laquelle les protéines des PcG et TrxG jouent un rôle déterminant dans la régulation de l'expression génique lors de l'embryogénèse de Ciona intestinalis tout en ayant une implication mineure dans la régulation de l'expression des gènes Hox à ce stade du développement. / Implications of Polycomb and Trithorax complexes in the early development of Ciona intestinalisPolycomb and Trithorax group (PcG and TrxG) proteins were discovered originally in Drosophila melanogaster. Both groups are classically known for their roles in the maintenance of silenced and active chromatin states over time, respectively. These factors regulate many target genes including the homeotic genes. During my PhD, I studied three components of these two groups: Enhancer of zest (E(z)), belonging to the PRC2 complex of PcG and responsible for H3K27me3 mark deposit for gene repression, Polyhomeotic (Ph), belonging to the PRC1 complex of PcG whose role remains to be determined, and Trithorax (Trx), belonging to the TAC1 complex of TrxG and responsible for H3K4me3 mark deposit for gene activation. Until now, no study addresses the epigenetic regulation mediated by PcG and TrxG in the solitary ascidian Ciona intestinalis. This specie has a disorganized Hox cluster and in which the Polycomb (Pc) protein of PRC1, responsible for the recognition of the repressive H3K27me3 mark, is absent.Our work shows that the E(z) protein is functional and retains its methyltransferase activity on H3K27 residue in Ciona intestinalis. Then, we demonstrated, by knockdown experiments with morpholino microinjection, that the inhibition of E(z), Ph and Trx has dramatic consequences on differentiation and on the establishment of different tissues during larval development, particularly on the notochord establishment since it is totally absent in E(z) and Ph morphants. E(z) morphant phenotypic defects are correlated with lack of H3K27me3 mark deposit and we highlighted that, during the E(z) inhibition, tissue-specific genes implied in early development are de-repressed while late-expressed genes are down-regulated. In addition among Hox genes, only Hox12 expression is significantly modified and found to be de-repressed in E(z) morphant context, as expected.Altogether, our results present the innovative idea that the PcG and TrxG proteins play a major role in the gene expression regulation during embryogenesis of Ciona intestinalis while having a minor involvement in the regulation of Hox genes expression at this stage of development.

La maturation des collagènes fibrillaires est régulée par la libération protéolytique des propeptides qui conduit à la formation spontanée de fibres, à partir des molécules de collagène mature. Les fibres de collagène ainsi formées confèrent résistance et solidité aux tissus. Les métalloprotéases Tolloïdes sont les principales enzymes responsables de la maturation C-terminale des procollagènes. Ces protéases possèdent de nombreux autres substrats et se distinguent par un mode de régulation original, l’utilisation de régulateurs « substrats-spécifiques », qui leur permet de moduler leur activité vis-à-vis de ces différents substrats. Parmi ces régulateurs, les Procollagen C-Proteinase Enhancers (PCPE-1 et 2) semblent jouer un rôle important car ils augmentent l’activité de clivage du C-propeptide des procollagènes jusqu’à 20 fois. Même si PCPE-1 a été découvert en 1985, son mode d’action n’est toujours pas compris. Le principal objectif de cette thèse était donc de mieux comprendre le mécanisme de l’activation. Nous avons commencé par identifier la région minimale de PCPE-1 capable d’activer les Tolloïdes et démontré une très forte coopérativité entre les domaines de cette région. Par ailleurs, nous avons mis en évidence, pour la première fois, un nouveau rôle physiologique pour le domaine C-terminal de PCPE-1 (NTR). Concernant les autres partenaires du complexe de maturation, nous avons observé une interaction d’affinité modérée entre PCPE-1 et la protéase Tolloïde appelée BMP-1 et montré que PCPE-1 se fixe uniquement sur la partie C-terminale du procollagène. Enfin, nous avons mis en évidence que d’autres métalloprotéases, les Méprines, pourraient également jouer un rôle dans la maturation de collagènes fibrillaires en étant régulées négativement par les PCPEs. L’ensemble de ces résultats nous a permis de proposer un nouveau