Erratum: Influence of oxygen plasma treatment parameters on poly(vinylidene fluoride) electrospun fiber mats wettability

Electrospun poly(vinylidene fluoride) (PVDF) fiber mats find applications in an increasing number of areas, such as battery separators, filtration and detection membranes, due to their excellent properties. However, there are limitations due to the hydrophobic nature and low surface energy of PVDF. In this work, oxygen plasma treatment has been applied in order to modify the surface wettability of PVDF fiber mats and superhydrophilic PVDF electrospun membranes have been obtained. Further, plasma treatment does not significantly influences fiber average size (~400 ± 200 nm), morphology, electroactive -phase content (~80-85%) or the degree of crystallinity (Xc of 42 ± 2%), allowing to maintain the excellent physical-chemical characteristics of PVDF. Plasma treatment mainly induces surface chemistry modifications, such as the introduction of oxygen and release of fluorine atoms that significantly changes polymer membrane wettability by a reduction of the contact angle of the polymer fibers and an overall decrease of the surface tension of the membranes.

Cathepsin D protects colorectal cancer cells from acetate-induced apoptosis through autophagy-independent degradation of damaged mitochondria

Acetate is a short-chain fatty acid secreted by Propionibacteria from the human intestine, known to induce mitochondrial apoptotic death in colorectal cancer (CRC) cells. We previously established that acetate also induces lysosome membrane permeabilization in CRC cells, associated with release of the lysosomal protease cathepsin D (CatD), which has a well-established role in the mitochondrial apoptotic cascade. Unexpectedly, we showed that CatD has an antiapoptotic role in this process, as pepstatin A (a CatD inhibitor) increased acetate-induced apoptosis. These results mimicked our previous data in the yeast system showing that acetic acid activates a mitochondria-dependent apoptosis process associated with vacuolar membrane permeabilization and release of the vacuolar protease Pep4p, ortholog of mammalian CatD. Indeed, this protease was required for cell survival in a manner dependent on its catalytic activity and for efficient mitochondrial degradation independently of autophagy. In this study, we therefore assessed the role of CatD in acetate-induced mitochondrial alterations. We found that, similar to acetic acid in yeast, acetate-induced apoptosis is not associated with autophagy induction in CRC cells. Moreover, inhibition of CatD with small interfering RNA or pepstatin A enhanced apoptosis associated with higher mitochondrial dysfunction and increased mitochondrial mass. This effect seems to be specific, as inhibition of CatB and CatL with E-64d had no effect, nor were these proteases significantly released to the cytosol during acetate-induced apoptosis. Using yeast cells, we further show that the role of Pep4p in mitochondrial degradation depends on its protease activity and is complemented by CatD, indicating that this mechanism is conserved. In summary, the clues provided by the yeast model unveiled a novel CatD function in the degradation of damaged mitochondria when autophagy is impaired, which protects CRC cells from acetate-induced apoptosis. CatD inhibitors could therefore enhance acetate-mediated cancer cell death, presenting a novel strategy for prevention or therapy of CRC.

Barramento delgado anti carbonatação

Dissertação de mestrado integrado em Engenharia Civil

A brincar e a aprender: a importância das atividades lúdicas em educação pré-escolar e ensino do 1.º ciclo do ensino básico

Relatório de estágio de mestrado em Educação Pré-Escolar e Ensino do 1.º Ciclo do Ensino Básico

Producción de factores de virulencia por fitopatógenos bacterianos de soja. Utilización de productos naturales como biocontrol. Production of virulence factors by soybean bacterial phytopathogens. The use of natural products as biocontrolers.

