Corrélation entre risque carieux et charge bactérienne : outil potentiel de décision thérapeutique

Many Oral infectious diseases such as dental caries or periodontitis are the result of an organised biofilm. Biofilm’s formation is a multifactorial event involving for example diet (sugar consumption), oral hygiene and oral microbiote composition. The risk to develop the disease is then specific to each individual. The clinician should be able to assess the patient risk in order to use a specific therapeutic in accordance. A simple to use, fast and low cost diagnostic tool doesn’t exist today. Rather than identifying specific species involved in carie development, we aim to measure the activity/quantity of microorganisms at specific sites. In doing so we are trying to find a link between these measurement and carie progression. We used the “2 MIN UNIT-ORAL® " from Clinident institute in our pilot experiment and found interesting results that will lead us to develop an exhaustive clinic study.La maladie carieuse ou les maladies parodontales sont des pathologies plurifactorielles liées aux microorganismes qui s’organisent en biofilm sur les surfaces dentaires. La virulence de ce biofilm dépend de divers facteurs tels que l’alimentation (quantité de sucre), l’hygiène dentaire, ainsi que la composition de la flore buccale. Le risque de développer ces maladies est donc propre à chaque patient. Afin d’individualiser au mieux sa prise en charge, le clinicien devrait posséder dans sa palette diagnostique un outil lui permettant d’évaluer ce risque de manière fiable, rapide et à bas coût. Malheureusement, la composition très hétérogène du microbiote oral, les techniques complexes d’identification des espèces microbiennes et notre incompréhension actuelle des interactions/coopérations inter-espèces au sein du biofilm rendent les différents tests existants inutilisables pour les praticiens. Nous avons donc cherché à savoir si en mesurant la quantité/activité globale de bactéries sur certains sites à risque (espaces interdentaires, poches parodontales), une prédiction du risque carieux du patient pouvait être faite. Pour cela nous avons utilisé le « 2 MIN UNIT-ORAL® » de l’institut Clinident. Nos premiers résultats semblent concluants et encouragent la mise en place d’une étude clinique

Le récepteur à activité tyrosine kinase ALK (signalisation et implication dans le développement du système nerveux)

Au cours du développement du système nerveux, les récepteurs à activité tyrosine kinase jouent un rôle prépondérant dans des processus variés. Parmi ces récepteurs, nous nous intéressons à ALK (Anaplastic Lymphoma Kinase) qui est actuellement considéré comme un récepteur orphelin chez les vertébrés. Ce dernier est essentiellement exprimé dans certaines régions du système nerveux central et périphérique au cours du développement. Sa fonction précise reste à être éclaircit chez les vertébrés. Mes travaux de thèse ont eu pour but de contribuer à mieux comprendre le rôle de ALK dans le développement du système nerveux. A cette fin, je me suis concentré sur deux axes de recherche. Etant donné la non-existence d un ligand clairement identifié, la première partie de mon étude s est intéressée à la mise en place de substituts de ligand qui nous ont permis d étudier les voies de signalisation et les effets régulés par ALK. La seconde partie de mes travaux a porté sur l expression et le rôle de ALK dans le développement du système nerveux en se focalisant sur les ganglions des racines dorsales.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Kinase activation dependent down-regulation was regulated by Cbl ubiquitin ligase and ALK ubiquitylation.

<p><b>A.</b> SH-SY5Y cells were treated or not with either agonist mAb 46 or antagonist mAb 30 at 6 nM during 15 or 60 min. Cbl immunoprecipitates from 2 mg total cell lysate proteins were submitted to western-blot analysis, and then immunoblotted with polyclonal anti Cbl and ALK recruitment with polyclonal REAB antibody. Phosphorylated ALK and Cbl were revealed with 4G10 antibody. <b>B.</b> SH-SY5Y cells were serum starved for 16 hours and non-treated or treated with either agonist mAb 46 or antagonist mAb 30 at 6 nM during 15 or 60 min. ALK immunoprecipitates from 2 mg total cell lysate proteins were submitted to western-blot analysis and then immunoblotted with polyclonal REAB. Cbl recruitment was revealed with polyclonal anti Cbl antibody. Phosphorylated ALK and Cbl were revealed with 4G10 antibody. <b>C.</b> SH-SY5Y were serum starved for 16 hours, and then non-treated or treated with either mAb 46 or mAb 30 at 6 nM for 15 minutes or 60 minutes. ALK immunoprecipitates were submitted to Western-blot analysis as described.</p

Agonist mAb treatment induced ALK degradation whereas antagonist mAb induced ALK internalization without down-regulation.

