In Nature, antibodies are capable of recognizing a huge variety of different molecular structures on the surface of antigens. The primary factor that defines the structural diversity of the antibody antigen combining site is the length variation of the complementarity determining region (CDR) loops. Following antigen stimulation, further diversification through the process called somatic hypermutation (SHM) leads to antibodies with improved affinity and specificity. Sequence diversification by SHM is mainly achieved by introduction of point substitutions and a small percentage of insertions/deletions (indels). Although the percentage of indels in affinity matured antibodies is low, probably due to the low rate incorporation of in-frame indels throughout the course of the SHM diversification process, it is likely that the antibody fold can accommodate higher diversity of affinity-enhancing indels. By in vitro evolution, other researchers have sampled either only restricted diversity of indels or extended diversity of insertions only in specific positions chosen based on structural information and natural length variation. The aim of this thesis was to study the impact of random and high diversity indels on antibody affinity by in vitro evolution. New approaches for construction of libraries with in-frame amino acid indels were applied to enable sampling of indels of different lengths across the entire antibody variable domains. I followed two different approaches for construction of indel libraries. Firstly, a recently developed random approach allowed the construction of libraries with random insertions and deletions. Secondly, a semi-random approach was developed to build libraries with different lengths of insertions that could be widely applied in future in vitro antibody affinity maturation campaigns. Libraries constructed by either of these approaches yielded variants with insertions with improved affinity. Overall, this thesis demonstrates that insertions besides offering alternative routes to affinity maturation can also be combined with point substitutions to take advantage of additive effects on function.

In wheat (Triticum aestivum L), high molecular weight glutenin subunits (HMW -GS) are synthesized by the loci Glu-A1, Glu-B1, and Glu-D1 on the long arm of group 1 chromosome, and their variants play a significant role in the functional properties of flour; hence dough properties and tortilla quality. This study was conducted to understand the effects of HMW-GS on dough properties and tortilla quality using 40 different wheat lines from two different locations; Texas Agrilife Experiment Station at McGregor, and at Castroville, Texas, in 2010. Wheat lines in which one or more of these loci were absent (deletion lines) and non-deletion lines were used. Flours were evaluated for insoluble polymeric protein (IPP) content and mixograph properties. Dough properties; compression force, stress relaxation test, and dough extensibility, were determined using a texture analyzer. Tortillas were produced by hot-pressed method and evaluated for physical properties and textural change during 16 days of storage. Flour from deletion lines had lower average IPP content (38.4%) than non-deletion lines (41.9%). Dough from deletion lines were more extensible (44.8 mm) and required lower equilibrium force from stress relaxation test (4.91 N) compared to non-deletion lines (34.2 mm, and 6.56 N, respectively). Deletion lines produced larger diameter tortillas (177 mm) than non-deletion lines (165 mm) and had lighter color (L* = 82.3) than tortillas from non-deletion lines (L* = 81.0). Most of the deletion lines interestingly produced tortillas with acceptable flexibility scores on day 16 of storage (>= 3.0). Flour IPP content (r = -0.57) and equilibrium force (r = -0.80) were negatively correlated with tortilla diameter, but positively correlated with 16 day flexibility scores (r = 0.72, and r = 0.68, respectively). In general, deletion at Glu-A1 or Glu-D1 or presence of 2+12 instead of 5+10 allelic pair at Glu-D1 locus produced large diameter tortillas, but with poor day 16 flexibility. However, combination of 7+9 at Glu-B1 locus with deletions at Glu-A1 or Glu-D1 or 2+12 at Glu-D1 consistently produced tortillas that had large diameter and retained good flexibility scores during 16 days of storage. The results indicate the presence of 7+9 at Glu-B1 may play a crucial role in selection of wheat varieties for tortilla making.

Little is known about dosage compensation in autosomal genes. Transcription-level compensation of deletions and other loss-of-function mutations may be a mechanism of dominance of wild-type alleles, a ubiquitous phenomenon whose nature has been a subject of a long debate. We measured gene expression in two isogenic Drosophila lines heterozygous for long deletions and compared our results with previously published gene expression data in a line heterozygous for a long duplication. We find that a majority of genes are at least partially compensated at transcription, both for 1/2-fold dosage (in heterozygotes for deletions) and for 1.5-fold dosage (in heterozygotes for a duplication). The degree of compensation does not vary among functional classes of genes. Compensation for deletions is stronger for highly expressed genes. In contrast, the degree of compensation for duplications is stronger for weakly expressed genes. Thus, partial transcriptional compensation appears to be based on regulatory mechanisms that insure high transcription levels of some genes and low transcription levels of other genes, instead of precise maintenance of a particular homeostatic expression level. Given the ubiquity of transcriptional compensation, dominance of wild-type alleles may be at least partially caused by of the regulation at transcription level.

