988 resultados para Ca2 influx
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ABSTRACT Preservation of mangroves, a very significant ecosystem from a social, economic, and environmental viewpoint, requires knowledge on soil composition, genesis, morphology, and classification. These aspects are of paramount importance to understand the dynamics of sustainability and preservation of this natural resource. In this study mangrove soils in the Suba river basin were described and classified and inorganic waste concentrations evaluated. Seven pedons of mangrove soil were chosen, five under fluvial influence and two under marine influence and analyzed for morphology. Samples of horizons and layers were collected for physical and chemical analyses, including heavy metals (Pb, Cd, Mn, Zn, and Fe). The moist soils were suboxidic, with Eh values below 350 mV. The pH level of the pedons under fluvial influence ranged from moderately acid to alkaline, while the pH in pedons under marine influence was around 7.0 throughout the profile. The concentration of cations in the sorting complex for all pedons, independent of fluvial or marine influence, indicated the following order: Na+>Mg2+>Ca2+>K+. Mangrove soils from the Suba river basin under fluvial and marine influence had different morphological, physical, and chemical characteristics. The highest Pb and Cd concentrations were found in the pedons under fluvial influence, perhaps due to their closeness to the mining company Plumbum, while the concentrations in pedon P7 were lowest, due to greater distance from the factory. For containing at least one metal above the reference levels established by the National Oceanic and Atmospheric Administration (United States Environmental Protection Agency), the pedons were classified as potentially toxic. The soils were classified as Gleissolos Tiomrficos rticos (slicos) sdico neofluvisslico in according to the Brazilian Soil Classification System, indicating potential toxicity and very poor drainage, except for pedon P7, which was classified in the same subgroup as the others, but different in that the metal concentrations met acceptable standards.
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RESUMO A matria orgnica do solo uma importante fonte primria de nutrientes s plantas e influencia a infiltrao, reteno de gua, suscetibilidade eroso e agregao do solo. No entanto, esse processo est condicionado qualidade e quantidade da matria orgnica aportada. O objetivo deste trabalho foi avaliar a influncia do desastre ambiental causado pela chuva em reas de produo de olerceas na regio Serrana do Rio de Janeiro. Foram selecionadas as seguintes reas: A1: sem impacto (testemunha); A2: soterramento total e cultivo de aveia + ervilhaca; A3: transbordamento do rio e cultivo de milheto + girassol; A4: transbordamento do rio e grande deposio de areia com cultivo de aveia-preta; A5: transbordamento do rio com grande deposio de areia e sem cultivo; e A6: transbordamento do rio em pequena escala e sem cultivo. As propriedades edficas analisadas foram: textura; densidade do solo (Ds); densidade de partculas (Dp); dimetro mdio ponderado (DMP) e dimetro mdio geomtrico (DMG) de agregados estveis em gua; pH(H2O), Al3+, Ca2++Mg2+, Na+, H+Al, K, P e carbono orgnico total (COT); e fraes granulomtricas e qumicas da matria orgnica. Os maiores valores de DMP e DMG foram observados nas reas A3 e A6, que seguem a mesma tendncia dos maiores teores de argila. Os teores de COT e suas fraes qumicas e fsicas apresentaram diferenas em relao testemunha com os menores valores verificados nas reas que foram submetidas ao impacto pelo transbordamento do rio e grande deposio de areia. As reas que foram impactadas pelo soterramento com predomnio da frao argila apresentaram valores semelhantes ou superiores aos da testemunha. As variveis foram selecionadas e avaliadas por meio da anlise de componentes principais, que evidenciou distino entre as reas estudadas, com separao entre elas associada textura do solo e densidade de partculas.
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ABSTRACT Soil solution samplers may have the same working principle, but they differ in relation to chemical and physical characteristics, cost and handling, and these aspects exert influence on the chemical composition of the soil solution obtained. This study was carried out to evaluate, over time, the chemical composition of solutions extracted by Suolo Acqua, with the hydrophilic membrane (HM) as a standard, using soils with contrasting characteristics, and to determine the relationship between electrical conductivity (EC) and concentration of ions and pH of soil solution samples. This study was carried out under laboratory conditions, using three soils samples with different clay and organic matter (OM) contents. Soil solution contents of F−, Cl−, NO−3, Br−, SO42−, Na+, NH4+, K+, Mg2+, Ca2+, were analyzed, as well as inorganic, organic, and total C contents, pH, and EC, in four successive sampling times. Soil solution chemical composition extracted by the Suolo Acqua sampler is similar to that collected by the HM, but the Suolo Acqua extracted more Na+ and soluble organic C than the HM solution. Solution EC, cation and anion concentrations, and soluble C levels are higher in the soil with greater clay and OM contents (Latossolo and Cambissolo in this case). Soil solution composition varied over time, with considerable changes in pH, EC, and nutrient concentrations, especially associated with soil OM. Thus, single and isolated sampling of the soil solution must be avoided, otherwise composition of the soil solution may not be correctly evaluated. Soil solution EC was regulated by pH, as well as the sum of cation and anion concentrations, and the C contents determined in the soil liquid phase.