En Argentina el cultivo de soja ocupa el primer lugar en superficie sembrada. El 90% de la producción se obtiene en la zona central del pais. La siembra directa favorece la multiplicación y supervivencia de fitopatógenos causantes de tizón y pústula bacterianos. El tizón es producido por Pseudomonas syringae pv. glycinea observándose manchas marrones en las hojas. Produce gran variedad de toxinas: coronatina, faseolotoxina, siringomicina, tabtoxina, proteínas “nucleation ice”, entre otras, las cuales contribuyen a la clorosis y necrosis. En la infección, además, están involucrados exopolisacáridos (levano y alginato). La celulosa ha sido relacionada en la adhesión bacteriana y en la formación de biofilm. La pústula es causada por Xanthomonas axonopodis pv. glycines. Produce manchas pequeñas con una pequeña pústula de color claro. Libera enzimas como α-amilasa, proteasa, endo β-mannanasa, actividad peptolítica, que degradan componentes vegetales. Xantan, producido por X. axonopodis es uno de los componentes necesarios para la formación de biofilm. Este último es considerado un importante factor de virulencia porque proporciona una estrategia de colonización que otorga mayor resistencia a ambientes desfavorables, tolerancia a antimicrobianos, producción de metabolitos y exoenzimas, etc. Actualmente el control de bacterias fitopatógenas se realiza mediante pesticidas con alta toxicidad para los consumidores y el ambiente. Para evitar las bacteriosis en la práctica se sugiere la rotación de cultivos y utilizar semillas certificadas. Se están probando compuestos naturales derivados de plantas medicinales como pesticidas; estos se pueden dividir en varias categorías fitoquímicas. Varios estudios confirman la actividad antibacteriana, antifúngica y antiviral de estos productos. Extractos vegetales con alto contenido de flavonoides y aceite esenciales poseen una importante actividad antibacteriana. Además, algunos aceites esenciales podrían estar incidiendo en la liberación y/o producción de biofilm, exopolisacáridos y exoproteínas. La gran incidencia de las infecciones por fitopatógenos y las pérdidas económicas que estas acarrean hacen que su control presente grandes dificultades para la agricultura sustentable en soja de nuestro país. En este trabajo se propone estudiar los diferentes factores de virulencia de cepas bacterianas fitopatógenas y evaluar el rol que cumplen en el proceso de la enfermedad en cultivos de soja y desarrollar estrategias para el control de bacteriosis vegetales aplicando productos naturales aislados de plantas aromáticas. La correcta utilización de productos antimicrobianos de origen natural aplicados sobre el cultivo y/o sobre las semillas evitaría la dispersión de la enfermedad y la eliminación al medio ambiente de productos contaminantes no deseados. In Argentina, soybean cultivation occupies the first place; 90% of this cereal is produced in the central region of the country. Intensive tillage practices favour multiplication and survival of bacterial phytopathogens causing blight and pustule diseases. Pseudomonas syringae pv. glycinea produce several toxins like coronatine, faseolotoxine, siringomicine, tabtoxine and proteins of nucleation ice that contribute to the develop of chlorosis and necrosis, characteristic of bacterial blight. It also produces levan and alginate, cellulose and biofilm. Pustule disease is caused by Xanthomonas axonopodis pv glycines, which produce enzymes like α-amilase, protease, endo β-mannanase, peptolitic activity, xanthan and biofilm. Nowadays the control of phytopathogenic bacteria consists in the application of pesticides that are toxic for the environment and man. Natural products from medicinal plants are a new alternative for the treatment of phytopathogens. Researches made with phytochemical compounds (flavonoids, phenols, quinones, cummarines, essential oils, terpenes) support the antimicrobial activity of these natural products. What is more, these substances could suppress the biofilm, exoproteins and exopolisaccharides formation and release of them. The infections caused by phytopathogens provoke economical loses and its control presents big difficulties in our country. The proposal of this work is the characterization of phytopatoghenic strains, its virulence factors and the role they play in the disease process. The development of a new alternative for the control of vegetable bacteriosis using natural products obtained from aromatic plants and the correct application of them on sown fields or on seeds is also an objective in this work.

Perinatal choline supplementation reveals a dissociation between two types of spatial strategies.

