Régulation de l'activation et de l'adhésion des leucocytes, des cellules endothéliales et des plaquettes par la protéine C-réactive

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal

PHEN : parkinson helper emergency notification system using Bayesian Belief Network

Context-aware systems are used to aid users in their daily lives. In the recent years, researchers are exploring how context aware systems can benefit humanity through assist patients, specifically those who suffer incurable diseases, to cope with their illness. In this paper, we direct our work to help people who suffer from Parkinson disease. We propose PHEN, Parkinson Helper Engine Network System, a context-aware system that aims to support Parkinson disease patients on m any levels. We use ontology is for context representation and modeling. Then the ontology based context model is used to learn with Bayesian Belief network (BBN) which is beneficial in handling the uncertainty aspect of context-aware systems

Opposing effects of monomeric and pentameric C-reactive protein on endothelial progenitor cells

C-reactive protein (CRP) has been linked to the pathogenesis of atherosclerosis. The dissociation of native, pentameric (p)CRP to monomeric (m)CRP on the cell membrane of activated platelets has recently been demonstrated. The dissociation of pCRP to mCRP may explain local pro-inflammatory reactions at the site of developing atherosclerotic plaques. As a biomarker, pCRP predicts cardiovascular adverse events and so do reduced levels and function of circulating endothelial progenitor cells (EPCs). We hypothesised that mCRP and pCRP exert a differential effect on EPC function and differentiation. EPCs were treated with mCRP or pCRP for 72 h, respectively. Phenotypical characterisation was done by flow cytometry and immunofluorescence microscopy, while the effect of mCRP and pCRP on gene expression was examined by whole-genome gene expression analysis. The functional capacity of EPCs was determined by colony forming unit (CFU) assay and endothelial tube formation assay. Double staining for acetylated LDL and ulex lectin significantly decreased in cells treated with pCRP. The length of tubuli in a matrigel assay with HUVECs decreased significantly in response to pCRP, but not to mCRP. The number of CFUs increased after pCRP treatment. RNA expression profiling demonstrated that mCRP and pCRP cause highly contradictory gene regulation. Interferon-responsive genes (IFI44L, IFI44, IFI27, IFI 6, MX1, OAS2) were among the highly up-regulated genes after mCRP, but not after pCRP treatment. In conclusion, EPC phenotype, genotype and function were differentially affected by mCRP and pCRP, strongly arguing for differential roles of these two CRP conformations. The up-regulation of interferon-inducible genes in response to mCRP may constitute a mechanism for the local regulation of EPC function

Acetylcholine Inhibits Monomeric C-Reactive Protein Induced Inflammation, Endothelial Cell Adhesion, and Platelet Aggregation; A Potential Therapeutic?

Objectives: In this study, we examined the possibility of using targeted antibodies and the potential of small molecular therapeutics (acetylcholine, nicotine and tacrine) to block the pro-inflammatory and adhesion-related properties of monomeric C-reactive protein (mCRP). Methods: We used three established models (platelet aggregation assay, endothelial leucocyte binding assay and monocyte inflammation via ELISA and Western blotting) to assess the potential of these therapeutics. Results: The results of this study showed that monocyte induced inflammation (raised tumor necrosis factor-alpha-TNF-α) induced by mCRP was significantly blocked in the presence of acetylcholine and nicotine, whilst tacrine and targeted antibodies (clones 8C10 and 3H12) had less of or no significant effects. Western blotting confirmed the ability of acetylcholine to inhibit mCRP-induced cell signaling phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2), p38 and nuclear factor-kappa B (NF-κB). There was no evidence of direct binding between small molecules and mCRP. mCRP also induced endothelial cell-monocyte adhesion in a dose dependent fashion, however, both acetylcholine and nicotine as well as targeting antibodies notably inhibited adhesion. Finally, we investigated their effects on mCRP-induced platelet aggregation. All three small molecules significantly attenuated platelet aggregation as did the antibody 8C10, although 3H12 had a weaker effect. Discussion: Acetylcholine and to a lesser extent nicotine show potential for therapeutic inhibition of mCRP-induced inflammation and cell and platelet adhesion. These results highlight the potential of targeted antibodies and small molecule therapeutics to inhibit the binding of mCRP by prevention of membrane interaction and subsequent activation of cellular cascade systems, which produce the pro-inflammatory effects associated with mCRP

