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The vertebrate neuromuscular junction (NMJ) is a specialised chemical synapse involved in the transmission of bioelectric signals between a motor neuron and a skeletal muscle fiber, leading to muscle contraction. Typically, the NMJ is a tripartite synapse comprising (a) a presynaptic region represented by the motor nerve ending, (b) a postsynaptic skeletal motor endplate area, and (c) perisynaptic Schwann cells (PSCs) that shield the motor nerve terminal. Increasing evidence points towards the role of PSCs in the maintenance and control of neuromuscular integrity, transmission, and plasticity. Acetylcholine (ACh) is the main neurotransmitter at the vertebrate skeletal NMJ, and its role is fine-tuned by co-released purinergic neuromodulators, like adenosine 5′-triphosphate (ATP) and its metabolite adenosine (ADO). Adenine nucleotides modulate transmitter release and expression of postsynaptic ACh receptors at motor synapses via the activation of P2Y and P2X receptors. Endogenously generated ADO modulates ACh release by acting via co-localised inhibitory A1 and facilitatory A2A receptors on motor nerve terminals, whose tonic activation depends on the neuronal firing pattern and their interplay with cholinergic receptors and neuropeptides. Thus, the concerted action of adenine nucleotides, ADO, and ACh/neuropeptide co-transmitters is paramount to adapting the neuromuscular transmission to the working load under pathological conditions, like Myasthenia gravis. Unravelling these functional complexities prompted us to review our knowledge about the way purines orchestrate neuromuscular transmission and plasticity in light of the tripartite synapse concept, emphasising the often-forgotten role of PSCs in this context.

Background Endogenously released adenine and uracil nucleotides favour the osteogenic commitment of bone marrow-derived mesenchymal stromal cells (BM-MSCs) through the activation of ATP-sensitive P2X7 and UDP-sensitive P2Y6 receptors. Yet, these nucleotides have their osteogenic potential compromised in post-menopausal (Pm) women due to overexpression of nucleotide metabolizing enzymes, namely NTPDase3. This prompted us to investigate whether NTPDase3 gene silencing or inhibition of its enzymatic activity could rehabilitate the osteogenic potential of Pm BM-MSCs. Methods MSCs were harvested from the bone marrow of Pm women (69 ± 2 years old) and younger female controls (22 ± 4 years old). The cells were allowed to grow for 35 days in an osteogenic-inducing medium in either the absence or the presence of NTPDase3 inhibitors (PSB 06126 and hN3-B3s antibody); pre-treatment with a lentiviral short hairpin RNA (Lenti-shRNA) was used to silence the NTPDase3 gene expression. Immunofluorescence confocal microscopy was used to monitor protein cell densities. The osteogenic commitment of BM-MSCs was assessed by increases in the alkaline phosphatase (ALP) activity. The amount of the osteogenic transcription factor Osterix and the alizarin red-stained bone nodule formation. ATP was measured with the luciferin-luciferase bioluminescence assay. The kinetics of the extracellular ATP (100 µM) and UDP (100 µM) catabolism was assessed by HPLC Results The extracellular catabolism of ATP and UDP was faster in BM-MSCs from Pm women compared to younger females. The immunoreactivity against NTPDase3 increased 5.6-fold in BM-MSCs from Pm women vs. younger females. Selective inhibition or transient NTPDase3 gene silencing increased the extracellular accumulation of adenine and uracil nucleotides in cultured Pm BM-MSCs. Downregulation of NTPDase3 expression or activity rehabilitated the osteogenic commitment of Pm BM-MSCs measured as increases in ALP activity, Osterix protein cellular content and bone nodule formation; blockage of P2X7 and P2Y6 purinoceptors prevented this effect. Conclusions Data suggest that NTPDase3 overexpression in BM-MSCs may be a clinical surrogate of the osteogenic differentiation impairment in Pm women. Thus, besides P2X7 and P2Y6 receptors activation, targeting NTPDase3 may represent a novel therapeutic strategy to increase bone mass and reduce the osteoporotic risk of fractures in Pm women.

