Dans les cellules eucaryotes, l'expression des gènes est régulée à de multiples étapes. Les régulations post-transcriptionnelles regroupent l'ensemble des contrôles qui s'exercent sur un ARN, en particulier sa maturation nucléaire, sa stabilité et son contrôle traductionnel. Les protéines de liaison aux ARN sont des acteurs majeurs de ces régulations post-transcriptionnelles. Les protéines CELF1 et ELAVL1, abondantes et quasiment ubiquitaires, participent à ces différents niveaux de régulation et sont susceptibles de modifier le devenir des transcrits. Au cours de ces travaux, nous avons étudié l'impact de CELF1 sur l'abondance et la maturation des ARN par des approches transcriptomiques. Nous avons classé les transcrits en fonction de leurs réponses aux différentes inactivations. L'ensemble de ces données montre que i) CELF1 et ELAVL1 ont des rôles bivalents, leur déplétion étant associée à une répression ou une activation de certains gènes ; ii) dans des cellules HeLa, les effets des différentes déplétions sont majoritairement des effets indirects ; iii) CELF1 et ELAVL1 ont très majoritairement le même effet sur l'abondance des ARNm qu'elles contrôlent directement ; iv) CELF1 et ELAVL1 ont le plus souvent des effets coopératifs ou redondants sur l'abondance des transcrits liés. L'effet combinatoire de CELF1 et ELAVL1 dépend de l'ARNm considéré, révélant une complexité des régulations post-transcriptionnelles critiques pour le devenir d'une cellule. / In eukaryotic cells, after transcription gene expression is controlled at multiple steps. These qualitative and quantitative post-transcriptional regulations are specified by RNA binding proteins (RBP). By combining transcriptomic analysis and binding site information for CELF1, we showed that CELF1 regulates both nuclear and cytoplasmic steps of gene expression. CELF1 directly controls the stability of cyclin D1 mRNA and the splicing of several RNA including KLC1 (light chain kinesin 1). Because mRNAs are in complexes consisting of multiple RBP we studied whether CELF1 and ELAVL1 would interact and control the abundance of their bound mRNAs. This analysis unravel a surprising redundancy or cooperativity of CELF1 and ELAVL1. The combinatorial effects of CELF1 and ELAVL1 were highly dependent on the considered RNA. Interestingly, we showed that both proteins cooperate and interact physically.

La production de viande à moindre coût nécessite de disposer d’animaux robustes, efficaces, capables de s’adapter aux contraintes d’élevage. Ceci requiert l’évaluation de la capacité de l’animal à constituer et restituer ses réserves énergétiques. Ce travail de thèse a donc pour objectif d’identifier de potentiels biomarqueurs sanguins en lien avec la composition corporelle. Pour cela, deux espèces monogastriques sont étudiées, le porc et le poulet. Au sein de chaque espèce, les animaux de deux lignées génétiques (sélection divergente sur la Consommation Moyenne Journalière Résiduelle chez le porc et sur la proportion de gras abdominal chez le poulet) ont reçu des régimes alimentaires contrastés (riches en lipides et en fibres vs. riches en amidon) mais isoprotéiques et isoénergétiques. Les paramètres plasmatiques et le transcriptome du sang ont été étudiés en réponse à ces régimes alimentaires.Les métabolites et hormones plasmatiques sont affectés par le régime chez le porc alors que ces paramètres sont affectés par le régime et la lignée chez le poulet. L’analyse du métabolome associée aux mesures ciblées des concentrations en métabolites et hormones montre que l’association de plusieurs paramètres sanguins explique entre 37 et 75% de la variabilité de la masse adipeuse chez le porc ou le poulet. Pour les deux espèces, le transcriptome du sang est plus affecté par la lignée génétique que par le régime alimentaire. Les porcs et les poulets nourris avec un régime riche en lipides et en fibres, surexpriment le gène codant la forme hépatique d’une enzyme mitochondriale : CP / Meat production at a lower cost requires robust and efficient animals able to adapt to different rearing conditions. This requires assessing animal’s abilities to store and restore its energy reserves. The objective of the current thesis was to identify potential blood markers of body composition. Two monogastric species were studied, pig and chicken. For each species, animals of two genetic lines (divergent selection on Residual Feed Intake in pigs and abdominal fat proportion in chicken) received two diets contrasted in energy sources (high vegetable oils and fibers vs. rich in starch) but isoproteic and isoenergetic. Plasma parameters and the blood transcriptome were studied in response to those diets.Plasma metabolites and hormones were affected by the diet in pigs whereas those parameters were affected by the diet and the genetic lines in chickens. Metabolome analysis, associated with targeted measurement of metabolites and hormones concentrations, shows that the combination of several blood parameters explained between 37 and 75% of the variability of body fat in pig or chicken. For both species, the blood transcriptome was more affected by the line than by the diet. Pigs and chickens fed a diet rich in lipids and fibers, overexpressed the gene encoding the hepatic form of a mitochondrial enzyme: CPT1A. In summary, this work supports the potential use of blood transcriptome to study variations of phenotypes in a dynamic way throughout the life of the animal and to highlight biomarkers for future selection process.

