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No description available.

BACKGROUND:General, breed- and diet-dependent associations between feed efficiency in beef cattle and single nucleotide polymorphisms (SNPs) or haplotypes were identified on a population of 1321 steers using a 50K SNP panel. Genomic associations with traditional two-step indicators of feed efficiency - residual feed intake (RFI), residual average daily gain (RADG), and residual intake gain (RIG) - were compared to associations with two complementary one-step indicators of feed efficiency: efficiency of intake (EI) and efficiency of gain (EG). Associations uncovered in a training data set were evaluated on independent validation data set. A multi-SNP model was developed to predict feed efficiency. Functional analysis of genes harboring SNPs significantly associated with feed efficiency and network visualization aided in the interpretation of the results.RESULTS:For the five feed efficiency indicators, the numbers of general, breed-dependent, and diet-dependent associations with SNPs (P-value<0.0001) were 31, 40, and 25, and with haplotypes were six, ten, and nine, respectively. Of these, 20 SNP and six haplotype associations overlapped between RFI and EI, and five SNP and one haplotype associations overlapped between RADG and EG. This result confirms the complementary value of the one and two-step indicators. The multi-SNP models included 89 SNPs and offered a precise prediction of the five feed efficiency indicators. The associations of 17 SNPs and 7 haplotypes with feed efficiency were confirmed on the validation data set. Nine clusters of Gene Ontology and KEGG pathway categories (mean P-value<0.001) including, 9nucleotide binding / ion transport, phosphorous metabolic process, and the MAPK signaling pathway were overrepresented among the genes harboring the SNPs associated with feed efficiency.CONCLUSIONS:The general SNP associations suggest that a single panel of genomic variants can be used regardless of breed and diet. The breed- and diet-dependent associations between SNPs and feed efficiency suggest that further refinement of variant panels require the consideration of the breed and management practices. The unique genomic variants associated with the one- and two-step indicators suggest that both types of indicators offer complementary description of feed efficiency that can be exploited for genome-enabled selection purposes.

Protein identification and quantitation using mass spectrometry has evolved as the dominant technique for studying the protein complement of a system: cell, tissue or organism. The proteomics of body fluids is a very active research area as there is great potential for protein biomarker discovery; application of such technologies would revolutionise medical practice and treatment. Saliva, through its non intrusive nature of sampling, is an ideal body fluid for disease diagnosis, screening and monitoring. Gingivitis is a gum disease with symptoms including bleeding, swollen, and receding gums. After dental decay, gingivitis is estimated to be the most common disease worldwide, and around 40% of the population in the US are reported to have gingivitis. The end point goal of this project was to identify salivary biomarkers for gingivitis. This dissertation presents an investigation of: 1) the salivary proteome; 2) developments and applications of a mass spectrometry kinase assay; and 3) salivary biomarkers for gingivitis using proteomics and kinase activities. The soluble portion of the human salivary proteome (saliva supernatant) has been studied by several research groups but very few proteomic studies have been performed on the insoluble, cellular and bacterial portion of saliva. Presented here, is the first global proteomics study performed on the saliva residue and supernatant from the same test subject. A total of 834 and 1426 proteins were identified in the saliva supernatant and residue, respectively. A global analysis of protein complexes in saliva was also performed and is the first study, to date, of such an analysis. KAYAK (‘Kinase ActivitY Assay for Kinome analysis’) was further developed for its application on a number of cell types, tissue types, and a variety of organisms. Proof of concept work for in-gel kinase activity/kinase abundance correlation profiling using blue native gels was performed, and experiments using anion exchange chromatographic kinase activity/kinase abundance correlation profiling were performed to identify kinase-substrate pairs. KAYAK applications included the analysis of kinase activities in Saccharomyces cervisiae, Drosophila, mouse, and human saliva in which significant kinase activity was detected in the saliva supernatant, a novel finding. Finally, gingivitis was induced in patients, and the saliva samples were analysed using proteomics and kinase activity profiling. Although this work is ongoing, preliminary data indicate that there are increases in various inflammatory proteins, certain bacteria and also in the activity of particular kinases as a result of the induction of gingivitis. The overall study provided insights into the salivary proteome for both the human and bacterial complement, as well as discovering the presence of significant kinase activity in saliva. In the induced gingivitis study, almost half of all the proteins identified in the residue were from bacteria (1274 bacterial proteins, 198 species identified) and there may be more potential for biomarker discovery for certain diseases in the saliva residue than in the supernatant. A very large overlap was observed between the human proteins in the saliva supernatant and residue, indicating that many of the salivary proteins originate from lysed cells. The origin of the kinase activity in the saliva supernatant is not known but is also proposed to originate predominantly from lysed cells. A range of novel KAYAK applications have been investigated, demonstrating that KAYAK has a wide variety of future uses ranging from target compound evaluation in Pharmaceutical companies to patient testing in the clinic.