Choline supplementation improving memory functions in rodents is assumed to increase the synthesis and release of acetylcholine in the brain. We have found that a combined pre- and postnatal supplementation results in long-lasting facilitation of spatial memory in juvenile rats when training was conducted in presence of a local salient cue. The present work was aimed at analysing the effects of peri- and postnatal choline supplementation on spatial abilities of naive adult rats. Rats given a perinatal choline supplementation were trained in various cued procedures of the Morris navigation task when aged 5 months. The treatment had a specific effect of reducing the escape latency of the rats when the platform was at a fixed position in space and surrounded by a suspended cue. This effect was associated with an increased spatial bias when the cue and platform were removed. In this condition, the control rats showed impaired spatial discrimination following the removal of the target cue, most likely due to an overshadowing of the distant environmental cues. This impairment was not observed in the treated rats. Further training with the suspended cue at unpredictable places in the pool revealed longer escape latencies in the control than in the treated rats suggesting that this procedure induced a selective perturbation of the normal but not of the treated rats. A special probe trial with the cue at an irrelevant position and no escape platform revealed a significant bias of the control rats toward the cue and of the treated rats toward the uncued spatial escape position. This behavioural dissociation suggests that a salient cue associated with the target induces an alternative "non spatial" guidance strategy in normal rats, with the risk of overshadowing of the more distant spatial cues. In this condition, the choline supplementation facilities a spatial reliance on the cue, that is an overall facilitation of learning a set of spatial relations between several visual cues. As a consequence, the improved escape in presence of the cue is associated with a stronger memory of the spatial position following disappearance of the cue. This and previous observations suggest that a specific spatial attention process relies on the buffering of highly salient visual cues.to facilitate integration of their relative position in the environment.

The effects of feeding Karlodinium veneficum (PLY # 103; Gymnodinium veneficum Ballantine) to the blue mussel Mytilus edulis

The effects of exposure to the type species for Karlodinium veneficum (PLY # 103) on immune function and histopathology in the blue mussel Mytilus edulis were investigated. Mussels from Whitsand Bay, Cornwall (UK) were exposed to K. veneficum (PLY # 103) for 3 and 6 days. Assays for immune function included total and differential cells counts, phagocytosis and release of extra cellular reactive oxygen species. Histology was carried out on digestive gland and mantle tissues. The toxin cell quota for K. veneficum (PLY #103) was measured by liquid chromatography-mass spectrometry detecting two separable toxins KvTx1 (11.6 ± 5.4 ng/ml) and KvTx2 (47.7 ± 4.2 ng/ml). There were significant effects of K. veneficum exposure with increasing phagocytosis and release of reactive oxygen species following 6 days exposure. There were no significant effects on total cell counts. However, differential cell counts did show significant effects after 3 days exposure to the toxic alga. All mussels produced faeces but not pseudofaeces indicating that algae were not rejected prior to ingestion. Digestive glands showed ingestion of the algae and hemocyte infiltration after 3 days of exposure, whereas mantle tissue did not show differences between treatments. As the effects of K. veneficum were not observed in the mantle tissue it can be hypothesized that the algal concentration was not high enough, or exposure long enough, to affect all the tissues. Despite being in culture for more than 50 years the original K. veneficum isolate obtained by Mary Parke still showed toxic effects on mussels.

Advances in pharmacological studies of silymarin

Silymarin is the flavonoids extracted from the seeds of Silybum marianum (L) Gearth as a mixture of three structural isomers: silybin, silydianin and silychristin, the former being the most active component. Silymarin protects liver cell membrane against hepatotoxic agents and improves liver function in experimental animals and humans. It is generally accepted that silymarin exerts a membrane-stabilizing action preventing or inhibiting membrane peroxidation. The experiments with soybean lipoxygenase showed that the three components of silymarin brought about a concentration-dependent non-competitive inhibition of the lipoxygenase. The experiments also showed an analogous interaction with animal lipoxygenase, thus showing that an inhibition of the peroxidation of the fatty acid in vivo was self-evident. Silybin almost completely suppressed the formation of PG at the highest concentration (0.3 mM) and proved to be an inhibitor of PG synthesis in vitro. In our experiments, silybin at lower dose (65 mg/Kg) decreased liver lipoperoxide content and microsomal lipoperoxidation to 84.5% and 68.55% of those of the scalded control rats respectively, and prevented the decrease of liver microsomal cytochrome p-450 content and p-nitroanisole-0-demethylase activity 24 h post-scalding. Effects of silymarin on cardiovascular systen have been studied in this university since 1980. O. O silymarin 800 mg/Kg/d or silybin 600 mg/Kg/d reduced plasma total cholesterol, LDL-C and VLDL-C. They however, enhanced HDL-C in hyperlipenic rats. Further studies showed that silymarin enhanced HDL-C in hyperlipemic rats. Further studies showed that silymarin enhanced HDL-C but didn't affect HDL-C, a property of this component which is beneficial to treatment of atherosclerosis. The results showed silymarin 80 mg or silybin 60 mg decreased in vitro platelet aggregation (porcentagem) in rats. The maximal platelet aggregation induced by ADP declined significantly, and time to reach maximal platelet aggregation and five-minute disaggregation didn't change. In our experiments, iv silybin 22,4 mg/kg lowered the amplitude and duration of diastolic blood pressure (DBP) more than those of systolic (SBP), but the descending aortic blood flow, cardiac contractility and ECG did not change significantly in anesthetized open-chest cats. The results indicated a reduction of peripheral resistance and dilatatory action on the resistant blood vessels. These effects are beneficial to coronary heart disease. We also observed the effects of silybin on morphological change, the release of glutamic oxaloacetate aminotrasferase (GOT) and lactate dehydrogenase (LDH) as well as the radioactivity of 3H-TdR incorporated into DNA in normal cardiac cells and cells infected by coxsackie B5, virus os newborn rats. The results showed that silynin did not affect the morphology of normal cell, and that the pathological change of cells infected by virus was delayed and reduced as compared to control. We have investigated the effect of silybin on synthesis and release of LTs in the cultured porcine cerebral basilar arteries (PCBA). Silybin 100 and 500 µmol/L declined the amounts of LTs released from the PCBA incubsated in the presence of A 23187, AA and indomenthacin. The result suggests that silybin can inhibit the activity of 5-lipoxygenase of cerebral blood vessel and may protect the brain from ischemia.

