The gammaretroviral p12 protein has multiple domains that function during the early stages of replication.

BACKGROUND: The Moloney murine leukaemia virus (Mo-MLV) gag gene encodes three main structural proteins, matrix, capsid and nucleocapsid and a protein called p12. In addition to its role during the late stages of infection, p12 has an essential, but undefined, function during early post-entry events. As these stages of retroviral infection remain poorly understood, we set out to investigate the function of p12. RESULTS: Examination of the infectivity of Mo-MLV virus-like particles containing a mixture of wild type and mutant p12 revealed that the N- and C-terminal regions of p12 are sequentially acting domains, both required for p12 function, and that the N-terminal activity precedes the C-terminal activity in the viral life cycle. By creating a panel of p12 mutants in other gammaretroviruses, we showed that these domains are conserved in this retroviral genus. We also undertook a detailed mutational analysis of each domain, identifying residues essential for function. These data show that different regions of the N-terminal domain are necessary for infectivity in different gammaretroviruses, in stark contrast to the C-terminal domain where the same region is essential for all viruses. Moreover, chimeras between the p12 proteins of Mo-MLV and gibbon ape leukaemia virus revealed that the C-terminal domains are interchangeable whereas the N-terminal domains are not. Finally, we identified potential functions for each domain. We observed that particles with defects in the N-terminus of p12 were unable to abrogate restriction factors, implying that their cores were impaired. We further showed that defects in the C-terminal domain of p12 could be overcome by introducing a chromatin binding motif into the protein. CONCLUSIONS: Based on these data, we propose a model for p12 function where the N-terminus of p12 interacts with, and stabilizes, the viral core, allowing the C-terminus of p12 to tether the preintegration complex to host chromatin during mitosis, facilitating integration

Etude de poudres et films mésoporeux par RMN du 129Xe hyperpolarisé

L objectif de ce travail est de montrer l apport de la RMN du 129Xe hyperpolarisé dans l étude des matériaux mésoporeux. Une attention particulière est portée à l étude de la stabilité de ces matériaux dans l eau ainsi qu au suivi de la distribution des groupements organiques dans les solides greffés. L originalité de ce travail réside dans l étude pionnière de la dynamique du xénon adsorbé sur des films minces par la RMN du xénon hyperpolarisé et dans l exploration de l hétérogénéité des surfaces de films de silice greffés. Gra ce au dispositif de pompage optique (couplage laser-RMN), nous avons effectué des expériences de RMN-129Xe hyperpolarisé en flux continu. Le transfert de la polarisation électronique du rubidium vers les spins nucléaires du xénon conduit à une augmentation considérable de la sensibilité de détection RMN (x 104) ce qui permet de travailler à des pressions faibles en xénon et d approcher au plus près l interaction entre celui ci et la surface interne du pore. Les résultats obtenus montrent que contrairement aux matériaux SBA-15 classiques (synthétisés à 100C) qui ne sont pas très stables dans l eau, (principalement en raison de la présence des micropores), l absence des micropores dans les SBA-15(130C) rend ces matériaux plus stables. Un transfert de polarisation du 129Xe vers le proton (CP) pour un matériau mésoporeux acide fonctionnalisé par des groupements arylsulfoniques a été effectué. Les résultats ont prouvé que ce transfert était sélectif et que seuls les protons alkyls en étaient affectés. La RMN 2D-échange (EXSY) du xénon hyperpolarisé appliquée sur les films minces greffés a permis de suivre la vitesse d échange du xénon diffusant parmi les différentes régions d'adsorption et de sonder les différents environnements chimiques du matériau.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Assimilation du fer chez Pseudomonas aeruginosa (Etude du mécanisme de transport de la Pyoverdine ferrique à travers la membrane externe)