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ABSTRACT Applications of phosphogypsum (PG) provide nutrients to the soil and reduce Al3+ activity, favoring soil fertility and root growth, but allow Mg2+ mobilization through the soil profile, resulting in variations in the PG rate required to achieve the optimum crop yield. This study evaluated the effect of application rates and splitting of PG on soil fertility of a Typic Hapludox, as well as the influence on annual crops under no-tillage. Using a (4 3) + 1 factorial structure, the treatments consisted of four PG rates (3, 6, 9, and 12 Mg ha-1) and three split applications (P1 = 100 % in 2009; P2 = 50+50 % in 2009 and 2010; P3 = 33+33+33 % in 2009, 2010 and 2011), plus a control without PG. The soil was sampled six months after the last PG application, in stratified layers to a depth of 0.8 m. Corn, wheat and soybean were sown between November 2011 and December 2012, and leaf samples were collected for analysis when at least 50 % of the plants showed reproductive structures. The application of PG increased Ca2+ concentrations in all sampled soil layers and the soil pH between 0.2 and 0.8 m, and reduced the concentrations of Al3+ in all layers and of Mg2+ to a depth of 0.6 m, without any effect of splitting the applications. The soil Ca/Mg ratio increased linearly to a depth of 0.6 m with the rates and were found to be higher in the 0.0-0.1 m layer of the P2 and P3 treatments than without splitting (P1). Sulfur concentrations increased linearly by application rates to a depth of 0.8 m, decreasing in the order P3>P2>P1 to a depth of 0.4 m and were higher in the treatments P3 and P2 than P1 between 0.4-0.6 m, whereas no differences were observed in the 0.6-0.8 m layer. No effect was recorded for K, P and potential acidity (H+Al). The leaf Ca and S concentration increased, while Mg decreased for all crops treated with PG, and there was no effect of splitting the application. The yield response of corn to PG rates was quadratic, with the maximum technical efficiency achieved at 6.38 Mg ha-1 of PG, while wheat yield increased linearly in a growing season with a drought period. Soybean yield was not affected by the PG rate, and splitting had no effect on the yield of any of the crops. Phosphogypsum improved soil fertility in the profile, however, Mg2+ migrated downwards, regardless of application splitting. Splitting the PG application induced a higher Ca/Mg ratio in the 0.0-0.1 m layer and less S leaching, but did not affect the crop yield. The application rates had no effect on soybean yield, but were beneficial for corn and, especially, for wheat, which was affected by a drought period during growth.
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Acid-sensing ion channels (ASICs) are non-voltage-gated sodium channels activated by an extracellular acidification. They are widely expressed in neurons of the central and peripheral nervous system. ASICs have a role in learning, the expression of fear, in neuronal death after cerebral ischemia, and in pain sensation. Tissue damage leads to the release of inflammatory mediators. There is a subpopulation of sensory neurons which are able to release the neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP). Neurogenic inflammation refers to the process whereby peripheral release of the neuropeptides CGRP and SP induces vasodilation and extravasation of plasma proteins, respectively. Our laboratory has previously shown that calcium-permeable homomeric ASIC1a channels are present in a majority of CGRP- or SP-expressing small diameter sensory neurons. In the first part of my thesis, we tested the hypothesis that a local acidification can produce an ASIC-mediated calcium-dependant neuropeptide secretion. We have first verified the co-expression of ASICs and CGRP/SP using immunochemistry and in-situ hybridization on dissociated rat dorsal root ganglion (DRG) neurons. We found that most CGRP/SP-positive neurons also expressed ASIC1a and ASIC3 subunits. Calcium imaging experiments with Fura-2 dye showed that an extracellular acidification can induce an increase of intracellular Ca2+ concentration, which is essential for secretion. This increase of intracellular Ca2+ concentration is, at least in some cells, ASIC-dependent, as it can be prevented by amiloride, an ASIC antagonist, and by Psalmotoxin (PcTx1), a specific ASIC1a antagonist. We identified a sub-population of neurons whose acid-induced Ca2+ entry was completely abolished by amiloride, an amiloride-resistant population which does not express ASICs, but rather another acid-sensing channel, possibly transient receptor potential vanillode 1 (TRPV1), and a population expressing both H+-gated channel types. Voltage-gated calcium channels (Cavs) may also mediate Ca2+ entry. Co-application of the Cavs inhibitors (ω-conotoxin MVIIC, Mibefradil and Nifedipine) reduced the Ca2+ increase in neurons expressing ASICs during an acidification to pH 6. This indicates that ASICs can depolarise the neuron and activate Cavs. Homomeric ASIC1a are Ca2+-permeable and allow a direct entry of Ca2+ into the cell; other ASICs mediate an indirect entry of Ca2+ by inducing a membrane depolarisation that activates Cavs. We showed with a secretion assay that CGRP secretion can be induced by extracellular acidification in cultured rat DRG neurons. Amiloride and PcTx1 were not able to inhibit the secretion at acidic pH, but BCTC, a TRPV1 inhibitor was able to decrease the secretion induced by an extracellular acidification in our in vitro secretion assay. In conclusion, these results show that in DRG neurons a mild extracellular acidification can induce a calcium-dependent neuropeptide secretion. Even if our data show that ASICs can mediate an increase of intracellular Ca2+ concentration, this appears not to be sufficient to trigger neuropeptide secretion. TRPV1, a calcium channel whose activation induces a sustained current - in contrary of ASICs - played in our experimental conditions a predominant role in neurosecretion. In the second part of my thesis, we focused on the role of ASICs in neuropathic pain. We used the spared nerve injury (SNI) model which consists in a nerve injury that induces symptoms of neuropathic pain such as mechanical allodynia. We have previously shown that the SNI model modifies ASIC currents in dissociated rat DRG neurons. We hypothesized that ASICs could play a role in the development of mechanical allodynia. The SNI model was performed on ASIC1a, -2, and -3 knock-out mice and wild type littermates. We measured mechanical allodynia on these mice with calibrated von Frey filaments. There were no differences between the wild-type and the ASIC1, or ASIC2 knockout mice. ASIC3 null mice were less sensitive than wild type mice at 21 day after SNI, indicating a role for ASIC3. Finally, to investigate other possible roles of ASICs in the perception of the environment, we measured the baseline heat responses. We used two different models; the tail flick model and the hot plate model. ASIC1a null mice showed increased thermal allodynia behaviour in the hot plate test at three different temperatures (49, 52, 55C) compared to their wild type littermates. On the contrary, ASIC2 null mice showed reduced thermal allodynia behaviour in the hot plate test compared to their wild type littermates at the three same temperatures. We conclude that ASIC1a and ASIC2 in mice can play a role in temperature sensing. It is currently not understood how ASICs are involved in temperature sensing and what the reason for the opposed effects in the two knockout models is.
