Three ultrasonic energy sources were studied to speed up the sample treatment for in-solution protein identification by peptide mass fingerprint using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Protein reduction, alkylation, and enzymatic digestion steps were done in 15 min. Nine proteins, including zinc resistance-associated protein precursor from Desulfovibrio desulfuricans strain G20 and split-soret cytochrome c from D. desulfuricans ATCC27774 were successfully identified with the new protocol.

Cognitive task automation may lead to over trust, complacency and loss of the necessary work environment situation awareness. This is a major constraint in complex work organizations teamwork, ending up into an operational gap, between system developments and its understanding and usability, by operators. This document presents a summary of the main results of author’s research on operational decision processes and occupational competences, applied to the air traffic control operational reality. Introducing a human/technological complementary approach to virtual team’s conceptualisation, the results show there is a dimension to be followed in human/machine integration, which stands beyond interface design, and calls for a deeper human comprehension of technological agent’s structure and functionalities, which will, ultimately, require the development of an operational cognitive framework, where work processes and technological behaviour are integrated in professional competences, as he two faces of the same coin.

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No abstract is available for this item.

The WORKS Project started two years ago (2005), involving the efforts of research institutes of 13 European countries with the main purpose of improving the understanding of the major changes in work in the knowledge-based society, taking account both of global forces and the regional diversity within Europe. This research meeting in Sofia (Bulgaria) aimed to present synthetically the massive amount of data collected in the case studies (occupational and organisational) and with the quantitative research during last year.

No abstract is available for this item.

The WORKS Project started two years ago (2005), involving the efforts of research institutes of 13 European countries with the main purpose of improving the understanding of the major changes in work in the knowledge-based society, taking account both of global forces and the regional diversity within Europe. This research meeting in Sofia (Bulgaria) aimed to present synthetically the massive amount of data collected in the case studies (occupational and organisational) and with the quantitative research during last year.

Various S = 3/2 EPR signals elicited from wild-type and variant Azotobacter vinelandii nitrogenase MoFe proteins appear to reflect different conformations assumed by the FeMo-cofactor with different protonation states. To determine whether these presumed changes in protonation and conformation reflect catalytic capacity, the responses (particularly to changes in electron flux) of the alpha H195Q, alpha H195N, and alpha Q191 K variant MoFe proteins (where His at position 195 in the alpha subunit is replaced by Gln/Asn or Gln at position alpha-191 by Lys), which have strikingly different substrate-reduction properties, were studied by stopped-flow or rapid-freeze techniques. Rapid-freeze EPR at low electron flux (at 3-fold molar excess of wild-type Fe protein) elicited two transient FeMo-cofactor-based EPR signals within 1 s of initiating turnover under N-2 with the alpha H195Q and alpha H195N variants, but not with the alpha Q191K variant. No EPR signals attributable to P cluster oxidation were observed for any of the variants under these conditions. Furthermore, during turnover at low electron flux with the wild-type, alpha H195Q or alpha H195N MoFe protein, the longer-time 430-nm absorbance increase, which likely reflects P cluster oxidation, was also not observed (by stopped-flow spectrophotometry); it did, however, occur for all three MoFe proteins under higher electron flux. No 430-nm absorbance increase occurred with the alpha Q191K variant, not even at higher electron flux. This putative lack of involvement of the P cluster in electron transfer at low electron flux was confirmed by rapid-freeze Fe-57 Mossbauer spectroscopy, which clearly showed FeMo-factor reduction without P cluster oxidation. Because the wild-type, alpha H195Q and alpha H195N MoFe proteins can bind N-2, but alpha Q195K cannot, these results suggest that P cluster oxidation occurs only under high electron flux as required for N-2 reduction. (C) 2007 Elsevier Inc. All rights reserved.

