A subgroup of non-small cell lung cancer (NSCLC) cases harbour mutations in classical oncogenes, which can affect therapy response and prognosis. By therapeutically targeting the corresponding proteins with inhibitory drugs, the clinical outcome for these lung cancer patients may be improved. One of these oncogenes is the phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) which encodes the catalytic subunit of the phosphatidylinositol 3 kinase (PI3K). PIK3CA is a central regulator in the PI3K/Akt/mTOR pathway, which controls cell growth and apoptosis. Mutations in the PIK3CA gene are considered to up-regulate the kinase activity in tumour cells and through that dysregulate fundamental cellular processes. PI3K inhibitors are currently tested in clinical trials and present a promising therapy option in lung cancer patients. In this study, a pyrosequencing assay for detection of PIK3CA mutations in tumours from patients with NSCLC was established. The three "hot-spot" codons 542, 545 and 1047 of the PIK3CA gene were analysed. The sensitivity of this assay was determined to the presence of 5 % of mutant alleles. In agreement with previous reports, three of the 60 lung cancer cases revealed PIK3CA mutations (5 %). All mutations occurred in exon 9 codon 542 or 545. In line with previous reports, two of the three samples harboured concurrent mutation in the EGFR or KRAS gene. The established pyrosequencing analysis for PI3KCA mutations provides a reliable and cost-effective assay for clinical diagnostics. The determination of the PI3KCA mutation status may help to distinguish patients for treatment targeting the PI3K pathway.

The pathogen C. jejuni is now recognised as the leading cause of bacterial foodborne enteritis in the industrial world. The yearly estimate for Campylobacter infections in the United States alone is 2.4 million people or 1% of the population. Illness caused by C. jejuni is self-limiting, however, some individuals develop complications resulting in autoimmune responses. Despite being a major health burden, the pathogenic process is not fully understood. One aspect of importance is the ability of C. jejuni to adhere to glycosaminoglycans (GAGs), such as heparin. GAGs, sulphated carbohydrates expressed on or in host cells, can serve as receptors for bacterial proteins. In the first study, five heparin-binding proteins of C. jejuni NCTC 11168H were identified. For PEB3 (Cj0289c), this work showed that native wild-type PEB3 and purified recombinant PEB3 produced in E. coli bind heparin. The location of two PEB3 heparin-binding clusters: 62KAKKD65 and 122NKKVRI127, was investigated via site-directed mutagenesis, resulting in impaired heparin-binding. These data suggest GAG-protein-binding may play a role in the pathogenesis of C. jejuni. As well as GAG-binding PEB3 binds 3-PG. Though its exact in vivo role remains unclear, it may act to deliver 3-PG. Scrutiny of the C. jejuni NCTC 11168H genome revealed an uncharacterised gene next to peb3 encoding glpT, or a putative 3-PG transporter. The location of glpT adjacent to peb3 may suggest a related function for the corresponding proteins with PEB3 as the periplasmic binding partner for the transport of 3-PG via GlpT. In this thesis, the roles of peb3 and glpT for two independent phenotypes, 3-PG dependent growth and fosfomycin sensitivity was studied in vitro. The findings indicate glpT has an effect on both, but not peb3. Furthermore, the NCTC 11168H glpT pseudogene, despite containing two frameshift mutations, has the capacity to encode a functional protein. Lastly, the NCTC 11168H peb3/glpT locus was compared with other C. jejuni strains and closely related species C. coli, C. lari and C. upsaliensis genome sequences. The majority of strains peb3/glpT locus followed the gene arrangement lpxB, peb3, glpT, surE. However, the findings indicate the gene loci between lpxB/surE in remaining strains to be hypervariable. Further analysis shows peb3 to be relatively conserved, whereas, the majority of glpT genes display genetic diversity due to interruptions such as indels and deletion. Lastly, I display the organisation of the peb3/glpT locus and glpT structure in their evolutionary context through MLST. In summary, the findings provide for further characterisation of the PEB3 protein and explores the importance of the uncharacterised GlpT of C. jejuni.

