Publications

Abstract This paper reports the values of the standard (p(o) = 0. 1 MPa) molar enthalpy of formation in the condensed, at T = 298.15 K, for 2-R-benzimidazoles (R = propyl, isopropyl), derived from, the respective enthalpies of combustion in oxygen, measured by static bomb combustion calorimetry and the standard molar enthalpies of sublimation, at T = 298.15 K, obtained using Calvet micro-calorimetry in the case of 2-isopropylbenzimidazole and, by the variation of vapour pressures, determined by the Knudsen effusion technique, with temperatures between (344 and 365) K for 2-propylbenzimidazole. Heat capacities, in the temperature ranges from T = 268 K to near their respective melting temperatures, T = 421 K for 2-propylbenzimidazole and T = 464 K for 2-isopropylbenzimidazole, were measured with a differential scanning calorimeter. These values were used to derive the standard molar enthalpies of formation, of the two 2-benzimidazole derivatives, in gaseous phase.

Abstract The photophysics of the single tyrosine in bovine ubiquitin (UBQ) was studied by picosecond time-resolved fluorescence spectroscopy, as a function of pH and along thermal and chemical unfolding, with the following results: First, at room temperature (25degreesC) and below pH 1.5, native UBQ shows single-exponential decays. From pH 2 to 7, triple-exponential decays were observed and the three decay times were attributed to the presence of tyrosine, a tyrosine-carboxylate hydrogen-bonded complex, and excited-state tyrosinate. Second, at pH 1.5, the water-exposed tyrosine of either thermally or chemically unfolded UBQ decays as a sum of two exponentials. The double-exponential decays were interpreted and analyzed in terms of excited-state intramolecular electron transfer from the phenol to the amide moiety, occurring in one of the three rotamers of tyrosine in UBQ. The values of the rate constants indicate the presence of different unfolded states and an increase in the mobility of the tyrosine residue during unfolding. Finally, from the pre-exponential coefficients of the fluorescence decays, the unfolding equilibrium constants (K-U) were calculated, as a function of temperature or denaturant concentration. Despite the presence of different unfolded states, both thermal and chemical unfolding data of UBQ could be fitted to a two-state model. The thermodynamic parameters T-m=54.6degreesC, DeltaH(Tm)=56.5 kcal/mol, and DeltaC(p)=890 cal/mol//K, were determined from the unfolding equilibrium constants calculated accordingly, and compared to values obtained by differential scanning calorimetry also under the assumption of a two-state transition, T-m=57.0degreesC, DeltaH(m)=51.4 kcal/mol, and DeltaC(p)=730 cal/mol//K.

Abstract The biocompatibility of implant materials used for substitution of bone tissue depends on its ability to induce the deposition of a hydroxyapatite layer when in contact with body fluids. In previous work, some of the authors found that bovine serum albumin (BSA) promotes calcium phosphate deposition if preadsorbed on hydroxyapatite and retards precipitation if preadsorbed on titania. In the present study, we investigated the adsorption of BSA upon particles of titania and hydroxyapatite in order to understand the different role played by the protein on the mineralization of both biomaterials. The adsorption isotherms were determined and the structural changes induced by adsorption at different surface coverages were investigated by circular dichroism spectroscopy and differential scanning microcalorimetry. At low surface coverages, the adsorbed BSA molecules lost part of their a-helix content. However, at high surface coverages, corresponding to the plateau values of the adsorption isotherms, the BSA molecules did not undergo structural rearrangements upon adsorption. In the latter circumstances, the availability of BSA calcium binding sites, which should be responsible for inducing mineralization, depends on the electrostatic interactions between BSA and the sorbent surface. A possible explanation for the different mineralization behavior of hydroxyapatite and titania is advanced. (C) 2004 Wiley Periodicals, Inc.

Abstract We report on a research/action project, to be developed on with a 9th form class, in a Portuguese school, in 2004/2005. The study focuses on ICT, emotional teacher-student relationship and cooperative learning. The research will focus on a class project, involving the community of students, teachers and parents and it integrates objectives from three non-disciplinary areas from the present curricula of the 3(rd) cycle of basic education. It will operate through regular e-mail communication among teachers, students and parents outside school activities. We will take advantage of the e-mail potential to facilitate the acquisition of emotional and cognitive proficiency, contributing to a better learning community. Teachers and parents will follow previous training in the use of email and in some theory or practical suggestions to promote collaborative learning and spelling out the emotional world.

Abstract VALIDATION OF SIMULTANEOUS DETERMINATION OF BROMIDES AND BROMATES BY ION CHROMATOGRAPHY IN DRINKING WATER. European Directive (98/83/CE), compulsory after 2008, states that bromate in drinking water must be controlled at levels below 10 mg L-1. Supporting implementation of the Directive, the European Comission has established project SMT4-CT96 2134, in collaboration with various european institutions, aiming at the identification of the interferents to the current analytical method (Ionic Chromatography with Conductimetric Detection - IC/CD), their removal and the automation of pretreatment and injection steps, as well as the development of alternative methods. EPAL, responsible for the water supply to a great deal of Portuguese regions, has taken steps to meet these requirements. Although not part of such project, this work (the result of a project conducted under a protocol of collaboration between EPAL SA and FOUL - Faculty of Sciences, University of Lisbon), reports on studies of usefulness to laboratories planning to monitor bromate in ozone treated waters, in conditions different from those described in EPA 300.1. Simultaneous determination of bromide is justified by its role as bromate precursor.

