Configuración e implementación de servicios de infraestructura IT, bajo Zentyal Server

En este artículo se busca abordar la importancia de Zentyal Server como plataforma para la administración de la Infraestructura IT dentro de una organización que quiera estar a la vanguardia tecnológica, donde se explicará de manera técnica la implementación de esta importante herramienta de acuerdo a lo solicitado por el curso e identificando los posibles errores.This article seeks to address the importance of Zentyal Server as a platform for the administration of IT Infrastructure within an organization that wants to be at the forefront of technology, where the implementation of this important tool will be explained in a technical manner according to what was requested by the course and identifying possible errors

Caractérisation structurale de TDP-43, une protéine de liaison à l’ARN, impliquée dans la Sclérose Latérale Amyotrophique (SLA)

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease without any cure. The prevalence of ALS accounts for 2.5 to 3 cases per 100.000 inhabitants. This thesis work is devoted to TDP-43, an RNA-binding protein involved in ALS and found in cytoplasmic inclusions of neurons and motor neurons from ALS patients. The targets of TDP-43 are primarily GU-rich RNA sequences. In physiological conditions, TDP-43 localizes predominantly in the nucleus. The functions of this protein are associated to the splicing of messenger RNAs as well as the maturation and transport of RNAs. TDP-43 is also involved in the neuronal plasticity and in the formation of membrane-less compartments called “liquid-liquid phases” through the formation of stress granules (SGs). While the high-order TDP-43 self-assembly by its structured N-terminal (NTD) and the intrinsically disordered C-terminal domains (CTD) has been extensively studied, the role of the RRM domains responsible for the RNA binding remains unaddressed. Therefore, the mechanism dissecting the role of high-order TDP-43/RNA complexes in maintaining TDP-43 functional, in normal and pathological conditions, needs to be investigated.Through an integrative approach, we undertook an in-deep study regarding the TDP-43 binding on long poly-GU sequences which are found in the intronic regions of many TDP-43 target pre-mRNAs. We have shown that TDP-43 binds on poly-GU targets in a cooperative manner by self-assembling into multimers. A 3D structural model has been also obtained which highlights an interaction interface between deux monomeric TDP-43 units leading to the protein multimerization. This intermolecular interface involves a pocket centered around the V220 residue, located in the RRM2 domain of the first TDP-43 monomer and the loop 3 of RRM1 of the second monomer. The amino acid residues and the intermolecular interactions essential for the interface stability have been identified. Additionally, we have investigated, by in cellulo methods, several mutant forms of TDP-43 in which the cooperativity is impaired. Thus, we demonstrate that the cooperative binding of TDP-43 on mRNAs is critical to maintain the solubility of nuclear TDP-43 and the miscibility of TDP-43 condensates within cytoplasmic SGs. Based on the results presented here, we propose a mechanistic model in which the high-order TDP-43 assemblies promoted by the RRM domains bound to the RNA, constitutes a steric barrier limiting short range self-interactions between consecutive NTDs and CTDs of adjacent monomeric TDP-43 units. These assemblies may therefore favorize the dynamics and solubility of TDP-43/mRNA complexes. Impairments in the cooperative interaction of these complexes may lead to an anarchic attachment of TDP-43 along mRNAs leading to an increased occurrence of self-attraction between the NTDs and CTDs. Impaired multimers may then promote the TDP-43 aggregation. In conclusion, the TDP-43 multimerization on target RNA platforms would play a crucial role in processes linked to the control of its aggregation and mRNA splicing.La Sclérose Latérale Amyotrophique (SLA), ou Maladie de Charcot, est une maladie neurodégénérative fatale et sans remède avec une prévalence de 2,5 à 3 cas par 100 000 habitants. Ce travail de thèse est consacré à TDP-43, une protéine de liaison à l'ARN, impliquée dans cette maladie et retrouvée dans des inclusions cytoplasmiques des neurones et motoneurones de malades SLA. Cette protéine cible principalement des longues séquences d'ARN riches en répétitions GU. En conditions normales, TDP-43 est localisée majoritairement dans le noyau. Elle est impliquée dans des processus d'épissage de l'ARN messager ainsi que dans la maturation et transport des ARNs. TDP-43 est impliquée dans la plasticité neuronale et dans la formation de compartiments appelés « phases liquides » par l'intermédiaire de granules de stress. Auparavant, il a été démontré que le domaine N-terminal (NTD) ainsi que le domaine C-terminal (CTD) intrinsèquement désordonné de TDP-43 sont impliqués dans des autoassemblages constitués de complexes de haut poids-moléculaire. Au cours de ce processus, le rôle des domaines RRMs responsables de la fixation de l'ARN reste à déterminer. Ainsi, le mécanisme déchiffrant le rôle de la fixation de cette protéine à l'ARN, dans son maintien à l'état normal ou pathologique, reste à élucider.Par une approche intégrative, nous avons entrepris une étude approfondie de la fixation de TDP-43 sur de longues séquences poly-GU, trouvées dans des régions introniques de certains pré-ARNm cibles de TDP-43. Nous avons montré que TDP-43 se fixe sur ces cibles poly-GU de manière coopérative en formant des multimères. Un modèle structural 3D a été obtenu lequel met en évidence une interface d'interaction entre deux unités monomériques de TDP-43 qui favorise la multimérisation. Cette interface intermoléculaire implique une poche centrée autour du résidu Val220, localisé dans le domaine RRM2 du premier monomère et la boucle 3 du domaine RRM1 de la seconde unité monomérique. Les résidus d'acides aminés et les interactions mises en jeu, pour la stabilité de cette interface, ont été identifiés. Par la suite, nous avons étudié in cellulo des formes mutées de TDP-43 dont la coopérativité est altérée. Ainsi, nous avons montré que la coopérativité de liaison de TDP-43 à l'ARNm est déterminante pour la solubilité de cette protéine aussi bien que pour la miscibilité de ses condensats dans les granules du stress au niveau du cytoplasme. Enfin, un modèle mécanistique a été proposé selon lequel des assemblages de TDP-43 mettant en jeu ses domaines RRM liés sur l'ARN, limitent des auto-interactions entre les domaines NTD et CTD des unités monomériques adjacentes. Ces assemblages favoriseraient ainsi la dynamique et la solubilité des complexes TDP-43/ARN. Une altération de la coopérativité d'interaction de ce complexe pourrait faciliter une fixation désordonnée de TDP-43 le long des ARNs ce qui favoriserait une abondance des interactions entre les domaines NTD et CTD et ainsi promouvoir l'agrégation de TDP-43. En conclusion, la multimérisation de TDP-43 opérée sur la plateforme ARN cible jouerait ainsi un rôle crucial dans le contrôle de son agrégation et dans certains processus d'épissage des ARN messagers