Two different cloned human DNA segments encompassing transfer RNA gene and pseudogene clusters have been isolated from a human gene library harbored in bacteriophage lambda Charon 4-A. One clone (designated as λhVal7) encompassing a 20.5-kilobase (Kb) human DNA insert was found to contain a valine transfer RNA_AAC gene and several Alu-like elements by Southern blot hybridization analysis and DNA sequencing with the dideoxyribonucleotide chain-termination method in the bacteriophage M13mp19 vector. Another lambda clone (designated as λhLeu8) encompassing a 14.3-Kb segment of human DNA was found to contain a methionine elongator transfer RNA_CAT pseudogene and other as yet unidentified transfer RNA pseudogenes.

The need for a more reliable method to select beef heifers to retain as replacement dams has become a concern in the beef industry. Two polymorphisms described in leptin (LEP), p.Arg25Cys, and pro-melanin concentrating hormone (PMCH), g.-134A>T, have already been shown to improve carcass quality in beef cattle. This study was designed to evaluate any additional advantages of these polymorphisms in terms of heifer conception and calving success and lactational milk yield measured indirectly by their calves’ early ADG while they were primarily on lactation. A dominant effect of the dam’s PMCH T allele was observed on improved calf early ADGs in Simmental heifer dams, although not in Angus heifer dams. This effect could be useful in cow-calf operations where calves were suckling their dams for a longer period before sending the cow-calf pairs out to summer pasture. The dam LEP genotype did not show an effect on their calves’ ADG. This was assumed to be due to low body fat reserves of the heifer dams at the age of two years, allowing for only low levels of leptin. Even though heifer conception was not affected by their LEP and PMCH genotypes, it would be worth evaluating their rebreeding success in the presence of these SNPs in the future. The Integral Membrane Protein 2A (ITM2A) was hypothesized as a candidate gene for frame size in cattle. DNA fragments from 20 cattle, matching the predicted exons of the cattle ITM2A gene, were sequenced to determine whether genetic variation existed. However, the sequence obtained based on the predicted cattle ITM2A sequences appeared to be a pseudogene, rather than the actual cattle ITM2A gene, because exons 1, 2, 3 and 5 contained stop codons. Since frame size has been reported to be associated with the reproductive performance of beef dams and their calves’ growth characteristics, it would be useful to characterize this gene once an improved cattle genome assembly is available.

Gene loss is the obverse of novel gene acquisition by a genome through a variety of evolutionary processes. It serves a number of functional and structural roles, compensating for the energy and material costs of gene complement expansion. A type of gene loss widespread in the lineages of plant genomes is ``fractionation" after whole genome doubling or tripling, where one of a pair or triplet of paralogous genes in parallel syntenic contexts is discarded. Based on previous mathematical work on the distribution of gap sizes caused by fractionation in synteny blocks, we studied fractionation in the evolutionary history of the allotetraploid Coffea arabica (CA) and its two diploid progenitors, C. canephora (CC) and C. eugenioides (CE), annotated genome assemblies being provided by the Arabica Coffee Genome Consortium. By taking advantage of synteny blocks produced by SynMap, we studied the fractionation process after speciation and tetraploidization events, including visualization and modelling the distribution of deletion segments, and mechanisms of deletion events. We also expanded the research to eight other plant species to verify the dominance of DNA excision over pseudigenization during the fractionation and other gene loss.

