Étude de la spécificité fonctionnelle des protéines adaptatrices NCK1 et NCK2

Plusieurs réponses cellulaires aux stimuli extracellulaires sont transmises par des voies de signalisation qui agissent en aval de récepteurs membranaires tels les récepteurs tyrosine kinase (RTK). Les signaux provenant des RTK sont souvent relayés via des protéines adaptatrices comme NCK1 (Non-Catalytic region of tyrosine Kinase 1) et NCK2 dont les fonctions sont considérées redondantes et indissociables. Toutefois, certaines études suggèrent que chacune de ces deux protéines pourraient avoir des cibles cellulaires spécifiques et des fonctions uniques. L’objectif de mon projet de doctorat était d’analyser la spécificité des protéines NCK1/2, c’est-à-dire d’identifier pour chacune des cibles uniques, pour ensuite caractériser ce qui génère cette spécificité tout en définissant la fonction de ces interactions. En premier lieu, j’ai utilisé deux techniques complémentaires de protéomique, soit (i) des purifications d’affinité (AP) et (ii) du marquage de proximité in vivo (BioID) suivies d’analyses en spectrométrie de masse (MS) afin de caractériser les interactomes respectifs de NCK1/2. La combinaison de ces deux approches m’a permis d’identifier plus d’une centaine d’interactions spécifiques pour chaque NCK. Des analyses bio-informatiques basées sur ces résultats m’ont permises de mettre en évidence que NCK2 semble plus spécifiquement impliquée que NCK1 dans la régulation de l’organisation du cytosquelette d’actine, structure essentielle lors de la division et de la cytokinèse. En comparant simultanément des cellules fibroblastiques murines (MEF) déplétées soit pour NCK1, soit pour NCK2, j’ai remarqué que les cellules Nck2-/-, à l’inverse des cellules Nck1-/- étaient plus multinucléées et présentent un midbody altéré en longueur. Également, j’ai remarqué une altération dans la composition du midbody des cellules Nck2-/- tel que suggéré par l’absence dans cette structure des protéines Polo-like kinase1 (PLK1), Epithelial cell transforming 2 (ECT2) ou encore Aurora B (AURKB), régulateurs clefs de la cytokinèse. Finalement, j’ai montré que la fonction de NCK2 durant la cytokinèse repose principalement sur son domaine SH2. Dans un deuxième temps, j’ai sélectionné 27 partenaires identifiés en MS et confirmé par une méthode orthogonale leurs interactions respectives avec NCK1 et/ou NCK2. Grâce à des tests de liaison in vitro, j’ai déterminé que plusieurs protéines dont la Plakophiline 4 (PKP4), un régulateur de la cytokinèse, lient directement et spécifiquement NCK2. Par différentes expériences in vitro, j’ai pu déterminer que NCK2 lie les portions N-terminale et centrale de PKP4 grâce à son domaine Src Homology (SH) 2 et que la spécificité de NCK2 envers PKP4 ne semble pas dépendre seulement des propriétés intrinsèques du SH2. L’association résulte plutôt de la combinaison d’une partie ou de l’ensemble des propriétés des domaines et régions interdomaines constituant les protéines NCK1/2. En conclusion, bien que les fonctions de NCK1/2 soient généralement considérées comme redondantes, mes résultats démontrent que ces protéines sont capables de lier des partenaires différentspour réguler des fonctions biologiques distinctes. Ainsi, mes travaux suggèrent que NCK2 semble spécifiquement requise lors du processus de cytokinèse. De plus, l’ensemble de mes expériences in vitro apporte une première idée du mécanisme de spécificité des protéines NCK1/2 en suggérant que leur spécificité ne semble pas entièrement provenir des propriétés intrinsèques de leurs domaines individuels, mais plutôt d’une combinaison des propriétés de leurs domaines et/ou régions interdomaines respectives.Signals from cell surface receptors are often relayed via adaptor proteins that can serve as hubs to recruit appropriate target signaling molecules and guide signals along specific pathways. Among these, adaptor proteins NCK1 (Non-Catalytic region of tyrosine Kinase 1) and NCK2 have functions that are often considered redundant and/or indistinguishable. The main goal of my work was to demonstrate that NCK1 and NCK2 are not fully redundant and may each display functional specificity. To achieve this, I delineated NCK1-and NCK2-specific signalling networks, identified for each unique target, then characterized what generates this specificity and obtained the function of these interactions. First, to identify the complement of interaction partners for NCK1 and NCK2, I used two unbiased mass spectrometry (MS)-based approaches: (i) epitope-tagged protein affinity purification (AP) followed by MS analysis and (ii) in vivo proximity labelling (BioID). The combination of these two approaches allowed me to identify more than one hundred specific interactions for each NCK. Bioinformatics analyzes based on the specific partners identified in MS enabled me to highlight that NCK2 was more specifically involved in the regulation of the actin cytoskeleton organization, structure essential for cell division and cytokinesis. By simultaneously comparing mouse embryo fibroblasts (MEF) depleted either for NCK1 or NCK2, I noticed that Nck2-/-, but not Nck1-/-cells are multi-nucleated and display extended protrusions reminiscent of intercellular bridges, which correlate with an extended time spent in cytokinesis as well as a failure of a significant proportion of cells to complete abscission. Further analysis of this phenotype revealed that the midbody of NCK2-deficient cells is not only increased in length, but also altered in composition, as judged by the mislocalization of the Polo-like kinase 1 (PLK1), Epithelial cell transforming 2 (ECT2) and Aurora B (AURKB) proteins. Moreover, I showed that NCK2 function during cytokinesis requires its SH2 domain. Second, to underline the molecular mechanism of specific protein complex formation, I selected based on my MS results 27 partners to confirm by an orthogonal method their respective interactions with NCK1 and/or NCK2. By using in vitro binding assays, I was able to determine that several proteins including Plakophilin 4(PKP4), a key regulator of the cytokinesis process, were able to bind directly and specifically to NCK2. Through various in vitro experiments, I was able to determine that NCK2 binds the N-terminal and central portions of PKP4 through its SH2 domain and that the specificity of PKP4 toward NCK2 does not appear to result from the intrinsic properties of its SH2 alone. This association seems to result from the combination of some or all of the properties of the individual domains and inter-regions constituting the NCK1/2 proteins. In conclusion, despite what is generally accepted, I showed that both NCK1 and NCK2 may form specific protein complexes, thus reflecting the functional specificity of these two adaptor proteins. I further demonstrated that NCK1 and NCK2 are not completely redundant. I also shed light on a previously uncharacterized function for the NCK2 adaptor protein in cell division. Finally, my in vitro experiments provide an explanation for the specificity mechanism of NCK1/2 adaptor proteins by suggesting that their specificity come from the combination of the properties of their respective domains and/or interdomain regions

