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<p>Ribosome biogenesis is a major metabolic expense of bacteria and a promising target for antibacterial drug discovery. <em>Trans-</em>acting proteins, called ribosome biogenesis factors, aid this complex and cooperative process. EngA (YfgK, Der) is a widely distributed bacterial GTPase that is shown here to be important for normal ribosome biogenesis. EngA is an attractive antibacterial target because it is essential for viability in bacteria but is absent in humans.</p> <p>The GTPase activity and cellular function of EngA was investigated in <em>Escherichia coli</em>. Depletion of EngA caused accumulation of 30S and 50S ribosomal subunits at the expense of 70S ribosomes, showing for the first time that EngA is important for normal ribosome biogenesis. Mutation of either of the tandem GTPase domains of EngA led to abnormal ribosome profiles, cell death and loss of GTPase activity, revealing that the two GTPase domains act cooperatively to carry out an essential function. EngA bound the 50S subunit of the ribosome in cells and <em>in vitro</em>. Depletion of EngA resulted in sensitization to aminoglycoside antibiotics, which bind at the aminoacyl-tRNA binding site of ribosomes. To search for an inhibitor of ribosome biogenesis, a high-throughput screen of the GTPase activity of EngA was developed. A specific inhibitor was not identified, however, this robust screen can be extended to other compound libraries. Thus, we showed that the GTPase domains of EngA have a cooperative function in ribosome biogenesis, probably in maturation of the 50S subunit, and that EngA is an amenable target for further inhibitor screens.</p> / Doctor of Philosophy (PhD)

Post-transcriptional control of gene expression in Eukaryotes plays a pivotal role in determining intricated networks defining physiological and pathological conditions among each organism. RNA Binding Proteins (RBPs), by exploiting RNA-protein and protein-protein interactions, have been recognized as the main actors in modulating these processes. As a consequence, RBPs aberrant expression, modulation or mis-localization, leads to the insurgence of complex phenotypes and diseases. Therefore, targeting and modulating the activity of RBPs found associated to different pathologies represents a new promising therapeutic strategy. During my PhD I aimed at identify, characterize and refine inhibitors targeting the RNA binding protein HuR. HuR belongs to the ELAVL protein family, it is ubiquitously expressed in the cells and among tissues and highly conserved throughout mammalian evolution. By binding AU/U rich elements (ARE) in the 3’UTRs of mRNAs, HuR mainly stabilizes its target transcripts, enhancing their translation. ARE sequences are found in 7% of the human mRNAs, coding for protein involved in key cellular processes as: immune response and inflammation, cell division and proliferation, angiogenesis, senescence and apoptosis. Hence, dysregulation in HuR expression and in its subcellular localization have been associated with the insurgence of several pathologies, mostly cancers and inflammation diseases. Notably, malignant transformations and poor prognosis in patients have been found characterized by highly nuclear or cytosolic HuR expression in a significant number of human cancers. Indeed, the majority of HuR regulated transcripts encode for protein responsible for the appearance of several cancerogenic traits. In particular, critical crosstalk established between cancer cells and inflammation processes play a pivotal role in worsening and compromising cancers development and onset. Moreover, considering that 90% of mRNAs coding for cytokines and chemokines contains repeated AREs sites in the 3’UTR, HuR plays a strong regulatory role in immune system (innate and adaptive) development and homeostasis as well as in pathogenic mechanisms. The searching for HuR inhibitors represents a challenging area, in the drug discovery field, due to its pleiotropic functions and its intrinsic structural complexity, which presents unfolded regions and sequences prone to aggregation. HuR disruptors have been reported in the literature, but without systematic studies, thus the identification of a new class of small molecules is still at the beginning. Among the molecules discovered so far, in 2015 our group identified through a High-throughput Screening a natural compound, DHTS, as a bona fide HuR inhibitor. Following that finding, we, me included, ascribed to the molecule a well-defined mechanism of action, identifying the specific binding sites on which HuR:DHTS interaction is based, defining that upon the mRNA binding DHTS interplays with HuR maintaining the protein in a closed conformation, thus inhibiting its function. Furthermore, we demonstrated DHTS anti-cancer activity in vitro, in cellular context and in vivo, in an HuR-dependent manner. In this way, DHTS represented the molecular scaffold, for the generation of a new class of highly potent HuR inhibitors, called Tanshinone Mimics (TMs). A functional oriented approach was applied for the synthesis of new molecules harboring only DHTS chemical elements responsible for HuR targeting, leading to a completely new molecular scaffold, not previously described in the literature, with respect to the ancestor molecule. I have characterized and identified more potent molecules, describing their anticancer properties, through the evaluation of their capabilities of downregulating the total expression level of well-known HuR targets, coding for proteins involved in tumor insurgence and progression, as VEGF, ERBB2 and CTNNB1, and reducing cancer cell migration, cell cycle progression in a minor extent. On the other end, I have explored TMs anti-inflammatory properties, counteracting the inflammatory response mediated by macrophages, directly impairing the binding between HuR and its pro-inflammatory targets, diminishing their expression and related protein secretion. Moreover, I have put evidences on TMs activity in vivo in acute inflammation mouse models. Lastly, I have evaluated TMs activity in affecting T-cells proliferation, on which HuR it is known to play a regulatory role. In conclusion, we identified TMs with Structure-Activity Relationships (SARs) towards HuR inhibition and its biological implications, aimed at ameliorating their specificity and bioavailability suitable for in vivo therapeutic strategies.

