65 research outputs found

    Étude du polymorphisme des gènes TAP : TAP1 et TAP2, en relation avec la susceptibilité au VIH chez les africains

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    Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal

    Le Scirpus cyperinus : germination, établissement et compétition en contexte de restauration de fen

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    L’évolution récente des méthodes d’extraction de la tourbe laisse les tourbières dans un état près d’un stade historique de développement de type minérotrophe plutôt qu’ombrotrophe. Conséquemment, les tourbières industrielles post-exploitation sont fréquemment envahies de colonies monospécifiques de Scirpus cyperinus. Cette étude porte sur l’élaboration de nouvelles méthodes d’intervention visant l’intégration du S. cyperinus dans une biodiversité représentative d’un écosystème de référence. Pour ce faire, deux expériences en serre ont été réalisées. La première expérience évalue la performance de quatre couverts végétaux pour limiter la germination du Scirpus cyperinus en fonction des conditions hydrologiques. Deux couverts végétaux, le Sphagnum warnstorfii et les plants de graminées, ont efficacement limité la germination du Scirpus cyperinus. La seconde expérience évalue le potentiel de deux espèces cibles de restauration à s’implanter parmi le Scirpus cyperinus sous deux régimes hydriques. Une seule espèce, le Calamagrostis canadensis, a réussi à se maintenir en présence du Scirpus cyperinus.Recent advances in peat extraction methods now leave the environment in a state closer to an historical development stage designated as minerotrophic (fen) rather than ombotrophic (bog). Consequently, industrial peatlands are commonly invaded monospecifically by Scirpus cyperinus soon after the end of operations. This study investigates new intervention techniques promoting biodiversity of the degraded ecosystem. In this context, two greenhouse experiments were carried out. The first one compared the performance of four plant covers to prevent S. cyperinus germination in relation to hydrologic conditions. Sphagnum warnstorfii and graminoid plants mats efficiently limited Scirpus cyperinus germination. The second experiment looked at the potential of two species targeted for reintroduction to grow and compete with Scirpus cyperinus under two hydrologic regimes. The biomass production of one of the two selected species (Calamagrostis canadensis) was able to maintain itself in presence of Scirpus cyperinus

    Étude des facteurs influençant la susceptibilité à l'infection au VIH chez des femmes africaines

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    Chez la femme, la majorité des cas d’infection au VIH sont acquis lors de relations hétérosexuelles. Cependant, très peu d’informations sont disponibles concernant l’immunité locale naturelle du tractus génital féminin, les facteurs influençant la susceptibilité à l’infection au VIH dans ce compartiment, ainsi que la réponse immunitaire de la muqueuse enclenchée après l’infection. Le but de notre projet est donc d’étudier certains facteurs pouvant être impliqués dans la susceptibilité à l’infection au VIH, afin de mieux comprendre l’immunité du tractus génital féminin. Nous avons, dans un premier temps, analysé le rôle du polymorphisme des gènes HLA-G et HLA-E sur la susceptibilité au VIH dans une population de femmes zimbabwéennes. La présence de l’allèle HLA-G*0105N, en combinaison avec le génotype HLA-EG/HLA-EG, était associée avec une diminution du risque d’infection. Puis, dans une étude cas-contrôle de travailleuses du sexe (TS) du Bénin, nous avons mesuré l’expression de HLA-G soluble au niveau du plasma. Nous avons observé une différence significative dans l’expression de HLA-G soluble, celle-ci étant plus faible dans le groupe des TS VIH positives comparé aux groupes de TS VIH négatives et de femmes VIH négatives de la population générale. Nous avons aussi analysé l’expression de cytokines et chimiokines dans le sérum et le tractus génital des participantes de l’étude du Bénin. Nous avons constaté que chez les TS VIH positives il y avait une expression plus élevée des chimiokines MPC-3, IP-10 et MIG dans le tractus génital et le sérum comparativement aux deux autres groupes. Les patrons d’expression des cytokines variaient selon les compartiments : le niveau de TNF-α et IFN-γ était plus élevé dans le tractus génital des TS VIH positives, alors que le niveau d’IL-2, d’IL-10 et de TNF-α était plus faible dans le sang des TS VIH positives, comparativement aux deux autres groupes. Ainsi, au niveau du tractus génital des femmes VIH positives, il semble y avoir une activation chronique du système immunitaire dans le but de favoriser la dissémination/perpétuation du virus. Les patrons d’expression différents entre le milieu systémique et génital nous montrent que l’immunité présente dans un compartiment n’est pas nécessairement le reflet de l’autre. Nous avons aussi observé une augmentation significative des niveaux d’IL-4, de MIP-1α, de MIP-1β et de MCP-1 dans le sérum des TS VIH négatives. Ces personnes, hautement exposées mais non infectées, semblent démontrer une plus grande capacité à enclencher une réponse immunitaire précoce pour empêcher la dissémination du virus. Notre étude a donc permis d’acquérir de nouvelles connaissances sur l’immunité du tractus génital féminin en relation avec l’infection au VIH.Initial exposure to HIV during heterosexual transmission occurs in the female genital tract. However, little is known about the local immunity, the factors influencing the susceptibility to HIV infection and the immune response in the female genital tract against HIV infection. The aim of this study is to analyse some factors that could be implicated in the susceptibility to HIV infection and to analyse, in part, the immunity present in the female genital tract. We investigated the role of HLA-G and HLA-E in the susceptibility to HIV infection in a cohort of Zimbabwean women. We found that the presence of HLA-G*0105N allele in combination with the genotype HLA-EG/HLA-EG was associated with a decrease in the risk of HIV infection. We also measured the expression of soluble HLA-G in a study of commercial sex workers (CSW) in Benin. Levels of soluble HLA-G were lower in the HIV-1-infected CSWs compared to those observed in both the HIV-1-uninfected CSWs and the HIV-1-uninfected women from the general population at low risk of infection. We also analysed the chemokine and cytokine expression patterns in the serum and female genital tract of the three groups of women. HIV-1-infected CSWs had significantly higher blood and genital levels of the chemokines IP-10, MCP-3 and MIG compared with those in both the HIV-1-uninfected CSW and non-CSW groups. HIV-1-infected CSWs had significantly higher genital mucosal levels of the cytokines TNF-α and IFN-γ compared with those in both the HIV-uninfected CSW and non-CSW groups. In contrast, the serum levels of the cytokines IL-2, IL-10 and TNF-α were lower in HIV-1-infected CSWs compared with those in the other groups. This suggests the presence of a constant immune cells recruitment and immune activation in the female genital tract in order to favour perpetuation and dissemination of the virus. Our results also demonstrate the important difference between the systemic and the mucosal immunity. We also observed a significant increase in the levels of IL-4, MIP-1α, MIP-1β and MCP-1 in the serum of the HIV-1-uninfected CSWs. It seems that these highly-exposed and yet uninfected women can have a better capacity to mount an early immune response against HIV. This study gives us new insights of the mucosal immunology of HIV infection

