Protection of pancreatic beta-cells by high density lipoproteins

SUMMARYThe incidence of type 2 diabetes (T2D) is increasing worldwide and is linked to the enhancement of obesity. The principal cause of T2D development is insulin resistance, which lead to the increase of insulin production by the pancreatic beta-cells. In a pathological environment, namely dyslipidaemia, hyperglycaemia and inflammation, beta-cell compensation will fail in more vulnerable cells and diabetes will occur. High Density Lipoproteins (HDLs), commonly named "good cholesterol" are known to be atheroprotective. Low levels of HDLs are associated with increased prevalence of cardiovascular disease but are also an independent risk factor for the development of T2D. HDLs were demonstrated to protect pancreatic beta-cells against several stresses. However the molecular mechanisms of the protection are unknown and the objectives of this work were to try to elucidate the way how HDLs protect. The first approach was a broad screening of genes regulated by the stress and HDLs. A microarray analysis was performed on beta-cells stressed by serum deprivation and rescued by HDLs. Among the genes regulated, we focused on 4E-BP1, a cap-dependent translational inhibitor. In addition, HDLs were also found to protect against several other stresses.Endoplasmic reticulum (ER) stress is a mechanism that may play a role in the onset of T2D. The unfolded protein response (UPR) is a physiological process that aims at maintaining ER homeostasis in conditions where the protein folding and secretion is perturbed. Specific signalling pathways are involved in the increase of folding, export and degradation capacity of the ER. However, in case where the stress is prolonged, this mechanism turns to be pathological, by inducing cell death effector pathways, leading to beta-cell apoptosis. In our study, we discovered that HDLs were protective against ER stress induced by drugs and physiological stresses such as saturated free fatty acids. HDLs protected beta-cells by promoting ER homeostasis via the improvement of the folding and trafficking od proteins from the ER to the Golgi apparatus.Altogether our results suggest that HDLs are important for beta-cell function and survival, by protecting them from several stresses and acting on ER homeostasis. This suggests that attempt in keeping normal HDLs levels or function in patients is crucial to lessen the development of T2D.RÉSUMÉL'incidence du diabète de type 2 est en constante augmentation et est fortement liée à l'accroissement du taux d'obésité. La cause principale du diabète de type 2 est la résistance à l'insuline, qui entraîne une surproduction d'insuline par les cellules bêta pancréatiques. Dans un environnement pathologique associé à l'obésité (dyslipidémie, hyperglycémie et inflammation), les cellules bêta les plus vulnérables ne sont plus capables de compenser en augmentant leur production d'insuline, dysfonctionnent, ce qui conduit à leur mort par apoptose. Les lipoprotéines de hautes densités (HDLs), communément appelées (( bon cholestérol », sont connues pour leurs propriétés protectrices contre l'athérosclérose. Des niveaux bas de HDLs sanguins sont associés au risque de développer un diabète de type 2. Les HDLs ont également montré des propriétés protectrices contre divers stresses dans la cellule bêta. Cependant, les mécanismes de protection restent encore inconnus et l'objectif de ce travail a été d'investiguer les mécanismes moléculaires de protection des HDLs. La première approche choisie a été une étude du profil d'expression génique par puce à ADN afin d'identifier les gènes régulés par le stress et les HDLs. Parmi les gènes régulés, notre intérêt s'est porté sur 4E-BP1, un inhibiteur de la traduction coiffe- dépendante, dont l'induction par le stress était corrélée avec une augmentation de l'apoptose. Suite à cette étude, les HDLs ont également montrés un rôle protecteur contre d'autres stresses. Il s'agit particulièrement du stress du réticulum endoplasmique (RE), qui est un mécanisme qui semble jouer un rôle clé dans le développement du diabète. L'UPR (« Unfolded Protein Response ») est un processus physiologique tendant à maintenir l'homéostasie du réticulum endoplasmique, organelle prépondérante pour la fonction des cellules sécrétrices, notamment lorsqu'elle est soumise à des conditions extrêmes telles que des perturbations de la conformation tertiaire des protéines ou de la sécrétion. Dans ces cas, des voies de signalisation moléculaires sont activées, ce qui mène à l'exportation des protéines mal repliées, à leur dégradation et à l'augmentation de l'expression de chaperonnes capables d'améliorer le repliement des protéines mal formées. Toutefois, en cas de stress persistant, ce mécanisme de protection s'avère être pathologique. En induisant des voies de signalisation effectrices de l'apoptose, il conduit finalement au développement du diabète. Dans cette étude, nous avons démontré que les HDLs étaient capables de protéger la cellule bêta contre le stress du RE induits par des inhibiteurs (thapsigargine, tunicamycine) ou des stresses physiologiques tels que les acides gras libres. Les HDLs ont la capacité d'améliorer l'homéostasie du RE, notamment en favorisant le repliement et le transfert des protéines du RE à l'appareil de Golgi.En résumé, ces données suggèrent que les HDLs sont bénéfiques pour la survie des cellules bêta soumises à des stresses impliqués dans le développement du diabète, notamment en restaurant l'homéostasie du RE. Ces résultats conduisent à soutenir que le maintien des taux de cholestérol joue un rôle important dans la limitation de l'incidence du diabète.