Production of a conjugate between the rK346 antigen from Leishmania infantum and the horseradish peroxidase C for the detection of rK346 antibodies.

International audienceIt was designed and characterized a reporter system to be captured by an- tibodies bound to ELISA plates. The system was designed with the rK346 from Leishmania infantum, a highly antigenic and specific protein. The rK346 was coupled to the horseradish peroxidase C (HRPc) from Armoracia rusticana using glutaraldehyde or sulfo-SMCC. Gluta- raldehyde conjugation was performed in two steps. Separation of conjugates was carried out using a Sepharose S-200 in size exclusion chromatography (SEC); fractions were analyzed via HRPc activity and through ELISA plates sensitized with polyclonal anti-rK346 IgG puri- fied from rabbit serum. A heterogeneous population of conjugates rK346-HRPc was obtained with molecular weights ranging between 109.7 ± 16.5 to 67.6 ± 10.1 kDa; with rK346-HRPe stoichiometries of 1:2; 2:1; 3:1; and 2:2. Conjugation using sulfo-SMCC was carried out first by introducing -SH groups onto the HRPc using the SATA reagent and the antigen was modi- fied with sulfo-SMCC during 45 min. Separation and analysis of conjugates was performed similarly as with glutaraldehyde, resulting in a heterogeneous population of conjugates rK346- HRPc with molecular weights between 150.5 ± 22.6 to 80.0 ± 12.0 kDa; with rK346-HRPC stoichiometries of 2:1; 1:2; 2:2; and 1:3, with an increased conjugation efficiency in compari- son with glutaraldehyde. This enables sulfo-SMCC to be used as a potential reagent for cou- pling the antigen to the HRPc, to design an economic, specific and easy method to apply as a reporter system, available to assess individuals at risk and/or at early and late stages of visceral leishmaniasis