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Trace element and isotopic compositions of marine fossils and sediment were analyzed from several Miocene deposits in the circum-Alpine region in order to reconstruct the paleoceanographic and paleoclimatic changes related to sea level changes, basin evolution and Alpine orogeny. To the north and the east the Alps were border by an epicontinental sea, the Paratethys, while to the south the Mediterranean surrounded the uplifting mountains during the Miocene. The thesis mainly focused on sediments and fossils sampled from Miocene beds of these two oceanic provinces. The north Alpine Molasse, the Vienna and Pannonian Basins were located in the Western and Central Paratethys. O-isotope compositions of well-preserved phosphatic fossils in these sediments support deposition under sub-tropical to warm-temperate climate with water temperatures between 14 to 28 C for the Miocene. δ18O values of fossil shark teeth from different horizons vary similarly to those of the global trend until the end of the Badenian, however the δ18O values show wider range, which indicates local effects iii the sub-basins. The trend of 87Sr/86Sr in the samples roughly agrees with an open ocean environment for the Miocene. Yet a number of samples deviate from typical open ocean compositions with higher ratios suggesting modification of seawater by local and old terrestrial sources. In contrast, two exceptional teeth from the locality of La Moliere have extremely low δ18O values and low 87Sr/86Sr. However, the REE patterns of their enameloid are similar to those of teeth having O and Sr isotopic compositions typical of a marine setting at this site. Collectively, this suggests that the two teeth formed while the sharks frequented a freshwater environment with very low 18O-content and 87Sr/86Sr controlled by Mesozoic calcareous rocks. This is consistent with a paleogeography of high-elevation (~2300m) Miocene Alps adjacent to a marginal sea. The local effects are also reflected in the εNd values of the Paratethyan fossils, which is compatible with input from ancient crystalline rocks and Mesozoic sediments, while other samples with elevated εNd values indicate an influence of Neogene volcanism on the water budget. Excluding samples whose isotopic compositions reflect a local influence on the water column, an average εNd value of -7.9 0.5 may be inferred for the Paratethys seawater. This value is indistinguishable from the Miocene value of the Indian Ocean, supporting a dominant role of ludo-Pacific water masses in the Paratethys. Regarding the Mediterranean, stable C-and O-isotope compositions of benthic and planktonic foraminifera from the Umbria-Marche region (UMC) have an offset typical for their habitats and the changes in composition mimic global changes, suggesting that the regional conditions of climate and the carbon cycle were controlled by global changes. The radiogenic isotope compositions of the fossil assemblages allow for distinction of periods. From 25 to 19 Ma, high εNd values and low 87Sr/86Sr of sediments and fossils support intense tectonism and volcanism, related to the opening of the western Mediterranean. Between 19 and 13 Ma the Mediterranean has εNd values that are largely controlled by incursion of Indian Ocean water. Brief periods of local hinterland control on seawater compositions are indicated by spikes in the εNd record, coinciding with volcanic events and a short sea-level decrease at about 15.2 Ma. Lower 87Sr/86Sr compared to the open ocean is compatible with rapid uplift of the hinterland and intense influx of Sr from Mesozoic carbonates of the western Apennines, while higher 87Sr/86Sr for other sites indicates erosion of old crustal silicate rocks. Finally, from 13 to 7 Ma the fossils have 87Sr/86Sr similar to those of Miocene seawater and their εNd values indicates fluctuating influence of Atlantic, and Indian Ocean or Paratethys sources of seawater entering the Mediterranean, driven by global sealevel changes and local tectonism. RSUM DE LA THSE Les compositions en lments traces et isotopiques de fossiles marins et de sdiments on t analyses partir de nombreux dpts marins dans la rgion circum Alpine dans le but de reconstruire les changements palocanographiques et paloclimatiques lis aux changements du niveau marin, l'volution en bassins et l'orognie alpine. Au nord et l'est des Alpes, une mer picontinentale appele Paratthys s'est ouverte, alors que plus au sud la mer Mditerrane bordait au Miocne les Alpes naissantes. Le but de cette recherche est de se concentrer sur les sdiments et les fossiles provenant des couches du Miocnes de ces deux provinces marines. Les bassins de la Molasse Alpine du nord, de Vienne et Pannonien taient situs au niveau de la Paratthys Occidentale et Centrale. Les compositions isotopiques de l'oxygne de fossiles phosphats bien prservs dans ces sdiments tayent la thorie d'un dpt sous un climat subtropical tempr chaud avec des tempratures entre 14 et 28C pendant le Miocne. Les valeurs δ18O des fossiles sont similaires la tendance globale jusqu' la fin du Badnien. Cependant les larges fluctuations en δ18O indiquent des effets locaux au niveau des sous bassins. En outre, deux dents de requin exceptionnelles prsentent des valeurs extrmement basses de δ18O. Ces donnes suggrent que ces deux dents se sont formes alors que les requins frquentaient un environnement d'eau douce avec de faibles valeurs de 18O. Le calcul de la composition isotopique de l'oxygne de cette eau douce permet d'obtenir une estimation de la palolvatian moyenne des Alpes du Miocne (~2300m). La tendance 87Sr/86Sr pour ces chantillons concorde approximativement avec un environnement d'ocan ouvert au cours du Miocne. Toutefois un nombre d'chantillons dvie des compositions d'ocan ouvert typiques, avec des rapports levs suggrant des modifications de l'eau de mer par des sources locales et terrestres. Les