Cytochromes c7 are periplasmic triheme proteins that have been reported exclusively in δ-proteobacteria. The structures of five triheme cytochromes identified in Geobacter sulfurreducens and one in Desulfuromonas acetoxidans have been determined. In addition to the hemes and axial histidines, a single aromatic residue is conserved in all these proteins - phenylalanine 15 (F15). PpcA is a member of the G. sulfurreducens cytochrome c7 family that performs electron/proton energy transduction in addition to electron transfer that leads to the reduction of extracellular electron acceptors. For the first time we probed the role of the F15 residue in the PpcA functional mechanism, by replacing this residue with the aliphatic leucine by site-directed mutagenesis. The analysis of NMR spectra of both oxidized and reduced forms showed that the heme core and the overall fold of the mutated protein were not affected. However, the analysis of 1H-15N heteronuclear single quantum coherence NMR spectra evidenced local rearrangements in the α-helix placed between hemes I and III that lead to structural readjustments in the orientation of heme axial ligands. The detailed thermodynamic characterization of F15L mutant revealed that the reduction potentials are more negative and the redox-Bohr effect is decreased. The redox potential of heme III is most affected. It is of interest that the mutation in F15, located between hemes I and III in PpcA, changes the characteristics of the two hemes differently. Altogether, these modifications disrupt the balance of the global network of cooperativities, preventing the F15L mutant protein from performing a concerted electron/proton transfer.

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Detailed thermodynamic and structural data measured in soluble monomeric multiheme cytochromes c provided the basis to investigate the functional significance of interactions between redox co-factors. The steep decay of intramolecular interactions with distance means that close proximity of the redox centers is necessary to modulate the intrinsic reduction potentials in a significant way. This ensures selection of specific populations during redox activity in addition to maintaining fast intramolecular electron transfer. Therefore, intramolecular interactions between redox co-factors play an important role in establishing the biological function of the protein by controlling how electrons flow through and are distributed among the co-factors.

Consider a set of methods implementing an Application Programming Interface (API) of a given library or program module that is to be used in a multithreaded setting. If those methods were not originally designed to be thread safe, races and deadlocks are expected to happen. This work introduces the novel concept of program closure and describes how it can be applied in a methodology used to make the library or module implementation thread safe, by identifying the high level data races introduced by interleaving the parallel execution of methods from the API. High-level data races result from the misspecification of the scope of an atomic block, by wrongly splitting it into two or more atomic blocks sharing a data dependency. Roughly speaking, the closure of a program P, clos(P), is obtained by incrementally adding new threads to P in such a way that enables the identification of the potential high level data races that may result from running P in parallel with other programs. Our model considers the methods implementing the API of a library of program module as concurrent programs and computes and analyses their closure in order to identify high level data races. These high level data races are inspected and removed to make the interface thread safe. We illustrate the application of this methodology with a simple use case.

The scientific controversy generated around the destiny given to the fraction of hazardous industrial waste produced in Portugal and how the country dealt with this situation was the stand out point in Souselas case. Here, the dominant aspect of the analysis focused on the implementation of a solution for the treatment of hazardous industrial waste. These wastes result from industrial processes contain or are contaminated, by substances that, at certain concentrations, represent a risk to human health or to the environment. Their treatment can be done using co-incineration in existing cement factories. Having in mind the environment analysis of a controversial process, through the statements made by the different actors involved, the case of Souselas was our object of study. Initially, the actors involved in the process were identified and characterized, in terms of position, interests and / or concerns. This analysis has strengthened with the gathering of documentary elements of analysis. In a second phase the historical process was prepared. Only then, the conditions to make an interpretation of what really happened in the process were gathered, then , it was possible to identify which parts were successful and unsuccessful, and to interpret “why” these successes and failures occurred. Thus, after the identification of key variables and leverage points, a causal diagram and a schematic simulation of the behaviour of reference in case Souselas was designed. We conclude that the process of Souselas was a significant milestone with regard to social organization and spontaneous local actors in situations of opposition to central government decisions with local impact. It was also a turning point in governance according to the model of representative democracy, whose technocratic and elitist character is called into question. The Souselas case emphasized itself as a microcosm on the conflict of interests that we find at a global level heightened since the 90s and that

A gene encoding Bfr (bacterioferritin) was identified and isolated from the genome of Desulfovibrio vulgaris cells, and overexpressed in Escherichia coli. In vitro, H2O2 oxidizes Fe2+ ions at much higher reaction rates than O-2. The H2O2 oxidation of two Fe2+ ions was proven by Mossbauer spectroscopy of rapid freeze-quenched samples. On the basis of the Mossbauer parameters of the intermediate species we propose that D. vulgaris Bfr follows a mineralization mechanism similar to the one reported for vertebrate H-type ferritins subunits, in which a diferrous centre at the ferroxidase site is oxidized to diferric intermediate species, that are subsequently translocated into the inner nanocavity. D. vulgaris recombinant Bfr oxidizes and stores up to 600 iron atoms per protein. This Bfr is able to bind DNA and protect it against hydroxyl radical and DNase deleterious effects. The use of H2O2 as an oxidant, combined with the DNA binding and protection activities, seems to indicate a DPS (DNA-binding protein from starved cells)-like role for D. vulgaris Bfr.