L'une des caractéristiques majeures des microorganismes, et donc des bactéries, est qu'ils sont en contact direct avec l'environnement et doivent donc percevoir et répondre rapidement à ses variations. Pour cela, plusieurs niveaux de contrôle existent, et récemment le rôle des ARN non codants régulateurs, ou riborégulateurs, a été mis en lumière comme un mécanisme de contrôle peu couteux et rapide pour la cellule. Chez le phytopathogène Agrobacterium fabrum (anciennement appelé Agrobaterium tumefaciens), la virulence est principalement régulée au niveau transcriptionnel par le système à deux composants VirANirG. L'implication des riborégulateurs dans la virulence d'A.fabrum est encore mal connue et ces travaux de thèse ont eu pour objectif de déterminer l'implication de riborégulateurs dans le cycle infectieux de cette bactérie.Pour cela, nous avons identifié l'ensemble des transcrits d 'A. fabrum C58 en combinant l'utilisation de plusieurs méthodes d'analyses globales et nous avons étudié la fonction de différents candidats transcrits à partir du plasmide Ti (plasmide de virulence). Des souches modifiées dans la production des riborégulateurs candidats ont été construites, Jeurs ARNm cibles ont été prédits et validés, et des tests phénotypiques, en particulier des tests de virulence, ont été réalisés. Ainsi, le séquençage des transcrits de petite taille a permis d'identifier plus d'un millier de riborégulateurs potentiels dont plusieurs sont exprimés à partir de régions en relation avec le cycle infectieux. Nous avons validé 4 de ces petits transcrits comme étant des riborégulateurs puisqu'ils sont de petite taille, non traduits en protéine et fortement structurés (RNAI 111, RNA1083, RNA1059 et RNA1051) . Plus particulièrement, nous avons montré que le riborégulateur RNAI 111 était nécessaire pour la virulence d'A.fabrum C58, et que son action semblait se faire au travers du contrôle post-transcriptionnel de gènes impliqués dans les fonctions de virulence et de transfert du plasmide Ti. Un rôle plus modéré du riborégulateur RNA1083 dans le contrôle du cycle infectieux a également été observé, potentiellement au travers de la modulation de la mobilité et du transfert conjugatif du plasmide Ti. D'autre part, nous avons mis en évidence deux autres riborégulateurs, RNA1059 et RNA1051, qui sont impliqués dans le contrôle du maintien du plasmide Ti via une implication dans la réplication du plasmide (RNA 1059) et via une implication dans un nouveau system de toxine-antitoxine présent sur le plasmide Ti (RNA1051). Ainsi à partir d'une analyse globale nous avons mis en évidence le rôle des riboregulateurs dans les systèmes de mise en place de l'infection bactérienne , soit via le contrôle de facteurs de virulence, soit via le contrôle de la persistance du plasmide responsable de la virulence / One of the main characteristics of microorganisms, including bacteria., is their direct interaction with their environment. They thus need to perceive and quickly answer to its variations. Several steps of control exist, and recently the role of regulatory non-coding RNA, or riboregulator, was highlighted as a fast and economic mechanism of regulation. In the phytopathogen Agrobacterium fabrum (previously named Agrobacterium tumefaciens), the virulence is mainly controlled transcriptionally by the two components system VirANirG. The implication of riboregulators in the virulence of this bacterium is still unknown . The objectives of this thesis were to identify A .fabrum riboregulators and to determine their involvement in the infectious cycle of the bacteria. To this end, we identified small transcripts of A . fabrum C58 strain by combining several global analyses, and we studied the function of different candidates transcribed from the Ti plasmid (the virulence plasmid). Strains modified in the production of these candidates were constructed, their mRNA targets were predicted and validated, and phenotypic analyses -especially virulence tests­ were realized.Thereby, small transcript deep-sequencing allowed the identification of a thousand potential riboregulators, some of them being transcribed from regions related to the infectious cycle. We validated 4 of these transcripts as riboregulators according to their small size, their strong secondary structure and their non-translation into protein (RNAIOS I, RNA1059, RNA1083 and RNAl ll l). In particular, we showed that RNA 1111 was necessary for the virulence of A. fabrum C58, and that it seems to act through the posttranscriptional control of genes implicated in virulence functions and in Ti plasmid conjugation. A more moderated role of RNA 1083 was also observed, potentially by the modulation of the bacterial mobility and of the plasmid conjugation. Furthermore, we highlighted two riboregulators, RNA1059 and RNA1051, involved in the control of the Ti plasmid persistence, through their implication in the replication of the plasmid (RNA1059) and in a toxin-antitoxin system present on the Ti plasmid (RNA1051) .Thus, from a global analysis, we brought out the role of riboregulators in the control of several steps of the infectious cycle of A. fabrum C58, through the control of virulence factors, or through the contrai of the persistence of the main actor of the virulence, the Ti plasmid