effets locaux sont aussi reflts au niveau des valeurs en εNd des fossiles paratthysiens. Ceci est cohrent avec un apport d'anciennes roches cristallines et de sdiments msozoques, tandis que d'autres chantillons avec des valeurs hautes de εNd indiquent une influence d'un volcanisme nogne dans le budget marin. En excluant les chantillons dont les compositions isotopiques confirment une influence locale, une valeur moyenne de εNd de 7.9 t 0.5 peut tre dduite pour l'eau de la Parathtys. Cette valeur est semblable la valeur correspondant l'Ocan Indien durant le Miocne, confirmant un rle dominant de cet ocan dans la Paratthys. Au niveau de la Mditerrane, les compositions en isotopes stables du Carbone et de l'Oxygne de foraminifres planctoniques et benthique de la rgion Umbria-Marche prsentent un offset typique leurs habitats. De plus les changements dans leurs compositions suivent les changements globaux, suggrant ainsi que les conditions climatiques rgionales et le cycle du carbone taient contrls par des phnomnes globaux. La composition en isotopes radiogniques d'assemblages fossiles permet une reconnaissance sur trois priodes distinctes. De 25 19 millions d'annes (Ma), des valeurs leves de εNd et un faible rapport 87Sr/86Sr dans les sdiments soutiennent l'ide d'une activit tectonique et volcanique intense, lie l'ouverture de la Mditerrane occidentale. Entre 19 et 13 Ma, la Mditerrane montre des valeurs de εNd qui sont largement contrles par une incursion d'eau provenant de l'Ocan Indien. En effet, aux alentours de 15,2 Ma, des pics dans l'enregistrement des valeurs de εNd, concidant avec des vnements volcaniques et de brves diminutions du niveau marin. Enfin, de 13 7 Ma, les fossiles ont des rapports 7Sr/8fiSr similaires ceux de l'eau de mer au Miocne. Leurs valeurs de εNd indiquent une influence changeante de l'ocan Atlantique, et de l'ocan Indien ou des sources d'eau de merde la Parathtys qui entrent dans les bassins mditerranens. Ce changement est guid par des modifications globales du niveau marin et par la tectonique locale. RSUM DE LA THSE (POUR LE GRAND PUBLIC) Les analyses des compositions en lments traces et isotopiques des fossiles marins sont un outil trs utile pour reconstruire les conditions ocaniques et climatiques anciennes. Ce travail de thse se concentre sur les sdiments dposs dans un environnement marin proches des Alpes au cours du Miocne, entre 23 et 7 millions d'annes (Ma). Cette priode est caractrise par une tectonique alpine active, ainsi que par des changements climatiques et ocanographiques globaux importants. Dans le but de tracer ces changements, les compositions isotopiques du Strontium, du Nodyme, de l'Oxygne et du Carbone ont t analyses dans des fossiles bien prservs ainsi que les sdiments contemporains. Les chantillons proviennent de deux provinces ocaniques distinctes, la premire est la Mer Mditerrane, et l'autre est une mer picontinentale appele Parathtys, qui existait au nord et l'est des Alpes durant le Miocne. Au niveau de la Parathtys Occidentale et Orientale, les compositions isotopiques d'oxygne de dents de requins confirment un dpt sous un climat subtropical tempr chaud avec des tempratures d'eau entre 14 et 28C au Miocne. En outre, deux dents de requins exceptionnelles ont enregistr des compositions isotopiques d'oxygne extrmement basses. Cela suggre que ces deux dents se sont formes alors que les requins entraient dans un systme d'eau douce. Le calcul de la composition isotopique de l'oxygne de cette eau douce permet d'obtenir une estimation de la palolvation des Alpes au Miocne qui est aussi leve que celle d'aujourd'hui. La tendance isotopique du Strontium pour ces chantillons concorde approximativement avec un environnement d'ocan ouvert. Cependant un certain nombre d'chantillons indique des modifications de l'eau de mer par des sources terrestres locales. Les effets locaux sont aussi visibles au niveau des compositions isotopiques du Nodyme, qui sont en accord avec un apport provenant de roches cristallines anciennes et de sdiments du Msozoque, alors que d'autres chantillons indiquent une influence volcanique nogne dans le budget marin. A l'exclusion des chantillons dont les compositions correspondent une influence locale, les compositions isotopiques du Nodyme de la Parathtys sont trs similaires aux valeurs de l'Ocan Indien, montrant ainsi un rle important des masses d'eau IndoPacifiques dans cette rgion. Au niveau de la Mditerrane, les compositions en isotopes stables du Carbone et de l'Oxygne de foraminifres planctoniques et benthique de la rgion Umbria-Marche prsentent un offset typique leurs habitats. De plus, les changements dans leurs compositions suivent les changements globaux, suggrant ainsi que les conditions climatiques rgionales et le cycle du carbone taient contrls par des phnomnes globaux. La composition en isotopes radiogniques d'assemblages fossiles permet une reconnaissance sur trois priodes distinctes. De 25 19 Ma, des rapport isotopiques levs pour le Nodyme et faibles pour le Strontium dans les sdiments et les fossiles soutiennent l'ide d'une activit tectonique et volcanique intense, lie l'ouverture de la Mditerrane occidentale. Entre 19 et 13 Ma, la Mditerrane prsente des rapports isotopiques du Nodyme qui sont largement contrls par une incursion d'eau provenant de l'Ocan Indien. En effet, aux alentours de 15,2 Ma, des pics dans l'enregistrement des valeurs des isotopes du Nodyme concident avec des vnements volcaniques et de brves diminutions du niveau marin. Finalement, de 13 7 Ma, les fossiles ont des rapports isotope Strontium similaires ceux de l'eau de mer au Miocne. Les rapports isotopiques du Nodyme indiquent une influence changeante de l'ocan Atlantique, et de l'ocan Indien ou des sources d'eau de mer de la Parathtys qui entrent dans les bassins mditerranens. Ce changement est guid par des modifications globales du niveau marin et par la tectonique locale.
