Structures de glace des cours d'eau à forte pente : cristallographie et processus de croissance

Réunissant une série de trois articles scientifiques, ce mémoire offre une description et une quantification des processus dynamiques de glace dans les cours d’eau à forte pente. Menée sur trois cours d’eau du bassin versant de la rivière Montmorency (à proximité de Québec, QC, Canada), cette recherche démontre grâce à l’analyse par CAT scan que la glace de fond et les barrages de glace se développent principalement par croissance in-situ des cristaux de frasil-fixé. La porosité des structures de frasil-fixé varie entre 23% et 52% alors que celle de des structures de glace colonnaire, uniquement retrouvées dans les barrages de glace, varie entre 0% et 1,5%. De plus, les taux de croissance et de fonte des barrages de glace varient de façon importante autour d’une moyenne d’environ 1 cm/h. Le développement complet d’un barrage de glace a été simulé avec succès à l’aide d’un modèle physique comportant plusieurs degrés de liberté.Uniting three scientific papers, this thesis offers a description and a quantification of dynamic ice processes taking place in steep channels. This research was led on three channels located in the Montmorency River watershed near Quebec City, Canada. CAT scan analyses showed that the development of anchor ice and ice dams is mainly caused by in-situ growth of fixed-frazil ice crystals. Porosity of fixed-frazil ice structures ranges between 23% and 52% while porosity of columnar ice structures, only found in ice dams, ranges from 0% to 1.5%. Moreover, ice dam growth and decay rates greatly vary around the mean value of about 1 cm/h. The complete development of a single ice dam was successfully simulated using a physical model that demonstrated the large number of degrees of freedom inherent in the process

Étude du mécanisme d’augmentation de la relâche virale par la protéine Vpu du VIH-1

Les différentes protéines accessoires du VIH-1, l’agent étiologique du SIDA, optimisent la réplication et la propagation du virus in vivo. Parmi ces dernières figure Vpu, l’antagoniste du facteur de restriction nommé Tetherin qui prévient la relâche des particules virales à partir de la surface de cellules infectées. En diminuant son expression de surface, Vpu prévient l’incorporation de ce facteur de restriction dans la particule virale en formation et conséquemment, empêche la formation d’une ancre protéique reliant le virus mature à la membrane plasmique de la cellule infectée. La mécanistique sous-jacente n’était cependant pas connue. Cette présente thèse relate nos travaux exécutés afin d’élucider la dynamique des mécanismes cellulaires responsables de cet antagonisme. Une approche de mutagénèse dirigée a d’abord permis d’identifier deux régions contenant des déterminants de la localisation de Vpu dans le réseau trans-Golgi (RTG), puis de démontrer la relation existante entre cette distribution et l’augmentation de la relâche des particules virales. Des expériences subséquentes de marquage métabolique suivi d’une chasse exécutées dans des systèmes cellulaires où Tetherin est exprimée de façon endogène ont suggéré le caractère dispensable de l’induction par Vpu de la dégradation du facteur de restriction lors de son antagonisme. En revanche, une approche de réexpression de Tetherin conduite en cytométrie en flux, confirmée en microscopie confocale, a mis en évidence une séquestration de Tetherin dans le RTG en présence de Vpu, phénomène qui s’est avéré nécessiter l’interaction entre les deux protéines. L’usage d’un système d’expression de Vpu inductible conjugué à des techniques de cytométrie en flux nous a permis d’apprécier l’effet majeur de Vpu sur la Tetherin néo-synthétisée et plus mineur sur la Tetherin de surface. En présence de Vpu, la séquestration intracellulaire de la Tetherin néo-synthétisée et la légère accélération de l’internalisation naturelle de celle en surface se sont avérées suffisantes à la réduction de son expression globale à la membrane plasmique et ce, à temps pour l’initiation du processus de relâche virale. À la lumière de nos résultats, nous proposons un modèle où la séquestration de la Tetherin néo-synthétisée dans le RTG préviendrait le réapprovisionnement de Tetherin en surface qui, combinée avec l’internalisation naturelle de Tetherin à partir de la membrane plasmique, imposerait l’établissement d’un nouvel équilibre de Tetherin incompatible avec une restriction de la relâche des particules virales. Cette thèse nous a donc permis d’identifier un processus par lequel Vpu augmente la sécrétion de virus matures et établit une base mécanistique nécessaire à la compréhension de la contribution de Vpu à la propagation et à la pathogénèse du virus, ce qui pourrait mener à l’élaboration d’une stratégie visant à contrer l’effet de cette protéine virale.All accessory proteins of HIV-1, the ethiologic agent of AIDS, are thought to optimize viral replication and propagation in vivo. Among them, Vpu antagonizes Tetherin, a cellular factor that inhibits viral particle release. Downregulation of cell-surface Tetherin by Vpu is believed to prevent incorporation of this restriction factor into nascent viral particles, which would impede the formation of a Tetherin-derived protein anchor that bridges the virus to the plasma membrane of the infected cell. This thesis presents our studies on cellular mechanisms governing Tetherin antagonism by Vpu. A directed mutagenesis approach first identified two regions encompassing determinants of the localization of Vpu in the trans-Golgi network, and it correlated this intracellular distribution with enhanced release viral particle. Pulse-chase experiments in cellular systems wherein Tetherin was endogenously expressed showed that Vpu-induced Tetherin degradation is dispensable for restriction. In contrast, both a flow cytometry-based Tetherin re-expression assay and confocal microscopy analyses demonstrated that Vpu-mediated sequestration of Tetherin in the trans-Golgi network, a phenomenon that appeared to be triggered by the transmembrane association of the two proteins, was necessary for release inhibition. Vpu inducible expression in flow cytometry-based experiments provided evidence for an optimal antagonism of Tetherin at 6h after Vpu expression, following the interruption of Tetherin re-supply and a to the modest acceleration of the natural clearance of surface-localized Tetherin. Our work supports a model in which Tetherin sequestration in the trans-Golgi network prevents its re-supply, which, combined with its clearance from the surface, imposes a new equilibrium at the plasma membrane that is incompatible with the restriction of viral particle release. Overall, this thesis sheds light on the processes by which Vpu enhances the secretion of mature viruses and it establishes a mechanistic basis that could serve as starting point for the development of strategies aimed at interfering with Tetherin functions

