Ce mémoire rapporte deux approches de la synthèse des protéines à l'échelle de la molécule unique. Nous utilisons la microscopie de fluorescence en onde évanescente pour sonder l'activité traductionnelle de deux types de ribosomes. Les premiers, issus d'E. Coli (organisme procaryote), sont mutés afin de les marquer d'un nanocristal semiconducteur (QD). La fin de la traduction, qui correspond au décrochage du ribosome de l'ARNm lorsque celui-ci atteint le codon stop, est alors mise en évidence par la disparition du QD de la surface de l'échantillon. Le deuxième type de ribosome étudié est quant à lui extrait de cellules de lapins (organisme eucaryote) et est dit "sauvage", c'est à dire qu'il n'a pas subi de modification, tandis qu'un oligonucléotide marqué d'un fluorophore est hybridé à l'ARNm. L'activité hélicase du ribosome lui permettant de séparer deux brins complémentaires, l'oligonucléotide et donc le fluorophore disparaissent en même temps que le ribosome parcourt l'ARNm, permettant ainsi de sonder l'activité du ribosome. Nous donnons pour ces deux types de ribosomes une vitesse moyenne de la traduction dans des milieux contenant les facteurs de la traduction issus d'extraits cellulaires.La deuxième approche de la synthèse des protéines porte sur les propriétés de l'ARNm, support de l'information génétique codant pour la séquence des protéines. Nous avons développé un montage de pince optique permettant de manipuler et caractériser les propriétés mécaniques d'oligonucléotides, ainsi qu'une méthode originale de calibration de ce piège optique. La cohérence de nos mesures sur l'étirement d'un double brin d'ADN avec la littérature nous permettra de poursuivre notre étude sur la mesure des forces nécessaires pour ouvrir une structure secondaire de l'ARNm. / We hereby report two approaches of the protein synthesis at the single molecule level. We use total internal reflection fluorescence microscopy to study the translation kinetic of two different types of ribosomes. The first ones, extracted from E. Coli (prokaryotic organism), are mutated in order to label them with a quantum dot (QD). The end of translation, which corresponds to the dissociation of the ribosome from the mRNA when the stop codon has been reached, is highlighted by the disparition of the QD from the surface. The second type of ribosome is extracted from rabbit cells (eukaryotic organism) and has not been modified (wild type), while a labeled oligonucleotide is hybridized on the mRNA. The helicase activity of the ribosome allowing the dissociation of two complementary strands, the oligonucleotide and so the label disappear at the same time while the ribosome moves along the mRNA and thus inform us about its activity. For these two types of ribosomes we measure their average translation speed in cell extracts.The second approach focuses on the properties of the mRNA, carrying the genetic code for the protein sequence. We developped an optical tweezer setup in order to manipulate and characterize the mechanical properties of nucleotides, as well as an original method to calibrate this optical trap. The consistency of our measurements with the litterature on the properties of a double stranded DNA will allow us to study secondary structures of mRNA.

La traduction est considérée comme une étape clé de l'expression des gènes permettant à la cellule de s'adapter aux variations de son environnement en réponse aux signaux internes ou externes. Des études bioinformatiques ont montrés que la moitié des ARN messagers chez l'homme portent, en amont de leur phase codante, des éléments régulateurs appelés uORF. Le laboratoire a montré qu'un défaut de terminaison de la traduction par déplétion du facteur de terminaison eRF3 modifie l'expression de gènes dont l'ARNm contient des uORF comme le gène ATF4. Cette modification se fait soit par un mécanisme de réinitiation après traduction de l'uORF soit par une augmentation de la stabilité de l'ARNm résultant d'un défaut de sa dégradation par la voie du "Nonsense-mediated mRNA Decay" (NMD). A travers leur association dans le même complexe et leur implication dans la terminaison de la traduction et la NMD, eRF3 et Upf1 contribuent à la régulation fine de l'expression des gènes. Cependant, on ne sait pas dans quelle mesure ces deux facteurs affectent la traduction et la stabilité des ARNm. Nous avons évalué la traduction par ribosome profiling et le taux de transcrits par RNA-seq dans les cellules humaines déplétées en eRF3 ou en Upf1. Ces analyses nous ont permis de dresser une carte des uORF traduites dans le transcriptome des cellules humaines HCT116. Nous avons également observé que peu de gènes cibles sont communs entre la déplétion en eRF3 ou en Upf1. Nos résultats appuient