Vérification de la corrélation entre la fonction, la structure et la dynamique sur un chemin évolutif recombinant les β-lactamases TEM-1 et PSE-4

La corrélation, déjà bien établie, entre la structure et la fonction des enzymes a permis d’expliquer le mécanisme catalytique de plusieurs d’entre elles. Toutefois, des lacunes entourant l’explication de leur capacité à accélérer, si drastiquement, la vitesse réactionnelle subsistent toujours. L’une des hypothèses propose l’inclusion d’un rôle fonctionnel aux mouvements présents dans les enzymes. Un nombre grandissant d’évidences montrant un lien entre la dynamique et la fonction des protéines nous poussent, ici, à vérifier la présence d’une telle corrélation dans un contexte évolutif et à vérifier si la dynamique devrait être prise en compte afin de mieux faire l’ingénierie des enzymes. Le modèle enzymatique étudié sera composé de deux β-lactamases naturellement évoluées et homologues, TEM-1 et PSE-4. Par la caractérisation de ces deux enzymes, structurellement et fonctionnellement similaires, nous avons observé que TEM-1 et PSE-4 possèdent une dynamique similaire, généralement restreinte, sur les échelles de temps allant des picosecondes aux millisecondes. Ces résultats supportent l’hypothèse selon laquelle la dynamique a été évolutivement maintenue dans ces enzymes, et suggèrent que celle-ci possède un rôle envers la fonction enzymatique, d’où son maintien au cours de l’évolution. En laboratoire, plusieurs approches peuvent être utilisées afin de faire l’ingénierie des protéines. La recombinaison d’homologues est grandement utilisée, puisque cette méthode, mimant le processus naturel d’évolution par recombinaison, permet d’intégrer un grand nombre de mutations simultanées qui ont déjà été sélectionnées au cours de l’évolution. Notre modèle enzymatique sera constitué de trois enzymes chimériques issues de la recombinaison des β-lactamases TEM-1 et PSE-4. Ces enzymes représentent de potentiels intermédiaires évolutifs entre ces deux enzymes naturellement évoluées. Ces trois chimères ont été sélectionnées en fonction de leur site actif ayant été recombiné. Nos résultats démontrent une conservation de l’efficacité catalytique, de la structure et de la dynamique rapide chez ces trois chimères. Toutefois, des changements importants de dynamique lente, coïncidant avec l’échelle de temps de la vitesse catalytique de ces enzymes, ont été observés. Une exploration in silico des conformations adoptées par les chaînes latérales des résidus catalytiques montre des variations corrélant aux légères variations fonctionnelles (diminution du kcat pour l’hydrolyse des céphalosporines) entre les β-lactamases TEM-1, PSE-4 et ces chimères. Ces résultats mettent en évidence l’importance de caractériser les mouvements ayant lieu sur plusieurs échelles de temps afin de pouvoir comprendre leur rôle et de considérer ceux-ci relativement à la fonction de la protéine. Nous proposons ici que la dynamique rapide de ces enzymes est impliquée au niveau de leur réactivité hydrolytique (c’est-à-dire hydrolyser un β-lactame) alors que la dynamique lente pourrait être impliquée au niveau de la grande adaptabilité évolutive (reconnaissance non native de nouveaux antibiotiques de type β-lactame) de ces enzymes de résistance bactérienne.The link between enzyme structure and function is well established and has led to the description of many catalytic mechanisms. However, how enzymes accelerate the catalytic rate so drastically is still not fully understood. A growing amount of evidence linking protein dynamics to function, suggests that protein motions might have functional implications. This compels us to verify this correlation in an evolutionary context and to verify if protein dynamics should be considered to better engineer enzymes. Our studied enzymatic model is composed of two homologous and naturally evolved bacterial resistance enzymes, the TEM-1 and PSE-4 β-lactamases. By characterizing these structurally and functionally similar enzymes, we verified if protein dynamics are evolutionarily conserved. Our results show that TEM-1 and PSE-4 maintain similar patterns of restricted dynamics on timescales ranging from picoseconds to milliseconds. This supports the hypothesis that dynamics have been evolutionarily maintained in these enzymes and is consistent with a contribution of dynamics to enzyme function resulting in the evolutionary maintenance of dynamics. Various approaches are used to engineer proteins. Homologous recombination is extensively applied, as it mimics natural evolution. This technique allows the incorporation of many simultaneous sequence variations selected over the course of evolution. Our enzymatic model includes three chimeric enzymes, resulting from the recombination of TEM-1 and PSE-4. These represent intermediates over a hypothetical evolutionary path from TEM-1 to PSE-4. These artificially-evolved enzymes were selected to compose this system of evolutionarily related protein on the basis of their active site cavity having been recombined. A conservation of function, structure and fast dynamics was found in these chimeras. Striking variations of slow dynamics, occurring on a timescale coinciding with the catalytic turnover rate, were also observed. In silico exploration of the conformations adopted by the catalytic residue side-chains showed variations correlating with the functional diversification between TEM-1, PSE-4, and the chimeric β-lactamases. These results highlight the importance of probing motions on various timescales to fully comprehend their role in function. We propose that dynamics on the faster timescales are associated with the maintenance of reactivity in these enzymes, while modification of the slow dynamics may be associated with the high evolvability of these bacterial resistance enzymes