In this paper we present a methodology for optimising the design of a metamaterial structure with one degree of freedom that is able to simultaneously bend and stretch. The structure is intended for assisting flexion-extension of the wrist joint. The metamaterial is comprised of serially connected, in- dividually designed cells. The design parameters can be chosen to optimally fit a desired planar curve such as the curvature of the skin on a plane normal to the flexion/extension axis of the wrist joint. A tool for the optimised design is described and the experimental validation of the output is conducted, to show the ability of the mechanism to conform to a 2D curve, also exhibiting a change in length, which is desirable for reducing sliding and shear on the skin. The design tool allows the generation of metamaterials optimised for a multitude of other applications where actuated mechanisms must be worn by a user for rehabilitation or assistance purposes. Agency for Science, Technology and Research (A*STAR) Nanyang Technological University Accepted version This research was partially supported by the Singapore Agency for Science, Technology and Research (A*STAR) under the project ”Intelligent Human-Robot interface for upper limb wearable robots” (Award Number SERC 1922500046) and by NTU SUG grant - ”Building blocks for next generation soft robots”.

In this paper, we present the modelling of a hyper-redundant mechanism for assisting fingers in achieving desired configurations. The device is composed of serially linked rotating rigid elements and is actuated by a cable-driven mechanism that can provide both flexion and extension. The modelling is based on the analysis of the static equilibrium configurations that the structure acquires, which are dependent on the design parameters of each segment and on the ratio of the tensions applied on the cables. We show how it is possible to obtain different configurations by properly shaping the geometry of each element. Such a relationship was theoretically investigated and experimentally validated to compare the actual configuration with the one predicted by the model. The prototype was designed so that its static equilibrium configuration approximates the geometry of a finger during flexion-extension. It was observed that the customised structure achieves a configuration more closely resembling the desired one than a non-customised actuator. By adapting the design of each element and properly adjusting the ratio of tensions, one is able to compute the final configuration achieved by the actuator so that it can approximate the desired posture.

Background/Aims: In this study, we evaluated the functional impact of facilitatory presynaptic adenosine A2A and muscarinic M1 receptors in the recovery of neuromuscular tetanic depression caused by the blockage of high-affinity choline transporter (HChT) by hemicholinium-3 (HC-3), a condition that mimics a myasthenia-like condition. Methods: Rat diaphragm preparations were indirectly stimulated via the phrenic nerve trunk with 50-Hz frequency trains, each consisting of 500–750 supramaximal intensity pulses. The tension at the beginning (A) and at the end (B) of the tetanus was recorded and the ratio (R) B/A calculated. Results: Activation of A2A and M1 receptors with CGS21680 (CGS; 2 nmol/L) and McN-A-343c (McN; 3 μmol/L) increased R values. Similar facilitatory effects were obtained with forskolin (FSK; 3 μmol/L) and phorbol 12-myristate 13-acetate (PMA; 10 μmol/L), which activate adenylate cyclase and protein kinase C respectively. HC-3 (4 μmol/L) decreased transmitter exocytosis measured by real-time videomicroscopy with the FM4-64 fluorescent dye and prevented the facilitation of neuromuscular transmission caused by CGS, McN, and FSK, with a minor effect on PMA. The acetylcholinesterase inhibitor, neostigmine (NEO; 0.5 μmol/L), also decreased transmitter exocytosis. The paradoxical neuromuscular tetanic fade caused by NEO (0.5 μmol/L) was also prevented by HC-3 (4 μmol/L) and might result from the rundown of the positive feedback mechanism operated by neuronal nicotinic receptors (blocked by hexamethonium, 120 μmol/L). Conclusion: Data suggest that the recovery of tetanic neuromuscular facilitation by adenosine A2A and M1 receptors is highly dependent on HChT activity and may be weakened in myasthenic patients when HChT is inoperative.