The Effect of C-Reactive Protein Isoforms on Nitric Oxide Production by U937 Monocytes/Macrophages

Inflammation is regulated by many endogenous factors including estrogen, a steroid hormone that declines with increasing age, leading to excessive inflammation in the elderly. C-reactive protein (CRP) is an acute phase inflammatory protein that exists in two forms, native CRP (nCRP) and monomeric CRP (mCRP), which mediate distinct biological activities. It is unclear how each CRP isoform mediates nitric oxide (NO), a signalling molecule generated by NO synthase (NOS). This study investigated whether CRP isoforms have distinct effects on NO production by unstimulated and lipopolysaccharide (LPS)-activated monocytes/macrophages and whether estrogen mediates CRP-induced NO production in an in vitro model of ageing. NO and inducible NOS (iNOS) were measured (n=12) by the Griess assay and an enzyme-linked immunosorbent assay (ELISA) respectively following incubation (24 hours) of human-derived U937 monocytes/macrophages with CRP isoforms ([nCRP] = 500 and 1000µg/ml; [mCRP] = 100 and 250µg/ml) in the absence or presence of 17 beta-estradiol (1x10-7, 1x10-8 and 1x10-9M). The response to each CRP isoform and estrogen was dependent on the differentiation and activation status of cells. Monocytes with or without prior LPS-activation significantly increased (P0.05) on NO/iNOS production by unactivated or LPS-activated macrophages whereas nCRP significantly (P<0.05) reduced NO/iNOS production by macrophages, with or without prior LPS-activation. The nCRP isoform had opposing actions on monocytes, significantly (p<0.01) increasing and reducing NO/iNOS by unactivated and LPS-activated monocytes respectively. Estrogen significantly (P<0.01) reversed nCRP-mediated NO inhibition by unactivated macrophages but decreased CRP-induced NO by unactivated monocytes treated with nCRP or mCRP and LPS-activated monocytes treated with mCRP. NO was differentially mediated by CRP isoforms in a cell-type/state specific manner, with production corresponding to concomitant changes in iNOS levels. Collectively, the findings indicate nCRP and estrogen predominantly reduce NO production whereas mCRP increases NO production. This supports growing evidence that mCRP exacerbates inflammation whilst nCRP and estrogen dampen the overall inflammatory response. Therapeutic strategies that restore estrogen levels to those found in youth and promote the stability of nCRP or/and prevent the formation of mCRP may reduce NO production in age-related inflammatory conditions

P2X7 nucleotide receptors mediate caspase-8/9/3-dependent apoptosis in rat primary cortical neurons

Apoptosis is a major cause of cell death in the nervous system. It plays a role in embryonic and early postnatal brain development and contributes to the pathology of neurodegenerative diseases. Here, we report that activation of the P2X7 nucleotide receptor (P2X7R) in rat primary cortical neurons (rPCNs) causes biochemical (i.e., caspase activation) and morphological (i.e., nuclear condensation and DNA fragmentation) changes characteristic of apoptotic cell death. Caspase-3 activation and DNA fragmentation in rPCNs induced by the P2X7R agonist BzATP were inhibited by the P2X7R antagonist oxidized ATP (oATP) or by pre-treatment of cells with P2X7R antisense oligonucleotide indicating a direct involvement of the P2X7R in nucleotide-induced neuronal cell death. Moreover, Z-DEVD-FMK, a specific and irreversible cell permeable inhibitor of caspase-3, prevented BzATP-induced apoptosis in rPCNs. In addition, a specific caspase-8 inhibitor, Ac-IETD-CHO, significantly attenuated BzATP-induced caspase-9 and caspase-3 activation, suggesting that P2X7R-mediated apoptosis in rPCNs occurs primarily through an intrinsic caspase-8/9/3 activation pathway. BzATP also induced the activation of C-jun N-terminal kinase 1 (JNK1) and extracellular signal-regulated kinases (ERK1/2) in rPCNs, and pharmacological inhibition of either JNK1 or ERK1/2 significantly reduced caspase activation by BzATP. Taken together, these data indicate that extracellular nucleotides mediate neuronal apoptosis through activation of P2X7Rs and their downstream signaling pathways involving JNK1, ERK and caspases 8/9/3