The cooperative binding of TDP-43 to GU-rich RNA repeats antagonizes TDP-43 aggregation

International audienceTDP-43 is a nuclear RNA-binding protein that forms neuronal cytoplasmic inclusions in two major neurodegenerative diseases, ALS and FTLD. While the self-assembly of TDP-43 by its structured N-terminal and intrinsically disordered C-terminal domains has been widely studied, the mechanism by which mRNA preserves TDP-43 solubility in the nucleus has not been addressed. Here, we demonstrate that tandem RNA Recognition Motifs of TDP-43 bind to long GU-repeats in a cooperative manner through intermolecular interactions. Moreover, using mutants whose cooperativity is impaired, we found that the cooperative binding of TDP-43 to mRNA may be critical to maintain the solubility of TDP-43 in the nucleus and the miscibility of TDP-43 in cytoplasmic stress granules. We anticipate that the knowledge of a higher order assembly of TDP-43 on mRNA may clarify its role in intron processing and provide a means of interfering with the cytoplasmic aggregation of TDP-43

FUS fibrillation occurs through a nucleation-based process below the critical concentration required for liquid–liquid phase separation

Abstract FUS is an RNA-binding protein involved in familiar forms of ALS and FTLD that also assembles into fibrillar cytoplasmic aggregates in some neurodegenerative diseases without genetic causes. The self-adhesive prion-like domain in FUS generates reversible condensates via the liquid–liquid phase separation process (LLPS) whose maturation can lead to the formation of insoluble fibrillar aggregates in vitro, consistent with the appearance of cytoplasmic inclusions in ageing neurons. Using a single-molecule imaging approach, we reveal that FUS can assemble into nanofibrils at concentrations in the nanomolar range. These results suggest that the formation of fibrillar aggregates of FUS could occur in the cytoplasm at low concentrations of FUS, below the critical ones required to trigger the liquid-like condensate formation. Such nanofibrils may serve as seeds for the formation of pathological inclusions. Interestingly, the fibrillation of FUS at low concentrations is inhibited by its binding to mRNA or after the phosphorylation of its prion-like domain, in agreement with previous models

FUS RRM regulates poly(ADP-ribose) levels after transcriptional arrest and PARP-1 activation on DNA damage

PARP-1 activation at DNA damage sites leads to the synthesis of long poly(ADP-ribose) (PAR) chains, whichserve as a signal for DNA repair. Here we show that FUS, an RNA-binding protein, is specifically directed toPAR through its RNA recognition motif (RRM) to increase PAR synthesis by PARP-1 in HeLa cells after genotoxicstress. Using a structural approach, we also identify specific residues located in the FUS RRM, whichcan be PARylated by PARP-1 to control the level of PAR synthesis. Based on the results of this work, we proposea model in which, following a transcriptional arrest that releases FUS from nascent mRNA, FUS can berecruited by PARP-1 activated by DNA damage to stimulate PAR synthesis. We anticipate that this modeloffers new perspectives to understand the role of FET proteins in cancers and in certain neurodegenerativediseases such as amyotrophic lateral sclerosis

FUS fibrillation occurs through a nucleation-based process below the critical concentration required for liquid–liquid phase separation

Abstract FUS is an RNA-binding protein involved in familiar forms of ALS and FTLD that also assembles into fibrillar cytoplasmic aggregates in some neurodegenerative diseases without genetic causes. The self-adhesive prion-like domain in FUS generates reversible condensates via the liquid–liquid phase separation process (LLPS) whose maturation can lead to the formation of insoluble fibrillar aggregates in vitro, consistent with the appearance of cytoplasmic inclusions in ageing neurons. Using a single-molecule imaging approach, we reveal that FUS can assemble into nanofibrils at concentrations in the nanomolar range. These results suggest that the formation of fibrillar aggregates of FUS could occur in the cytoplasm at low concentrations of FUS, below the critical ones required to trigger the liquid-like condensate formation. Such nanofibrils may serve as seeds for the formation of pathological inclusions. Interestingly, the fibrillation of FUS at low concentrations is inhibited by its binding to mRNA or after the phosphorylation of its prion-like domain, in agreement with previous models