Requirements for the selective degradation of CD4 receptor molecules by the human immunodeficiency virus type 1 Vpu protein in the endoplasmic reticulum

BACKGROUND: HIV-1 Vpu targets newly synthesized CD4 receptor for rapid degradation by a process reminiscent of endoplasmic reticulum (ER)-associated protein degradation (ERAD). Vpu is thought to act as an adaptor protein, connecting CD4 to the ubiquitin (Ub)-proteasome degradative system through an interaction with beta-TrCP, a component of the SCFbeta-TrCP E3 Ub ligase complex. RESULTS: Here, we provide direct evidence indicating that Vpu promotes trans-ubiquitination of CD4 through recruitment of SCFbeta-TrCP in human cells. To examine whether Ub conjugation occurs on the cytosolic tail of CD4, we substituted all four Ub acceptor lysine residues for arginines. Replacement of cytosolic lysine residues reduced but did not prevent Vpu-mediated CD4 degradation and ubiquitination, suggesting that Vpu-mediated CD4 degradation is not entirely dependent on the ubiquitination of cytosolic lysines and as such might also involve ubiquitination of other sites. Cell fractionation studies revealed that Vpu enhanced the levels of ubiquitinated forms of CD4 detected in association with not only the ER membrane but also the cytosol. Interestingly, significant amounts of membrane-associated ubiquitinated CD4 appeared to be fully dislocated since they could be recovered following sodium carbonate salt treatment. Finally, expression of a transdominant negative mutant of the AAA ATPase Cdc48/p97 involved in the extraction of ERAD substrates from the ER membrane inhibited Vpu-mediated CD4 degradation. CONCLUSION: Taken together, these results are consistent with a model whereby HIV-1 Vpu targets CD4 for degradation by an ERAD-like process involving most likely poly-ubiquitination of the CD4 cytosolic tail by SCFbeta-TrCP prior to dislocation of receptor molecules across the ER membrane by a process that depends on the AAA ATPase Cdc48/p97.This work was performed by JB in partial fulfillment of her doctoral thesis and was supported by grants from the Canadian Institutes of Health Research (CIHR) (MOP-14228) and from the Fonds de la Recherche en Santé du Québec (FRSQ) to EAC. JB is the recipient of a studentship from the FRSQ and EAC holds the Canada Research Chair in Human Retrovirology

Modulation of HIV-1-host interaction: role of the Vpu accessory protein

Viral protein U (Vpu) is a type 1 membrane-associated accessory protein that is unique to human immunodeficiency virus type 1 (HIV-1) and a subset of related simian immunodeficiency virus (SIV). The Vpu protein encoded by HIV-1 is associated with two primary functions during the viral life cycle. First, it contributes to HIV-1-induced CD4 receptor downregulation by mediating the proteasomal degradation of newly synthesized CD4 molecules in the endoplasmic reticulum (ER). Second, it enhances the release of progeny virions from infected cells by antagonizing Tetherin, an interferon (IFN)-regulated host restriction factor that directly cross-links virions on host cell-surface. This review will mostly focus on recent advances on the role of Vpu in CD4 downregulation and Tetherin antagonism and will discuss how these two functions may have impacted primate immunodeficiency virus cross-species transmission and the emergence of pandemic strain of HIV-1

Cleavage of a Neuroinvasive Human Respiratory Virus Spike Glycoprotein by Proprotein Convertases Modulates Neurovirulence and Virus Spread within the Central Nervous System.