The EPR characterization of the molybdenum(V) forms obtained on formate reduction of both as-prepared and inhibited formate dehydrogenase from Desulfovibrio desulfuricans ATCC 27774, an enzyme that catalyzes the oxidation of formate to CO(2), is reported. The Mo(V) EPR signal of the as-prepared formate-reduced enzyme is rhombic (g(max)=2.012, g(mid)=1.996, g(min)=1.985) and shows hyperfine coupling with two nuclear species with I=1/2. One of them gives an anisotropic splitting and is not solvent exchangeable (A(max)=11.7, A(mid)=A(min)=non-detectable, A-values in cm(-1)x10(-4)). The second species is exchangeable with solvent and produces a splitting at the three principal g-values (A(max)=7.7, A(mid)=10.0, A(min)=9.3). The hyperfine couplings of the non-solvent and solvent exchangeable nuclei are assigned to the hydrogen atoms of the beta-methylene carbon of a selenocysteine and to a Mo ligand whose nature, sulfydryl or hydroxyl, is still in debate. The Mo(V) species obtained in the presence of inhibitors (azide or cyanide) yields a nearly axial EPR signal showing only one detectable splitting given by nuclear species with I=1/2 (g(max)=2.092, g(mid)=2.000, g(min)=1.989, A(max)=non-detectable, A(mid)=A(min)=7.0), which is originated from the alpha-proton donated by the formate to a proximal ligand of the molybdenum. The possible structures of both paramagnetic molybdenum species (observed upon formate reduction in presence and absence of inhibitors) are discussed in comparison with the available structural information of this enzyme and the structural and EPR properties of the closely related formate dehydrogenase-H from Escherichia coli.

In this working paper is presented information on the Portuguese labour market developed with the support of the European project WORKS-“Work organisation and restructuring in the knowledge society”. Is still a on the process article and thus commentaries are welcome. The structure is based on the following topics: a) The employment policy (Time regimes - time use, flexibility, part-time work, work-life balance -, and the work contracts regimes – wages, contract types, diversity); b) Education and training (skilling outcomes, rules on retraining and further training, employability schemes, transferability of skills); c) Equal opportunities (relevance of equal opportunity regulation for restructuring outcomes, the role of gender and age regulation); d) Restructuring effects (policy on transfer of personnel, policy on redundancies, and participation or voice in restructuring).

In this working paper is presented information on the Portuguese labour market developed with the support of the European project WORKS-“Work organisation and restructuring in the knowledge society”. Is still a on the process article and thus commentaries are welcome. The structure is based on the following topics: a) The employment policy (Time regimes - time use, flexibility, part-time work, work-life balance -, and the work contracts regimes – wages, contract types, diversity); b) Education and training (skilling outcomes, rules on retraining and further training, employability schemes, transferability of skills); c) Equal opportunities (relevance of equal opportunity regulation for restructuring outcomes, the role of gender and age regulation); d) Restructuring effects (policy on transfer of personnel, policy on redundancies, and participation or voice in restructuring).

This work describes the construction and voltammetric characterization of a nitrite biosensor based on a cytochrome c-type nitrite reductase (ccNiR) and the Nafion ionomeric matrix loaded with methyl viologen as redox mediator. Despite the potential electrostatic repulsions between the anionic substrate and the Nafion sulfonate groups, the resulting bioelectrode exhibited electrocatalytic activity toward nitrite. This phenomenon must be due to the nonuniformity of the enzyme/Nafion membrane, which allows the direct interaction between the substrate and numerous enzyme molecules. Nevertheless, the anionic nature of Nafion exerted a certain diffusion barrier to nitrite, as revealed by the unusually elevated limits of the linear dynamic range and k(m)(app). The irregularity of the composite membrane also contributed to slow down the rate of charge transfer throughout the Nafion polymer. The level of viologens incorporated within the Nafion membrane had a strong influence in the analytical parameters: as much mediator was present, lower was the sensitivity and wider was the linear range. For an optimized ratio enzyme/mediator the sensitivity was 445+/-8 mA M(-1)cm(-2), within the linear range 75-800 microM; the lowest detected nitrite concentration was 60 microM. The operational stability of the biosensor and the influence of some possible interferences were evaluated.

A method is reported for the determination of acaricides (amitraz, bromopropylate, coumaphos and fluvalinate) from honey by gas chromatography mass spectrometry after a new fast solid phase micro-extraction, SPME, procedure. Six different fibers were assessed for micro-extraction purpose studying the following variables: (i) SPME coating, (ii) extraction temperature, (iii) extraction time, (iv) desorption conditions and (v) agitation conditions. The new ultrasonic bath technology providing different sonication frequencies (35 and 130 kHz) and different working modes (Sweep, Standard and Degas) was studied and optimized for speeding up the acaricide micro-extraction. The best extraction results were achieved with the polyacrylate fiber. The extraction process was done in 30 min using the ultrasonic bath at 130 kHz in the Standard mode. Quality parameters of the proposed method show a good precision (<11%) and detection and quantitation limits lower than 6 and 15 ng/g, respectively, except for fluvalinate. Eleven Portuguese commercial honey samples were analyzed with the developed method in order to assess the performance of the method with real samples and to determine whether the concentration of acaricides in honey exceed their maximum residue levels (MRLs). Acaricide residues detected were lower than those established by the legislation. (c) 2006 Elsevier B.V. All rights reserved.