Le fer est un ÈlÈment essentiel la croissance et la virulence de Pseudomonas aeruginosa, mais son acquisition est limitÈe en raison de la faible solubilitÈ de ce mÈtal pH physiologique. Pour contourner cette faible biodisponibilitÈ, P. aeruginosa sÈcrËte un sidÈrophore majeur appelÈ pyoverdine (Pvd), composÈ d un chromophore reliÈ un peptide de 8 acides aminÈs. Les sidÈrophores sont caractÈrisÈs par une trËs grande affinitÈ pour le fer et leur rÙle est de solubiliser et chÈlater ce mÈtal dans le milieu extracellulaire. Le complexe sidÈrophore-ferrique est ensuite transportÈ dans la bactÈrie gr ce des transporteurs membranaires spÈcifiques. Le transport de la pyoverdine-ferrique travers la membrane externe de P. aeruginosa est assurÈ par le rÈcepteur FpvA, composÈ de 3 domaines: un tonneau b, un domaine globulaire appelÈ bouchon qui obture le pore du tonneau et enfin un domaine de signalisation localisÈ dans le pÈriplasme. Une premiËre partie de ce travail de thËse a permis de caractÈriser par des approches structurales et fonctionnelles, les mÈcanismes d interactions de FpvA avec diffÈrentes pyoverdines qui diffËrent dans la sÈquence du peptide. Par mutagenËse dirigÈe, l importance de certains rÈsidus proches du site actif dans le mÈcanisme de liaison de la pyoverdine-ferrique avec FpvA a ÈtÈ ÈtudiÈe. Le mÈcanisme de transport a ÈtÈ suivi avec un rÈcepteur reconstituÈ partir des domaines tonneau et bouchon n appartenant pas la mÍme chaÓne peptidique. Ce rÈcepteur reconstituÈ prÈsent au niveau des membranes externes de la bactÈrie, est capable de lier et de transporter la pyoverdine-ferrique avec la mÍme affinitÈ que le rÈcepteur sauvage, mais avec une efficacitÈ moindre.La deuxiËme partie de ce travail de thËse a concernÈ un rÈcepteur alternatif de la pyoverdine appelÈ FpvB. Les Ètudes de liaison ont montrÈ une affinitÈ de FpvB pour la pyoverdine-ferrique Èquivalente celle du rÈcepteur FpvA. En revanche, nos donnÈes indiquent que les mÈcanismes de dissociation du mÈtal de la pyoverdine seraient probablement diffÈrents selon que le complexe siderophore-fer est transportÈ par le rÈcepteur FpvA ou FpvB. Dans le cas de la voie FpvA, une rÈduction du fer est impliquÈe. Pour la voie FpvB, un mÈcanisme diffÈrent serait utilisÈ. Gr ce aux propriÈtÈs de fluorescence de la pyoverdine, les ÈlÈments impliquÈs dans la dissociation du complexe Pvd-Fe et dans le transport travers la membrane interne devraient Ítre caractÈrisÈs par une approche alliant la biologie molÈculaire et la biochimie.Pseudomonas aeruginosa is Gram negative opportunistic human pathogen that infects injured, immunodeficient and otherwise compromised patients. Under conditions of iron limitation, Pseudomonas aeruginosa secretes a fluorescent siderophore named pyoverdine (Pvd) that chelates iron in the extracellular medium. The Pvd-Fe complex is then transported into the bacterium via the outer membrane TonB dependent receptor termed FpvA. This receptor consists of a membrane-spanning b-barrel occluded by a plug domain, which in its resting state leaves no space for an iron-loaded siderophore to pass through. The N-terminal extension of FpvA is involved in a signaling pathway that regulates the expression of the FpvA protein and its siderophore. Although the crystal structure of FpvA receptor is solved, the transfer mechanism of Pvd-Fe complex across the outer membrane of the bacteria has not been determined so far. A two-gated system has been proposed for the translocation of ferric-siderophore across the outer membrane. The first gate consists of the extracellular loops trapping Pvd-Fe in its binding site, and the second consists of the plug closing the barrel. The opening of the second gate is an energy-consuming process involving the inner membrane TonB complex.During my Ph.D thesis I studied the transport mechanism through the TonB dependent outer membrane receptor FpvA, and I characterized a second receptor termed FpvB The molecular determinants for specificity in Pvd-FpvA interactions were investigated through structural and biochemical approaches. The extracellular loops involved in the recognition of the Pvd-iron complex were identified using site directed mutagenesis, time-resolved fluorescence spectroscopy, and Fluorescence Resonance Energy Transfer (FRET). To get more insight on the translocation mechanism through the FpvA receptor, the ability of a reconstituted FpvA receptor obtained by cosynthesis of separately encoded plug and barrel domains to bind and transports Pvd-Fe was studied.Furthermore, the FpvA signaling, plug and barrel domains were characterized in vivo and the in vitro interactions between the purified FpvA domains and the proteins involved in the iron uptake machinery notably the periplasmic part of the TonB protein were studied.An additional pyoverdine receptor termed FpvB was described recently. Little is known about the Pvd-Fe uptake mechanism via this receptor, therefore FpvB was characterized at the molecular and functional level. FpvA and FpvB receptors are differentially expressed, and their expression is regulated differently. We showed that FpvB receptor transports Pvd-Fe with the same affinity as FpvA but, the identification of the elements involved in the uptake across the inner membrane remains to be identified combining molecular and functional approaches.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Understanding the stability in water of mesoporous SBA-15 and MCM-41