    New insights into Legionella pneumophila biofilm regulation by c-di-GMP signaling

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    The waterborne pathogen Legionella pneumophila grows as a biofilm, freely or inside amoebae. Cyclic-di-GMP (c-di-GMP), a bacterial second messenger frequently implicated in biofilm formation, is synthesized and degraded by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), respectively. To characterize the c-di-GMP-metabolizing enzymes involved in L. pneumophila biofilm regulation, the consequences on biofilm formation and the c-di-GMP concentration of each corresponding gene inactivation were assessed in the Lens strain. The results showed that one DGC and two PDEs enhance different aspects of biofilm formation, while two proteins with dual activity (DGC/PDE) inhibit biofilm growth. Surprisingly, only two mutants exhibited a change in global c-di-GMP concentration. This study highlights that specific c-di-GMP pathways control L. pneumophila biofilm formation, most likely via temporary and/or local modulation of c-di-GMP concentration. Furthermore, Lpl1054 DGC is required to enable the formation a dense biofilm in response to nitric oxide, a signal for biofilm dispersion in many other species

    A Tool for Automatic Correction of Endogenous Concentrations: Application to BHB Analysis by LC–MS-MS and GC-MS

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    Several substances relevant for forensic toxicology purposes have an endogenous presence in biological matrices: beta-hydroxybutyric acid (BHB), gamma-hydroxybutyric acid (GHB), steroids and human insulin, to name only a few. The presence of significant amounts of these endogenous substances in the biological matrix used to prepare calibration standards and quality control samples (QCs) can compromise validation steps and quantitative analyses. Several approaches to overcome this problem have been suggested, including using an analog matrix or analyte, relying entirely on standard addition analyses for these analytes, or simply ignoring the endogenous contribution provided that it is small enough. Although these approaches side-step the issue of endogenous analyte presence in spiked matrix-matched samples, they create serious problems with regards to the accuracy of the analyses or production capacity. We present here a solution that addresses head-on the problem of endogenous concentrations in matrices used for calibration standards and quality control purposes. The endogenous analyte concentration is estimated via a standard-addition type process. This estimated concentration, plus the spiked concentration are then used as the de facto analyte concentration present in the sample. These de facto concentrations are then used in data analysis software (MultiQuant, Mass Hunter, etc.) as the sample’s concentration. This yields an accurate quantification of the analyte, free from interference of the endogenous contribution. This de facto correction has been applied in a production setting on two BHB quantification methods (GC-MS and LC–MS-MS), allowing the rectification of BHB biases of up to 30 μg/mL. The additional error introduced by this correction procedure is minimal, although the exact amount will be highly method-dependent. The endogenous concentration correction process has been automated with an R script. The final procedure is therefore highly efficient, only adding four mouse clicks to the data analysis operations

