Dans un contexte de réduction des intrants agricoles, la création de variétés de blé qui utilisent l’azote de manière plus efficiente est aujourd’hui nécessaire. Cette thèse, issue d'un partenariat public-privé entre l'Institut National de la Recherche Agronomique et Biogemma, avait pour but d'apporter des outils nécessaires à la création de variétés répondant à cette exigence. Pour ce faire, nous avons analysé 225 variétés commerciales génotypées avec 24K SNP et testées dans huit combinaisons d’année, lieu et régime azoté. Nous avons montré que même si la sélection a amélioré l’efficience d’utilisation de l’azote en condition optimale et sub-optimale, ce progrès génétique doit être accéléré et mieux réparti entre les différents traits. Nous proposons pour cela de mixer sélection phénotypique et sélection assistée par marqueurs. Dans ce sens, nous avons développé une méthode pour définir les régions chromosomiques associées à nos 28 traits. Parmi les 333 régions identifiées, nous avons notamment localisé le gène NAM-A1 et avons pu caractériser ses variants naturels. Nous avons aussi montré que la sélection génomique pourrait être plus efficace si les SNP étaient présélectionnés en fonction de leurs significativités en génétique d’association multi-environnementale. Les réseaux d’interactions épistatiques furent aussi étudiés, mettant en évidence un sous-réseau particulièrement intéressant. Nos résultats et méthodes sont discutés au regard des stratégies d’amélioration variétale et de découverte de gènes. Des pistes de recherche complémentaires et des améliorations ont aussi été suggérées. / In a context of fertiliser reduction, breeding for enhanced nitrogen use efficiency in bread wheat is necessary. This PhD thesis resulting from private-public collaboration between the French National Institute for Agricultural Research and Biogemma aimed providing necessary tools. Analyses were conducted using a dataset of 225 commercial varieties genotyped with 24K SNP and tested in eight combinations of year, location, and nitrogen regimes. We showed that even if past selection increased nitrogen use efficiency at high and moderate nitrogen regimes, genetic progresses need to be accelerated and better balanced between traits. This could be achieved by mixing phenotypic and marker assisted selections. In this sense, we developed a method to define quantitative trait locus from genome-wide association study: 333 chromosomal regions involved in 28 NUE-related traits have been identified. The NAM-A1 gene was located in one of these regions and its natural variants were characterized. We also showed that genomic selection could be improved by pre-selecting SNP based on their significance in a multi-environmental genome-wide association study. Networks of epistasis interactions were also studied and an interesting sub-network was identified. Results and methods are discussed regarding breeding and gene discovery strategy. Further investigations and improvements are suggested.

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Les cellules sont la plus petite forme de vie individuelle qui forme un organisme. La structure et la santé de tous les organismes est essentiellement définie par le nombre, le type et la taille de leurs cellules. Composé d'environ 30 trillions de cellules, l'homme possède des cellules aux fonctions et aux tailles remarquablement variées, allant d'un neurone pouvant atteindre un mètre à une cellule lymphoïde d'environ 16 µm de diamètre. Il est connu que la taille est fondamentalement l'équilibre entre la croissance cellulaire et la division cellulaire. Néanmoins, les questions sur les réseaux moléculaires qui contrôlent et déterminent le maintien de la taille optimale des cellules restent à déchiffrer. D'innombrables travaux ont caractérisé mTORC1 comme une voie régulatrice majeure de la croissance cellulaire jouant un rôle central, intégrant des stimuli intra et extracellulaires. Ce travail porte sur l'investigation et la caractérisation des acteurs moléculaires et des processus qui orchestrent la taille des cellules humaine déterminées par l'épistase chimique. J'ai entrepris une bibliothèque CRISPR / Cas9 à inactivation prolongée (EKO) dans NALM-6 (lignée cellulaire de lymphome pré-B), suivie d'un fractionnement de la taille des cellules par élutriation à contre-courant en présence de rapamycine (inhibiteur de mTOR), et comparé aux données non publiées données du laboratoire utilisant les mêmes méthodes sans rapamycine. Cette analyse de l'étude indique que dans le contexte amont de mTOR, la perte de gènes liés à la détection des nutriments entraîne une perte de taille en présence d'inhibition de mTOR. En outre, plusieurs knockouts géniques dans la biogenèse des ribosomes et l'homéostasie du calcium ont conduit à une perte ou un gain de taille, montrant un rôle pivot possible de ces processus dans le contrôle de la taille des cellules d'une manière dépendante de mTOR. Ce travail a fourni des informations sur les gènes et réseaux connus et inconnus qui peuvent réguler la taille des cellules d'une manière dépendante de mTOR. Ces résultats doivent être validés et approfondis. / All organisms are essentially structured and fitness defined by cell number, type and size. Composed of around 30 trillion cells, humans have cells with remarkably varied functions and size, ranging from a neuron that can reach one meter in length to a lymphoid cell that is around 16 μm in diameter. At a fundamental level, size is determined by the balance between cell growth and cell division. The molecular networks that control and maintain optimal cell size are yet to be deciphered. The mTORC1 pathway is a major regulator of cell growth that plays a central role in integrating intra- and extra-cellular stimuli. This study addresses the investigation and characterization of the molecular players and processes that orchestrate cell size in human cells, as determined by chemical-genetic size screens and epistasis analysis. I undertook a CRISPR/Cas9 extended-knockout (EKO) genome-wide library screen in the NALM-6 pre-B lymphoma cell line, followed by cell size fractionation by counter flow elutriation in the presence of the mTOR inhibitor rapamycin, and compared the screen data to a similar screen performed in the absence of rapamycin. The analysis indicates that upstream of mTOR, the loss of genes that are related to nutrient sensing, results in size changes in the presence of mTOR inhibition. Also, several gene knockouts in ribosome biogenesis and calcium homeostasis led to size alterations, suggesting a possible a pivotal role of these processes in cell size control in a mTOR-dependent fashion. This study provides insights into the genetic networks that regulate cell size in a mTOR-dependent fashion and establishes new hypotheses for future experimental tests.

