Nos travaux sur 26 gènes de la famille des WDR a permis d’en identifier sept (Atg16l1, Coro1c, Dmxl2, Herc1, Kif21b, Wdr47, Wdr89) associés à des anomalies cérébrales majeures. Cette grande famille de protéines reste pourtant peu explorée quant à ses rôles dans le développement du système nerveux central. Nous avons choisi d’étudier WDR47, dont la fonction est totalement inconnue en dépit d’une très grande similarité structurale avec LIS1, protéine à l’origine de la lissencéphalie. En combinant trois modèles expérimentaux (souris, siRNA et levure), nous avons démontré que Wdr47 est essentiel pour la survie de l’organisme et est impliqué dans la coordination motrice et le maintien de l’homéostasie énergétique avec une origine probablement centrale. Au niveau cellulaire, Wdr47 assure un rôle clé dans la dynamique des microtubules et la stabilisation du cône de croissance au travers d’interaction protéiques avec Reelin et SCG10. En outre, Wdr47 est aussi impliqué dans la prolifération neuronale et la macroautophagie. Ces résultats ont permis d’établir un lien de causalité entre une duplication de 200 kb contenant Wdr47 et des troubles de coordination motrice et une obésité hyperphagique chez un jeune patient. / WD40-repeat (WDR) proteins are one of largest eukaryotic family, however little is known about their role in neurodevelopment. We investigated 26 WDR genes, and found 7 (Atg16l1, Coro1c, Dmxl2, Herc1, Kif21b, Wdr47, Wdr89) with a major impact in brain structure when inactivated in mice. We chose WDR47 for further investigation, as it is a completely unknown protein that shares striking domain similarity with LIS1. Using three independent model systems (mice, siRNA and yeast), we found an essential role of Wdr47 in survival, and key neuronal processes involving microtubule dynamics such as proliferation, autophagy and growth cone stabilization. Next we identified Reelin and superior cervical ganglion 10 (SCG10) as top interacting proteins of WDR47. Interestingly, a 200-kb duplication encompassing WDR47 was linked to poor coordination in one patient, recapitulating mouse behavioural anomalies. Together our data help unravel for the first time a key role of Wdr47 in brain.

Les lignines sont des hétéropolymères phénoliques de la paroi cellulaire, principalement à base de p-coumaryl, coniférylique et sinapylique alcools. Ces monolignols sont synthétisées dans le cytoplasme de la voie des phénylpropanoïdes, peut ensuite transportées vers les parois des cellules où ils sont polymérisés par oxydation en p-hydroxyphényl (H), guaiacyle (G) et syringyle (S) des unités de lignine. Cette étape de polymérisation par oxydation est conduite par les peroxydases dépendantes H₂O₂ et / ou laccases dépendantes O₂. Dans cette étude, nous avons signalé pour la première fois que les laccases sont également impliqués dans la lignification du les herbes. Un gène de laccase spécifique (BdLAC5) a été identifié parmi les 29 gènes de laccase non redondants dans Brachypodium génome, qui est responsable de la lignification dans les tiges de Brachypodium distachyon, une plante modèle pour les graminées. BdLAC5 gène a été retrouvé fortement exprimé dans les organes lignifiées (internodal, nœud et le pédoncule) et mal exprimé dans les organes avec faible niveau de lignine (jeunes feuilles et épillet), ni dans les tissus non-lignifiée (endosperme). Deux autres laccases BdLAC6 et BdLAC8 sont également trouvés coexprimés avec BdLAC5 et curieusement ils appartiennent à la même clade phylogénétique. BdLAC6 et BdLAC8 sont orthologues de Arabidopsis LAC4 et LAC2 respectivement. Dans les expériences d'hybridation in situ ont démontré le signal le plus intense dans les fibres interfasciculaires de l'entre-nœud a été détectée avec des sondes BdLAC5. En outre, des essais ont révélé que les protéines immunomarquages BdLAC5 pourraient être sécrétés dans la matrice de la paroi cellulaire, car nous avons détecté des particules fluorescentes dans ou à proximité de la paroi cellulaire. Le double mutant laccase touchée BdLAC5 et BdLAC8 a clairement montré que la lignification dans les fibres interfasciculaires impliqué différents gènes / protéines que la lignification dans les cellules métaxylème de Brachypodium. Métaxylème cellules ont été que faiblement affectés dans le double mutant lorsque les fibres interfasciculaires montré diminution dramatique de Wiesner coloration. Les différents mécanismes de lignification entre xylème et fibres est