fortement l'hypothèse qu'il y a au moins deux classes de transcrits portant des uORF, l'une dont la régulation implique la terminaison de la traduction et l'autre dont la régulation implique la NMD. / Regulation of gene expression at the translational level is increasingly being recognized as a key mechanism by which cells can rapidly change their gene expression pattern in response to internal or external stimuli. Bioinformatic studies revealed that half of human transcripts present at least one expression regulatory element uORF in the 5’ leader sequence preceding the main ORF. We have previously shown that translation termination disruption caused by eRF3a depletion induces upregulation of the transcriptional activator ATF4 and its targeted genes partly by a translational control at uORFs, and partly in relation to a defect in Nonsense-mediated mRNA Decay activation, increasing ATF4 mRNA stability. Through their physical association and their involvement in translation termination and NMD, eRF3 and Upf1 are regulating the protein and mRNA levels of a significant number of genes and thus contribute to the fine-tuning of their expression. It is not known yet, in what extent both of these factors affect translational control and what is the subset of genes that are regulated by these factors. In this study, we evaluated translation by ribosome profiling and mRNA level by RNA-seq in human cells subjected to either eRF3a or Upf1 depletion. These analyses allowed us to draw a transcriptome-wide map of uORFs and obtained a list of functional uORFs in our reference HCT116 transcriptome. We also observe that only a small fraction of these are common targets for both eRF3a and Upf1. Our results provide strong support for the notion that different classes of transcripts bearing uORFs are regulated either by translational processes involving translation termination or by NMD.

Des techniques variées de visualisation de molécules d’intérêt sur cellules vivantes ou fixées ont permis de suivre leur synthèse, localisation, dégradation et autres activités. Dans cette étude, nous avons développé deux outils de fluorescence pour étudier la synthèse des protéines sur bactéries vivantes. Le premier décrit l’utilisation du système Spinach pour l’imagerie du ribosome. Cette approche diffère des méthodes conventionnelles qui utilisent des protéines fluorescentes puisque l’ARN ribosomal 16S contient un aptamère qui rend fluorescent un composé fluorogène. Une étude comparative de la performance de différents aptamères Spinach a été réalisée. Un deuxième outil se focalise sur l’accumulation d’un antibiotique de la famille des aminoglycosides (ligand du ribosome) conjugué à un fluorophore. Ce nouveau conjugué, qui a conservé son activité bactéricide permet pour la première fois de visualiser l’accumulation de l’antibiotique sur bactérie vivante. Cela permet une analyse au niveau de la cellule unique d’une population bactérienne exposée à l’antibiotique. Nous avons également obtenu des données sur la localisation de l’antibiotique une fois qu’il a pénétré dans la bactérie à une résolution inégalée par microscopie super-résolutive. Nous espérons que ces deux méthodes vont maintenant permettre une meilleure compréhension de la synthèse des protéines et fournir une vue nouvelle de la pénétration des antibiotiques dans les bactéries pour y produire leur action bactéricide. / Various visualizing techniques have previously enabled monitoring the fate of molecules of interest: their expression, localization, degradation and other activities in live or fixed cells. In this study, we have developed two fluorescent tools to study protein synthesis in live bacterial cell. The first one describes the application of Spinach system to ribosomes imaging. This is different from conventional methods (that use fluorescent proteins) in that 16S rRNA contains an inserted RNA aptamer that elicits fluorescence of a fluorogenic compound. A comparative study of the performance of different Spinach aptamers was performed here. A second system focuses on the uptake of a fluorescently labeled ligand of the ribosome, an antibiotic of the class of aminoglycosides. This novel conjugate, which kept its bactericidal activity allows for the first time imaging of aminoglycoside uptake on live bacteria. This opened the door to a single cell analysis of bacterial cell populations. We also obtained data about the localization of the antibiotic once inside the bacteria to an unprecedented resolution using super resolution microscopy. We hope that both of these methods will contribute to a better understanding of protein synthesis as well as provide a novel view on the way antibiotics penetrate into cells and perform their bactericidal action.

