Endocannabinoid regulation of acute and protracted nicotine withdrawal: effect of FAAH inhibition.

Evidence shows that the endocannabinoid system modulates the addictive properties of nicotine. In the present study, we hypothesized that spontaneous withdrawal resulting from removal of chronically implanted transdermal nicotine patches is regulated by the endocannabinoid system. A 7-day nicotine dependence procedure (5.2 mg/rat/day) elicited occurrence of reliable nicotine abstinence symptoms in Wistar rats. Somatic and affective withdrawal signs were observed at 16 and 34 hours following removal of nicotine patches, respectively. Further behavioral manifestations including decrease in locomotor activity and increased weight gain also occurred during withdrawal. Expression of spontaneous nicotine withdrawal was accompanied by fluctuation in levels of the endocannabinoid anandamide (AEA) in several brain structures including the amygdala, the hippocampus, the hypothalamus and the prefrontal cortex. Conversely, levels of 2-arachidonoyl-sn-glycerol were not significantly altered. Pharmacological inhibition of fatty acid amide hydrolase (FAAH), the enzyme responsible for the intracellular degradation of AEA, by URB597 (0.1 and 0.3 mg/kg, i.p.), reduced withdrawal-induced anxiety as assessed by the elevated plus maze test and the shock-probe defensive burying paradigm, but did not prevent the occurrence of somatic signs. Together, the results indicate that pharmacological strategies aimed at enhancing endocannabinoid signaling may offer therapeutic advantages to treat the negative affective state produced by nicotine withdrawal, which is critical for the maintenance of tobacco use.

Tumor necrosis-like weak inducer of apoptosis as a proinflammatory cytokine in human adipocyte cells: up-regulation in severe obesity is mediated by inflammation but not hypoxia.

CONTEXT Adipose tissue hypoxia and endoplasmic reticulum (ER) stress may link the presence of chronic inflammation and macrophage infiltration in severely obese subjects. We previously reported the up-regulation of TNF-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible 14 (Fn14) axis in adipose tissue of severely obese type 2 diabetic subjects. OBJECTIVES The objective of the study was to examine TWEAK and Fn14 adipose tissue expression in obesity, severe obesity, and type 2 diabetes in relation to hypoxia and ER stress. DESIGN In the obesity study, 19 lean, 28 overweight, and 15 obese nondiabetic subjects were studied. In the severe obesity study, 23 severely obese and 35 control subjects were studied. In the type 2 diabetes study, 11 type 2 diabetic and 36 control subjects were studied. The expression levels of the following genes were analyzed in paired samples of sc and visceral adipose tissue: Fn14, TWEAK, VISFATIN, HYOU1, FIAF, HIF-1a, VEGF, GLUT-1, GRP78, and XBP-1. The effect of hypoxia, inflammation, and ER stress on the expression of TWEAK and Fn14 was examined in human adipocyte and macrophage cell lines. RESULTS Up-regulation of TWEAK/Fn14 and hypoxia and ER stress surrogate gene expression was observed in sc and visceral adipose tissue only in our severely obese cohort. Hypoxia modulates TWEAK or Fn14 expression in neither adipocytes nor macrophages. On the contrary, inflammation up-regulated TWEAK in macrophages and Fn14 expression in adipocytes. Moreover, TWEAK had a proinflammatory effect in adipocytes mediated by the nuclear factor-kappaB and ERK but not JNK signaling pathways. CONCLUSIONS Our data suggest that TWEAK acts as a pro-inflammatory cytokine in the adipose tissue and that inflammation, but not hypoxia, may be behind its up-regulation in severe obesity.

Schistosomiasis differentially affects vasoconstrictor responses: up-regulation of 5-HT receptor-mediated aorta contraction

Schistosomiasis, classified by the World Health Organization as a neglected tropical disease, is an intravascular parasitic disease associated to a chronic inflammatory state. Evidence implicating inflammation in vascular dysfunction continues to mount, which, broadly defined, reflects a failure in the control of intracellular Ca2+ and consequently, vascular contraction. Therefore, we measured aorta contraction induced by 5-hydroxytryptamine (5-HT) and endothelin-1 (ET-1), two important regulators of vascular contraction. Isometric aortic contractions were determined in control and Schistosoma mansoni-infected mice. In the infected animals, 5-HT induced a 50% higher contraction in relation to controls and we also observed an increased contraction in response to Ca2+ mobilisation from sarcoplasmic reticulum. Nevertheless, Rho kinase inhibition reduced the contraction in response to 5-HT equally in both groups, discarding an increase of the contractile machinery sensitivity to Ca2+. Furthermore, no alteration was observed for contractions induced by ET-1 in both groups. Our data suggest that an immune-vascular interaction occurs in schistosomiasis, altering vascular contraction outside the mesenteric portal system. More importantly, it affects distinct intracellular signalling involved in aorta contraction, in this case increasing 5-HT receptor signalling.