Dynamiques de genre et pratiques professionnelles : le cas des journalistes québécoises correspondantes à l'étranger

Ce mémoire de maîtrise porte sur les dynamiques de genre dans la pratique professionnelle des journalistes québécoises correspondantes à l’étranger. S’appuyant sur une démarche qualitative et constructiviste, l’auteure examine l’influence du genre sur l’expérience des journalistes correspondantes. La théorie du point de vue et l’analyse critique de discours servent à décrire leur situation particulière. L’analyse met en lumière la manière dont les dynamiques de genre teintent leur expérience. Assignations genrées et perception d’une écriture « féminine » sont particulièrement marquantes, déclenchant un malaise certain. Dans la salle de rédaction, harcèlement sexuel, « double standard » et culture masculine désavantagent les femmes. À l’étranger, elles ont accès à plus de sources mais rencontrent des désagréments, dont le harcèlement sexuel. Après de nombreux progrès à ces différents égards, des reculs apparaissent. Globalement, les femmes semblent porteuses, de par leur expérience située, d’un nouveau journalisme riche en humanité, en complexité et en équité.This memoir aims to explore gender dynamics in the specific context of the professional practice of Quebec female journalists working as international correspondents. Starting from a qualitative and constructivist research approach, this study seeks to understand how gender influences the lived experience of female correspondents. Standpoint theory is used to understand their specific experience. Starting from semi-structured interviews, the author uses critical discourse analysis to underline gender dynamics in the respondents’ discourse. Gendered beats and perceived “feminine” writing take a specific importance, generating a strong malaise for women. In the newsroom, sexual harassment, “double standard” and masculine organizational culture are disadvantages to them. In the field, women gain access to more sources, but are also prey to sexual harassment. After significant progress in the past years, there seems to be significant steps backwards. Globally, women seem to be carrying a new kind of journalism, rich in humanity, complexity and equity

Liberté et précarité comme nouvelles valeurs ?

Les transformations contemporaines traversant les sphères professionnelles de la communication, et se construisant notamment autour du numérique, paraissent nombreuses et potentiellement de grande ampleur, bien qu’il demeure difficile d’en délimiter les contours avec certitude. Dans ces métiers, les conditions d’emploi et les conditions de travail changent graduellement, non sans faire écho à des changements touchant l’ensemble des sociétés, et semblent s’accompagner de réactions diverses, parfois équivoques de la part des différentes catégories d’acteurs. Dans cet article, nous interrogeons les métiers de la communication comme résistances, reflets et accélérations de ces transformations qui les dépassent, afin d’aborder trois propositions principales de travail : le transfert de responsabilités – économiques comme éthiques – à la charge des travailleurs et travailleuses ; la construction de la flexibilité comme idéal au sein d’un entrepreneuriat de soi ; la déstabilisation des carrières au profit d’une mise en avant d’une valeur ajoutée à l’offre de travail. Ensemble, ces mutations au sein des sphères de la communication apparaissent comme une traduction, à la fois reprise et interprétation spécifique, des exigences du capitalisme néolibéral. Contemporary transformations of labour in the different professional spheres of communication, which are increasingly taking ground in the digital landscape, appear to be emerging on a potentially large scale, although the precise nature and forms of these transformations remain to be studied more closely. Working conditions and modes of employment are gradually changing, echoing larger societal trends, and are met with different, sometimes equivocal reactions. In this article, we look at communication work as a site where these transformations are reflected, resisted and accelerated. We formulate three research avenues: 1) a transfer of responsibility – both of an economic and ethical nature – from the organisations to the workers; 2) the presentation of flexibility as a new ideal in a movement favouring self-entrepreneurship; 3) a destabilization of careers and employment trajectories that puts forward the need for workers to demonstrate their added value and expertise. We argue that, taken together, these transformations that are taking place in communication spheres of work can be considered as translations – both picking up and offering specific interpretations of – neoliberal capitalism and its pressures