La réponse stringente est un processus mis en place lors d'une carence nutritionnelle qui permet l'arrêt coordonné de la croissance. Cette réponse essentielle à la survie des bactéries est très conservée. Elle se caractérise par la production et l'accumulation de guanosine tretra- et pentaphosphate (ppGpp). Le ppGpp, en se fixant sur l'ARN polymérase modifie ses propriétés cinétiques et affecte ainsi de manière globale la transcription de très nombreux gènes. Principalement, l'accumulation de ppGpp inhibe la biosynthèse des ARNs stables (ARNr et ARNt) et en conséquence inhibe la biogenèse des ribosomes. Chez Escherichia coli, le niveau de ppGpp est régulé par les deux enzymes RelA et SpoT. Lors d'une carence en acides aminés, RelA fixée au ribosome détecte le blocage de la machinerie traductionnelle causée par la fixation d'un ARNt déacylé au site A du ribosome, et synthétise du ppGpp. SpoT quant à elle serait capable de détecter et de synthétiser le ppGpp en réponse à d'autres carences nutritionnelles notamment en source de carbone, mais les mécanismes et les signaux détectés sont inconnus. Il a été proposé que la protéine CgtA serait impliquée dans le contrôle de la réponse stringente, en interagissant avec SpoT au niveau des ribosomes. CgtA est une GTPase conservée et essentielle de la famille Obg, mais sa fonction précise est inconnue. Elle a été impliquée à la fois dans la maturation des ribosomes et dans la ségrégation des chromosomes et la division. Le gène cgtA est situé en aval des gènes rplU, rpmA, et yhbE codant respectivement pour les protéines L21 et L27 de la sous-unité 50S du ribosome et pour une protéine intégrale de membrane interne de fonction inconnue. / The stringent response is a physiological process that occurs when bacterial cells encounter nutritional stresses, and allowing coordinated growth arrest. This conserved response is characterized by the accumulation of tetra- and pentaphosphate guanosine (ppGpp). ppGpp bind to RNA polymerase and modifies its kinetic properties, thereby affecting the transcription of many genes. Prinicpaly, ppGpp accumulation inhibits stable RNAs (rRNA and tRNA) biosynthesis, which in consequence inhibits ribosome biogenesis. Escherichia coli contains two enzymes involved in ppGpp metabolism, RelA and SpoT. During amino acid starvation, RelA bound to ribosomes produces ppGpp in response to the presence of uncharged tRNA in the ribosomal A-site. In contrast, SpoT produces ppGpp in response to other types of nutrient limitations, such as carbon starvation, but the detected signals and mechanism involved are still unknown. It has been proposed that the CgtA protein is involved in the stringent response control by interacting with SpoT at the ribosome. CgtA is a conserved and essential small GTPase of the Obg family. CgtA has also been implicated in ribosome maturation, chromosome segregation and division, but its precise function remains unknown. The cgtA gene is located downstream of rplU, rpmA and yhbE genes coding respectively for L21 and L27 proteins of the 50S subunit of the ribosome, and an integral inner membrane protein of unknown function. This genetic proximity with rplU and rpmA genes is highly conserved in bacteria. My thesis work was therefore organized around three questions. First, understanding the role of CgtA in growth control and in the stringent response.

Isolé d’une bactérie, Streptomyces alboniger, la puromycine est un nucléoside antibiotique naturel du fait de sa ressemblance structurale avec l’adénosine terminale de l’extrémité 3’ de l’ARNt - aminoacyle. Grâce à cette similarité, cette petite molécule, qui peut diffuser au sein du site A (actif) du ribosome, a la faculté d’inhiber la synthèse protéique. La puromycine est exploitée seulement comme outils pour la recherche fondamentale. En effet, elle n’a encore jamais été exploitée à des fins thérapeutiques chez l’homme car elle est à l’origine de la métabolisation d’un produit toxique. A la suite des travaux du Dr Benoit Michel sur l’analogue nord de la puromycine, l’objectif du projet a été de confectionner un analogue carbocyclique de la puromycine, dont le ribofuranose est bloqué en conformation sud à l’aide d’un pont cyclopropane. L’étape ultérieure serait le test et la comparaison de l’activité dans le ribosome de la molécule obtenue avec celle des analogues précédemment synthétisés. De plus, ces études biochimiques pourraient démontrer si l’équilibre conformationnel du ribofuranose participe ou non à la catalyse du transfert peptidique au même titre que l’hydroxyle 2’ de l’A2451 du peptidyle transférase center. Dans la synthèse énantiosélective de l’analogue sud de la puromycine, la plus grande difficulté vient de l’introduction stéréosélective de l’azote se trouvant en jonction de cycle à 3 et 5 chainons. Dans le but de mettre au point une synthèse la plus courte possible nous avons décidé de former le carbocycle contenant l’azote correctement installé en une seule étape dite de cyclisation intramoléculaire