Chromatin regulation in schistosomes and histone modifying enzymes as drug targets

Only one drug is currently available for the treatment and control of schistosomiasis and the increasing risk of selecting strains of schistosome that are resistant to praziquantel means that the development of new drugs is urgent. With this objective we have chosen to target the enzymes modifying histones and in particular the histone acetyltransferases and histone deacetylases (HDAC). Inhibitors of HDACs (HDACi) are under intense study as potential anti-cancer drugs and act via the induction of cell cycle arrest and/or apoptosis. Schistosomes like other parasites can be considered as similar to tumours in that they maintain an intense metabolic activity and rate of cell division that is outside the control of the host. We have shown that HDACi can induce apoptosis and death of schistosomes maintained in culture and have set up a consortium (Schistosome Epigenetics: Targets, Regulation, New Drugs) funded by the European Commission with the aim of developing inhibitors specific for schistosome histone modifying enzymes as novel lead compounds for drug development.

Le Mahā-Bhārata : poème épique. Septième volume, [Bhīshma-Parva, suite] / de Krishna-Dwaipayana,... ; traduit du sanscrit en français par Hippolyte Fauche,..

[Mahābhārata (français). 1863-]

Status of radiation protection at different hospitals in Nepal.

Nepal has a long history of medical radiology since1923 but unfortunately, we still do not have any Radiation Protection Infrastructure to control the use of ionizing radiations in the various fields. The objective of this study was an assessment of the radiation protection in medical uses of ionizing radiation. Twenty-eight hospitals with diagnostic radiology facility were chosen for this study according to patient loads, equipment and working staffs. Radiation surveys were also done at five different radiotherapy centers. Questionnaire for radiation workers were used; radiation dose levels were measured and an inventory of availability of radiation equipment made. A corollary objective of the study was to create awareness in among workers on possible radiation health hazard and risk. It was also deemed important to know the level of understanding of the radiation workers in order to initiate steps towards the establishment of Nepalese laws, regulation and code of radiological practice in this field. Altogether, 203 Radiation workers entertained the questionnaire, out of which 41 are from the Radiotherapy and 162 are from diagnostic radiology. The radiation workers who have participated in the questionnaire represent more than 50% of the radiation workers working in this field in Nepal. Almost all X-ray, CT and Mammogram installations were built according to protection criteria and hence found safe. Radiation dose level at the reference points for all the five Radiotherapy centers are within safe limit. Around 65% of the radiation workers have never been monitored for radiation. There is no quality control program in any of the surveyed hospitals except radiotherapy facilities.

A key role of TRPC channels in the regulation of electromechanical activity of the developing heart.

Aims It is well established that dysfunction of voltage-dependent ion channels results in arrhythmias and conduction disturbances in the foetal and adult heart. However, the involvement of voltage-insensitive cationic TRPC (transient receptor potential canonical) channels remains unclear. We assessed the hypothesis that TRPC channels play a crucial role in the spontaneous activity of the developing heart.Methods and results TRPC isoforms were investigated in isolated hearts obtained from 4-day-old chick embryos. Using RT-PCR, western blotting and co-immunoprecipitation, we report for the first time that TRPC1, 3, 4, 5, 6, and 7 isoforms are expressed at the mRNA and protein levels and that they can form a macromolecular complex with the alpha 1C subunit of the L-type voltage-gated calcium channel (Cav1.2) in atria and ventricle. Using ex vivo electrocardiograms, electrograms of isolated atria and ventricle and ventricular mechanograms, we found that inhibition of TRPC channels by SKF-96365 leads to negative chrono-, dromo-, and inotropic effects, prolongs the QT interval, and provokes first-and second-degree atrioventricular blocks. Pyr3, a specific antagonist of TRPC3, affected essentially atrioventricular conduction. On the other hand, specific blockade of the L-type calcium channel with nifedipine rapidly stopped ventricular contractile activity without affecting rhythmic electrical activity.Conclusions These results give new insights into the key role that TRPC channels, via interaction with the Cav1.2 channel, play in regulation of cardiac pacemaking, conduction, ventricular activity, and contractility during cardiogenesis.