C-reactive protein in atherothrombosis and angiogenesis

© 2018 Badimon, Peña, Arderiu, Padró, Slevin, Vilahur and Chiva-Blanch. C-reactive protein (CRP) is a short pentraxin mainly found as a pentamer in the circulation, or as non-soluble monomers CRP (mCRP) in tissues, exerting different functions. This review is focused on discussing the role of CRP in cardiovascular disease, including recent advances on the implication of CRP and its forms specifically on the pathogenesis of atherothrombosis and angiogenesis. Besides its role in the humoral innate immune response, CRP contributes to cardiovascular disease progression by recognizing and binding multiple intrinsic ligands. mCRP is not present in the healthy vessel wall but it becomes detectable in the early stages of atherogenesis and accumulates during the progression of atherosclerosis. CRP inhibits endothelial nitric oxide production and contributes to plaque instability by increasing endothelial cell adhesion molecules expression, by promoting monocyte recruitment into the atheromatous plaque and by enzymatically binding to modified low-density lipoprotein. CRP also contributes to thrombosis, but depending on its form it elicits different actions. Pentameric CRP has no involvement in thrombogenesis, whereas mCRP induces platelet activation and thrombus growth. In addition, mCRP has apparently contradictory pro-angiogenic and anti-angiogenic effects determining tissue remodeling in the atherosclerotic plaque and in infarcted tissues. Overall, CRP contributes to cardiovascular disease by several mechanisms that deserve an in-depth analysis

Dbp6, une ARN hélicase requise pour les étapes précoces de la synthèse de la grande sous-unité du ribosome eucaryotes