Abstract Isothermal titration calorimetry was used to characterize and quantify the partition of indomethacin and acemetacin between the bulk aqueous phase and the membrane of egg phosphatidylcholine vesicles. Significant electrostatic effects were observed due to binding of the charged drugs to the membrane, which implied the use of the Gouy-Chapman theory to calculate the interfacial concentrations. The binding/ partition phenomenon was quantified in terms of the partition coefficient (K-p), and/or the equilibrium constant (K-b). Mathematical expressions were developed, either to encompass the electrostatic effects in the partition model, or to numerically relate partition coefficients and binding constants. Calorimetric titrations conducted under a lipid/drug ratio [ 100: 1 lead to a constant heat release and were used to directly calculate the enthalpy of the process, DeltaH, and indirectly, DeltaG and DeltaS. As the lipid/drug ratio decreased, the constancy of reaction enthalpy was tested in the fitting process. Under low lipid/drug ratio conditions simple partition was no longer valid and the interaction phenomenon was interpreted in terms of binding isotherms. A mathematical expression was deduced for quanti. cation of the binding constants and the number of lipid molecules associated with one drug molecule. The broad range of concentrations used stressed the biphasic nature of the interaction under study. As the lipid/drug ratio was varied, the results showed that the interaction of both drugs does not present a unique behavior in all studied regimes: the extent of the interaction, as well as the binding stoichiometry, is affected by the lipid/drug ratio. The change in these parameters reflects the biphasic behavior of the interaction - possibly the consequence of a modification of the membrane's physical properties as it becomes saturated with the drug.

Abstract 2-O-alpha-Mannosylglycerate, a negatively charged osmolyte widely distributed among (hyper) thermophilic microorganisms, is known to provide notable protection to proteins against thermal denaturation. To study the mechanism responsible for protein stabilization, picosecond time-resolved fluorescence spectroscopy was used to characterize the thermal unfolding of a model protein, Staphylococcus aureus recombinant nuclease A ( SNase), in the presence or absence of mannosylglycerate. The fluorescence decay times are signatures of the protein state, and the pre-exponential coefficients are used to evaluate the molar fractions of the folded and unfolded states. Hence, direct determination of equilibrium constants of unfolding from molar fractions was carried out. Van't Hoff plots of the equilibrium constants provided reliable thermodynamic data for SNase unfolding. Differential scanning calorimetry was used to validate this thermodynamic analysis. The presence of 0.5 M potassium mannosylglycerate caused an increase of 7 degreesC in the SNase melting temperature and a 2-fold increase in the unfolding heat capacity. Despite the considerable degree of stabilization rendered by this solute, the nature and population of protein states along unfolding were not altered in the presence of mannosylglycerate, denoting that the unfolding pathway of SNase was unaffected. The stabilization of SNase by mannosylglycerate arises from decreased unfolding entropy up to 65 degreesC and from an enthalpy increase above this temperature. In molecular terms, stabilization is interpreted as resulting from destabilization of the denatured state caused by preferential exclusion of the solute from the protein hydration shell upon unfolding, and stabilization of the native state by specific interactions. The physiological significance of charged solutes in hyperthermophiles is discussed.

Abstract A novel amperometric glucose sensor was developed based on the facilitated proton transfer across microinterfaces between two immiscible electrolyte solutions. The combination of a 1,3:2,4-dibenzylidene sorbitol/2-nitrophenyl octyl ether gel membrane and 3-(2-pyridyl)-5,6diphenyl-1,2,4-triazine as the ionophore allows the transfer of protons from water to the gellified organic phase; the gel membrane is supported on arrays of microholes drilled on a polyester film. The protons are generated as the result of the dissociation of gluconic acid produced during the enzymatic degradation of glucose by glucose oxidase. The characteristics of the glucose sensor were investigated using several experimental conditions, namely, the concentration of ligand and enzyme. The electrochemical response is typical of an enzymatic electrode and displays a linear behavior in the range 0.2-3 mM glucose. The effect of the experimental parameters of the voltammetric technique was also optimized with the aimof improving sensor sensitivity.

Abstract Electrochemical impedance spectroscopy, Fourier transform infrared reflection-absorption spectroscopy, and cyclic voltammetry were employed to characterize polyelectrolyte multilayers (PEMs) fabricated with poly-(styrenesulfonate) as the polyanion and the polypeptides poly-L-histidine, poly-L-lysine, and poly-L-arginine as polycations. The layer-by-layer electrostatic assembly was produced onto alkanethiol-modified gold surfaces. The frequency response reveals that the effect of the number of layers seems to be related to a progressive reduction in the active area of the PEM-modified electrodes. The active area after the deposition of seven layers can be lower than 10% of its original value. The film surface is then inhomogeneous with respect to the transport of the electroactive species and has spots through which transport is quite favored. These structural features of the PEM have been taken into account in the theoretical model of ion transport and very good agreement with the experimental impedance results has been found.

Abstract The firefly luciferase reaction intermediate luciferyl adenylate was detected by RP-HPLC analysis when the luciferase reaction was performed under a nitrogen atmosphere. Although this compound is always specified as an intermediate in the light-production reaction, this is the first report of its identification by HPLC in a luciferase assay medium. Under a low-oxygen atmosphere, luciferase can catalyze the synthesis of luciferyl coenzyme A from luciferin, ATP, and coenzyme A, but in air dehydroluciferyl coenzyme A was produced. The luciferase-catalyzed synthesis of these coenzyme A derivatives may be a consequence of the postulated recent evolutionary origin of firefly luciferases from an ancestral acyl-coenzyme A synthetase.