    Natural Killer Cells Adapt to Cytomegalovirus Along a Functionally Static Phenotypic Spectrum in Human Immunodeficiency Virus Infection

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    Events related to HCMV infection drive accumulation of functionally enhanced CD57posNKG2Cpos adapted NK cells. We investigated NK cell adaptation to HCMV along a proposed continuum progressing from acute activation through maturation and memory formation towards functional exhaustion. Acute exposure to conditioned medium collected 24 h after HCMV infection (HCMVsn) increased NK cell cytotoxicity for all HCMV-seronegative and seropositive donors tested, with mean 38 and 29% boosts in natural and antibody-dependent cell-mediated cytotoxicity (ADCC), respectively. Increases in NK cell cytotoxicity were completely abrogated by blocking type I interferon (IFN) receptors and equivalent responses occurred with exposure to IFN-α2 alone at the same concentration present in HCMVsn. To study longer term effects of HCMV infection, we focused on three groups of human immunodeficiency virus (HIV)-infected subjects distinguished as HCMV-seronegative or HCMV-seropositive with either high (>20%) or low (<6%) fractions of their NK cells expressing NKG2C. The NK cells of all three HIV-infected groups responded to HCMVsn and IFN-α2 in a manner similar to the NK cells of either HCMV-seronegative or seropositive controls. Neither HCMV status, nor the extent of phenotypic evidence of adaptation to HCMV infection significantly affected mean levels of ADCC or CD16-mediated NK cell degranulation and IFN-γ production compared between the HIV-infected groups. Levels of IFN-γ production correlated significantly with the fraction of NK cells lacking FcεRIγ (FcRγ), but not with the fraction of NK cells expressing NKG2C. There was negligible expression of exhaustion markers Lag-3 and PD-1 on NK cells in any of the groups and no significant difference between groups in the fraction of NK cells expressing Tim-3. The fraction of NK cells expressing Tim-3 was unaffected by CD16 stimulation. Relative to the total NK cell population, responses of Tim-3-expressing cells to CD16 stimulation were variably compromised in HCMV seronegative and seropositive groups. In general, NK cell function in response to signaling through CD16 was well preserved in HIV infection and although HCMV had a clear effect on NK cell FcRγ and NKG2C expression, there was little evidence that the level of adaptation to HCMV infection affected CD16-dependent NK cell signaling in HIV infection

    Phosphorylation by Cdk1 Increases the Binding of Eg5 to Microtubules In Vitro and in Xenopus Egg Extract Spindles

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    BACKGROUND:Motor proteins from the kinesin-5 subfamily play an essential role in spindle assembly during cell division of most organisms. These motors crosslink and slide microtubules in the spindle. Kinesin-5 motors are phosphorylated at a conserved site by Cyclin-dependent kinase 1 (Cdk1) during mitosis. Xenopus laevis kinesin-5 has also been reported to be phosphorylated by Aurora A in vitro. METHODOLOGY/PRINCIPAL FINDINGS:We investigate here the effect of these phosphorylations on kinesin-5 from Xenopus laevis, called Eg5. We find that phosphorylation at threonine 937 in the C-terminal tail of Eg5 by Cdk1 does not affect the velocity of Eg5, but strongly increases its binding to microtubules assembled in buffer. Likewise, this phosphorylation promotes binding of Eg5 to microtubules in Xenopus egg extract spindles. This enhancement of binding elevates the amount of Eg5 in spindles above a critical level required for bipolar spindle formation. We find furthermore that phosphorylation of Xenopus laevis Eg5 by Aurora A at serine 543 in the stalk is not required for spindle formation. CONCLUSIONS/SIGNIFICANCE:These results show that phosphorylation of Eg5 by Cdk1 has a direct effect on the interaction of this motor with microtubules. In egg extract, phosphorylation of Eg5 by Cdk1 ensures that the amount of Eg5 in the spindle is above a level that is required for spindle formation. This enhanced targeting to the spindle appears therefore to be, at least in part, a direct consequence of the enhanced binding of Eg5 to microtubules upon phosphorylation by Cdk1. These findings advance our understanding of the regulation of this essential mitotic motor protein

    Qualitative method validation and uncertainty evaluation via the binary output: II - Application to a multi-analyte LC-MS/MS method for oral fluid