discutée. L'interaction physique et la synergie entre réglementation spécifique R2R3-MYB, bHLH et WDR protéines est bien étudiée dans la biosynthèse des flavonoïdes, racine des cheveux, trichomes et le développement en mucilage de graines de différentes espèces de plantes. Dans cette étude, nous avons essayé de comprendre les rôles de MYB-bHLH-WDR pour la régulation de la biosynthèse de la lignine. / Lignins are cell wall phenolic heteropolymers, mainly made from p-coumaryl, coniferyl, and sinapyl alcohols. These monolignols are synthesized in the cytoplasm from the phenylpropanoid pathway, then may transported to the cell walls where they are oxidatively-polymerized into p_hydroxyphenyl (H), guaiacyl (G) and syringyl (S) lignin units. This oxidative polymerization step is driven by H₂O₂-dependent peroxidases and/or O₂-dependent laccases. In this study we reported for the first time that laccases are also involved in lignification in grasses. A specific laccase gene (BdLAC5) was identified among 29 non-redundant laccase genes in Brachypodium genome, which is responsible for the lignification in stems of Brachypodium distachyon, a model plant for grasses. BdLAC5 gene was found highly expressed in lignified organs (internode, node and peduncle) and poorly expressed in organs with low lignin level (young leaf and spikelet) nor in non-lignified tissue (endosperm). Two other laccases BdLAC6 and BdLAC8 are also found coexpressed with BdLAC5 and interestingly enough they belong to the same phylogenetic clade. BdLAC6 and BdLAC8 are close orthologues of Arabidopsis LAC4 and LAC2 respectively. In situ hybridization experiments demonstrated the most intense signal in the interfascicular fibers of the internode was detected with BdLAC5 probes and then for BdLAC8 and BdLAC6 probes. Furthermore, immunolabelling assays revealed that BdLAC5 proteins might be secreted into the cell wall matrix because we detected some fluorescent particles close to or in the cell wall. The double laccase mutant affected in BdLAC5 and BdLAC8 (5ho8ho) clearly showed that the lignification in interfascicular fibers involved different genes/proteins than the lignification in metaxylem cells of Brachypodium. Metaxylem cells were only poorly affected in the double mutant when interfascicular fibers showed dramatic decrease of Wiesner staining. The different mechanisms of lignification between xylem and fibers is discussed. The physical interaction and regulatory synergy between specific R2R3-MYB, bHLH and WD repeat protein is well studied in the biosynthesis of flavonoids, root hair, trichome and seed mucilage development in different plant species. In this study, we were trying to figure out the roles of MYB- bHLH-WDR for the regulation of lignin biosynthesis.

Master of Science / Department of Plant Pathology / Karen A. Garrett / Ecological clines often result in gradients of disease pressure in natural plant communities, imposing a gradient of selection on disease resistance genes. We describe the diversity of a resistance gene homolog in natural populations of the dominant tallgrass prairie grass, Andropogon gerardii, across a precipitation gradient ranging from 47.63 cm/year in western Kansas to 104.7 cm/year in central Missouri. Since moisture facilitates infection by foliar bacterial pathogens, plants along this precipitation gradient will tend to experience heavier bacterial disease pressure to the east. In maize, the gene Rxo1 confers resistance to the pathogenic bacterium Burkholderia andropogonis. Rxo1 homologs have been identified in A. gerardii and B. andropogonis is known to infect natural populations of A. gerardii. The spatial genetic structure of A. gerardii was assessed from central Missouri to western Kansas by genotyping with AFLP markers. Samples were also genotyped for Rxo1 homologs by amplifying an 810 base pair region of the leucine-rich repeat and digesting with restriction enzymes. We compared Rxo1 homolog diversity to AFLP diversity across different spatial scales. Genetic dissimilarity based on AFLP markers was lower than would have occurred by chance at distances up to 30 m, and different prairies were more dissimilar than would have occurred by chance, but there was not a longitudinal trend in within-prairie dissimilarity as measured by AFLP markers. Dissimilarity of the Rxo1 homologs was higher in the east suggesting the presence of diversifying selection in the more disease-conducive eastern environments.