schéma d’interaction pour les PCPEs et de faire de nouvelles hypothèses concernant leur mécanisme d’action / The maturation of fibrillar collagens is a tightly regulated process controlled by two proteolytic cleavages that remove the propeptide regions from procollagen precursors leading to spontaneous assembly of mature collagen molecules into fibrils. These fibrils provide tensile strength and toughness to connective tissues and consequently to the organism itself. The group of extracellular metalloproteinases mostly responsible for procollagen processing are the tolloids. As these enzymes have several functions other than procollagen processing, their activities on different substrates are controlled by a growing number of substrate-specific regulators. The most prominent of these regulators are the procollagen C-proteinase enhancers (PCPEs), of which PCPE-1 is a 55 kDa glycoprotein composed of two CUB and a C-terminal NTR domain, which is capable of enhancing the proteolytic activity of tolloids up to 20-fold. Even though PCPE-1 has been known since 1985 the molecular mechanism of enhancement is still unclear. The aim of this thesis was to understand and characterize this mechanism with the aid of biochemical and biophysical methods. We have identified the minimal unit responsible for enhancing activity. In addition, we propose a mechanism for how the subdomains responsible for enhancement cooperatively bind to procollagen substrates. Furthermore, we have for the first time been able to identify a possible physiological function of the NTR domain. Also, we have identified meprins as new players involved in procollagen processing and this has given valuable insights in the mechanism of action of PCPEs. Finally, we have been able to demonstrate that the interaction of PCPE-1 with procollagen is mostly limited to the C-propeptide region. Based on these findings we propose a new hypothetical interaction mechanism for PCPE-1

Analyse fonctionnelle de la protéine Enhancer of Zeste, SlEZ2, chez la tomate, Solanum lycopersicumLes protéines Polycomb, initialement découvertes chez la drosophile, ont récemment caractérisées chez les plantes où elles remplissent des fonctions essentielles au cours du développement de la plante. Chez la drosophile, les protéines polycomb (PcG) agissent sous forme de trois complexes multi-protéiques : PRC1, PRC2 et PhoRC. Seulement, deux de ces complexes ont été identifiés chez les plantes : un orthologue fonctionnel du complexe PRC1 (PRC1-like) et PRC2. Le complexe PRC2 maintien la chromatine dans un état condensé et intervient dans le contrôle du développement des fleurs, des graines, des fruits et des feuilles. Chez la tomate Solanum lycopersicum, le complexe PRC2 est composé de trois protéines polycomb : SlEMF2 (EMbryotic Flower), SlFIE (Fertilization Independent Endosperm) and SlE(Z) (Enhancer of Zeste). Les protéines SlE(Z) portent l’activité histone méthyl transférase qui permet la mise en place de la marque répressive H3K27me3. Chez la plante modèle, Arabidopsis thaliana, cette marque joue un rôle essentiel au cours du développement de la plante Afin d’étudier le rôle du complexe PRC2 dans le développement du fruit et de la plante de tomate, et plus particulièrement de la protéine SlE(Z), nous avons identifié trois gènes codant les protéines SlE(Z) : SlEZ1, SlEZ2 et SlEZ3. Au laboratoire, il a récemment été montré que la protéine SlEZ1 intervient au cours du développement floral (How Kit et al., 2010). L’objectif de ce travail est de déterminer la fonction de la protéine SlEZ2 au cours du développement du fruit et de la plante de tomate. Pour cela, nous avons analysé des plantes transgéniques sous exprimant le gène SlEZ2, orthologue au gène CURLY LEAF d’A. thaliana, par stratégie RNAi. Ce travail indique que la protéine SlEZ2 est impliquée dans la croissance de la plante de tomate, ainsi que dans le développement des feuilles, des fleurs et des fruits. Les plantes transgéniques présentent des phénotypes pléiotropes tels que des fleurs et des feuilles modifiées, un fort taux d’avortement des fruits, des fruits de texture et de couleur altérées ainsi qu’une réduction de la taille des plantes. De plus, nous avons identifiés quatre gènes ciblés par la protéine SlEZ2 dont l’expression est dérégulée dans les feuilles. Il s’agit de deux gènes à MADS box, TAG1 et TAGL1, ainsi que de