Acetylcholine (ACh) spillover from motor endplates occurs after neuronal firing bursts being potentiated by cholinesterase inhibitors (e.g., neostigmine). Nicotinic α7 receptors (α7nAChR) on perisynaptic Schwann cells (PSCs) can control ACh spillover by unknown mechanisms. We hypothesized that adenosine might be the gliotransmitter underlying PSCs-nerve terminal communication. Rat isolated hemidiaphragm preparations were used to measure (1) the outflow of [3 H]ACh, (2) real-time transmitter exocytosis by video-microscopy with the FM4-64 fluorescent dye, and (3) skeletal muscle contractions during high-frequency (50 Hz) nerve stimulation bursts in the presence of a selective α7nAChR agonist, PNU 282987, or upon inhibition of cholinesterase activity with neostigmine. To confirm our prediction that α7nAChR-mediated effects require direct activation of PSCs, we used fluorescence video-microscopy in the real-time mode to measure PNU 282987-induced [Ca2+ ]i transients from Fluo-4 NW loaded PSCs in non-stimulated preparations. The α7nAChR agonist, PNU 282987, decreased nerve-evoked diaphragm tetanic contractions. PNU 282987-induced inhibition was mimicked by neostigmine and results from the reduction of ACh exocytosis measured as decreases in [3 H]ACh release and FM4-64 fluorescent dye unloading. Methyllycaconitine blockage of α7nAChR and the fluoroacetate gliotoxin both prevented inhibition of nerve-evoked ACh release and PSCs [Ca2+ ]i transients triggered by PNU 282987 and neostigmine. Adenosine deamination, inhibition of the ENT1 nucleoside outflow, and blockage of A1 receptors prevented PNU 282987-induced inhibition of transmitter release. Data suggest that α7nAChR controls tetanic-induced ACh spillover from the neuromuscular synapse by promoting adenosine outflow from PSCs via ENT1 transporters and retrograde activation of presynaptic A1 inhibitory receptors.

Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into bone forming cells. Such ability is compromised in elderly individuals resulting in bone disorders such as osteoporosis, also limiting their clinical usage for cell transplantation and bone tissue engineering strategies. In bone marrow niches, adenine and uracil nucleotides are important local regulators of osteogenic differentiation of MSCs. Nucleotides can be released to the extracellular milieu under both physiological and pathological conditions via (1) membrane cell damage, (2) vesicle exocytosis, (3) ATP-binding cassette transporters, and/or (4) facilitated diffusion through maxi-anion channels, hemichannels or ligand-gated receptor pores. Nucleotides and their derivatives act via adenosine P1 (A1 , A2A , A2B , and A3 ) and nucleotide-sensitive P2 purinoceptors comprising ionotropic P2X and G-protein-coupled P2Y receptors. Purinoceptors activation is terminated by membrane-bound ecto-nucleotidases and other ecto-phosphatases, which rapidly hydrolyse extracellular nucleotides to their respective nucleoside 5'-di- and mono-phosphates, nucleosides and free phosphates, or pyrophosphates. Current knowledge suggests that different players of the "purinome" cascade, namely nucleotide release sites, ecto-nucleotidases and purinoceptors, orchestrate to fine-tuning regulate the activity of MSCs in the bone microenvironment. Increasing studies, using osteoprogenitor cell lines, animal models and, more recently, non-modified MSCs from postmenopausal women, raised the possibility to target chief components of the purinergic signaling pathway to regenerate the ability of aged MSCs to differentiate into functional osteoblasts. This review summarizes the main findings of those studies, prompting for novel therapeutic strategies to control ageing disorders where bone destruction exceeds bone formation, like osteoporosis, rheumatoid arthritis, and fracture mal-union. J. Cell. Physiol. 231: 1852-1861, 2016. © 2016 Wiley Periodicals, Inc.

