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Agrégation plaquettaire in vitro : effets anticoagulants du CTAD et utilisation à des fins diagnostiques dans les espèces sensibles / In vitro platelet aggregation : anticoagulant effects of CTAD and its use for diagnostic investigation in sensitive speciesGranat, Fanny 13 April 2016 (has links)
La numération plaquettaire est une analyse délicate et le résultat est souvent erroné notamment du fait d’une tendance à l’agrégation in vitro dans certaines espèces animales. Il a ainsi pu être démontré chez le Chat que ce phénomène peut être inhibé par l’association d’un anticoagulant avec des inhibiteurs plaquettaires : le CTAD (Citrate, Théophylline, Adénosine et Dipyridamole). Cette association permet ainsi l’obtention de numérations plaquettaires fiables sans affecter les autres populations sanguines, mais également d’effectuer des analyses d’hémostase et de biochimie. De nouveaux intervalles de référence ont dû être établis pour certaines variables hématologiques avec les analyseurs utilisés en laboratoire et dans les cliniques vétérinaires. Par ailleurs, si les effets antiagrégants du CTAD sont moins nets chez le Chien, il peut également servir d’anticoagulant « universel », permettant de réduire le nombre de prélèvements et d’améliorer ainsi le bien-être des animaux. / The platelet count is a delicate measurement, which may often be erroneous because of the tendency of platelets from some animal species to aggregate in vitro. This study demonstrated that this effect can be inhibited in cats using CTAD (Citrate, Theophylline, Adenosine and Dipyridamole) composed of an anticoagulant and platelet inhibitors. This association provides reliable platelet counts without affecting other blood populations and also allows hemostasis and biochemical analyses. New hematological reference intervals have been established for some variables with analyzers used in clinical pathology laboratories and veterinary clinics. Furthermore, if the antiplatelet clumping effects of CTAD are less marked in canine species, the CTAD can also serve as "universal" anticoagulant, reducing the number of blood samples and thus improving animal welfare.
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Etude et modélisation de la précipitation de la silice selon le procédé Stöber en phase homogène et en émulsion / Study and modelling of the silica precipitation by the Stöber process in homogeneous phase and in emulsionGabsi-Wolf, Nadia 04 July 2007 (has links)
L’étude de la précipitation de la silice amorphe à partir de TétraEthylOrtoSilicate (TEOS) et d’eau, selon le procédé Stöber, a permis de quantifier la cinétique de réaction par spectroscopie Infra Rouge à Transformée de Fourrier. Nous avons montré que la croissance des particules se fait par agrégation des nuclei, qui sont formés tout au long de la réaction. La transposition de ce procédé en microémulsion eau-dans-huile a permis de synthétiser des nanoparticules de silice de tailles contrôlées. L’hydrolyse du TEOS est ici dix fois plus faible qu’en phase homogène. Lors de l’ajout du TEOS à la microémulsion, on observe une coalescence des gouttelettes accompagnée d’échanges inter-micellaires. Ce phénomène a pu être décrit par un noyau d’agrégation qui dépend des concentrations en tensioactif et en TEOS non hydrolysé. L’étude de ce procédé en macroémulsion a montré l’effet déstabilisateur de l’éthanol et la nécessité de stabiliser l’émulsion pour synthétiser des particules sphériques / The study of the precipitation of amorphous silica starting from Tetraethoxysilane (TEOS) and water according to the Stöber process, made it possible to quantify the kinetics of the reaction by Fourier-Transform Infrared spectroscopy. We demonstrated that the particles grow by aggregation of the nuclei, which are formed throughout the reaction.The transposition of this process in water-in-oil microemulsion made it possible to synthesize silica nanoparticules with controlled sizes. The hydrolysis of the TEOS is here ten times weaker than in homogeneous phase. When we add the TEOS to the microemulsion, we observe a coalescence of the droplets accompanied by inter-micellar exchanges. This coalescence is well described by a core of aggregation which depends on the concentrations of the surfactant and the non-hydrolysed TEOS. The study of this process in macroemulsions showed the destabilizing effect of ethanol and the need for stabilizing the emulsion to synthesize spherical particles
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Alpha-synuclein aggregation: visualization by X-ray techniques and its modulation by ironCarboni, Eleonora 19 October 2016 (has links)
No description available.