Endothelial Cell-Derived Nitric Oxide Enhances Aerobic Glycolysis in Astrocytes via HIF-1α-Mediated Target Gene Activation.

Astrocytes exhibit a prominent glycolytic activity, but whether such a metabolic profile is influenced by intercellular communication is unknown. Treatment of primary cultures of mouse cortical astrocytes with the nitric oxide (NO) donor DetaNONOate induced a time-dependent enhancement in the expression of genes encoding various glycolytic enzymes as well as transporters for glucose and lactate. Such an effect was shown to be dependent on the hypoxia-inducible factor HIF-1α, which is stabilized and translocated to the nucleus to exert its transcriptional regulation. NO action was dependent on both the PI3K/Akt/mTOR and MEK signaling pathways and required the activation of COX, but was independent of the soluble guanylate cyclase pathway. Furthermore, as a consequence of NO treatment, an enhanced lactate production and release by astrocytes was evidenced, which was prevented by downregulating HIF-1α. Several brain cell types represent possible sources of NO. It was found that endothelial cells, which express the endothelial NO synthase (eNOS) isoform, constitutively produced the largest amount of NO in culture. When astrocytes were cocultured with primary cultures of brain vascular endothelial cells, stabilization of HIF-1α and an enhancement in glucose transporter-1, hexokinase-2, and monocarboxylate transporter-4 expression as well as increased lactate production was found in astrocytes. This effect was inhibited by the NOS inhibitor l-NAME and was not seen when astrocytes were cocultured with primary cultures of cortical neurons. Our findings suggest that endothelial cell-derived NO participates to the maintenance of a high glycolytic activity in astrocytes mediated by astrocytic HIF-1α activation.

Host innate immune responses to microbial pathogens.

Sepsis is among the leading causes of death worldwide and its incidence is increasing. Defined as the host response to infection, sepsis is a clinical syndrome considered to be the expression of a dysregulated immune reaction induced by danger signals that may lead to organ failure and death. Remarkable progresses have been made in our understanding of the molecular basis of host defenses in recent years. The host defense response is initiated by innate immune sensors of danger signals designated under the collective name of pattern-recognition receptors. Members of the family of microbial sensors include the complement system, the Toll-like receptors, the nucleotide-binding oligomerization domainlike receptors, the RIG-I-like helicases and the C-type lectin receptors. Ligand-activated pattern-recognition receptors kick off a cascade of intracellular events resulting in the expression of co-stimulatory molecules and release of effector molecules playing a fundamental role in the initiation of the innate and adaptive immune responses. Fine tuning of proinflammatory and anti-inflammatory reactions is critical for keeping the innate immune response in check. Overwhelming or dysregulated responses induced by infectious stimuli may have dramatic consequences for the host as shown by the profound derangements observed in sepsis. Unfortunately, translational research approaches aimed at the development of therapies targeting newly identified innate immune pathways have not held their promises. Indeed, all recent clinical investigations of adjunctive anti-sepsis treatments had little, if any, impact on morbidity and all-cause mortality of sepsis. Dissecting the mechanisms underlying the transition from infection to sepsis is essential for solving the sepsis enigma. Important components of the puzzle have already been identified, but the hunt must go on in the laboratory and at the bedside.

