Étude du mécanisme de transport des cations par la Na, K-ATPase et de son implication dans la migraine familiale hémiplégique de type 2

Résumé La Na,K-ATPase est une protéine transmembranaire, présente dans toutes les cellules de mammifères et indispensable à la viabilité cellulaire. Elle permet le maintien des gradients sodiques et potassiques à l'origine du potentiel membranaire en transportant 3 Na+ en dehors de la cellule contre 2 K+, grâce à l'énergie fournie par l'hydrolyse d'une molécule d'ATP. Le potentiel membranaire est indispensable au maintien de l'excitabilité cellulaire et à la transmission de l'influx nerveux. Il semblerait que la Na,K-ATPase soit liée à l'hypertension et à certains troubles neurologiques comme la Migraine Familiale Hémiplégique (1VIFH). La MFH est une forme de migraine avec aura, qui se caractérise par une hémiparésie. Cette forme de migraine est très rare. Elle se transmet génétiquement sur un mode autosomique dominant. Plusieurs mutations localisées dans le gène de la Na,K-ATPase ont été identifiées durant ces 3 dernières années. C'est la première fois qu'une maladie génétique est associée au gène de la Na,K-ATPase. La compréhension du fonctionnement de cette protéine peut donner des informations sur les mécanismes conduisant à ces pathologies. On sait que la fonction d'une protéine est liée à sa structure. L'étude de sa fonction nécessite donc l'étude de sa structure. Alors que la structure de la SERCA a été déterminée à haute résolution, par cristallographie, celle de la Na,K-ATPase ne l'est toujours pas. Mais ces 2 ATPases présentent une telle homologie qu'un modèle de la Na,K-ATPase a pu être élaboré à partir de la structure de la SERCA. Les objectifs de cette étude sont d'une part, de comprendre le contrôle de l'accessibilité du K+ extracellulaire àses sites de liaison. Pour cela, nous avons ciblé cette étude sur la 2ìème et la 31eme boucle extracellulaire, qui relient respectivement les segments transmembranaires (STM) 3-4 et 5-6. Le choix s'est porté sur ces 2 boucles car elles bordent le canal des cations formés des 4ième' Sième et 6'ème hélices. D'autre part, nous avons également essayer de comprendre les effets des mutations, liées à la Migraine Familiale Hémiplégique de type 2 (MFH2), sur la fonctionnalité de la Na,K-ATPase. Alors que les STM et les domaines cytoplasmiques sont relativement proches entre la Na,KATPase et la SERCA, les boucles extracellulaires présentent des différences. Le modèle n'est donc pas une approche fiable pour déterminer la structure et la fonction des régions extracellulaires. Nous avons alors utilisé une approche fonctionnelle faisant appel à la mutation dirigée puis à l'étude de l'activité fonctionnelle de la Na,K ATPase par électrophysiologie sur des ovocytes de Xenopus. En conclusion, nous pouvons dire que la troisième boucle extracellulaire participerait à la structure de la voie d'entrée des cations et que la deuxième boucle extracellulaire semble impliquée dans le contrôle de l'accessibilité des ions K+àses sites de liaison. Concernant les mutations associées à la MFH2, nos résultats ont montré une forte diminution de l'activité fonctionnelle de la pompe Na,K, inférieure aux conditions physiologiques de fonctionnement, et pour une des mutations nous avons observés une diminution de l'affmité apparente au K+ externe. Nous poumons faire l'hypothèse que l'origine pathologique de la migraine est liée à une diminution de l'activité de la pompe à Na+. Summary The Na,K-ATPase is a transmembrane protein, present in all mammalian cells and is necessary for the viability of the cells. It maintains the gradients of Na+ and K+ involved in the membrane potential, by transporting 3Na+ out the cell, and 2K+ into the cell, using the energy providing from one ATP molecule hydrolysis. The membrane potential is necessary for the cell excitability and for the transmission of the nervous signal. Some evidence show that Na,K-ATPase is involved in hypertension and neurological disorders like the Familial Hemiplegic Migraine (FHM). La FHM is a rare form of migraine characterised by aura and hemiparesis and an autosomal dominant transmission. Several mutations linked to the Na,KATPase gene have been identified during these 3 last years. It's the first genetic disorder associated with the Na,K-ATPase gene. Understand the function of this protein is important to elucidate the mechanisms implicated in these pathologies. The function of a protein is linked with its structure. Thus, to know the function of a protein, we need to know its structure. While the Ca-ATPase (SERCA) has been crystallised with a high resolution, the structure of the Na,K-ATPase is not known. Because of the great homology between