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EXPECTATIONS IN MACROECONOMICS: PERSPECTIVES, LABORATORY EXPERIMENTS AND AB MODELSTETTAMANZI, MICHELE 22 December 2017 (has links)
La presente tesi studia le aspettative in macroeconomia contribuendo alla letteratura esistente sia indagando circa il meccanismo di formazione delle aspettative, sia analizzando come le aspettative a razionalità limitata influenzino la dinamica economica.
Nel primo capitolo viene presentato un esperimento nel quale ai soggetti viene chiesto di predire il valore futuro dell'inflazione: a seconda del trattamento, i soggetti possono venire esposti ad un segnale, che mira a stabilizzare l'economia, che fungendo quindi da indicazione prospettica (Forward Guidance). I risultati vengono poi studiati sottolineando il meccanismo di formazione delle aspettative soprattutto in funzione della credibilità del segnale; inoltre viene studiata l'efficacia dello strumento di politica monetaria nella stabilizzazione del sistema economico: si evidenzia come un segnale informativo permetta una sensibile stabilizzazione dell'economia, prevenendo spirali deflazionistiche.
Nel secondo capitolo viene sviluppato un modello ad agenti il quale incorpora un meccanismo di formazione delle aspettative a razionalità limitata, derivato da esperimenti precedenti. Inoltre, grazie ad un peculiare processo di aggregazione, viene derivato un modello analiticamente trattabile che permette di studiare il meccanismo di trasmissione di uno shock, isolando gli effetti dovuti all'eterogeneità fra gli agenti e alle aspettative: entrambi gli effetti sono considerevoli ed aiutano nello spiegare la dinamica economica. / The present dissertation analyses expectations in macroeconomics, contributing to the existing literature both studying the expectation formation process, and inquiring how economic dynamic is influenced by boundedly rational expectations.
The first chapter presents a learn to forecast experiment in which subject are asked to form expectation regarding the future value of inflation: depending on the treatment, subjects might be exposed to a signal, which possibly aim at stabilizing economy, mimicking the non conventional monetary policy instrument called Delphic Forward Guidance. The collected data are studied trying to recover the underlying expectation formation process highlighting especially the role of credibility of the signal; moreover from the data emerges that informative Forward Guidance helps in stabilizing economy, drastically reducing the probability of deflationary spirals.
The second chapter develops an agent-based model, encapsulating a boundedly rational expectation formation process, which had been extrapolated in previous experiments. Moreover benefiting from a specific aggregation procedure, we derive a model characterized by high analytical tractability, allowing hence to study the transmission mechanisms of a shock by insulating the effects due to the heterogeneity among agents and due to expectations: both the effects are sizable and help in understanding the dynamics of the economic system.
