Les ARN constituent des cibles thérapeutiques extrêmement intéressantes bien qu’encore assez peu exploitées. En effet, les obstacles pour la conception de ligands spécifiques de ces cibles non traditionnelles, polyanioniques et très flexibles, sont encore loin d’être levés. Les aminoglycosides, utilisés depuis longtemps pour leurs propriétés antibiotiques, sont souvent décrits comme des « ligands universels » d’ARN. Leur structure constitue donc une architecture favorable pour l’élaboration de nouveaux ligands spécifiques des ARN.Le but de cette thèse a été de développer une méthode systémique originale combinant chimie organique et microbiologie pour synthétiser de nouvelles molécules de structure analogue aux aminoglycosides, se fixant de façon spécifique sur des cibles ARN. Ce travail repose sur la compréhension récente des voies de biosynthèse des aminoglycosides permettant leur ingénierie rationnelle selon une stratégie de mutasynthèse. Cette approche expérimentale s’appuie sur la conception de mimes de métabolites naturels pouvant être transformés par des bactéries génétiquement modifiées. Le développement de méthodologies novatrices en ingénierie métabolique, synthèse organique et chimie analytique nous a permis de concevoir des analogues d’aminoglycosides fluorés qui se sont avérées être d’excellentes sondes dans l’étude des ARN par RMN du fluor. / Pas de résumé en anglais

Objectives: The discovery of new antibiotic targets is important to stem the increase in antibiotic resistance to most currently used antimicrobials. The bacterial ribosome is a major target for a large number of antibiotics that inhibit different aspects of translation. Most of these antimicrobial agents also inhibit ribosomal subunit formation as a second cellular target. Precise subunit assembly requires the activity of several distinct RNases for proper rRNA processing. The present work shows that the vanadyl ribonucleoside complex (VRC) inhibited RNases in Staphylococcus aureus involved in ribosomal subunit formation without an effect on translation. Methods: Methicillin-susceptible and -resistant strains of S. aureus were examined for the inhibitory effects of VRC on cell viability by colony counting. Protein synthesis rates were measured by isotopic methionine incorporation. Ribosome synthesis was measured by radiolabelled uridine incorporation into ribosomal subunits as displayed on sucrose gradients. Pulse and chase radiolabelling was used to measure subunit synthesis rates. RNA turnover was determined by a gel on a chip assay. Results: The rates of subunit synthesis and the amounts of both subunits were significantly reduced in the presence of the compound. Ribosomal RNA was degraded and cell viability was reduced as a consequence. VRC also stimulated the inhibitory effects of a macrolide and an aminoglycoside antibiotic on ribosome formation. Conclusions: Bacterial ribosomal subunit synthesis was specifically impaired in VRC-treated cells, with the rates and amounts of both subunits reduced. Cell viability was significantly reduced and rRNA turnover was stimulated.

We tested 42 strains of amikacin-resistant gram-negative bacilli with amikacin in combination with six beta-lactam antibiotics using the checkerboard and time kill curve techniques. Synergism was demonstrated with time-killing curve in 43-68% of the strains tested. Ceftazidime plus amikacin was the most active combination by the checkerboard technique, while amikacin-cefoperazone was the most active combination by the time-killing curve technique against Pseudomonas aeruginosa. Discrepancies were found between the results of the two methods used.