de Kulinkovich – De Meijere. Dans le cas idéal ou la réaction marche convenablement, l’ensemble des 5 centres asymétriques présents de la molécule serait dans la stéréochimie optimale pour la suite de la synthèse. Après de nombreux problèmes, l’objectif directeur du projet a été quelque peu modifié mais est resté l’essai, voir l’optimisation de cette réaction intramoléculaire dans le but de confirmer ou d’infirmer la validité du chemin réactionnel choisi dès le départ / Isolated by a bacterium, Streptomyces Alboniger, the puromycine is a natural nucleoside antibiotic because of its structural similarity with the terminal adenosine of the extremity 3’ of the ARNt-aminoacyle. Thanks to this resemblance, this small molecule, which can spread within the site A of the ribosome, is able to inhibit the protein synthesis. The puromycine is used only as tools for the basic research. Indeed, it was still never run in therapeutics purposes insofar as it is at the origin of the metabolization of a toxic product. Following the work of Dr Benoit Michel on the north analogue of the puromycine, the aim of the project is to synthesize a carbocyclique analogue of the puromycine, whose ribofuranose is blocked in south conformation by means of cyclopropyl bridge. The later step would be the test and the comparison of the activity previously synthesized. Furthermore, these biochemical studies could demonstrate if the conformational equilibrium of the ribofuranose participates or not in the catalysis of the peptide transfer as well as the hydroxyl 2’ of A2451 of the peptidyle transferase center. In the enantioselective synthesis of the south analogue of the puromycine, the highest difficulty comes from the stereoselective introduction of the nitrogen which is found in junction of cycle in 3 and 5 links. With the aim of working out the shortest possible synthesis we decided to form the carbocycle bicyclo[3,1,0]hexane, with the nitrogen correctly set up, in a single step called intramolecular Kulinkovich-De Meijere cyclization. In the ideal case the reaction works suitably, all five asymmetrics contiguous centers of the carbocycle would be obtained in the optimal stereochemistry for next step of the synthesis. After numerous problems, the guiding objective of the project was a little modified but stayed the try and the optimization of this intramolecular reaction with the aim of confirming or invalidating the cogency of the reactive pathway chosen from the beginning

Type-II Ribosome Inactivating Proteins from Abrus precatorius: Cytotoxicity and Mechanism of Cell Death A/B toxins produced by bacteria and plants are among the deadliest molecules known. The plant type-II ribosome inactivating proteins (RIPs) are prototype of A/B toxins. They are two subunit proteins with a toxic A subunit that harbors an RNA N-glycosidase activity and a lectin like B subunit which allows toxin entry into cells. The toxicity of A chain is due to its RNA-N-glycosidase activity which cleaves the bond between the ribose sugar and the adenine at position 4324 as demonstrated in rat liver ribosomes. The B- chain, a lectin, binds to the cell surface receptors terminating in galactose sugars and allows toxin entry into cells. The seeds of the subtropical climber Abrus precatorius contain two RIPs: the potent toxic lectin abrin and the relatively less toxic Abrus agglutinin. The toxic property of RIPs has widespread applications in the field of agriculture and medicine. The cells of our body commit suicide in response to genetic or environmental cues by the process, apoptosis or programmed cell death which results in the safe clearance of the dead cells without affecting the extra-cellular milieu. Apoptosis is essential for development, tissue homeostasis, and defense against pathogens. It involves the interplay of multiple pathways that are initiated and executed by a family of proteases termed caspases. Several plant type-I and type-II RIPs as well as bacterial toxins have been shown to induce apoptosis in cultured cell lines. Though many agents that inhibit macromolecular synthesis in cells induce DNA fragmentation and morphological changes associated with apoptosis, the link between protein synthesis inhibition by these toxins and apoptosis remains elusive. Though extensive studies have been carried out on several RIPs for e.g. ricin and shiga toxin, only few reports are available in literature on the mechanisms of toxicity exhibited by abrin, a type-II RIP, of South-East Asian origin. Earlier studies from the laboratory have focused on the sensitivity and mechanism of abrin induced cell death in Jurkat, a cell line of haematopoietic lineage and its variants. In the same direction, the objectives of my study were: (1) To delineate the structure-function relationship of Abrus agglutinin-I in comparison with abrin, (2) To establish monoclonal antibodies to the A subunit of abrin, analyzing their neutralizing effect on abrin toxicity in vitro and in vivo and (3) To delineate the