Les protéines kinases « Aurora » sont des régulatrices clés du cycle cellulaire. Alors que l'activité de Aurora-A est requise en début de la mitose, Aurora-B et -C sont nécessaires pour la fin de mitose. Toute perturbation de leur activité peut conduire au processus de tumorisation. Plus spécifiquement, Aurora-A se comporte à la fois comme oncogène et suppresseur de tumeur. Par conséquent, la connaissance du rôle de Aurora-A durant cycle cellulaire est essentielle. Toutefois, peu d'études ont exploré, jusque là, le rôle de Aurora-A dans les phases tardives de la mitose. En fait, l'inhibition de Aurora-A conduit à l'arrêt du cycle rendant impossible de voir ce qui se passe au delà de la transition métaphase/anaphase. Néanmoins, en combinant le couple génétique chimique et inhibiteur spécifique, j'ai pu identifier une nouvelle fonction de la kinase Aurora-A liée au checkpoint (SAC). En effet, mes résultats montrent que l'inhibition de l'activité de Aurora-A induit un défaut de congression et une chute de l'index mitotique. Ce résultat paradoxal suggère un défaut du SAC. J'ai donc pu montré que cette inhibition outrepassait le SAC en perturbant la localisation aux kinétochores de Mad2 et BubR1. Cependant, ma tentative pour sauver le phénotype du SAC par le mutant S19D-P150Glued n'a pas réussi malgré que le mutant S19AP150Glued se soit comporté comme un vrai dominant négatif. Enfin, j'ai pu montré que l'activité de Aurora-A est requise pour maintenir le SAC actif durant la prométaphase. / Protein kinases "Aurora " are the key regulators of the cell cycle. While the activity of Aurora-A is required at the beginning of mitosis, Aurora-B and -C are required for the end of mitosis. Any disruption of their activity can lead to process tumorisation. Specifically, Aurora-A acts as both oncogene and tumor suppressor. Therefore, knowledge of the role of Aurora-A is essential for cell cycle. However, few studies have explored so far, the role of Aurora-A in the late stages of mitosis. In fact, inhibition of Aurora-A leads to cell cycle arrest making it impossible to see what happens beyond the transition metaphase / anaphase. However, by combining chemical genetics couple and specific inhibitor, I have identified a new function of Aurora-A kinase -related checkpoint (SAC). Indeed, my results show that inhibition of the activity of Aurora-A induces a congression defect and the mitotic index decrease. This paradoxical result suggests a defect in the SAC. So I have shown that this inhibition was beyond the SAC disrupting kinetochore localization of Mad2 and BubR1. However, my attempt to rescue the phenotype of the SAC by the S19D-P150Glued mutant failed despite the fact that S19A-P150Glued mutant behaved like a true negative dominant. Finally, I have shown that the activity of Aurora-A is required to maintain the active SAC during prometaphase.