Macromolecular complexes of anionic-nonionic block copolymers and cationic antibiotic aminoglycosides have been formed by electrostatic condensation. Amphiphilicity of the complexes was introduced into the shells by incorporating a hydrophobic poly(propylene oxide) segment into the block copolymer. The resulting particles have an average hydrodynamic diameter of ~ 200 nm and contain up to 30-40 % of the drug payload. In vitro efficacies of such nanostructures in reduction of intracellular pathogens like Salmonella, Listeria, and Brucella were demonstrated. Current effort focuses on translation of this nano-drug delivery concept to in vivo model of intracellular infectious diseases. Atom transfer radical polymerization (ATRP) was utilized to prepare well-defined polymeric dispersion stabilizers that readily adsorb onto metal oxide surfaces. Two unimolecular bis(phosphonate) ATRP initiators were designed and prepared in good yield. These special initiators were successfully used to initiate polymerization of poly(N-isopropylacrylamide) (PNIPAM) in a controlled manner yielding PNIPAM with a bis(phosphonate) moiety at one terminus. The polymers readily adsorbed onto magnetite nanoparticle surfaces, thus creating thermosensitive magnetic nanostructures that form nanosized clusters upon heating above the lower critical solution temperature of PNIPAM. It is envisioned that modularity of this approach, relying on the applicability of ATRP to polymerize a vast array of monomers, could be used to prepare a library of polymeric shells for magnetic iron oxide nanoparticles. Medical intervention in drug delivery that includes detectability of drug carriers is greatly desirable. A real-time assessment of disease prognosis could be highly beneficial for developing personalized treatment strategies. As an example of this conceptual innovation, block ionomer functionalized magnetite complexes were synthesized and investigated as carriers for delivery of aminoglycosides into phagocytic cells for treatment of intracellular bacterial infections. The ionic block of copolymer contains multiple carboxylates for binding onto the iron oxide surface. The remaining unbound carboxylate anions were used to complex with cationic gentamicin in nanoshells of these complexes. The iron oxide particle core provides an imaging modality and serves as a pseudo-crosslinking site to enhance stabilities of the polyelectrolyte complexes, thus preventing them from disintegrating in the physiological environment. Currently, these hybrid complexes are being investigated in possible pharmaceutical formulations to eradicate intracellular pathogens in animal models. / Ph. D.

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.

Entre 10% et 30% des maladies humaines sont liées à l'apparition d'une mutation non-sens (PTC). La synthèse protéique est alors arrêté prématurément. Cet arrêt peut être inhibé par des molécules inductrices de translecture qui permettent l’incorporation d’un ARNt suppresseur naturel au niveau du PTC (translecture). Le ribosome peut alors franchir le PTC et restaurer l’expression de la protéine.Au cours de ma thèse, je me suis intéressé à la suppression des codons stop en caractérisant de nouvelles molécules inductrices de translecture et en analysant les mécanismes de la fidélité de la traduction.J’ai tout d’abord mis au point un système de criblage innovant avec lequel j’ai testé plus de 17 000 molécules et identifié la molécule TLN468. J’ai pu mettre en évidence que cette molécule est capable d’induire la réexpression d’une protéine p53 active.J'ai aussi caractérisé de nouveaux composés dérivés d’aminoglycosides. J’ai pu montré que le NB124 est capable d’induire l’apoptose de cellules tumorales via la réexpression de la protéine p53 tout ayant une toxicité bien plus faible que la gentamicine.En parallèle, j’ai développé une approche en molécule unique permettant d’étudier les erreurs programmées du ribosome (recodage). J’ai ainsi pu analyser la cinétique d’élongation des ribosomes eucaryotes et montré que l’initiation de la traduction sur un site d’entrée interne (IRES) ralentit le ribosome lors des premiers cycles d’élongation. / Nonsense mutations, also known as premature termination codons (PTCs) are responsible for 10% to 30% of all human genetic diseases. Nonsense translation suppression can be induced by readthrough inducers. The presence of such PTC leads to premature translation termination. These stop therapeutic strategies have emerged which attempt to use molecules that facilitate tRNA incorporation at the PTC (readthrough). The, translation continue in the same reading frame until the next stop codon. I first developed an innovative screening system I used to test more than 17,000 molecules and have identified one hit, TLN468 molecule. I have shown that this molecule is able to induce re-expression of an active p53 protein.I also characterized new compounds derived from aminoglycosides. I have shown that the NB124 induces apoptosis of tumor cells by re-expressing p53 protein while having a much lower toxicity than gentamicin.I developed a single molecule approach for studying the ribosome programmed errors (recoding). I was able to analyze the kinetics of elongation eukaryotic ribosomes and showed that the initiation of translation at an internal entry site (IRES) slows the ribosome during the first elongation cycle.