Targeting RNA:protein interactions with an integrative approach leads to the identification of potent YBX1 inhibitors

International audienceRNA-protein interactions (RPIs) are promising targets for developing new molecules of therapeutic interest. Nevertheless, challenges arise from the lack of methods and feedback between computational and experimental techniques during the drug discovery process. Here, we tackle these challenges by developing a drug screening approach that integrates chemical, structural and cellular data from both advanced computational techniques and a method to score RPIs in cells for the development of small RPI inhibitors; and we demonstrate its robustness by targeting Y-box binding protein 1 (YB-1), a messenger RNA-binding protein involved in cancer progression and resistance to chemotherapy. This approach led to the identification of 22 hits validated by molecular dynamics (MD) simulations and nuclear magnetic resonance (NMR) spectroscopy of which 11 were found to significantly interfere with the binding of messenger RNA (mRNA) to YB-1 in cells. One of our leads is an FDA-approved poly(ADP-ribose) polymerase 1 (PARP-1) inhibitor. This work shows the potential of our integrative approach and paves the way for the rational development of RPI inhibitors

Crystal structure and biochemical characterization of the transmembrane PAP2 type phosphatidylglycerol phosphate phosphatase from Bacillus subtilis.

Type 2 phosphatidic acid phosphatases (PAP2s) can be either soluble or integral membrane enzymes. In bacteria, integral membrane PAP2s play major roles in the metabolisms of glycerophospholipids, undecaprenyl-phosphate (C55-P) lipid carrier and lipopolysaccharides. By in vivo functional experiments and biochemical characterization we show that the membrane PAP2 coded by the Bacillus subtilis yodM gene is the principal phosphatidylglycerol phosphate (PGP) phosphatase of B. subtilis. We also confirm that this enzyme, renamed bsPgpB, has a weaker activity on C55-PP. Moreover, we solved the crystal structure of bsPgpB at 2.25 A resolution, with tungstate (a phosphate analog) in the active site. The structure reveals two lipid chains in the active site vicinity, allowing for PGP substrate modeling and molecular dynamic simulation. Site-directed mutagenesis confirmed the residues important for substrate specificity, providing a basis for predicting the lipids preferentially dephosphorylated by membrane PAP2s

SVC-onGoing: Signature verification competition

This article presents SVC-onGoing1, an on-going competition for on-line signature verification where researchers can easily benchmark their systems against the state of the art in an open common platform using large-scale public databases, such as DeepSignDB2 and SVC2021_EvalDB3, and standard experimental protocols. SVC-onGoing is based on the ICDAR 2021 Competition on On-Line Signature Verification (SVC 2021), which has been extended to allow participants anytime. The goal of SVC-onGoing is to evaluate the limits of on-line signature verification systems on popular scenarios (office/mobile) and writing inputs (stylus/finger) through large-scale public databases. Three different tasks are considered in the competition, simulating realistic scenarios as both random and skilled forgeries are simultaneously considered on each task. The results obtained in SVC-onGoing prove the high potential of deep learning methods in comparison with traditional methods. In particular, the best signature verification system has obtained Equal Error Rate (EER) values of 3.33% (Task 1), 7.41% (Task 2), and 6.04% (Task 3). Future studies in the field should be oriented to improve the performance of signature verification systems on the challenging mobile scenarios of SVC-onGoing in which several mobile devices and the finger are used during the signature acquisitionThis work has been supported by projects: PRIMA (H2020- MSCA-ITN-2019-860315), TRESPASS-ETN (H2020-MSCA-ITN-2019- 860813), INTER-ACTION (PID2021-126521OB-I00 MICINN/FEDER), Orange Labs, and by UAM-Cecaban