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Au cours de son cycle sexuel, le cilié Paramecium tetraurelia procède à de massifs réarrangements programmés de son génome (RPG). Ils consistent, entre autres choses, en l’excision de 45 000 séquences précisément délimitées, appelées IES (Internal Eliminated Sequences). La transposase domestiquée Piggymac (Pgm) introduit les cassures double-brin (CDB) à l’extrémité des IES. La réparation très précise de ces dommages est réalisée par la voie de réparation des extrémités non-homologues (NHEJ). Un des acteurs de cette voie est l’hétérodimère Ku70/Ku80. Suite à des duplications globales du génome, la paramécie possède trois gènes KU80, Un seul de ces gènes est induit lors des RPG (KU80c) et une expérience d’ARN interférence (ARNi) contre KU80c montre une complète inhibition de l’introduction des CDB. De plus, des expériences de Co-IP en système hétérologue montrent que Ku70/Ku80c interagit avec Pgm. Ces résultats prouvent le rôle essentiel de Ku dans l’introduction des CDB lors des RPG et soulèvent la question du mécanisme impliqué. Au cours de ma thèse j’ai caractérisé le couplage entre Ku et Pgm en analysant des expériences d’immunofluorescence avec ou sans pré-extraction, permettant de déterminer les interdépendances de ces protéines pour leur localisation et pour leur stabilité nucléaire. Ces approches ont permis de démontrer que Pgm requiert la présence de Ku pour être stablement localisé dans les noyaux lors des RPG. Ku80c partage 74% de sa séquence protéique avec Ku80a. Des expériences de complémentations fonctionnelles surexprimant Ku80a lors des RPG ont montré que Ku80a n’est pas capable ni de se localiser stablement dans les noyaux ni de participer à la stabilisation nucléaire de Pgm. De plus, les RPG sont inhibés. Ces résultats montrent que Ku80c s’est spécialisé dans le couplage avec Pgm pour l’introduction des CDB lors des RPG. L’utilisation de protéines chimériques a permis de déterminer que la spécialisation de Ku80c est portée par son domaine N-terminal ∝-β. / During its sexual cycle, the ciliate Paramecium tetraurelia undergoes massive Programmed Genome Rearrangements (PGR). They consist, among others, in excision of 45,000 precisely delimited sequences, called IES (Internal Eliminated Sequences). A domesticated transposase, PiggyMac (Pgm), introduces double-strand DNA breaks (DSB) at IES ends. The Non Homologous End Joining pathway (NHEJ) handles highly precise repair of DSB. One of the actors of this pathway is the heterodimer Ku70/Ku80. In P. tetraurelia, the KU80 gene is present in three paralogous copies. Only KU80c is specifically expressed during PGR and RNA interferences against KU80c showed a complete inhibition of DNA cleavage. Furthermore, a Co-IP experiment in a heterologous system showed that both Ku70/Ku80c interact with Pgm. These results provide evidence that Ku is an essential partner of Pgm for DSB introduction; raising the question of the activating mechanism involved. During my PhD, I characterized the coupling between Ku and Pgm by analyzing immunofluorescence experiments, with or without pre-extraction, allowing the determination of inter-dependencies between those proteins for their nuclear localization and stability. Those methods demonstrated that Pgm requires the presence of Ku for a stable nuclear localization during the PGR. Ku80c shares 74% of the protein sequence with Ku80a. Functional complementation assays overexpressing Ku80a during the PGR showed that Ku80a is not capable to stably localize in nuclei nor to participate in Pgm nuclear stability. Furthermore, PGR are inhibited. Those results show that Ku80c has specialized for the DSB introduction during PGR. The use of chimeric proteins allowed to determine that Ku80c specialization was carried out by its N terminal domain.

