Calcium signaling and the novel anti-proliferative effect of the UTP-sensitive P2Y11 receptor in rat cardiac myofibroblasts

During myocardial ischemia and reperfusion both purines and pyrimidines are released into the extracellular milieu, thus creating a signaling wave that propagates to neighboring cells via membrane-bound P2 purinoceptors activation. Cardiac fibroblasts (CF) are important players in heart remodeling, electrophysiological changes and hemodynamic alterations following myocardial infarction. Here, we investigated the role UTP on calcium signaling and proliferation of CF cultured from ventricles of adult rats. Co-expression of discoidin domain receptor 2 and -smooth muscle actin indicate that cultured CF are activated myofibroblasts. Intracellular calcium ([Ca2+]i) signals were monitored in cells loaded with Fluo-4 NW. CF proliferation was evaluated by the MTT assay. UTP and the selective P2Y4 agonist, MRS4062, caused a fast desensitizing [Ca2+]i rise originated from thapsigargin-sensitive internal stores, which partially declined to a plateau providing the existence of Ca2+ in the extracellular fluid. The biphasic [Ca2+]i response to UTP was attenuated respectively by P2Y4 blockers, like reactive blue-2 and suramin, and by the P2Y11 antagonist, NF340. UTP and the P2Y2 receptor agonist MRS2768 increased, whereas the selective P2Y11 agonist NF546 decreased, CF growth; MRS4062 was ineffective. Blockage of the P2Y11receptor or its coupling to adenylate cyclase boosted UTP-induced CF proliferation. Confocal microscopy and Western blot analysis confirmed the presence of P2Y2, P2Y4 and P2Y11 receptors. Data indicate that besides P2Y4 and P2Y2 receptors which are responsible for UTP-induced [Ca2+]i transients and growth of CF, respectively, synchronous activation of the previously unrecognized P2Y11 receptor may represent an important target for anti-fibrotic intervention in cardiac remodeling.

On the use of IEEE 802.15.4/Zigbee for time-sensitive wireless sensor network applications

Mestrado em Engenharia Electrotécnica e de Computadores

Molecular imprinted nanoelectrodes for ultra sensitive detection of ovarian cancer marker

The relentless discovery of cancer biomarkers demands improved methods for their detection. In this work, we developed protein imprinted polymer on three-dimensional gold nanoelectrode ensemble (GNEE) to detect epithelial ovarian cancer antigen-125 (CA 125), a protein biomarker associated with ovarian cancer. CA 125 is the standard tumor marker used to follow women during or after treatment for epithelial ovarian cancer. The template protein CA 125 was initially incorporated into the thin-film coating and, upon extraction of protein from the accessible surfaces on the thin film, imprints for CA 125 were formed. The fabrication and analysis of the CA 125 imprinted GNEE was done by using cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) techniques. The surfaces of the very thin, protein imprinted sites on GNEE are utilized for immunospecific capture of CA 125 molecules, and the mass of bound on the electrode surface can be detected as a reduction in the faradic current from the redox marker. Under optimal conditions, the developed sensor showed good increments at the studied concentration range of 0.5–400 U mL−1. The lowest detection limit was found to be 0.5 U mL−1. Spiked human blood serum and unknown real serum samples were analyzed. The presence of non-specific proteins in the serum did not significantly affect the sensitivity of our assay. Molecular imprinting using synthetic polymers and nanomaterials provides an alternative approach to the trace detection of biomarker proteins.

Salt content in bread and dough from northern Portugal: method development and comparison

In the initial stage of this work, two potentiometric methods were used to determine the salt (sodium chloride) content in bread and dough samples from several cities in the north of Portugal. A reference method (potentiometric precipitation titration) and a newly developed ion-selective chloride electrode (ISE) were applied. Both methods determine the sodium chloride content through the quantification of chloride. To evaluate the accuracy of the ISE, bread and respective dough samples were analyzed by both methods. Statistical analysis (0.05 significance level) indicated that the results of these methods did not differ significantly. Therefore the ISE is an adequate alternative for the determination of chloride in the analyzed samples. To compare the results of these chloride-based methods with a sodium-based method, sodium was quantified in the same samples by a reference method (atomic absorption spectrometry). Significant differences between the results were verified. In several cases the sodium chloride content exceeded the legal limit when the chloride-based methods were used, but when the sodium-based method was applied this was not the case. This could lead to the erroneous application of fines and therefore the authorities should supply additional information regarding the analytical procedure for this particular control.