Current-density-functional approach to large quantum dots in intense magnetic fields

Within current-density-functional theory, we have studied a quantum dot made of 210 electrons confined in a disk geometry. The ground state of this large dot exhibits some features as a function of the magnetic field (Beta) that can be attributed in a clear way to the formation of compressible and incompressible states of the system. The orbital and spin angular momenta, the total energy, ionization and electron chemical potentials of the ground state, as well as the frequencies of far-infrared edge modes are calculated as a function of Beta, and compared with available experimental and theoretical results.

Spin and density longitudinal response of quantum dots in the time-dependent local-spin-density approximation

The longitudinal dipole response of a quantum dot has been calculated in the far-infrared regime using local-spin-density-functional theory. We have studied the coupling between the collective spin and density modes as a function of the magnetic field. We have found that the spin dipole mode and single-particle excitations have a sizable overlap, and that the magnetoplasmon modes can be excited by the dipole spin operator if the dot is spin polarized. The frequency of the dipole spin edge mode presents an oscillation which is clearly filling factor (v) related. We have found that the spin dipole mode is especially soft for even-n values. Results for selected numbers of electrons and confining potentials are discussed.

Density-functional calculations of magnetoplasmons in quantum rings

We have studied the structure and dipole charge-density response of nanorings as a function of the magnetic field using local-spin-density-functional theory. Two small rings consisting of 12 and 22 electrons confined by a positively charged background are used to represent the cases of narrow and wide rings. The results are qualitatively compared with experimental data existing on microrings and on antidots. A smaller ring containing five electrons is also analyzed to allow for a closer comparison with a recent experiment on a two-electron quantum ring.

Vertically coupled quantum dots in the local spin-density functional theory

We have investigated the structure of double quantum dots vertically coupled at zero magnetic field within local-spin-density functional theory. The dots are identical and have a finite width, and the whole system is axially symmetric. We first discuss the effect of thickness on the addition spectrum of one single dot. Next we describe the structure of coupled dots as a function of the interdot distance for different electron numbers. Addition spectra, Hund's rule, and molecular-type configurations are discussed. It is shown that self-interaction corrections to the density-functional results do not play a very important role in the calculated addition spectra

Method to estimate soil macroporosity and microporosity based on sand content and bulk density