La publication du génome de la paramécie (Aury, 2006) a révélé une séquence atypique particulièrement intéressante pour les études de génomique évolutive. Au cours de cette thèse, j’ai mené une analyse bioinformatique détaillée de ce génome en me concentrant particulièrement sur les trois points suivants : 1) Le rôle de deux classes distinctes de petits ARN fonctionnels non codants, l’une intervenant dans les processus de régulation de l’expression des gènes tandis que l’autre participe aux réarrangements génomiques (élimination de fragments d’ADN) associés au cycle sexuel de la paramécie. 2) L’évolution des paires de gènes après une duplication globale de génome (WGD). En effet, avec une WGD relativement récente précédée de deux autres WGDs plus anciennes encore bien visibles, la paramécie est un modèle de choix pour cette étude. Nous avons pu montrer que la rétention des deux copies d’un gène après une WGD est fortement corrélée au niveau d’expression des gènes. Nous proposons un modèle basé sur les coûts et bénéfices de l’expression des gènes pour expliquer cette observation. 3) L’analyse de contraintes sélectives sur les introns pour produire des messagers détectables par le Nonsense-Mediated mRNA Decay (NMD). Ces contraintes sélectives, mises en évidence initialement chez la paramécie, se sont avérées être présentes chez tous les eucaryotes que nous avons pu analyser, ce qui nous amène à questionner l’efficacité des mécanismes d’épissage chez les eucaryotes et le rôle du NMD dans la prévention des erreurs d’épissage. L’ensemble de ces analyses a permis de mieux comprendre un certain nombre de mécanismes évolutifs universels / This work presents a detailed analysis of the paramecium genome, with focusing more precisely on the 3 following topics : 1) The role of two distinct classes of small non-coding RNAs. The first one (siRNAs) being involved in post-transcriptional gene silencing while the other (scanRNAs) plays a crucial role during the massive genomic rearrangements that occur in ciliates after sexual reproduction (Lepère et al. 2009). 2) The evolution of duplicated genes following Whole-Genome Duplications (WGDs). Indeed, the paramecium genome contains evidences for 3 successive WGDs (Aury et al. 2006), which explains why this organisms is perfectly well suited for such an analysis. We show that retention of duplicated genes is strongly correlated to their expression level and we propose a model based on cost and benefit of gene expression to explain this pattern. 3) The analysis of the extremely tiny introns in paramecium (99% of introns are less than 20-33nt in length) revealed the presence of a translational control of splicing in eukaryotes. This work suggests that splicing errors are frequent and that eukaryotic cells rely on the Nonsense-mediated mRNA Decay to detect aberrant transcripts produced by splicing errors (Jaillon et al. 2008). These analyses provide new insights on several evolutionary mechanisms that shape the genomes of eukaryotes

Mycobacteriosis is a common disease of laboratory zebrafish (Danio rerio). Different infection patterns occur in zebrafish depending on mycobacterial species. Mycobacterium marinum and M. haemophilum produce virulent infections associated with high mortality, whereas M. chelonae is more wide spread and not associated with high mortality. Identification of mycobacterial infections to the species level provides important information for making management decisions. Observation of acid-fast bacilli in histological sections or tissue imprints is the most common diagnostic method for mycobacteriosis in fish, but only allows for diagnosis to the genus level. Mycobacterial culture, followed by molecular or biochemical identification is the traditional approach for species identification, but recently it has been shown that DNA of diagnostic value can be retrieved from paraffin blocks. Type of fixative, time in fixative before processing, species of mycobacteria, and severity of infection were investigated as parameters to determine if the hsp gene PCR assay (primer set HS5F/hsp667R) could detect and amplify mycobacterial DNA from paraffin-embedded zebrafish. Whole zebrafish were experimentally infected with either M. chelonae or M. marinum, and then preserved in 10% neutral buffered formalin or Dietrich's fixative for 3, 7, 21 and 45 days. Subsequently, fish were evaluated by H&E and Fite's acid-fast stains to detect mycobacteria within granulomatous lesions. The PCR assay was quite effective, and obtained PCR product from 75% and 88% of the M. chelonae and M. marinum infected fish, respectively. Fixative type, time in fixative, and mycobacterial species showed no statistical relationship with the efficacy of the PCR test. Regarding natural transmission, zebrafish are capable of contracting mycobacterial infections by feeding on infected fish tissue, but other natural routes have not been clearly elucidated. Free living amoebae have been shown to be vectors for mycobacteria and their virulence is enhanced when residing in these protozoans. Paramecium caudatum are commonly used as a first food for zebrafish, and I investigated this ciliate's potential to serve as a vector of Mycobacterium marinum and M. chelonae. The ability of live P. caudatum to transmit these mycobacteria to larval, juvenile and adult zebrafish was evaluated. Infections were defined by histologic observation of granulomas containing acid-fast bacteria in extraintestinal locations. In both experiments, fish fed paramecia containing mycobacteria became infected at a higher incidence than controls. Larvae (exposed at 4 days post hatch) fed paramecia with M. marinum exhibited an incidence of 30% (24/80) and juveniles (exposed at 21 days post hatch) showed 31% incidence (14/45). Adult fish fed gelatin diets containing bacteria within paramecia or mycobacteria alone for 2 wk resulted in infections when examined 8 wk after exposure: M. marinum OSU 214; in paramecia 47% (21/45; 3.5 x 10⁵ dose/fish/day), M. marinum CH in paramecia 47% (9/19; 3.6 x 10⁵ dose/fish/day), M. chelonae in paramecia 38% (5/13; 3.5 x 10⁵ dose/fish/day). I investigated the ability of mycobacteria to persist within paramecia, as this has previously been demonstrated in amoebae. Gram negative bacteria ingested by paramecia were processed within an hour. In contrast, I determined using GFP-labeled Mycobacterium marinum that mycobacteria can persist within paramecia digestive vacuoles. The concentration of M. marinum at 1 hour was similar to that at the time of ingestion. Twenty-four hours post-ingestion and later there was significant decline in M. marinum concentrations compared to time of ingestion, but M. marinum continued to persist inside digestive vacuoles for up to one week. My results demonstrate for the first time that Paramecium caudatum can act as a vector for mycobacteria. This provides a useful animal model for evaluation of natural mycobacterial infections and demonstrates the possibility of mycobacterial transmission in zebrafish facilities via contaminated paramecia cultures. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from April 2, 2013 - April 2, 2015