The geometry of the axial ligands of the hemes in the triheme cytochrome PpcA from Geobacter sulfurreducens was determined in solution for the ferric form using the unambiguous assignment of the NMR signals of the α-substituents of the hemes. The paramagnetic 13C shifts of the hemes can be used to define the heme electronic structure, the geometry of the axial ligands, and the magnetic susceptibility tensor. The latter establishes the magnitude and geometrical dependence of the pseudocontact shifts, which are crucial to warrant reliable structural constraints for a detailed structural characterization of this paramagnetic protein in solution.

The periplasmic sensor domains encoded by genes gsu0582 and gsu0935 are part of methyl accepting chemotaxis proteins in the bacterium Geobacter sulfurreducens (Gs). The sensor domains of these proteins contain a heme-c prosthetic group and a PAS-like fold as revealed by their crystal structures. Biophysical studies of the two domains showed that nitric oxide (NO) binds to the heme in both the ferric and ferrous forms, whereas carbon monoxide (CO) binds only to the reduced form. In order to address these exogenous molecules as possible physiological ligands, binding studies and resonance Raman (RR) spectroscopic characterization of the respective CO and NO adducts were performed in this work. In the absence of exogenous ligands, typical RR frequencies of five-coordinated (5c) high-spin and six-coordinated (6c) low-spin species were observed in the oxidized form. In the reduced state, only frequencies corresponding to the latter were detected. In both sensors, CO binding yields 6c low-spin adducts by replacing the endogenous distal ligand. The binding of NO by the two proteins causes partial disruption of the proximal Fe-His bond, as revealed by the RR fingerprint features of 5cFe-NO and 6cNO-Fe-His species. The measured CO and NO dissociation constants of ferrous GSU0582 and GSU0935 sensors reveal that both proteins have high and similar affinity toward these molecules (Kd ≈ 0.04−0.08 μM). On the contrary, in the ferric form, sensor GSU0582 showed a much higher affinity for NO (Kd ≈ 0.3 μM for GSU0582 versus 17 μM for GSU0935). Molecular dynamics calculations revealed a more open heme pocket in GSU0935, which could account for the different affinities for NO. Taken together, spectroscopic data and MD calculations revealed subtle differences in the binding properties and structural features of formed CO and NO adducts, but also indicated a possibility that a (5c) high-spin/(6c) low-spin redox-linked equilibrium could drive the physiological sensing of Gs cells.

Increasing levels of environmental pollution and destruction of the planet, as
well as the future of societies, have been prominent themes in our time.
Concern about the sustainable future of ecosystems and future generations
brings to each sector of activity the need to define a process of sustainable development
and measures that ought to be implemented.
In the construction sector this is a major issue, since it consumes a great part of
natural resources, and leads to high emissions of pollutants. Likewise, this sector
determines the mode of living of human beings, as well as their comfort and health.
The work herein presented has the purpose to study how, and what systems and
procedures should be implemented to ensure techniques for sustainable construction,
and consequently achieve the principles of sustainable development into its
components: environment, society and economy.
The national reality of professionals will also be referred to, thus contributing to
a practice of sustainable construction, complementary to what currently exists both
national and internationally.
Thus, this study aims to reflect on the importance of certification, systems and
procedures, to ensure compliance of construction with the sustainability goals.

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Previous studies have demonstrated that Geobacter sulfurreducens requires the c-type cytochrome OmcZ, which is present in large (OmcZL; 50-kDa) and small (OmcZS; 30-kDa) forms, for optimal current production in microbial fuel cells. This protein was further characterized to aid in understanding its role in current production. Subcellular-localization studies suggested that OmcZS was the predominant extracellular form of OmcZ. N- and C-terminal amino acid sequence analysis of purified OmcZS and molecular weight measurements indicated that OmcZS is a cleaved product of OmcZL retaining all 8 hemes, including 1 heme with the unusual c-type heme-binding motif CX14CH. The purified OmcZS was remarkably thermally stable (thermal-denaturing temperature, 94.2°C). Redox titration analysis revealed that the midpoint reduction potential of OmcZS is approximately −220 mV (versus the standard hydrogen electrode [SHE]) with nonequivalent heme groups that cover a large reduction potential range (−420 to −60 mV). OmcZS transferred electrons in vitro to a diversity of potential extracellular electron acceptors, such as Fe(III) citrate, U(VI), Cr(VI), Au(III), Mn(IV) oxide, and the humic substance analogue anthraquinone-2,6-disulfonate, but not Fe(III) oxide. The biochemical properties and extracellular localization of OmcZ suggest that it is well suited for promoting electron transfer in current-producing biofilms of G. sulfurreducens.

Rail corrugation is a phenomenon that leads to a waving in the rails with wavelengths typically between 3 cm and 100 cm and amplitude levels of several microns. The genesis of this waving is complex. Rail corrugation is a recognized problem that leads to excess vibration on the rails and vehicles to a point of reducing their life span and compromising safety. In urban areas excess vibration noise is also a problem. A software tool was developed to analyze accelerometer signals acquired in the boggies of rail vehicles in order to quantify the rail corrugation according to their frequency and amplitude. A wavelet packet methodology was used in this work and compared with the One Third Octave Filter (OTOF) power representations, which is currently used in the industry. It is shown that the former produces better results.

