Publications

Abstract The use of a good chemical calibration or test reaction in Isothermal Titration Calorimetry is crucial for getting reliable enthalpy values that can be compared across different laboratories. Indeed most titration calorimeters are used to measure both equilibrium constants and molar enthalpies of reaction. But a necessary prerequisite for such measurements is to first perform the enthalpy measurement accurately and precisely. The values of the equilibrium constant(s) are then calculated by regression from an appropriate model. As such, we found it timely to extensively test a previously proposed test reaction, the dilution of propan-1-ol into water, using two calorimeters of different design (heat conduction and power compensation calorimeters) and sensitivity. Experiments were performed at 298.15 K for the previously suggested 10% mass fraction propan-1-ol solution, as well as for the lower concentrations of 5% and 2% mass fractions. Due to our capacity to use insertion heaters with one of the used calorimeters, which allows for very accurate calibration constants to be obtained, we also determined a value for the enthalpy of dilution of 10% mass fraction solution at 308.15 K, previously not available, and closer to the temperatures commonly used in titration experiments involving biological samples. The observed change in the enthalpy of dilution was found to decrease in absolute value, but to an extent that did not justify the determination of new values for the less concentrated solutions. The values obtained with the two calorimeters are in excellent agreement, as well as with the values from the literature for the 10% mass fraction solution at 298.15 K. This reaction is thus again proposed as an excellent test reaction and the detailed conditions of their use depending on instrument sensitivity are suggested. In summary, the values for the enthalpies of dilution to infinite dilution Delta H-dil(m)infinity at 298.15 K are -(1.540 +/- 0.021) kJ . mol (1), -(0.604 +/- 0.020) kJ . mol (1), and -(0.186 +/- 0.011) kJ . mol (1) for the 10%, 5%, and 2% mass fraction solutions, respectively, and at 308.15 K -(1.486 +/- 0.017) kJ . mol (1) for the 10% mass fraction solution.

Abstract Liposome dispersions obtained from the mixture of gemini surfactants of the type alkane-alpha,omega-diyl-bis(alkyl-dimethylammonium bromide) and helper lipid DOPC create complexes with DNA showing a regular inner microstructure, identified by small angle X-ray diffraction as condensed lamellar phase (L-alpha(c)). In addition to the L-alpha(c) phase, a coexisting lamellar phase L-B was also identified in the complexes formed, with periodicities in the range similar to 8.8-5.7 nm, at ionic strengths corresponding to 50-200 mM NaCl. The periodicities of L-B phase did not correspond to those identified in liposome dispersion without DNA using small angle neutron scattering. The observed phase separation is shown to depend on the interplay between the surface charge density of cationic liposomes, ionic strength and method of complex preparation. The effect of ionic strength on complex formation was studied by isothermal titration calorimetry and zeta potential measurements. High ionic strength reduces the fraction of bound DNA in the complexes, and the isoelectric point is attained at a ratio of DNA/gemini surfactant which is lower than the one that can be estimated by calculation based on nominal charges of CLs and DNA.

Abstract The use of deep eutectic solvents for metal electrodeposition has become an area of interest in the recent years. In this study, ethaline, propeline, and reline were used as solvents for the electrodeposition of Sn-Zn alloys. Ethaline, propeline, and reline displayed identical voltammetric profiles for the reduction of Zn(II) and Sn(II). Further studies were carried out in ethaline which is the liquid with lowest viscosity. To improve physical and morphological properties of the Sn-Zn deposits, additives were added to the ionic liquid solution. In this study, the addition of three chelators (EDTA, HEDTA, and Idranal VII) and their effects on the voltammetric behavior of zinc and tin and the resultant morphology was described. The structure and composition of the Zn-Sn deposit was largely affected by the additives with the largest effect being obtained in the presence of Idranal VII.

Abstract The treatment of a highly concentrated pesticide and systemic fungicide solution, containing metalaxyl as active compound (150 mg L-1), was investigated by using the photo-Fenton process in combination with Solanum nigrum L weed plants. High concentration of metalaxyl has a destructive effect on S. nigrum plants although this pesticide can be successfully degraded by the photo-Fenton process. S. nigrum seed germination and seedling development were not affected by iron species at concentrations lower than 11 mg L-1, which is well above the optimal dosage for the photo-Fenton process (4 mg L-1). In addition, at the referred optimal dosage, S. nigrum growth was not affected and those plants were able to extract large amounts of iron species from the solution (between 1.5-2.0 and 0.2-0.5 mg of Fe per gram of dry weight of plant in their roots and shoots, respectively). Negative effects on biomass production and chlorophyll and carotenoids contents, used as stress biomarkers, were not detected. When S. nigrum plants were directly exposed to the solution pre-treated by the photo-Fenton process, an increase in glutathione-S-transferase activity in shoots, some toxicity towards the plant physiology and a decrease in the total soluble protein content, in both shoots and roots, were observed; nevertheless, the plant growth was still remarkable. These results suggest that the photo-Fenton process can be used as a chemical primary treatment, followed by S. nigrum plants biological remediation (as post-treatment) for waste waters that are contaminated with concentrations of metalaxyl as high as 150 mg L-1.

Abstract The activity of two phenolic acids, gallic acid (GA) and ferulic acid (FA) at 1000 mu g ml(-1), was evaluated on the prevention and control of biofilms formed by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes. In addition, the effect of the two phenolic acids was tested on planktonic cell susceptibility, bacterial motility and adhesion. Biofilm prevention and control were tested using a microtiter plate assay and the effect of the phenolic acids was assessed on biofilm mass (crystal violet staining) and on the quantification of metabolic activity (alamar blue assay). The minimum bactericidal concentration for P. aeruginosa was 500 mu g ml(-1) (for both phenolic acids), whilst for E. coli it was 2500 mu g ml(-1) (FA) and 5000 mu g ml(-1) (GA), for L. monocytogenes it was >5000 mu g ml(-1) (for both phenolic acids), and for S. aureus it was 5000 mu g ml(-1) (FA) and 45000 mu g ml(-1) (GA). GA caused total inhibition of swimming (L. monocytogenes) and swarming (L. monocytogenes and E. coli) motilities. FA caused total inhibition of swimming (L. monocytogenes) and swarming (L. monocytogenes and E. coli) motilities. Colony spreading of S. aureus was completely inhibited by FA. The interference of GA and FA with bacterial adhesion was evaluated by the determination of the free energy of adhesion. Adhesion was less favorable when the bacteria were exposed to GA (P. aeruginosa, S. aureus and L. monocytogenes) and FA (P. aeruginosa and S. aureus). Both phenolics had preventive action on biofilm formation and showed a higher potential to reduce the mass of biofilms formed by the Gram-negative bacteria. GA and FA promoted reductions in biofilm activity >70% for all the biofilms tested. The two phenolic acids demonstrated the potential to inhibit bacterial motility and to prevent and control biofilms of four important human pathogenic bacteria. This study also emphasizes the potential of phytochemicals as an emergent source of biofilm control products.

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.