Magister Scientiae - MSc / Sexual assault is a significant problem facing the South African society. In this context, efficient but also affordable genotyping systems are needed for positive identification of criminals in incidences of sexual violence. The aim of this study was therefore to develop non-commercial Y-STR genotyping systems suitable for sexual assault cases in South Africa. Y-chromosome STR loci constituting the minimal haplotype are still the most widely used loci in investigating sexual assault cases despite the fact that DYS391 and DYS392 have shown low levels of polymorphism in Xhosa populations in Cape Town. The minimal haplotype was, therefore, further investigated in the Cape Muslim population. The Cape Muslim population generally exhibited high GD values among all the South African populations. These values were higher than 0.5 for most loci, and ranged from 0.447 for DYS391 to 0.957 for DYS385. The highest number of alleles in most loci was also recorded in this population. The overall assessment of the minimal haplotype has shown that this system is still a useful in investigating sexual assault case in many South African subpopulations. Therefore the exercise of internal validation of the minimal haplotype system was successfully carried out in the laboratory. The properties of additional novel and widely used STRs were also investigated in this study. Loci were successfully sequenced and allele nomenclature was assigned to them according to the ISFG guidelines. / South Africa

La dystrophie myotonique de type 1 (DM1) est une maladie à transmission autosomale dominante causée par une répétition trinucléotidique CTG située dans la région 3’ non-traduite du gène dystrophia myotonica protein kinase (DMPK). La prévalence mondiale de la DM1 est de 8,26 personnes atteintes par 100 000 habitants : celle-ci est presque 20 fois plus importante au Saguenay-Lac-St-Jean en raison d’un effet fondateur. La présentation clinique de la DM1 peut comprendre divers symptômes dont de la faiblesse musculaire, de la myotonie, des cataractes, de l’insuffisance respiratoire, de l’arythmie cardiaque, de l’hypersomnolence et des troubles cognitifs et endocriniens. Par ailleurs, une grande variation dans la présence et la sévérité de ces symptômes est observée chez les patients et celle-ci n’est qu’en partie expliquée par la longueur des répétitions CTG. Plusieurs mécanismes pourraient expliquer la variabilité inexpliquée dont les défauts d’épissage, la mauvaise régulation des facteurs de transcription, la traduction non-ATG associée aux répétitions et les modifications épigénétiques, en particulier la méthylation de l’ADN. L’objectif de ce projet était donc d’évaluer l’impact de la méthylation de l’ADN au locus DMPK sur la variabilité phénotypique des patients atteints de DM1. Nous rapportons que la méthylation de l’ADN mesurée en amont et en aval de la répétition CTG est respectivement corrélée négativement et positivement avec la longueur de la répétition CTG. La présence d’une interruption de la répétition est associée à un niveau plus élevé de méthylation de l’ADN. À l’aide de modèles de régression linéaire multiple, nous démontrons que la méthylation de l’ADN contribue significativement et indépendamment de la longueur des répétitions CTG, à expliquer la variabilité́ de la force des dorsifléchisseurs de la cheville, de la force de préhension, de la force des pinces, de la capacité́ vitale forcée, du débit expiratoire de pointe, de la pression expiratoire et inspiratoire maximale. La méthylation de l’ADN explique une fraction de la variabilité phénotypique en DM1 et en association avec la longueur de la répétition CTG pourrait aider à améliorer la prédiction de la progression de la maladie chez ces patients. / Abstract : Myotonic dystrophy type 1 (DM1) is an autosomal dominant disorder caused by a CTG repeat extension in the 3’ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Worldwide, the prevalence of DM1 is 8.26 affected persons per 100 000 persons, but it goes up to 158 affected persons per 100 000 in the Saguenay-Lac-St-Jean region of the province of Quebec (Canada) due to a founder effect. Clinical presentation includes muscular weakness, myotonia, cataracts, respiratory insufficiency, cardiac arrhythmia, hypersomnolence and endocrine and cognitive problems. There is a large variability in the presence and severity of these symptoms that is only partially explained by the CTG repeat length. Many mechanisms such as splicing defects, impaired regulation of transcription factors, repeat-associated non-ATG translation and epigenetic modifications, including DNA methylation, may explain this variability. The objective of this study was to assess the impacts of DNA methylation measured at the DMPK gene locus on phenotypic variability in DM1. We report that DNA methylation upstream of the repeat was negatively correlated with CTG repeat length whereas downstream DNA methylation was positively correlated. The presence of a variant repeat within the CTG repeat was associated with a higher level of DNA methylation. Linear multiple regression models support that DNA methylation contributes significantly and independently of the CTG repeat length to the variability of the ankle dorsiflexor, grip and pinch strengths, as well as forced vital capacity, peak expiratory flow and maximal inspiratory and expiratory pressures. DNA methylation could thus explain part of the phenotypic variability in DM1 and, together with CTG repeat length, could help improve the prediction of the progression of the disease.