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The epithelial Na+ channel (ENaC) belongs to a new class of channel proteins called the ENaC/DEG superfamily involved in epithelial Na+ transport, mechanotransduction, and neurotransmission. The role of ENaC in Na+ homeostasis and in the control of blood pressure has been demonstrated recently by the identification of mutations in ENaC beta and gamma subunits causing hypertension. The function of ENaC in Na+ reabsorption depends critically on its ability to discriminate between Na+ and other ions like K+ or Ca2+. ENaC is virtually impermeant to K+ ions, and the molecular basis for its high ionic selectivity is largely unknown. We have identified a conserved Ser residue in the second transmembrane domain of the ENaC alpha subunit (alphaS589), which when mutated allows larger ions such as K+, Rb+, Cs+, and divalent cations to pass through the channel. The relative ion permeability of each of the alphaS589 mutants is related inversely to the ionic radius of the permeant ion, indicating that alphaS589 mutations increase the molecular cutoff of the channel by modifying the pore geometry at the selectivity filter. Proper geometry of the pore is required to tightly accommodate Na+ and Li+ ions and to exclude larger cations. We provide evidence that ENaC discriminates between cations mainly on the basis of their size and the energy of dehydration.
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Rsum La Na,K-ATPase est une protine transmembranaire, prsente dans toutes les cellules de mammifres et indispensable la viabilit cellulaire. Elle permet le maintien des gradients sodiques et potassiques l'origine du potentiel membranaire en transportant 3 Na+ en dehors de la cellule contre 2 K+, grce l'nergie fournie par l'hydrolyse d'une molcule d'ATP. Le potentiel membranaire est indispensable au maintien de l'excitabilit cellulaire et la transmission de l'influx nerveux. Il semblerait que la Na,K-ATPase soit lie l'hypertension et certains troubles neurologiques comme la Migraine Familiale Hmiplgique (1VIFH). La MFH est une forme de migraine avec aura, qui se caractrise par une hmiparsie. Cette forme de migraine est trs rare. Elle se transmet gntiquement sur un mode autosomique dominant. Plusieurs mutations localises dans le gne de la Na,K-ATPase ont t identifies durant ces 3 dernires annes. C'est la premire fois qu'une maladie gntique est associe au gne de la Na,K-ATPase. La comprhension du fonctionnement de cette protine peut donner des informations sur les mcanismes conduisant ces pathologies. On sait que la fonction d'une protine est lie sa structure. L'tude de sa fonction ncessite donc l'tude de sa structure. Alors que la structure de la SERCA a t dtermine haute rsolution, par cristallographie, celle de la Na,K-ATPase ne l'est toujours pas. Mais ces 2 ATPases prsentent une telle homologie qu'un modle de la Na,K-ATPase a pu tre labor partir de la structure de la SERCA. Les objectifs de cette tude sont d'une part, de comprendre le contrle de l'accessibilit du K+ extracellulaire ses sites de liaison. Pour cela, nous avons cibl cette tude sur la 2me et la 31eme boucle extracellulaire, qui relient respectivement les segments transmembranaires (STM) 3-4 et 5-6. Le choix s'est port sur ces 2 boucles car elles bordent le canal des cations forms des 4ime' Sime et 6'me hlices. D'autre part, nous avons galement essayer de comprendre les effets des mutations, lies la Migraine Familiale Hmiplgique de type 2 (MFH2), sur la fonctionnalit 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 prsentent des diffrences. Le modle n'est donc pas une approche fiable pour dterminer la structure et la fonction des rgions extracellulaires. Nous avons alors utilis une approche fonctionnelle faisant appel la mutation dirige puis l'tude de l'activit fonctionnelle de la Na,K ATPase par lectrophysiologie sur des ovocytes de Xenopus. En conclusion, nous pouvons dire que la troisime boucle extracellulaire participerait la structure de la voie d'entre des cations et que la deuxime boucle extracellulaire semble implique dans le contrle de l'accessibilit des ions K+ses sites de liaison. Concernant les mutations associes la MFH2, nos rsultats ont montr une forte diminution de l'activit fonctionnelle de la pompe Na,K, infrieure aux conditions physiologiques de fonctionnement, et pour une des mutations nous avons observs une diminution de l'affmit apparente au K+ externe. Nous poumons faire l'hypothse que l'origine pathologique de la migraine est lie une diminution de l'activit de la pompe 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. Rsum au large publique De la mme manire 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 tres vivants. La fonction d'une cellule dans le corps humain peut se rapprocher de celle d'une usine hydrolectrique. La matire premire apporte est l'eau, l'usine lectrique va ensuite convertir l'eau en nergie hydraulique pour fournir de l'lectricit. Le fonctionnement de base d'une cellule suit le mme processus. La cellule a besoin de matires premires (oxygne, nutriments, eau...) pour produire une nergie sous forme chimique, l'ATP. Cette nergie est utilise par exemple pour contracter les muscles et permet donc l'individu de se dplacer. Morphologiquement la cellule est une sorte de petit sac rempli de liquide (milieu intracellulaire) baignant elle-mme dans le liquide (milieu extracellulaire) composant le corps humain (un adulte est constitu environ de 65 % d'eau). La composition du milieu intracellulaire est diffrente de celle du milieu extracellulaire. Cette diffrence doit tre maintenue pour que l'organisme fonctionne correctement. Une des diffrences majeures est la quantit de sodium. En effet