deux gènes KNOX, LeT6 et TKN4. / Functional analysis SlEZ2, a tomato Enhancer of zeste proteinPolycomb proteins, first discovered in Drosophila, have been identified in plants and play essential functions in plant development. In Drosophila, polycomb proteins (PcG) acts as a complex and three have been identified: PRC1, PRC2 and PhoRC. However, only two polycomb complexes have been identified in plants: like-PCR1 and PRC2. The PCR2 complex maintain chromatin in a closed state and control flower, seed, fruit and leaf development.In tomato Solanum lycopersicum, PRC2 is composed by three polycomb proteins SlEMF2 (EMbryotic Flower), SlFIE (Fertilization Independent Endosperm) and SlE(Z) (Enhancer of Zeste)(Enhancer of Zeste). SlE(Z) proteins have a methyltransferase activity that puts in place an repressive epigenetic mark a trimethylation of lysine 27 histone 3. In plant model, Arabidopsis thaliana, this mark plays an essential role in plant development but little is known about PRC2 role in plant and fruit development of tomato. In order to unravel the function of the E(z) protein in the control of tomato fruit and plant development, we have characterized three E(z) encoding genes, namely SlEz1, SlEz2 and SlEZ3. In a recent work, we reported that SlEZ1 protein plays a role in flower development (How Kit at al., 2010). The aim of this present study was to determine the function of the SlEZ2 protein in plant and fruit development. We present our results focusing on RNAi transgenic plants which underexpressed SlEZ2 gene, homologue of Curly Leaf Arabidopsis gene. This analysis indicates that SlEZ2 protein is implicated in tomato plant growth and affects also leaf, flower and fruit development. Phenotypes include abnormal flowers and leafs, fruit development abortion, altered fruit colour and texture and plant of reduced size. Moreover, we characterize four target genes of SlEZ2 genes in leaves which present a deregulated expression : TAG1, TAGL1, LeT6 and TKN4.

No description available.

Tombusviruses are single, positive strand RNA viruses of plants, often associated with parasitic defective interfering (DI) RNAs. Two viral- coded gene products, namely p33 and p92, are required for tombusvirus replication. The overlapping domains of p33 and p92 contain an arginine/proline-rich (RPR) RNA binding motif. In this study, the role of RPR motif and viral RNA in tombusvirus replication and recombination, as well as involvement of viral RNA in tombusvirus replicase assembly was examined. Using site-directed mutagenesis I generated a series of RPR mutants of Cucumber necrosis tombusvirus (CNV). Analysis of RPR mutants defined that wild type RPR motif, especially two of the four arginines, were required for efficient RNA binding in vitro, for replication of tombusviruses, their associated DI RNAs, subgenomic (sg)RNA synthesis and DI RNA recombination in vivo. Experiments using a two-component tombusvirus replication system showed that RPR motif is critical for functions of both p33 and p92 in replication, but its role in these proteins might not be identical. Recombination studies using a novel tombusvirus three-component system revealed that mutations in RPR motif of p33 replicase protein resulted in an altered viral RNA recombination rate. Identified DI RNA recombinants were mostly imprecise, with recombination sites clustered around a replication enchancer and an additional putative cis-acting element that might facilitate the template switching events by the tombusvirus replicase. To study the role of RNA during the assembly of functional tombusvirus replicase, recombinant CNV replicase that showed similar properties to plant-derived CNV replicase was purified from Saccharomyces cerevisiae. When in addition to p33 and p92 proteins DI RNA was co-expressed in yeast cells, the isolated replicase activity was increased ~40 fold. Further studies defined RNA motifs within two short DI RNA regions that enhanced active CNV replicase formation. In summary, this study showed that the conserved RNA binding motif of the tombusvirus replicase proteins and viral RNA are involved in replicase assembly, viral RNA replication, subgenomic RNA synthesis and RNA recombination. This data shed new light on the complex roles of the viral elements in replication, and will help future studies aimed at interfering with viral infections.