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The evolutionary options of a population are strongly influenced by the avail- ability of adaptive mutants. In this thesis, I use the concept of neutral networks to show that neutral drift can actually increase the accessibility of adaptive mu- tants, and therefore facilitate adaptive evolutionary change. Neutral networks are groups of unique genotypes which all code for the same phenotype, and are connected by simple point mutations. I calculate the size and shape of the networks in a small but exhaustively enumerated space of RNA genotypes by mapping the sequences to RNA secondary structure phenotypes. The qual- itative results are similar to those seen in many other genotype–phenotype map models, despite some significant methodological differences. I show that the boundary of each network has single point–mutation connections to many more phenotypes than the average individual genotype within that network. This means that paths involving a series of neutral point–mutation steps across a network can allow evolution to adaptive phenotypes which would otherwise be extremely unlikely to arise spontaneously. This can be likened to walking along a flat ridge in an adaptive landscape, rather than traversing or jumping across a lower fitness valley. Within this model, when a genotype is made up of just 10 bases, the mean neutral path length is 1.88 point mutations. Furthermore, the map includes some networks that are so convoluted that the path through the network is longer than the direct route between two sequences. A minimum length adaptive walk across the genotype space usually takes as many neutral steps as adaptive ones on its way to the optimum phenotype. Finally I show that the shape of a network can have a very important affect on the number of generations it takes a population to drift across it, and that the more routes between two sequences, the fewer generations required for a population to find an advantageous sequence. My conclusion is that, within the RNA map at least, the size, shape and connectivity of neutral networks all have a profound effect on the way that sequences change and populations evolve, and by not considering them, we risk missing an important evolutionary mechanism.

Directed evolution is generally regarded as a useful approach in protein engineering. By subjecting members of a mutant library to the power of Darwinian evolution, desired protein properties are obtained. Numerous reports have appeared in the literature showing the success of tailoring proteins for various applications by this method. Is it a one-way track that protein practitioners can only learn from nature to enable more efficient protein engineering? A structure-and-mechanism-based approach, supplemented with the use of reduced amino acid alphabets, was proposed as a general means for semi-rational enzyme engineering. Using human GST A2-2*E, the most active human enzyme in the bioactivation of azathioprine, as a parental enzyme to test this approach, a L107G/L108D/F222H triple-point mutant of GST A2-2*E (thereafter designated as GDH) was discovered with 70-fold increased activity, approaching the upper limit of specific activity of the GST scaffold. The approach was further experimentally verified to be more successful than intuitively choosing active-site residues in proximity to the bound substrate for the improvement of enzyme performance. By constructing all intermediates along all putative mutational paths leading from GST A2-2*E to mutant GDH and assaying them with nine alternative substrates, the fitness landscapes were found to be “rugged” in differential fashions in substrate-activity space. The multidimensional fitness landscapes stemming from functional promiscuity can lead to alternative outcomes with enzymes optimized for other features than the selectable markers that were relevant at the origin of the evolutionary process. The results in this thesis suggest that in this manner an evolutionary response to changing environmental conditions can readily be mounted. In summary, the thesis demonstrates the attractive features of the structure-and-mechanism-based semi-rational directed evolution approach for optimizing enzyme performance. Moreover, the results gained from the studies show that laboratory evolution may refine our understanding of evolutionary process in nature.