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Les usages sociaux et les logiques économiques de l'audiovisuel sur smartphone / Social uses and business logics of audiovisual media on smartphoneSonet, Virginie 14 November 2014 (has links)
L’audiovisuel sur smartphone est un phénomène complexe et en construction. La mobilité et l’hybridation caractérisent ce nouveau territoire audiovisuel et cette thèse cherche à comprendre en quoi ces deux dimensions contribuent à la construction des usages sociaux et des logiques économiques de l’audiovisuel sur smartphone. À partir de plusieurs enquêtes qualitatives et d’une observation des offres des chaînes sur ce nouvel écran, ce travail analyse comment les utilisateurs et les acteurs de l’audiovisuel particulièrement les chaînes de télévision se saisissent de ce nouvel écran.Ce territoire audiovisuel est d’abord contextualisé, puis une lecture originale de l’évolution de l’audiovisuel est présentée, au prisme de disruptions d’ordre technologique, économique et d’usage. La construction de la posture scientifique reposant sur une approche interdisciplinaire est ensuite exposée. Dès lors, cette thèse met en évidence que la mobilité participe de l’appropriation du smartphone en tant qu’écran audiovisuel, notamment à travers les dimensions de contexte et de contraintes technologiques, commerciales, sociales. Quant au déploiement des modèles économiques des chaînes de télévision sur cet écran, il est contraint par un environnement technoéconomique imposé par les plateformes mobiles (comme Apple et Google). Avec la dimension d’hybridation, la thèse montre que les usages se construisent dans une logique d’entrelacement des pratiques audiovisuelles et des pratiques de communication et de connexion. Les chaînes de télévision réagissent en proposant des applications enrichies, en se déployant dans les réseaux socionumériques et en développant des dispositifs interactifs entre le smartphone et le téléviseur. Autant de nouvelles modalités d’agrégation susceptibles de drainer des implications économiques défavorables aux chaînes. / Watching and delivering video contents on smartphone is a complex phenomenon because it is forming as we investigate it. Both dimensions of mobility and hybridization characterize this new media territory and this thesis explains how their contribution leads to the construction of the social uses and the economic logics. Through several qualitative surveys based on interviews with users and professionals and a long term observation of the TV offers on this new screen, this research analyzes how the users on one hand and the broadcasting industry (TV networks) on the other hand, seize this new screen.This new audiovisual field is put into perspective by drawing up its offers overview and the first observed uses. An original reading of the audiovisual field evolution with the prism of technological, economical and use disruptions is then presented, and the construction of our scientific position is described, by explaining the interdisciplinary approach.Therefore, this research highlights that the mobility contributes to the appropriation of the smartphone as an audiovisual screen, essentially through the dimensions of context, as well as technological, commercial and social constraints. It also analyzes how the techno-economic environment, generated by the mobile platforms (Apple and Google), constrains the deployment of French TV networks’ business models on this new screen. With the dimension of hybridization, this thesis explains that the uses are expanding through the interlacing of communication, connection and audiovisual uses. Therefore, Television Networks try to conquer the attention of smartphone users by providing enhanced applications, by spreading in Social Networking Sites and by developing interactive systems between the smartphone and theTV set. But, these new aggregations often come with adverse economic implications.
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Compréhension des mécanismes synergiques pour l'extraction de l'uranium des mines de phosphates / Comprehension of synergistic mechanisms for uranium extraction from phosphate minesPecheur, Olivia 06 November 2014 (has links)
Le procédé hydrométallurgique le plus utilisé pour extraire l'uranium VI des minerais phosphoriques met en jeu un mélange synergique de deux molécules extractantes : HDEHP et TOPO. Si les combinaisons synergiques sont connues dans le domaine de l'extraction liquide/liquide, les mécanismes qui pilotent la synergie d'extraction sont encore mal décrits. Une approche multi-échelle a été utilisée pour décrire ces mécanismes, combinant deux descriptions qui diffèrent par leur centre d'intérêt, respectivement l'ion pour l'approche moléculaire et les agrégats d'extractants pour l'approche supramoléculaire. Ces deux approches ont parallèlement été rationnalisées par des calculs de dynamique moléculaire. La description obtenue permet de rendre compte de la synergie via la structure des complexes et des agrégats formés pour différents ratios HDEHP/TOPO. De la même façon, des composés bifonctionnels, qui combinent deux fonctions extractantes au sein de la même molécule ont été étudiés et comparés au système HDEHP/TOPO pour identifier l'origine de l'augmentation des performances d'extraction et de sélectivité. / Uranium VI is commonly extracted from phosphoric ores by a well-known process exploiting the synergistic mixture of two extractant molecules : HDEHP and TOPO. In the field of liquid-liquid extraction, synergistic combinations are common but the mechanisms at the origin of the synergy are not well understood. A multi-scale approach has been used to describe these mechanisms, combining two different descriptions : the molecular scale focuses on the ion point of view, while the supramolecular scale focuses on extractants' aggregation. These two approaches have been rationalized by molecular dynamics computations. The results allow describing the synergy through the structure of the complexes and aggregates. With the same approach, some bifunctional compounds, combining the two extracting sites in one molecule, have been studied and compared to the HDEHP/TOPO system in order to identify the origin of their increased capacities in extraction and selectivity.