Like many angiosperms, Brassica rapa underwent several rounds of whole genome duplication during its evolutionary history. Brassica rapa is particularly valuable for studying genome evolution because it also experienced whole genome triplication shortly after it diverged from the common ancestor it shares with Arabidopsis thaliana about 17-20 million years ago. While many B. rapa genes appear resistant to paralog retention, close to 50% of B. rapa genes have retained multiple, paralogous loci for millions of years and appear to be multi-copy tolerant. Based on previous studies, gene function may contribute to the selective pressure driving certain genes back to singleton status. It is suspected that other factors, such as gene expression patterns, also play a role in determining the fate of genes following whole genome triplication. Published RNA-seq data was used to determine if gene expression patterns influence the retention of extra gene copies. It is hypothesized that retention of genes in duplicate and triplicate is more likely if those genes are expressed in a tissue-specific manner, as opposed to being expressed globally across all tissues. This study shows that genes expressed specifically in flowers and roots in B. rapa are more resistant to fractionation than globally expressed genes following whole genome triplication. In particular, there appears to have been selection on genes expressed specifically in flower tissues to retain higher copy numbers and for all three copies to exhibit the same flower-specific expression pattern. Future research to determine if these observations in Brassica rapa are consistent with other angiosperms that have undergone recent whole genome duplication would confirm that retention of flower-specific-expressed genes is a general feature in plant genome evolution and not specific to B. rapa.

La bactérie Salmonella enterica sérovar Typhi (S. Typhi) provoque la fièvre typhoïde chez les humains et constitue un problème de santé publique important. La majorité de nos connaissances sur la pathogenèse de cette bactérie provient du modèle de fièvre entérique chez la souris causée par le sérovar Typhimurium. Peu d’études se sont penchées sur les facteurs de virulence uniques au sérovar Typhi, ni sur la possibilité que les pseudogènes retrouvés dans son génome puissent être fonctionnels. Le fimbria stg, unique au sérovar Typhi, renferme un codon d’arrêt TAA prématuré dans le gène stgC qui code pour le placier responsable de l’assemblage des sous-unités fimbriaires à la surface de la bactérie. Ainsi, le fimbria stg a été classifié dans la liste des pseudogènes non-fonctionnels. Les objectifs de cette étude étaient d’évaluer l’implication du fimbria stg lors de l’interaction avec les cellules humaines, puis de vérifier l’importance du pseudogène stgC lors de la biogenèse fimbriaire. Dans une première partie, la transcription de stg a été évaluée à l’aide d’une fusion lacZ. Malgré des niveaux d’expression observés généralement faibles en milieu riche, la croissance en milieu minimal a favorisé la transcription de l’opéron. La délétion complète de l’opéron fimbriaire stgABCD du génome de S. Typhi a été réalisée par échange allélique, puis a été complémentée sur un plasmide. Il a été démontré que la présence de stg chez S. Typhi, S. Typhimurium et E. coli contribue à une adhérence accrue sur les cellules épithéliales humaines. De plus, ce fimbria semble agir comme une structure anti-phagocytaire lors de l’interaction avec des macrophages humains. Ainsi, l’opéron stg semble fonctionnel, malgré son codon d’arrêt prématuré, puisque des phénotypes ont été observés. La seconde partie de cette étude consistait à vérifier le rôle joué par le pseudogène stgC dans la biogenèse du fimbria. Différentes variantes de l’opéron ont été générées, clonées dans un vecteur inductible à l’arabinose, puis transformées dans la souche afimbriaire d’E. coli ORN172. La translocation de la sous-unité fimbriaire StgD à la surface de la bactérie a été évaluée chez ces différents mutants par immunobuvardage de type Western. Cette expérience a permis de démontrer que le pseudogène stgC est essentiel pour l’exportation de la sous-unité StgD à la surface. L’ajout d’une étiquette de 6-histidines en C-terminal de StgC a permis de confirmer la traduction complète du gène, malgré le codon d’arrêt TAA prématuré. Le séquençage peptidique a révélé l’insertion d’une tyrosine à ce codon. Une fusion traductionnelle avec la protéine verte fluorescente a révélé qu’environ 0.8% de l’ARNm peut être traduit et permet la production complète du placier. Ce projet a permis la caractérisation d’un facteur de virulence unique à S. Typhi et constitue une étape de plus vers la compréhension de ses mécanismes de pathogenèse. Il s’agit de la première démonstration chez les bactéries de la fonctionnalité d’un gène interrompu prématurément par un codon d’arrêt TAA. / Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever in humans and is considered as an important health problem. Most of our knowledge on the pathogenesis of this bacterium comes from an enteric fever model in mice caused by serovar Typhimurium. Few studies have examined the virulence factors unique to serovar Typhi or the possibility that pseudogenes harbored in its genome may be functional. stg fimbriae are found only within the serovar Typhi genome and contain a premature TAA stop codon in the stgC gene encoding the usher responsible for the assembly of fimbrial subunits at the bacterial surface. Thus, the stg fimbria has been classified among the list of non-functional pseudogenes. The objectives of this study were to assess the involvement of stg fimbriae during interaction with human cells, and then to evaluate the importance of the stgC pseudogene in fimbrial biogenesis. First, stg transcription was evaluated using a lacZ fusion. Despite low expression levels generally observed in rich medium, growth in minimal medium promoted transcription of the operon. Complete deletion of the stgABCD fimbrial operon from S. Typhi was performed by allelic exchange and was complemented on a plasmid. It has been shown that the presence of stg in S. Typhi, S. Typhimurium and E. coli contributes to increased adherence to human epithelial cells. In addition, the fimbriae seem to act as an anti-phagocytic structure during the interaction with macrophages. Thus, the stg operon appears to be functional despite its premature codon, as phenotypes were observed. The second part of this study involved testing the role of the stgC pseudogene in fimbrial biogenesis. Different variants of the operon were generated, cloned into an arabinose inducible vector, and then transformed into afimbriated E. coli strain ORN172. Translocation of the StgD subunit to the cell surface of the different mutants was evaluated using Western blot. This experiment demonstrated that stgC is essential for export of the StgD subunit to the cell surface. The addition of a 6-histidine tag at the C-terminal end of StgC confirmed the complete translation of the gene, despite the premature TAA stop codon. Peptide sequencing revealed the insertion of a tyrosine at this codon. A translational fusion with the green fluorescent protein demonstrated that approximately 0.8% of the mRNA can be translated to allow full production of the usher. This project allowed characterization of a virulence factor unique to S. Typhi and is a step closer towards better understanding of its pathogenesis mechanisms. This is the first demonstration in bacteria of the functionality of a gene which is interrupted by a premature TAA stop codon.