Immune activation is generally acknowledged to be costly. These costs are frequently assumed to result from trade-offs arising due to the reallocation of resources from other life-history traits to be invested in immunity. Here, I investigated the energetic basis of the costs associated with immune activation in Drosophila melanogaster. I found that immune activation significantly reduced fly fecundity (45%) and also caused a decline in metabolic rate (6%) but had no effect on body weight. To understand the factors behind reduced fecundity and metabolic rate I measured feeding and found that food intake was reduced by almost 31% in immune-challenged D. melanogaster. These findings suggest that fecundity costs of immune activation result not from the commonly accepted resource reallocation hypothesis but probably because resource acquisition is impaired during immune responses. The individuals of any animal population generally vary greatly in their ability to resist infectious disease. This variation arises due to both environmental heterogeneity and genetic diversity. Genetic variation in disease susceptibility has generally been considered to lie in the nuclear genome. Here, for the first time, I explored the influence of mitochondrial genetic (mtDNA) variation on disease susceptibility. I crossed 22 mitochondrial haplotypes onto a single nuclear genome and also studied epistasis interactions between mitochondrial and nuclear genomes (mitonuclear epistasis) by crossing five haplotypes onto five different genetic backgrounds. I found that fly susceptibility to Serratia marcescens was influenced significantly by mtDNA allelic variation. Furthermore, the effect of mitonuclear epistasis on fly susceptibility to S. marcescens was twice as great as the individual effects of either mitochondrial or nuclear genome. However, susceptibility to Beauveria bassiana was not affected by mtDNA allelic variation. These findings suggest the mitochondrial genome may play an important role in host-parasite coevolution. The Mother’s Curse hypothesis suggests that sex-specific selection due to maternal mitochondrial inheritance means that mitochondria are poorly adapted to function in males, resulting in impaired male fitness. Mother’s Curse effects have previously only been studied for two phenotypic traits (sperm-infertility and ageing) and their generality for broader life-history has not been explored. I investigated the impact of mtDNA allelic variation on 10 phenotypic traits and tested whether the patterns of phenotypic variation in males and females conformed to the expectations of the Mother’s Curse hypothesis. I found that seven of the 10 traits were significantly influenced by mtDNA allelic variation. However, there was no evidence that the effects of this variation differed between males and females. I therefore concluded that Mother’s Curse is unlikely to be a general phenomenon, nor to provide a general explanation for sexual dimorphism in life-history traits. Overall, this thesis explored the impacts of immunity costs, mitochondrial genetic variation, mitonuclear epistasis and sex-specific mitochondrial selection on D. melanogaster life-history.

Submitted by (ana.araujo@ufrpe.br) on 2016-07-05T14:12:49Z No. of bitstreams: 1 Elias Dias Coelho Neto.pdf: 2570519 bytes, checksum: 45c9ec25f4446657c86b65924c054a2d (MD5) / Made available in DSpace on 2016-07-05T14:12:49Z (GMT). No. of bitstreams: 1 Elias Dias Coelho Neto.pdf: 2570519 bytes, checksum: 45c9ec25f4446657c86b65924c054a2d (MD5) Previous issue date: 2012-12-20 / The rate at which species accumulate with increased sample size - the species-area relationship - is one of the most basic and fundamental problem in biogeography. This relationship has profound significance in understanding the generation and maintenance of biodiversity in an ecosystem. In this sense, this thesis introduces two models of community dynamics within two different frameworks: The first investigate the spatial patterns of species distribution in fragmented landscapes within the framework of the neutral theory. In the second approach the spatial patterns of species distribution on the genetic variability is studied under the assumption that natural selection has a prominent role in driving the evolution of such populations. Additionally, the model assumes that the environment is heterogeneous, such that the strength of natural selection depends on the localization of the species in the lattice. Our results for the neutral community model show that fragmentation has an important influence in shaping the specie-area relationship. In particular, the level of fragmentation than changes the size of the area interval when the species-area relation is well described by a power-law, S ∼ Az. We also investigate the biodiversity on the percolating cluster. In the non-neutral model our simulation results demonstrate that the level of heterogeneity of the environment affects the shape of the genetic-area relationships. But it is possible to recover the triphasic scenario for low and intermediate level of heterogeneity. / A taxa com que as espécies acumulam com o crescimento da área de amostragem - a relação espécie-área - é um dos problemas mais básicos e fundamentais em biogeografia. Esta relação tem significado profundo na compreensão da geração e manutenção da biodiversidade no ambiente. Nesse sentido, nesta tese introduzimos dois modelos para populações de organismos que interagem em ambiente finito e saturado: O primeiro para populações que sofrem variação em suas abundâncias de forma nula - modelo neutro; o segundo, as populações estão sob seleção natural e variabilidade genética - modelo não neutro. Em ambas as abordagens, para caracterizarmos a relação espécie-área, realizamos simulações computacionais para gerar diversidade de espécies em comunidades em equilíbrio. Na abordagem neutra utilizamos o método da coalescência na versão estendida a habitats fragmentados. Enquanto que na abordagem com seleção natural utilizamos o modelo NK para o ambiente com níveis de heterogeneidade entre habitats controlados pelo parâmetro λ . Nossos resultados para comunidade neutra mostram que o aumento da fragmentação do habitat influência o padrão da curva espécie-área, principalmente em áreas pequenas e intermediárias, aonde ocorre o encurtamento do comprimento do intervalo de áreas em que o regime de lei de potências é verificado. Nós notamos também que um pequeno valor da taxa de especiação ν , o expoente z da relação espécie-área se eleva com o crescimento da fragmentação. Por outro lado, quando pressão de seleção é considerada, o parâmetro de correlação λ também exerce uma importante influência sobre a formação do tamanho do regime intermediário da relação espécie-área, que decresce com o aumento do nível de correlação entre habitats. Quanto maior for a epistasia, mais pronunciado é esse efeito.