Inflammatory cutaneous reaction induced by the lectin of Dioclea grandiflora (Mart. )

The lectin from Dioclea grandiflora (Mart.) that selectively binds glucose and mannose, when subcutaneously injected in mouse induces an inflammatory cutaneous reaction whose histological analysis reveals an hemorrhagic ulceration with exudative reaction accompanied by an influx of polymorphonuclear leukocytes and giant cells. The presence of lymphocytes and plasma cells in the lesion was insignificant. In order to characterize the in vivo action of inflammatory factors generated by this lesion, distinct lines of mice were used: high and low antibody responder mice; the genetically selected mice to the acute phase of inflammatory reaction; lines of mice deficient in C5, a protein of the complement system. It is shown that the lectin of D. grandiflora acts as an inflammatory agent probably promoting exocytosis and release of mediators.

Paclitaxel-eluting biodegradable synthetic vascular prostheses: a step towards reduction of neointima formation?

BACKGROUND: Clinical small-caliber vascular prostheses are unsatisfactory. Reasons for failure are early thrombosis and late intimal hyperplasia. We thus prepared biodegradable small-caliber vascular prostheses using electrospun polycaprolactone (PCL) with slow-releasing paclitaxel (PTX), an antiproliferative drug. METHODS AND RESULTS: PCL solutions containing PTX were used to prepare nonwoven nanofibre-based 2-mm ID prostheses. Mechanical morphological properties and drug loading, distribution, and release were studied in vitro. Infrarenal abdominal aortic replacement was carried out with nondrug-loaded and drug-loaded prostheses in 18 rats and followed for 6 months. Patency, stenosis, tissue reaction, and drug effect on endothelialization, vascular remodeling, and neointima formation were studied in vivo. In vitro prostheses showed controlled morphology mimicking extracellular matrix with mechanical properties similar to those of native vessels. PTX-loaded grafts with suitable mechanical properties and controlled drug-release were obtained by factorial design. In vivo, both groups showed 100% patency, no stenosis, and no aneurysmal dilatation. Endothelial coverage and cell ingrowth were significantly reduced at 3 weeks and delayed at 12 and 24 weeks in PTX grafts, but as envisioned, neointima formation was significantly reduced in these grafts at 12 weeks and delayed at 6 months. CONCLUSIONS: Biodegradable, electrospun, nanofibre, polycaprolactone prostheses are promising because in vitro they maintain their mechanical properties (regardless of PTX loading), and in vivo show good patency, reendothelialize, and remodel with autologous cells. PTX loading delays endothelialization and cellular ingrowth. Conversely, it reduces neointima formation until the end point of our study and thus may be an interesting option for small caliber vascular grafts.