these 2 ATPases, a model of the Na,K-ATPase was realised by comparing with the structure of the SERCA. The aim of this study is on one side, understand the control of the extracellular K+ accessibility to their binding sites. Because of theirs closed proximity with the cation pathway, located between the 4th, 5th and 6th helices, we have targeted this study on the 2nd and the 3rd extracellular loops linking respectively the transmembrane segment (TMS) 3 and 4, and the TMS 5 and 6. And on the other side, we have tried to understand the functional effects of mutations linked with the Familial Hemiplegic Migraine Type 2 (FHM2). In contrast with the transmembrane segments and the cytoplasmic domains, the extracellular loops show lots of difference between Na,K-ATPase and SERCA, the model is not a good approach to know the structure and the function of the extracellular loops. Thus, we have used a functional approach consisting in directed mutagenesis and the study of the functional activity of the Na,K-ATPase by electrophysiological techniques with Xenopus oocytes. In conclusion, we have demonstrated that the third extracellular loop could participate in the structure of the entry of the cations pathway and that the second extracellular loop could control the K+ accessibility to their binding sites. Concerning the mutations associated with the FHM2, our results showed a strong decrease in the functional activity of the Na,K-pump under physiological conditions and for one of mutations, induce a decrease in the apparent external K+ affinity. We could make the hypothesis that the pathogenesis of migraine is related to the decrease in Na,K-pump activity. Résumé au large publique De la même manière que l'assemblage des mots forme des phrases et que l'assemblage des phrases forme des histoires, l'assemblage des cellules forme des organes et l'ensemble des organes constitue les êtres vivants. La fonction d'une cellule dans le corps humain peut se rapprocher de celle d'une usine hydroélectrique. La matière première apportée est l'eau, l'usine électrique va ensuite convertir l'eau en énergie hydraulique pour fournir de l'électricité. Le fonctionnement de base d'une cellule suit le même processus. La cellule a besoin de matières premières (oxygène, nutriments, eau...) pour produire une énergie sous forme chimique, l'ATP. Cette énergie est utilisée par exemple pour contracter les muscles et permet donc à l'individu de se déplacer. Morphologiquement la cellule est une sorte de petit sac rempli de liquide (milieu intracellulaire) baignant elle-même dans le liquide (milieu extracellulaire) composant le corps humain (un adulte est constitué environ de 65 % d'eau). La composition du milieu intracellulaire est différente de celle du milieu extracellulaire. Cette différence doit être maintenue pour que l'organisme fonctionne correctement. Une des différences majeures est la quantité de sodium. En effet il y a beaucoup plus de sodium à l'extérieur qu'à l'intérieur de la cellule. Bien que l'intérieur de la cellule soit isolé de l'extérieur par une membrane, le sodium arrive à passer à travers cette membrane, ce qui a tendance à augmenter la quantité de sodium dans la cellule et donc à diminuer sa différence de concentration entre le milieu extracellulaire et le milieu intracellulaire. Mais dans les membranes, il existe des pompes qui tournent et dont le rôle est de rejeter le sodium de la cellule. Ces pompes sont des protéines connues sous le nom de pompe à sodium ou Na,K-ATPase. On lui attribue le nom de Na,K-ATPase car en réalité elle rejette du sodium (Na) et en échange elle fait entrer dans la cellule du potassium (K), et pour fonctionner elle a besoin d'énergie (ATP). Lorsque les pompes à sodium ne fonctionnent pas bien, cela peut conduire à des maladies. En effet la Migraine Familiale Hémiplégique de type 2, est une migraine très rare qui se caractérise par l'apparition de la paralysie de la moitié d'un corps avant l'apparition du mal de tête. C'est une maladie génétique (altération qui modifie la fonction d'une protéine) qui touche la pompe à sodium située dans le cerveau. On a découvert que certaines altérations (mutations) empêchent les pompes à sodium de fonctionner correctement. On pense alors que le développement des migraines est en partie dû au fait que ces pompes fonctionnent moins bien. Il est important de bien connaître la fonction de ces pompes car cela permet de comprendre des mécanismes pouvant conduire à certaines maladies, comme les migraines. En biologie, la fonction d'une protéine est étudiée à travers sa structure. C'est pourquoi l'objectif de cette thèse a été d'étudier la structure de la Na,K-ATPase afin de mieux comprendre son mécanisme d'action.