Staphylococcus xylosus est couramment utilisé comme ferment dans les produits carnés pour son rôle dans le développement de la flaveur et de la couleur. Beaucoup de propriétés technologiques ont été caractérisées in vitro. Cependant, les mécanismes moléculaires mis en place par cette bactérie pour s’adapter à une matrice carnée et aux composés nitrosés, fréquemment ajoutés dans ces produits, étaient méconnus. Pour identifier ces mécanismes, des approches de transcriptomique globale ont été mises en œuvre. S. xylosus survit dans un modèle viande en modulant l’expression de 55 % de ses gènes. Il surexprime des gènes codant des protéines impliqués dans le catabolisme du glucose et du gluconate et des gènes codant des peptidases. En parallèle, il sous exprime de nombreux gènes impliqués dans la synthèse des acides aminés probablement en raison de leur disponibilité dans le modèle viande. Le modèle viande est un milieu riche en divers substrats et la bactérie pourrait adapter sa physiologie via les régulateurs transcriptionnels CcpA et CodY. S. xylosus répond au sel ajouté au modèle viande en surexprimant des gènes impliqués dans des mécanismes d’osmoprotection, d’extrusion de Na + et de protons. S. xylosus répond aux composés nitrosés dans le modèle viande en modulant 24 % de son génome. Ces composés nitrosés génèrent un stress nitrosant et S. xylosus répond à ce stress par la surexpression de gènes impliqués dans l’homéostasie du fer via la dérépression du régulateur Fur. S. xylosus surexprime aussi des gènes codant des enzymes antioxydants via la dérépression du régulateur PerR. De plus, il surexprime des gènes impliqués dans la réparation de l’ADN et des protéines. La viande est un aliment riche en fer hémique et non hémique. Ainsi, S. xylosus est capable d’acquérir du fer à partir de ferritine, de transferrine et potentiellement des hémoprotéines. La ferritine est une source préférentielle de fer pour S. xylosus. Un opéron codant potentiellement un complexe membranaire impliqué dans des réactions d’oxydo-réduction a été identifié. Un mutant de délétion/insertion dans le premier gène de l’opéron confirme que ce système pourrait jouer un rôle dans l’acquisition du fer de la ferritine chez S. xylosus. Cette étude révèle un changement global dans l’expression des gènes de S. xylosus dans un modèle viande, elle souligne la capacité de S. xylosus à s’adapter à un stress osmotique ou nitrosant et elle caractérise pour la première fois la capacité d’un staphylocoque à utiliser du fer de la ferritine. / Staphylococcus xylosus is used as starter culture in meat product for its role in the development of flavor and color. S. xylosus is characterized for its technological properties in vitro. However, the molecular mechanisms for its adaptation in meat with or without nitrate and nitrite, frequently added in meat product, remained unknown. Global transcriptomic approaches were carried out to determine the molecular mechanisms. S. xylosus modulated the expression of 55 % of the genes to survive in a meat model. Many genes encoding proteins involved in glucose and gluconate catabolisms and peptidases were up expressed. In parallel, a lot of genes involved in amino acids synthesis were down regulated, probably due to their availability in the meat model. The meat model is a rich medium composed of various substrates and S. xylosus adapted its physiology through the transcriptional regulators CcpA and CodY. Finally, it responded to salt added in the meat model in overexpressing genes involved in mechanisms of osmoprotection, Na + and H + extrusion. S. xylosus modulated the expression of 24 % of the genes in presence of nitroso compounds in the meat model. These compounds generated a nitrosative stress. S. xylosus responded to this stress by over expressing genes involved in iron homeostasis through the derepression of the regulator Fur. It over expressed also genes encoding antioxidant enzymes through the derepression of the regulator PerR. Moreover, it over expressed genes involved in DNA and proteins repairs. Meat is rich in hemic and non-hemic iron. S. xylosus is able to grow in presence of ferritin, transferrin and potentially hemoproteins. Ferritin is one of preferential iron sources. An operon encoding potentially a membranous complex involved in oxydo-reduction reactions has been identified. A strain defective in the first gene of the operon confirmed that this complex could contribute to the iron acquisition from ferritin. This study revealed a global change in the gene expression of S. xylosus in the meat model; it highlighted ability of S. xylosus to mitigate nitrosative or osmotic stress, it characterised for the first time the capacity of a Staphylococcus to acquire ferritin-iron.