Defining secretion processes in astrocytes

During the last decade, evidence that release of chemical transmitters from astrocytes might modulate neuronal activity (the so-called "gliotransmission") occurs in situ has been extensively provided. Nevertheless, gliotransmission remains a highly debated topic because of the lack of direct morphological and functional evidence. Here we provided new information supporting gliotransmission, by i) deepen knowledge about specific properties of regulated secretion of glutamatergic SLMVs, and ii) investigating the involvement of astrocytes in the transmission of dopamine, a molecule whose interaction with astrocytes is likely to occur, but it's still not proven.¦VGLUT-expressing glutamatergic SLMVs have been previously identified both in situ and in vitro, but description of kinetics of release were still lacking. To elucidate this issue, we took advantage of fluorescent tools (styryl dyes and pHluorin) and adapted experimental paradigms and analysis methods previously developed to study exo-endocytosis and recycling of glutamatergic vesicles at synapses. Parallel use of EPIfluorescence and total internal reflection (TIRF) imaging allowed us to find that exo-endocytosis processes in astrocytes are extremely fast, with kinetics in the order of milliseconds, able to sustain and follow neuronal signalling at synapses. Also, exocytosis of SLMVs is under the control of fast, localized Ca2+ elevations in close proximity of SLMVs and endoplasmatic reticulum (ER) tubules, the intracellular calcium stores. Such complex organization supports the fast stimulus-secretion coupling we described; localized calcium elevations have been recently observed in astrocytes in situ, suggesting that these functional microdomains might be present in the intact tissue. In the second part of the work, we investigated whether astrocytes possess some of the benchmarks of brain dopaminergic cells. It's been known for years that astrocytes are able to metabolize monoamines by the enzymes MAO and COMT, but to date no clear information that glial cells are able to uptake and store monoamines have been provided. Here, we identified a whole apparatus for the storage, degradation and release of monoamines, at the ultrastructural level. Electron microscopy immunohistochemistry allowed us to visualize VMAT2- and dopamine-positive intracellular compartments within astrocytic processes, i.e. dense -core granules and cisterns. These organelles might be responsible for dopamine release and storage, respectively; interestingly, this intracellular distribution is reminiscent of VMAT2 expression in dendrites if neurons, where dopamine release is tonic and plays a role in the regulation of its a basal levels, suggesting that astrocytic VMAT2 is involved in the homeostasis of dopamine in healthy brains of adult mammals.¦Durant cette dernière décennie, de nombreux résultats sur le relâchement des transmetteurs par les astrocytes pouvant modulé l'activité synaptique (gliotransmission) ont été fournis. Néanmoins, la gliotransmission reste un processus encore très débattu, notamment à cause de l'absence de preuves directes, morphologique et fonctionnelle démontrant ce phénomène. Nous présentons dans nos travaux de nombreux résultats confortant l'hypothèse de la gliotransmission, dont i) une étude approfondie sur les propriétés spatiales et temporelles de la sécrétion régulée du glutamate dans les astrocytes, et ii) une étude sur la participation des astrocytes dans la transmission de la dopamine, une neuromodulateur dont l'interaction avec les astrocytes est fortement probable, mais qui n'a encore jamais été prouvée. L'expression des petites vésicules (SLMVs - Synaptic Like Micro Vesicles) glutamatergiques exprimant les transporteurs vésiculaires du glutamate (VGLUTs) dans les astrocytes a déjà été prouvé tant in situ qu'in vitro. Afin de mettre en évidence les propriétés précises de la sécrétion de ces organelles, nous avons adapté à nos études des méthodes expérimentales conçues pour observer les processus de exocytose et endocytose dans les neurones. Les résolutions spatiale et temporelle obtenues, grâce a l'utilisation en parallèle de l'épi fluorescence et de la fluorescence a onde évanescente (TIRF), nous ont permis de montrer que la sécrétion régulée dans les astrocytes est un processus extrêmement rapide (de l'ordre de la milliseconde) et qu'elle est capable de soutenir et de suivre la transmission de signaux entre neurones. Nous avons également découvert que cette sécrétion a lieu dans des compartiments subcellulaires particuliers où nous observons la présence du reticulum endoplasmique (ER) ainsi que des augmentations rapides de calcium. Cette organisation spatiale complexe pourrait être la base morphologique du couplage rapide entre le stimulus et la sécrétion. Par ailleurs, plusieurs études récentes in vivo semblent confirmer l'existence de ces compartiments. Depuis des années nous savons que les astrocytes sont capables de métaboliser les monoamines par les enzymes MAO et COMT. Nous avons donc fourni de nouvelles preuves concernant la présence d'un appareil de stockage dans les astrocytes participant à la dégradation et la libération de monoamines au niveau ultrastructurelle. Grâce à la microscopie électronique, nous avons découvert la présence de compartiments intracellulaires exprimant VMAT2 dans les processus astrocytaires, sous forme de granules et des citernes. Ces organelles pourraient donc être responsables à la fois du relâchement et du stockage de la dopamine. De manière surprenante, cette distribution intracellulaire est similaire aux dendrites des neurones exprimant VMAT2, où la dopamine est libérée de façon tonique permettant d'agir sur la régulation de ses niveaux de base. Ces résultats, suggèrent une certaine participation des VMAT2 présents dans les astrocytes dans le processus d'homéostase de la dopamine dans le cerveau.¦A de nombreuses reprises, dans des émissions scientifiques ou dans des films, il est avancé que les hommes n'utilisent que 10% du potentiel de leur cerveau. Cette légende provient probablement du fait que les premiers chercheurs ayant décrit les cellules du cerveau entre le XIXème et le XXeme siècle, ont montré que les neurones, les cellules les plus connues et étudiées de cet organe, ne représentent seulement que 10% de la totalité des cellules composant du cerveau. Parmi les 90% restantes, les astrocytes sont sans doute les plus nombreuses. Jusqu'au début des années 90, les astrocytes ont été plutôt considérés peu plus que du tissu conjonctif, ayant comme rôles principaux de maintenir certaines propriétés physiques du cerveau et de fournir un support métabolique (énergie, environnement propre) aux neurones. Grace à la découverte que les astrocytes ont la capacité de relâcher des substances neuro-actives, notamment le glutamate, le rôle des astrocytes dans le fonctionnement cérébral a été récemment reconsidérée.¦Le rôle du glutamate provenant des astrocytes et son impact sur la fonctionnalité des neurones n'a pas encore été totalement élucidé, malgré les nombreuses publications démontrant l'importance de ce phénomène en relation avec différentes fonctions cérébrales. Afin de mieux comprendre comment les astrocytes sont impliqués dans la transmission cérébrale, nous avons étudié les propriétés spatio-temporelles de cette libération grâce à l'utilisation des plusieurs marqueurs fluorescents combinée avec différentes techniques d'imagerie cellulaires. Nous avons découvert que la libération du glutamate par les astrocytes (un processus maintenant appelé "gliotransmission") était très rapide et contrôlée par des augmentations locales de calcium. Nous avons relié ces phénomènes à des domaines fonctionnels subcellulaires morphologiquement adaptés pour ce type de transmission. Plus récemment, nous avons concentré nos études sur un autre transmetteur très important dans le fonctionnement du cerveau : la dopamine. Nos résultats morphologiques semblent indiquer que les astrocytes ont la capacité d'interagir avec ce transmetteur, mais d'une manière différente comparée au glutamate, notamment en terme de rapidité de transmission. Ces résultats suggèrent que le astrocytes ont la capacité de modifier leurs caractéristiques et de s'adapter à leur environnement par rapport aux types de transmetteur avec lequel ils doivent interagir.