No description available.

<p>For the purpose of this descriptive translation study, a translation corpus was built from roughly the first 20,000 words of each of the first four Harry Potter books by J.K. Rowling, and their respective translations into Swedish. I*Link, a new type of word alignment tool, was used to align the samples on a word level and to investigate and analyse the aligned corpus. The purpose of the study was threefold: to investigate manifestations of translation universals, to search for evidence of translator development and to study the efficiency of different strategies for using the alignment tools.</p><p>The results show that all three translation universals were manifested in the corpus, both on a general pattern level and on a more specific lexical level. Additionally, a clear pattern of translator development was discovered, showing that there are differences between the four different samples. The tendency is that the translations become further removed from the original texts, and this difference occurs homogeneously and sequentially. In the word alignment, four different ways of using the tools were tested, and one strategy was found to be more efficient than the others. This strategy uses dynamic resources from previous alignment sessions as input to I*Trix, an automatic alignment tool, and the output file is manually post-edited in I*Link.</p><p>In conclusion, the study shows how new tools and methods can be used in descriptive translation studies to extract information that is not readily obtainable with traditional tools and methods.</p>

For the purpose of this descriptive translation study, a translation corpus was built from roughly the first 20,000 words of each of the first four Harry Potter books by J.K. Rowling, and their respective translations into Swedish. I*Link, a new type of word alignment tool, was used to align the samples on a word level and to investigate and analyse the aligned corpus. The purpose of the study was threefold: to investigate manifestations of translation universals, to search for evidence of translator development and to study the efficiency of different strategies for using the alignment tools. The results show that all three translation universals were manifested in the corpus, both on a general pattern level and on a more specific lexical level. Additionally, a clear pattern of translator development was discovered, showing that there are differences between the four different samples. The tendency is that the translations become further removed from the original texts, and this difference occurs homogeneously and sequentially. In the word alignment, four different ways of using the tools were tested, and one strategy was found to be more efficient than the others. This strategy uses dynamic resources from previous alignment sessions as input to I*Trix, an automatic alignment tool, and the output file is manually post-edited in I*Link. In conclusion, the study shows how new tools and methods can be used in descriptive translation studies to extract information that is not readily obtainable with traditional tools and methods.

One in six couples wishing to start a family are infertile. The many causes of infertility include genetic defects that can be single gene, multifactorial or chromosomal (including Y deletions, karyotype abnormalities and gamete aneuploidy). This thesis is concerned with the association between infertility and increased sperm aneuploidy. Specific questions are: should males be screened for sperm aneuploidy before intracytoplasmic sperm injection (ICSI)? Is there a relationship between individual semen parameters and sperm aneuploidy for specific chromosome pairs? What is the role of genome organisation in male gametes and its association with infertility? Whether use of alternative therapy (in this case, traditional Chinese Medicine (TCM)) can be used to improve sperm disomy levels. Statistical analysis of questionnaire data revealed that infertility specialists believed there to be merit in screening sperm aneuploidy levels before ICSI. Evidence is presented for possible chromosome-specific and semen parameter specific mechanisms for sperm aneuploidy as is evidence of genome organisation that may be perturbed in infertile males. Finally, in six males studied, sperm aneuploidy levels improved significantly coincident with TCM. Closer investigation of the biological activity of individual therapeutic herbs and treatment cocktails revealed strong anti-oestrogenic and anti-oxidant properties. This suggests a possible mechanism of action of the herbs and provides the basis from which future placebo controlled clinical trials might continue. Possible criticisms of the work presented here include the unavailability of blood samples from many of the patients (thus preventing karyotype analysis) and the absence of a second control group in our studies on semen parameters. Nevertheless significant steps have been made towards establishing the need for, and the implementation of, a pre-ICSI screening test. Moreover progress has been made towards further understanding the aetiology of sperm aneuploidy and towards the implementation of a new treatment that may, ultimately, augment, or even replace ICSI.