The close structural similarity between the two cyclooxygenase (COXs) isoforms and the absence of selective inhibitors without side effects continues to stimulate the development of novel approaches towards selective anti-inflammatory drugs. In the present study a small library of new indolic compounds involving two different substitutions patterns at the indole scaffold was synthesized. In order to establish a relation between the spatial distribution of known functional groups related with inhibitory activity, two substitution patterns were explored: one with substituents at N-1, C-3, C-5 positions and another at C-2, C-3 and C5 positions. Accordingly, indole positions C-5, C-3 and N-1 were substituted with: sulfonamide or methylsulfone at C-5, p-halo-benzyl group at C-3, and an alkyl chain with a trifluoromethyl group at N-1. Alternatively, a p-halo-benzyl group was introduced at C-2, leaving the indolic nitrogen free. Inhibitory studies were performed and the activity results obtained against both COXs isoforms were rationalized based on docking and NMR studies. Docking studies show that dialkyation at C-2 and C-3 favors a binding with an orientation similar to that of the known selective inhibitor SC-558. From the tested compounds, this substitution pattern is correlated with the highest inhibitory activity and selectivity: 70% COX-2 inhibition at 50 M, and low COX-1 inhibition (18±9%). Additionally, Saturation Transfer Difference NMR experiments reveal different interaction patterns with both COXs isoforms that may be related with different orientations of the sulfonamide group in the binding pocket. Despite the moderated inhibitory activities found, this study represents an innovative approach towards COXs inhibitory activity rationalization and to the design of anti-inflammatory drugs.

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This paper describes for the first time the intimate molecular details of the association between a platinated oligonucleotide and a zinc-finger peptide. Site-specific platination of the guanine in a ss hexanucleotide gave {[Pt(dien)d(5’-TACGCC-3’)], Pt(dien)(6-mer)}, II, characterized by mass spectrometry and 1H-NMR spectroscopy. The work extends the study of platinum-nucleobase complex-zinc finger interactions using small molecules such as [Pt(dien)(9-EtGua)]2+, I . The structure of the (34-52) C-terminal finger of the HIV nucleocapsid protein HIVNCp7 (ZF1) was characterized by 1H-NMR spectroscopy and compared with that of the N-terminal single finger and the 2-finger “intact” NCp7. Interaction of II with ZF1 results in significant changes in comparison to the “free” uncomplexed hexanucleotide – the major shifts occur for Trp37 resonances are broadened and shifted upfield and other major shifts are for Gln45 (H21, H3, Q), Met46 (NH, H2), Lys47 (NH, Q) and Glu50 (H2, H3). The Zn-Cys/His chemical shifts show only marginal deviations. The solution structure of ZF1, the 6-mer/ZF1 and II/ZF1 adducts were calculated from the NOESY-derived distance constraints. The DNA position in II/ZF1 is completely different than in the absence of platinum. Major differences are the appearance of new Met46-Cyt6H5 and Trp37-Cyt5H5 contacts but severe weakening of the Trp37-Gua4 contact, attributed to the steric effects caused by Gua4 platination, accompanied by a change in the position of the aromatic ring. The results demonstrate the feasibility of targetting specific ZF motifs with DNA-tethered coordination compounds, such as Pt compounds and Co-macrocycles – with implications for drug targetting and indeed the intimate mechansims of DNA repair of platinated DNA.

The function of prion-like protein Doppel was suggested to be related to male fertility. In this study, the importance of ovine Doppel polypeptide on spermatozoa capacitation and fertilization was evaluated. After refolding, recombinant Doppel (rDpl) was supplemented with different concentrations (40, 80 or 190 ng/ml) to ovine spermatozoa during the capacitation process. In experiment 1, post-thawed ovine spermatozoa were incubated with different concentrations of rDpl during 1 h for swim-up, and changes in sperm motility, concentration, vigour, viability and capacitation were monitored (10 replicates). In experiment 2, the fertilization ability of post-swim-up spermatozoa incubated as above was tested through heterologous fertilization of bovine in vitro matured oocytes (n = 423, three replicates). Regardless of dosage, rDpl improved (p = 0.03) spermatozoa viability. Sperm individual motility and vigour were the highest (p = 0.04) for the group receiving 190 ng/ml rDpl. Sperm supplemented with the highest doses of rDpl achieved higher (p = 0.02) fertilization rates (56.0 +/- 3.0%) than control (39.1 +/- 2.2%) and 40 ng/ml rDpl (39.8 +/- 2.7%). Preliminary data suggest that Doppel protein may enhance in vitro spermatozoa fertilizing ability.