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Controlling the morphology of nanoparticle-polymer composite films for potential use in solar cellsRhodes, Rhys William January 2011 (has links)
This thesis presents an investigation into the factors affecting the morphology of hybrid inorganic/organic photoactive layers used in photovoltaic cells. Although optimisation of the organic (polymer) phase has received substantial attention, research into the morphology of the inorganic phase (semiconducting nanocrystals) remains limited. It is believed that there is a strong link between the morphology of the final photoactive film and the quality of the initial nanocrystal dispersion. To this end, two nanocrystal systems were investigated; zinc oxide (ZnO) and lead sulphide (PbS). ZnO nanocrystals were synthesised and found to possess reproducible characteristics. It was determined that colloid stability was initially dependent upon the presence of acetate groups bound to the surface, which in turn required a small quantity of methanol to be present in the organic dispersant. It was also discovered that while methanol evaporated readily from the surface of the nanocrystals, another molecule, 1-propylamine (1-PA), did not. Further investigations showed that while methanol only weakly physisorbed to the surface of ZnO nanocrystals, 1-PA formed strong, dative covalent bonds with Zn2+, preventing evaporation despite a low boiling point. Subsequent investigations into the effects of different ligands upon colloid stability found that amine-based groups typically possessed superior stabilising capabilities compared to alcohol-based analogues. The characteristics of nanocrystal / polymer blends were also investigated. It was determined that the nanocrystal dispersion became unstable at higher concentrations of polymer due to depletion aggregation. Films of nanocrystal / polymer blends were cast from dispersions containing either alcohol or amine-based ligands, and it was observed that dispersions stabilised with 1-PA possessed smooth morphologies on the micrometer scale. Investigations at the nanometer scale, however, revealed aggregates large enough to favour recombination.The latter half of this thesis regards the characterisation of PbS nanocrystals and investigations into triggered aggregation. It was determined that while PbS nanocrystals possessed reproducible characteristics, the stabilising molecule, oleic acid (OA) was insulating. The effects of exchanging the OA groups for a shorter ligand, butylamine (BA) were investigated.Finally, PbS nanocrystals were treated with a bidentate ligand, 1,2-ethanedithiol (EDT) to induce triggered aggregation. It was observed that the system was highly sensitive to the concentration of EDT in dispersion, forming small, relatively dispersed aggregates at low [EDT], and micrometer-sized crystalline structures at high [EDT]. The characterisation and entrapment of these nanocrystal structures within semi-conducting polymer films is also discussed.
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Redundance v datových sítích / Redundancy in data networksŠoun, Jan January 2010 (has links)
This thesis focuses on redundancy in data networks and on technologies which are used to achieve high availability in network infrastructure. This thesis is based on industry standards published by well-known standardization authorities. One proprietary technology which can effectively replace these standards and simplify the whole network is also presented in more detail. The theoretical part individually describes all such technologies and standards. The practical part deals with an appropriate combination of these technologies and their application in a specific product environment. This work is meant to describe the whole concept of high availability in network infrastructure and should be instrumental in understanding how these technologies are deployed in practice.