This work describes the construction and evaluation of lactate sol-gel biosensors to accomplish the determination of lactate in pharmaceutical products. Lactate oxidase was incorporated in a porous sol-gel film placed onto a platinum-based electrode. Acid and basic catalysis were assessed. When coupled to a sequential injection system (SIA) the biosensor, based on (3-aminopropyl)trimethoxysilane, 2-(3,4-epoxycyclohexyl)ethyl-trimethoxysilane, deionised water, polyethylene glycol 6000 and acid catalyst, presented a range of linearity of 5x10(-5) to 5x10(-3)M. The analytical usefulness of the developed biosensor was evaluated through analysis of commercial pharmaceutical products containing lactate with a sampling rate of 40 samples h(-1). The enzyme remained active for at least 30 days, enabling about 700 determinations without sensitivity decrease.

Two arsenite-inhibited forms of each of the aldehyde oxidoreductases from Desulfovibrio gigas and Desulfovibrio desulfuricans have been studied by X-ray crystallography and electron paramagnetic resonance (EPR) spectroscopy. The molybdenum site of these enzymes shows a distorted square-pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. Arsenite addition to active as-prepared enzyme or to a reduced desulfo form yields two different species called A and B, respectively, which show different Mo(V) EPR signals. Both EPR signals show strong hyperfine and quadrupolar couplings with an arsenic nucleus, which suggests that arsenic interacts with molybdenum through an equatorial ligand. X-ray data of single crystals prepared from EPR-active samples show in both inhibited forms that the arsenic atom interacts with the molybdenum ion through an oxygen atom at the catalytic labile site and that the sulfido ligand is no longer present. EPR and X-ray data indicate that the main difference between both species is an equatorial ligand to molybdenum which was determined to be an oxo ligand in species A and a hydroxyl/water ligand in species B. The conclusion that the sulfido ligand is not essential to determine the EPR properties in both Mo-As complexes is achieved through EPR measurements on a substantial number of randomly oriented chemically reduced crystals immediately followed by X-ray studies on one of those crystals. EPR saturation studies show that the electron transfer pathway, which is essential for catalysis, is not modified upon inhibition.

Some programs and research networks are dealing with topics associated to the automtive sector and they observe one of the most significative sectores of modern economies. Because of that is so interesting to forecast some possible changes in an horizon of 10 to 20 years. But this exercse must not be centred only on technical aspects of automobile construction, or on the design, or even on the mobility systems. How one can face these problems, and how it can be so decisivelly important, is what we will try to answer in this paper that resumes some of the debates on the foresight exercises in Germany (Futur) and on the automotive sector in Portugal (WorTiS).

Some of the phenomena where the “globalization” concept is applied include the internationalization of markets, globalization of culture, pol{\'ıtical hegemony of world by some states, or groups of states, the increasing power of supranational institutions, and the development of a global division of labour. A starting point to understand the global division of work is the study of how companies are re-structuring, once they are the key-actors in the decision on which work should be found and where. The “value chains” describe each step in the productive process of a final product or service. Separated units of value chains can be in the same company (in-house) or in different companies (outsourced). Similarly they can be in a same local, or in other location. Normalization of business processes, combined with digitalization of information and the development of telecommunication networks made possible the tele-mediated work. This paper presents results from the European WORKS project, where are studied Portuguese cases of firms that integrate globalized value chain, and are analized the implications on work organization models and the (new) professional structures.

Advances in nanoscience are having a significant impact on many scientific fields, boosting the development of a variety of important technologies. The impact of these new technologies is particularly large in biodiagnostics, where a number of nanoparticle-based assays have been introduced for biomolecular detection. The physicochemical malleability and high surface areas of nanoparticle surfaces make them ideal candidates for developing biomarker platforms. Given the variety of strategies afforded through nanoparticle technologies, a significant goal is to tailor nanoparticle surfaces to selectively bind a subset of biomarkers, either for direct detection and characterization or to sequester the target molecules for later study using other available techniques. To date, applications of nanoparticles have largely focused on DNA- or protein-functionalized gold nanoparticles used as the target-specific probes. These unique biophysical properties displayed by gold nanoparticles have huge advantages over conventional detection methods (e.g., molecular fluorophores, microarray technologies). These gold-nanoparticle based systems can then be used for the detection of specific sequences of DNA (pathogen detection, characterization of mutation and/or SNPs) or RNA (without previous retro-transcription and amplification.