Cette thèse porte sur l'histoire évolutive des gènes de vertébrés. Deux types de phénomènes évolutifs peuvent perturber l’organisation des gènes dans les génomes eucaryotes : les modifications du contenu en gènes des génomes par duplications ou délétions de gènes, et les changements dans l’ordre des gènes par réarrangements. L’impact fonctionnel et sélectif de ces processus sur les génomes est encore mal connu.Ce travail de thèse s’articule autour de trois projets portant sur les événements de duplication et de délétion qui modifient le nombre de copies d'un gène. Nous nous sommes intéressés à ces événements à partir d’arbres phylogénétiques reconstruits à l’échelle de génomes entiers. Dans une première partie nous avons examiné le cas où ces événements seraient sous sélection négative, en étudiant les arbres de gènes ou aucune duplication ou délétion ne s'est fixée. Nous avons observé que ces gènes avaient des propriétés particulières et nous proposons des hypothèses pour les expliquer. Dans une deuxième partie nous nous sommes intéressés aux duplications de gènes et aux corrélations que l'on observe avec l'évolution des fonctions biologiques. Enfin en dernière partie nous avons étudié en détail la famille de gènes ROBO dont une des copies aurait acquis une fonction différente dans le développement du système nerveux des mammifères sous l’influence de la sélection positive. Dans leur ensemble ces résultats apportent de nouveaux éléments pour mesurer et comprendre l’impact global des contraintes ou des avantages que les duplications de gènes en particulier, et le changement du nombre de copies des gènes en général, peuvent exercer sur un génome de vertébré. / This thesis is about the evolutionary history of vertebrates genes. Two categories of evolutionary processes can disrupt the gene organization in eukaryotic genomes: changes in the content of genomes by gene duplications or gene deletions, and changes in the order of the genes by rearrangements. Functional and selective impacts of these processes on genomes are poorly understood.This thesis covers three different projects about duplication and deletion events that change the number of gene copies. We were interested in these events from phylogenetic trees reconstructed at the scale of whole genomes. In the first part we examined the case where these events would be under negative selection, by studying phylogenetic gene trees where no duplication or deletion was fixed. We found that these genes have special properties and propose hypotheses to explain them. In the second part we looked at gene duplications and correlated these events with the evolution of biological functions. Finally in the last part we have studied in detail the ROBO genes family in which one copy has acquired a different function in the developing nervous system of mammals under the influence of positive selection.Taken together these results provide new elements to measure and understand the global impact of constraints or advantages that gene duplications in particular, and the change of genes copy number in general, can have on a vertebrate genome.