L'activité de traduction des ribosomes est portée directement par les ARN ribosomiques (ARNr) qui composent ses deux sous-unités. La grande sous-unité (60S) est formée par les ARNr 25S, 5.8S et 5S et la petite sous-unité (40S) est formée par l'ARNr 18S. Un des principaux buts de la biogenèse des ribosomes est de convertir les ARNr en molécules correctement repliées et donc actives. La production des sous-unités ribosomiques est le résultat d'étapes successives de maturation de particules précurseurs, les pré-60S et les pré-40S, précurseurs de la grande (60S) et la petite (40S) sous-unité ribosomique, respectivement. Des protéines ribosomiques, des facteurs d'assemblage (FA) et des petites particules ribonucléoprotéique (snoRNPs) sont impliquées dans ces étapes successives. Ces facteurs jouent des rôles importants dans l'organisation spatiale et le maintien de l'intégrité structurelle des ARNr. Les ARN hélicases forment le plus grand groupe des FA et peuvent moduler les interactions ARN-ARN et ARN-protéine. Elles forment des candidates potentielles du repliement tridimensionnelle des ARNr. Cependant, les mécanismes par lesquels ces enzymes participent à la production des ribosomes restent vagues. Dans cette étude, on se focalise sur la fonction de l'ARN hélicase à boîte DEAD Dbp6 dans la structuration précoce de l'ARNr de la grande sous-unité ribosomique (60S). Dbp6 est essentielle pour la production de la grande sous-unité ribosomique. En son absence la production de la première particule pré-60S est inhibée. Néanmoins, l'importance des activités enzymatiques de Dbp6 pour la production de la première particule pré-60S n'ont pas été évalué et ses substrats ARN n'ont pas été déterminé. Dans notre étude, nous avons démontré que Dbp6 montre les activités biochimiques attendues, tels que l'hydrolyse d'ATP et la liaison à l'ARN. Dbp6 n'a pas montré une activité de dissociation de brins d'ARN (hélicase) dans les conditions testées dans le laboratoire. Nous avons pu identifier et étudier une activité d'association de brins (annealing) qui est contrôlée par l'ATP. En étudiant des mutants de Dbp6 qui ciblent les motifs conservés du cœur hélicase, nous avons établi que l'hydrolyse ATP est importante mais pas essentielle pour la survie cellulaire. Cependant, l'activité d'annealing semble jouer un rôle clé dans la fonction moléculaire de l'enzyme. Nous avons ensuite identifié les substrats in vivo de Dbp6 par l'expérience de pontage aux UV et analyse de l'ADNc (CRAC). Cela a montré que Dbp6 interagie principalement avec des snoARN qui se lient dans la région 5' de l'ARNr 25S et parmi lesquels certains sont des snoARN orphelins qui ne guident pas de modifications chimiques de nucléotides. Ces résultats soutiennent la notion que Dbp6 pourrait participer à l'organisation spatiale de cette région de l'ARNr de la grande sous-unité par l'intermédiaire des snoARN chaperons.The translation activity of ribosomes is directly held by the ribosomal RNAs (rRNAs) composing its two subunits. The large ribosomal subunit (60S) is formed of the 25S, 5.8S and 5S rRNAs and the small ribosomal subunit (40S) of the 18S rRNA. One of the main goals of ribosome biogenesis is to turn the rRNAs into correctly folded and active molecules. The production of the ribosomal subunits is the result of successive processing and maturation steps of precursor particles, the pre-60S and the pre-40S particles, precursors of the large (60S) and small (40S) ribosomal subunits, respectively. Ribosomal proteins (RPs), assembly factors (AFs) and small ribonucleoprotein particles (snoRNPs) are implicated in these successive steps. These factors play important roles in the spatial organization and in maintaining the structural integrity of the rRNAs. RNA helicases form the largest group of AFs and can modulate RNA-RNA and RNA-protein interactions. They form potential candidates for the tridimensional folding of the rRNAs. However, the mechanisms by which these enzymes participate in ribosomal particles production remain vague. In this study, we focus on the DEAD-box RNA helicase Dbp6's function in the early structuring of rRNAs of the large ribosomal subunit (60S). Dbp6 is essential for the production of the large ribosomal subunits. In its absence the production of the first pre-60S particle is impaired. Nevertheless, Dbp6 enzymatic activities' importance for the first pre-60S particle production has not been assessed nor have its RNA substrates been determined. In our study, we demonstrated that Dbp6 displays expected biochemical activities, such as ATP hydrolysis and RNA binding. Dbp6 did not show any RNA strands dissociation activity (helicase activity) in the conditions tested in the laboratory. We were able to identify and study a strand association activity (annealing activity) that is controlled by ATP. By studying Dbp6's mutants targeting the conserved helicase core motifs, we established that ATP hydrolysis is important but not essential for cell survival. However, the annealing activity seems to play a key role in the molecular function of the enzyme. We then identified Dbp6 in vivo substrates by in vivo cross-linking and analysis of cDNA experiment (CRAC). This showed that Dbp6 mostly interacts with snoRNAs that bind the 5' region of the 25S rRNA of which several are orphan snoRNA that do not guide the chemical modification of nucleotides. These findings support the notion that Dbp6 might participate in the spatial organization of this region of the large subunit rRNA by the intermediate of chaperoning snoRNAs