Les RTKs sont impliqués dans le dialogue au sein des tissus par la régulation de nombreuses réponses cellulaires dont la survie, la prolifération ou la mobilité. Dans les cancers, ces récepteurs sont fréquemment dérégulés notamment par des mutations activatrices. Ainsi, la suractivation des RTKs induit la transformation cellulaire et la tumorigenèse en favorisant par exemple la survie cellulaire. Depuis le début des années 2000, le développement de molécules inhibitrices de l’activité tyrosine kinase (TKI) et d’anticorps bloquant l’interaction ligand/récepteur ont montré que les RTKs représentent des cibles thérapeutiques majeures dans le traitement des cancers.MET est un RTK exprimé par les cellules épithéliales, dont le ligand est l’Hepatocyte Growth Factor/Scatter Factor (HGF/SF). En plus de son rôle pro-survie, MET peut également favoriser l’apoptose en absence de ligand et sous l’effet d’un stress. MET est alors clivé par les caspases et libère dans le cytosol un fragment de 40 kDa nommé p40MET. Ce fragment active la voie intrinsèque de l’apoptose en causant la perméabilisation des mitochondries. Cependant, les mécanismes moléculaires responsables de cette perméabilisation et l’impact physiologique de la fonction pro-apoptotique de MET étaient encore inconnus.Mon travail de thèse a permis de démontrer que le fragment p40MET se localise dans la région des MAMs, constituant l’interface entre le réticulum endoplasmique et les mitochondries, où il favorise un transfert de calcium entre les deux organites. Ce transfert déclenche une surcharge de calcium dans les mitochondries, responsable de leur perméabilisation. De plus, nous avons développé une lignée de souris transgéniques dans lesquelles MET est muté sur l’un des sites caspases. Ces souris sont incapables de produire le fragment p40MET pro-apoptotique. Ce modèle nous a permis de démontrer l'importance du clivage de MET dans l’amplification de l’apoptose in vivo. Ainsi, nos travaux apportent les premières preuves de la fonction de MET en tant que récepteur à dépendance au sein d’un organisme et décrivent un nouveau mécanisme de signalisation pro-apoptotique par la dérégulation des flux calciques.Ces dernières années, la découverte de mutations touchant les RTKs dans les cancers a augmenté de façon exponentielle. Toutefois, pour une grande majorité de mutations, leurs conséquences fonctionnelles sont totalement inconnues. Ainsi, en parallèle de mon principal sujet de thèse nous avons évalué la pertinence biologique et clinique des mutations de RTK identifiées par séquençage haut débit à partir d’échantillons de patients. Le séquençage de tissus sains, de tumeurs colorectales et de métastases hépatiques de 30 patients a permis d'identifier de nombreuses mutations somatiques. Parmi elles, certaines affectent le domaine kinase des récepteurs et sont présentes à la fois dans les tumeurs et les métastases. L’analyse fonctionnelle que j’ai menée sur 7 de ces mutations révèle qu’elles ne provoquent ni la suractivation de la kinase ni la transformation des fibroblastes NIH3T3. Au contraire, deux mutations de RTKs provoquent une inhibition drastique de leur activité kinase. Ces résultats démontrent que ces variants de RTK ne sont pas des cibles appropriées pour l’utilisation de TKI à des fins thérapeutiques et démontre l’intérêt de coupler la recherche de variants à des études fonctionnelles [...] / RTKs are involved in tissue dialogue by regulating many cellular mechanisms such as survival, proliferation or mobility. In cancers, these receptors are frequently deregulated, as a result of various molecular alterations leading to their activation. RTKs overactivation induces cell transformation and tumorigenesis notably by promoting survival. Since the early 2000s, the development of tyrosine kinase inhibitors (TKI) demonstrated that RTKs represent major therapeutic targets in cancer treatment.MET receptor and its ligand the Hepatocyte Growth Factor/Scatter Factor (HGF/SF) are known to promote survival of many epithelial structures during embryogenesis and later during adulthood. Besides pro-survival role of the ligand-activated MET, the receptor is also able to promote apoptosis, which has led to classify it within the dependence receptor family. Indeed, in absence of its ligand and under stress conditions, MET is cleaved by caspases leading to the production of an intracellular fragment of nearly 40 kDa named p40MET able to amplify apoptosis. This fragment activates the intrinsic pathway of apoptosis by causing mitochondrial permeabilization. However, the molecular mechanisms involved in this permeabilization and the physiological impact of the pro-apoptotic function of MET were still unknown.My PhD work has evidenced p40MET localization at the MAM microdomain and characterized a calcium transfer from the endoplasmic reticulum to the mitochondria triggered by p40MET. This calcium transfer triggers a calcium overload in mitochondria leading to their membrane permeabilization and apoptosis. In addition, we engineered a knock-in mouse model expressing mutated MET at the C-terminal caspase site. These mice are unable to produce the pro-apoptotic p40MET fragment. This model allowed us to assess the importance of MET cleavage in physiological apoptosis in vivo. Altogether, our work brings the first evidence for MET function as a dependence receptor in an organism and demonstrates a new signaling mechanism involved in apoptosis