The present work deals with the use of innovation indicators in the decision-making process. It intends to contribute to the discussion on the construction, use and analysis of indicator systems and also to evaluate its weight on decision-making in innovation. The goal is to help understand how innovation indicators can influence technology policy and through it, society at large. This work will start by analysing the use of indicators (their problems and consistency) and other sources of information that contribute to build the opinions of innovation decision makers. This will be followed by a survey and interviews with main innovation actors. The results will shed light on the impact of the use of indicators by the innovation community – both in terms of technology policy and in the social sphere. Proposals and implications for the future will be advanced, hopefully adding new contributions to the governance of the science, technology and innovation field.

In order to understand the decision-making process in a Radiology Department, taking the Magnetic Resonance Equipment as an example, this paper reports a project to be followed. It is a guideline for future work development regarding Technology Assessment in Radiology. The Theoretical Framework is divided is three big issues. The first is “Technology Assessment”. Starting with the definition of some important concepts, the history and development of Technology Assessment will be addressed. The aim of this issue is to give a general main idea concerning TA contextualization. Doing a transposition of this subject to health area, it is also important to understand the particularities of Health Technology Assessment, second issue. Portugal framework on this subject will also be addressed. As so, the Portuguese National Health System is characterized and the decision-making stakeholders identified, has well as the competences for the decision-making process in general. The third issue is Decision-Making and its aim is to give a general elucidation on decision-making matters. To accomplish this, a research methodology was outlined, so that six research questions could be answered and five hypotheses could be accepted or refuted, in the future. With this research methodology, the Portuguese state of the art Magnetic Resonance equipment existence will be studied, using a survey as a resource. In the future, a mapping stakeholder technique will be used to identify the decision making key stakeholders and a survey will be applied to map theirs skills and competences in the process, where a pre-test was already applied. The results of this pre-test are presented.

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The present article posits constructive technology assessment as the dominant perspective of societal embedding practices in the technical development process by the high-speed train manufacturing industry, resulting from a research study conducted in 2011 (Moretto 2011). The article covers the main elements of the study, being the high-speed train manufacturing industry’s strategic intelligence, technology pattern, knowledge exchange, technology trajectories; and finally presents the arguments justifying constructive technology assessment as the dominant approach.

Two cyclen-derived Gd probes, [Gd-DOTAM](3+) and [Gd-DOTP](5-) (DOTAM = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetamide; DOTP = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylenephosphonate)), were assessed as paramagnetic relaxation enhancement (PRE)-inducing probes for characterization of protein-protein interactions. Two proteins, Desulfovibrio gigas rubredoxin and Desulfovibrio gigas cytochrome c(3), were used as model partners. In a (1)H NMR titration it was shown that [Gd-DOTP](5-) binds to cytochrome c(3) near heme IV, causing pronounced PREs, characterized by line width broadenings of the heme methyl resonances at ratios as low as 0.08. A K(d) of 23 +/- 1 muM was calculated based on chemical shift perturbation of selected heme methyl resonances belonging to three different heme groups, caused by allosteric effects upon [Gd-DOTP](5-) binding to cytochrome c(3) at a molar ratio of 2. The other probe, [Gd-DOTAM](3+), caused PREs on a well-defined patch near the metal center of rubredoxin (especially the patch constituted by residues D19-G23 and W37-S45, which broaden beyond detection). This effect was partially reversed for some resonances (C6-Y11, in particular) when cytochrome c(3) was added to this system. Both probes were successful in causing reversible PREs at the partner binding site, thus showing to be good probes to identify partners' binding sites and since the interaction is reversible to structurally characterize protein complexes by better defining the complex interface.

We present a new approach for real-time monitoring of PCR amplification of a specific sequence from the human c-MYC proto-oncogene using a Ta(2)O(5) electrolyte-insulator-semiconductor (EIS) sensor. The response of the fabricated EIS sensor to cycle DNA amplification was evaluated and compared to standard SYBR-green fluorescence incorporation, showing it was possible to detect DNA concentration variations with 30 mV/μM sensitivity. The sensor's response was then optimized to follow in real-time the PCR amplification of c-MYC sequence from a genomic DNA sample attaining an amplification profile comparable to that of a standard real-time PCR. Owing to the small size, ease of fabrication and low-cost, the developed Ta(2)O(5) sensor may be incorporated onto a microfluidic device and then used for real-time PCR. Our approach may circumvent the practical and economical obstacles posed by current platforms that require an external fluorescence detector difficult to miniaturize and incorporate into a lab-on-chip system.