il y a beaucoup plus de sodium l'extrieur qu' l'intrieur de la cellule. Bien que l'intrieur de la cellule soit isol de l'extrieur par une membrane, le sodium arrive passer travers cette membrane, ce qui a tendance augmenter la quantit de sodium dans la cellule et donc diminuer sa diffrence de concentration entre le milieu extracellulaire et le milieu intracellulaire. Mais dans les membranes, il existe des pompes qui tournent et dont le rle est de rejeter le sodium de la cellule. Ces pompes sont des protines connues sous le nom de pompe sodium ou Na,K-ATPase. On lui attribue le nom de Na,K-ATPase car en ralit elle rejette du sodium (Na) et en change elle fait entrer dans la cellule du potassium (K), et pour fonctionner elle a besoin d'nergie (ATP). Lorsque les pompes sodium ne fonctionnent pas bien, cela peut conduire des maladies. En effet la Migraine Familiale Hmiplgique de type 2, est une migraine trs rare qui se caractrise par l'apparition de la paralysie de la moiti d'un corps avant l'apparition du mal de tte. C'est une maladie gntique (altration qui modifie la fonction d'une protine) qui touche la pompe sodium situe dans le cerveau. On a dcouvert que certaines altrations (mutations) empchent les pompes sodium de fonctionner correctement. On pense alors que le dveloppement des migraines est en partie d au fait que ces pompes fonctionnent moins bien. Il est important de bien connatre la fonction de ces pompes car cela permet de comprendre des mcanismes pouvant conduire certaines maladies, comme les migraines. En biologie, la fonction d'une protine est tudie travers sa structure. C'est pourquoi l'objectif de cette thse a t d'tudier la structure de la Na,K-ATPase afin de mieux comprendre son mcanisme d'action.
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Many would argue that the dramatic rise in autism has reached critical mass, and this council echoes that statement. Iowa, like many states in the nation, is currently ill equipped to handle the large influx of children and adults with autism. When this council was initially formed we were facing diagnosis rates of 1 in 150 and currently the diagnosis rate is 1 in 91. Current resource strains in education, qualified trained professionals, access to care, and financial services are rapidly deteriorating Iowas ability to deliver quality services to children, adults, and families affected by autism. If Iowa leadership fails to act quickly the already strained system will face a breaking point in the following areas: financing, coordination of care, educational resources, early identification, adult services, and access to service delivery - just to name a few. This council has taken the past 12 plus months hearing testimony from state officials, providers, and caregivers to ensure that care for those with autism is effective, cost efficient, and accessible. This council will be making recommendations on three major areas; early identification, seamless support/coordination of care, and financing of care. While these areas will be highlighted in this first annual report it in no way minimizes other areas that need to be addressed such as early intervention, special education, training, in-home support services, financing options, and data collection. Implementing the initial recommendations of this council will lay foundational support for the areas mentioned above. Often those in position to help ask what can be done to help families in Iowa. This council has provided a roadmap to help facilitate effective and proven treatments to children and adults with autism.
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The cytokine tumor necrosis factor-alpha (TNFalpha) induces Ca2+-dependent glutamate release from astrocytes via the downstream action of prostaglandin (PG) E2. By this process, astrocytes may participate in intercellular communication and neuromodulation. Acute inflammation in vitro, induced by adding reactive microglia to astrocyte cultures, enhances TNFalpha production and amplifies glutamate release, switching the pathway into a neurodamaging cascade (Bezzi, P., Domercq, M., Brambilla, L., Galli, R., Schols, D., De Clercq, E., Vescovi, A., Bagetta, G., Kollias, G., Meldolesi, J., and Volterra, A. (2001) Nat. Neurosci. 4, 702-710). Because glial inflammation is a component of Alzheimer disease (AD) and TNFalpha is overexpressed in AD brains, we investigated possible alterations of the cytokine-dependent pathway in PDAPP mice, a transgenic model of AD. Glutamate release was measured in acute hippocampal and cerebellar slices from mice at early (4-month-old) and late (12-month-old) disease stages in comparison with age-matched controls. Surprisingly, TNFalpha-evoked glutamate release, normal in 4-month-old PDAPP mice, was dramatically reduced in the hippocampus of 12-month-old animals. This defect correlated with the presence of numerous beta-amyloid deposits and hypertrophic astrocytes. In contrast, release was normal in cerebellum, a region devoid of beta-amyloid deposition and astrocytosis. The Ca2+-dependent process by which TNFalpha evokes glutamate release in acute slices is distinct from synaptic release and displays properties identical to those observed in cultured astrocytes, notably PG dependence. However, prostaglandin E2 induced normal glutamate release responses in 12-month-old PDAPP mice, suggesting that the pathology-associated defect involves the TNFalpha-dependent control of secretion rather than the secretory process itself. Reduced expression of DENN/MADD, a mediator of TNFalpha-PG coupling, might account for the defect. Alteration of this neuromodulatory astrocytic pathway is described here for the first time in relation to Alzheimer disease.