Macroporosity is often used in the determination of soil compaction. Reduced macroporosity can lead to poor drainage, low root aeration and soil degradation. The aim of this study was to develop and test different models to estimate macro and microporosity efficiently, using multiple regression. Ten soils were selected within a large range of textures: sand (Sa) 0.07-0.84; silt 0.03-0.24; clay 0.13-0.78 kg kg-1 and subjected to three compaction levels (three bulk densities, BD). Two models with similar accuracy were selected, with a mean error of about 0.02 m³ m-3 (2 %). The model y = a + b.BD + c.Sa, named model 2, was selected for its simplicity to estimate Macro (Ma), Micro (Mi) or total porosity (TP): Ma = 0.693 - 0.465 BD + 0.212 Sa; Mi = 0.337 + 0.120 BD - 0.294 Sa; TP = 1.030 - 0.345 BD 0.082 Sa; porosity values were expressed in m³ m-3; BD in kg dm-3; and Sa in kg kg-1. The model was tested with 76 datum set of several other authors. An error of about 0.04 m³ m-3 (4 %) was observed. Simulations of variations in BD as a function of Sa are presented for Ma = 0 and Ma = 0.10 (10 %). The macroporosity equation was remodeled to obtain other compaction indexes: a) to simulate maximum bulk density (MBD) as a function of Sa (Equation 11), in agreement with literature data; b) to simulate relative bulk density (RBD) as a function of BD and Sa (Equation 13); c) another model to simulate RBD as a function of Ma and Sa (Equation 16), confirming the independence of this variable in relation to Sa for a fixed value of macroporosity and, also, proving the hypothesis of Hakansson & Lipiec that RBD = 0.87 corresponds approximately to 10 % macroporosity (Ma = 0.10 m³ m-3).

Optical response of two-dimensional few-electron concentric double quantum rings: A local-spin-density-functional theory study

We have investigated the dipole charge- and spin-density response of few-electron two-dimensional concentric nanorings as a function of the intensity of a erpendicularly applied magnetic field. We show that the dipole response displays signatures associated with the localization of electron states in the inner and outer ring favored by the perpendicularly applied magnetic field. Electron localization produces a more fragmented spectrum due to the appearance of additional edge excitations in the inner and outer ring.

Density matrices and momentum distributions in 3He-4He mixtures

We report variational calculations, in the hypernetted-chain (HNC)-Fermi-HNC scheme, of one-body density matrices and one-particle momentum distributions for 3He-4He mixtures described by a Jastrow correlated wave function. The 4He condensate fractions and the 3He strength poles are examined and compared with the Monte Carlo available results. The agreement has been found to be very satisfactory. Their density dependence is also studied.

MODELING OF SOIL LOAD-BEARING CAPACITY AS A FUNCTION OF SOIL MECHANICAL RESISTANCE TO PENETRATION

Estimation of soil load-bearing capacity from mathematical models that relate preconsolidation pressure (σp) to mechanical resistance to penetration (PR) and gravimetric soil water content (U) is important for defining strategies to prevent compaction of agricultural soils. Our objective was therefore to model the σp and compression index (CI) according to the PR (with an impact penetrometer in the field and a static penetrometer inserted at a constant rate in the laboratory) and U in a Rhodic Eutrudox. The experiment consisted of six treatments: no-tillage system (NT); NT with chiseling; and NT with additional compaction by combine traffic (passing 4, 8, 10, and 20 times). Soil bulk density, total porosity, PR (in field and laboratory measurements), U, σp, and CI values were determined in the 5.5-10.5 cm and 13.5-18.5 cm layers. Preconsolidation pressure (σp) and CI were modeled according to PR in different U. The σp increased and the CI decreased linearly with increases in the PR values. The correlations between σp and PR and PR and CI are influenced by U. From these correlations, the soil load-bearing capacity and compaction susceptibility can be estimated by PR readings evaluated in different U.

Extent and limitations of density functional theory in describing magnetic systems

The performance of density-functional theory to solve the exact, nonrelativistic, many-electron problem for magnetic systems has been explored in a new implementation imposing space and spin symmetry constraints, as in ab initio wave function theory. Calculations on selected systems representative of organic diradicals, molecular magnets and antiferromagnetic solids carried out with and without these constraints lead to contradictory results, which provide numerical illustration on this usually obviated problem. It is concluded that the present exchange-correlation functionals provide reasonable numerical results although for the wrong physical reasons, thus evidencing the need for continued search for more accurate expressions.