Respiratory nitric oxide reductase (NOR) was purified from membrane extract of Pseudomonas (Ps.) nautica cells to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a heterodimer with subunits of molecular masses of 54 and 18 kDa. The gene encoding both subunits was cloned and sequenced. The amino acid sequence shows strong homology with enzymes of the cNOR class. Iron/heme determinations show that one heme c is present in the small subunit (NORC) and that approximately two heme b and one non-heme iron are associated with the large subunit (NORB), in agreement with the available data for enzymes of the cNOR class. Mossbauer characterization of the as-purified, ascorbate-reduced, and dithionite-reduced enzyme confirms the presence of three heme groups (the catalytic heme b(3) and the electron transfer heme b and heme c) and one redox-active non-heme Fe (Fe(B)). Consistent with results obtained for other cNORs, heme c and heme b in Ps. nautica cNOR were found to be low-spin while Fe(B) was found to be high-spin. Unexpectedly, as opposed to the presumed high-spin state for heme b(3), the Mossbauer data demonstrate unambiguously that heme b(3) is, in fact, low-spin in both ferric and ferrous states, suggesting that heme b(3) is six-coordinated regardless of its oxidation state. EPR spectroscopic measurements of the as-purified enzyme show resonances at the g approximately 6 and g approximately 2-3 regions very similar to those reported previously for other cNORs. The signals at g = 3.60, 2.99, 2.26, and 1.43 are attributed to the two charge-transfer low-spin ferric heme c and heme b. Previously, resonances at the g approximately 6 region were assigned to a small quantity of uncoupled high-spin Fe(III) heme b(3). This assignment is now questionable because heme b(3) is low-spin. On the basis of our spectroscopic data, we argue that the g = 6.34 signal is likely arising from a spin-spin coupled binuclear center comprising the low-spin Fe(III) heme b(3) and the high-spin Fe(B)(III). Activity assays performed under various reducing conditions indicate that heme b(3) has to be reduced for the enzyme to be active. But, from an energetic point of view, the formation of a ferrous heme-NO as an initial reaction intermediate for NO reduction is disfavored because heme [FeNO](7) is a stable product. We suspect that the presence of a sixth ligand in the Fe(II)-heme b(3) may weaken its affinity for NO and thus promotes, in the first catalytic step, binding of NO at the Fe(B)(II) site. The function of heme b(3) would then be to orient the Fe(B)-bound NO molecules for the formation of the N-N bond and to provide reducing equivalents for NO reduction.

The periplasmic nitrate reductase (NapAB) from Cupriavidus necator is a heterodimeric protein that belongs to the dimethyl sulfoxide reductase family of mononuclear Mo-containing enzymes and catalyzes the reduction of nitrate to nitrite. The protein comprises a large catalytic subunit (NapA, 91 kDa) containing the molybdenum active site plus one [4Fe-4S] cluster, as well as a small subunit (NapB, 17 kDa), which is a diheme c-type cytochrome involved in electron transfer. Crystals of the oxidized form of the enzyme diffracted beyond 1.5 A at the European Synchrotron Radiation Facility. This is the highest resolution reported to date for a nitrate reductase, providing true atomic details of the protein active center, and this showed further evidence on the molybdenum coordination sphere, corroborating previous data on the related Desulfovibrio desulfuricans NapA. The molybdenum atom is bound to a total of six sulfur atoms, with no oxygen ligands or water molecules in the vicinity. In the present work, we were also able to prepare partially reduced crystals that revealed two alternate conformations of the Mo-coordinating cysteine. This crystal form was obtained by soaking dithionite into crystals grown in the presence of the ionic liquid [C(4)mim]Cl(-). In addition, UV-Vis and EPR spectroscopy studies showed that the periplasmic nitrate reductase from C. necator might work at unexpectedly high redox potentials when compared to all periplasmic nitrate reductases studied to date.

The 275GHz electron-paramagnetic-resonance spectrometer we reported on in 2004 has been equipped with a new probe head, which contains a cavity especially designed for operation in continuous-wave mode. The sensitivity and signal stability that is achieved with this new probe head is illustrated with 275GHz continuous-wave spectra of a 1mM frozen solution of the complex Fe(III)-ethylenediamine tetra-acetic acid and of 10mM frozen solutions of the protein rubredoxin, which contains Fe(3+) in its active site, from three different organisms. The high quality of the spectra of the rubredoxins allows the determination of the zero-field-splitting parameters with an accuracy of 0.5GHz. The success of our approach results partially from the enhanced absolute sensitivity, which can be reached using a single-mode cavity. At least as important is the signal stability that we were able to achieve with the new probe head.

