Systemic hypoxia frequently occurs in patients with cardiopulmonary diseases. Maintenance of vascular reactivity and endothelial viability is essential to preserving oxygen delivery in these patients. The role of matrix metalloproteinase-2 (MMP-2) and heme oxygenase-2 (HO-2) in the vascular response to hypoxia were investigated. In the first part of the thesis, the role of MMP-2 in regulating systemic arterial contraction after prolonged hypoxia was investigated. MMP-2 inhibition with cyclic peptide CTTHWGFTLC (CTT) reduced phenylephrine (PE)-induced contraction in aortae and mesenteric arteries harvested from rats exposed to hypoxia for 7 d. Responses to PE were reduced in MMP-2-/- mice exposed to hypoxia for 7 d compared to wild-type controls. CTT reduced contraction induced by big endothelin-1 (big ET-1) in aortae harvested from rats exposed to hypoxia. Increased contraction to big ET-1 after hypoxia was observed in wild-type controls, but not MMP-2-/- mice. Rat aortic MMP-2 and MT1-MMP protein levels and MMP activity were increased after 7 d of hypoxia. Rat aortic MMP-2 and MT1-MMP mRNA levels were increased in the deep medial vascular smooth muscle. These results suggest that hypoxic induction of MMP-2 activity potentiates contraction in systemic conduit and resistance arteries through proteolytic activation of big ET-1. The second part of the thesis investigated oxygen regulation of HO-2 protein and whether it plays a role in preserving endothelial cell viability during hypoxia. HO-2, but not HO-1, protein level was maintained during hypoxia in human endothelial cells through enhanced translation of HO-2 transcripts. Inhibition of HO-2 expression increased the production of reactive oxygen species, decreased mitochondrial membrane potential, and enhanced apoptotic cell death and activated caspases during hypoxia, but not during normoxia. These data indicate that HO-2 is translationally regulated and important in maintaining endothelial viability and function during hypoxia. In summary, the thesis demonstrates the importance of MMP-2 and HO-2 in preserving vascular function during prolonged systemic hypoxia. These enzymatic pathways may, therefore, represent novel therapeutic targets that may be exploited to ameliorate the effects of hypoxia in patients with cardiopulmonary disease.

The synthesis of proteins bearing unnatural amino acids has the potential to enhance and elucidate many processes in biochemistry and molecular biology. There are two primary methods for site specific unnatural amino acid incorporation, both of which use the cell’s native protein translating machinery: in vitro chemical acylation of suppressor tRNAs and the use of orthogonal amino acyl tRNA synthetases. Total chemical synthesis is theoretically possible, but current methods severely limit the maximum size of the product protein. In vivo orthogonal synthetase methods suffer from the high cost of the unnatural amino acid. In this thesis I sought to address this limitation by increasing cell density, first in shake flasks and then in a bioreactor in order to increase the yield of protein per amount of unnatural amino acid used. In a parallel project, I used the in vitro chemical acylation system to incorporate several unnatural amino acids, key among them the fluorophore BODIPYFL, with the aim of producing site specifically fluorescently labeled protein for single molecule FRET studies. I demonstrated successful incorporation of these amino acids into the trial protein GFP, although incorporation was not demonstrated in the final target, FEN1. This also served to confirm the effectiveness of a new procedure developed for chemical acylation.

Covalent nucleotide modifications in RNAs affect numerous biological processes, and novel functions are continually being revealed even for well-known modifications. Among all RNA species, transfer RNAs (tRNAs) are highly enriched with diverse modifications, which are known to play roles in decoding and tRNA stability, charging, and cellular trafficking. However, studies of tRNA modifications have been limited in a small scale and performed by groups with different methodologies. To systematically compare the functions of a large set of noncoding RNA modifications in translational regulation, I carried out ribosome profiling in 57 budding yeast mutants lacking nonessential genes involved in tRNA modifications. Deletion mutants with enzymes known to modify the anticodon loop or non-tRNA substrates such as rRNA exhibited the most dramatic translational perturbations, including altered dwell time of ribosomes on relevant codons, and altered ribosome density in protein-coding regions or untranslated regions of specific genes. Several mutants that result in loss of tRNA modifications in locations away from the anticodon loop also exhibited altered dwell time of ribosomes on relevant codons. Translational upregulation of the nutrient-responsive transcription factor Gcn4 was observed in roughly half of the mutants, consistent with the previous studies of Gcn4 in response to numerous tRNA perturbations. This work also discovered unexpected roles for tRNA modifying enzymes in rRNA 2’-O-methylation, and in transcriptional regulation of TY retroelements. Taken together, this work revealed the importance and novel functions of tRNA modifications, and provides a rich resource for discovery of additional links between tRNA modifications and gene regulation.