Nesta comunica{\c c}ão em que se discutem, numa perspectiva de complementaridade, os conceitos de qualifica{\c c}ão e de competência profissional, dá-se conta da metodologia adoptada para a identifica{\c c}ão e valida{\c c}ão de um conjunto de competências profissionais num sistema complexo de trabalho. O estudo incidiu sobre os Servi{\c c}os de Controlo de Tráfego Aéreo da Região de Informa{\c c}ão de Voo de Lisboa e permitiu concluir pela existência de uma dimensão integrativa das componentes trabalho e tecnologia, enquanto elementos estruturantes dos mapas de competências profissionais, indispensáveis à integra{\c c}ão sistémica dos diferentes agentes (humanos e tecnológicos) no processo produtivo.

Some programs and research networks are dealing with topics associated to the automtive sector and they observe one of the most significative sectores of modern economies. Because of that is so interesting to forecast some possible changes in an horizon of 10 to 20 years. But this exercse must not be centred only on technical aspects of automobile construction, or on the design, or even on the mobility systems. How one can face these problems, and how it can be so decisivelly important, is what we will try to answer in this paper that resumes some of the debates on the foresight exercises in Germany (Futur) and on the automotive sector in Portugal (WorTiS).

Some of the phenomena where the “globalization” concept is applied include the internationalization of markets, globalization of culture, polítical hegemony of world by some states, or groups of states, the increasing power of supranational institutions, and the development of a global division of labour. A starting point to understand the global division of work is the study of how companies are re-structuring, once they are the key-actors in the decision on which work should be found and where. The “value chains” describe each step in the productive process of a final product or service. Separated units of value chains can be in the same company (in-house) or in different companies (outsourced). Similarly they can be in a same local, or in other location. Normalization of business processes, combined with digitalization of information and the development of telecommunication networks made possible the tele-mediated work. This paper presents results from the European WORKS project, where are studied Portuguese cases of firms that integrate globalized value chain, and are analized the implications on work organization models and the (new) professional structures.

Nesta comunicação em que se discutem, numa perspectiva de complementaridade, os conceitos de qualificação e de competência profissional, dá-se conta da metodologia adoptada para a identificação e validação de um conjunto de competências profissionais num sistema complexo de trabalho. O estudo incidiu sobre os Serviços de Controlo de Tráfego Aéreo da Região de Informação de Voo de Lisboa e permitiu concluir pela existência de uma dimensão integrativa das componentes trabalho e tecnologia, enquanto elementos estruturantes dos mapas de competências profissionais, indispensáveis à integração sistémica dos diferentes agentes (humanos e tecnológicos) no processo produtivo.

New findings in sample treatment based on high-intensity focused ultrasound (HIFU) for protein digestion after polyacrylamide gel electrophoresis separation are presented. The following variables were studied: (i) sample volume; (ii) sonotrode diameter; (iii) previous protein denaturation; (iv) cooling; (v) enzyme concentration; and (vi) protein concentration. Results showed that positive protein identification could be done after protein separation by gel electrophoresis through peptide mass fingerprint (PMF) in a volume as low as 25 mu L. The time needed was less than 2 min and no cooling was necessary. The importance of the sonotrode diameter was negligible. On the other hand, protein denaturation before sonication was a trade-off for the success of procedure here described. The protein coverage was raised from 5 to 30%, and the number of peptides matching the proteins was also increased in a percentage ranging 10-100% when the classical overnight treatment is compared with the proposed HIFU procedure. The minimum amount of protein that can be identified using the HIFU sample treatment by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was 0.06 mu g. The lower concentration of trypsin successfully used to obtain an adequate protein digestion was 3.6 mu g/mL. (c) 2006 Elsevier B.V. All rights reserved.

The periplasmic nitrate reductase from Cupriavidus necator (also known as Ralstonia eutropha) is a heterodimer that is able to reduce nitrate to nitrite. It comprises a 91 kDa catalytic subunit (NapA) and a 17 kDa subunit (NapB) that is involved in electron transfer. The larger subunit contains a molybdenum active site with a bis-molybdopterin guanine dinucleotide cofactor as well as one [4Fe-4S] cluster, while the small subunit is a di-haem c-type cytochrome. Crystals of the oxidized form of this enzyme were obtained using polyethylene glycol 3350 as precipitant. A single crystal grown at the High Throughput Crystallization Laboratory of the EMBL in Grenoble diffracted to beyond 1.5 A at the ESRF (ID14-1), which is the highest resolution reported to date for a nitrate reductase. The unit-cell parameters are a = 142.2, b = 82.4, c = 96.8 A, beta = 100.7 degrees, space group C2, and one heterodimer is present per asymmetric unit.