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A cross-layer approach for optimizing the efficiency of wireless sensor and actor networksKohlmeyer, Eckhard Bernhard 25 June 2009 (has links)
Recent development has lead to the emergence of distributed Wireless Sensor and Actor Networks (WSAN), which are capable of observing the physical environment, processing the data, making decisions based on the observations and performing appropriate actions. WSANs represent an important extension of Wireless Sensor Networks (WSNs) and may comprise a large number of sensor nodes and a smaller number of actor nodes. The sensor nodes are low-cost, low energy, battery powered devices with restricted sensing, computational and wireless communication capabilities. Actor nodes are resource richer with superior processing capabilities, higher transmission powers and a longer battery life. A basic operational scenario of a typical WSAN application follows the following sequence of events. The physical environment is periodically sensed and evaluated by the sensor nodes. The sensed data is then routed towards an actor node. Upon receiving sensed data, an actor node performs an action upon the physical environment if necessary, i.e. if the occurrence of a disturbance or critical event has been detected. The specific characteristics of sensor and actor nodes combined with some stringent application constraints impose unique requirements for WSANs. The fundamental challenges for WSANs are to achieve low latency, high energy efficiency and high reliability. The latency and energy efficiency requirements are in a trade-off relationship. The communication and coordination inside WSANs is managed via a Communication Protocol Stack (CPS) situated on every node. The requirements of low latency and energy efficiency have to be addressed at every layer of the CPS to ensure overall feasibility of the WSAN. Therefore, careful design of protocol layers in the CPS is crucial in attempting to meet the unique requirements and handle the abovementioned trade-off relationship in WSANs. The traditional CPS, comprising the application, network, medium access control and physical layer, is a layered protocol stack with every layer, a predefined functional entity. However, it has been found that for similar types of networks with similar stringent network requirements, the strictly layered protocol stack approach performs at a sub-optimal level with regards to network efficiency. A modern cross-layer paradigm, which proposes the employment of interactions between layers in the CPS, has recently attracted a lot of attention. The cross-layer approach promotes network efficiency optimization and promises considerable performance gains. It is found that in literature, the adoption of this cross-layer paradigm has not yet been considered for WSANs. In this dissertation, a complete cross-layer enabled WSAN CPS is developed that features the adoption of the cross-layer paradigm towards promoting optimization of the network efficiency. The newly proposed cross-layer enabled CPS entails protocols that incorporate information from other layers into their local decisions. Every protocol layer provides information identified as beneficial to another layer(s) in the CPS via a newly proposed Simple Cross-Layer Framework (SCLF) for WSANs. The proposed complete cross-layer enabled WSAN CPS comprises a Cross-Layer enabled Network-Centric Actuation Control with Data Prioritization (CL-NCAC-DP) application layer (APPL) protocol, a Cross-Layer enabled Cluster-based Hierarchical Energy/Latency-Aware Geographic Routing (CL-CHELAGR) network layer (NETL) protocol and a Cross-Layer enabled Carrier Sense Multiple Access with Minimum Preamble Sampling and Duty Cycle Doubling (CL-CSMA-MPS-DCD) medium access control layer (MACL) protocol. Each of these protocols builds on an existing simple layered protocol that was chosen as a basis for development of the cross-layer enabled protocols. It was found that existing protocols focus primarily on energy efficiency to ensure maximum network lifetime. However, most WSAN applications require latency minimization to be considered with the same importance. The cross-layer paradigm provides means of facilitating the optimization of both latency and energy efficiency. Specifically, a solution to the latency versus energy trade-off is given in this dissertation. The data generated by sensor nodes is prioritised by the APPL and depending on the