La découverte d’un candidat médicament repose sur l’identification de hits, présentant des propriétés physico-chimiques adéquates pour leur optimisation. Le criblage à haut débit et l’approche par fragments sont deux techniques couramment utilisées lors de cette étape d’identification et elles ont été mises en œuvre au cours de ma thèse dans le but de découvrir de nouveaux composés ciblant d’une part le complexe CD81/CLDN-1 pour empêcher l’entrée du virus de l’hépatite C (VHC) dans les hépatocytes et d’autre part EthR2, un régulateur transcriptionnel mycobactérien, afin de potentialiser l’activité d’un antituberculeux sur les souches résistantes de M. tuberculosis.Dans une première partie, un criblage à haut débit sur le complexe CD81/CLDN-1 a permis d’identifier des modulateurs en série thiéno[2,3-c]pyrazole. Ces composés ont été pharmacomodulés et un composé spécifique de l’étape d’entrée du VHC, non toxique et présentant une activité submicromolaire a pu être ainsi identifié. Cette sonde pharmacologique permettra de mieux comprendre les mécanismes impliqués dans le processus d’entrée virale.Dans une deuxième partie, nous nous sommes intéressés à la conception de nouveaux fragments dits privilégiés. Ainsi, le développement des voies de synthèse, sous irradiation micro-onde, de deux entités moléculaires, le noyau 1,4-benzodiazepine-2,5-dione et le noyau spirohydantoïne, nous a permis d’obtenir 34 composés originaux. Afin d’évaluer le potentiel de cette stratégie, une librairie virtuelle de fragments a été générée et son criblage in silico sur la protéine MDM2 a été effectué. La mesure in vitro de l’activité des hits identifiés permettra de valider l’intérêt de cette approche pour la découverte de nouveaux ligands ciblant les interactions protéine-protéine.Dans une troisième partie, des inhibiteurs d’un répresseur transcriptionnel mycobactérien impliqué dans la potentialisation de l’activité de l’éthionamide ont été développés. A l’issue d’un criblage de 960 fragments, l’identification d’un hit en série tropinone, et sa cocristallisation avec la protéine EthR2, a permis d’entamer une optimisation rationnelle qui a conduit à l’obtention rapide de composés présentant de meilleures activités. / The discovery of drug candidates is based on the identification of hits with appropriated physico-chemical properties for further development. High throughput screening and fragment-based drug discovery approaches are two strategies commonly used for this identification. These strategies were applied during my PhD research work for identifying not only new modulators of the CD81/CLDN-1 complex to prevent entry of the Hepatitis C virus (HCV) into hepatocytes but also inhibitors of the mycobacterial transcriptional repressor, called EthR2, to boost ethionamide antibacterial activity against resistant strains of M. tuberculosis.Firstly, a high throughput screening assay was developed to identify molecules bearing a thieno[2,3-c]pyrazole scaffold that modulate the CD81/CLDN-1 complex. The structure-activity relationships allowed us to design and synthesize one non-toxic compound that inhibits viral entry with an IC50 in the submicromolar range. This best analog will be used as pharmacological tool to understand the molecular mechanism involving the CD81/CLDN-1 interaction during virus entry.Secondary, we worked on the design and synthesis of a new generation of fragments called privileged fragments. We focused our interest on the 1,4-benzodiazepine-2,5-dione and spirohydantoin scaffolds and using microwave-assisted conditions 44 original privileged fragments have been synthesized. To further illustrate the potential of our privileged fragments, a virtual focused library has been generated and screened in silico on MDM2 protein. The in vitro evaluation of the identified hits will allow us to validate our approach and to show the potential of our privileged fragments for the discovery of new hits against protein-protein interactions.Finally, inhibitors of a new mycobacterial transcriptional repressor involved in the boosting of ethionamide activity have been developed. Screening of 960 fragments allowed us to identify a hit bearing a tropinone scaffold which was cocrystallized with EthR2. A rational design from this cocrystal structure led rapidly to more potent ligands.