Extent and limitations of density functional theory in describing magnetic systems

The performance of density-functional theory to solve the exact, nonrelativistic, many-electron problem for magnetic systems has been explored in a new implementation imposing space and spin symmetry constraints, as in ab initio wave function theory. Calculations on selected systems representative of organic diradicals, molecular magnets and antiferromagnetic solids carried out with and without these constraints lead to contradictory results, which provide numerical illustration on this usually obviated problem. It is concluded that the present exchange-correlation functionals provide reasonable numerical results although for the wrong physical reasons, thus evidencing the need for continued search for more accurate expressions.

Nuclear receptor PPARS function in stem cell differentiation and bone physiology

In adult, bone remodeling is a permanent process, reaching an annual turnover of about 10% of the skeleton. Bone remodeling requires the sequential and coordinated actions of the hematopoietic origin osteoclasts, to remove bone and the mesenchymal origin osteoblasts to replace it. An increased level of bone resorption is the primary cause of age-related bone loss often resulting in osteopenia, and is the major cause of osteoporosis.¦Peroxisome proliferator-activated receptors (PPARs), which are expressed in three isotypes, PPARa, PPARp and PPARy, are ligand-activated transcription factors that control many cellular and metabolic processes, more particularly linked to lipid metabolism. In bone, previous works has shown that PPARy inhibits osteogenesis by favoring adipogenesis from common mesenchymal progenitors. In addition, the pro-osteoclastogenesis activity of PPARy results in an increased bone resorption. Accordingly, treatment with PPARy agonist such as the anti-diabetic drug TZD causes bone loss and accumulation of marrow adiposity in mice as well as in postmenopausal women. The aim of the present thesis work was to elucidate the PPARs functions in bone physiology.¦The initial characterization of the PPARP" bone phenotype mainly revealed a decreased BMD. In vitro studies exploring the potency of mesenchymal stem cells to differentiate in osteoblast showed no differences depending on the genotype. However, we could demonstrate an effect of PPARp in partially inhibiting osteoclastogenesis. These results are further sustained by a study made in collaboration with the group of Dr Kronke, which showed an impressive protection against ovariectomy-generated bone loss when the females are treated with a PPARp agonist.¦Observations in PPARy null mice are more complex. The lab has recently been able to generate mice carrying a total deletion of PPARy. Intriguingly, the exploration of the bone phenotype of these mice revealed paradoxical findings. Whereas short bones such as vertebrae exhibit an elevated BMD as expected, long bones (tibia and femur) are clearly osteoporotic. According to their activity when set in culture, osteoblast differentiation normally occurs. Indeed the phenotype can be mainly attributed to a high density of osteoclasts in the cortical bone of PPARy null mice, associated to large bone resorption areas.¦Our explorations suggest a mechanism that involves regulatory processes linking osteoclastogenesis to adipogenesis, the latter being totally absent in PPARy null mice. Indeed, the lack of adipose tissue creates a favorable niche for osteoclastogenesis since conditioned medium made from differentiated adipocyte 3T3L1 inhibited osteoclastogenesis from both PPARy-/- and WT cells. Thus, adipokines deficiency in PPARy-/- mice contributes to de- repress osteoclastogenesis. Using specific blocking antibody, we further identified adiponectin as the major player among dozens of adipokines. Using flow cytometry assay, we explored the levels at which the osteoclastic commitment was perturbed in the bone marrow of PPARy-/- mice. Intriguingly, we observe a general decrease for hematopoietic stem cell and lineage progenitors but increased proportion of osteoclast progenitor in PPARy-/- bone marrow. The general decrease of HSC in the bone marrow is however largely compensated by an important extra-medullary hematopoeisis, taking place in the liver and in the spleen.