Conditional tests for elliptical symmetry using robust estimators

This paper presents a procedure for testing the hypothesis that the underlying distribution of the data is elliptical when using robust location and scatter estimators instead of the sample mean and covariance matrix. Under mild assumptions that include elliptical distributions without first moments, we derive the test statistic asymptotic behaviour under the null hypothesis and under special alternatives. Numerical experiments allow to compare the behaviour of the tests based on the sample mean and covariance matrix with that based on robust estimators, under various elliptical distributions and different alternatives. This comparison was done looking not only at the observed level and power but we rather use the size-corrected relative exact power which provides a tool to assess the test statistic skill to detect alternatives. We also provide a numerical comparison with other competing tests.Comment: In press in Communications in Statistics: Theory and Methods, 201

Implication of AMPK in glucose-evoked modulation of Na,K-ATPase

Background and aims: Na,K-ATPase is an integral membrane protein that maintains the gradients of Na+ and K+, using the energy of ATP hydrolysis, maintaining the ionic gradients that allow electrical activity to occur. It has been demonstrated that, in pancreatic β-cells, Na,K-ATPase is regulated by glucose and that this phenomenon is impaired in glucose intolerant subjects. However, the mechanism underlying glucose-induced modulation of Na,K-ATPase is still unclear. The AMP-activated protein kinase (AMPK) is a molecular key player in energy homeostasis, providing exquisite sensitivity to small changes in intracellular AMP levels and thus to intracellular [ATP]/[ADP] ratio, that is known to activate protein regulatory pathways. Since in pancreatic β-cell, glucose has marked effects on oxidative metabolism and total intracellular ATP and AMP levels, the involvement of AMPK in the cascade of events regulating Na,K-ATPase regulation in pancreatic β-cells was postulated. The aim of this work was to evaluate the putative role of AMPK in the glucose-evoked regulation of Na,K-ATPase activity in the pancreatic β-cell. Materials and methods: Pancreatic -cells from normal (control) or glucose-intolerant Wistar rats (GIR) were isolated and cultured (48h). Cell batches were pre-incubated (30min) with 2.1mM glucose to reach basal activity. Afterwards cells were challenged to 8.4mM glucose for 20min, in the presence or absence of AMPK agonists (AICAR) and antagonists (compound C; CC). ATPase activity was assessed in intact cells by colorimetric quantification of Pi formed in 30min. Na,K-ATPase activity was calculated by the difference between the activities obtained in the absence and in presence the of 1mM ouabain. Results: In basal conditions the activity of Na,K-ATPase from normal and GIR pancreatic β-cell was similar (0.184±0.030 and 0.186±0.020 molPi/min/mgProt, respectively). Challenging the control β-cells with glucose 8.4mM evoked a 62% reduction of Na,K-ATPase activity whereas in GIR β-cells a significantly lower inhibition (40%) was observed. The addition of AICAR 1mM abolished glucose-induced Na,K-ATPase inhibition (0,166±0.011 molPi/min/mg). In control β-cell, the addition of CC 10 μM had no effect on glucose-induced inhibition of Na,K-ATPase. In the contrary, in GIR β-cells it significantly potentiated glucose-evoked inhibition of Na,K-ATPase reaching values similar to that observed in the controls (66%). Conclusions: The AMPK agonist AICAR counteracts the inhibitory action of glucose on Na,K-ATPase of control β-cells whereas CC amplified the glucose-induced inhibition of Na,K-ATPase in GIR β-cells. These results suggest that AMPK plays a central role in the cascade of events underlying glucose-induced modulation of Na,K-ATPase and that the defect must be upstream of AMPK. Finally, abnormal glucose-induced regulation of Na,K-ATPase occurs prior to overt type 2 diabetes and might be a feature in the disease development

Abnormal regulation of Na,K-ATPase in Glucose Intolerant Rats.