A inclusão de óleos essenciais em suplementos concentrados de bovinos de corte recriados e terminados em pastejo pode melhorar a eficiência da fermentação ruminal, devido às suas propriedades antimicrobianas e terapêuticas. Dois experimentos foram conduzidos para investigar a inclusão de uma mistura de óleos essenciais (MOE; Crina ®) composta por Timol, Eugenol, Vanilina e Limoneno sobre o consumo de forragem e total, digestibilidade aparente dos nutrientes, parâmetros ruminais e o crescimento microbiano. Para a realização do do Experimento 1, 12 bovinos machos não castrados, da raça Nelore com 436 ± 34,14 kg de peso corporal inicial médio (PCI) foram distribuídos em três Quadrados Latinos 4 X 4. Foram avaliadas doses da MOE e os tratamentos consistuídos por: Controle (0 mg), 100 mg, 200 mg e 400 mg de MOE para cada 100 kg peso corporal (PC). Todos os animais receberam suplemento proteico-energético na dose de 0,3% do PC (matéria natural) com os respectivos tratamentos. Os animais foram alocados em um piquete de 2,2 ha de Brachiaria brizantha cv. Xaraés, sob lotação contínua. No Experimento 2, foram utilizados oito bovinos, machos não castrados da raça Nelore com 481,8 ± 28,2 kg de PCI, dispostos em 2 Quadrados Latinos 4 X 4. Foram avaliados 2 aditivos, monensina sódica (Rumensin ®) e MOE+AM (Crina ® combinado com &#945- amilase) para bovinos na fase de terminação em pasto com dois níveis de suplementação (1% x 1,5% do PC). Os tratamentos testados foram: T1) monensina + 1% do PC; T2) monensina + 1,5% do PC; T3) MOE+AM + 1,0% do PC; T4) MOE+AM + 1,5% do PC de suplemento proteico-energético. Os animais foram alocados em um piquete de 2.2 ha de Brachiaria brizantha cv. Xaraés, sob lotação contínua. As análises estatísticas foram conduzidas com a utilização do programa estatístico SAS. No Experimento 1 a inclusão de doses da MOE na dieta não afetou (P>0,10) o consumo e a digestibilidade aparente dos nutrientes, a concentração de AGCC totais, as proporções molares de acetato, propionato, butirato, isobutirato, isovalerato, a relação acetato:propionato e a concentração de N-NH3 no fluido ruminal. Houve efeito linear positivo de doses crescentes da MOE na dieta sobre o pH ruminal e efeito quadrático na proporção molar de valerato. A inclusão de doses de MOE na dieta também não afetou (P>0,10) o N ingerido, N excretado (fezes), N absorvido e a síntese microbiana em bovinos mantidos em pastagens tropicais suplementados com suplemento proteico-energético na dose de 0,3% do PC. No Experimento 2, houve interação entre aditivo e nível de suplemento para todas as variaveis de consumo (kg e %PC) avaliadas (P<0,10). A suplementação com 1,5% do PC aumentou o CMSt, o CMO, CMSD e o CMOD dos animais suplementados com monensina, mas não nos animais suplementados com MOE+AM. Na presença de MOE+AM o nível alto de suplementação (1,5% do PC) causou redução no CMS de forragem e consequentemente no consumo de FDN. As digestibilidades da MS e da PB foram maiores nas dietas com nível de suplementação de 1,5% do PC que nas de 1% do PC (P<0,10) independente do aditivo fornecido. As digestibilidades da MS, MO, FDN e PB foram maiores nas dietas contendo monensina em comparação com MOE+AM, independentemente do nível de suplemento fornecido. A suplementação com MOE+AM resultou em menor pH ruminal, maior concentração 7 molar de AGCC e maior proporção molar de C2 que a monensina (P<0,10). A suplementação com 1,5% do PC reduziu o pH ruminal, a proporção molar de C2, e aumentou a concentração ruminal de N-NH3, a concentração molar de AGCC e a proporção molar de valerato (P<0,10). Houve interação (P<0,10) entre nível de suplemento e aditivo para a proporção molar de C3, de isobutírico, de isovalérico e para a relação C2:C3. Tanto a monensina quanto o nível alto (1,5% PC) de suplementação aumentaram a proporção molar de C3, entretanto, não houve diferença entre os tratamentos monensina + 1,0% PC e MOE+AM + 1,5% PC. A relação C2:C3 foi reduzida tanto pela monensina quanto pelo nível de suplementação de 1,5% PC, entretanto, o