L'étude comparative de l'architecture des génomes mammaliens a révélé l'association des séquences répétées et des réarrangements. Cette thèse porte sur la dynamique et le rôle dans les remaniements de deux types de séquences répétées: les clusters ribosomiques et les satellites. Ces séquences sont analysées par une approche cytogénomique (FISH, CO-FISH) dans le genre Mus connu pour sa diversité chromosomique, et pour lequel les phylogénies moléculaires et chromosomiques sont disponibles.1) La distribution chromosomique des clusters ribosomiques, établie chez 19 espèces, a permis de reconstruire les états ancestraux des clusters. Cette analyse montre que les clusters (24%) sont associés à des points de cassures, mais présentent également une grande labilité en l'absence de réarrangements. De plus, une forte association entre les clusters et les centromères est mise en évidence. 2) Le sous-genre Mus se caractérise par un caryotype très conservé excepté chez une sous-espèce de la souris domestique (M. musculus domesticus), qui est connue pour son extraordinaire radiation chromosomique impliquant les séquences satellites du centromère. Afin de rechercher les spécificités génomiques responsables de ce patron d'évolution contrasté, la dynamique évolutive des séquences satellites a été analysée chez 11 taxons. Révélant des différences qualitatives entre taxons, cette étude a permis de proposer un scénario évolutif de ces séquences. Toutefois, aucune des caractéristiques étudiées (composition, orientation) n'est propre à M. m. domesticus, et ne permet de rendre compte de sa plasticité chromosomique. De même, chez cette dernière, aucun lien entre la quantité de séquences satellites et la fréquence d'implication des chromosomes dans les réarrangements n'est mis en évidence.Cette étude confirme que les séquences répétées participent à l'évolution chromosomique, mais ne constituent pas à elles seules l'élément clef de cette dernière. / Comparative analyses of the architecture of mammalian genomes have highlighted the association between repetitive sequences and rearrangements. This thesis focuses on the evolutionary dynamics of two repeat sequences (ribosomal clusters and satellites) and explores their role in chromosomal change. These sequences are analyzed by a cytogenomic approach (FISH, CO-FISH) in the genus Mus that is known for its chromosomal diversity and for which molecular and chromosomal phylogenies are available.1) The chromosomal distribution of ribosomal clusters, established in 19 species, allowed us to reconstruct the ancestral states of clusters. This analysis demonstrated that 24% of clusters were associated with breakpoints, whereas others showed high lability in the absence of rearrangements. Moreover, a strong association between clusters and centromeres was retrieved.2) The subgenus Mus is characterized by a highly conserved karyotype except for one subspecies of the house mouse (M. musculus domesticus), that displays an extraordinary chromosomal radiation involving centromeric satellite sequences. To determine the genomic traits related to this difference in rate, the evolutionary dynamics of satellite sequences was analyzed in 11 taxa. From the qualitative differences evidenced between taxa, an evolutionary scenario of these sequences is proposed. None of the studied features (composition, orientation) of these sequences was found to be specific to M. m. domesticus, and could explain its chromosomal plasticity. Similarly, in the latter, no relationship between satellite sequence quantity and the rearrangement frequency of chromosomes was found.This study confirms that although repeated sequences are involved in chromosomal evolution, they aren't in themselves the key element of the latter.