Obesity is a worldwide health problem which have reached pandemic proportions, now also including low and middle-income countries. Excessive or abnormal fat deposition in the abdomen especially in the visceral compartment is tightly associated with a high metabolic risk for arterial hypertension, type II diabetes, cardiovascular diseases, musculoskeletal disorders (especially articular degeneration) and some cancers. Contrariwise, accumulation of fat in the subcutaneous compartment has been associated with a neutral metabolic impact, favoring a lower risk of insulin resistance. Obesity results more often from an avoidable imbalance between food consumption and energy expenditure. There are several recommended strategies for dealing with obesity, including pharmacological therapies, but their success remains incomplete and may not compensate the associated adverse effects. Purinergic signaling operated by ATP and its metabolite, adenosine, has attracted increasing attention in obesity. The extracellular levels of purines often reflect the energy status of a given cell population. Adenine nucleotides and nucleosides fine tuning control adipogenesis and mature adipocytes function via the activation of P2 and P1 purinoceptors, respectively. These features make the purinergic signaling cascade a putative target for therapeutic intervention in obesity and related metabolic syndromes. There are, however, gaps in our knowledge regarding the role of purines in adipocyte precursors differentiation and mature adipocytes functions, as well as their impact among distinct adipose tissue deposits (e.g. white vs. brown, visceral vs. subcutaneous), which warrants further investigations before translation to clinical trials can be made.

The mechanisms underlying improvement of neuromuscular transmission deficits by glucocorticoids are still a matter of debate despite these compounds have been used for decades in the treatment of autoimmune myasthenic syndromes. Besides their immunosuppressive action, corticosteroids may directly facilitate transmitter release during high-frequency motor nerve activity. This effect coincides with the predominant adenosine A 2A receptor tonus, which coordinates the interplay with other receptors ( e.g. muscarinic) on motor nerve endings to sustain acetylcholine (ACh) release that is required to overcome tetanic neuromuscular depression in myasthenics. Using myographic recordings, measurements of evoked [ 3 H]ACh release and real-time video microscopy with the FM4-64 fluorescent dye, results show that tonic activation of facilitatory A 2A receptors by endogenous adenosine accumulated during 50 Hz bursts delivered to the rat phrenic nerve is essential for methylprednisolone (0.3 mM)-induced transmitter release facilitation, because its effect was prevented by the A 2A receptor antagonist, ZM 241385 (10 nM). Concurrent activation of the positive feedback loop operated by pirenzepine-sensitive muscarinic M 1 autoreceptors may also play a role, whereas the corticosteroid action is restrained by the activation of co-expressed inhibitory M 2 and A 1 receptors blocked by methoctramine (0.1 μM) and DPCPX (2.5 nM), respectively. Inhibition of FM4-64 loading (endocytosis) by methylprednisolone following a brief tetanic stimulus (50 Hz for 5 s) suggests that it may negatively modulate synaptic vesicle turnover, thus increasing the release probability of newly recycled vesicles. Interestingly, bulk endocytosis was rehabilitated when methylprednisolone was co-applied with ZM241385. Data suggest that amplification of neuromuscular transmission by methylprednisolone may involve activation of presynaptic facilitatory adenosine A 2A receptors by endogenous adenosine leading to synaptic vesicle redistribution.