delay-sensitivity, handled in a specialised manor by every layer of the CPS. Delay-sensitive data packets are handled in order to achieve minimum latency. On the other hand, delay-insensitive non critical data packets are handled in such a way as to achieve the highest energy efficiency. In effect, either latency minimization or energy efficiency receives an elevated precedence according to the type of data that is to be handled. Specifically, the cross-layer enabled APPL protocol provides information pertaining to the delay-sensitivity of sensed data packets to the other layers. Consequently, when a data packet is detected as highly delay-sensitive, the cross-layer enabled NETL protocol changes its approach from energy efficient routing along the maximum residual energy path to routing along the fastest path towards the cluster-head actor node for latency minimizing of the specific packet. This is done by considering information (contained in the SCLF neighbourhood table) from the MACL that entails wakeup schedules and channel utilization at neighbour nodes. Among the added criteria, the next-hop node is primarily chosen based on the shortest time to wakeup. The cross-layer enabled MACL in turn employs a priority queue and a temporary duty cycle doubling feature to enable rapid relaying of delay-sensitive data. Duty cycle doubling is employed whenever a sensor node’s APPL state indicates that it is part of a critical event reporting route. When the APPL protocol state (found in the SCLF information pool) indicates that the node is not part of the critical event reporting route anymore, the MACL reverts back to promoting energy efficiency by disengaging duty cycle doubling and re-employing a combination of a very low duty cycle and preamble sampling. The APPL protocol conversely considers the current queue size of the MACL and temporarily halts the creation of data packets (only if the sensed value is non critical) to prevent a queue overflow and ease congestion at the MACL By simulation it was shown that the cross-layer enabled WSAN CPS consistently outperforms the layered CPS for various network conditions. The average end-to-end latency of delay-sensitive critical data packets is decreased substantially. Furthermore, the average end-to-end latency of delay-insensitive data packets is also decreased. Finally, the energy efficiency performance is decreased by a tolerable insignificant minor margin as expected. The trivial increase in energy consumption is overshadowed by the high margin of increase in latency performance for delay-sensitive critical data packets. The newly proposed cross-layer CPS achieves an immense latency performance increase for WSANs, while maintaining excellent energy efficiency. It has hence been shown that the adoption of the cross-layer paradigm by the WSAN CPS proves hugely beneficial with regards to the network efficiency performance. This increases the feasibility of WSANs and promotes its application in more areas. / Dissertation (MEng)--University of Pretoria, 2009. / Electrical, Electronic and Computer Engineering / unrestricted
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Efeito do fator de necrose tumoral alfa na agregação plaquetária / Effect of tumor necrosis factor alpha on platelet aggregationBonfitto, Pedro Henrique Leite, 1987- 26 August 2018 (has links)
Orientador: Sisi Marcondes Paschoal / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T18:31:06Z (GMT). No. of bitstreams: 1
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Previous issue date: 2015 / Resumo: As plaquetas são importantes células na inflamação, entretanto, os trabalhos que estudam as citocinas na reatividade plaquetária são raros. O objetivo do presente trabalho foi estudar os efeitos do fator de necrose tumoral-alfa (TNF-?) em plaquetas. Ensaios de agregação foram realizados incubando-se plaquetas com crescentes concentrações de TNF-? (1 - 3000 pg/ml) por diferentes intervalos de tempo (5 - 60 min), na ausência ou presença do antagonista não seletivo dos receptores TNFR1 e TNFR2, o R7050. Também foi estudado o efeito do TNF-? na viabilidade plaquetária utilizando-se o MTT. O efeito do TNF-? na mobilização de Ca2+ em plaquetas foi investigado através de ensaios de fluorescência utilizando-se o fluo-3-AM; os ensaios de western blotting foram realizados para o estudo da ativação da enzima c-Src e do receptor de fibrinogênio. Finalmente, foram determinados os níveis intraplaquetários de AMPc e GMPc por ELISA. O