Bien que de nombreuses séquences génomiques soient maintenant connues, les mécanismes évolutifs qui déterminent la taille des génomes, et notamment leur part d’ADN non codant, sont encore débattus. Ainsi, alors que de nombreux mécanismes faisant grandir les génomes (prolifération d’éléments transposables, création de nouveaux gènes par duplication, ...) sont clairement identifiés, les mécanismes limitant la taille des génomes sont moins bien établis. La sélection darwinienne pourrait directement défavoriser les génomes les moins compacts, sous l’hypothèse qu’une grande quantité d’ADN à répliquer limite la vitesse de reproduction de l’organisme. Cette hypothèse étant cependant contredite par plusieurs jeux de données, d’autres mécanismes non sélectifs ont été proposés, comme la dérive génétique et/ou un biais mutationnel rendant les petites délétions d’ADN plus fréquentes que les petites insertions. Dans ce manuscrit, nous montrons à l’aide d’un modèle matriciel de population que la taille du génome peut aussi être limitée par la dynamique spontanée des duplications et des grandes délétions, qui tend à raccourcir les génomes même si les deux types de réarrangements se produisent à la même fréquence. En l’absence de sélection darwinienne, nous prouvons l’existence d’une distribution stationnaire pour la taille du génome même si les duplications sont deux fois plus fréquentes que les délétions. Pour tester si la sélection darwinienne peut contrecarrer cette dynamique spontanée, nous simulons numériquement le modèle en choisissant une fonction de fitness qui favorise directement les génomes contenant le plus de gènes, tout en conservant des duplications deux fois plus fréquentes que les délétions. Dans ce scénario où tout semblait pousser les génomes à grandir infiniment, la taille du génome reste pourtant bornée. Ainsi, notre étude révèle une nouvelle force susceptible de limiter la croissance des génomes. En mettant en évidence des comportements contre-intuitifs dans un modèle pourtant minimaliste, cette étude souligne aussi les limites de la simple « expérience de pensée » pour penser l’évolution. / Even though numerous genome sequences are now available, evolutionary mechanisms that determine genome size, notably their fraction of non-coding DNA, are still debated. In particular, although several mechanisms responsible for genome growth (proliferation of transposable elements, gene duplication and divergence, etc.) were clearly identified, mechanisms limiting the overall genome size remain unclear. Darwinian selection could directly disadvantage less compact genomes, under the hypothesis that a larger quantity of DNA could slow down the speed of reproduction of the organism. Because this hypothesis was proven wrong by several datasets, non selective mechanisms have been proposed, e.g. genetic drift and/or a mutational bias towards small DNA deletions compared to small DNA insertions. In this manuscript, we use a matrix model to show that genome size can also be limited by the spontaneous dynamics of duplications and large deletions, which tends to decrease genome size even if the two types of rearrangements occur at the same rate. In the absence of Darwinian selection, we prove the existence of a stationary distribution of genome size even if duplications are twice as frequent as large deletions. To test whether selection can overcome this spontaneous dynamics, we simulate our model numerically and choose a fitness function that directly favors genomes containing more genes, while keeping duplications twice as frequent as large deletions. In this scenario where, at first sight, everything seems to favor infinite genome growth, genome size remains nonetheless bounded. As a result, our study reveals a new pressure that could be responsible for limiting genome growth. By illustrating counter-intuitive behaviors in a minimal model, this study also underlines the limits of simple "thought experiments" to understand evolution.