Real-World Normal Map Capture for Nearly Flat Reflective Surfaces

Although specular objects have gained interest in recent years, virtually no approaches exist for markerless reconstruction of reflective scenes in the wild. In this work, we present a practical approach to capturing normal maps in real-world scenes using video only. We focus on nearly planar surfaces such as windows, facades from glass or metal, or frames, screens and other indoor objects and show how normal maps of these can be obtained without the use of an artificial calibration object. Rather, we track the reflections of real-world straight lines, while moving with a hand-held or vehicle-mounted camera in front of the object. In contrast to error-prone local edge tracking, we obtain the reflections by a robust, global segmentation technique of an ortho-rectified 3D video cube that also naturally allows efficient user interaction. Then, at each point of the reflective surface, the resulting 2D-curve to 3D-line correspondence provides a novel quadratic constraint on the local surface normal. This allows to globally solve for the shape by integrability and smoothness constraints and easily supports the usage of multiple lines. We demonstrate the technique on several objects and facades

Substandard Quality of the Antimicrobials Sold in the Street Markets in Haiti

This pilot study was conducted to analyze the quality of the antimicrobials sold in the street markets in Port-au-Prince, Haiti. A total of 258 packs containing antimicrobials were bought in 28 street markets in Port-au-Prince (Haiti). Tablets and contents of capsules included in 196 packs were analyzed using a Raman handheld spectrometer (NanoRAM of BWTEK, Model: BWS456-785) during the first quarter of 2019. Three out of 11 antimicrobials (Amoxicillin, Metronidazole, and Cotrimoxazole) had a high spectral match with an HQI ≥ 90 to the respective authentic medicine for more than 95% of their tablets/capsules. For six antimicrobials (Tetracycline, Erythromycin, Cloxacillin, Azithromycin, Clarithromycin, and the combination Amoxicillin + Clavulanic Acid) none of their tablets/capsules showed a sufficient spectral match with the authentic medicine. This finding indicates that these products sold in the markets did not contain the labeled drug and/or contained a degraded drug. In addition to the fact that prescription antimicrobials can be purchased in street markets, the present field study found that for most of them (including "Watch" antimicrobials according to the AWaRe classification) were substandard, which contributes to the present antimicrobials resistance epidemic