Concurrent programs may suffer from concurrency anomalies that may lead to erroneous and unpredictable program behaviors. To ensure program correctness, these anomalies must be diagnosed and corrected. This paper addresses the detection of both low- and high-level anomalies in the Transactional Memory setting. We propose a static analysis procedure and a framework to address Transactional Memory anomalies. We start by dealing with the classic case of low-level dataraces, identifying concurrent accesses to shared memory cells that are not protected within the scope of a memory transaction. Then, we address the case of high-level dataraces, bringing the programmer's attention to pairs of memory transactions that were misspecified and should have been combined into a single transaction. Our framework was applied to a set of programs, collected form different sources, containing well known low- and high-level anomalies. The framework demonstrated to be accurate, confirming the effectiveness of using static analysis techniques to precisely identify concurrency anomalies in Transactional Memory programs.

Mammalian xanthine oxidase (XO) and Desulfovibrio gigas aldehyde oxidoreductase (AOR) are members of the XO family of mononuclear molybdoenzymes that catalyse the oxidative hydroxylation of a wide range of aldehydes and heterocyclic compounds. Much less known is the XO ability to catalyse the nitrite reduction to nitric oxide radical (NO). To assess the competence of other XO family enzymes to catalyse the nitrite reduction and to shed some light onto the molecular mechanism of this reaction, we characterised the anaerobic XO- and AOR-catalysed nitrite reduction. The identification of NO as the reaction product was done with a NO-selective electrode and by electron paramagnetic resonance (EPR) spectroscopy. The steady-state kinetic characterisation corroborated the XO-catalysed nitrite reduction and demonstrated, for the first time, that the prokaryotic AOR does catalyse the nitrite reduction to NO, in the presence of any electron donor to the enzyme, substrate (aldehyde) or not (dithionite). Nitrite binding and reduction was shown by EPR spectroscopy to occur on a reduced molybdenum centre. A molecular mechanism of AOR- and XO-catalysed nitrite reduction is discussed, in which the higher oxidation states of molybdenum seem to be involved in oxygen-atom insertion, whereas the lower oxidation states would favour oxygen-atom abstraction. Our results define a new catalytic performance for AOR-the nitrite reduction-and propose a new class of molybdenum-containing nitrite reductases.

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Formate dehydrogenases (FDHs) are enzymes that catalyze the formate oxidation to carbon dioxide and that contain either Mo or W in a mononuclear form in the active site. In the present work, the influence of Mo and W salts on the production of FDH by Desulfovibrio alaskensis NCIMB 13491 was studied. Two different FDHs, one containing W (W-FDH) and a second incorporating either Mo or W (Mo/W-FDH), were purified. Both enzymes were isolated from cells grown in a medium supplemented with 1 muM molybdate, whereas only the W-FDH was purified from cells cultured in medium supplemented with 10 muM tungstate. We demonstrated that the genes encoding the Mo/W-FDH are strongly downregulated by W and slightly upregulated by Mo. Metal effects on the expression level of the genes encoding the W-FDH were less significant. Furthermore, the expression levels of the genes encoding proteins involved in molybdate and tungstate transport are downregulated under the experimental conditions evaluated in this work. The molecular and biochemical properties of these enzymes and the selective incorporation of either Mo or W are discussed.

This work examines a recent study that used various forecasting methods and in particular the Delphi method, to understand how the indicators were selected during the development of the prospective study. It can be concluded that the indicators in the study were selected through discussion on existing knowledge (formal and informal) and the broad consensus of the respective community, which established and confirmed the choice of indicators as the most relevant to prospectively examine the matter concerned. The technical support provided to choose certain forecasting methods as well as to choose the methods that could not be used throughout the development of the work, contributed to the strength of the list of indicators.

In this paper we present MoTh, a tool that uses static analysis to enable the automatic verification of concurrency anomalies in Transactional Memory Java programs. Currently MoTh detects high-level dataraces and stale-value errors, but it is extendable by plugging-in sensors, each sensor implementing an anomaly detecting algorithm. We validate and benchmark MoTh by applying it to a set of well known concurrent buggy programs and by close comparison of the results with other similar tools. The results achieved so far are very promising, yielding good accuracy while triggering only a very limited number of false warnings.