Le récent développement des méthodes de séquençage permet aujourd’hui d’identifier des variations structurales chez de nombreuses espèces. Chez le maïs, des milliers de grandes insertions et délétions (InDel) de quelques pb à plusieurs centaines de Kbp ont été découvertes entre le génome de référence B73 et de nombreux autres génomes reséquencés. Ces InDel peuvent changer la composition des gènes entre les individus et donc être impliquées dans la variation du phénotype, mais cet effet sur le phénotype reste mal connu. L’objectif de cette thèse était d'étudier la contribution des InDel à l'adaptation, aux variations phénotypiques et aux performances hybrides chez le maïs. Nous avons développé une puce de génotypage des InDel Affymetrix® Axiom® capable de génotyper 105 927 InDel de 35bp à 129,7Kbp. 79 969 de ces InDel ont leur séquences absentes du génome de référence B73 et ont été identifiées par l’assemblage 3 génomes (F2, C103, and PH207). Nous avons sélectionné 61 492 InDel polymorphiques pour génotyper 362 lignées de maïs représentant une large gamme de diversité pour étudier la contribution des InDel à la diversité génétique, l’adaptation et la variation des caractères. Nous avons également génotypé 1 million de SNP à partir de deux puces de génotypage et du génotypage par séquençage pour étudier la complémentarité entre les InDel et les SNP. Qu’ils soient calculés avec les InDel ou les SNP la structuration génétique et les valeurs d’apparentement entre les lignées sont très similaires, ce qui suggère que la plupart des InDel ont suivi la même trajectoire évolutive que les SNP. 51% des InDel ne sont pas en déséquilibre de liaison élevé (>0.8) avec aucun SNP proche donc l’effet de ces InDel n’est donc a priori pas capturé pas des SNP à cette densité. Parmi les 294 régions génomiques associées au phénotype (QTL), 13 nouveaux QTL ont été détectés grâce aux InDel par rapport aux SNP par une approche de génétique d’association (GA). Nous avons détecté un enrichissement en InDel sous sélection entre les lignées tropicales, cornées et dentées par rapport aux SNP, avec 56 sur 188 régions sous sélection détectées avec les InDel. Ces régions contiennent des gènes impliqués dans l’adaptation et/ou la tolérance aux stress. De plus, le plus grand nombre d’associations a été découvert pour la floraison, caractère adaptatif chez le maïs. Ces résultats suggèrent que les InDel sont plus souvent impliquées dans l’adaptation et la tolérance aux stress. Nous avons enfin testé l’effet des InDel sur les performances hybride en analysant un panel de 287 hybrides issus du croisement de 210 lignées tempérées du panel précédent. Nous avons décomposé la variance des performances hybrides en distinguant les effets de dominance et d’additivité pour la floraison femelle (FF), la hauteur (PH) et le rendement (GY). La plus forte part de dominance et d’interaction génotype-environnement a été observée pour le GY et la plus faible pour la FF. L’effet additif et de dominance de 51,844 InDel et 469 267 SNP a été testé pour 4 combinaisons d’environnements par une approche de GA. 78 et 133 QTL avec un effet additif et dominant respectivement ont été identifiés, dont 6 et 11 avec des InDel. 83% de ces QTL ont été identifiés dans une seule combinaison d’environnements. Un des QTL de rendement identifié avec des InDel est situé dans un large cluster d’InDel sur le chromosome 6 et colocalise avec un QTL déjà identifié avec des SNP avec un effet fort dans l’augmentation du rendement sous des températures élevées. L’ajout de l’effet de dominance en plus de l’effet additif permet d’augmenter la précision des prédictions génomiques jusqu’à 5,6% pour le rendement. Cependant, l’ajout du génotypage des InDel en plus de celui des SNP n’a pas permis d’améliorer les prédictions des phénotypes hybrides. / In the last decades, the rapid development of genome sequencing allowed to identify structural variations in many species. In maize, thousands of large insertions and deletions (InDels) from few bp to hundreds of Kbp were discovered by comparing the reference genome B73 and many other resequenced genomes. These InDel sequences can carry genes and therefore be involved in phenotypic variation by changing the gene composition between individuals, but their effect on phenotype was not well studied. The aim of this thesis was to study the contribution of InDels to adaptation, phenotypic variations and hybrid performances in maize. We developed an Affymetrix® Axiom® genotyping array that allowed to genotype 105,947 InDels sequences ranging from 35bp to 129,7Kbp of size. 79,969 out 105,947 sequences of these InDels were not present in B73 reference genome and have been discovered by assembling three genomes (F2, C103, and PH207). We selected 61,492 polymorphic InDels to genotype a 362 maize inbred lines panel representing a broad range of diversity to study the contribution of InDels to genetic diversity, adaptation and trait variation. We also assembled one million of SNPs from two genotyping arrays and genotyping by sequencing to study the complementarity between InDels and SNPs. Genetic structuration and relatedness between inbred lines displayed by SNPs or by InDels were highly similar suggesting that almost all indels and SNPs followed a similar evolutionary trajectory. 51% of InDels were not in high linkage disequilibrium (LD>0.8) with any nearby SNP suggesting that the effect of these InDels was not be well captured using this density of SNP. Thanks to InDels, we detected 13 new quantitative trait loci (QTLs) among 294 QTLs identified for 23 traits by a genome wide association studies (GWAS). Similarly, 56 out 188 regions under selection between tropical, dent and flint maize lines were identified by InDels leading to an enrichment of genomic regions under selection detected by InDels compared to SNPs. These InDels include genes involved in tolerance to biotic and abiotic stress and/or adaptive traits as flowering time. Accordingly, the highest number of associated InDels was found for flowering time. These results suggest that InDels were often involved in adaptation and stress tolerance. In order to study the effect of InDels on hybrid performances, we analyzed a panel of 287 hybrids derived from the crossing of 210 maize temperate inbred lines from the previous panel. We decomposed the variance of female flowering (FF), plant height (PH) and grain yield (GY) by distinguishing the additive and dominant genetic effects. We observed the highest dominance and genotype by environment effects for GY and the lowest for FF. We performed GWAS on this panel by testing additive and dominance effects of 51,844 InDels and 469,267 SNPs on these three traits in 4 different environment combinations. We identified 78 and 133 QTLs with an additive and dominance effect, respectively including 6 and 11 QTLs discovered only by InDels. 83% of all QTLs were found with only one environment combination. One QTL for GY detected with InDels was located in a large cluster of InDels on chromosome 6, previously identified to have a strong effect on GY in heat conditions. We finally used InDels and/or SNPs genotyping to predict hybrid performances. Whereas including a dominance effect in genomic prediction models increased by 1.5 to 5.6% predictive abilities (PA) for GY, including InDels genotyping did not increased PA.