Inverse Solidification Induced by Active Janus Particles

Crystals melt when thermal excitations or the concentration of defects in the lattice is sufficiently high. Upon melting, the crystalline long‐range order vanishes, turning the solid to a fluid. In contrast to this classical scenario of solid melting, here a counter‐intuitive behavior of the occurrence of crystalline long‐range order in an initially disordered matrix is demonstrated. This unusual solidification is demonstrated in a system of passive colloidal particles accommodating chemically active defects—photocatalytic Janus particles. The observed crystallization occurs when the amount of active‐defect‐induced fluctuations (which is the measure of the effective temperature) reaches critical value. The driving mechanism behind this unusual behavior is purely internal and resembles a blast‐induced solidification. Here, the role of “internal micro‐blasts” is played by the photochemical activity of defects residing in the colloidal matrix. The defect‐induced solidification occurs under non‐equilibrium conditions: the resulting solid exists as long as a constant supply of energy in the form of ion flow is provided by the catalytic photochemical reaction at the surface of active Janus particle defects. The findings could be useful for the understanding of the phase transitions of matter under extreme conditions far from thermodynamic equilibrium.Inverse solidification driven by active colloids provides novel insight into the collective effects in mixed colloidal systems. It offers versatile possibilities to address the processes of solidification in various systems out of equilibrium, including the formation of bio‐molecular condensates or biomineralization, transitions from amorphous to polycrystalline state in condensed matter, or synthesis of materials under extreme conditions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/162773/3/adfm202003851.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162773/2/adfm202003851-sup-0001-SuppMat.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162773/1/adfm202003851_am.pd

A broadly cross-reactive antibody neutralizes and protects against sarbecovirus challenge in mice

Severe acute respiratory syndrome coronaviruses 1 (SARS-CoV) and 2 (SARS-CoV-2), including SARS-CoV-2 variants of concern, can cause deadly infections. The mortality associated with sarbecovirus infection underscores the importance of developing broadly effective countermeasures against them, which could be key in the prevention and mitigation of current and future zoonotic events. Here, we demonstrate the neutralization of SARS-CoV, bat coronaviruses WIV-1, RsSHC014, and SARS-CoV-2 variants D614G, B.1.1.7, B.1.351, P.1, B.1.429, B.1.526, B.1.617.1, and B.1.617.2 by a receptor-binding domain (RBD)-specific human antibody, DH1047. Prophylactic and therapeutic treatment with DH1047 was protective against SARS-CoV, WIV-1, RsSHC014, and SARS-CoV-2 B.1.351 infection in mice. Binding and structural analysis showed high affinity binding of DH1047 to an epitope that is highly conserved among sarbecoviruses. Thus, DH1047 is a broadly protective antibody that can prevent infection and mitigate outbreaks caused by SARS-related strains and SARS-CoV-2 variants. Our results also suggest that the conserved RBD epitope bound by DH1047 is a rational target for a universal sarbecovirus vaccin

In vitro and in vivo functions of SARS-CoV-2 infection-enhancing and neutralizing antibodies

SARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-g (FcgR)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated FcgR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Three of 46 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can rarely occur in SARS-CoV-2 antibody-infused macaques

Les pratiques professionnelles genrées : le cas des journalistes québécoises correspondantes à l’étranger