Saturation transfer difference (STD) NMR has emerged as one of the most popular ligand-based NMR techniques for the study of protein-ligand interactions. The success of this technique is a consequence of its robustness and the fact that it is focused on the signals of the ligand, without any need of processing NMR information about the receptor and only using small quantities of nonlabeled macromolecule. Moreover, the attractiveness of this experiment is also extendable to the classroom. In the context of a practical NMR class, this experiment is ideal to illustrate some fundamental NMR concepts, such as the nuclear Overhauser effect and relaxation in a multidisciplinary context, bridging chemistry and biochemistry with a taste of medicinal chemistry.

We use the readily available human serum albumin (HSA), 6-d,l-methyl-tryptophan (6-CH3-Trp), and 7- d,l-methyl-tryptophan (7-CH3-Trp) to introduce the STD-NMR experiment and to illustrate its applicability for ligand screening, mapping of binding moieties, and determination of the dissociation constant, in a context that can be explored or adapted to the student’s course level and topic (chemistry or biochemistry). We also cover the most important theoretical aspects of the STD experiment, calling attention to some of its limitations and drawbacks without a complex theoretical approach.

Analysis of sequenced bacterial genomes revealed that the genomes encode more than 30% hypothetical and conserved hypothetical proteins of unknown function. Among proteins of unknown function that are conserved in anaerobes, some might be determinants of the anaerobic way of life. This study focuses on two divergent clusters specifically found in anaerobic microorganisms and mainly composed of genes encoding conserved hypothetical proteins. We show that the two gene clusters DVU2103-DVU2104-DVU2105 (orp2) and DVU2107-DVU2108-DVU2109 (orp1) form two divergent operons transcribed by the sigma(54)-RNA polymerase. We further demonstrate that the sigma(54)-dependent transcriptional regulator DVU2106, located between orp1 and orp2, collaborates with sigma(54)-RNA polymerase to orchestrate the simultaneous expression of the divergent orp operons. DVU2106, whose structural gene is transcribed by the sigma(70)-RNA polymerase, negatively retrocontrols its own expression. By using an endogenous pulldown strategy, we identify a physiological complex composed of DVU2103, DVU2104, DVU2105, DVU2108, and DVU2109. Interestingly, inactivation of DVU2106, which is required for orp operon transcription, induces morphological defects that are likely linked to the absence of the ORP complex. A putative role of the ORP proteins in positioning the septum during cell division is discussed.

We report a new strategy that combines a Forster Resonance Energy Transfer (FRET) based spectral codification tool with a single base extension (SBE) reaction for rapid and medium-throughput analysis of single nucleotide polymorphisms (SNPs). This strategy is based on the spectral codification - a donor (fluorophore labeled probe complementary to the region adjacent to an SNP) is used to induce specific FRET signatures from an acceptor fluorophore revealing the SNP variant. Using an SBE reaction and differently labeled ddNTPs, we can directly question each donor probe and retrieve information about which allele variant is present at that locus. The potential of the method is demonstrated by application to simultaneous questioning of two loci in the same reaction tube. Following calibration with all possible combinations of FRET pairs, an evaluation algorithm was calibrated so as to optimize base calling and allow unequivocal allele scoring with more than 80% confidence (for two simultaneous loci being questioned, one homo-and one heterozygous). In conclusion, this spectral codification approach may constitute a solution towards increasing throughput capability of single base extension based assays.

Adenylate kinases (AK) from Gram-negative bacteria are generally devoid of metal ions in their LID domain. However, three metal ions, zinc, cobalt, and iron, have been found in AK from Gram-negative bacteria. Crystal structures of substrate-free AK from Desulfovibrio gigas with three different metal ions (Zn(2+), Zn-AK; Co(2+), Co-AK; and Fe(2+), Fe-AK) bound in its LID domain have been determined by X-ray crystallography to resolutions 1.8, 2.0, and 3.0 A, respectively. The zinc and iron forms of the enzyme were crystallized in space group I222, whereas the cobalt-form crystals were C2. The presence of the metals was confirmed by calculation of anomalous difference maps and by X-ray fluorescence scans. The work presented here is the first report of a structure of a metal-containing AK from a Gram-negative bacterium. The native enzyme was crystallized, and only zinc was detected in the LID domain. Co-AK and Fe-AK were obtained by overexpressing the protein in Escherichia coli. Zn-AK and Fe-AK crystallized as monomers in the asymmetric unit, whereas Co-AK crystallized as a dimer. Nevertheless, all three crystal structures are very similar to each other, with the same LID domain topology, the only change being the presence of the different metal atoms. In the absence of any substrate, the LID domain of all holoforms of AK was present in a fully open conformational state. Normal mode analysis was performed to predict fluctuations of the LID domain along the catalytic pathway.