Blastocladiella emersonii é um fungo aquático da classe dos quitridiomicetos, notável pelas mudanças morfogenéticas que ocorrem durante o seu ciclo de vida. Neste trabalho isolamos 8.495 seqüências expressas (ESTs) deste fungo, que representam 3.226 seqüências únicas putativas. Destas seqüências, 37% foram classificadas segundo o processo biológico onde estariam envolvidas, de acordo com o sistema de anotação do Gene Ontology (GO). Analisamos os perfis de expressão in silico das ESTs usando estatística Bayesiana e os resultados obtidos foram validados por Northern blot para sete perfis de expressão selecionados. Pudemos encontrar boa correlação entre vários padrões de expressão e determinados processos biológicos. Foram selecionadas algumas seqüências potencialmente envolvidas com a esporulação do fungo para melhor caracterização. Analisamos a expressão de dois genes codificando centrinas (BeCenl e BeCen2) pertencentes a subfamílias distintas. Centrinas são proteínas ligantes de cálcio envolvidas em diferentes processos como o direcionamento do aparelho flagelar e a duplicação dos centros organizadores de microtúbulos (MTOCs). Observamos que os níveis da proteína BeCenl, que não havia sido descrita em fungos, apresentam um máximo aos 150 min da esporulação, defasado do pico de expressão do seu mRNA que ocorre aos 90 min deste estágio. A proteína BeCen2 está presente em níveis constantes durante todo a ciclo de vida do fungo, embora o seu mRNA apresente um pico de expressão aos 120 min da esporulação. Experimentos de imunofluorescência localizaram a proteína BeCenl no citoplasma e no corpo basal do zoósporo. Estes dados sugerem que BeCenl atue na re-orientação e movimento dos corpos basais e BeCen2 na duplicação dos MTOCs. Investigamos também a expressão de dois genes codificando proteína-quinases dependentes de ciclina putativas (BeCdkl e BeCdk2). Apenas uma Cdk (Cdkl) foi descrita em fungos como diretamente envolvida no controle do ciclo celular. Ambos os genes apresentam expressão diferencial, com níveis máximos de mRNA para os dois casos aos 90 min da esporulação. Por outro lado, a proteína BeCdkl está presente durante todo o ciclo de vida do fungo e foi localizada no núcleo e no capacete nuclear dos zoósporos. Um transportador putativo de hexose (Bemst) foi também analisado, com base na regulação por glicose de genes envolvidos com o ciclo celular observada em eucariotos. Verificou-se que os níveis do mRNA de Bemst diminuem drasticamente durante a esporulação, mas glicose ou outras hexoses não afetaram a expressão de Bemst. / Blastocladiella emersonii is an aquatic fungus that belongs to the class of chytridiomycetes, notable for the morphogenetic processes which occur during its life cycle. In this work we have isolated 8,495 expressed sequence tags (ESTs) from this fungus, representing 3,226 putative unigenes. From these unigenes, 37% were classified into a biological process, as a result of Gene Ontology (GO) annotation. Furthermore, we analyzed the expression profile in silico of each transcript using Bayesian Statistics and seven of these profiles were validated by Northern blot analysis. In addition, we found a good correlation between several of these expression patterns and certain biological processes. Some ESTs potentially involved in the sporulation of the fungus were selected to be further characterized. We analyzed the expression of two genes encoding two centrins (BeCenl and BeCen2) of distinct subfamilies. Centrins are calcium-binding proteins involved in different processes such as basal body orientation and duplication of the microtubuleorganizing centers (MTOCs). The amount of BeCenl, a centrin ortholog not previously described in fungi, presents a maximum at 150 min of sporulation, whereas the peak of its mRNA occurs at 90 min of this stage. Protein BeCen2 presents constant levels during the entire life cycle of the fungus, even though its mRNA shows a peak of expression at 120 min of sporulation. In addition, immunofluorescent studies localized BeCenl in the cytoplasm and the basal body of zoospores. These results suggest that BeCenl plays a role in re-orientation and movement of basal bodies and BeCen2 in MTOCs duplication. We also investigated the expression of two genes encoding putative cyclin-dependent protein kinases (BeCdkl and BeCdk2). Only one type of Cdk (Cdkl) directly involved in cell cycle control has been described in other fungi. Both genes were found to be differentially expressed, with maximum mRNA levels being detected in either case at 90 min of sporulation. In contrast, BeCdk1 is present throughout the life cycle of the fungus and was immunolocalized in the nuc1eus and the nuclear cap of zoospores. A putative hexose transporter (Bemst) was also investigated, taking into account the regulation by glucose of cell cycle controlled genes in eukaryotes. We found that Bemst mRNA levels decrease drastically during sporulation, but glucose and other hexoses had no effect on Bemst expression.