¦These specific characteristics emphasize the key role of PPARy on a cross road of osteogenesis, adipogenesis and hematopoiesis/osteoclastogenesis. They underline the complexity of the bone marrow niche, and demonstrate the inter-dependance of different cell types in defining bone homeostasis, that may be overseen when experimental design single out pure cell populations.¦Chez l'adulte, même après la fin de la croissance, le renouvellement des os se poursuit et porte sur environ 10% de l'ensemble du squelette adulte, par année. Ce renouvellement implique à la fois des mécanismes séquentiels et coordonnés des ostéoclastes d'origine hématopoïetique, qui dégradent l'os, et des ostéoblastes d'origine mésenchymale, qui permettent la régénération de l'os. La perte en densité osseuse due à l'âge entraîne un fort niveau de résorption, conduisant souvent à une ostéopénie, elle-même cause de l'ostéoporose.¦Les trois isotypes PPAR (Peroxisome proliferator-activated receptor, PPARa, PPARp, et PPARy) sont des récepteurs nucléaires qui contrôlent de nombreux mécanismes cellulaires et métaboliques, plus particulièrement liés au métabolisme lipidique. Au niveau osseux, des travaux précédents ont montré que PPARy inhibe l'ostéoblastogenèse en favorisant la formation d'adipocytes à partir de la cellule progénitrice commune. De plus, l'activité pro- ostéoclastogénique de PPARy induit une résorption osseuse accrue. Condormément à ces observations, les patients diabétiques traités par les thiazolidinediones qui agissent sur PPARy, ont un risque accrue d'ostéoporose liée à une perte osseuse accrue et un accroissement de l'adiposité au niveau de la moelle osseuse. Dans ce contexte, l'objectif de mon travail de thèse a été d'élucider le rôle des PPAR dans la physiologie osseuse, en s'appuyant sur le phénotype des souris porteuses de mutation pour PPAR.¦La caractérisation initiale des os des souris porteuses d'une délétion de ΡΡΑΕφ a principalement révélé une diminution de la densité minérale osseuse (DMO). Alors que l'ostéogenèse n'est pas significativement altérée chez ces souris, l'ostéoclastogenèse est elle augmentée, suggérant un rôle modérateur de ce processus par ΡΡΑΕΙβ. Ces résultats sont par ailleurs soutenus par une étude menée par le groupe du Dr Krônke en collaboration avec notre groupe, et qui monte une protection très importante des souris traitées par un activateur de PPARP contre l'ostéoporose provoquée par l'ovariectomie.¦Les observations concernant PPARy donnent des résultats plus complexes. Le laboratoire a en effet été capable récemment de générer des souris portant une délétion totale de PPARy. Alors que les os courts chez ces souris présentent une augmentation de la DMO, comme attendu, les os longs sont clairement ostéoporotiques. Ce phénotype corrèle avec une densité élevée d'ostéoclastes dans l'os cortical de ces os longs. Deux processus semblent contribuer à ce phénotype. En premier lieu, nous démontrons qu'un milieu conditionné provenant de cultures de cellules 3T3-L1 différenciées en adipocytes contiennent une forte activité inhibitrice d'osteoclastogenesis. L'utilisation d'anticorps neutralisant permet d'identifier l'adiponectine comme l'un des facteurs principaux de cette inhibition. Les souris PPARy étant totalement dépourvues d'adipocytes et donc de tissu adipeux, la sécrétion locale d'adiponectine dans la moelle osseuse est donc également absente, entraînant une désinhibition de l'ostéoclastogenèse. En second lieu, des analyses par FACS révèle une proportion accrue des cellules progénitrices d'ostéoclastes dans la moelle osseuse. Cela s'accompagne par une diminution globale des cellules souches hématopoïétiques, qui est cependant largement compensée par une importante hématopoëise extra-médullaire, dans le foie comme dans la rate.¦L'ensemble de notre travail montre toute l'importance de PPARy au carrefour de l'ostéogenèse, adipogenèse, et hématopoëise/osteoclastogenèse. Il souligne la complexité de la niche que représente la moelle osseuse et démontre l'inter-dépendance des différents types cellulaires définissant l'homéostasie osseuse, complexité qui peut facilement être masqué lorsque le travail expérimental se concentre sur le comportement d'un type cellulaire donné.