Introduction: Glucose is the most important physiological insulin secretagogue. However, the mechanisms underlying glucose-induced insulin release are not fully understood. The role of electrogenic systems such as ionic pumps, to these events remains essentially uninvestigated. Na,K-ATPase, responsible for maintaining Na+ and K+ gradients across the plasma membrane and generates a net outward current, thus changes in its activity may contribute to the early ionic events regulating insulin secretion (Therien and Blostein, 2000). Objective: The aim of this work was to evaluate the regulation of Na,K-ATPase activity by glucose in intact -cells of normal and glucose intolerant (GI) rats and its putative contribution to the regulation of insulin secretion. Material and Methods: Pancreatic -cells, from normal or control or GI rats, were isolated and cultured (48h). Cell batches were pre-incubated (30min) with 2mM glucose to reach basal. Afterwards cells were challenged with glucose in the interval 0-11mM for 60min, for dose-dependence evaluation, or with 8mM glucose for 5-120min, for time-dependence evaluation. ATPase activity was assessed in intact cells by colorimetric quantification of Pi formed in 30min. Na,K-ATPase activity was calculated by the difference between the activities obtained in the absence and in presence the of 1mM ouabain (Costa et al., 2009). Results: In β-cells from normal rats, glucose induced a bimodal regulation of Na,K-ATPase. In the absence of glucose, Na,K-ATPase activity was 0.056±0.015 U/mg. Stimulation with 2mM glucose induced an increase of Na,K-ATPase activity of ~4 fold whereas for [glucose] above 2mM it was observed a significant inhibition of Na,K-ATPase activity (0.061±0.013, 0.080±0.009 and 0.064±0.005 U/mg for 5.6, 8.4 and 11mM glucose, respectively, compared to 0.188±0.035 U/mg observed in 2mM G; n=3-8). β-cells from GI rats does not present this profile; in the absence of glucose, Na,K-ATPase activity was 0.202±0.036 U/mg and no significant differences from this value were observed with the other glucose concentration tested. Addicionally, in β-cells from normal rats, glucose (8mM) induced a time-dependent inhibition, with a biphasic profile, of Na,K-ATPase - it was observed a decrease in the pump activity between 0 and 20min stimulation where it reached a minimum value (77%). For incubation periods over 20min, the pump activity slowly and partially recovered (54%, 55% and 52%, for 30, 60 and 120min, respectively; n=7). In β-cells from GI animals, an less accentuated decrease of Na,K-ATPase activity between 0 ans 20min was also observed (34%), and is not observed further recover in activity. Conclusions: This work demonstrates there Na,K-ATPase is strictly regulated by glucose in pancreatic β-cell. This regulation is unpaired in GI animals. Na,K-ATPase contribution to glucose-induced ionic events and insulin secretion might be relevant and must be explored as a possible therapeutic target in TD2 . 1. Therien AG, Blostein R (2000) Mechanisms of sodium pump regulation. Am J Physiol Cell Physiol 279:C541-C566 2. Costa AR, Real J, Antunes CM, Cruz-Morais J (2009) A new approach for determination of Na,K-ATPase activity: application to intact pancreatic beta-cells. In Vitro Cell Dev Biol Ani

Inhalable fucoidan microparticles combining two antitubercular drugs with potential application in pulmonary tuberculosis therapy

The pulmonary delivery of antitubercular drugs is a promising approach to treat lung tuberculosis. This strategy not only allows targeting the infected organ instantly, it can also reduce the systemic adverse effects of the antibiotics. In light of that, this work aimed at producing fucoidan-based inhalable microparticles that are able to associate a combination of two first-line antitubercular drugs in a single formulation. Fucoidan is a polysaccharide composed of chemical units that have been reported to be specifically recognised by alveolar macrophages (the hosts of Mycobacterium). Inhalable fucoidan microparticles were successfully produced, effectively associating isoniazid (97%) and rifabutin (95%) simultaneously. Furthermore, the produced microparticles presented adequate aerodynamic properties for pulmonary delivery with potential to reach the respiratory zone, with a mass median aerodynamic diameter (MMAD) between 3.6-3.9 mu m. The formulation evidenced no cytotoxic effects on lung epithelial cells (A549), although mild toxicity was observed on macrophage-differentiated THP-1 cells at the highest tested concentration (1 mg/mL). Fucoidan microparticles also exhibited a propensity to be captured by macrophages in a dose-dependent manner, as well as an ability to activate the target cells. Furthermore, drug-loaded microparticles effectively inhibited mycobacterial growth in vitro. Thus, the produced fucoidan microparticles are considered to hold potential as pulmonary delivery systems for the treatment of tuberculosis.Portuguese Foundation for Science and Technology [PTDC/DTP-FTO/0094/2012, UID/Multi/04326/2013, UID/BIM/04773/2013]; CAPES-Brazil [BEX 1168/13-4

Electrically conductive bacterial cellulose for tissue-engineered neural interfaces