efeito da monensina só ocorreu em combinação com suplementação a 1,0% PC. A suplementação com MOE+AM aumentou (P<0,10) a eficiência e a produção de proteína microbiana em comparação com a monensina, enquanto o nível de suplementação não teve efeito (P>0,10) sobre esses parâmetros. / The inclusion of essential oils in supplements for growing and finishing grazing cattle can improve the efficiency of ruminal fermentation due to its antimicrobial and therapeutic properties. Two experiments were conducted to investigate the effects of of the inclusion of a mixture of essential oils (EOM; Crina®) composed of Timol, Eugenol, Vanillin and Limonene in supplements for grazing beef cattle on forage intake, total dry matter intake, apparent digestibility of nutrients, ruminal parameters and microbial growth. In Experiment 1, twelve Nellore bulls with IBW of 436 ± 34.14 kg were alloted to three 4 X 4 Latin Squares. Four doses of EOM were evaluated: Controll (0 mg), 100 mg, 200 mg e 400 mg for every 100 kg of body weight (BW). All the animals were fed an energy-protein supplement at 0.3% BW (AF). The animals grazed a 2.2 ha paddock of Brachiaria brizantha cv. Xaraés, under continuous stocking. In Experiment 2, eight Nellore bulls with IBW of 481.8 ± 28.2 kg, were allotted to two 4 x 4 Latin Squares. Two feed additives, monensin (MON; Rumensin®) and a mixture of essential oil (EOM+AM; Crina® combined with exogenous &#945-amylase), were compared for grazing cattle fed an energy-protein supplement at 1.0 or 1.5% of BW (AF). Treatments were: MON+1%, M+1.5%, EOM+1.0% and EOM+1.5%. The animals grazed a 2.2 ha paddock of Brachiaria brizantha cv. Xaraés, under continuous stocking. Statistical analyzes were performed using the SAS statistical program. In Experiment 1, doses of EOM had no effect (P>0.10) on DMI, apparent digestiibility of nutrientes, ruminal concentration of SCFA, molar concentrations of acetate, propionate, butirate, isobutirate, isovalerate, the C2:C3 ratio and on the rumen concentration of N-NH3. There was a positive linear effect (P<0.10) of doses of EOM on rumen pH and a quadratic effect (P<0.10) on proportion of valerate. The EOM had no effect (P<0.10) on N ingested, N excreted, N absorbed and on microbial synthesis and efficiency. In Experiment 2, there was interaction between feed additive and supplement level for all consumption variables (kg and% BW) evaluated (P <0.10). Supplementation with 1.5% of BW increased DMI, OMI, DDMI and DOMI of animals supplemented with monensin, but not in animals supplemented with EOM+AM. In the presence of EOM+AM, the high level of supplementation (1.5% of CP) caused a reduction in forage DMI and consequently in the consumption of NDF. The digestibilities of DM and CP were greater for cattle supplemented at 1.5% of BW than at 1.0% of BW (P<0.10) regardless of the feed additive. The digestibilities of DM, OM, NDF and CP were greater in diets containing monensin compared to EOM+AM, regardless of the supplement level provided. The supplementation with EOM+AM resulted in lower ruminal pH, greater molar concentration of total VFA and greater molar proportion of C2 than monensin (P<0.10). Feeding supplement at 1.5% of BW decreased rumen pH, molar proportion of C2, and increased the concentration of rumen N-NH3, total VFA and the molar proportion of valerate (P<0.10). There was interaction (P<0.10) between feed additive and level of supplement for molar proportion of C3, isobutyrate, isovalerate and for the C2:C3 ratio. Both monensin and the high level of supplement (1.50% of BW) increased ruminal proportion of C3, however, there was no difference between the treatments monensin + 1.0% of BW and EOM+AM + 1.5% of BW. The ratio C2:C3 was decreased by both monensin and supplement fed at 1.50% of BW, 9 however, the monensin effect occurred only when combined with supplement fed at 1.0% of BW. The EOM+AM increased (P<0.10) the efficiency and production of microbial protein compared with monensin, while the level of supplement had no effect (P>0.10) on those parameters.