La tomate (Solanum lycopersicum) [...] présente de nombreux avantages : facilité de culture, temps de génération court, connaissances et ressources importantes, génome séquencé, facilité de transformation... Le développement du fruit est un procédé complexe hautement régulé et divisible en quatre étapes principales : la division cellulaire, l'expansion cellulaire, l’étape appelé « turning » et la maturation. Chaque étape est associée à un phénotype, qui lui-même découle de changements à différents niveaux cellulaires. [...]. Grâce aux récents progrès technologiques et en particulier au développement des «techniques omiques», comme la génomique, la transcriptomique, la protéomique, la métabolomique, les principaux composants cellulaires peuvent désormais être étudiés à haut densité. Dans ce contexte, l'objectif de mon doctorat était d'effectuer une analyse protéomique quantitative du développement du fruit de tomate puis d’intégrer les données «omiques» à la fois par des analyses statistiques et par la modélisation mathématique. Le premier chapitre rapporte les résultats de quantification du protéome de fruit de tomate réalisé en collaboration avec la plateforme PAPPSO (INRA, Gif-sur-Yvette). Des échantillons collectés à neuf stades de développement du fruit de tomate ont été extraits et le protéome quantifié, en absence de marquage, par chromatographie liquide couplée à la spectrométrie de masse (LC-MS/MS). Ensuite, j'ai cherché la méthode la plus adaptée, testant un ensemble de filtres sur les données, pour obtenir une quantification précise des protéines à partir des intensités ioniques (XIC). Au total, j’ai pu obtenir la quantification absolue de 2494 protéines en utilisant une méthode basée sur la modélisation de l'intensité des peptides. [...] Le deuxième chapitre est consacré aux résultats obtenus par analyses combinées d’«omiques» au cours du développement du fruit de tomate. La transcriptomique a été réalisée en collaboration avec Genotoul GeT (Toulouse) et le groupe Usadel (RWTH Aachen University, Allemagne). Grâce à l’ajout d’étalons internes, plus de 20000 transcrits ont été quantifiés de manière absolue à chacune des neuf étapes de développement. Cette quantification a ensuite été validée par comparaison avec des données de concentration de 71 transcrits précédemment obtenues par PCR quantitative. Enfin, nous avons cherché à intégrer les quatre niveaux de données - transcriptome, protéome, métabolome et activome- afin d‘identifier les principales variables associées au développement. Pour ces quatre niveaux, les analyses ont confirmé que l’entrée en maturation s’accompagne de changements majeurs et révélé une grande similarité entre la fin et le début du développement, notamment au niveau du métabolisme énergétique. Le troisième chapitre porte sur les résultats de modélisation de la traduction protéique obtenus grâce à la quantification absolue du transcriptome et du protéome. Afin d’expliquer la corrélation décroissante observée au cours du développement entre les concentrations en protéines et celles des transcrits correspondants, nous avons résolu un modèle mathématique de la traduction protéique basé sur une équation différentielle ordinaire et impliquant deux constantes de vitesse: pour la synthèse et la dégradation de la protéine. La résolution de cette équation, validée par un critère de qualité basé un intervalle de confiance fermé, a conduit à l'estimation de ces constantes pour plus de 1000 protéines. [...] Enfin le dernier chapitre décrit l’ensemble du matériel et des méthodes utilisées pour obtenir les différents résultats présentés dans le manuscrit. Dans le domaine de la biologie des systèmes, ce travail illustre comment l'intégration de multiples données «omiques» et la modélisation mécanistique basée sur la quantification absolue des «omiques» peut révéler de nouvelles propriétés des composants cellulaires. / The interest of the tomato (Solanum lycopersicum) fruit has spread in plant science where it is used as the model for fleshy fruit. The valuable advantages of the tomato fruit are numerous: an ease of culture, a short generation time, a high knowledge with important resources, a sequenced genome, an ease for transforming…. The development of tomato fruit is a complex regulated process, divided in four main steps: cell division, cell expansion, turning and ripening. Each step is characterized by a phenotype resulting from changes at different cellular levels. Thus, gene expression, protein abundance, enzyme activities, metabolic fluxes and metabolite concentrations show significant changes during these steps. Thanks to recent technologies advances and in particular the development of ‘omics techniques’, such as genomic, transcriptomic, proteomic, metabolomic, the main cell components can now be analysed by high-throughput. In this context, the objective of my PhD was to perform a quantitative proteomic analysis of the tomato fruit development and then integrate omics data both by statistical analyses and by mathematical modelling. The first chapter focused on results obtained for the quantitative proteomic developed in collaboration with the PAPPSO platform (INRA, Gif-sur-Yvette). Samples were harvested at nine stages