The majority of papers published in the last decades on European Union policy strongly stress the importance of the so-called Lisbon Strategy approved in the year 2000. The same applies to studies and reports on the shift of the European countries towards modernisation and restructuring policy in recent years. This EU development strategy defines a new direction for the coordination of national policies. But why has it become so important? One of the reasons is the fact that many of the papers are based on the concept of “knowledge society” as the key driver for an increased competitiveness of all political and economic regions of Europe. In this context, the term “knowledge” means the inter-linkage of education (including training, qualification, skills) and innovation (including research, information and communication). The use of the concept represents an important shift in the European strategy: further development would not only be based on investment in material infrastructures, but also more on the immaterial ground. However, this Lisbon Strategy was criticised by many politicians and opinion-makers in the first years of this century because the European structures were not prepared for such a quick change. At the same time, the focus for investment moved away from the traditional support of industrial sectors (manufacturing, agriculture and fisheries, construction) towards the “new economy” sectors. The vision of a knowledge society remained appealing also in a changing international context: the Middle East wars (Afghanistan, Iraq and Israel-Palestine) and the fast growth of the Chinese economy. However, the shadows of new recessions have strongly questioned the options made by the European Council. New challenges have emerged with the need to redefine collective strategies in terms of European development as set by the Lisbon strategy. “Europe 2020” is one more attempt to define a new strategy. But at present no clear path has been identifi

Nanotechnology has prompted researchers to develop new and improved materials aimed at biomedical applications with particular emphasis in diagnostics and therapy. Special interest has been directed at providing enhanced biomolecular diagnostics, including SNP detection gene expression profiles and biomarker characterisation. These strategies have focused on the development of nanoscale devices and platforms that can be used for single molecule characterisation of nucleic acid, DNA or RNA, and protein at an increased rate when compared to traditional techniques. Also, several advances have been reported on DNA analysis in real time, at both high resolution and very high throughputs, suitable for biomedical diagnostics. Here, we shall provide a review of available nanotechnology-based platforms for biomolecular recognition, and their application to molecular diagnostics and genome analysis, with emphasis on the use of noble metal nanoparticles for simple and specific analysis systems. Particular focus will be put on those already being translated into clinical settings. This article is part of a Special Issue entitled: Clinical Proteomics.

In this paper we report on the fabrication and performance of a portable and low cost optoelectronic platform integrating a double color tuned light emitting diode as light source, an amorphous/nanocrystalline silicon photodetector with a flat spectral response in the wavelength range from 520. nm to 630. nm and integrated electronic for signal acquisition and conditioning constituted by current to voltage converter, a filter and an amplification stage, followed by an analog to digital converter, with appropriate software for full automation to minimize human error. Incorporation of the double color tuned light emitting diode provides for a simple yet innovative solution to signal acquisition independently from the light intensity and/or solution concentration, while considerably decreasing production costs. Detection based on Au-nanoprobes constitutes the biorecognition step and allowed identification of specific sequences of Mycobacterium tuberculosis complex, namely Mycobacterium bovis and M. tuberculosis in biological samples.

The majority of papers published in the last decades on European Union policy strongly stress the importance of the so-called Lisbon Strategy approved in the year 2000. The same applies to studies and reports on the shift of the European countries towards modernisation and restructuring policy in recent years. This EU development strategy defines a new direction for the coordination of national policies. But why has it become so important? One of the reasons is the fact that many of the papers are based on the concept of “knowledge society” as the key driver for an increased competitiveness of all political and economic regions of Europe. In this context, the term “knowledge” means the inter-linkage of education (including training, qualification, skills) and innovation (including research, information and communication). The use of the concept represents an important shift in the European strategy: further development would not only be based on investment in material infrastructures, but also more on the immaterial ground. However, this Lisbon Strategy was criticised by many politicians and opinion-makers in the first years of this century because the European structures were not prepared for such a quick change. At the same time, the focus for investment moved away from the traditional support of industrial sectors (manufacturing, agriculture and fisheries, construction) towards the “new economy” sectors. The vision of a knowledge society remained appealing also in a changing international context: the Middle East wars (Afghanistan, Iraq and Israel-Palestine) and the fast growth of the Chinese economy. However, the shadows of new recessions have strongly questioned the options made by the European Council. New challenges have emerged with the need to redefine collective strategies in terms of European development as set by the Lisbon strategy. “Europe 2020” is one more attempt to define a new strategy. But at present no clear path has been identified. Whether the