Made available in DSpace on 2017-06-01T18:29:26Z (GMT). No. of bitstreams: 1 esperidiao_barbosa_neto.pdf: 1375062 bytes, checksum: 895b299c670d82741485df3d45482fc6 (MD5) Previous issue date: 2015-01-15 / The theme of repetition crosses the entire psychoanalytic theory, having become one of its fundamental concepts. Freud developed the concept of repetition from his clinical experience and Lacan placed the order of the Real. In the field of Philosophy, repetition was defined by the philosopher Kierkegaard, a Danish thinker of the nineteenth century. The psychoanalytic concept of repetition is distinct from philosophy; however, they are intertwined in some points. The psychoanalytic clinic is presented as a place of repetition and speech; therein, according to the articulation we made, the subject is also thought from the existence, according to Kierkegaard's conception. This research is of theoretical nature and uses clinical fragments; has as main objective to analyze the concept of repetition in psychoanalysis, with the contribution of Kierkegaardian concept of repetition, highlighting the clinical implications at the interface of the two concepts. The specific objectives are: to investigate the concept of repetition in Freud and Lacan; study the concept of repetition in Kierkegaard; situate the psychoanalytic clinic as a place of repetition and speech; highlight the creativity in the clinical process, considering the positivity of repetition from the point of view of Kierkegaard articulated with the Psychoanalysis. As theoretical reference we use the theories of Freud, Lacan and Kierkegaard. The text is presented in three chapters: in the first chapter, we work with the concept of repetition from the theoretical elaborations of Freud and Lacan's theoretical contribution; in the second chapter, we present the concept of repetition in Kierkegaard; in the third chapter, we situate the clinical implications of the psychoanalytic concept of repetition in the interface with the Kierkegaardian concept, considering the demand of the suffering in the subject‟s present context. In conclusion, we present a reflection on the subject as capable of developing an affective experience of trauma and all the excitement received as inheritance, to build something that belongs to him. We hope that the approach between psychoanalysis and Kierkegaard's philosophy contributes to a better understanding of the concept of repetition and, above all, enriches the clinical work. / O tema da repetição atravessa toda a teoria psicanalítica, tendo se constituído um dos seus conceitos fundamentais. Freud elaborou o conceito de repetição a partir de sua experiência clínica e Lacan o situou na ordem do Real. No campo da Filosofia, a repetição foi conceituada pelo filósofo Kierkegaard, pensador dinamarquês do Século XIX. O conceito psicanalítico de repetição é distinto do da Filosofia, no entanto, eles se entrelaçam em alguns pontos. A clínica psicanalítica se apresenta como lugar da repetição e da fala; nela, conforme a articulação que fazemos, o sujeito é pensado, também, a partir da existência, segundo a concepção kierkegaardiana. Esta pesquisa é de natureza teórica e utiliza alguns fragmentos clínicos; tem como objetivo geral analisar o conceito de repetição na Psicanálise, com o aporte do conceito kierkegaardiano de repetição, ressaltando as repercussões clínicas na interface dos dois conceitos. Os objetivos específicos são: investigar o conceito de repetição em Freud e Lacan; estudar o conceito de repetição em Kierkegaard; situar a clínica psicanalítica como lugar da repetição e da fala; destacar a criatividade no processo clínico, considerando a positividade da repetição segundo o ponto de vista de Kierkegaard articulado ao da Psicanálise. Como referencial teórico, utilizaremos as teorias de Freud, Lacan e Kierkegaard. O texto é apresentado em três capítulos: no primeiro, trabalhamos o conceito de repetição a partir das elaborações teóricas de Freud e a contribuição teórica de Lacan; no segundo capítulo, apresentamos o conceito de repetição em Kierkegaard; no terceiro capítulo, situamos as repercussões clínicas do conceito psicanalítico de repetição na interface com o conceito kierkegaardiano, considerando a demanda do sofrimento no contexto do sujeito na atualidade. Como conclusão, apresentamos uma reflexão a respeito do sujeito enquanto capaz de elaborar a experiência afetiva do trauma e toda a excitação recebida como herança, no sentido de construir algo que lhe pertença. Esperamos que a aproximação entre a Psicanálise e a Filosofia kierkegaardiana contribua para uma melhor compreensão do conceito de repetição e, sobretudo, para enriquecer o trabalho clínico.