Facile synthesis of freestanding Si nanowire arrays by one-step template-free electro-deoxidation of SiO2 in molten salt

This communication presents a novel kind of silicon nanomaterial: freestanding Si nanowire arrays (Si NWAs), which are synthesized facilely by one-step template-free electro-deoxidation of SiO2 in molten CaCl2. The self-assembling growth process of this material is also investigated preliminarily.

An implicit GTS allocation mechanism in IEEE 802.15.4 for time-sensitive wireless sensor networks: theory and practice

Timeliness guarantee is an important feature of the recently standardized IEEE 802.15.4 protocol, turning it quite appealing for Wireless Sensor Network (WSN) applications under timing constraints. When operating in beacon-enabled mode, this protocol allows nodes with real-time requirements to allocate Guaranteed Time Slots (GTS) in the contention-free period. The protocol natively supports explicit GTS allocation, i.e. a node allocates a number of time slots in each superframe for exclusive use. The limitation of this explicit GTS allocation is that GTS resources may quickly disappear, since a maximum of seven GTSs can be allocated in each superframe, preventing other nodes to benefit from guaranteed service. Moreover, the GTS may be underutilized, resulting in wasted bandwidth. To overcome these limitations, this paper proposes i-GAME, an implicit GTS Allocation Mechanism in beacon-enabled IEEE 802.15.4 networks. The allocation is based on implicit GTS allocation requests, taking into account the traffic specifications and the delay requirements of the flows. The i-GAME approach enables the use of one GTS by multiple nodes, still guaranteeing that all their (delay, bandwidth) requirements are satisfied. For that purpose, we propose an admission control algorithm that enables to decide whether to accept a new GTS allocation request or not, based not only on the remaining time slots, but also on the traffic specifications of the flows, their delay requirements and the available bandwidth resources. We show that our approach improves the bandwidth utilization as compared to the native explicit allocation mechanism defined in the IEEE 802.15.4 standard. We also present some practical considerations for the implementation of i-GAME, ensuring backward compatibility with the IEEE 801.5.4 standard with only minor add-ons. Finally, an experimental evaluation on a real system that validates our theoretical analysis and demonstrates the implementation of i-GAME is also presented

IEEE 802.15.4: a Federating Communication Protocol for Time-Sensitive Wireless Sensor Networks

Wireless Sensor Networks (WSNs) have been attracting increasing interests for developing a new generation of embedded systems with great potential for many applications such as surveillance, environment monitoring, emergency medical response and home automation. However, the communication paradigms in WSNs differ from the ones attributed to traditional wireless networks, triggering the need for new communication protocols. In this context, the recently standardised IEEE 802.15.4 protocol presents some potentially interesting features for deployment in wireless sensor network applications, such as power-efficiency, timeliness guarantees and scalability. Nevertheless, when addressing WSN applications with (soft/hard) timing requirements some inherent paradoxes emerge, such as power-efficiency versus timeliness, triggering the need of engineering solutions for an efficient deployment of IEEE 802.15.4 in WSNs. In this technical report, we will explore the most relevant characteristics of the IEEE 802.15.4 protocol for wireless sensor networks and present the most important challenges regarding time-sensitive WSN applications. We also provide some timing performance and analysis of the IEEE 802.15.4 that unveil some directions for resolving the previously mentioned paradoxes.

i-GAME: an implicit GTS allocation mechanism in IEEE 802.15.4 for time-sensitive wireless sensor networks