Dans ce travail nous avons élaboré une nouvelle méthode de l'analyse de réseau à définir des membres possibles des voies moléculaires qui sont important pour ce phénotype en utilisant la « hit-liste » des expériences « omics » qui travaille dans le réseau intégré (le réseau comprend des interactions protéine-protéine, de transcription, l’acide ribonucléique micro-l’acide ribonucléique messager et celles métaboliques). La méthode tire des sous-réseaux qui sont construit des voies de quatre types les plus courtes (qui ne se composent des interactions protéine-protéine, ayant au minimum une interaction de transcription, ayant au minimum une interaction l’acide ribonucléique micro-l’acide ribonucléique messager, ayant au minimum une interaction métabolique) entre des hit –gènes et des soi-disant « exécuteurs terminaux » - les composants biologiques qui participent à la réalisation du phénotype finale (s’ils sont connus) ou entre les hit-gènes (si « des exécuteurs terminaux » sont inconnus). La méthode calcule la valeur de la centralité de chaque point culminant et de chaque voie dans le sous-réseau comme la quantité des voies les plus courtes trouvées sur la route précédente et passant à travers le point culminant et la voie. L'importance statistique des valeurs de la centralité est estimée en comparaison avec des valeurs de la centralité dans les sous-réseaux construit des voies les plus courtes pour les hit-listes choisi occasionnellement. Il est supposé que les points culminant et les voies avec les valeurs de la centralité statistiquement signifiantes peuvent être examinés comme les membres possibles des voies moléculaires menant à ce phénotype. S’il y a des valeurs expérimentales et la P-valeur pour un grand nombre des points culminant dans le réseau, la méthode fait possible de calculer les valeurs expérimentales pour les voies (comme le moyen des valeurs expérimentales des points culminant sur la route) et les P-valeurs expérimentales (en utilisant la méthode de Fischer et des transpositions multiples).A l'aide de la méthode masterPATH on a analysé les données de la perte de fonction criblage de l’acide ribonucléique micro et l'analyse de transcription de la différenciation terminal musculaire et les données de la perte de fonction criblage du procès de la réparation de l'ADN. On peut trouver le code initial de la méthode si l’on suit le lien https://github.com/daggoo/masterPATH / In this work we developed a new exploratory network analysis method, that works on an integrated network (the network consists of protein-protein, transcriptional, miRNA-mRNA, metabolic interactions) and aims at uncovering potential members of molecular pathways important for a given phenotype using hit list dataset from “omics” experiments. The method extracts subnetwork built from the shortest paths of 4 different types (with only protein-protein interactions, with at least one transcription interaction, with at least one miRNA-mRNA interaction, with at least one metabolic interaction) between hit genes and so called “final implementers” – biological components that are involved in molecular events responsible for final phenotypical realization (if known) or between hit genes (if “final implementers” are not known). The method calculates centrality score for each node and each path in the subnetwork as a number of the shortest paths found in the previous step that pass through the node and the path. Then, the statistical significance of each centrality score is assessed by comparing it with centrality scores in subnetworks built from the shortest paths for randomly sampled hit lists. It is hypothesized that the nodes and the paths with statistically significant centrality score can be considered as putative members of molecular pathways leading to the studied phenotype. In case experimental scores and p-values are available for a large number of nodes in the network, the method can also calculate paths’ experiment-based scores (as an average of the experimental scores of the nodes in the path) and experiment-based p-values (by aggregating p-values of the nodes in the path using Fisher’s combined probability test and permutation approach). The method is illustrated by analyzing the results of miRNA loss-of-function screening and transcriptomic profiling of terminal muscle differentiation and of ‘druggable’ loss-of-function screening of the DNA repair process. The Java source code is available on GitHub page https://github.com/daggoo/masterPATH