Global Emergency Medicine: A Review of the Literature From 2014

ObjectivesThe Global Emergency Medicine Literature Review (GEMLR) conducts an annual search of peer‐reviewed and gray literature relevant to global emergency medicine (EM) to identify, review, and disseminate the most important new research in this field to a worldwide audience of academics and clinical practitioners.MethodsThis year 6,376 articles written in six languages were identified by our search. These articles were distributed among 20 reviewers for initial screening based on their relevance to the field of global EM. An additional two reviewers searched the gray literature. A total of 477 articles were deemed appropriate by at least one reviewer and approved by the editor for formal scoring of overall quality and importance.ResultsOf the 477 articles that met our predetermined inclusion criteria, 63% were categorized as emergency care in resource‐limited settings, 13% as EM development, and 23% as disaster and humanitarian response. Twenty‐five articles received scores of 17.5 or higher and were selected for formal summary and critique. Inter‐rater reliability for two reviewers using our scoring system was good, with an intraclass correlation coefficient of 0.657 (95% confidence interval = 0.589 to 0.713). Studies and reviews focusing on infectious diseases, trauma, and the diagnosis and treatment of diseases common in resource‐limited settings represented the majority of articles selected for final review.ConclusionsIn 2014, there were fewer total articles, but a slightly higher absolute number of articles screening in for formal scoring, when compared to the 2013 review. The number of EM development articles decreased, while the number of disaster and humanitarian response articles increased. As in prior years, the majority of articles focused on infectious diseases and trauma.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/113141/1/acem12733.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/113141/2/acem12733_am.pd

A Crucial Role for Infected-Cell/Antibody Immune Complexes in the Enhancement of Endogenous Antiviral Immunity by Short Passive Immunotherapy

Antiviral monoclonal antibodies (mAbs) represent promising therapeutics. However, most mAbs-based immunotherapies conducted so far have only considered the blunting of viral propagation and not other possible therapeutic effects independent of virus neutralization, namely the modulation of the endogenous immune response. As induction of long-term antiviral immunity still remains a paramount challenge for treating chronic infections, we have asked here whether neutralizing mAbs can, in addition to blunting viral propagation, exert immunomodulatory effects with protective outcomes. Supporting this idea, we report here that mice infected with the FrCasE murine retrovirus on day 8 after birth die of leukemia within 4–5 months and mount a non-protective immune response, whereas those rapidly subjected to short immunotherapy with a neutralizing mAb survive healthy and mount a long-lasting protective antiviral immunity with strong humoral and cellular immune responses. Interestingly, the administered mAb mediates lysis of infected cells through an antibody-dependent cell cytotoxicity (ADCC) mechanism. In addition, it forms immune complexes (ICs) with infected cells that enhance antiviral CTL responses through FcγR-mediated binding to dendritic cells (DCs). Importantly, the endogenous antiviral antibodies generated in mAb-treated mice also display the same properties, allowing containment of viral propagation and enhancement of memory cellular responses after disappearance of the administered mAb. Thus, our data demonstrate that neutralizing antiviral mAbs can act as immunomodulatory agents capable of stimulating a protective immunity lasting long after the end of the treatment. They also show an important role of infected-cells/antibody complexes in the induction and the maintenance of protective immunity through enhancement of both primary and memory antiviral T-cell responses. They also indicate that targeting infected cells, and not just viruses, by antibodies can be crucial for elicitation of efficient, long-lasting antiviral T-cell responses. This must be considered when designing antiviral mAb-based immunotherapies