Esta tese é estruturada em dois capítulos. O primeiro capítulo explora os retrotransposons com LTR (LTR-RT) em cana-de-açúcar e grande parte de seus resultados foram publicados no artigo \'\'Analysis of plant LTR-retrotransposons at the fine-scale family level reveals individual molecular patterns\'\'. Nossos resultados mostraram que as diferentes famílias de LTR-RT em cana-de-açúcar possuem estruturas e regulação distintas. O segundo capítulo desta tese visa responder a perguntas que surgiram durante a primeira metade deste trabalho, mas ao invés de focar no genoma de uma planta optamos por trabalhar com linhagem Del de LTR-RT em dez genomas de angiospermas sequenciados. Os resultados desta parte do trabalho foram submetidos para publicação no artigo intitulado \'\'Virus-like attachment sites and plastic CpG islands: landmarks of diversity in plant Del retrotransposons\'\'. Os resultados mostraram que a LTR é uma região dinâmica e importante para a evolução dos LTR-RTs. Nós especulamos que mudanças nas LTR atuem como gatilhos para a diversificação dos LTR-RTs. / This doctoral thesis is structured in two chapters. In the first chapter we explore the LTRretrotransposons (LTR-RT) in sugarcane, these results were published in an article entitled \'\'Analysis of plant LTR-etrotransposons at the fine-scale family level reveals individual molecular patterns\'\'. In this paper we show that different sugarcane LTR-RT families have distinct structure and are differentially regulated. In the second chapter we try to find answers to questions that came up in the first half of this work, but instead of focusing in one plant genome we chose to work with the Del lineage of LTR-RT in tem angiosperm sequenced genomes. These results are submitted to publication as an article entitled \'\'Virus-like attachment sites and plastic CpG islands: landmarks of diversity in plant Del retrotransposons\'\'. Our results indicate that the LTR region is dynamic and important in the evolution of LTR-retrotransposons, we speculate that it is a trigger for retrotransposon diversification.

Mutant mRNA and protein both contribute to the clinical manifestation of many repeat-associated neurodegenerative and neuromuscular disorders. The presence of nuclear RNA clusters is a feature shared amongst these diseases, such as C9ORF72/ALS and myotonic dystrophy 1/2 (DM1/2); however, this pathological hallmark has not been conclusively demonstrated in Huntington’s disease (HD) in vivo. Investigations into HD – caused by a CAG repeat expansion in exon 1 of the huntingtin (HTT) gene – have largely focused on toxic protein gain-of-function as a disease-causing feature, with fewer studies investigating the role of mutant HTT mRNA in pathology or pathogenesis. Here we report that in two HD mouse models, YAC128 and BACHD-97Q-ΔN17, mutant HTT mRNA is preferentially retained in the nucleus in vivo. Furthermore, we observed the early, widespread formation of large mutant HTT mRNA clusters (approximately 0.6 to 5 µm3 in size) present in over 50-75% of striatal and cortical neurons. Affected cells were limited to one cluster at most. Endogenous wild-type mouse Htt or human HTT mRNA containing 31 or fewer repeats did not form clusters. Additionally, the aberrantly spliced N-terminal exon 1-intron 1 RNA fragment, HTT1a, also formed clusters that fully co-localized with the mutant HTT mRNA clusters. These results suggest that multiple repeat-containing transcripts can coalesce to form a single cluster in a given cell. Treating YAC128 mice with antisense oligonucleotides efficiently silenced individual HTT mRNA foci but had limited impact on clusters. Our findings identify mutant HTT mRNA clustering as an early, robust molecular signature of HD, further supporting HD as a repeat expansion disease with suspected mRNA involvement.

Cílem bylo navrhnout dva simulační scénáře v prostředí NS-3. První scénář obsahuje ARQ (Automatic Repeat Request) metody v TCP (Transmission Control Protocol). Je v něm porovnání Stop-and-Wait, Go-Back-N a Selective-Repeat metod. Teoretická část obsahuje TCP a ARQ. Druhý scénář je o způsobech přenosu zpráv. Vytvořený scénář převážně s komutací paketů a buněk a teoretické základy jsou obsaženy v práci. Je v něm porovnání metod komutací s různou velikostí paketu/buňky, počtem uzlů a důsledek zpoždění v každé metodě. Scénáře jsou ve formě laboratorní úlohy s instrukcemi k vypracování.