Bacterial cellulose (BC) with its high crystallinity, tensile strength, degree of polymerisation, and water holding capacity (98%) becomes increasingly attractive as 3D nanofibrillar material for biomedical applications. Such multi-scale fibrillary BC networks can be potentially functionalised with electrically conductive moieties to facilitate the conductive properties required for various smart biomedical devices, in particular, in the construction of bioelectronic neural interfaces. In this thesis, BC fibres are chemically modified with poly(4-vinylaniline) (PVAN) interlayer for further enhancement of electrical conductivity and cell viability of subsequent polyaniline (PANI) coatings as a bilayer grafted BC nanocomposite. This functional poly(4-vinylaniline)/polyaniline (PVAN/PANI) bilayer can be efficiently anchored onto BC fibrils through successive surface-initiated atom transfer radical polymerisation and in situ chemical oxidative polymerisation. PVAN is found to have promoted the formation of a uniform PANI layer with 1D nanofiber- and nanorod-like supramolecular structures, with an overall augmentation of PANI yield, hence further improved electrical performance. Compositional and microstructural analysis reveals such a PVAN/PANI bilayer with a thickness up to ~2 µm on BC formed through a significant growth of PANI with rough surface morphology due to the insertion of PVAN, which has improved the functional properties of the BC nanocomposites. Successful impregnation of both layers onto BC fibrils was corroborated with systematic microstructural and chemical analysis. The solid-state electrical conductivity of such synthesised BC nanocomposites with PVAN interlayer reaches as high as (4.5±2.8)×10-2 S.cm-1 subject to the amounts of PVAN chemically embraced. Electrochemical examination evinces the switching in the electrochemical behaviour of BC/PVAN/PANI nanocomposites at -0.70/0.74 V (at 100 mV.s-1 scan rate) due to the existence of PANI, where the maximal electrical performance can be achieved at charge transfer resistance of as low as 21 Ω and capacitance of as high as 39 μF. Both electrochemical and mechanical properties can be tailored onto an incomplete BC dehydration, where a mathematical model is herein developed to predict BC water loss accordingly. BC/PVAN/PANI nanocomposites are thermally stable up to 200 ºC. Furthermore, further improvement of the electrical conductivity has been achieved through grafting Carbon Nano Tubes (CNTs) into the BC/PVAN/PANI nanocomposites, where the interactions between PANI and CNTs present new electrochemical characteristics with enhanced capacity. PANI/CNTs coatings with a nanorod-like morphology can promote the efficient ions diffusion and charge transfer, resulting in the increased electrical conductivity up to (1.0±0.3)×10-1 S.cm-1. An escalating amplification of the double charge capacity to ~54 mF of the CNTs grafted BC nanocomposites was also detected through electrochemical analysis. In addition, the thermal stability of CNTs grafted BC/PVAN/PANI nanocomposites are improved, and they become stable up to 234 ºC. Cytocompatibility tests conducted using two neuronal cell linages show non-cytotoxic effects for PC-12 Adh cells and SVZ neural stem cells, confirming cell viability that can be over 80 % and neuronal differentiation capability of the electrically functionalised BC-based nanocomposite membranes, which can induce neurites outgrowth up to 115±24 μm long. These voltage-sensible nanocomposites can hence interact with neural cells, thereby significantly stimulate specialised response. These findings pave the path to the new tissue engineered neural interfaces which embraces electronic functions into the tissue regeneration, to enable full functional neural tissue recovery

Chemical composition and anti-diabetic properties of Cytisus multiflorus

Bakground and aims: The interest on plants with potential medicinal properties has been increasing worldwide. In the Iberian Peninsula there are some endemic species known by the population for their pharmacologic activity with valorization potential that have not been yet characterized. The white Spanish broom (Cytisus multiflorus) is described as having anti-diabetic effect [1] and in a preliminary the hypoglycemic and hyper-insulinemic effect of an aqueous extract has been shown [2]. The aim of this work was to fractionate and analyse the composition of the aqueous extract of C. multiflorus flowering parts and evaluate its potential as an anti-diabetic agent. Materials and methods: The aqueous extract was primarily fractionated by SPE using water:methanol (W:Me) eluent (a 10% step-wise gradient W:Me from 100:0 to 0:100) followed by high performance liquid chromatography with diode array detector (HPLC-DAD). The most relevant fraction were analysed by LC-MS to determine the chemical composition. Total fenol content was determined by a modified Folin-Ciocalteau method and the anti-oxidant activity was evaluated by the DPPH mehod. Finally, the hipoglicemic potential was evaluated in vivo using glucose intolerant rats (GIR). Results: Eleven fractions of the bulk extract were obtained. Seven of these fractions (10, 30, 40, 50, 60, 70 e 80% Me) were found to have a relevant compounds, mostly flavonoid compounds, namely, rutin (50, 60 and 70% Me fractions), ferrulic acid (30% Me), referred as having hypoglicemic effect. The fractions obtained with 50 and 70% Me showed the highest content in phenol equivalents and the highest anti-oxidant effect were found in the 50 and 60% Me fractions. The 30 and 60% Me fraction had no effect on the post-prandial glicemia. Conclusions: The 30, 50, 60 and 70% Me fractions, due to their chemical composition and anti-oxidant effects were the most promising to have anti-diabetic effect. However, the 30 and 60% Me were found to be ineffective. The 50% Me fraction showed both a high content of flavonoid compounds and the highest anti-oxidant power which suggest that it may constitute the most promising one. The anti-diabetic properties of this fraction should be investigated. [1] Camejo-Rodrigues J. et al. (2003). J. Ethnopharmacol, 89, 199-209 [2] Célia M. Antunes, Laurinda R. Areias, Inês P. Vieira, Ana C. Costa, M. Teresa Tinoco, & Júlio Cruz-Morais (2009). Rev. Fitoterapia 9 (Supl.1): 91