pathway and determine the kinetics of apoptosis induced by abrin on cell lines of epithelial lineage. The thesis will be presented in three four chapters. The first chapter, ‘Introduction’, begins with a brief history of RIPs, followed by the description of their distribution and classification. The transport of toxins which is a unique property of this class of proteins is discussed in detail and supported with appropriate figures. Also, information pertaining to the structure of abrin and apoptosis induced by RIPs is written in brief. In the second chapter of the thesis the structural and functional studies of Abrus agglutinin-I (APA-I) as compared to abrin are discussed. Abrin and APA-I share a high degree of homology, however, previous reports by Liu et al., indicate that APA-I is many fold less toxic in cell free systems as compared to abrin. In our studies, APA-I was found to be less toxic on cultured cell lines. The IC50 value of protein synthesis inhibition by abrin was found to be 0.4 ng/ml for both Jurkat and MCF-7 cell lines. A 20-1000 fold difference was observed in the sensitivity of these cell lines to APA-I. The extent of apoptosis induced by APA-I in A3I9.2 a caspases-8 mutant Jurkat variant cell line was comparable to abrin indicating that the apoptosis induction by APA-I might not be through the extrinsic pathway. instead, our studies showed that APA-I induced apoptosis followed the mitochondrial pathway of cell death, in a caspase dependent manner similar to that of abrin. Unlike other agglutinins like wheat germ agglutinin, the agglutinating ability of the agglutinin-I had no role in the apoptosis induced. Protein synthesis inhibition appeared to be mandatory for the apoptosis induced by APA-I. The reason for the decreased toxicity of agglutinin-I became apparent on the analysis of the crystal structure of agglutinin-I obtained by us in comparison to that of the reported structure of abrin. The substitution of Asn200 in abrin with Pro199 in agglutinin-I seems to be a major cause for the decreased toxicity. This perhaps is not a consequence of any kink formation by Pro residue in the helical segment, as reported by others earlier but due to fewer interactions that proline can possibly have with the bound substrate. Passive immuno-neutralization by administration of neutralizing antibodies is widely used as therapy against poisoning by various toxins. In case of type-II RIPs like ricin, antibodies to the toxic subunit were proven to have better protective efficacy than those to the lectin subunit. Neutralizing antibodies to abrin are not reported in literature. Therefore, a panel of monoclonal antibodies (mAbs) to the recombinant A chain of abrin was developed in our laboratory and characterized, which is presented in the third chapter of the thesis. Of these, D6F10 a high affinity antibody, exhibited neutralizing effect on abrin induced cytotoxicity on different cell lines tested. Antibodies may neutralize biological toxins in multiple ways; our studies suggested that mAb D6F10 interferes in the earliest event i.e. attachment of the toxin to the cell surface. Significantly, with the administration of mice with mAb D6F10 the prophylactic effect of the mAb could be demonstrated. In chapter 4, the sensitivity, kinetics of proteins synthesis inhibition and the mechanism of abrin induced cell death in cell lines of epithelial lineage is presented. Both sensitivity and kinetics of MCF-7/pv, Ovcar3, and T47D cells appeared comparable while, a variant culture of MCF-7 over-expressing caspases-3 was 50 times more sensitive to abrin. There was no significant difference in the binding of abrin between MCF-7/pv and MCF-7/C3+ cells. Previous studies in our laboratory indicated that abrin induced apoptosis is a caspases-3 dependent process. Also, in several systems it has been shown that caspases-3 is an indispensable molecule for apoptotic cell death. To test the absolute requirement of caspase-3, we examined abrin-induced apoptosis in a human breast cancer cell line MCF-7/pv reportedly deficient in caspases-3. Unlike other molecules like cisplatin, apoptosis induced by abrin in the MCF- 7/pv cells was found to be caspase -3 independent. However faster kinetics of apoptosis is observed, indicating that there is amplification of the apoptotic signals in the presence of caspases-3 resulting in an early onset of DNA fragmentation. The kinetics of protein synthesis inhibition and apoptosis follows similar kinetics in Jurkat cells while there is a time lapse between the two events in epithelial cells. Even with very high concentrations of abrin no detectable apoptosis was observed within 24 h in epithelial cells. The onset of fragmentation occurs after 24 h in the cell lines tested as opposed to Jurkat where it is observed as early as 6 h. Inhibition of caspases rescued the toxins from DNA fragmentation suggesting that the toxin does not cause direct nuclear damage in the cell line which does not involve the activation of caspases.