<p><b>A.</b> SH-SY5Y cells were untreated (-) or treated with agonist mAb 46 for 15 min, 60 min or 3 hours. At the end of the agonist treatment, cells were subjected to cell surface protein biotinylation. ALK immunoprecipates from 1.5 mg of total cells lysate proteins were analyzed by western blot. Biotinalyted cell surface ALK were detected with streptavidin coupled to IRdye 800, and total ALK with REAB antibody. Experiments were done in triplicates, total and biotinylated (cell surface) ALK were quantified with LiCor Odyssey software. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033581#s2" target="_blank">Results</a> are expressed in percentage of control +/− s.e.m. <b>B.</b> SH-SY5Y cells were untreated or treated with agonist mAb 30 for 15 min, 1 h or 3 h. At the end of mAb treatment, cells were subjected to cell surface protein biotinylation. ALK immunoprecipates from 1.5 mg of total cells lysate proteins were analyzed by western blot. Biotinalyted cell surface ALK were detected with streptavidin coupled to IRdye 800, and total ALK with REAB antibody. Experiments were done in triplicates, total and biotinylated (cell surface) ALK were quantified with LiCor Odyssey software. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033581#s2" target="_blank">Results</a> are expressed in percentage of control +/− s.e.m.</p

ALK down-regulation after agonist mAb 46 treatment.

<p><b>A. IMR-32 (WT) and B. SH-SY5Y (WT/F1174L)</b> were treated with mAb 46 for 15 min to 6 h. ALK immunoprecipitates from 1.5 mg of total cell lysate proteins were submitted to western blot. Total ALK were immunoblotted with polyclonal REAB and phosphorylated ALK were detected with monoclonal phosphotyrosine antibody 4G10 platinium. Tubulin acted as a loading control. <b>C.</b> SH-SY5Y cells were pre-treated or not with 50 nM NVP-TAE684 one hour before cell stimulation by agonist mAb 46 at 6 nM in time course manner (0 min to 6 h). ALK immunoprecipitates from 1.5 mg of total cell lysate proteins were submitted to western-blot analysis and immunoblotted as described. <i>Lower panel</i>: Experiments were done in triplicates and the 220 kD forms (the doublet was impossible to separate) and the 140 kD form of ALK were quantified with LiCor Odyssey software. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033581#s2" target="_blank">Results</a> are expressed in percentage of control +/− s.e.m. <b>D.</b> SH-SY5Y cells were pre-treated with either bafilomycin (0.25 µM) or lactacystin (10 µM) for 15′, then non treated or treated with agonist mAb 46 at 6 nM during 6 hours. ALK immunoprecipitates from 1.5 mg of total cell lysate proteins were submitted to Western-blot analysis as described. <i>Lower panel:</i> Experiments were done in triplicates total ALK (220 kD forms+140 kD form) was quantified with LiCor Odyssey software. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033581#s2" target="_blank">Results</a> are expressed in percentage of control +/− s.e.m.</p

Proteasome dependent degradation of receptor retained in intracellular compartment.

<p><b>A.</b> NIH3T3 cells stably expressing either the WT ALK or F1174L mutated ALK were non-treated or treated with Bafilomycin A1 (0.25 µM) or with Lactacystin (10 µM) for 16 hours. ALK immunoprecipitates from 1 mg of total cell lysate proteins were subjected to western blot analysis. ALK was immunoblotted with polyclonal REAB antibody. <b>B.</b> SH-SY5Y were non-treated or treated with Bafilomycin A1 (0.25 µM) or with Lactacystin (10 µM) for 16 hours. ALK immunoprecipitates from 1.5 mg of total cell lysate proteins were subjected to western blot analysis using polyclonal REAB antibody. The experiment was done in triplicates and quantified with LiCor Odyssey system. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033581#s2" target="_blank">Results</a> were expressed in percentage of control +/− s.e.m.</p

Internalization and Down-Regulation of the ALK Receptor in Neuroblastoma Cell Lines upon Monoclonal Antibodies Treatment

Recently, activating mutations of the full length ALK receptor, with two hot spots at positions F1174 and R1275, have been characterized in sporadic cases of neuroblastoma. Here, we report similar basal patterns of ALK phosphorylation between the neuroblastoma IMR-32 cell line, which expresses only the wild-type receptor (ALK(WT)), and the SH-SY5Y cell line, which exhibits a heterozygous ALK F1174L mutation and expresses both ALK(WT) and ALK(F1174L) receptors. We demonstrate that this lack of detectable increased phosphorylation in SH-SY5Y cells is a result of intracellular retention and proteasomal degradation of the mutated receptor. As a consequence, in SH-SY5Y cells, plasma membrane appears strongly enriched for ALK(WT) whereas both ALK(WT) and ALK(F1174L) were present in intracellular compartments. We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALK(WT). We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation. In contrast, antagonist mAb induced ALK internalization and recycling to the plasma membrane. Importantly, we correlate this differential trafficking of ALK in response to mAb with the recruitment of the ubiquitin ligase Cbl and ALK ubiquitylation only after agonist stimulation. This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization

Riquiqui and Minibrain are regulators of the Hippo pathway downstream of Dachsous

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