of tomato fruit development, total proteome was extracted and quantified by label-free LC-MS/MS. Then I searched for the most appropriate method, testing a set of filters on the data, to obtain an absolute label-free protein quantification from ion intensities (XIC). Finally, I obtained the absolute quantification of 2494 proteins using a method based on peptides intensity modelling. The quantification of proteins by LC-MS/MS was then validated by comparison with 32 enzymatic capacities used as proxy for protein abundance. The second chapter was dedicated to the results of integrative omics analyses throughout tomato fruit development. First, transcriptomic has been performed in collaboration with Genotoul GeT (Toulouse) and Usadel‘lab (RWTH Aachen University, Germany). Using spikes in the experimental design, more than 20000 transcripts have been quantitatively determined at the nine stages of development. Then, this absolute quantification of the tomato transcriptome has been cross-validated with 71 transcripts previously measured by qRT-PCR. Finally, we integrated the four omics datasets- transcriptome, proteome, metabolome and activome – in order to identify key variables of the tomato fruit development. For the four levels, analyses confirmed that the entrance in maturation phase was accompanied by major changes, and revealed a great similarity between the end and the beginning of development, especially in the energy metabolism. The third chapter focuses on modelling results of the protein translation based on the absolute quantification of transcriptomic and proteomic. To explain the decreasing correlation observed between proteins and transcripts concentration throughout development, we proposed a mathematical model of protein translation based on an ordinary differential equation and involving two rate constants (for synthesis and degradation of the protein). The resolution of this equation, validated by a quality criteria based on a closed confidence interval, led to the estimation of the rate constants for more than 1000 proteins. These results were then compared with previous published data reported for plants and more widely in eukaryotic cells. Finally, the last chapter describes all the materials and methods used to obtain the results presented in the manuscript.In the systems biology context, this work illustrates how integration of multiple omics datasets and mechanistic modelling based on absolute omics quantification can reveal new properties of cellular component.

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<p>This thesis details the development of a kinetic model of translation which takes into account codon usage. The process of translation involves ribosomes decoding a sequence of codons to produce a protein. Codon usage is important in the kinetics of translation since experiments have shown that codons are processed at different rates. Codons which code for the same amino acid appear with unequal frequencies and certain synonymous codons are preferred by high expression genes. The relationship between translational efficiency and codon adaptation is explored in this thesis.</p> <p>We use a simple physics model called the totally asymmetric exclusion process (TASEP) to emulate the action of ribosomes, and the decoding of mRNA in protein elongation. The simple model is parameterized by an initiation rate that determines how quickly new ribosomes are introduced onto the lattice, and the rate of motion for ribosomes associated with a site on the lattice (codon message). Based on bioinformatics studies, we assign codon speeds so that codons preferred by high expression genes are translated more quickly.</p> <p>The model captures important aspects of translation like ribosome collision and codons of different speeds, and simulating it allows us to see details in dynamics which are inaccessible to experiments. TASEP has non-trivial behaviour when codon rates, and the rate of ribosome binding is varied. Slow codons can cause ribosomes to pause and may lead to a queue. We approximated real genes with its average rate, and with its slowest codons to test the salient features of how codons are used on mRNAs. We found that codon selection is important in determining when queues occur, and the ribosome density on genes. The model also shows that highly expressed genes queue later than low expression genes. The simple model gives us general insights into the translational selection of codons, and the important kinetic parameters.</p> / Master of Science (MSc)