Unavailability of CD147 leads to selective erythrocyte trapping in the spleen.

Adhesive interactions with stromal cells and the extracellular matrix are essential for the differentiation and migration of hematopoietic progenitors. In the erythrocytic lineage, a number of adhesion molecules are expressed in the developing erythrocytes and are thought to play a role in the homing and maturation of erythrocytic progenitors. However, many of these molecules are lost during the final developmental stages leading to mature erythrocytes. One of the adhesion molecules that remains expressed in mature, circulating erythrocytes is CD147. This study shows that blockade of this molecule on the cell surface by treatment with F(ab')(2) fragments of anti-CD147 monoclonal antibody disrupts the circulation of erythrocytes, leading to their selective trapping in the spleen. Consequently, mice develop an anemia, and de novo, erythropoietin-mediated erythropoiesis in the spleen. In contrast, these changes were not seen in mice similarly treated with another antierythrocyte monoclonal antibody with a different specificity. These results suggest that the CD147 expressed on erythrocytes likely plays a critical role in the recirculation of mature erythrocytes from the spleen into the general circulation. (Blood. 2001;97:3984-3988)

Hybrids and Regulation in the Global Political Economy

While regulation theory literature has made important contributions to the much-debated domain of globalisation by focusing on various aspects of post-Fordism, it has not yet fully engaged with the implications that can be drawn from critical approaches in international political economy. Recent studies have explored the transnational bases of new patterns and agents of change beyond states, firms and institutions traditionally involved in regulatory practices. Hybrid is often used as a default attribute reflecting lack of clear understanding of the breadth of this new type of influence and the opacity of the means involved. Drawing on the insights of philology and mythology, the paper argues that the notion of hybrid is relevant in elucidating the ontological ambiguity between imaginary and real aspects of globalisation. Furthermore, it specifies the categories involved in the analysis of emerging forms of hybrid regulation. Recent scholarship on globalisation tends to focus on the private-public nexus of the subjects involved in new forms of institutional arrangements and authority. Here, subjects, objects and space are analysed as joint issues. By focusing particularly on transformations affecting the role of the state, forms of competition, and their rescaling on a transnational basis, the concept of global hybrid is seen as complementary to the emancipation of regulation approaches from early emphasis on national levels of compromises.

Update of the FANTOM web resource: from mammalian transcriptional landscape to its dynamic regulation.

The international Functional Annotation Of the Mammalian Genomes 4 (FANTOM4) research collaboration set out to better understand the transcriptional network that regulates macrophage differentiation and to uncover novel components of the transcriptome employing a series of high-throughput experiments. The primary and unique technique is cap analysis of gene expression (CAGE), sequencing mRNA 5'-ends with a second-generation sequencer to quantify promoter activities even in the absence of gene annotation. Additional genome-wide experiments complement the setup including short RNA sequencing, microarray gene expression profiling on large-scale perturbation experiments and ChIP-chip for epigenetic marks and transcription factors. All the experiments are performed in a differentiation time course of the THP-1 human leukemic cell line. Furthermore, we performed a large-scale mammalian two-hybrid (M2H) assay between transcription factors and monitored their expression profile across human and mouse tissues with qRT-PCR to address combinatorial effects of regulation by transcription factors. These interdependent data have been analyzed individually and in combination with each other and are published in related but distinct papers. We provide all data together with systematic annotation in an integrated view as resource for the scientific community (http://fantom.gsc.riken.jp/4/). Additionally, we assembled a rich set of derived analysis results including published predicted and validated regulatory interactions. Here we introduce the resource and its update after the initial release.

Epigenetic regulation of human cancer/testis antigen gene, HAGE, in chronic myeloid leukemia.