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    A study of impaired driving rates in the province of Québec is currently planned following the legalization of recreational cannabis in Canada. Oral fluid (OF) samples are to be collected with a Quantisal device and sent to the laboratory for analysis. In order to prepare for this project, a qualitative decision point analysis method monitoring for the presence of 97 drugs and metabolites in OF was validated according to the guidelines presented in the first part of this paper (I – Validation guidelines and statistical foundations). This high throughput method uses incubation with a precipitation solvent (acetone:acetonitrile 30:70 v:v) to boost drug recovery from the collecting device and improve stability of benzodiazepines (e.g. α-hydroxyalprazolam, clonazepam, 7-aminoclonazepam, flunitrazepam, 7-aminoflunitrazepam, N-desmethylflunitrazepam, nitrazepam). The Quantisal device has polyglycol in its stabilizing buffer but timed use of the mass spectrometer waste valve proved sufficient to avoid the glycol interferences for nearly all analytes. Interferences from OF matrices and 140 potentially interfering compounds, carryover, ion ratios, stability, recovery, reproducibility, robustness, false positive rate, false negative rate, selectivity, sensitivity and reliability rates were tested in the validation process. Five of the targeted analytes (olanzapine, oxazepam, 7-aminoclonazepam, flunitrazepam and nitrazepam) did not meet the set validation criteria but will be monitored for identification purposes (no comparison to a cut-off level). Blind internal proficiency teting was performed, where six OF samples were tested and analytes were classified as “negative”, “likely positive” or “positive” with success. The final validated OF qualitative decision point method covers 92 analytes, and the presence of 5 additional analytes is screened in this high hroughput analysis

    A threshold LC–MS/MS method for 92 analytes in oral fluid collected with the Quantisal® device

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    A study of impaired driving rates in the province of Québec is currently planned following the legalization of recreational cannabis in Canada. Oral fluid (OF) samples are to be collected with a Quantisal® device and sent to the laboratory for analysis. In order to prepare for this project, a qualitative decision point analysis method monitoring for the presence of 97 drugs and metabolites in OF was developed and validated. This high throughput method uses incubation with a precipitation solvent (acetone:acetonitrile 30:70 v:v) to boost drug recovery from the collecting device and improve stability of benzodiazepines (e.g., α-hydroxyalprazolam, clonazepam, 7-aminoclonazepam, flunitrazepam, 7-aminoflunitrazepam, N-desmethylflunitrazepam, nitrazepam). The Quantisal® device has polyglycol in its stabilizing buffer, but timed use of the mass spectrometer waste valve proved sufficient to avoid the glycol interferences for nearly all analytes. Interferences from OF matrices and 140 potentially interfering compounds, carryover, ion ratios, stability, recovery, reproducibility, robustness, false positive rate, false negative rate, selectivity, sensitivity and reliability rates were tested in the validation process. Five of the targeted analytes (olanzapine, oxazepam, 7-aminoclonazepam, flunitrazepam and nitrazepam) did not meet the set validation criteria but will be monitored for identification purposes (no comparison to a cut-off level). Blind internal proficiency testing was performed, where six OF samples were tested and analytes were classified as “negative”, “likely positive” or “positive” with success. The final validated OF qualitative decision point method covers 92 analytes, and the presence of 5 additional analytes is screened in this high throughput analysis

    High Level of Soluble HLA-G in the Female Genital Tract of Beninese Commercial Sex Workers Is Associated with HIV-1 Infection

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    Most HIV infections are transmitted across mucosal epithelium. Understanding the role of innate and specific mucosal immunity in susceptibility or protection against HIV infection, as well as the effect of HIV infection on mucosal immunity, are of fundamental importance. HLA-G is a powerful modulator of the immune response. The aim of this study was to investigate whether soluble HLA-G (sHLA-G) expression in the female genital tract is associated with HIV-1 infection.Genital levels of sHLA-G were determined in 52 HIV-1-uninfected and 44 antiretroviral naïve HIV-1-infected female commercial sex workers (CSWs), as well as 71 HIV-1-uninfected non-CSW women at low risk of exposure, recruited in Cotonou, Benin. HIV-1-infected CSWs had higher genital levels of sHLA-G compared with those in both the HIV-1-uninfected CSW (P = 0.009) and non-CSW groups (P = 0.0006). The presence of bacterial vaginosis (P = 0.008), and HLA-G*01:01:02 genotype (P = 0.002) were associated with higher genital levels of sHLA-G in the HIV-1-infected CSWs, whereas the HLA-G*01:04:04 genotype was also associated with higher genital level of sHLA-G in the overall population (P = 0.038). When adjustment was made for all significant variables, the increased expression of sHLA-G in the genital mucosa remained significantly associated with both HIV-1 infection (P = 0.02) and bacterial vaginosis (P = 0.03).This study demonstrates that high level of sHLA-G in the genital mucosa is independently associated with both HIV-1 infection and bacterial vaginosis
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