Les antibiotiques sont des polluants présents dans les écosystèmes aquatiques, réceptacles ultimes des substances anthropiques. L’étude de ces composés porte sur leur rémanence dans le milieu ou leurs effets sur des organismes naturels. De nombreux efforts ont été faits à l’échelle mondiale pour l’évaluation de la qualité environnementale des différentes ressources en eau pour la survie des espèces aquatiques mais aussi pour la consommation humaine et le risque sanitaire lié. Dans ce but, l’optimisation des techniques analytiques pour ces composés dans les systèmes aquatiques demeure une nécessité. Notre objectif est de développer des méthodes d’extraction et de détection pour 12 molécules appartenant à la famille des aminoglycosides et de la colistine dans les eaux des stations d’épuration et les eaux hospitalières. L’absence des méthodes d’analyse pour ces composés ainsi que le manque des études permettant leur détection dans l’eau sont les raisons de leur étude. L’Extraction sur Phase Solide (SPE) en mode classique (hors ligne) ou en ligne, suivie d’une analyse par la Chromatographie Liquide couplée à la Spectrométrie de Masse (LC/MS/MS) est la méthode la plus couramment employée pour ce type d’analyse. Les paramètres sont optimisés et validés afin d’assurer les meilleures conditions utilisées dans les analyses environnementales. Cette technique a été appliquée sur des échantillons réels des eaux des stations d’épuration à Bordeaux et au Liban. / Antibiotics are pollutants present in aquatic ecosystems ultimate receptacles of anthropogenic substances. These compounds are studied as their persistence in the environment or their effects on natural organisms. Numerous efforts have been made worldwide to assess the environmental quality of different water resources for the survival of aquatic species, but also for human consumption and health risk related. Towards goal, the optimization of analytical techniques for these compounds in aquatic systems remains a necessity. Our objective is to develop extraction and detection methods for 12 molecules of aminoglycosides and colistin in sewage treatment plants and hospitals waters. The lack of analytical methods for analysis of these compounds and the deficiency of studies for their detection in water is the reason for their study. Solid Phase Extraction (SPE) in classic mode (offline) or online followed by Liquid Chromatography analysis coupled with Mass Spectrometry (LC/MS/ MS) is the most method commonly used for this type of analysis. The parameters are optimized and validated to ensure the best conditions for the environmental analysis. This technique was applied to real samples of wastewater treatment plants in Bordeaux and Lebanon.

En réanimation, les facteurs de risque d’infection à bactéries résistantes sont nombreux et il est nécessaire d’instaurer une antibiothérapie rapide et adéquate. Cela conduit donc souvent au choix empirique d’antibiotiques à large spectre. Ce travail de thèse regroupant quatre études porte sur les éléments de ce choix. Dans la première étude, les critères de "pneumopathie associée aux soins" sont discutés. Dans la seconde, il est rapporté que le traitement antibiotique prescrit au début du séjour en réanimation est associé à l’acquisition de Pseudomonas aeruginosa. Dans le diagnostic d’une pneumopathie acquise sous ventilation, la troisième étude décrit une technique rapide d’antibiogramme permettant une désescalade antibiotique plus précoce. La néphrotoxicité des aminoglycosides dans le traitement empirique des patients en sepsis sévère est présentée dans la dernière étude. Ces travaux participent à la bonne gestion des antibiotiques à la phase empirique du traitement des infections sévères en réanimation. / Intensive care units (ICU) are a niche for risk factors of infection due to multidrug resistant bacteria. ICU patients are in a need for a rapid and adequate antibiotic therapy. This leads ICU physicians to use empirical broad spectrum antibiotics. This thesis comprises four studies which focus on the empirical step of the treatment. In the first study, the criteria for "health-care-associated pneumonia" are discussed. The second shows that the antibiotic selection pressure administered early during the ICU stay could lead to Pseudomonas aeruginosa acquisition. In the third study, a rapid direct specimen testing method was assessed for ventilator-associated pneumonia diagnosis in order to hasten antibiotic de-escalation. Finally, a review on aminoglycosides’ nephrotoxicity in the severe sepsis setting represents the fourth study. These studies bring a loop forward into the understanding of the antibiotic stewardship of patients with severe sepsis, with particular focus on the empirical antibiotic treatment.