Mechanism of HFR1 function in adaptive growth responses in "Arabidopsis thaliana"

AbstractPlants are sessile organisms, which have evolved an astonishing ability to sense changes in their environment. Depending on the surrounding conditions, such as changes in light and temperature, plants modulate the activity of important transcriptional regulators. The shade avoidance syndrome (SAS) is one important mechanism for shade-intolerant plants to adapt their growth in high vegetative density. In shaded conditions plants sense a diminished red/far-red ratio via the phytochrome system and respond with morphological changes such as elongation growth of stems and petioles. The Phytochrome Interacting Factors 4 and 5 (PIF4 and PIF5) are positive regulators of the SAS and required for a full response (Lorrain et al, 2008). They regulate the SAS by inducing the expression of shade avoidance marker genes such as PIL1, ATHB2, XTR7 and HFR1 (Hornitschek et al, 2009; Lorrain et al, 2008).I investigated the molecular mechanism underlying the regulation of the SAS by HFR1 (long Hypocotyl in FR light). Although HFR1 is a PIF-related bHLH transcription factor, we discovered that HFR1 is a non-DNA binding protein. Moreover, we revealed that HFR1 inhibits an exaggerated SAS by forming non-DNA binding heterodimers with PIF4 and PIF5 (Hornitschek et al, 2009). This negative feedback loop is an important mechanism to limit elongation growth also in elevated temperatures. HFR1 accumulation and activity are highly temperature-dependent and the increased activity of HFR1 at warmer temperatures also provides an important restraint on PIF4-driven elongation growth (Foreman et al, 2011).Finally we performed a genome-wide analysis to determine how PIF4 and PIF5 regulate growth in response to shade. We identified potential PIF5- target genes, which represent many well-known shade-responsive genes. Our analysis of gene expression also revealed a role of PIF4 and PIF5 in simulated sun possibly via the regulation of auxin sensitivity.RésuméLes plantes sont des organismes sessiles ayant développé une capacité surprenante à détecter des changements dans leur environnement. En fonction des conditions extérieures, telles que les variations de lumière ou de température, elles adaptent l'activité d'importants régulateurs transcriptionnels. Le syndrome d'évitement de l'ombre (SAS), est un mécanisme important pour les plantes intolérantes à l'ombre leur permettant d'adapter leur croissance lorsqu'elles se développent dans des conditions de végétations très denses. Dans ces conditions, les plantes détectent une réduction de la quantité relative de lumière rouge par rapport à la lumière rouge-lointain (rapport R/FR). Ce changement, perçu via le système des phytochromes, induit des modifications morphologiques telle qu'une élongation des tiges et des pétioles. Les protéines PIF4 et PIF5 (Phytochrome Interacting Factors) sont des régulateurs positifs du SAS et sont nécessaires pour une réponse complète (Lorrain et al, 2008). Ces facteurs de transcription régulent le SAS en induisant l'expression de gènes marqueurs de cette réponse tels que PIL1, ATHB2, XTR7 et HFR1 (Hornitschek et al, 2009; Lorrain et al, 2008).J'ai étudié les mécanismes moléculaires sous-jacents à la régulation du SAS par HFR1 (long Hypocotyl in FR light). HFR1 est un facteur de transcription type bHLH de la famille des PIF, quoique nous ayons découvert que HFR1 est une protéine ne se liant pas à Γ ADN. Nous avons montré que HFR1 inhibe un SAS exagéré en formant des heterodimères avec PIF4 et PIF5 (Hornitschek et al, 2009). Nous avons également montré que cette boucle de régulation négative est également un mécanisme important pour limiter la croissance de l'élongation dans des conditions de fortes températures. De plus l'accumulation et l'activité de HFR1 augmentent avec la température ce qui permet d'inhiber plus fortement l'effet activateur de PIF4 sur la croissance.Enfin, nous avons effectué une analyse génomique à large échelle afin de déterminer comment PIF4 et PIF5 régulent la croissance en réponse à l'ombre. Nous avons identifié les gènes cibles potentiels de PIF5, correspondant en partie à des gènes connus dans la réponse de l'évitement de l'ombre. Notre analyse de l'expression des gènes a également révélé un rôle important de PIF4 et PIF5 dans des conditions de croissance en plein soleil, probablement via la régulation de la sensibilité à l'auxine.