Zu den höheren kognitiven Fähigkeiten des menschlichen Neokortex gehören abstraktes Denken, komplexes Verstehen, Sprache und Lernfähigkeit. Die Bildung der Großhirnrinde beginnt in der mittleren Phase der Embryogenese und ist ein hochgradig organisierter und streng regulierter Prozess. Durch asymmetrische Teilung neuronaler Stammzellen entstehen unreife Neuronen, die im Anschluss an ihre Migration ihre spezifische Position innerhalb des Cortex einnehmen. Der korrekte Erwerb der axonalen Morphologie und die Spezifizierung des Dendritenbaums bilden die Grundlage für die Etablierung der kortikalen Konnektivität. Diese morphologischen Merkmale werden durch intrinsische genetische Programme der postmitotischen Differenzierung kodiert sowie durch entwicklungsbedingte Einflüsse im extrazellulären Milieu reguliert. Das im endoplasmatischen Retikulum lokalisierte Inositol-Requiring Enzyme 1α (Ire1α) ist einer der Hauptregulatoren der entfalteten Proteinantwort. In dieser Studie zeigen wir, dass Ire1α für die Spezifizierung der Neurone der oberen Cortexschichten sowie den Erwerb der neuronalen Morphologie von zentraler Bedeutung ist, indem es mRNA-Translationsraten reguliert. Diese Arbeit zeigt auch, dass frühe und späte kortikale neuronale Vorläuferzellen sowie früh und spät geborene postmitotische Neurone unterschiedliche Translationsraten aufweisen, was auf differenzierte Anforderungen an die Proteom-Synthesemaschinerie bezüglich der Entwicklung der kortikalen Schichten hinweist. Störungen in allen Phasen der kortikalen Entwicklung, welche entweder auf Umweltfaktoren oder Genmutationen zurückzuführen sind, können zu einer abweichenden Physiologie der kortikalen Schaltkreise führen. Eine große Anzahl solcher Anomalien kann zu schweren neurologischen Erkrankungen wie Epilepsie oder komplexen Störungen mit Epilepsie wie Rett-Syndrom, Angelman-Syndrom, Mowat-Wilson-Syndrom, Lafora-Krankheit und/oder Kaufman-Okulozerebrofazial-Syndrom führen. / Higher cognitive abilities of human neocortex comprise abstract thinking, complex comprehension, language and learning capacity. Formation of the cerebral cortex begins in the middle of embryogenesis and is a tightly organized and highly regulated process. Asymmetric divisions of neuronal stem cells give rise to immature neurons that migrate to consequently assume their specific position in the cortical plate. Correct acquisition of a single-axon morphology and specification of the dendritic tree complexity sets grounds for establishment of cortical connectivity. These morphological characteristics are encoded by intrinsic genetic programs of postmitotic differentiation and regulated by developmental cues in the extracellular milieu. Endoplasmic Reticulum resident Inositol-Requiring Enzyme 1α (Ire1α) is one of the main regulators of the unfolded protein response. In this study, we demonstrate that Ire1α is pivotal for specification of upper layer cortical neurons and the acquisition of the neuronal morphology by regulating mRNA translation rates. This work also shows that early and late cortical neuronal progenitors and early- and late-born postmitotic neurons exhibit different translation rates, indicative of the specific requirements for the proteome synthesis machinery for the development of cortical layers. Disturbances of any cortical developmental milestones due to either environmental factors or gene mutations may result in aberrant physiology of cortical circuits. High number of such abnormalities can lead to serious neurological diseases such as epilepsy or complex disorders with epilepsy such as Rett syndrome, Angelman syndrome, Mowat-Wilson syndrome, Lafora disease and/or Kaufman oculocerebrofacial syndrome. One major hypothesis of the causes of epilepsy links its molecular pathology to alterations in excitation/inhibition (E/I) balance in the neuronal networks.

Post-transcriptional regulatory mechanisms are widely used to influence cell fate decisions in germ cells, early embryos, and neurons. Many conserved cytoplasmic RNA regulatory proteins associate with each other and assemble on target mRNAs, forming ribonucleoprotein (RNP) complexes, to control the mRNAs translational output. How these RNA regulatory networks are orchestrated during development to regulate cell fate decisions remains elusive. We addressed this problem by focusing on Caenorhabditis elegans germline development, an exemplar of post-transcriptional control mechanisms. Here, we report the discovery of GLS-1, a new factor required for many aspects of germline development, including the oocyte cell fate in hermaphrodites and germline survival. We find that GLS-1 is a cytoplasmic protein that localizes in germ cells dynamically to germplasm (P) granules. Furthermore, its functions depend on its ability to form a protein complex with the RNA-binding Bicaudal-C ortholog GLD-3, a translational activator and P granule component important for similar germ cell fate decisions. Based on genetic epistasis experiments and in vitro competition experiments, we suggest that GLS-1 releases FBF/Pumilio from GLD-3 repression. This facilitates the sperm-to-oocyte switch, as liberated FBF represses the translation of mRNAs encoding spermatogenesis-promoting factors. Our proposed molecular mechanism is based on the GLS-1 protein acting as a molecular mimic of FBF/Pumilio. Furthermore, we suggest that a maternal GLS-1/GLD-3 complex in early embryos promotes the expression of mRNAs encoding germline survival factors. Our work identifies GLS-1 as a fundamental regulator of germline development. GLS-1 directs germ cell fate decisions by modulating the availability and activity of a single translational network component, GLD-3. Hence, the elucidation of the mechanisms underlying GLS-1 functions provides a new example of how conserved machinery can be developmentally manipulated to influence cell fate decisions and tissue development.