amplification by p40MET through calcium flux deregulation. This process may be relevant in the physio-pathology of organs where MET is expressed.In recent years, the discovery of mutations affecting RTKs in cancers has increased exponentially. However, for a large majority of mutations, their functional consequences are totally unknown. Thus, in parallel of my main thesis topic, we evaluated the biological and clinical relevance of RTKs mutations identified by high throughput sequencing from patient samples. Sequencing of healthy tissues, colorectal tumours and liver metastases of 30 patients has identified many somatic mutations. Some of them affect the receptor kinase domain and are present in both tumors and metastases. Functional analysis of 7 of these mutations shows that they do not cause neither kinase overactivation nor transformation of NIH3T3 fibroblasts. On the contrary, two RTK mutations cause drastic inhibition of the corresponding kinase activity. These findings indicate that these RTK variants are not suitable targets for TKI. Therefore, it appears important to set up reliable functional assays to interpret identified variants and classify them as pathogenic or neutral.In conclusion, my work opens up new perspectives on therapeutic strategies targeting RTKs in cancers. First of all, the pro-apoptotic capacities of some RTKs are undoubtedly a brake to tumorigenesis, and their stimulations could reinforce the effectiveness of anti-cancer therapies. On the other hand, we have shown that RTKs mutations in the kinase domain do not necessarily lead to overactivation of the receptor suggesting that they are probably not involved in tumorigenesis and that treatment with TKIs targeting them would be ineffective. This functional information could notably influence the choice of a suitable targeted therapy.

Afin de corriger la suractivation de la voie de signalisation Raf/MEK/ERK observée dans 30 % des cancers,nous avons choisi d’inhiber la kinase Raf-1. Les inhibiteurs potentiels de Raf-1 ont été conçus avec un cyclecentral original 4-azaindolique. Pour apporter de la diversité fonctionnelle au niveau des sommets C-2 et C-5lors de la synthèse, nous avons optimisé deux méthodes à partir du synthon 5-méthoxy-4-azaindole-Nphénylsulfonyle.La première est une réaction de lithiation du sommet C-2 suivie de la condensation dedifférents électrophiles et la deuxième est la C-arylation ou N-arylation du sommet C-5 à partir du dérivétriflate en 5 via des réactions de couplage pallado-catalysées de type Suzuki et Buchwald, respectivement.Ces deux méthodes ont permis d’aboutir à 2 séries de composés : une première série fonctionnalisée enposition N-1 et C-5 du noyau 4-azaindole et une deuxième série substituée en position C-5 et C-2.Pour tester les nouveaux inhibiteurs synthétisés et un inhibiteur naturel appelé PEBP, nous avons mis aupoint des tests d’activité in vitro sur l’ensemble de la cascade Raf/MEK/ERK et sur chacune des 3 kinases.Les tests ont été développés avec 2 méthodes de TR-FRET, Lance UltraTM et LanthascreenTM, et ont étévalidés avec des inhibiteurs commerciaux et comparés par rapport à la méthode radioactive PFBA.Au total, 30 produits finaux ont été évalués in vitro sur la kinase Raf-1. Grâce aux plateformes duCancéropôle GO, les produits ont aussi été testés sur 6 lignées de cellules cancéreuses et sur les kinasesdu cycle cellulaire DYRK1A, GSK3 et CDK5. Plusieurs molécules ont montré une activité antitumoraleencourageante de l’ordre du μM et un composé a été identifié comme inhibiteur de Raf-1 avec une valeurd’IC50 de 9,8 μM et une cytotoxicité sélective vis-à-vis des cellules du foie Huh7 (IC50 = 3 μM). / Considering the implication of the Raf/MEK/ERK pathway deregulation in 30 % of human cancers, wedecided to synthesize potential Raf-1 inhibitors. They were synthesized using the original 4-azaindole as thecentral core. To introduce various types of substituents in C-2 and C-5, we developed two methodologiesfrom the building block N-benzenesulfonyl-5-methoxy-4-azaindole. The first one is the C-2 lithiation followedby the addition of various electrophiles. The second methodology is the C-5 C-arylation or the N-arylationusing palladium-catalysed cross-couplings from the C-5 triflate derivative through Suzuki or Buchwald crosscoupling reactions, respectively. The two methodologies ended up to the synthesis of two series ofcompounds. The first one is functionalized in N-1 and C-5 of the 4-azaindole core. The second one issubstituated in C-2 and C-5.In order to test the newly synthesized inhibitors as well as the natural inhibitor PEBP, we have developedseveral tests to measure in vitro the effect of inhibitors on the activity of the complete cascade Raf/MEK/ERKand also on the activities of each individual kinase. The assays were developed by using 2 TR-FRETmethods, Lance UltraTM and LanthascreenTM, and were validated with commercially available inhibitors andcompare to the radioactive PFBA method.Finally, 30 compounds were evaluated through in vitro Raf-1 assays. By using the facilities offered by theCanceropôle GO platforms, the compounds were also tested on six different tumor cell lines and on thekinases DYRK1A, GSK3 and CDK5. Several compounds displayed encouraging antitumoral activity and onecompound was identified as a Raf-1 inhibitor with an IC50 value of 9.8 μM and a selective cytotoxicity activitytowards liver tumor cells Huh7 (IC50 = 3 μM).

