Publications

Abstract A fluorescence flow injection analysis (FIA) methodology for nitric oxide (NO) quantification was optimized by factorial analysis for the lowest limit of detection of nitric oxide. This methodology is based on the reaction of the NO with the non-fluorescent reduced fluoresceinamine given a high fluorescent oxidized fluoresceinamine. Box-Behnken and central composite optimization experimental design methodologies were used. The factors initially analysed by a screening experimental design methodology were the flow rate of the pump (Q), loop volume (L), reactor length (R), reduced fluoresceinamine concentration (C-Fl) and cobalt chloride concentration (C-CoCl2). The response variables under analysis were the maximum fluorescence intensity, response repeatability and peak width. The optimum conditions were: one flow stream FIA configuration, Q = 0.60 mL min(-1), L = 100 mu L, R = 2 m, C-Fl = 1.50 mM and without CoCl2. A linear working range between 5 to 40 mu M was evaluated with a limit of detection of 1.20 mu M. Hydrogen peroxide, superoxide, nitrite and nitrate did not interfere with the NO detection. Good results were found in the quantification of NO liberated by a NO donor at pH 7.4 and in fortified serum samples.

Abstract Infections caused by Legionella spp. are considered at the present time, an emerging public health problem andare linked to high rates of mortality and morbidity, if not properly treated. In this study were analyzed 54 samples of waterfrom 8 counties at Northern Portugal, with the aim of obtaining a collection of strains of the genus Legionella and to characterizethem genetically and phenotypically. Another objective of this study was to evaluate the effectiveness of the techniqueof cultivation, a standard method according to International Organization for Standardization ISO 11731:1998, fordetection and enumeration of species of Legionella. For laboratory processing, after the filtration of samples (1 L), the filtratewas resuspended in sterile distilled water (5 ml). Heat treatment for selective inhibition of non-Legionella bacteria wasperformed. Subsequently, 100 l of the suspension was spread in GVPC selective agar medium, and incubated (7 to 10 days)at 37 . Colonies that were morphologically characteristic of the genus were sub-cultured onto BCYE agar and blood agarfor verification. According to the procedure recommended by the standard method, only the colonies which grew in BCYEagar and not on blood agar were considered as suspected Legionella strains. The identification of these initially selectedcolonies was performed by sequencing the 16S rRNA gene, which revealed that none of the isolates were identified as belongingto the genus Legionella. However, through the ISO 11731:1998 they were interpreted as positive, correspondingtherefore to false-positive results. The methods used in this study allowed the isolation of a number of isolates (40), whichform an independent group of all genus of the family Chitinophagaceae outlined so far, and that by their phylogenetic distancemight be a genus not yet described and therefore a new species. The results obtained, highlighted the importance ofusing culture and genetic methods in parallel for the proper identification of microorganisms

Abstract A new fluorescent analytical methodology for the quantification of peroxynitrite (ONOO-) in the presence of nitric oxide (NO) was developed. The quantification of ONOO- is based in the oxidation of the non-fluorescent reduced fluoresceinamine to a high fluorescent oxidized fluoresceinamine in reaction conditions where the interference of NO is minimized. Screening factorial experimental designs and optimization Box-Behnken experimental design methodologies were used in order to optimize the detection of ONOO- in the presence of NO. The factors analysed were: reduced fluoresceinamine concentration (C (Fl) ); cobalt chloride concentration (C (CoCl2) ); presence of oxygen (O (2) ); and, the pH (pH). The concentration of sodium hydroxide (C (NaOH) ) needed to diluted the initially solution of ONOO- was also evaluated. An optimum region for ONOO- quantification where the influence of NO is minimal was identified - C (Fl) from 0.50 to 1.56 mM, C (CoCl2) from 0 to 1.252 x 10(-2) M, pH from 6 to 8 and C (NaOH) 0.10 M. Better results were found in the presence of NO at pH 7.4, C (Fl) 0.5 mM, without oxygen, without cobalt chloride and with a previous dilution of peroxynitrite solution with C (NaOH) 0.1 M. This methodology shows a linear range from 0.25 to 40 mu M with a limit of detection of 0.08 mu M. The bioanalytical methodology was successfully applied in the ONOO- quantification of fortified serum and macrophage samples.

Abstract In this work, ion transfer and facilitated ion transfer of ammonium ion by a lipophilic cyclodextrin is investigated at the water/1,6-dichlorohexane micro-interface, using electrochemical approaches (cyclic voltammetry, differential pulse voltammetry and square wave voltammetry). The association constant has been obtained for the complex between ammonium ion and the cyclodextrin. Experimental conditions for the analytical determination of ammonium ion were established and a detection limit of 0.12 mu M was obtained. The amperometric sensor gave a current response proportional to the ammonium ion concentration in the range from 4.2 to 66 mu M.

Abstract Finite element simulations were used to investigate the effect of a smooth variation of permittivity across a polarized liquid/liquid interface on the differential capacitance. The results show that a relative permittivity profile can account for the variation of ion solvation in the interfacial region, and therefore upon the diffuse double layer itself. The width and the symmetry of this profile across the interface are shown to be crucial parameters for interfacial distributions and fitting of capacitance data has been used to estimate the width of the interfacial region.

Abstract Stroke is a problem that affects more than 15 million people worldwide. It is the third most common cause of death and is also an important cause of morbidity and of severe long-term disability. In this chapter, stroke mechanisms are outlined and particular attention is given to the oxidative and nitrosative stress, essentially to the role of the reactive oxygen (ROS) and nitrogen (RNS) species in stroke events. Following this approach, the most recent literature relative to success and pitfalls of the use of antioxidants of natural (including endogenous and those from diet) or synthetic origin in stroke is herein reviewed. Antioxidant therapies have enjoyed general success in preclinical studies, across disparate animal models, but little benefit in human intervention studies or clinical trials. This mismatch has been often attributed both to limitations of the animal models and to pitfalls in the clinical trial design. Finally, some ADME/Tox problems related to a number of drawbacks were also discussed. © Springer Science+Business Media, LLC 2012.

Abstract Direct and continuous measurement of dissolved CO2 (dCO(2)) is crucial for intensive aquaculture, especially in shallow raceway systems (SRS). In this work the performance of a portable dissolved CO2 probe analyzer was tested for the effects of different aqueous solutions, pure oxygen injection and agitation. Laboratory results showed significant (p < 0.05) solution effects on probe performance for low (10-20 mg L-1) and high (30-50 mg L-1) dCO(2) concentrations. Globally performance was better in deionized water, followed by marine fish farm water and artificial seawater. Accuracy and response time were the parameters most affected by the type of solution tested. Linearity was always observed (R-2 = 0.995-0.999). The probe was sensitive to 1 mg L-1 dCO(2) increments for concentrations <6 mg L-1 in artificial seawater. Pure oxygen injection did not affect probe readouts, and agitation was needed for better accuracy and response time. In real marine SRS with tanks in series dCO(2) dynamics was revealed using the probe coupled to a developed flow cell. A prototype SRS was built and used to study dCO(2) dynamics without endangering cultivated fish. Generally, results obtained indicate that the probe tested although precise, is better suited for discrete, single-point dCO(2) monitoring, being a limited resource for the special needs of shallow raceway systems. As SRS represent a paradigm change in aquaculture, new water quality monitoring strategies and instrumentation are needed, especially for dCO(2). Fiber optic sensors can be a solution for continuous, multipoint monitoring, thus contributing to the understanding of water quality dynamics in hyperintensive aquaculture systems.