BACKGROUND: Organelle retention is a form of mixotrophy that allows organisms to reap metabolic benefits similar to those of photoautotrophs through capture of algal prey and sequestration of their plastids. Mesodinium rubrum is an abundant and broadly distributed photosynthetic marine ciliate that steals organelles from cryptophyte algae, such as Geminigera cryophila. M. rubrum is unique from most other acquired phototrophs because it also steals a functional nucleus that facilitates genetic control of sequestered plastids and other organelles. We analyzed changes in G. cryophila nuclear gene expression and transcript abundance after its incorporation into the cellular architecture of M. rubrum as an initial step towards understanding this complex system. METHODS: We compared Illumina-generated transcriptomes of the cryptophyte Geminigera cryophila as a free-living cell and as a sequestered nucleus in M. rubrum to identify changes in protein abundance and gene expression. After KEGG annotation, proteins were clustered by functional categories, which were evaluated for over- or under-representation in the sequestered nucleus. Similarly, coding sequences were grouped by KEGG categories/ pathways, which were then evaluated for over- or under-expression via read count strategies. RESULTS: At the time of sampling, the global transcriptome of M. rubrum was dominated (~58-62 %) by transcription from its stolen nucleus. A comparison of transcriptomes from free-living G. cryophila cells to those of the sequestered nucleus revealed a decrease in gene expression and transcript abundance for most functional protein categories within the ciliate. However, genes coding for proteins involved in photosynthesis, oxidative stress reduction, and several other metabolic pathways revealed striking exceptions to this general decline. CONCLUSIONS: Major changes in G. cryophila transcript expression after sequestration by M. rubrum and the ciliate's success as a photoautotroph imply some level of control or gene regulation by the ciliate and at the very least reflect a degree of coordination between host and foreign organelles. Intriguingly, cryptophyte genes involved in protein transport are significantly under-expressed in M. rubrum, implicating a role for the ciliate's endomembrane system in targeting cryptophyte proteins to plastid complexes. Collectively, this initial portrait of an acquired transcriptome within a dynamic and ecologically successful ciliate highlights the remarkable cellular and metabolic chimerism of this system.

Thesis (PhD (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / ENGLISH ABSTRACT: The slow growth rate of Haliotis midae impedes the optimal commercial production of this most profitable South African aquaculture species. To date, no comprehensive effort has been made to identify genes associated with growth variation in farmed H. midae. The aim of this study was therefore to investigate growth variation in H. midae and to identify and quantify the expression of selected growth-related genes. Towards this aim, molecular methodologies and cell cultures were combined as a time-efficient and economical way of studying abalone transcriptomics and cell biology. Modern Illumina sequencing-by-synthesis technology and subsequent sequence annotation were used to elucidate differential gene expression between two sibling groups of abalone demonstrating significant growth variation. Following transcriptome sequencing, genes involved in growth and metabolism, previously unknown in H. midae, were identified. The expression of selected target genes involved in growth was subsequently analyzed by quantitative real-time PCR (qPCR). The feasibility of primary cell cultures for H. midae was furthermore investigated by targeting embryo, larval and haemolymph tissues for the initiation of primary cell culture. Larval cells and haemocytes could be successfully maintained in vitro for limited periods. Primary haemocyte cultures demonstrated to be a suitable in vitro system for studying gene expression and were subsequently used for RNA extraction and qPCR, to evaluate differential growth induced by bovine insulin and epidermal growth factor (EGF). Gene expression was thus quantified in fast and slow growing abalone and in in vitro primary haemocyte cultures treated with different growth stimulating factors. The results obtained from transcriptome analysis and qPCR revealed significant differences in gene expression between large and small abalone, and between treated and untreated haemocyte cell cultures. Throughout in vivo and in vitro qPCR experiments, the up-regulation of genes involved in the insulin signaling pathway provides evidence for the involvement of insulin in enhanced growth rate for various H. midae tissues. Besides the regulation of target genes, valuable knowledge was also gained in terms of reference genes, during qPCR experimentation. By quantifying the stable expression of two genes (8629, ribosomal protein S9 and 12621, ornithine decarboxylase) in various tissues and under various conditions, suitable reference genes, that