Made available in DSpace on 2018-12-11T17:14:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-11-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação para a Ciência e a Tecnologia Background and purpose Crotoxin (CTX), a heterodimeric phospholipase A2 (PLA2) neurotoxin from Crotalus durissus terrificus snake venom, promotes irreversible blockade of neuromuscular transmission. Indirect electrophysiological evidence suggests that CTX exerts a primary inhibitory action on transmitter exocytosis, yet contribution of a postsynaptic action of the toxin resulting from nicotinic receptor desensitization cannot be excluded. Here, we examined the blocking effect of CTX on nerve-evoked transmitter release measured directly using radioisotope neurochemistry and video microscopy with the FM4-64 fluorescent dye. Experimental approach Experiments were conducted using mice phrenic-diaphragm preparations. Real-time fluorescence video microscopy and liquid scintillation spectrometry techniques were used to detect transmitter exocytosis and nerve-evoked [3H]-acetylcholine ([3H]ACh) release, respectively. Nerve-evoked myographic recordings were also carried out for comparison purposes. Key results Both CTX (5 μg/mL) and its basic PLA2 subunit (CB, 20 μg/mL) had biphasic effects on nerve-evoked transmitter exocytosis characterized by a transient initial facilitation followed by a sustained decay. CTX and CB reduced nerve-evoked [3H]ACh release by 60% and 69%, respectively, but only the heterodimer, CTX, decreased the amplitude of nerve-evoked muscle twitches. Conclusion and implications Data show that CTX exerts a presynaptic inhibitory action on ACh release that is highly dependent on its intrinsic PLA2 activity. Given the high safety margin of the neuromuscular transmission, one may argue that the presynaptic block caused by the toxin is not enough to produce muscle paralysis unless a concurrent postsynaptic inhibitory action is also exerted by the CTX heterodimer. Departamento de Farmacologia Instituto de Ciências Biológicas, UFMG, Av. Antônio Carlos Laboratório de Farmacologia e Neurobiologia Instituto de Ciências Biomédicas Abel Salazar - Universidade do Porto (ICBAS-UP), R. Jorge Viterbo Ferreira, 228 Center for Drug Discovery and Innovative Medicines (MedInUP) Instituto de Ciências Biomédicas Abel Salazar - Universidade do Porto (ICBAS-UP), R. Jorge Viterbo Ferreira, 228 Departamento de Física e Biofísica Instituto de Biociências UNESP, Distrito de Rubião Jr. Departamento de Farmacologia Instituto de Biociências UNESP, Distrito de Rubião Jr. Departamento de Física e Biofísica Instituto de Biociências UNESP, Distrito de Rubião Jr. Departamento de Farmacologia Instituto de Biociências UNESP, Distrito de Rubião Jr. CAPES: 1592/2011 CNPq: 300596/2013-8 Fundação para a Ciência e a Tecnologia: OE/SAU/UI0215/2014 Fundação para a Ciência e a Tecnologia: UID/BIM/4308/2016

The ability of robotic rehabilitation devices to support paralysed end-users is ultimately limited by the degree to which human-machine-interaction is designed to be effective and efficient in translating user intention into robotic action. Specifically, we evaluate the novel possibility of binocular eye-tracking technology to detect voluntary winks from involuntary blink commands, to establish winks as a novel low-latency control signal to trigger robotic action. By wearing binocular eye-tracking glasses we enable users to directly observe their environment or the actuator and trigger movement actions, without having to interact with a visual display unit or user interface. We compare our novel approach to two conventional approaches for controlling robotic devices based on electromyo-graphy (EMG) and speech-based human-computer interaction technology. We present an integrated software framework based on ROS that allows transparent integration of these multiple modalities with a robotic system. We use a soft-robotic SEM glove (Bioservo Technologies AB, Sweden) to evaluate how the 3 modalities support the performance and subjective experience of the end-user when movement assisted. All 3 modalities are evaluated in streaming, closed-loop control operation for grasping physical objects. We find that wink control shows the lowest error rate mean with lowest standard deviation of (0.23 ± 0.07, mean ± SEM) followed by speech control (0.35 ± 0. 13) and EMG gesture control (using the Myo armband by Thalamic Labs), with the highest mean and standard deviation (0.46 ± 0.16). We conclude that with our novel own developed eye-tracking based approach to control assistive technologies is a well suited alternative to conventional approaches, especially when combined with 3D eye-tracking based robotic end-point control.