The IEEE 802.15.4 Medium Access Control (MAC) protocol is an enabling technology for time sensitive wireless sensor networks thanks to its Guaranteed-Time Slot (GTS) mechanism in the beacon-enabled mode. However, the protocol only supports explicit GTS allocation, i.e. a node allocates a number of time slots in each superframe for exclusive use. The limitation of this explicit GTS allocation is that GTS resources may quickly disappear, since a maximum of seven GTSs can be allocated in each superframe, preventing other nodes to benefit from guaranteed service. Moreover, the GTSs may be only partially used, resulting in wasted bandwidth. To overcome these limitations, this paper proposes i-GAME, an implicit GTS Allocation Mechanism in beacon-enabled IEEE 802.15.4 networks. The allocation is based on implicit GTS allocation requests, taking into account the traffic specifications and the delay requirements of the flows. The i-GAME approach enables the use of a GTS by multiple nodes, while all their (delay, bandwidth) requirements are still satisfied. For that purpose, we propose an admission control algorithm that enables to decide whether to accept a new GTS allocation request or not, based not only on the remaining time slots, but also on the traffic specifications of the flows, their delay requirements and the available bandwidth resources. We show that our proposal improves the bandwidth utilization compared to the explicit allocation used in the IEEE 802.15.4 protocol standard. We also present some practical considerations for the implementation of i-GAME, ensuring backward compatibility with the IEEE 801.5.4 standard with only minor add-ons.

Sensitive bi-enzymatic biosensor based on polyphenoloxidases–gold nanoparticles–chitosan hybrid film–graphene doped carbon paste electrode for carbamates detection

A bi-enzymatic biosensor (LACC–TYR–AuNPs–CS/GPE) for carbamates was prepared in a single step by electrodeposition of a hybrid film onto a graphene doped carbon paste electrode (GPE). Graphene and the gold nanoparticles (AuNPs) were morphologically characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, dynamic light scattering and laser Doppler velocimetry. The electrodeposited hybrid film was composed of laccase (LACC), tyrosinase (TYR) and AuNPs entrapped in a chitosan (CS) polymeric matrix. Experimental parameters, namely graphene redox state, AuNPs:CS ratio, enzymes concentration, pH and inhibition time were evaluated. LACC–TYR–AuNPs–CS/GPE exhibited an improved Michaelis–Menten kinetic constant (26.9 ± 0.5 M) when compared with LACC–AuNPs–CS/GPE (37.8 ± 0.2 M) and TYR–AuNPs–CS/GPE (52.3 ± 0.4 M). Using 4-aminophenol as substrate at pH 5.5, the device presented wide linear ranges, low detection limits (1.68×10− 9 ± 1.18×10− 10 – 2.15×10− 7 ± 3.41×10− 9 M), high accuracy, sensitivity (1.13×106 ± 8.11×104 – 2.19×108 ± 2.51×107 %inhibition M− 1), repeatability (1.2–5.8% RSD), reproducibility (3.2–6.5% RSD) and stability (ca. twenty days) to determine carbaryl, formetanate hydrochloride, propoxur and ziram in citrus fruits based on their inhibitory capacity on the polyphenoloxidases activity. Recoveries at two fortified levels ranged from 93.8 ± 0.3% (lemon) to 97.8 ± 0.3% (orange). Glucose, citric acid and ascorbic acid do not interfere significantly in the electroanalysis. The proposed electroanalytical procedure can be a promising tool for food safety control.