No description available.

La maladie de Parkinson (MP) est une pathologie neurodégénérative fréquente. Différents mécanismes moléculaires ont été mis en cause, dont une dysfonction mitochondriale. Des mutations du gène LRRK2, codant la protéine leucine-rich repeat kinase 2, sont responsables de formes autosomiques dominantes. La substitution la plus fréquente, G2019S, confère à la protéine un gain de fonction. LRRK2 semble réguler l’homéostasie mitochondriale, rôle initialement attribué aux protéines Parkine et PINK1, qui régulent en particulier la mitophagie. Nous avons étudié le rôle de LRRK2 et de son variant LRRK2-G2019S dans la régulation de la mitophagie dépendante de PINK1/Parkine. Nous avons également évalué l’effet de l’activité kinase sur ce processus dans un modèle cellulaire et dans des fibroblastes de patients. Nous avons exploré l’effet de LRRK2 sur la régulation d’interactions protéiques essentielles dans la mitophagie. Enfin, nous avons comparé l’efficacité de la mitophagie dans les formes familiales de la MP liées aux gènes LRRK2 et PARK2. Nous avons montré que LRRK2 et son variant LRRK2 G2019S retardent la mitophagie. Au travers de son activité kinase, LRRK2 compromet des interactions protéiques clefs impliquant Parkine et la GTPase Drp1. Nous avons mis en évidence un défaut de ce processus dans les fibroblastes de patients porteurs de mutations du gène PARK2. Ce défaut est également retrouvé dans les fibroblastes de patients porteurs de la substitution G2019S, dans lesquels il est corrigé par l’inhibition de l’activité kinase de la protéine. Ces résultats mettent en évidence un rôle de LRRK2 et de sa substitution pathogène dans la mitophagie dépendante de la voie PINK1/Parkine. / Parkinson’s disease (PD) is a frequent neurodegenerative disorder. Different molecular mechanisms are suspected, among which mitochondrial dysfunction stands out. Mutations in LRRK2, encoding leucine-rich repeat kinase 2 (LRRK2), cause autosomal dominant PD forms. The most frequent G2019S substitution leads to a gain of function. LRRK2 seems to modulate mitochondrial homeostasis, initially associated with Parkin and PINK1 proteins, which regulate in particular mitophagy. Here, we explored the involvement of LRRK2 and its kinase activity in the regulation of PINK1/Parkin-dependent mitophagy, and evaluated the consequence of the G2019S substitution, both in a cell line (COS7) and in primary fibroblasts from PD patients. In particularly, we studied the impact of LRRK2 on the regulation of protein-protein interactions essential for mitophagy initiation. We also compared the efficiency of PINK1/Parkin-dependent mitochondrial clearance in familial PD forms linked to LRRK2 and PARK2. Our results show that LRRK2 and its LRRK2 G2019S variant delay mitophagy. Moreover, these proteins compromised key interactions involving Parkin and the GTPase dynamin related protein 1 (Drp1) on the outer mitochondrial membrane. We confirmed a defect in this process in fibroblasts from patients with PARK2 mutations and found a similar alteration in fibroblasts from patients with the G2019S substitution. Inhibition of LRRK2 kinase activity restored mitophagy induction in cells from LRRK2 patients, but not in cells from PARK2 patients. Altogether, these results highlight a role of LRRK2 and its pathogenic substitution in the regulation of PINK1/Parkin-dependent mitophagy.