Effect of atazanavir versus other protease inhibitor-containing antiretroviral therapy on endothelial function in HIV-infected persons: randomised controlled trial.

OBJECTIVE: Impaired endothelial function was demonstrated in HIV-infected persons on protease inhibitor (PI)-containing antiretroviral therapy, probably due to altered lipid metabolism. Atazanavir is a PI causing less atherogenic lipoprotein changes. This study determined whether endothelial function improves after switching from other PI to atazanavir. DESIGN: Randomised, observer-blind, treatment-controlled trial. SETTING: Three university-based outpatient clinics. PATIENTS: 39 HIV-infected persons with suppressed viral replication on PI-containing regimens and fasting low-density lipoprotein (LDL)-cholesterol greater than 3 mmol/l. INTERVENTION: Patients were randomly assigned to continue the current PI or change to unboosted atazanavir. MAIN OUTCOME MEASURES: Endpoints at week 24 were endothelial function assessed by flow-mediated dilation (FMD) of the brachial artery, lipid profiles and serum inflammation and oxidative stress parameters. RESULTS: Baseline characteristics and mean FMD values of the two treatment groups were comparable (3.9% (SD 1.8) on atazanavir versus 4.0% (SD 1.5) in controls). After 24 weeks' treatment, FMD decreased to 3.3% (SD 1.4) and 3.4% (SD 1.7), respectively (all p = ns). Total cholesterol improved in both groups (p<0.0001 and p = 0.01, respectively) but changes were more pronounced on atazanavir (p = 0.05, changes between groups). High-density lipoprotein and triglyceride levels improved on atazanavir (p = 0.03 and p = 0.003, respectively) but not in controls. Serum inflammatory and oxidative stress parameters did not change; oxidised LDL improved significantly in the atazanavir group. CONCLUSIONS: The switch from another PI to atazanavir in treatment-experienced patients did not result in improvement of endothelial function despite significantly improved serum lipids. Atherogenic lipid profiles and direct effects of antiretroviral drugs on the endothelium may affect vascular function. Trial registration number: NCT00447070.

Novel action of a wake-promoting neuropeptide in hippocampus : inhibition of nmda receptor function at excitatory synapses through orexin-a