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ATP, released by both neurons and glia, is an important mediator of brain intercellular communication. We find that selective activation of purinergic P2Y1 receptors (P2Y1R) in cultured astrocytes triggers glutamate release. By total internal fluorescence reflection imaging of fluorescence-labeled glutamatergic vesicles, we document that such release occurs by regulated exocytosis. The stimulus-secretion coupling mechanism involves Ca2+ release from internal stores and is controlled by additional transductive events mediated by tumor necrosis factor-alpha (TNFalpha) and prostaglandins (PG). P2Y1R activation induces release of both TNFalpha and PGE2 and blocking either one significantly reduces glutamate release. Accordingly, astrocytes from TNFalpha-deficient (TNF(-/-)) or TNF type 1 receptor-deficient (TNFR1(-/-)) mice display altered P2Y1R-dependent Ca2+ signaling and deficient glutamate release. In mixed hippocampal cultures, the P2Y1R-evoked process occurs in astrocytes but not in neurons or microglia. P2Y1R stimulation induces Ca2+ -dependent glutamate release also from acute hippocampal slices. The process in situ displays characteristics resembling those in cultured astrocytes and is distinctly different from synaptic glutamate release evoked by high K+ stimulation as follows: (a) it is sensitive to cyclooxygenase inhibitors; (b) it is deficient in preparations from TNF(-/-) and TNFR1(-/-) mice; and (c) it is inhibited by the exocytosis blocker bafilomycin A1 with a different time course. No glutamate release is evoked by P2Y1R-dependent stimulation of hippocampal synaptosomes. Taken together, our data identify the coupling of purinergic P2Y1R to glutamate exocytosis and its peculiar TNFalpha- and PG-dependent control, and we strongly suggest that this cascade operates selectively in astrocytes. The identified pathway may play physiological roles in glial-glial and glial-neuronal communication.
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About sixty small water bodies (coastal lagoons, marshes, salt pans, channels, springs, etc.) of the Spanish Mediterranean coast were sampled seasonally for one year (1979-1980), in order to study different aspects of their chemical composition. The concentrations of major ions (alkalinity, Cl-, Ca2+, Mg2+, Na+, and K+), nutrients (N.NO-3, N.NO2-, TRP and Si), oxygen and pH were determined for this purpose. The salt concentrations measured range between 0.4 and 361.3 g l-1. The samples have been divided into four classes of salinity (in g l-1): Cl, S < 5; C2, 5 40. Within these classes, the pattern of ionic dominance recorded is remarkably constant and similar to that found in most coastal lagoons (Cl- > So42- > Alk., for the anions, and Na+ > Mg2+ > Ca2+ > K+, for the cations), although other models occur especially in the first class. The dominance of Na+ and Cl-, as well as the molar ratios Mg2+/Ca2+ and Cl- / SO42- ,clearly increase from class Cl to class C4. The hyperhaline waters include different subtypes of the major brine type"c",, of EUGSTER & HARDIE (1978), the Na+ - (Mg2+) - Cl- - (SO42-) being the most frequent. Nutrient concentrations fall within a wide range (N.NO3 from 0.1 to 1100 mg-at 1-1; PRT from 0.01 to 23.56 mg-at l-1 and Si from 1.0 to 502.0 mg-at l-1). The oxygen values are very variable too, ranging between 0 and 14.4 ml l-1. Four different patterns of nutrient distribution have been distinguished based on the mean concentrations of N.NO3-, and TRP (mean values in mg-at l-1): A, N.NO3- < 10, TRP > l ; B, N.NO3- > 100, TRP < 1; C, 10 < N.NO3- < 100, TRP < 1; C, D, N.NO3- < 10, TRP < 1. As a rule, lagoons of low salinity (C1 and C2 classes) display the nutrient pattern C, and lagoons of high salinity (C3 and C4) show the nutrient pattern D. Model A only appears in waters of very low salinity, whereas model B does not seem to be related to salinity.
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The hippocampal formation is essential for the processing of episodic memories for autobiographical events that happen in unique spatiotemporal contexts. Interestingly, before 2 years of age, children are unable to form or store episodic memories for recall later in life, a phenomenon known as infantile amnesia. From 2 to 7 years of age, there are fewer memories than predicted based on a forgetting function alone, a phenomenon known as childhood amnesia. Here, we discuss the postnatal maturation of the primate hippocampal formation with the goal of characterizing the development of the neurobiological substrates thought to subserve the emergence of episodic memory. Distinct regions, layers and cells of the hippocampal formation exhibit different profiles of structural and molecular development during early postnatal life. The protracted period of neuronal addition and maturation in the dentate gyrus is accompanied by the late maturation of specific layers in different hippocampal regions that are located downstream from the dentate gyrus, particularly CA3. In contrast, distinct layers in several hippocampal regions, particularly CA1, which receive direct projections from the entorhinal cortex, exhibit an early maturation. In addition, hippocampal regions that are more highly interconnected with subcortical structures, including the subiculum, presubiculum, parasubiculum and CA2, mature even earlier. These findings, together with our studies of the development of human spatial memory, support the hypothesis that the differential maturation of distinct hippocampal circuits might underlie the differential emergence of specific "hippocampus-dependent" memory processes, culminating in the emergence of episodic memory concomitant with the maturation of all hippocampal circuits.
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O relevo um importante fator de formao dos solos, condicionando o fluxo de gua na paisagem. Com o objetivo de avaliar a influncia desse fator sobre a distribuio da matria orgnica, analisou-se uma toposseqncia de solos localizada no campus da Universidade Federal Rural do Rio de Janeiro, Seropdica, RJ. Os seis perfis estudados, Podzlico Vermelho-Amarelo (perfis 1 e 2) localizados no tero superior da encosta, Podzlico Amarelo (perfil 3) situado no tero mdio, Planossolo (perfis 4 e 5) no tero inferior e Glei Pouco Hmico (perfil 6) situado ao p da encosta, apresentaram baixos teores de carbono orgnico total. O fluxo de gua condicionou a distribuio das fraes da matria orgnica, principalmente as fraes cidos flvicos livres e cidos flvicos. A matria orgnica apresentou correlao significativa com o valor da cor do solo, com a densidade, porosidade total, contedo de Ca2+, K+, Na+, H+, soma de bases e valor T. A via de humificao identificada nos perfis 1, 2, 3, 4 e 5 foi a da insolubilizao. No perfil 6, situado ao p da encosta, a humificao dos compostos orgnicos ocorre pela lenta transformao dos restos vegetais, caracterizando a via de herana como a principal rota de humificao.