Currently, the buildings are one of the elements that mostly contribute to
environmental degradation.
Regarding the natural resources consumption and the environmental comfort, the
building efficiency level means that, actually, the same residential built environment can be
considered unsustainable.
Thereby, it is important to know, determine and identify the specific residential
buildings problems in order to contribute in solving this situation.
After knowing the major residential buildings problems and those causes, it is needed
to adapt the rehabilitation process to the traditional concept of sustainable construction, which
can be achieved by the evaluation and certification systems of the construction sustainability.
This thesis aims at contributing to the buildings rehabilitation process which it is
intended to be conducted in a sustainable method, based on the presented issue.

The effect of kefir grains on the proteolysis of major milk proteins in milk kefir and in a culture of kefir grains in pasteurized cheese whey was followed by reverse {phase-HPLC} analysis. The reduction of kappa-, alpha-, and beta-caseins {(CN)}, alpha-lactalbumin {(alpha-LA)}, and beta-lactoglobulin {(beta-LG)} contents during 48 and 90 h of incubation of pasteurized milk {(100mL)} and respective cheese whey with kefir grains (6 and 12 g) at 20 degrees C was monitored. Significant proteolysis of {alpha-LA} and kappa-, alpha-, and beta-caseins was observed. The effect of kefir amount (6 and 12 {g/100mL)} was significant for {alpha-LA} and alpha- and {beta-CN.} {alpha-Lactalbumin} and {beta-CN} were more easily hydrolyzed than {alpha-CN.} No significant reduction was observed with respect to {beta-LG} concentration for 6 and 12 g of kefir in {100mL} of milk over 48 h, indicating that no significant proteolysis was carried out. Similar results were observed when the experiment was conducted over 90 h. Regarding the cheese whey kefir samples, similar behavior was observed for the proteolysis of {alpha-LA} and {beta-LG:} {alpha-LA} was hydrolyzed between 60 and 90% after 12h (for 6 and 12 g of kefir) and no significant {beta-LG} proteolysis occurred. The proteolytic activity of lactic acid bacteria and yeasts in kefir community was evaluated. Kefir milk prepared under normal conditions contained peptides from proteolysis of {alpha-LA} and kappa-, alpha-, and beta-caseins. Hydrolysis is dependent on the kefir:milk ratio and incubation time. {beta-Lactoglobulin} is not hydrolyzed even when higher hydrolysis time is used. Kefir grains are not appropriate as adjunct cultures to increase {beta-LG} digestibility in whey-based or whey-containing foods.

A family of five periplasmic triheme cytochromes (PpcA-E) was identified in Geobacter sulfurreducens, where they play a crucial role by driving electron transfer from the cytoplasm to the cell exterior and assisting the reduction of extracellular acceptors. The thermodynamic characterization of PpcA using NMR and visible spectroscopies was previously achieved under experimental conditions identical to those used for the triheme cytochrome c7 from Desulfuromonas acetoxidans. Under such conditions, attempts to obtain NMR data were complicated by the relatively fast intermolecular electron exchange. This work reports the detailed thermodynamic characterization of PpcB, PpcD, and PpcE under optimal experimental conditions. The thermodynamic characterization of PpcA was redone under these new conditions to allow a proper comparison of the redox properties with those of other members of this family. The heme reduction potentials of the four proteins are negative, differ from each other, and cover different functional ranges. These reduction potentials are strongly modulated by heme-heme interactions and by interactions with protonated groups (the redox-Bohr effect) establishing different cooperative networks for each protein, which indicates that they are designed to perform different functions in the cell. PpcA and PpcD appear to be optimized to interact with specific redox partners involving e−/H+ transfer via different mechanisms. Although no evidence of preferential electron transfer pathway or e−/H+ coupling was found for PpcB and PpcE, the difference in their working potential ranges suggests that they may also have different physiological redox partners. This is the first study, to our knowledge, to characterize homologous cytochromes from the same microorganism and provide evidence of their different mechanistic and functional properties. These findings provide an explanation for the coexistence of five periplasmic triheme cytochromes in G. sulfurreducens.

Mining massive amounts of transcript data for alternative splicing information is paramount to help understand how the maturation of RNA regulates gene expression. We developed an algorithm to cluster transcript data to annotated genes to detect unannotated splice variants. A higher number of alternatively spliced genes and isoforms were found compared to other alternative splicing databases. Comparison of human and mouse data revealed a marked increase, in human, of splice variants incorporating novel exons and retained introns. Previously unannotated exons were validated by tiling array expression data and shown to correspond preferentially to novel first exons. Retained introns were validated by tiling array and deep sequencing data. The majority of retained introns were shorter than 500 nt and had weak polypyrimidine tracts. A subset of retained introns matching small RNAs and displaying a high GC content suggests a possible coordination between splicing regulation and production of noncoding RNAs. Conservation of unannotated exons and retained introns was higher in horse, dog and cow than in rodents, and 64% of exon sequences were only found in primates. This analysis highlights previously bypassed alternative splice variants, which may be crucial to deciphering more complex pathways of gene regulation in human.