Despite their surprisingly thick walls, SBA-15 mesoporous silicas are not as stable as expected in water, even at room temperature. The microporosity of SBA-15, synthesized at temperature under 110°C, is lost during water treatment, leading to a strong decrease in specific surface area and to an increase in mesopore size. Only SBA-15s without microporosity, as the ones synthesized at 130°C, are stable under water treatment. Investigations by nitrogen adsorption isotherms and hyperpolarized 129Xe NMR spectroscopy have been performed to understand the stabilities of three SBA-15s synthesized at different temperature (60, 100 and 130°C) and treated in water at room temperature and at the boiling point. The different behaviour are explained by a mechanism of dissolution/redeposition of silica based on the local curvatures of silica in the different structures. Similar experiments on MCM-41 silica lead to a totally different dissolution/redeposition behaviour, due to the thinner walls and lower pore size, surface area and pore volume of the material

Masculinity and secessionism in Italy: An assessment

The masculinist rhetoric of the northern Italian separatist party, the Lega Nord, is an example of nationalist status affirmation. The stereotyped contrast between effeminate southerners and masculine northerners developed at the end of last century is used to affirm the superiority of the masculine North. The Lega's rhetoric parallels mainstream concerns on Italy's international status, and proposals for reforms to make Italy more ‘masculine’ and European. The use of a gendered language for the affirmation of national status reveals a belief in a normative hierarchy of nations, but also introduces a tension between a ‘masculinist’ status affirmation and a model of modernity which includes the emancipation of women as one of its principles.FLWINinfo:eu-repo/semantics/publishe

Low incidence of SARS-CoV-2, risk factors of mortality and the course of illness in the French national cohort of dialysis patients

International audienceThe aim of this study was to estimate the incidence of COVID-19 disease in the French national population of dialysis patients, their course of illness and to identify the risk factors associated with mortality. Our study included all patients on dialysis recorded in the French REIN Registry in April 2020. Clinical characteristics at last follow-up and the evolution of COVID-19 illness severity over time were recorded for diagnosed cases (either suspicious clinical symptoms, characteristic signs on the chest scan or a positive reverse transcription polymerase chain reaction) for SARS-CoV-2. A total of 1,621 infected patients were reported on the REIN registry from March 16th, 2020 to May 4th, 2020. Of these, 344 died. The prevalence of COVID-19 patients varied from less than 1% to 10% between regions. The probability of being a case was higher in males, patients with diabetes, those in need of assistance for transfer or treated at a self-care unit. Dialysis at home was associated with a lower probability of being infected as was being a smoker, a former smoker, having an active malignancy, or peripheral vascular disease. Mortality in diagnosed cases (21%) was associated with the same causes as in the general population. Higher age, hypoalbuminemia and the presence of an ischemic heart disease were statistically independently associated with a higher risk of death. Being treated at a selfcare unit was associated with a lower risk. Thus, our study showed a relatively low frequency of COVID-19 among dialysis patients contrary to what might have been assumed