Isolée d'une bactérie, Streptomyces alboniger, la puromycine est un nucléoside antibiotique naturel présentant une analogie structurale avec l'adénosine terminale de l’extrémité 3’ de l'ARNt aminoacylé. Cette similarité confère à cette molécule la faculté de pouvoir s'insérer dans le site A (actif) du ribosome et d'inhiber la synthèse des protéines. Cependant, du fait de la formation d'un produit toxique lors de sa métabolisation, la puromycine n'a jamais été employée à des fins thérapeutiques chez l'homme. Néanmoins, utilisée en tant qu'outil synthétique, elle a largement contribué à une meilleure compréhension du mécanisme du transfert peptidique. Au travers de cette thèse, six analogues carbobicycliques (deux en série ribo et quatre en série 2'-désoxy), mimant de façon optimale les conformations extrêmes nord ou sud de la puromycine, ont été synthétisés puis testés dans le ribosome. Outre confirmer que la présence d'un groupement 2'-hydroxyle améliorait l'activité inhibitrice, ces expériences in vitro ont apporté une preuve que, dans le site actif, le déplacement de l'équilibre conformationnel du ribofuranose de l'adénosine terminale de l'ARNt aminoacylé – analogue structural de la puromycine – en faveur de son conformère nord pourrait être directement impliqué dans la catalyse ribosomale du transfert peptidique. Par ailleurs, un projet annexe sur le développement de nouveaux antipaludiques potentiels a permis la synthèse, en série xylo, de la puromycine et de son métabolite naturel le puromycine aminonucléoside. Ces composés ont été testés sur les souches 3D7 et Dd2 du parasite Plasmodium falciparum. / Puromycin, a natural antibiotic nucleoside isolated from the bacterium Streptomyces alboniger, has been used to approach and to clear up the understanding of the mechanism of protein biosynthesis. In fact, its structural similarity to the 3' terminal 3'-O-aminoacyl adenylate moiety of aminoacyl-tRNA explains its activity in the ribosomal A site causing the inhibition of the protein synthesis. Since its metabolism generates a toxic product, puromycin cannot be used as therapeutical purposes for humans. During this PhD work, six carbobicyclic analogues of puromycin, conformationally restricted into the northern or southern conformations with the help of a cyclopropane moiety, were synthesized (two ribo-derivatives and four in the 2'-deoxy ribo-series) then tested for pep¬tidyl transfer efficiency in ribosomes. In addition to confirming that the 2'-hydroxyl function is necessary to improve the inhibition properties, these enzymological tests brought an evidence that the conformational switching: southern to northern, occurring in the A site, could directly be involved in the ribosomal catalysis of the peptidyl transfer. Besides, a side project on the elaboration of potential antimalarial compounds provided new xylo-analogues of puromycin and its natural metabolite PAN. These derivatives were tested on the 3D7 and Dd2 strains of the Plasmodium falciparum parasite

Protein synthesis is a complex process performed by sophisticated cellular particles known as ribosomes. Although RNA constitutes the major structural and functional component, ribosomes from all kingdoms contain an extensive array of proteins with largely undefined functional roles. The work presented in this thesis addresses ribosomal complexity using mutants of Salmonella typhimurium to examine the physiological effects of ribosomal protein (r-protein) removal and orthologous replacement on bacterial fitness and ribosome function. The results of paper I demonstrate that removal of small subunit protein S20 conferred two independent translation initiation defects: (i) a significant reduction in the rate and extent of mRNA binding and (ii) a drastic decrease in the yield of 70S complexes caused by an impairment in subunit association. The topographical location of S20 in mature 30S subunits suggests that these perturbations are the result of improper orientation of helix 44 of the 16S rRNA when S20 is absent. In paper II we show that the major functional impairment associated with loss of large subunit protein L1 manifested as an increase in free ribosomal subunits at the expense of translationally active 70S particles. Furthermore, the formation of free ribosomal subunits was imbalanced suggesting that L1 is required to suppress degradation or promote formation of 30S subunits. Compensatory evolution revealed that mutations in other large subunit proteins mitigate the cost of L1 removal, in one case seemingly via an increase in 70S complex formation. As shown in paper III, the large fitness costs associated with complete removal of r-proteins is in contrast to the generally mild costs of orthologous protein replacement, even in the absence of a high degree of homology to the native protein. This clearly demonstrates the robustness and plasticity of the ribosome and protein synthesis in general and it also implies that functional constraints are highly conserved between these proteins. The findings of paper III also allowed us to examine the barriers that constrain horizontal gene transfer and we find that increased gene dosage of the sub-optimal heterologous protein may be an initial response to stabilize deleterious transfer events. Overall the results highlight the requirement of r-proteins for the maintenance of ribosomal structural integrity.