L’auteure examine les pratiques professionnelles genrées des journalistes québécoises correspondantes à l’étranger. Dans une démarche constructiviste alliant théorie du point de vue et analyse de discours, elle considère les rapports sociaux de sexe dans l’expérience des correspondantes. Au pays, affectations et attentes genrées, harcèlement sexuel, système « à deux poids, deux mesures » et valorisation de comportements « masculins » les désavantagent. Plus sympathiques et moins menaçantes, à l’étranger, elles accèdent à davantage de sources, mais elles sont aux prises avec le harcèlement sexuel. Malgré un malaise à l’idée d’un journalisme « féminin », leur vision du métier semble porter humanité, équité et complexité.The author aims to explore gender dynamics in the context of the professional practice of Quebec female journalists working as international correspondents. Starting from a qualitative and constructivist research approach, she seeks to understand how gender influences the lived experience of female correspondents, using standpoint theory and critical discourse analysis. In the newsroom, gendered beats, « feminine writing », sexual harassment, double standard and masculine organizational culture are disadvantages to them. In the field, women gain access to more sources, but they are also prey to sexual harassment. Globally, women seem to carry a journalism rich in humanity, complexity and equity.Este artículo examina las prácticas profesionales por el género de las periodistas quebequenses corresponsales en el extranjero. Desde una iniciativa constructivista que combina perspectiva de la teoría y análisis del discurso, la autora examina las relaciones de género en el experimento de las corresponsales. En su propio país, las tareas y expectativas de género, el acoso sexual, la doble moral y la valoración del comportamiento identificado como « masculino » las perjudican mucho. Más amables y menos amenazantes, en el extranjero, tienen acceso a más fuentes, pero se enfrentan a acoso sexual. A pesar de la incomodidad con respecto a la idea de un periodismo « femenino », su visión de la profesión parece ser humanidad, equidad y complejidad

Janus Particles: Inverse Solidification Induced by Active Janus Particles (Adv. Funct. Mater. 39/2020)

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/162804/1/adfm202070260.pd

Anisotropic exclusion effect between photocatalytic Ag/AgCl Janus particles and passive beads in a dense colloidal matrix

Synthetic nano- and micromotors interact with each other and their surroundings in a complex manner. Here, we report on the anisotropy of active-passive particle interaction in a soft matter system containing an immobile yet photochemical Ag/AgCl-based Janus particle embedded in a dense matrix of passive beads in pure water. The asymmetry in the chemical gradient around the Janus particle, triggered upon visible light illumination, distorts the isotropy of the surrounding electric potential and results in the repulsion of adjacent passive beads to a certain distance away from the Janus particle. This exclusion effect is found to be anisotropic with larger distances to passive beads in front of the Ag/AgCl cap of the Janus particle. We provide insight into this phenomenon by performing the angular analysis of the radii of exclusion and tracking their time evolution at the level of a single bead. Our study provides a novel fundamental insight into the collective behavior of a complex mixture of active and passive particles and is relevant for various application scenarios, e.g., particle transport at micro- and nanoscale and local chemical sensing

Inverse Solidification Induced by Active Janus Particles

Crystals melt when thermal excitations or the concentration of defects in the lattice is sufficiently high. Upon melting, the crystalline long‐range order vanishes, turning the solid to a fluid. In contrast to this classical scenario of solid melting, here a counter‐intuitive behavior of the occurrence of crystalline long‐range order in an initially disordered matrix is demonstrated. This unusual solidification is demonstrated in a system of passive colloidal particles accommodating chemically active defects—photocatalytic Janus particles. The observed crystallization occurs when the amount of active‐defect‐induced fluctuations (which is the measure of the effective temperature) reaches critical value. The driving mechanism behind this unusual behavior is purely internal and resembles a blast‐induced solidification. Here, the role of “internal micro‐blasts” is played by the photochemical activity of defects residing in the colloidal matrix. The defect‐induced solidification occurs under non‐equilibrium conditions: the resulting solid exists as long as a constant supply of energy in the form of ion flow is provided by the catalytic photochemical reaction at the surface of active Janus particle defects. The findings could be useful for the understanding of the phase transitions of matter under extreme conditions far from thermodynamic equilibrium.Inverse solidification driven by active colloids provides novel insight into the collective effects in mixed colloidal systems. It offers versatile possibilities to address the processes of solidification in various systems out of equilibrium, including the formation of bio‐molecular condensates or biomineralization, transitions from amorphous to polycrystalline state in condensed matter, or synthesis of materials under extreme conditions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/162773/3/adfm202003851.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162773/2/adfm202003851-sup-0001-SuppMat.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162773/1/adfm202003851_am.pd