Descending pain modulation in a Kaolin-induced hydrocephalus rat model

Pain transmission at the spinal cord is modulated by descending actions that arise from supraspinal areas which collectively form the endogenous pain control system. Two key areas involved of the endogenous pain control system have a circunventricular location, namely the periaqueductal grey (PAG) and the locus coeruleus (LC). The PAG plays a crucial role in descending pain modulation as it conveys the input from higher brain centers to the spinal cord. As to the LC, it is involved in descending pain inhibition by direct noradrenergic projections to the spinal cord. In the context of neurological defects, several diseases may affect the structure and function of the brain. Hydrocephalus is a congenital or acquired disease characterized by an enlargement of the ventricles which leads to a distortion of the adjacent tissues, including the PAG and LC. Usually, patients suffering from hydrocephalus present dysfunctions in learning and memory and also motor deficits. It remains to be evaluated if lesions of the periventricular brain areas involved in pain control during hydrocephalus may affect descending pain control and, herein, affect pain responses. The studies included in the present thesis used an experimental model of hydrocephalus (the rat injected in the cisterna magna with kaolin) to study descending modulation of pain, focusing on the two circumventricular regions referred above (the PAG and the LC). In order to evaluate the effects of kaolin injection into the cisterna magna, we measured the degree of ventricular dilatation in sections encompassing the PAG by standard cytoarquitectonic stanings (thionin staining). For the LC, immunodetection of the noradrenaline-synthetizing enzyme tyrosine hydroxylase (TH) was performed, due to the noradrenergic nature of the LC neurons. In general, rats with kaolin-induced hydrocephalus presented a higher dilatation of the 4th ventricle, along with a tendency to a higher area of the PAG. Due to the validated role of detection the c-fos protooncogene as a marker of neuronal activation, we also studied neuronal activation in the several subnuclei which compose the PAG, namely the dorsomedial, dorsolateral, lateral and ventrolateral (VLPAG) parts. A decrease in the numbers of neurons immunoreactive for Fos protein (the product of activation of the c-fos protooncogene) was detected in rats injected with kaolin, whereas the remaining PAG subnuclei did not present changes in Fos-immunoreactive nuclei. Increases in the levels of TH in the LC, namely at the rostral parts of the nucleus, were detected in hydrocephalic animals. The following pain-related parameters were measured, namely 1) pain behavioural responses in a validated pain inflammatory test (the formalin test) and 2) the nociceptive activation of spinal cord neurons. A decrease in behavioral responses was detected in rats with kaolin-induced hydrocephalus was detected, namely in the second phase of the test (inflammatory phase). This is the phase of the formalin test in which the motor behaviour is less important, which is important since a semi-quantitative analysis of the motor performance of rats injected with kaolin indicates that these animals may present some motor impairments. Collectively, the results of the behavioral studies indicate that rats with kaolin-induced hydrocephalus exhibit hypoalgesia. A decrease in Fos expression was detected at the superficial dorsal layers of the spinal cord in rats with kaolin-induced hydrocephalus, further indicating that hydrocephalus decreases nociceptive responses. It remains to be ascertained if this is due to alterations in the PAG and LC in the rats with kaolin-induced hydrocephalus, which may affect descending pain modulation. It remains to be evaluated what are the mechanisms underlying the increased pain inhibition at the spinal dorsal horn in the hydrocephalus rats. Regarding the VLPAG, the decrease in neuronal activity may impair descending modulation. Since the LC has higher levels of TH in rats with kaolininduced hydrocephalus, which also appears to increase the noradrenergic innervation in the spinal dorsal horn, it is possible that an increase in the release of noradrenaline at the spinal cord accounts for pain inhibition. Our studies also determine the need to study in detail patients with hydrocephalus namely in what concerns their thresholds to pain and to perform imaging studies focused on the structure and function of pain control areas in the brain.

Electrospun Polyaniline-Reduced Graphene Oxide Composite Nanofibers Based High Sensitive Ammonia Gas Sensor

