Raquel Sousa

Dose-dependent expression of neuronal injury markers during experimental osteoarthritis induced by monoiodoacetate in the rat

BackgroundIt was recently reported that the mono-iodoacetate (MIA) experimental model of osteoarthritis (OA) courses with changes of neurons innervating the affected joints that are commonly interpreted as a neuronal response to axonal injury. To better characterize these changes, we evaluated the expression of two markers of neuronal damage, ATF-3 and NPY, and the growth associated protein GAP-43, in primary afferent neurons of OA animals injected with three different doses of MIA (0.3, 1 or 2u2009mg). Measurements were performed at days 3, 7, 14, 21 and 31 post-MIA injection.ResultsOA animals showed the characteristic histopathological changes of the joints and the accompanying nociceptive behaviour, evaluated by the Knee-Bed and CatWalk tests. An increase of ATF-3 expression was detected in the DRG of OA animals as early as 3u2009days after the injection of 1 or 2u2009mg of MIA and 7u2009days after the injection of 0.3u2009mg. NPY expression was increased in animals injected with 1 or 2u2009mg of MIA, at day 3 or in all time-points, respectively. From day 7 onwards there was a massive increase of GAP-43 expression in ATF-3 cells.ConclusionsThe expression of the neuronal injury markers ATF-3 and NPY as well as an up-regulation of GAP-43 expression, indicative of peripheral fibre regeneration, suggests that axonal injury and a regeneration response may be happening in this model of OA. This opens new perspectives in the unravelling of the physiopathology of the human disease.

In vitro exposure of acer negundo pollen to atmospheric levels of SO2 and NO2: effects on allergenicity and germination

In the last years, a rising trend of pollen allergies in urban areas has been attributed to atmospheric pollution. In this work, we investigated the effects of SO(2) and NO(2) on the protein content, allergenicity, and germination rate of Acer negundo pollen. A novel environmental chamber was assembled to exposure pollen samples with SO(2) or NO(2) at two different levels: just below and two times the atmospheric hour-limit value acceptable for human health protection in Europe. Results showed that protein content was lower in SO(2)-exposed pollen samples and slightly higher in NO(2)-exposed pollen compared to the control sample. No different polypeptide profiles were revealed by SDS-PAGE between exposed and nonexposed pollen, but the immunodetection assays indicated higher IgE recognition by all sera of sensitized patients to Acer negundo pollen extracts in all exposed samples in comparison to the nonexposed samples. A decrease in the germination rate of exposed in contrast to nonexposed pollen was verified, which was more pronounced for NO(2)-exposed samples. Our results indicated that in urban areas, concentrations of SO(2) and NO(2) below the limits established for human protection can indirectly aggravate pollen allergy on predisposed individuals and affect plant reproduction.

Optimisation and application of dispersive liquid–liquid microextraction for simultaneous determination of carbamates and organophosphorus pesticides in waters

A very simple and sensitive method was developed for the simultaneous determination of carbamate and organophosphorus pesticides in water samples, using dispersive liquid–liquid microextraction (DLLME) coupled with gas chromatography-mass spectrometry (GC-MS). Carbofuran and chlorfenvinphos were used as model molecules in this study. The optimisation of some important DLLME parameters (type and volume of extraction and dispersive solvents, sample volume, extraction time, and use of NaCl) was performed using a design of experiments approach. Under optimised conditions of the DLLME methodology (500 μL of methanol, 80 μL of chlorobenzene, 10 mL of aqueous sample, 4 minutes of vortexing and no salt addition), the calibration curve was linear in the concentration range of 0.1–250 μg L−1 for carbofuran and 0.1–200 μg L−1 for chlorfenvinphos. The limits of detection (LODs) were 0.04 μg L−1 for the carbamate and 0.02 μg L−1 for the organophosphorus pesticide. The proposed method was successfully applied to the analysis in river, mineral and tap water samples, yielding satisfactory results.