can also be used in future H. midae qPCR studies, were identified. By providing evidence at the transcriptional level for the involvement of insulin, insulin-like growth factors (IGFs) and insulin-like growth factor binding proteins (IGFBPs) in improved growth rate of H. midae, the relevance of investigating ways to stimulate insulin/IGF release in aquaculture species was again emphasized. As nutritional administration remains the most probable route of introducing agents that can stimulate the release of insulin-related peptides, continuous endeavours to stimulate abalone growth through a nutritional approach is encouraged. This is the first time next generation sequencing is used towards the large scale transcriptome sequencing of any haliotid species and also the first time a comprehensive investigation is launched towards the establishment of primary cell cultures for H. midae. A considerable amount of sequence data was furthermore annotated for the first time in H. midae. The results obtained here provide a foundation for future genetic studies exploring ways to optimise the commercial production of H. midae. / AFRIKAANSE OPSOMMING: Die stadige groeitempo van Haliotis midae belemmer die optimale kommersiele produksie van hierdie mees winsgewende Suid-Afrikaanse akwakultuur spesie. Tot op hede is geen omvattende poging aangewend om gene verwant aan groeivariasie in H. midae te identifiseer nie. Die doel van hierdie studie was dus om groeivariasie in H. midae te ondersoek en om spesifieke groei-gekoppelde gene te identifiseer en hul uitdrukking te kwantifiseer. Ter bereiking van hierdie doel is molekulêre metodes en selkulture gekombineer as 'n en tydsbesparende en ekonomiese manier om perlemoen transkriptomika en selbiologie te bestudeer. Moderne Illumina volgordebepaling-deur-sintese tegnologie en daaropvolgende annotasie is gebruik om verskille in geenuitdrukking tussen naby-verwante groepe perlemoen, wat noemenswaardige groeivariasie vertoon, toe te lig. Na afloop van die transkriptoom volgordebepaling is gene betrokke by groei en metabolisme, vantevore onbekend in H. midae, geïdentifiseer. Die uitdrukking van uitgesoekte teikengene betrokke by groei is vervolgens ge-analiseer deur kwantitatiewe "real-time PCR" (qPCR). die lewensvatbaarheid van 'n primêre selkulture vir H. midae is ook ondersoek deur embrio, larwe en hemolimf weefsels te teiken vir die daarstelling van primêre selkulture. Larweselle en hemosiete kon in vitro suksesvol onderhou word vir beperkte periodes. Primêre hemosietkulture het geblyk 'n gepaste in vitro sisteem te wees om geenuitdrukking te bestudeer en dit is vervolgens gebruik vir RNS ekstraksie en qPCR, om differensiële groei, geïnduseer deur insulien en epidermale groeifaktor (EGF), te evalueer. Geenuitdrukking is dus gekwantifiseer in vinnig- en stadiggroeiende perlemoen en in in vitro primêre hemosiet selkulture wat behandel is met verskillende groei stimulante. Die resultate wat verkry is van transkriptoomanalise en qPCR het noemenswaardige verskille in geenuitdrukking tussen groot en klein perlemoen, en tussen behandelde en onbehandelde hemosiet selkulture uitgelig. Die op-regulering van gene betrokke by die insulien sein-padweg, tydens in vivo en in vitro qPCR eksperimente, bied getuienis vir die betrokkenheid van insulien in die verhoogde groeitempo van verskeie H. midae weefsels. Benewens die regulering van teikengene is waardevolle kennis ook ingewin in terme van verwysingsgene tydens qPCR eksperimentering. Deur die stabiele uitdrukking van twee gene (8629, ribosomale proteien S9 en 12621, ornitien dekarboksilase) te kwantifiseer in verskeie weefsels en onder verskeie kondisies is gepaste verwysingsgene, wat ook in toekomstige H. midae qPCR eksperimente aangewend kan word, geïdentifiseer. Deur getuienis vir die betrokkenheid van insulien, insuliensoortige groeifaktor en insuliensoortige groeifaktor-bindingsproteïene by verbeterde groei van H. midae op transkripsievlak te bied, is die toepaslikheid van bestudering van maniere om insulienvrystelling in akwakultuurspesies te stimuleer, beklemtoon. Aangesien voeding die mees waarskynlike roete is om middele wat insuliensoortige peptiedvrystelling stimuleer daar te stel, word vogehoue pogings om perlemoengroei deur die regte voeding te stimuleer, aangemoedig. Hierdie is die eerste studie wat volgende generasie volgordebepaling (“next generation sequencing”) gebruik vir die grootskaalse transkriptoom volgordebepaling van enige haliotied spesie. Dit is ook die eerste keer dat ‘n omvattende ondersoek geloods word na die daarstelling van primêre selkulture vir H.midae. ‘n Aansienlike hoeveelheid volgorde data is ook vir die eerste keer geannoteer in H. midae. Die resultate wat hier verkry is bied ‘n basis vir toekomstige genetiese studies wat maniere ondersoek om die kommersiële produksie van perlemoen te optimiseer.