Evaluation of uptake and transport of ultrasmall superparamagnetic iron oxide nanoparticles by human brain-derived endothelial cells.

Aim: Ultrasmall superparamagnetic iron oxide nanoparticles (USPIO-NPs) are under development for imaging and drug delivery; however, their interaction with human blood-brain barrier models is not known. Materials & Methods: The uptake, reactive oxygen species production and transport of USPIO-NPs across human brain-derived endothelial cells as models of the blood-brain tumor barrier were evaluated for either uncoated, oleic acid-coated or polyvinylamine-coated USPIO-NPs. Results: Reactive oxygen species production was observed for oleic acid-coated and polyvinylamine-coated USPIO-NPs. The uptake and intracellular localization of the iron oxide core of the USPIO-NPs was confirmed by transmission electron microscopy. However, while the uptake of these USPIO-NPs by cells was observed, they were neither released by nor transported across these cells even in the presence of an external dynamic magnetic field. Conclusion: USPIO-NP-loaded filopodia were observed to invade the polyester membrane, suggesting that they can be transported by migrating angiogenic brain-derived endothelial cells.

Cholesterol transport from late endosomes to the Golgi regulates t-SNARE trafficking, assembly, and function

Cholesterol regulates plasma membrane (PM) association and functioning of syntaxin-4 and soluble N-ethylmaleimide-sensitive fusion protein 23 (SNAP23) in the secretory pathway. However, the molecular mechanism and cellular cholesterol pools that determine the localization and assembly of these target membrane SNAP receptors (t-SNAREs) are largely unknown. We recently demonstrated that high levels of annexin A6 (AnxA6) induce accumulation of cholesterol in late endosomes, thereby reducing cholesterol in the Golgi and PM. This leads to an impaired supply of cholesterol needed for cytosolic phospholipase A2 (cPLA2) to drive Golgi vesiculation and caveolin transport to the cell surface. Using AnxA6-overexpressing cells as a model for cellular cholesterol imbalance, we identify impaired cholesterol egress from late endosomes and diminution of Golgi cholesterol as correlating with the sequestration of SNAP23/syntaxin-4 in Golgi membranes. Pharmacological accumulation of late endosomal cholesterol and cPLA2 inhibition induces a similar phenotype in control cells with low AnxA6 levels. Ectopic expression of Niemann-Pick C1 (NPC1) or exogenous cholesterol restores the location of SNAP23 and syntaxin-4 within the PM. Importantly, AnxA6-mediated mislocalization of these t-SNAREs correlates with reduced secretion of cargo via the SNAP23/syntaxin-4¿dependent constitutive exocytic pathway. We thus conclude that inhibition of late endosomal export and Golgi cholesterol depletion modulate t-SNARE localization and functioning along the exocytic pathway.

Measuring conduct and cost parameters in the Spanish air transport market

This paper estimates a model of airline competition for the Spanish air transport market. I test the explanatory power of alternative oligopoly models with capacity constraints. In addition, I analyse the degree of density economies. Results show that Spanish airlines conduct follows a price-leadership scheme so that it is less competitive than the Cournot solution. I also find evidence that thin routes can be considered as natural monopolies

Differences in glucose transporter gene expression between rat pancreatic alpha- and beta-cells are correlated to differences in glucose transport but not in glucose utilization.

Glucose exerts inverse effects upon the secretory function of islet alpha- and beta-cells, suppressing glucagon release and increasing insulin release. This diverse action may result from differences in glucose transport and metabolism between the two cell types. The present study compares glucose transport in rat alpha- and beta-cells. beta-Cells transcribed GLUT2 and, to a lesser extent, GLUT 1; alpha-cells contained GLUT1 but no GLUT2 mRNA. No other GLUT-like sequences were found among cDNAs from alpha- or beta-cells. Both cell types expressed 43-kDa GLUT1 protein which was enhanced by culture. The 62-kDa beta-cell GLUT2 protein was converted to a 58-kDa protein after trypsin treatment of the cells without detectable consequences upon glucose transport kinetics. In beta-cells, the rates of glucose transport were 10-fold higher than in alpha-cells. In both cell types, glucose uptake exceeded the rates of glucose utilization by a factor of 10 or more. Glycolytic flux, measured as D-[5(3)H]glucose utilization, was comparable in alpha- and beta-cells between 1 and 10 mmol/liter substrate. In conclusion, differences in glucose transporter gene expression between alpha- and beta-cells can be correlated with differences in glucose transport kinetics but not with different glucose utilization rates.

Diffusive transport in spatially periodic hydrodynamic flows

We calculate the effective diffusion coefficient in convective flows which are well described by one spatial mode. We use an expansion in the distance from onset and homogenization methods to obtain an explicit expression for the transport coefficient. We find that spatially periodic fluid flow enhances the molecular diffusion D by a term proportional to D-1. This enhancement should be easy to observe in experiments, since D is a small number.

Regulation by aldosterone of Na+,K+-ATPase mRNAs, protein synthesis, and sodium transport in cultured kidney cells.