Understanding the biological activity profile of the snake venom components is fundamental for improving the treatment of snakebite envenomings and may also contribute for the development of new potential therapeutic agents. In this work, we tested the effects of BthTX-I, a Lys49 PLA2 homologue from the Bothrops jararacussu snake venom. While this toxin induces conspicuous myonecrosis by a catalytically independent mechanism, a series of in vitro studies support the hypothesis that BthTX-I might also exert a neuromuscular blocking activity due to its ability to alter the integrity of muscle cell membranes. To gain insight into the mechanisms of this inhibitory neuromuscular effect, for the first time, the influence of BthTX-I on nerve-evoked ACh release was directly quantified by radiochemical and real-time video-microscopy methods. Our results show that the neuromuscular blockade produced by in vitro exposure to BthTX-I (1 μM) results from the summation of both pre- and postsynaptic effects. Modifications affecting the presynaptic apparatus were revealed by the significant reduction of nerve-evoked [3H]-ACh release; real-time measurements of transmitter exocytosis using the FM4-64 fluorescent dye fully supported radiochemical data. The postsynaptic effect of BthTX-I was characterized by typical histological alterations in the architecture of skeletal muscle fibers, increase in the outflow of the intracellular lactate dehydrogenase enzyme and progressive depolarization of the muscle resting membrane potential. In conclusion, these findings suggest that the neuromuscular blockade produced by BthTX-I results from transient depolarization of skeletal muscle fibers, consequent to its general membrane-destabilizing effect, and subsequent decrease of evoked ACh release from motor nerve terminals.

The synthesis and structure elucidation of three new chiral xanthone (9H-xanthon-9-one) derivatives (2-4) are fully reported. The coupling reactions of the synthesized building block 6-methoxy-9-oxo-9H-xanthene-2-carboxylic acid (1) with two enantiomerically pure amino alcohols ((S)-(+)-valinol and (S)-(+)-leucinol) and one amine ((S)-(-)-α-4-dimethylbenzylamine), were carried out using the coupling reagent O-(benzotriazol-1-yl-)-N,N,N',N'-tetramethylluronium tetrafluoroborate (TBTU). The coupling reactions were performed with yields higher than 97% and enantiomeric excess higher than 99%. The structures of the compounds were established by IR, MS, and NMR ((1)H, (13)C, HSQC, and HMBC) techniques. Taking into account that these new chiral xanthone derivatives have molecular moieties structurally very similar to local anaesthetics, the ability to block compound action potentials (CAP) at the isolated rat sciatic nerve was also investigated. Nerve conduction blockade might result from a selective interference with Na(+) ionic currents or from a non-selective modification of membrane stabilizing properties. Thus, the mechanism, by which the three chiral xanthone derivatives cause conduction blockade in the rat sciatic nerve and their ability to prevent hypotonic haemolysis, given that erythrocytes are non-excitable cells devoid of voltage-gated Na(+) channels, are also described. Data suggest that nerve conduction blockade caused by newly-synthesized xanthone derivatives might result predominantly from an action on Na(+) ionic currents. This effect can be dissociated from their ability to stabilize cell membranes, which became apparent only upon increasing the concentration of compounds 2-4 to the higher micromolar range.

This study aimed at investigating the expression and function of uracil nucleotide-sensitive receptors (P2Y2, P2Y4, and P2Y6) on osteogenic differentiation of human bone marrow stromal cells (BMSCs) in culture. Bone marrow specimens were obtained from postmenopausal female patients (68 ± 5 years old, n = 18) undergoing total hip arthroplasty. UTP and UDP (100 µM) facilitated osteogenic differentiation of the cells measured as increases in alkaline phosphatase (ALP) activity, without affecting cell proliferation. Uracil nucleotides concentration-dependently increased [Ca2+]i in BMSCs; their effects became less evident with time (7 > 21 days) of the cells in culture. Selective activation of P2Y6 receptors with the stable UDP analog, PSB 0474, mimicked the effects of both UTP and UDP, whereas UTPγS was devoid of effect. Selective blockade of P2Y6 receptors with MRS 2578 prevented [Ca2+]i rises and osteogenic differentiation caused by UDP at all culture time points. BMSCs are immunoreactive against P2Y2, P2Y4, and P2Y6 receptors. While the expression of P2Y6 receptors remained fairly constant (7∼21 days), P2Y2 and P2Y4 became evident only in less proliferative and more differentiated cultures (7