BACKGROUND AND OBJECTIVES Cancer testis antigens (CTA) provide attractive targets for cancer-specific immunotherapy. Although CTA genes are expressed in some normal tissues, such as the testis, this immunologically protected site lacks MHC I expression and as such, does not present self antigens to T cells. To date, CTA genes have been shown to be expressed in a range of solid tumors via demethylation of their promoter CpG islands, but rarely in chronic myeloid leukemia (CML) or other hematologic malignancies. DESIGN AND METHODS In this study, the methylation status of the HAGE CTA gene promoter was analyzed by quantitative methylation-specific polymerase chain reaction (MSP) and sequencing in four Philadelphia-positive cell lines (TCC-S, K562, KU812 and KYO-1) and in CML samples taken from patients in chronic phase (CP n=215) or blast crisis (BC n=47). HAGE expression was assessed by quantitative reverse transcriptase-polymerase chain reaction. RESULTS The TCC-S cell line showed demethylation of HAGE that was associated with over-expression of this gene. HAGE hypomethylation was significantly more frequent in BC (46%) than in CP (22%) (p=0.01) and was correlated with high expression levels of HAGE transcripts (p<0.0001). Of note, in CP-CML, extensive HAGE hypomethylation was associated with poorer prognosis in terms of cytogenetic response to interferon (p=0.01) or imatinib (p=0.01), molecular response to imatinib (p=0.003) and progression-free survival (p=0.05). INTERPRETATIONS AND CONCLUSION: The methylation status of the HAGE promoter directly correlates with its expression in both CML cell lines and patients and is associated with advanced disease and poor outcome.

Analyse fonctionnelle et moléculaire d'un nouveau gène (VIT32) impliqué dans le mécanisme d'action de la vasopressine