La biologie a connu une extraordinaire révolution avec l'arrivée de nombreux génomes entièrement séquencés. L'analyse de la quantité d'informations disponibles nécessite la création et l'utilisation d'outils informatiques automatisés. L'interprétation des données biologiques prend tout son sens à la lumière de l'évolution. En ce sens, les études évolutives sont incontestablement nécessaires pour donner un sens aux données biologiques. Dans ce contexte, le laboratoire développe des outils pour étudier l'évolution des génomes (et protéomes) à travers les mutations subies. Cette thèse porte sur l'étude spécifique des événements de pertes de gènes unitaires. Ces événements peuvent révéler des pertes de fonctions très instructives pour comprendre l'évolution des espèces. En premier lieu, j'ai développé l'outil GLADX qui mime l'expertise humaine afin d'étudier automatiquement et avec précision les événements de pertes de gènes unitaires. Ces études se basent sur la création et l'interprétation de données phylogénétiques, de BLAST, de prédictions protéiques, etc., dans un contexte automatisé. Ensuite, j'ai développé une stratégie utilisant l'outil GLADX pour étudier à grande échelle les pertes de gènes unitaires au cours de l'évolution du protéome humain. La stratégie utilise d'abord comme filtre l'analyse de groupes d'orthologues fabriqués par un outil de clustérisation à partir du protéome complet de nombreuses espèces. Cette analyse a permis de détecter 6237 pertes de gènes unitaires putatives dans la lignée humaine. L'étude approfondie de ces pertes avec GLADX a mis en évidence de nombreux problèmes liés à la qualité des données disponibles dans les bases de données. / Biology has undergone an extraordinary revolution with the appearance of numerous whole genomes sequenced. Analysis of the amount of information available requires creation and use of automated tools. The interpretation of biological data becomes meaningful in light of evolution. In view of all this, evolutionary studies are undoubtedly necessary to highlight the biological data. In this context, the laboratory develops tools to study the genomes (and proteomes) evolution through all the undergone mutations. The project of this thesis focuses specifically on the events of unitary gene losses. These events may reveal loss of functions very instructive for understanding the evolution of species. First, I developed the GLADX tool that mimics human expertise to automatically and accurately investigate the events of unitary gene losses. These studies are based on the creation and interpretation of phylogenetic data, BLAST, predictions of protein, etc., in an automated environment. Secondly, I developed a strategy using GLADX tool to study, at large-scale, the loss of unitary genes during the evolution of the human proteome. The strategy uses, in the first step, the analysis of orthologous groups produced by a clustering tool from complete proteomes of numerous species. This analysis used as a filter, allowed detecting 6237 putative losses in the human lineage. The study of these unitary gene loss cases has been deepened with GLADX and allowed to highlight many problems with the quality of available data in databases.