Les ribosomes sont des complexes ribo-nucléoprotéiques qui lisent l'ARNm pour synthétiser les protéines. Il a été démontré que les inhibiteurs de la synthèse des protéines eucaryotes ont un potentiel thérapeutique important pour traiter un large éventail de cancers humains. Récemment, nous avons publié une étude multidisciplinaire dans laquelle nous démêlons le mécanisme d'action du chlorolissoclimide (CL), un composé qui partage la similarité chimique avec le cycloheximide (CHX), mais qui présente une cytotoxicité inférieure prometteuse. Nous nous sommes intéressés aux nouveaux types d'interactions de CL avec le ribosome et nous avons donc résolu une nouvelle structure cristalline du complexe S.cerevisiae 80S en complexe avec un autre composé de lissoclimide à une résolution de 3.1 Å. Le couplage de structures à haute résolution avec la conception de médicaments axée sur le calcul et l'analyse FRET guidera davantage la conception d'inhibiteurs plus sélectifs et moins cytotoxiques. / Ribosomes are ribo-nucleoprotein complexes that read mRNA to synthesize proteins. This is important to consider in the case of cancer cells, which show a global increase in protein synthesis to support their hyper-proliferative behaviour. Inhibitors of eukaryotic protein synthesis have been shown to have significant therapeutic potential to treat a wide range of human cancers. Recently we published a multidisciplinary study in which we unravel the mechanism of action of chlorolissoclimide (CL), a compound sharing chemical similarity to cycloheximide (CHX), but showing promising lower cytotoxicity. We were interested in the new types of interaction of CL with the ribosome and therefore we solved a new crystal structure of the S.cerevisiae 80S in complex with another lissoclimide compound at 3.1 Å resolution. The coupling of high-resolution structures with computationally-driven drug design and FRET analysis will further guide the design of more selective and less cytotoxic inhibitors.

Submitted by Karin Goebel (karing@fiocruz.br) on 2014-11-25T18:05:56Z No. of bitstreams: 1 Dissertação Bruna Hilzendeger Marcon.pdf: 6455497 bytes, checksum: 8ea632ce91cdf16edd8b86a624972dba (MD5) / Approved for entry into archive by Karin Goebel (karing@fiocruz.br) on 2014-11-25T18:06:29Z (GMT) No. of bitstreams: 1 Dissertação Bruna Hilzendeger Marcon.pdf: 6455497 bytes, checksum: 8ea632ce91cdf16edd8b86a624972dba (MD5) / Made available in DSpace on 2014-11-25T18:06:29Z (GMT). No. of bitstreams: 1 Dissertação Bruna Hilzendeger Marcon.pdf: 6455497 bytes, checksum: 8ea632ce91cdf16edd8b86a624972dba (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / As células-tronco (CTs) caracterizam-se por possuírem a capacidade de se autorrenovar e de dar origem a um ou mais tipos celulares diferenciados. Nos últimos anos, diversos trabalhos mostraram a existência de CTs em tecidos adultos, tornando-as uma alternativa interessante para uso em terapias celulares. Contudo, para melhor utilizar as CTs, é preciso primeiramente compreender como ocorre a diferenciação em um tipo celular específico e, principalmente, como é regulada a expressão gênica durante este processo. Em 2009, Ingolia e colaboradores apresentaram uma nova técnica conhecida como ribosome profiling, a qual consiste no isolamento e sequenciamento em larga escala dos fragmentos de RNA associados e protegidos pelos ribossomos, os quais têm um tamanho aproximado de 30 nucleotídeos (conhecido com footprint ribossomal). Ao mapear as sequências obtidas, é possível obter informações não apenas sobre quais sequências estão sendo traduzidas, mas também sobre a cinética da tradução e sua extensiva rede de regulação. Assim, o objetivo deste trabalho foi aplicar a técnica de ribosome profiling ao estudo do processo de diferenciação de CTs adultas. Como modelo de estudo, foram utilizadas CTs obtidas de tecido adiposo antes (t=0) e após a indução para diferenciação adipogênica por 3 dias (t=72h). O primeiro passo do trabalho foi a adaptação do protocolo de ribosome profiling para o estudo de CTs adultas, o qual consiste na lise celular, digestão do lisado com uma RNA nuclease (a qual irá degradar o RNA exposto, preservando os fragmentos protegidos pelo ribossomo), ultracentifrugação do homogenato sobre colchão de sacarose 1 M para sedimentação dos ribossomos, extração de RNA e isolamento