Over-expression of PHO1 in Arabidopsis leaves reveals its role in mediating phosphate efflux.

Inorganic phosphate (Pi) homeostasis in multi-cellular eukaryotes depends not only on Pi influx into cells, but also on Pi efflux. Examples in plants for which Pi efflux is crucial are transfer of Pi into the xylem of roots and release of Pi at the peri-arbuscular interface of mycorrhizal roots. Despite its importance, no protein has been identified that specifically mediates phosphate efflux either in animals or plants. The Arabidopsis thaliana PHO1 gene is expressed in roots, and was previously shown to be involved in long-distance transfer of Pi from the root to the shoot. Here we show that PHO1 over-expression in the shoot of A. thaliana led to a two- to threefold increase in shoot Pi content and a severe reduction in shoot growth. (31) P-NMR in vivo showed a normal initial distribution of intracellular Pi between the cytoplasm and the vacuole in leaves over-expressing PHO1, followed by a large efflux of Pi into the infiltration medium, leading to a rapid reduction of the vacuolar Pi pool. Furthermore, the Pi concentration in leaf xylem exudates from intact plants was more than 100-fold higher in PHO1 over-expressing plants compared to wild-type. Together, these results show that PHO1 over-expression in leaves leads to a dramatic efflux of Pi out of cells and into the xylem vessel, revealing a crucial role for PHO1 in Pi efflux.

Cytotoxins of the human pathogen Aeromonas hydrophila trigger, via the NLRP3 inflammasome, caspase-1 activation in macrophages.

Aeromonas hydrophila is a Gram-negative pathogen that causes serious infectious disease in humans. A. hydrophila induces apoptosis in infected macrophages, but the host proinflammatory responses triggered by macrophage death are largely unknown. Here, we demonstrate that the infection of mouse macrophages with A. hydrophila triggers the activation of caspase-1 and release of IL-1β. Caspase-1 activation was abrogated in macrophages deficient in Nod-like receptor family, pyrin domain containing 3 (NLRP3) and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), but not NLR family, CARD domain containing 4 (NLRC4). The activation of the NLRP3 inflammasome was mediated by three cytotoxins (aerolysin, hemolysin and multifunctional repeat-in-toxin) produced by A. hydrophila. Our results indicated that the NLRP3 inflammasome senses A. hydrophila infection through the action of bacterial cytotoxins.