Résumé Les mécanismes de régulation de la réabsorption fine du sodium dans la partie distale (tube distal et tube collecteur) du néphron ont un rôle essentiel dans le maintien de l'homéostasie de la composition ionique et du volume des fluides extracellulaires. Ces mécanismes permettent le maintien de la pression sanguine. Dans la cellule principale du tube collecteur cortical (CCD), le taux de réabsorption de sodium dépend essentiellement de l'activité du canal épithélial à sodium (ENaC) à la membrane apicale et de la pompe sodium-potassium-adénosine-triphosphatase (Na+-K±ATPase) à la membrane basolatérale. L'activité de ces deux molécules de transport est en partie régulée par des hormones dont l'aldostérone, la vasopressine et l'insuline. Dans les cellules principales de CCD, la vasopressine régule le transport de sodium en deux étapes : une étape précoce dite « non-génomique » et une étape tardive dite « génotnique ». Durant l'étape précoce, la vasopressine régule l'expression de gènes, dont certains peuvent être impliqués dans le transport de sodium, comme ENaC et la Na+ -K+ATP ase. Le but de mon travail a été d'étudier l'implication d'une protéine appelée VIP32 (vasopressin induced protein : VIP) dans le transport de sodium. L'expression de VIP32 est augmentée par la vasopressine dans les cellules principales de CCD. Dans l'ovocyte de Xenopus laevis utilisé comme système d'expression hétérologue, nous avons montré que l'expression de VIP32 provoque la maturation méiotique de l'ovocyte par l'activation de la voie des MAPK (mitogen-activated protein kinase : MAPK) et du facteur de promotion méiotique (MPF). La co-expression d'ENaC et de VIP32 diminue l'activité d'ENaC de façon sélective, par l'activation de la voie des MAPK, sans affecter l'expression du canal à la surface membranaire. Nous avons également montré que la régulation de l'activité d'ENaC par la voie des MAPK est dépendante du mécanisme de régulation d'ENaC lié à un motif du canal appelé PY. Ce motif est impliqué dans le contrôle de la probabilité d'ouverture ainsi que l'expression à la surface membranaire d'ENaC. Dans les cellules principales, VIP32 par l'activation de la voie des MAPK peut être impliqué dans la régulation négative du transport transépithélial qui a lieu après plusieurs heures de traitement à la vasopressine. Le tube collecteur de reins normaux présente un taux basal significatif d'activité de la voie MAPK MEK1/2-ERK1/2. Dans la lignée mpkCCDc14 de cellules principales de CCD de souris, que nous avons utilisé pour cette partie du travail, nous avons montré la présence d'un taux basal d'activité d'ERK1/2 (pERK1/2). L'aldostérone et la vasopressine, connus pour stimuler le courant sodique transépithélial dans le CCD, ne changeaient pas le taux basal de pERK1/2. Le transport de sodium transépithélial basal, ou stimulé par l'aldostérone ou la vasopressine est diminué par l'effet de PD98059, un inhibiteur de MEK1/2 qui diminue parallèlement le taux de pERK1/2. Nous avons également montré dans des cellules non stimulées, ou stimulées par de l'aldostérone ou de la vasopressine, que l'activité de la Na+-K+ ATPase, mais pas celle d'ENaC est inhibée par des traitements avec différents inhibiteurs de MEK1/2. Par un marquage de la Na±-K+ATPase à la surface membranaire nous avons montré que la voie d'ERK1/2 contrôle l'activité intrinsèque de la Na+-K+ ATPase, plutôt que son expression à la surface membranaire. Ces données ont montré que l'activité de la Na+-K+ATPase et le transport transépithélial de sodium sont contrôlés par l'activité basal et constitutive de la voie d'ERK1/2. Summary The regulation of sodium reabsorption in the distal nephron (distal tubule and cortical collecting duct) in the kidney plays an essential role in the control of extracellular fluids composition and volume, and thereby blood pressure. In the principal cell of the collecting duct (CCD), the level of sodium reabsorption mainlly depends on the activity of both epithlial sodium channel (ENaC) and sodium-potassium-adenosine-triphosphatase (Na+-K+ATPase). The activity of these two transporters is regulated by hormones especially