Transepithelial Na+ reabsorption across tight epithelia is regulated by aldosterone. Mineralocorticoids modulate the expression of a number of proteins. Na+,K+-ATPase has been identified as an aldosterone-induced protein (Geering, K., M. Girardet, C. Bron, J. P. Kraehenbuhl, and B. C. Rossier, 1982, J. Biol. Chem., 257:10338-10343). Using A6 cells (kidney of Xenopus laevis) grown on filters we demonstrated by Northern blot analysis that the induction of Na+,K+-ATPase was mainly mediated by a two- to fourfold accumulation of both alpha- and beta-subunit mRNAs. The specific competitor spironolactone decreased basal Na+ transport, Na+,K+-ATPase mRNA, and the relative rate of protein biosynthesis, and it blocked the response to aldosterone. Cycloheximide inhibited the aldosterone-dependent sodium transport but did not significantly affect the cytoplasmic accumulation of Na+,K+-ATPase mRNA induced by aldosterone.

Astrocyte-neuron lactate transport is required for long-term memory formation.

We report that, in the rat hippocampus, learning leads to a significant increase in extracellular lactate levels that derive from glycogen, an energy reserve selectively localized in astrocytes. Astrocytic glycogen breakdown and lactate release are essential for long-term but not short-term memory formation, and for the maintenance of long-term potentiation (LTP) of synaptic strength elicited in vivo. Disrupting the expression of the astrocytic lactate transporters monocarboxylate transporter 4 (MCT4) or MCT1 causes amnesia, which, like LTP impairment, is rescued by L-lactate but not equicaloric glucose. Disrupting the expression of the neuronal lactate transporter MCT2 also leads to amnesia that is unaffected by either L-lactate or glucose, suggesting that lactate import into neurons is necessary for long-term memory. Glycogenolysis and astrocytic lactate transporters are also critical for the induction of molecular changes required for memory formation, including the induction of phospho-CREB, Arc, and phospho-cofilin. We conclude that astrocyte-neuron lactate transport is required for long-term memory formation.

Antigen binding to secretory immunoglobulin A results in decreased sensitivity to intestinal proteases and increased binding to cellular Fc receptors.

In intestinal secretions, secretory IgA (SIgA) plays an important sentinel and protective role in the recognition and clearance of enteric pathogens. In addition to serving as a first line of defense, SIgA and SIgA x antigen immune complexes are selectively transported across Peyer's patches to underlying dendritic cells in the mucosa-associated lymphoid tissue, contributing to immune surveillance and immunomodulation. To explain the unexpected transport of immune complexes in face of the large excess of free SIgA in secretions, we postulated that SIgA experiences structural modifications upon antigen binding. To address this issue, we associated specific polymeric IgA and SIgA with antigens of various sizes and complexity (protein toxin, virus, bacterium). Compared with free antibody, we found modified sensitivity of the three antigens assayed after exposure to proteases from intestinal washes. Antigen binding further impacted on the immunoreactivity toward polyclonal antisera specific for the heavy and light chains of the antibody, as a function of the antigen size. These conformational changes promoted binding of the SIgA-based immune complex compared with the free antibody to cellular receptors (Fc alphaRI and polymeric immunoglobulin receptor) expressed on the surface of premyelocytic and epithelial cell lines. These data reveal that antigen recognition by SIgA triggers structural changes that confer to the antibody enhanced receptor binding properties. This identifies immune complexes as particular structural entities integrating the presence of bound antigens and adds to the known function of immune exclusion and mucus anchoring by SIgA.

Aplicació per a la gestió d'una empresa de transport

La finalitat d'aquest projecte és la creació d'una aplicació web per gestionar una empresa d'autobusos. El resultat que s'obtingui anirà dirigit a Agobe Tours SL. Permetrà reservar places als usuaris i l'administrador podrà gestionar tots els esdeveniments relacionats amb el negoci. Es desenvoluparà de zero per crear una aplicació a mida amb el propòsit de complir els objectius de l'empresa.

Silicon quantum dots embedded in a SiO2 matrix: From structural study to carrier transport properties

We study the details of electronic transport related to the atomistic structure of silicon quantum dots embedded in a silicon dioxide matrix using ab initio calculations of the density of states. Several structural and composition features of quantum dots (QDs), such as diameter and amorphization level, are studied and correlated with transport under transfer Hamiltonian formalism. The current is strongly dependent on the QD density of states and on the conduction gap, both dependent on the dot diameter. In particular, as size increases, the available states inside the QD increase, while the QD band gap decreases due to relaxation of quantum confinement. Both effects contribute to increasing the current with the dot size. Besides, valence band offset between the band edges of the QD and the silica, and conduction band offset in a minor grade, increases with the QD diameter up to the theoretical value corresponding to planar heterostructures, thus decreasing the tunneling transmission probability and hence the total current. We discuss the influence of these parameters on electron and hole transport, evidencing a correlation between the electron (hole) barrier value and the electron (hole) current, and obtaining a general enhancement of the electron (hole) transport for larger (smaller) QD. Finally, we show that crystalline and amorphous structures exhibit enhanced probability of hole and electron current, respectively.