One of the most intriguing functions of the brain is the ability to learn and memorize. The mechanism through which memory and learning are expressed requires the activation of NMDA receptors (NMDARs). These molecular entities are placed at the postsynaptic density of excitatory synapses and their function is tightly controlled by the actions of several modulators at the extracellular, intracellular and pore sites. A large part of the intracellular modulation comes from the action of G-protein coupled receptors (GPCRs). Through intracellular cascades typically involving kinases and phosphatases, GPCRs potentiate or inhibit NMDARs, controlling the conductive state but also the trafficking within the synapse. The GPCRs are involved in the modulation of a variety of brain functions. Many of them control cognition, memory and learning performance, therefore, their effects on NMDARs are extensively studied. The orexinergic system signals through GPCRs and it is well known for the regulation of waking, feeding, reward and autonomic functions. Moreover, it is involved in potentiating hippocampus-related cognitive tasks. Orexin receptors and fibers are present within the hippocampus, but whether these directly modulate hippocampal cells and synapses has not yet been determined. During my thesis, I studied orexinergic actions on excitatory synaptic transmission via whole-cell patch-clamp recordings in rat acute hippocampal slices. I observed that exogenously applied orexin-A (ox-A) exerted a strong inhibitory action on NMDAR-mediated synaptic potentials at mossy fiber (MF)-CA3 synapses, by postsynaptically activating orexin-2 receptors, a minor inhibition at Schaffer collateral-CAl synapses and did not affect other synapses with the CA3 area. Moreover, I demonstrated that the susceptibility of NMDARs to ox- A depends on the tone of endogenous orexin known to fluctuate during the day-night cycle. In fact, in slices prepared during the active period of the rats, when endogenous orexin levels are high, NMDAR-currents were not affected by exogenously applied ox-A. The inhibitory effect of ox-A was, however, reverted when interfering with the orexinergic system through intraperitoneal injections of almorexant, a dual orexin receptor antagonist, during the active phase prior to slice preparation. This thesis work suggests that the orexinergic system regulates NMDAR-dependent information flow through select hippocampal pathways depending on the time-of-day. The specific orexinergic modulation of NMDARs at MFs dampens the excitability of the hippocampal circuit and could impede the mechanisms related to memory formation, possibly also following extended periods of waking. -- La capacité d'apprentissage et de mémorisation est une des fonctions les plus intrigantes de notre cerveau. Il a été montré qu'elles requièrent l'activation des récepteurs NMDA (NMDARs). Ces entités moléculaires sont présentes au niveau de la densité post-synaptique des synapses excitatrices et leur fonction est étroitement contrôlée par l'action de nombreux modulateurs au niveau extracellulaire, intracellulaire et membranaire de ces récepteurs. Une grande partie de la modulation intracellulaire s'effectue via l'action de récepteurs couplés aux protéines G (GPCRs). Grace à leurs cascades intracellulaires typiquement impliquant des kinases et des phosphatases, les GPCRs favorisent l'activation ou l'inhibition des NMDARs, contrôlant ainsi leur perméabilité mais aussi leur mouvement à la synapse. Les GPCRs sont impliquées dans de nombreuses fonctions cérébrales telles que la cognition, la mémoire ainsi que la capacité d'apprentissage c'est pour cela que leurs effets sur les NMDARs sont très étudiés. Le système orexinergique fait intervenir ces GPCRs et est connu par son rôle dans la régulation de fonctions physiologiques telles que l'éveil, la prise alimentaire, la récompense ainsi que d'autres fonctions du système nerveux autonome. De plus, ce système est impliqué dans la régulation de tâches cognitives liées à l'hippocampe. Bien que les fibres et les récepteurs à l'orexine soient présents dans l'hippocampe, leur mécanisme d'action sur les cellules et les synapses de l'hippocampe n'a pas encore été élucidé. Durant ma thèse, je me suis intéressée aux effets de l'orexine sur la transmission synaptique excitatrice en utilisant la méthode d'enregistrement en patch-clamp en configuration cellule entière sur des tranches aiguës d'hippocampes de rats. J'ai observé que l'application exogène d'orexine A d'une part inhibe fortement les courants synaptiques dépendants de l'activation des NMDARs au niveau de la synapse entre les fibres moussues et CA3 via l'activation post-synaptique des orexine récepteurs 2 mais d'autre part n'inhibe que de façon mineure la synapse entre les collatérales de Schaffer et CAI et n'affecte pas les autres synapses impliquant CA3. J'ai également démontré que la sensibilité des NMDARs à l'orexine A dépend de sa concentration endogène qui fluctue durant le cycle éveil-sommeil. En effet, lorsque les coupes d'hippocampes sont préparées durant la période active de l'animal correspondant à un niveau endogène d'orexine élevé, l'application exogène d'orexine A n'a aucun effet sur les courants dépendants de l'activation des NMDARs. Cependant, l'injection dans le péritoine, durant la phase active de l'animal, d'un antagoniste des orexine récepteurs, l'almorexant, va supprimer l'effet inhibiteur de l'orexine A. Les résultats de ma thèse suggèrent donc que le système orexinergique module les informations véhiculées par les NMDARs via des voies de signalisation sélectives de l'hippocampe en fonction du moment de la journée. La modulation orexinergique des NMDARs au niveau des fibres moussues diminue ainsi l'excitabilité du circuit hippocampal et pourrait entraver les mécanismes liés à la formation de la mémoire, potentiellement après de longues périodes d'éveil.