aldosterone, vasopressin and insuline.In the principal cell of the CCD, vasopressin regulates sodium transport via a short-term effect and a late genomic effect. During the genomic effect vasopressin activates a complex network of vasopressin-dependent genes involved in the regulation of sodium transport as ENaC and Na+-K+ATPase. We were interested in the role of a recently identified vasopressin induced protein (VIP32) and its implication in the regulation of sodium transport in principal cell of the CCD. The Xenopus oocyte expression system revealed two functions : expressed alone VIP32 rapidly induces oocyte meiotic maturation through the activation of the mitogen-activated protein kinase (MAPK) pathway and the meiotic promoting factor and when co-expressed with ENaC, V1P32 selectively dowrn-egulates channel activity, but not channel cell surface expression. We have shown that the ENaC downregulation mediated by the activation of the MAPK pathway is related to the PY motif of ENaC. This motif is implicated in ENaC cell surface expression and open probability regulation. In the kidney principal cell, VIP32 through the activation of MAPK pathway may be involved in the downregulation of transepithelial sodium transport observed within a few hours after vasopressin treatment. The collecting duct of normal kidney exhibits significant activity of the MEK1/2-ERK1/2 MAPK pathway. Using in vitro cultured mpkCCDc14 principal cells we have shown a significant basal level of ERK1/2 activity (pERK1/2). Aldosterone and vasopressin, known to upregulate sodium reabsorption in CCDs, did not change ERK1/2 activity. Basal and aldosterone- or vasopressin-stimulated sodium transport were downregulated by the MEK1/2 inhibitor PD98059 in parallel with a decrease in pERK1/2 in vitro. The activity of Na+-K+ATPase but not that of ENaC was inhibited by MEK1/2 inhibitors in both, unstimulated and aldosterone- or vasopressin-stimulated CCDs in vitro. Cell surface labelling showed that intrinsic activity rather than cell surface expression of Na+-K+ATPase was controlled by pERK1/2. Our data demonstrate that basal constitutive activity of ERK1/2 pathway controls Na+-K+ATPase activity and transepithelial sodium transport in the principal cell. Résumé tout public Les mécanismes de régulation de la réabsorption fine du sodium dans la partie distale du néphron (l'unité fonctionnelle du rein) ont un rôle essentiel dans le maintien de l'homéostasie de la composition et du volume des fluides extracellulaires. Ces mécanismes permettent de maintenir une pression sanguine effective. Dans les cellules principales du tube collecteur, une région spécifique du néphron distal, le transport de sodium dépend essentiellement de l'activité de deux transporteurs de sodium : le canal épithélial à sodium (ENaC) et la pompe sodium-potassium-adénosine-triphosphatase (Na+-K+ATPase). Afin de répondre aux besoins de l'organisme, l'activité de ces deux molécules de transport est en partie régulée par des hormones dont l'aldostérone, la vasopressine et l'insuline. Dans les cellules principales du tube collecteur, la vasopressine régule le transport de sodium en deux étapes : une étape rapide et une étape lente dite « génomique ». Durant l'étape lente, la vasopressine régule l'expression de gènes pouvant être impliqués dans le transport de sodium, dont notamment ceux d'ENaC et de la Na+-K+ATPase. Parmi les gènes dont l'expression est augmentée par la vasopressine, celui de VIP32 (vasopressin induced protein : VIP) fait l'objet de cette étude. Le but de mon travail a été d'étudier, dans un système d'expression hétérologue (l'ovocyte de Xenopus leavis), l'implication de VIP32 dans le transport de sodium. Nous avons montré que VIP32 est capable d'activer un mécanisme moléculaire en cascade appelé MAPK (mitogen-activated protein kinase : MAPK) et est aussi capable de diminuer l'activité d'ENaC. Parallèlement, dans une lignée de cellules principales de tube collecteur les mpkCCDc14, nous avons montré que le taux basal d'activité de la cascade MAPK est capable de réguler l'activité de la Na+-K+ATPase, tandis qu'il n'influence pas l'activité d'ENaC.