J. Neurochem. (2011) 117, 797–811. Abstract In healthy motor endplates, tetanic depression is overcome by tonic adenosine A2A-receptor-mediated facilitation of transmitter release. The A2A receptor operates a coordinated shift from fast-desensitizing Cav2.1 (P/Q) calcium influx to long-lasting CaV1 (L) channels on motor nerve terminals. This study aimed at investigating whether A2A receptors-operated Ca2+ influx via CaV1 (L)-type channels contribute to sustain acetylcholine release evoked by 50 Hz-bursts in toxin-induced Myasthenia gravis (TIMG) rats. In contrast to control animals, inhibition of [3H]acetylcholine (ACh) release by the CaV2.1 (P/Q) channel blocker, ω-Agatoxin IVA (100 nM), in TIMG rats had a higher magnitude than that observed with the CaV1 (L) channel blocker, nifedipine (1 μM). Adenosine deaminase (0.5 U/mL) and the A2A receptor antagonist, ZM 241385 (50 nM), decreased [3H]ACh release by a similar amount in control rats, but their effects were smaller in magnitude in myasthenic animals. The adenosine precursor, AMP (100 μM), increased (∼40%) ACh release in both control and TIMG animals. Blockade of A2A, but not of A1, receptors prevented AMP-induced facilitation of transmitter release; nifedipine (1 μM) mimicked the effect of the A2A receptor antagonist. Video-microscopy studies designed to measure real-time transmitter exocytosis using the FM4-64 fluorescent dye fully supported radiochemical data. Thus, impairment of the adaptive shift from CaV2.1 (P/Q) to CaV1 (L) channels may contribute to tetanic failure in myasthenic rats. This parallels the reduction of adenosine A2A receptor tonus in TIMG animals, which might be restored by exogenous application of AMP.

Polymorphisms of the P2X7 receptor have been associated with increased risk of fractures in postmenopausal women. Although both osteoblasts and osteoclasts express P2X7 receptors, their function in osteogenesis remains controversial. Here, we investigated the role of the P2X7 receptor on osteogenic differentiation and mineralization of bone marrow mesenchymal stem cell (BMSC) cultures from postmenopausal women (age 71±3 yr, n=18). We focused on the mechanisms related to intracellular [Ca(2+)]i oscillations and plasma membrane-dynamics. ATP, and the P2X7 agonist BzATP (100 μM), increased [Ca(2+)]i in parallel to the formation of membrane pores permeable to TO-PRO-3 dye uptake. ATP and BzATP elicited reversible membrane blebs (zeiosis) in 38 ± 1 and 70 ± 1% of the cells, respectively. P2X7-induced zeiosis was Ca(2+) independent, but involved phospholipase C, protein kinase C, and Rho-kinase activation. BzATP (100 μM) progressively increased the expression of Runx-2 and Osterix transcription factors by 452 and 226% (at d 21), respectively, alkaline phosphatase activity by 88% (at d 28), and mineralization by 329% (at d 43) of BMSC cultures in a Rho-kinase-dependent manner. In summary, reversible plasma membrane zeiosis involving cytoskeleton rearrangements due to activation of the P2X7-Rho-kinase axis promotes osteogenic differentiation and mineralization of BMSCs, thus providing new therapeutic targets for postmenopausal bone loss.