Regeneration is a relatively widespread phenomenon in nature, although different organisms exhibit different abilities to reconstitute missing structures. Due to the diversity in the extent of damage the organisms can repair it has been debated for a long time whether those abilities are evolutionary traits that arose independently in multiple organisms or whether they represent a by-product of more basic processes. To date, due to constant increase in the amount of available genomic information this question can be approached by means of comparative genomics by comparing several taxa that have different regenerative capabilities. Two relatively closely related salamander species, newt, Notophthalmus viridescens, and the Mexican axolotl, Ambystoma mexicanum, offer a unique opportunity to compare two organisms with well-known regenerative capabilities. Despite their importance for regeneration research, relatively little sequence information was available until recently, owing mainly to the large sizes of the respective genomes. In this work I aimed to create a comprehensive transcriptome assembly of the axolotl by sequencing and then assembling the sequence data from a number of tissues and developmental stages. I also incorporated available sequence information that mostly comes from cDNA libraries sequenced previously. I assessed the completeness of the transcriptome by comparing it to a set of available axolotl sequences and found that 96% of those have homologs in the assembly. Additionally, I found that 7,568 of 7,695 protein families common to vertebrates are also represented in the transcriptome. In order to turn the assembly from a merely collection of sequences into a valuable and useful resource for the entire research community I first annotated the sequences, predicted the open reading frames and protein domains and additionally put together multiple bits of information available for each sequence including but not limited to time-course and tissue- specific expression data and in situ hybridization results. The assembly was thereafter made available for the entire axolotl research community through a web portal I developed. Not only does the web portal provide access to the transcriptome data, it is also equipped with an engine for automated data retrieval, which could facilitate automated cross-species bioinformatics analyses. The study crossed the boundary between pure bioinformatics and biology as the transcriptome allowed for computational comparison of the axolotl and the newt in order to identify salamander-specific genes possibly implicated in regeneration and subsequent functional analysis thereof in the lab. Since regeneration closely resembles embryonic development in terms of genes involved in both processes, I first identified approximately 200 homologous contigs in axolotl and newt, which had a predicted open reading frame, but did not have homologs in non-regenerating species. The expression profile of one of those candidate genes suggested that it had a role in regeneration. I studied the molecular function of that gene using CRISPR/Cas system to confirm that it was protein-coding and to create knock-out animals to study the effect of gene knock-down and knock-out. Knock-out animals exhibited significant delays in both, limb development and tail regeneration. The exact mechanism causing this delay is currently being investigated.

La myostatine, un facteur de croissance de la superfamille des TGF-béta, régule négativement la croissance et la masse du muscle squelettique en contrôlant le nombre de fibres par son action anti-proliférative, et la croissance individuelle de chacune d'entre elles. Dans le but de compléter les connaissances sur ces mécanismes d'action, cette thèse (2006-2009) avait pour objectif d'identifier de nouvelles cibles moléculaires de la myostatine impliquées dans l'hypertrophie musculaire. Ce travail de thèse a révélé de nouvelles cibles de la myostatine, notamment une activation de la cascade PI3K/Akt, une expression différentielle des gènes et des protèines impliquées dans la régulation de la survie cellulaire. L'inhibition de l'apoptose des noyaux musculaires en l'absence de myostatins fonctionnelle, et par conséquent , l'augmentation du nombre de noyaux et de la taille du domaine nucléo-cytoplasmique pourraient ainsi contribuer au phénomène d'hypertrophie