International audienceHuman coronaviruses (HCoV) are respiratory pathogens that may be associated with the development of neurological diseases, in view of their neuroinvasive and neurotropic properties. The viral spike (S) glycoprotein is a major virulence factor for several coronavirus species, including the OC43 strain of HCoV (HCoV-OC43). In an attempt to study the role of this protein in virus spread within the central nervous system (CNS) and neurovirulence, as well as to identify amino acid residues important for such functions, we compared the sequence of the S gene found in the laboratory reference strain HCoV-OC43 ATCC VR-759 to S sequences of viruses detected in clinical isolates from the human respiratory tract. We identified one predominant mutation at amino acid 758 (from RRSR↓ G758 to RRSR↓R758), which introduces a putative furin-like cleavage (↓) site. Using a molecular cDNA infectious clone to generate a corresponding recombinant virus, we show for the first time that such point mutation in the HCoV-OC43 S glycoprotein creates a functional cleavage site between the S1 and S2 portions of the S protein. While the corresponding recombinant virus retained its neuroinvasive properties, this mutation led to decreased neurovirulence while potentially modifying the mode of virus spread, likely leading to a limited dissemination within the CNS. Taken together, these results are consistent with the adaptation of HCoV-OC43 to the CNS environment, resulting from the selection of quasi-species harboring mutations that lead to amino acid changes in viral genes, like the S gene in HCoV-OC43, which may contribute to a more efficient establishment of a less pathogenic but persistent CNS infection. This adaptative mechanism could potentially be associated with human encephalitis or other neurological degenerative pathologies

Effectiveness of serious games and impact of design elements on engagement and educational outcomes in healthcare professionals and students : a systematic review and meta-analysis protocol

Introduction Serious games (SGs) are interactive and entertaining digital software with an educational purpose. They engage the learner by proposing challenges and through various design elements (DEs; eg, points, difficulty adaptation, story). Recent reviews suggest the effectiveness of SGs in healthcare professionals’ and students’ education is mixed. This could be explained by the variability in their DEs, which has been shown to be highly variable across studies. The aim of this systematic review is to identify, appraise and synthesise the best available evidence regarding the effectiveness of SGs and the impact of DEs on engagement and educational outcomes of healthcare professionals and students. Methods and analysis A systematic search of the literature will be conducted using a combination of medical subject headings terms and keywords in Cumulative Index of Nursing and Allied Health, Embase, Education Resources Information Center, PsycInFO, PubMed and Web of Science. Studies assessing SGs on engagement and educational outcomes will be included. Two independent reviewers will conduct the screening as well as the data extraction process. The risk of bias of included studies will also be assessed by two reviewers using the Effective Practice and Organisation of Care criteria. Data regarding DEs in SGs will first be synthesised qualitatively. A meta-analysis will then be performed, if the data allow it. Finally, the quality of the evidence regarding the effectiveness of SGs on each outcome will be assessed using the Grading of Recommendations Assessment, Development and Evaluation approach. Ethics and dissemination As this systematic review only uses already collected data, no Institutional Review Board approval is required. Its results will be submitted in a peerreviewed journal by the end of 2018

Efficacy of adaptive e-learning for health professionals and students : a systematic review and meta-analysis

Objective Although adaptive e-learning environments (AEEs) can provide personalised instruction to health professional and students, their efficacy remains unclear. Therefore, this review aimed to identify, appraise and synthesise the evidence regarding the efficacy of AEEs in improving knowledge, skills and clinical behaviour in health professionals and students. Design Systematic review and meta-analysis. Data sources CINAHL, EMBASE, ERIC, PsycINFO, PubMed and Web of Science from the first year of records to February 2019. Eligibility criteria Controlled studies that evaluated the effect of an AEE on knowledge, skills or clinical behaviour in health professionals or students. Screening, data extraction and synthesis Two authors screened studies, extracted data, assessed risk of bias and coded quality of evidence independently. AEEs were reviewed with regard to their topic, theoretical framework and adaptivity process. Studies were included in the meta-analysis if they had a non-adaptive e-learning environment control group and had no missing data. Effect sizes (ES) were pooled using a random effects model. Results From a pool of 10 569 articles, we included 21 eligible studies enrolling 3684 health professionals and students. Clinical topics were mostly related to diagnostic testing, theoretical frameworks were varied and the adaptivity process was characterised by five subdomains: method, goals, timing, factors and types. The pooled ES was 0.70 for knowledge (95% CI −0.08 to 1.49; p.08) and 1.19 for skills (95% CI 0.59 to 1.79; p<0.00001). Risk of bias was generally high. Heterogeneity was large in all analyses. Conclusions AEEs appear particularly effective in improving skills in health professionals and students. The adaptivity process within AEEs may be more beneficial for learning skills rather than factual knowledge, which generates less cognitive load. Future research should report more clearly on the design and adaptivity process of AEEs, and target higher-level outcomes, such as clinical behaviour