Ammonia is an important gas in many power plants and industrial processes so its detection is of extreme importance in environmental monitoring and process control due to its high toxicity. Ammonia’s threshold limit is 25 ppm and the exposure time limit is 8 h, however exposure to 35 ppm is only secure for 10 min. In this work a brief introduction to ammonia aspects are presented, like its physical and chemical properties, the dangers in its manipulation, its ways of production and its sources. The application areas in which ammonia gas detection is important and needed are also referred: environmental gas analysis (e.g. intense farming), automotive-, chemical- and medical industries. In order to monitor ammonia gas in these different areas there are some requirements that must be attended. These requirements determine the choice of sensor and, therefore, several types of sensors with different characteristics were developed, like metal oxides, surface acoustic wave-, catalytic-, and optical sensors, indirect gas analyzers, and conducting polymers. All the sensors types are described, but more attention will be given to polyaniline (PANI), particularly to its characteristics, syntheses, chemical doping processes, deposition methods, transduction modes, and its adhesion to inorganic materials. Besides this, short descriptions of PANI nanostructures, the use of electrospinning in the formation of nanofibers/microfibers, and graphene and its characteristics are included. The created sensor is an instrument that tries to achieve a goal of the medical community in the control of the breath’s ammonia levels being an easy and non-invasive method for diagnostic of kidney malfunction and/or gastric ulcers. For that the device should be capable to detect different levels of ammonia gas concentrations. So, in the present work an ammonia gas sensor was developed using a conductive polymer composite which was immobilized on a carbon transducer surface. The experiments were targeted to ammonia measurements at ppb level. Ammonia gas measurements were carried out in the concentration range from 1 ppb to 500 ppb. A commercial substrate was used; screen-printed carbon electrodes. After adequate surface pre-treatment of the substrate, its electrodes were covered by a nanofibrous polymeric composite. The conducting polyaniline doped with sulfuric acid (H2SO4) was blended with reduced graphene oxide (RGO) obtained by wet chemical synthesis. This composite formed the basis for the formation of nanofibers by electrospinning. Nanofibers will increase the sensitivity of the sensing material. The electrospun PANI-RGO fibers were placed on the substrate and then dried at ambient temperature. Amperometric measurements were performed at different ammonia gas concentrations (1 to 500 ppb). The I-V characteristics were registered and some interfering gases were studied (NO2, ethanol, and acetone). The gas samples were prepared in a custom setup and were diluted with dry nitrogen gas. Electrospun nanofibers of PANI-RGO composite demonstrated an enhancement in NH3 gas detection when comparing with only electrospun PANI nanofibers. Was visible higher range of resistance at concentrations from 1 to 500 ppb. It was also observed that the sensor had stable, reproducible and recoverable properties. Moreover, it had better response and recovery times. The new sensing material of the developed sensor demonstrated to be a good candidate for ammonia gas determination.

A molecularly imprinted sensor for sensitive detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG) oxidative stress biomarker

6th Graduate Student Symposium on Molecular Imprinting

A new biomimetic sensor for detecting carnitine, a potential biomarker in ovarian cancer

1st ASPIC International Congress

QuEChERS: a sample preparation for extraction of carbaryl from rat feces

Carbamate compounds are an important group of cholinesterase inhibitors. There is a need for creating awareness regarding the risks of the inadequate carbamate use in the residential areas due to potential adverse human effects. Carbaryl is a commonly used pesticide worldwide. A simple, fast, and high throughput method was developed employing liquid chromatography with fluorescence detector to determine carbaryl residues in rat feces. The extraction was performed by using a rapid, easy, cheap, effective, reliable, and safe (QuEChERS) method, using acetonitrile as the extracting solvent. The parameters for the performance of the extraction method were optimized, such as ratio of mass of sample per volume of extraction solvent, QuEChERS content, and cleanup columns. Linear response was obtained for all calibration curves (solven and matrix-matched) over the established concentration range (5 500 mg/L) with a correlation coefficients higher than 0.999. The achieved recovery was 97.9% with relative standard deviation values of 1.1% (n D 4) at 167 mg/kg fortified concentration level and the limits of detection and quantification were 27.7 and 92.3 mg/kg respectively.

Optical measurements of rat muscle samples under treatment with ethylene glycol and glucose

With the objective to study the variation of optical properties of rat muscle during optical clearing, we have performed a set of optical measurements from that kind of tissue. The measurements performed were total transmittance, collimated transmittance, specular reflectance and total reflectance. This set of measurements is sufficient to determine diffuse reflectance and absorbance of the sample, also necessary to estimate the optical properties. All the performed measurements and calculated quantities will be used later in inverse Monte Carlo (IMC) simulations to determine the evolution of the optical properties of muscle during treatments with ethylene glycol and glucose. The results obtained with the measurements already provide some information about the optical clearing treatments applied to the muscle and translate the mechanisms of turning the tissue more transparent and sequence of regimes of optical clearing.