Regulation of the cardiac voltage-gated sodium channel Nav1.5 : regulation of Nav1.5 by ubiquitin-protein ligases of the Nedd4/Nedd4-like family and characterisation of two novel mutations in the gene encoding Nav1.5 (SCN5A) implicated in the Brugada syndrome triggered by fever

Abstract: The genesis of the cardiac action potential, which accounts for the cardiac contraction, is due to the sodium current INa mediated by the voltage-gated sodium channel Nav1.5. Several cardiac arrhythmias such as the Brugada syndrome are known te be caused by mutations in SCN5A, the gene encoding Nav1.5. Studies of these mutations allowed a better understanding of biophysical and functional properties of Nav1.5. However, only few investigations have been performed in order to understand the regulation of Nav1.5. During my thesis, I investigated different mechanisms of regulation of Nav1.5 using a heterologous expression system, HEK293 cells, coupled with a technique of sodium current recording: the patch clamp in whole cell configuration. In previous studies it has been shown that an enzyme of the Nedd4 family (Nedd4-2) regulates an epithelial sodium channel via the interaction with PY-motifs present in the latter. Interestingly, Nav1.5 contains a similar PY-motif, which motivated us to study the role of Nedd4-2 expressed in heart for the regulation of Nav1.5. In a second study, we investigated the implication of two Nav1.5 mutants, which were either less functional or net functional (Nav1.5 R535X and Nav1.5 L325R respectively) implied in the genesis of the Brugada syndrome by fever. Our results established two mechanisms implied in Nav1.5 regulation. The first one implies that following the interaction between the PY-motif of Nav1.5 and Nedd4- 2 Nav1.5 is ubiquitinated by Nedd4-2. This ubiquitination leads to the internalization of Nav1 .5. The second mechanism is a phenomenon called the "dominant negative" effect of Nav1.5 L325R on Nay1.5 where the decrease of 'Na is potentially due to the retention of Nav1.5 by Nav1.5 L325R in an undefined intracellular compartment. These studies defined two mechanisms of Nav1.5 regulation, which could play an important role for the genesis of cardiac arrhythmias where molecular processes are still poorly understood. Résumé La genèse du potentiel d'action cardiaque, permettant la contraction cardiaque, est due au courant sodique INa issu des canaux sodiques cardiaques dépendants du voltage Nav1.5. Nombreuses arythmies cardiaques telles que le syndrome de Brugada sont connues pour être liées à des mutations du gène SCN5A, codant pour Nav1.5. L'étude de ces mutations a permis une meilleure compréhension des propriétés structurelles et fonctionnelles de Nav1.5 et leurs implications dans la genèse de ces pathologies. Néanmoins peu d'études ont été menées afin de comprendre les mécanismes de régulation de Nav1.5. Mon travail de thèse a consisté à étudier des mécanismes de régulation de Nav1.5 en utilisant un système d'expression hétérologue, les cellules HEK293, couplé à une technique d'enregistrement des courants sodiques, le "patch clamp" en configuration cellule entière. La présence sur Nav1.5 d'un motif-PY similaire à ceux nécessaires pour la régulation d'un canal épithélial sodique par une enzyme de la famille de Nedd4, nous a amenée à étudier le rôle de ces ubiquitine-ligases, en particulier Nedd4-2, dans la régulation de Nav1.5. La seconde étude s'est intéressée aux conséquences de deux mutations de SCN5A codant pour deux mutants peu ou pas fonctionnels (Nav1.5 L325R et Nav1.5 R535X respectivement) retrouvées chez des patients présentant un syndrome de Brugada exacerbé par un état fébrile. Nos résultats ont permis d'établir deux mécanismes de régulation de Nav1.5 L'un par Nedd4-2 qui implique rubiquitination de Nav1.5 par cette ligase suite à l'interaction entre le motif-PY de Nav1.5 et Nedd4-2. Cette modification déclenche l'internalisation du canal impliquée dans la diminution d'INa. Le second mécanisme quant à lui est un effet "dominant négatif" de Nav1.5 L325R sur Nav1.5 aboutissant à une diminution d'INa suite à la séquestration intracellulaire potentielle de Nav1.5 par Nav1.5 L325R. Ces études ont mis en évidence deux mécanismes de régulation de Nav1.5 pouvant jouer un rôle majeur dans la genèse et/ou l'accentuation des arythmies cardiaques dont les processus moléculaires au sein des cardiomyocytes, impliquant des modifications du courant sodiques, sont encore mal compris. Résumé destiné à un large public La dépolarisation électrique de la membrane des cellules cardiaques permet la contraction du coeur. La génèse de cette activité électrique est due au courant sodique issu d'un type de canal à sodium situé dans la membrane des cellules cardiaques. De nombreuses pathologies provoquant des troubles du rythme cardiaque sont issues de mutations du gène qui code pour ce canal à sodium. Ces canaux mutants, entrainant diverses pathologies cardiaques telles que le syndrome de Brugada, ont été largement étudiées. Néanmoins, peu de travaux ont été réalisés sur les mécanismes de régulation de ce canal à sodium non muté. Mon travail de thèse a consisté à étudier certains des mécanismes de régulation de ce canal à sodium en utilisant une technique permettant l'enregistrement des courants sodiques issus de l'expression de ces canaux à sodium à la membrane de cellules mammifères. La présence sur ce canal à sodium d'une structure spécifique, similaire à celle nécessaire pour la régulation d'un canal épithélial à sodium par une enzyme appelée Nedd4-2, nous a amenée à étudier le rôle de cette enzyme dans la régulation de ce canal à sodium. La seconde étude s'est intéressée aux rôles de deux mutations du gène codant pour ce canal à sodium retrouvées chez des patients présentant un syndrome de Brugada exacerbé par la fièvre. Nos résultats nous ont permis d'établir deux mécanismes de régulation de ce canal à sodium diminuant le courant sodique l'un par l'action de l'enzyme Nedd4-2, suite à son interaction avec ce canal, qui modifie ce canal à sodium (ubiquitination) diminuant de ce fait la densité membranaire du canal. L'autre par un mécanisme suggérant un effet négatif de l'un des canaux mutants sur l'expression à la membrane du canal à sodium non muté. Ces études ont mis en évidence deux mécanismes de régulation de ce canal à sodium pouvant jouer un rôle majeur dans la genèse et/ou l'accentuation des troubles du rythme cardiaques dont les mécanismes cellulaires sont encore incompris.

Regulatory volume decrease in Leishmania mexicana: effect of anti-microtubule drugs

The trypanosomatid cytoskeleton is responsible for the parasite's shape and it is modulated throughout the different stages of the parasite's life cycle. When parasites are exposed to media with reduced osmolarity, they initially swell, but subsequently undergo compensatory shrinking referred to as regulatory volume decrease (RVD). We studied the effects of anti-microtubule (Mt) drugs on the proliferation of Leishmania mexicana promastigotes and their capacity to undergo RVD. All of the drugs tested exerted antiproliferative effects of varying magnitudes [ansamitocin P3 (AP3)> trifluoperazine > taxol > rhizoxin > chlorpromazine]. No direct relationship was found between antiproliferative drug treatment and RVD. Similarly, Mt stability was not affected by drug treatment. Ansamitocin P3, which is effective at nanomolar concentrations, blocked amastigote-promastigote differentiation and was the only drug that impeded RVD, as measured by light dispersion. AP3 induced 2 kinetoplasts (Kt) 1 nucleus cells that had numerous flagella-associated Kts throughout the cell. These results suggest that the dramatic morphological changes induced by AP3 alter the spatial organisation and directionality of the Mts that are necessary for the parasite's hypotonic stress-induced shape change, as well as its recovery.