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Rsum : La scrtion de l'insuline en rponse au glucose circulant dans le sang est la fonction principale de la cellule β. La perte de cette fonction est une des caractristiques du diabte de type 2. L'exocytose est une fonction cellulaire indispensable au renouvellement des composants lipidiques et protiques de la membrane cellulaire, la communication entre les cellules et au maintien d'un environnement adquat. On peut distinguer deux types d'exocytose : l'exocytose constitutive et l'exocytose rgule. Cette dernire est dclenche par des stimuli externes. L'exocytose rgule est contrle au niveau de la fusion des vsicules de scrtion avec la membrane plasmique. Certains composants molculaires impliqus dans ce processus font partie de la famille des GTPases Rab. Les deux membres de cette famille impliqus sont Rab3 et Rab27. Nous avons tudi le rle de la GTPase Rab27 dans les cellules INS-1E, une ligne cellulaire pancratique β qui scrte de l'insuline de faon rgule. Nous avons trouv que la diminution d'expression de la protine en utilisant le technique de RNA interference diminue la scrtion stimule, mais que la distribution des granules n'est nullement affectes par ce changement d'activit intrinsque. Un des effecteurs identifis de cette GTPase est Slac2c/MyRIP. Cette protine possde plusieurs domaines fonctionnels dont un qui lui permet de se lier l'actine, constituant du cytosquelette cellulaire. L'ensemble de nos rsultats suggrent que Rab27 et MyRIP font partie d'un complexe permettant l'interaction de la granule de scrtion avec le cytosquelette d'actine corticale et participent la rgulation des dernires tapes de l'exocytose d'insuline. Ensuite, nous avons tudi les phosphoinositides (PI). Les phosphoinositides sont d'importantes molcules impliques dans le rgulation du trafic vsiculaire. Nous avons trouv que le phosphatidylinosito1-4-phosphate (PI4P) et le phosphatidylinositol-4,5-biphosphate (PI(4,5)P2) augmentent la scrtion sous l'action de 10M de Ca2+ dans les cellules INS-1E permabilises avec la streptolysine-O. En plus, nous avons dmontr que l'exocytose est diminue dans les cellules intactes exprimant une protine qui squestre le PI(4,5)P2. Une diminution similaire est observe en diminuant l'expression de deux enzymes impliques dans la production du PI(4,5)P2, la PI4Kinase β type III et la PIP5Kinase γ type I. Pour clarifier le mcanisme d'action des PI, nous avons investigu l'implication de trois cibles potentielles des PI, la PLD1, CAPS1 et Mint1. Pour ce faire, nous avons rduit le niveau d'expression endogne de ces protines, ce qui inhibe la libration d'hormones provoque par le glucose. Tout ceci indique donc que la production du PI(4,5)P2 est ncessaire pour le contrle de la scrtion et suggre qu'une partie de l'effet du PI sur la scrtion pourrait tre exerc par l'activation de la PLD1, CAPS1 et Mint1. Abstract Insulin release from pancreatic β-cells plays an essential role in the achievement of blood glucose homeostasis and defects in the regulation of this process lead to profound metabolic disorders and hyperglycaemia (eg. type 2 diabetes). Almost every cell in our organism releases proteins and other biological compounds using a fundamental cellular process known as constitutive exocytosis. In exocrine and endocrine glands, the cells are endowed with an additional and more refined release mechanism directly tuned by extracellular signals. This process, referred to as regulated exocytosis, ensures the timely delivery of molecules such as peptide hormones and digestive enzymes to match the moment-to-moment requirements of the organism. Some of the molecular components involved in this process have been identified, including Rab3 and Rab27, two GTPases that regulate the final steps of secretion in many cells. We investigated the involvement of Rab27 GTPase in the secretory process of the insulin-secreting cell line INS-1E. We found that selective reduction of Rab27 expression by RNA interference did not alter granule distribution but impaired exocytosis triggered by insulin secretagogues. Screening for potential effectors revealed that Slac2c/MyRIP is associated with granules and attenuation of Slac2c expression severely impaired hormone release. This protein contains several functional domains, including, a binding domain for the cellular cytoskeleton constituent actin. Taken together our data suggest the Rab27 and MyRIP are part of a complex mediating the interaction of secretory granules with cortical cytoskeleton and participate to the regulation of the final steps in insulin exoctytosis. In the second part of the thesis, we studied phosphoinositides (PI). Phosphoinositides are important molecules involved in the regulation of vesicular trafficking. We found that phosphatidylinosito1-4-phosphate (PI4P) and phosphatidylinosito1-4,5-biphosphate (PI(4,5)P2) increase the secretory response triggered by 10M Ca2+ in streptolysin-O permeabilized insulin-secreting INS-1E cells. In addition, nutrient-induced exocytosis was diminished in intact cells expressing constructs that sequester PI(4,5)P2. A similar decrease was observed after silencing of two enzymes involved in PI(4,5)P2 production, type III PI4Kinase β and type I PIP5Kinase γ, by RNA interference. To clarify the mechanism of action of PI, we investigated the involvement in the regulation of exocytosis of three potential PI targets, PLD1, CAPS1 and Mint1. Transfection of cells with silencers capable of reducing the endogenous levels of these proteins inhibited hormone release elicited by glucose. Our data indicate that the production PI(4,5)P2 is necessary for proper control of p-cell secretion and suggest that at least part of the effects of PI on insulin exocytosis could be exerted through the activation of PLD1, CAPS1 and Mint1.