dos fragmentos de 30 nucleotídeos. Também foi feita extração do RNA poliA. As amostras foram sequenciadas (SOLiD™) e os dados obtidos foram triados e mapeados contra um banco de dados de RNAm, utilizando-se a ferramenta CLC Genomics Workbench. Foram identificados mais de 8.000 transcritos para as amostras de ribosome profiling e mais de 17.000 para as de poliA. Ao calcular o fold change entre as condições t=0 e t=72h, foi possível verificar que mais de 50% dos genes foram detectados como diferencialmente expressos apenas por ribosome profiling. Observou-se que genes relacionados com vias de diferenciação adipogênica e de metabolismo de lipídeos encontravam-se regulados positivamente em ambas as amostras de RNA. Por outro lado, observou-se que vias de regulação do citoesqueleto de actina e de adesão focal estavam reguladas negativamente apenas nas amostras de ribosome profiling. Isso é interessante, uma vez que a inibição destas vias já foi descrita como importante para o processo de adipogênese. Além disso, foi observada uma forte redução na eficiência de tradução de genes relacionados com a tradução após 72 horas de indução para diferenciação. Os resultados obtidos no presente trabalho reforçam as evidências de que os mecanismos de regulação pós-transcricionais e traducionais têm um papel muito importante na regulação da diferenciação celular de CTs, sendo que a técnica de ribosome profiling permitiu obter informações mais detalhadas de como este processo pode estar acontecendo. / Stem cells (SC) are characterized by their capacity of both self-renewing and giving rise to new differentiated cells. SC are found in adult tissues, which are considered a putative source for cell therapy. However, little is known about the mechanisms involved in the trigger of SCs differentiation into a specific cell type. Understanding adult SCs differentiation process is a fundamental step to better use and to take advantage of their potential. In 2009, Ingolia and collaborators presented a new methodology of transcriptome analysis named ribosome profiling, which consists on the isolation and deep-sequencing of the mRNA fragments enclosed by ribosomes. When lysed cells are submitted to nuclease digestion, unprotected mRNA is degraded, while fragments within ribosomes are preserved and have a known footprint of 30 nucleotides. Sequencing these ribosome-protected fragments results in a high-precision measurement of in vivo translation, providing precise information about translation kinetics and its extensive regulation. The objective of this work was to apply the ribosome profiling methodology to the study of adipogenic differentiation in adult SCs. SCs were isolated from human adipose tissue from three donors and were cultured in a control medium (t=0) and induced to adipogenic differentiation for 72 hours (t=72h). The first step was to adapt and optimize the ribosome profiling protocol to the SC model, which consists in cell lysis, cell lysate digestion by nuclease (to degrade unprotected RNA, preserving ribosome-protected fragments), ultracentrifugation over a 1M sucrose cushion to pellet ribosomes, RNA extraction and 30 nucleotides fragments isolation. poliA RNA was also isolated. Samples were submitted to deep-sequencing (SOLiD™) and the reads obtained were trimmed and mapped onto the reference mRNA database using the CLC Genomics Workbench. Over 8000 transcripts were identified in ribosome profiling samples and over 17000 in poliA samples. Fold change analysis between t=0 and t=72h of both RNA samples showed that differential expression of more than 50% of the genes was identified only by ribosome profiling. Pathways related to adipogenesis and lipid metabolism were upregulated in both RNA samples. However, regulation of the actin cytoskeleton and focal adhesion proteins were downregulated only in ribosome profiling samples. Interestingly, downregulation of these pathways was already described as an important phenomenon to cell adipogenesis. Besides, we observed a strong reduction of translational efficiency of genes involved in translation at t=72h. Our results reinforce previous data, suggesting that posttranscriptional and translational regulation play a fundamental role in the regulation of SC differentiation process and that ribosome profiling is an important tool to better understand this process.