In contrast to the well-known signaling role of urothelial ATP to control bladder function, the hypothesis that uracil nucleotides (UTP and/or UDP) also exert autocrine/paracrine actions only recently gained experimental support. Urothelial cells express UDP-sensitive P2Y6 receptors, yet their role in the control of bladder activity has been mostly neglected. This study was designed to investigate the ability of PSB0474, a stable UDP analogue which exhibits selectivity for P2Y6 receptors, to modulate urodynamic responses in the anaesthetized rat in vivo. Instillation of PSB0474 into the bladder increased the voiding frequency (VF) without affecting the amplitude (A) and the duration (Δt) of bladder contractions. PSB0474-induced bladder overactivity was prevented by the selective P2Y6 antagonist, MRS2578. The increase in the VF produced by PSB0474 was also blocked by inhibitors of pannexin-1 hemichannels, (10)Panx or carbenoxolone, when these drugs were applied inside the bladder lumen but not when they were administered intravenously. Reduction of hemichannels pore permeability with H1152 also prevented PSB0474-induced bladder overactivity, but the exocytosis inhibitor, Exo-1, was inactive. PSB0474 increased by 3-fold the urinary ATP content. Implication of hemichannels permeability on PSB0474-induced ATP release was demonstrated by real-time fluorescence video-microscopy measuring the uptake of propidium iodide by intact urothelial cells in the absence and in the presence of MRS2578 or carbenoxolone. Confocal microscopy studies confirmed the co-localization of pannexin-1 and P2Y6 receptors in the rat urothelium. Data indicate that activation of P2Y6 receptors causes bladder overactivity in the anaesthetized rat indirectly by releasing ATP from the urothelium via pannexin-1 hemichannels.

O conhecimento tem sido estudado há bastante tempo dentro da administração. Entretanto, a partir da década de 1990, com as mudanças nos fundamentos das economias industriais dos recursos naturais para os ativos intelectuais, as empresas passaram a preocupar-se com o conhecimento existente em suas organizações e seu gerenciamento. Um dos aspectos mais relevantes para o gerenciamento do conhecimento é a sua transferência. O conhecimento tácito, por não poder ser estruturado e codificado, é difícil de ser transferido pela organização. O gerenciamento de ativos intangíveis como o conhecimento tácito é percebido como importante capacitação para a competição e como fonte de vantagem competitiva sustentável. O objetivo desse trabalho é identificar, através de um estudo de caso, se os fatores relevantes à transferência do conhecimento tácito estão presentes em uma grande empresa petrolífera brasileira. A presente pesquisa analisa parte relevante da bibliografia acerca da transferência do conhecimento tácito em organizações e coleta evidências para propor um modelo heurístico que possa explicar como ocorre essa transferência de conhecimento tácito, baseado em fatores idiossincráticos e nos fatores organizacionais cultura organizacional, estrutura organizacional e estratégia de gestão do conhecimento. Como resultado, o fator idiossincrático e os fatores estrutura organizacional e estratégia de gestão do conhecimento foram corroborados pelas evidências empíricas, entretanto o fator cultura organizacional não foi identificado. A partir dos resultados alcançados, um novo modelo é proposto. Verifica-se que alguns fatores identificados ajudam a transferência do conhecimento tácito enquanto outros criam barreiras a essa transferência. São sugeridos mecanismos para auxiliar as organizações na transferência do conhecimento tácito. Knowledge has long been studied in the management field. However, since the shift of the principles of industrial economy from natural resources to intellectual assets in the 1990’s, firms have been concerned about the management of its internal knowledge. The transfer of knowledge is one of the most important issues in knowledge management. Tacit knowledge, that cannot be codified, is more difficult to be transferred through the organization. The management of intangible assets like tacit knowledge is an important source if sustained competitive advantage. The objective of this study is to identify the presence in a major Brazilian oil company of key factors to the tacit knowledge transfer. This research revises relevant part of tacit knowledge transfer literature and gathers evidences to suggest a heuristic model to explaining how tacit knowledge is transferred within the organization, relied on idiosyncratic aspects and organizational aspects like organizational culture, organizational structure and knowledge management strategy. As a result, this work showed that idiosyncratic aspects, organizational structure and knowledge management strategy aspects were corroborated by empirical findings. However, organizational culture aspects were not identified. A new model of tacit knowledge transfer is drawn from the empirical results. Some of aspects identified in the organization studied help the transfer of tacit knowledge while others hamper its flow. Some tools to improve tacit knowledge transfer within organizations are proposed.