Chitosan/carrageenan nanoparticles: effect of cross-linking with tripolyphosphate

Chitosan/carrageenan/tripolyphosphate nanoparticles were prepared by polyelectrolyte complexation/ionic gelation, the latter compound acting as cross-linker. The incorporation of the three components in the nanoparticle matrix was assessed by analytical techniques (FTIR, XPS and TOF-SIMS). Using chitosan/carrageenan nanoparticles as control, the effect of the cross-linker in the particles properties was studied. A decrease in size (from 450-500 nm to 150-300 nm) and in zeta potential (from +75 - +85 mV to +50 - +60 mV), and an increase in production yield (from 15-20% to 25-35%), and in stability (from one week to up to 9 months) were observed. Also, a correlation between positive to negative charge ratios in the formulations and the above characteristics was established. The small size and high positive surface charge make the developed chitosan/carrageenan/tripolyphosphate nanoparticles potential tools for an application in mucosal delivery of macromolecules

Extractability and mobility of mercury from agricultural soils surrounding industrial and mining contaminated areas

This study focussed on a comparison of the extractability of mercury in soils with two different contamination sources (a chlor-alkali plant and mining activities) and on the evaluation of the influence of specific soil properties on the behaviour of the contaminant. The method applied here did not target the identification of individual species, but instead provided information concerning the mobility of mercury species in soil. Mercury fractions were classified as mobile, semi-mobile and non-mobile. The fractionation study revealed that in all samples mercury was mainly present in the semi-mobile phase (between 63 and 97%). The highest mercury mobility (2.7 mg kg-1) was found in soils from the industrial area. Mining soils exhibited higher percentage of non-mobile mercury, up to 35%, due to their elevated sulfur content. Results of factor analysis indicate that the presence of mercury in the mobile phase could be related to manganese and aluminum soil contents. A positive relation between mercury in the semi-mobile fraction and the aluminium content was also observed. By contrary, organic matter and sulfur contents contributed to mercury retention in the soil matrix reducing the mobility of the metal. Despite known limitations of sequential extraction procedures, the methodology applied in this study for the fractionation of mercury in contaminated soil samples provided relevant information on mercury's relative mobility

Rock engravings of Fieiral, Castro Laboreiro, Melgaço

Artigo bilingue, em português e inglêsNotice about the schemathic rock carving of Fieiral, in Northwestern Portugal.This work was develloped in the scope of the project Espaços naturais, arquiteturas, arte rupestre e deposições na pré-história recente da fachada ocidental do centro-norte português: das acções aos significados – ENARDAS / Natural spaces, architecture, rock art and depositions from the Late Prehistory of the Western front of Central and Northern Portugal: from actions to meanings (reference PTDC/HIS-ARQ/112983/2009) financed by the Operational Programme “Thematic Factors of Competitiveness” (COMPETE) and by the European Regional Development Fund (Fundo Europeu de Desenvolvimento Regional - FEDER).info:eu-repo/semantics/publishedVersio

Unit root tests for panel data: a survey and an application

The importance of a priori check of the existence of unit roots in the panel data comes from the already known effect that the presence of unit roots in time series may cause a misinterpretation of estimated results. Adding the cross-section dimension to the time series dimension offers an advantage in testing for nonstationary and cointegration since cross-section increases the data set used in those tests, thus improving their power. However, the cross-section dimension also brings some new problems into question, namely the existence of cross-section dependency which can bias usual panel data unit root test results in small samples. This paper presents a survey of panel unit root tests, evidencing the most recent developments on the issue, including those that account for the presence of contemporaneous cross-correlation as well as for the presence of heterogeneous serial correlation. Parallel to the developments of panel unit root tests, great attention has also been given to cointegration tests. We briefly review the most widely referred cointegration tests. We apply the reviewed panel unit root tests on an EU social variable which represents the population weight over than 65 years of age. We consider data running from 1970 to 2001. The panel unit root test results reveal to be sensitive to the prior assumptions regarding contemporaneous cross-correlation and heterogeneous serial correlation in small samples. The usual battery of panel unit root tests appear not to be adequate when a panel is composed by a mix of a stationary and nonstationary time series