The thesis is focused on high-temperature superconducting (HTS) ceramics series of bismuth. Specifically, there is studied Bi2Sr2CaCu2O8+x phase, known as the 2212 phase. The theoretical part describes the basic properties of superconductivity and superconductors, the properties of cuprate ceramics and description of LBCO, YBCO and BSCCO structures. The experimental part deals with the preparation of the precursor own Bi-2212 phase. It is synthesized by a process called sol-gel. Feedstock Bi2O3, CaCO3, SrCO3 and CuSO4 was dissolved in HNO3 and transferred to a complex with ethylenediaminetetraacetic acid (EDTA, Chelaton II). With NH3 pH > 9 was maintained due to the stability of complex. The obtained gel was concentrated, calcinated in a furnace at 860 °C and crushed into powder. Pure powder was subjected to analysis dipping microscope, SEM, XRD, FT-IR, TG-DTA at different atmospheres argon, oxygen and air. Then 1 wt.% of the oxide (La, Y, Sc, Sm) was added to part of the powder precursor and the samples were examined using TG-DTA.

The diploma thesis is focused on possible customs issues during order processing in the manufacturing company. The theoretical part of this thesis contains definition of the world trade basic terms and a description of the customs clearance process. In the practical part, there is a description of the ordering procedure with attention to customs issues and rules in international trade. It includes a project draft which should provide the company with the improvement of the monitored indicators such as the quantity of delayed deliveries for distribution centers or the number of customs inspections.

Le thème principal de cette thèse est la caractérisation des supraconducteurs à haute température critique (SHTc). Dans un premier temps, nous avons présenté des généralités des SHTc. L'utilisation possible dans l'avenir, des SHTc pour le transport de courant, nous a mené à étudier plus particulièrement les pertes en champ propre, donc parcouru par un courant sinusoïdal. Puis nous avons rappelé les calculs de pertes basés sur le modèle de l'état critique Bean pour différentes formes d'échantillon, comme une plaque, un cylindre, un tube cylindrique et un câble SHTc. Dans un deuxième temps, nous avons caractérisé des SHTc qui permet d'obtenir les caractéristiques E(J), U(I), Jc(B), et n(B) d'un tube cylindrique SHTc. La caractérisation a été effectuée à l'aide de la méthode électrique. Puis nous avons tenté la compensation du champ magnétique propre du tube par deux méthodes différentes. Ensuite nous avons mesuré la diffusion du champ magnétique dans une plaque SHTc et de la détermination du Jc de la plaque par la mesure de champ de pénétration complète. Dans un dernier temps, nous avons calculé analytiquement des pertes dans un tube SHTc en champ propre, à l'aide du modèle de l'état critique de Bean. Nous avons également montré qu'en champ propre, la pénétration du champ magnétique à l'intérieur du matériau SHTc, se passe en deux temps. Tout d'abord il y a pénétration incomplète du champ magnétique de l'extérieur vers l'intérieur du matériau, puis quand la pénétration est complète, le champ magnétique augmente uniformément dans tout le matériau. Ces résultats de calcul de pertes ont été comparés aux celles mesurées, cette comparaison montre une concordance manifeste. Pour la dernière expérience, nous avons mesuré et analysé des pertes dans une bobine SHTc alimentée en courant sinusoïdal de fréquence 50 Hz. Ces résultats nous ont montré que les pertes dans cette bobine sont principalement les pertes dans le matériau supraconducteur et non les pertes dans la matrice des supraconducteurs / The main of this thesis is the characterization of high critical temperature superconductors (HTS). First, we have presented the generality of the HTS. The possible use in the future, of the HTS for the transport current, involves to study more particularly the losses in self-field, or fed by a sinusoidal current. Then we have recalled the losses calculations based on the Bean model critical state for various forms of the sample, as a plate, a cylinder, a tube cylindrical hollow and an HTS cable. For the second time, we have characterized the HTS which allows make the characteristics E(J), U(I), Jc(B), and n(B) of an HTS tube cylindrical hollow. The characterization has