In the process of pre-mRNA splicing introns are removed from pre-mRNA and exons are joined together. Current studies show, that about 95 % of genes, which contain more than two exons, can undergo alternative splicing. In this process some exons are included in or excluded from the final mRNA. Majority of pre-mRNA splicing take place co- transcriptionaly at this time RNA polymerase II is still attached to pre-mRNA. Alternative splicing is complex process that takes place in a close proximity of DNA and histones that might modulate alternative splicing decisions. Futher studies have validated fibronectin gene (FN1) and his alternative exons EDA and EDB (extra domain A and B) as suitably model for studying alternative splicing. Study using FN1 minigene reporter system, which is composed from EDA exon and two surrounding introns and exons, has proved that insertion of transcription enhancer SV40 infront of promotor, the level of EDA inclusion is decreased. So far, has not been prooved if this mechanism can function in real genome context and if distal transcription elements can influence alternative splicing. In this study, we have predicted transcription enhancer for FN1 gene by using The Ensemble Regulatory Build and FANTOM 5. The predicted transcription enhancer, is located 23,5 kbp upstream of TSS...

O objetivo desse experimento foi verificar diferentes planos nutricionais através da utilização de diferentes tipos de levedura na alimentação de frangos de corte no período de 1 a 42 dias. Foram utilizadas leveduras autolizada, hidrolizada e levedura íntegra e sua influência sobre parâmetros de desempenho (ganho de peso, consumo de ração e conversão alimentar) e características de carcaça (rendimento de carcaça, rendimento de peito e rendimento de pernas). Foram utilizados 1.080 pintinhos de corte, machos de um dia de idade, da linhagem Cobb, distribuídos em delineamento experimental inteiramente casualizado, em esquema fatorial 3 x 3: 3 formas de suplementação de levedura no período de 1 a 14 dias (sem levedura, levedura autolisada e levedura hidrolisada) e 3 formas de suplementação no período de 15 a 42 dias (sem suplementação, levedura autolisada e levedura íntegra) mais um tratamento controle positivo, com a adição de um antibiótico promotor de crescimento (APC), totalizando 10 tratamentos, 9 repetições e 12 aves por unidade experimental. As dietas utilizadas foram formuladas a base de milho e farelo de soja, sendo utilizado como APC a bacitracina de zinco na inclusão de 0,5 kg/ton em todas as dietas. No período de 1 a 14 dias foi utilizado o nível de inclusão de 10kg/ton das leveduras autolisada e hidrolisada e no período de 15 a 42 dias, 5kg/ton das leveduras autolisada e íntegra. As análises estatísticas dos dados foram realizadas pelo método da análise de variância com o auxílio do procedimento GLM do SAS (2002) e em caso de significância estatística, as médias foram comparadas pelo teste de Tukey, no nível de 5% de probabilidade. No período de 1 a 14 dias a levedura hidrolisada apresentou melhor viabilidade de uso na dieta. Para a utilização de um programa contínuo, a combinação do uso da levedura hidrolisada no período de 1 a 14 dias e de levedura autolizada no período de 15 a 42 dias, apresenta-se como melhor resposta pelas aves. / The objective of this experiment was to evaluate different nutritional plans using various types of yeasts in broiler chicken feed during a 42-day period. We used autolyzed, hydrolyzed, and whole yeasts and evaluated their influence on performance parameters (weight gain, feed intake, and food conversion) and carcass features (whole chicken yield, breast yield, and leg yield). A total of 1080 1-day-old male Cobb lineage chicks were used and distributed across a completely randomized 3 x 3 factorial experimental design. The treatments were comprised of 3 yeast supplement types during days 1 to 14 (i.e., without yeast, autolyzed yeast, and hydrolyzed yeast), 3 supplement types during days 15 to 42 (as above), and growth-promoting antibiotic (GPA) as positive control for a total of 10 treatments, 9 replications, and 12 birds per experimental unit. The diets were formulated based on corn and soybean meal, and 0.5 Kg/ton of zinc bacitracin was included as the GPA in the control diets. We used 10 Kg/ton of autolyzed and hydrolyzed yeast in days 1 to 14, and 5 Kg/ton of autolyzed and whole yeast during days 15 to 42. Statistical data analyses were conducted using an analysis of variance and the SAS GLM procedure (2002), and statistically significant means were compared using Tukey´s test at the 5% probability level. In days 1 - 14, the hydrolyzed yeast presented the best viability of use in the chicken diet. As an ongoing program, the birds responded best to the combination of hydrolyzed yeast in days 1 to 14 and autolyzed yeast in days 15 to 42.