<p>Neuropeptide Y (NPY) is an abundant neurotransmitter in the nervous system and forms a family of evolutionarily related peptides together with peptide YY (PYY), pancreatic polypeptide (PP) and polypeptide Y (PY). These peptides are ligands to a family of receptors that mediate a wide range of physiological effects including stimulation of appetite. This work describes the molecular cloning of four novel NPY receptors.</p><p>In rat a receptor called PP1, later renamed Y₄, was cloned and characterized. It displays the highest amino acid sequence identity to the Y₁ receptor. Rat Y₄ differs extensively from human Y₄, cloned subsequently, in both pharmacological properties, tissue distribution, and amino acid sequence with only 75% identity. Rat and human Y₄are the most diverged orthologues in the NPY receptor family.</p><p>In guinea pig, the y₆ receptor gene was found to be a pseudogene with several frameshift mutations. The gene is a pseudogene in human and pig too, but seems to give rise to a functional receptor in mouse and rabbit. This unusual evolutionary situa- tion may be due to inactivation of the gene in a mammalian ancestor and then restoration of expression in mouse and rabbit, but perhaps more likely due to independent inactivations in guinea pig, human and pig.</p><p>In zebrafish, two new intronless receptor genes were cloned. Sequence comparisons suggest that both receptors are distinct from the mammalian receptors Y₁, Y₄ and y₆, hence they were named Ya and Yb. Chromosomal localization provides further support that Ya and Yb may be distinct subtypes. </p><p>The discoveries of the rat Y₄ and zebrafish Ya and Yb receptors were unexpected and show that the NPY receptor family is larger than previously thought.</p>

Neuropeptide Y (NPY) is an abundant neurotransmitter in the nervous system and forms a family of evolutionarily related peptides together with peptide YY (PYY), pancreatic polypeptide (PP) and polypeptide Y (PY). These peptides are ligands to a family of receptors that mediate a wide range of physiological effects including stimulation of appetite. This work describes the molecular cloning of four novel NPY receptors. In rat a receptor called PP1, later renamed Y4, was cloned and characterized. It displays the highest amino acid sequence identity to the Y1 receptor. Rat Y4 differs extensively from human Y4, cloned subsequently, in both pharmacological properties, tissue distribution, and amino acid sequence with only 75% identity. Rat and human Y4 are the most diverged orthologues in the NPY receptor family. In guinea pig, the y6 receptor gene was found to be a pseudogene with several frameshift mutations. The gene is a pseudogene in human and pig too, but seems to give rise to a functional receptor in mouse and rabbit. This unusual evolutionary situa- tion may be due to inactivation of the gene in a mammalian ancestor and then restoration of expression in mouse and rabbit, but perhaps more likely due to independent inactivations in guinea pig, human and pig. In zebrafish, two new intronless receptor genes were cloned. Sequence comparisons suggest that both receptors are distinct from the mammalian receptors Y1, Y4 and y6, hence they were named Ya and Yb. Chromosomal localization provides further support that Ya and Yb may be distinct subtypes. The discoveries of the rat Y4 and zebrafish Ya and Yb receptors were unexpected and show that the NPY receptor family is larger than previously thought.