been made by the electrical method. Then we have tried a self-field compensation of an HTS tube by two different methods. After we have measured the magnetic field diffusion in an HTS plate and we have determined its Jc by the magnetic field measurement in complete penetration. In the last time, we have calculated analytically the losses in self-field of the HTS tube, using the Bean model critical state. We have also showed that in self-field, the magnetic field penetration inside the HTS material happens in order. First of all, there is magnetic field incomplete penetration from outside to inside the material, then when the penetration is complete, the magnetic field increase uniformly throughout the material. These losses calculation results have been compared to measurement results, this comparison shows a clear coincidence. For the last experiment, we have measured and analysed the losses in an HTS coils fed by a sinusoidal current 50Hz frequency. These results have showed that the losses of this HTS coils are mainly losses in the superconducting material and not in the superconducting matrix.

Bacterial infections are again becoming difficult to treat because the microbes are growing increasingly resistant to the antibiotics in use today. The need for novel antimicrobial compounds is urgent and to achieve this new targets are crucial. In this thesis we present a strategy for identification of such targets via a bioinformatics approach. In our first study we compared proteins with unknown and hypothetical function of the spirochete Treponema pallidum to five other pathogens also causing chronic or persistent infections in humans (Yersinia pestis, Neisseria gonorrhoeae, Helicobacter pylori, Borrelia burgdorferi and Streptococcus pneumoniae). T. pallidum was used as a starting point for the comparisons since this organism has a condensed genome (1.1 Mb). As we aimed at identifying conserved proteins important for in vivo survival or virulence of the pathogens we reasoned that T. pallidum would have deleted genes not important in the human host. This comparison yielded 17 ORFs conserved in all six pathogens, these were deleted in our model organism, Yersinia pseudotuberculosis, and the virulence of these mutant strains was evaluated in a mouse model of infection. Five genes were found to be essential for virulence and thus constitute possible antimicrobial drug targets. We have studied one of these virulence associated genes (vags), vagH, in more detail. Functional and phenotypic analysis revealed that VagH is an S-adenosyl-methionine dependent methyltransferase targeting Release factor 1 and 2 (RF1 and RF2). The analysis also showed that very few genes and proteins were differentially expressed in the vagH mutant compared to wild-type Yersinia. One major finding was that expression of the Type III secretion system effectors, the Yops, were down regulated in a vagH mutant. We dissected this phenotype further and found that the down regulation was due to lowered amounts of the positive regulator LcrF. This can be suppressed either by a deletion of yopD or by over expression of the Ribosomal Recycling Factor (RRF). These results indicate that YopD in addition to its role in translational regulation of the Yops also plays a part in the regulation of LcrF translation. We suggest also that the translation of LcrF is particularly sensitive to the amount of translation competent ribosomes and that one effect of a vagH mutation in Y. pseudotuberculosis is that the number of free ribosomes is reduced; this in turn reduces the amount of LcrF produced thereby causing a down regulation of the T3SS. This down regulation is likely the cause of the attenuated virulence of the vagH mutant. Finally, we set up a high throughput screening assay to screen a library of small molecules for compounds with inhibiting the VagH methyltransferase activity. Five such compounds were identified and two were found to inhibit VagH also in bacterial culture. Furthermore, analogues to one of the compounds showed improved inhibitory properties and inhibited the T3SS-dependent cytotoxic response induced by Y. pseudotuberculosis on HeLa cells. We have successfully identified five novel targets for antimicrobial compounds and in addition we have discovered a new class of molecules with antimicrobial properties.