TNF-? inibiu a agregação plaquetária induzida por ADP ou trombina de forma dependente da concentração da citocina e do tempo de incubação. O efeito inibitório máximo do TNF-? na agregação induzida por ADP (5 ?M) foi obtido com a concentração de 300 pg/ml por um tempo de incubação de 30 min (90 ± 7% de inibição), o qual foi significativamente prevenido pela pré-incubação das plaquetas com o R7050. A viabilidade plaquetária não foi modificada pela incubação por 60 min com o TNF-? (30 e 3000 pg/ml). A incubação de plaquetas com TNF-? (300 pg/ml, 30 min) reduziu em 53% o aumento da concentração de Ca2+ total causado pela adição de trombina (200 mU/ml). A queda da concentração de Ca2+ citosólica plaquetária causada pelo TNF-? foi em decorrência da diminuição em 1,8 e 3,4 vezes da mobilização interna do íon e do influxo do mesmo, respectivamente. O TNF-? reduziu em 60% os níveis de AMPc em plaquetas ativadas com ADP. Por outro lado, o TNF-? aumentou significativamente os níveis de GMPc em plaquetas ativadas por ADP (aumento de 51%). A pré-incubação de plaquetas com o inibidor da guanilil ciclase ODQ não reduziu o efeito inibitório do TNF-? na agregação induzida por ADP. Os ensaios de western blotting mostraram que o TNF-? reduziu significativamente a fosforilação do resíduo de Tyr416 da c-Src em plaquetas ativadas. Da mesma forma, o TNF-? reduziu em 37% a fosforilação do resíduo de Tyr773 da subunidade ?3 da integrina ?IIb?3 (receptor de fibrinogênio) em plaquetas ativadas por ADP. Portanto concluímos que o TNF-? inibe a agregação plaquetária via receptores TNFR1 e/ou TNFR2, sem reduzir a viabilidade das plaquetas. O efeito inibitório do TNF-? na agregação é acompanhado pela redução de Ca2+ citosólico e inibição de c-Src e do receptor de fibrinogênio em plaquetas, sendo estes independentes de AMPc ou GMPc / Abstract: Platelets have been described as important cells in inflammation; however, the effects of cytokines on platelet reactivity are rarely studied. The objective of the present work was to investigate the effects of the tumor necrosis factor-alpha (TNF-?) in platelets. Aggregation assays were carried out incubating platelets with increasing TNF-? concentrations (1 - 3000 pg/ml) for different intervals of times (5 - 60 min), in the absence or in presence of the non-selective antagonist of TNFR1 and TNFR2, R7050. Effect of TNF-? on platelet viability was determined using MTT. The effect of TNF-? on the Ca2+ mobilization in platelets was investigated through fluorescence assays using fluo-3AM and Western blotting assays were carried out to determine the activation of c-Src and the fibrinogen receptor. Finally, the cAMP and cGMP levels in platelets were determined by ELISA. TNF-? dose- and time-dependently inhibited ADP or thrombin-induced platelet aggregation. The inhibitory effect of TNF-? on ADP(5 ?M)-induced platelet aggregation was maximum in a concentration of 300 pg/ml incubated with platelets for 30 min (90 ± 7% of inhibition), which was significantly prevented by the incubation of platelets with R7050. Platelet viability was not modified by TNF-? (30 and 3000 pg/ml) incubated for 5 to 60 min. Incubation of platelets with TNF-? (300 pg/ml, 30 min) reduced the increased total Ca2+ concentration induced by thrombin (200 mU/ml) by 53%. Decreasing Ca2+ internal mobilization (1,8 fold) and decreasing in external Ca2+ influx (3,4 fold) led to a reduction of total cytosolic Ca2+ in TNF-? activated platelets. TNF-? reduced the cAMP levels in ADP-activated platelets by 60%. On the other hand, TNF-? significantly increased cGMP levels in ADP-activated platelets (51% increase). Pre-incubation of platelets with the guanylyl cyclase inhibitor ODQ did not modify the inhibitory effect of TNF-? on ADP-induced platelet aggregation. Western blotting analysis showed that TNF-? significantly reduced phosphorylation on Tyr416 of c-Src in activated platelets. Similarly, TNF-? reduced by 37% the Tyr773 phosphorylation of ?3 subunit of ?IIb?3 integrin (fibrinogen receptor) in ADP-activated platelets. Therefore, our results show that TNF-? inhibits platelet aggregation via TNFR1 and/or TNFR2 receptors, without affecting platelet viability. The inhibitory effect of TNF-? on aggregation is accompanied by a reduction in cytosolic Ca2+ and the inhibition of c-Src and fibrinogen receptor activation, which are cAMP and cGMP-independent effects / Mestrado / Farmacologia / Mestre em Farmacologia
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