Impact of DOTS expansion on tuberculosis related outcomes and costs in Haiti

BACKGROUND: Implementation of the World Health Organization's DOTS strategy (Directly Observed Treatment Short-course therapy) can result in significant reduction in tuberculosis incidence. We estimated potential costs and benefits of DOTS expansion in Haiti from the government, and societal perspectives. METHODS: Using decision analysis incorporating multiple Markov processes (Markov modelling), we compared expected tuberculosis morbidity, mortality and costs in Haiti with DOTS expansion to reach all of the country, and achieve WHO benchmarks, or if the current situation did not change. Probabilities of tuberculosis related outcomes were derived from the published literature. Government health expenditures, patient and family costs were measured in direct surveys in Haiti and expressed in 2003 US$. RESULTS: Starting in 2003, DOTS expansion in Haiti is anticipated to cost$4.2 million and result in 63,080 fewer tuberculosis cases, 53,120 fewer tuberculosis deaths, and net societal savings of $131 million, over 20 years. Current government spending for tuberculosis is high, relative to the per capita income, and would be only slightly lower with DOTS. Societal savings would begin within 4 years, and would be substantial in all scenarios considered, including higher HIV seroprevalence or drug resistance, unchanged incidence following DOTS expansion, or doubling of initial and ongoing costs for DOTS expansion. CONCLUSION: A modest investment for DOTS expansion in Haiti would provide considerable humanitarian benefit by reducing tuberculosis-related morbidity, mortality and costs for patients and their families. These benefits, together with projected minimal Haitian government savings, argue strongly for donor support for DOTS expansion

Cost-effectiveness of novel vaccines for tuberculosis control: a decision analysis study

<p>Abstract</p> <p>Background</p> <p>The development of a successful new tuberculosis (TB) vaccine would circumvent many limitations of current diagnostic and treatment practices. However, vaccine development is complex and costly. We aimed to assess the potential cost effectiveness of novel vaccines for TB control in a sub-Saharan African country - Zambia - relative to the existing strategy of directly observed treatment, short course (DOTS) and current level of bacille Calmette-Guérin (BCG) vaccination coverage.</p> <p>Methods</p> <p>We conducted a decision analysis model-based simulation from the societal perspective, with a 3% discount rate and all costs expressed in 2007 US dollars. Health outcomes and costs were projected over a 30-year period, for persons born in Zambia (population 11,478,000 in 2005) in year 1. Initial development costs for single vaccination and prime-boost strategies were prorated to the Zambian share (0.398%) of global BCG vaccine coverage for newborns. Main outcome measures were TB-related morbidity, mortality, and costs over a range of potential scenarios for vaccine efficacy.</p> <p>Results</p> <p>Relative to the status quo strategy, a BCG replacement vaccine administered at birth, with 70% efficacy in preventing rapid progression to TB disease after initial infection, is estimated to avert 932 TB cases and 422 TB-related deaths (prevention of 199 cases/100,000 vaccinated, and 90 deaths/100,000 vaccinated). This would result in estimated net savings of $3.6 million over 30 years for 468,073 Zambians born in year 1 of the simulation. The addition of a booster at age 10 results in estimated savings of$5.6 million compared to the status quo, averting 1,863 TB cases and 1,011 TB-related deaths (prevention of 398 cases/100,000 vaccinated, and of 216 deaths/100,000 vaccinated). With vaccination at birth alone, net savings would be realized within 1 year, whereas the prime-boost strategy would require an additional 5 years to realize savings, reflecting a greater initial development cost.</p> <p>Conclusions</p> <p>Investment in an improved TB vaccine is predicted to result in considerable cost savings, as well as a reduction in TB morbidity and TB-related mortality, when added to existing control strategies. For a vaccine with waning efficacy, a prime-boost strategy is more cost-effective in the long term.</p

Search for gravitational-lensing signatures in the full third observing run of the LIGO-Virgo network

Gravitational lensing by massive objects along the line of sight to the source causes distortions of gravitational wave-signals; such distortions may reveal information about fundamental physics, cosmology and astrophysics. In this work, we have extended the search for lensing signatures to all binary black hole events from the third observing run of the LIGO--Virgo network. We search for repeated signals from strong lensing by 1) performing targeted searches for subthreshold signals, 2) calculating the degree of overlap amongst the intrinsic parameters and sky location of pairs of signals, 3) comparing the similarities of the spectrograms amongst pairs of signals, and 4) performing dual-signal Bayesian analysis that takes into account selection effects and astrophysical knowledge. We also search for distortions to the gravitational waveform caused by 1) frequency-independent phase shifts in strongly lensed images, and 2) frequency-dependent modulation of the amplitude and phase due to point masses. None of these searches yields significant evidence for lensing. Finally, we use the non-detection of gravitational-wave lensing to constrain the lensing rate based on the latest merger-rate estimates and the fraction of dark matter composed of compact objects