Changes in the IgE-reacting protein profiles of Acer negundo, Platanus x acerifolia and Quercus robur pollen in response to ozone treatment

This study aims to investigate the effects of O3 in protein content and immunoglobulin E (IgE)-binding profiles of Acer negundo, Platanus x acerifolia and Quercus robur pollen. Pollen was exposed to O3 in an environmental chamber, at half, equal and four times the limit value for the human health protection in Europe. Pollen total soluble protein was determined with Coomassie Protein Assay Reagent, and the antigenic and allergenic properties were investigated by SDS-PAGE and immunological techniques using patients’ sera. O3 exposure affected total soluble protein content and some protein species within the SDS-PAGE protein profiles. Most of the sera revealed increased IgE reactivity to proteins of A. negundo and Q. robur pollen exposed to the pollutant compared with the non-exposed one, while the opposite was observed in P. x acerifolia pollen. So, the modifications seem to be species dependent, but do not necessarily imply that increase allergenicity would occur in atopic individuals.

Ozone effects on soluble protein content of Acer negundo, Quercus robur and Platanus spp. pollen

In this study, the effect of three different ozone (O3) levels on soluble protein content of Acer negundo, Quercus robur and Platanus spp. pollen was investigated. Pollen was directly collected from each plant and exposed to O3 values below, equal and four times the limit for human health protection in an environmental chamber system. With this chamber, it was possible to control and reproduce the O3 levels between different experiments. Total protein content was determined colorimetrically with Coomassie protein assay reagent (Pierce) by the Bradford method. Results showed that O3 has significant effects on pollen causing a decrease in soluble proteins that seem to be dependent on species and gas concentration. Also, the existence of reproducibility in the effects of O3, within the same concentration, on the protein content of pollen was verified.

Contribution of EPMA to airborne pollen analysis

As a component of aerosol, pollen is found in suspension with other mineralogical and anthropogenic particles that can adhere to the pollen wall. The aim of this study was to determine possible alterations that pollen grains suffer under different meteorological conditions in the coastal city of Porto, Portugal. For this study, 2 airborne pollen types were taken into account: Poaceae and Alnus spp sampled in July 2010 and February 2011, respectively. Quantitative analysis and X-ray map analysis were performed with a field emission electron probe microanalyzer (EPMA) equipped with 5 WDS spectrometers. Samples were mounted over adhesive carbon tape and carbon coated. Control pollen showed mainly the presence of C, O, N, P, K, Mg, S, Cl and Ca in different quantities, depending on the considered species. Airborne pollen consistently revealed a higher content of Mg and Cl than control pollen, while Si, Ca, K and P contents varied differently according to the samples. Our results show that during their flight, pollen grains acquire an external coating, become heavier and change significantly their composition.

Response surface optimisation applied to a headspace-solid phase microextraction-gas chromatography-mass spectrometry method for the analysis of volatile organic compounds in water matrices

A new method for the simultaneous determination of 12 volatile organic compounds (trans-1,2-dichloroethene, 1,1,1-trichloroethane, benzene, 1,2-dichloroethane, trichloroethene, toluene, 1,1,2-trichloroethane, tetrachloroethene, ethylbenzene, m-, p-, o-xylene) in water samples by headspace solid phase microextraction (HS–SPME)–gas chromatography mass spectrometry (GC–MS) was described, using a 100u2009µm PDMS (polydimethylsiloxane) coated fibre. The response surface methodology was used to optimise the effect of the extraction time and temperature, as well as the influence of the salt addition in the extraction process. Optimal conditions were extraction time and temperature of 30u2009min and −20°C, respectively, and NaCl concentration of 4u2009molu2009L−1. The detection limits were in the range of 1.1u2009×u200910−3–2.3u2009µgu2009L−1 for the 12 volatile organic compounds (VOCs). Global uncertainties were in the range of 4–68%, when concentrations decrease from 250u2009µgu2009L−1 down to the limits of quantification. The method proved adequate to detect VOCs in six river samples.

Airborne Platanus Pollen Analysis by EPMA

Particles of anthropogenic and biological origin, minerals and gaseous elements are the major constituents of the aerosol. Pollen is a biological component of the aerosol, found in suspension with other pollutants. In recent years, research has been conducted on the crossed effects between non-biological and biological pollutants on allergy worsening in susceptible individuals or on the development of symptoms in non-symptomatic ones. Platanus spp pollen is abundantly present in the atmosphere of Porto in early spring and is known to promote allergic respiratory symptoms. The aim of this study was to characterize possible alterations of the main elements present on the Platanus spp. pollen wall under different atmospheric conditions in the coastal city of Porto, Portugal, using EPMA.