Doctor Educationis / Ototoxic induced hearing loss is a common adverse event related to aminoglycosides used in Multi Drug Resistant -Tuberculosis treatment. Exposure to ototoxic drugs damages the structures of the inner ear. Symptomatic hearing loss presents as tinnitus, decreased hearing, a blocked sensation, difficulty understanding speech, and perception of fluctuating hearing, dizziness and hyperacusis/recruitment. The World Health Organization (1995) indicated that most cases of ototoxic hearing loss globally could be attributed to treatment with aminoglycosides. The aim of the study was to determine the proportion of DR-TB patients initiated on treatment at three decentralized sites during a defined period (1st October to 31st December 2015) who developed ototoxic induced hearing loss and the corresponding risk factors, whilst receiving audiological monitoring with an extended high frequency audiometer (KUDUwave). A retrospective cross-sectional study was conducted. Cumulatively across the three decentralized sites, 69 patient records were reviewed that met the inclusion criteria of the study. The mean age of the patients was 36.1, with a standard deviation (SD) of 10.7 years; more than half (37) were female. Ototoxicity , a threshold shift, placing patients at risk of developing a hearing loss was detected in 56.5% (n=39)of patients and not detected in 30.4%(n=21).The remaining 13,1% (n=9)is missing data. As a result, the regimen was adjusted in 36.2% of patients. . From the 53 patients who were tested for hearing loss post completion of the injectable phase of treatment, 22.6% (n=12) had normal hearing, 17.0 % (n=9) had unilateral hearing loss, and 60.4% (n=32) had bilateral hearing loss. Therefore, a total of 41 patients had a degree of hearing loss: over 30% (n=22)had mild to moderate hearing loss, and only about 15% (n=11)had severe to profound hearing loss. Analysis of risk factors showed that having ototoxicity detected and not adjusting regimen significantly increases the risk of patients developing a hearing loss. The key findings of the study have shown that a significant proportion of DR-TB patients receiving an aminoglycoside based regimen are at risk of developing ototoxic induced hearing loss, despite receiving audiological monitoring with an extended high frequency audiometer that allows for early detection of ototoxicity (threshold shift).

Este trabalho propõe estudos sobre a caracterização do comportamento térmico dos aminoglicosídeos estreptomicina e tobramicina, que são antibióticos bactericidas, utilizados no combate a microorganismos patogênicos, que agem interrompendo a síntese de proteínas. Após a caracterização espectroscópica dos analitos, foram realizadas medidas termogravimétricas, em atmosfera de ar e nitrogênio, que possibilitaram a determinação da estabilidade térmica do fármaco e o reconhecimento das etapas de decomposição. A calorimetria exploratória diferencial forneceu informações a respeito de processos físicos com variação de entalpia. Os gases envolvidos foram analisados usando termogravimetria acoplada à espectroscopia vibracional na região do infravermelho (TG-FTIR), possibilitando a proposta de um mecanismo para sua decomposição térmica. Intermediários de decomposição térmica foram caracterizados por CG-MS e o conjunto de todas essas informações forneceu um possível mecanismo para o comportamento térmico dessas drogas. Também foi sintetizado, caracterizado e analisado por TG, DSC e TG-FTIR, o complexo de tobramicina com o íon Cu+2. / A study regarding the thermal behavior characterization of the aminoglycosides streptomycin and tobramycin which are bactericidal antibiotics is presented. These antibiotics are widely against pathogenic microorganisms and act by interrupting the synthesis of proteins. Thermogravimetric measurements were performed under air and nitrogen conditions. To evaluate thermal stability of the drugs and their decomposition steps. A differential scanning calorimetry provided information on physical processes with enthalpy change. Evolved gas analysis was performed using thermogravimetry coupled to infrared spectroscopy (TG-FTIR), and was used to characterize the gases released during the thermal heating of the samples. Decomposition intermediates were characterized by CG-MS and the set of all these resultsallowed the proposition of a mechanism for the thermal behavior of drugs. The complex of tobramycin with the Cu+2 ion was also synthesized, characterized and analyzed by TG, DSC and TG-FTIR.