Electron transfer proteins and redox enzymes containing paramagnetic redox centers with different relaxation rates are widespread in nature. Despite both the long distances and chemical paths connecting these centers, they can present weak magnetic couplings produced by spin-spin interactions such as dipolar and isotropic exchange. We present here a theoretical model based on the Bloch-Wangsness-Redfield theory to analyze the dependence with temperature of EPR spectra of interacting pairs of spin 1/2 centers having different relaxation rates, as is the case of the molybdenum-containing enzyme aldehyde oxidoreductase from Desulfovibrio gigas. We analyze the changes of the EPR spectra of the slow relaxing center (Mo(V)) induced by the faster relaxing center (FeS center). At high temperatures, when the relaxation time T(1) of the fast relaxing center is very short, the magnetic coupling between centers is averaged to zero. Conversely, at low temperatures when T(1) is longer, no modulation of the coupling between metal centers can be detected.

“Theory and Practice” of TA, which is referred to in the title of this journal “TATuP”, is usually addressed as a question of TA research. But science is more than research: the field of teaching requires just as much attention, both practically and theoretically. Therefore, a mere collection of individual teaching experiences and best practice examples does not provide a strong enough basis to discuss questions of TA teaching, these must also be embedded in a theoretical context and discussed in their relation to research. In this special issue, we aim to contribute to a combination of theoretical and practical approaches to the relation of TA and “Bildung”.

“Theory and Practice” of TA, which is referred to in the title of this journal “TATuP”, is usually addressed as a question of TA research. But science is more than research: the field of teaching requires just as much attention, both practically and theoretically. Therefore, a mere collection of individual teaching experiences and best practice examples does not provide a strong enough basis to discuss questions of TA teaching, these must also be embedded in a theoretical context and discussed in their relation to research. In this special issue, we aim to contribute to a combination of theoretical and practical approaches to the relation of TA and “Bildung”.

The catalytic mechanism of nitrate reduction by periplasmic nitrate reductases has been investigated using theoretical and computational means. We have found that the nitrate molecule binds to the active site with the Mo ion in the +6 oxidation state. Electron transfer to the active site occurs only in the proton-electron transfer stage, where the Mo(V) species plays an important role in catalysis. The presence of the sulfur atom in the molybdenum coordination sphere creates a pseudo-dithiolene ligand that protects it from any direct attack from the solvent. Upon the nitrate binding there is a conformational rearrangement of this ring that allows the direct contact of the nitrate with Mo(VI) ion. This rearrangement is stabilized by the conserved methionines Met141 and Met308. The reduction of nitrate into nitrite occurs in the second step of the mechanism where the two dimethyl-dithiolene ligands have a key role in spreading the excess of negative charge near the Mo atom to make it available for the chemical reaction. The reaction involves the oxidation of the sulfur atoms and not of the molybdenum as previously suggested. The mechanism involves a molybdenum and sulfur-based redox chemistry instead of the currently accepted redox chemistry based only on the Mo ion. The second part of the mechanism involves two protonation steps that are promoted by the presence of Mo(V) species. Mo(VI) intermediates might also be present in this stage depending on the availability of protons and electrons. Once the water molecule is generated only the Mo(VI) species allow water molecule dissociation, and, the concomitant enzymatic turnover.

The present invention relates to a system and process for detection and/or qualitative and quantitative identification of the biological material, such as specific sequences of nucleic acids or proteins as antibodies, present in biological samples. The system is composed by one or more light sources (1) combined with one or more integrated optical photo sensors, or not, and various electronic components (4), necessary for obtaining/ processing of the signal emitted by the metal nanoprobes functionalized with a solution of biological composite, as well as also a micro-controller and a microprocessor, fixed or portable. This photosensor structure is able to detect and to quantify the colour variations produced by metal nanoprobes, being this preferentially gold, functionalized by oligonucleotides complementary to specific DNA/RNA sequences, proteins, as for instance antibodies and/or antigens related with certain disease, or other sample or solution of biological composite, that are to be investigated. The detection and quantification process is based on the response of a photosensor, singular or integrated, based on thin film technology of amorphous, nanocrystalline or microcrystalline silicon and their alloys, as well as the new active ceramic semiconductors, amorphous and not amorphous.

O presente invento relaciona-se com um método para controlo de reac{\c c}ões enzimáticas de síntese de ácidos nucleicos, recorrendo a nucleótidos funcionalizados com derivados de 4-metilcumarinas (1) protectores e fotolábeis. Quando ligado aos nucleótidos (2), o grupo cumarinico (3) impede que estes sejam utilizados como substrato por parte das enzimas, impossibilitando a ocorrência de reac{\c c}ão. Através de irradia{\c c}ão com radia{\c c}ão electromagnética, o grupo cumarinico é libertado, ficando o nucleótido disponível para a reac{\c c}ão. Desta forma, as reac{\c c}ões enzimáticas de síntese de ácidos nucleicos podem ser controladas através da luz.

Neste trabalho pretende-se estudar a variabilidade da frequência cardíaca através da implementação de vários métodos de análise de electrocardiogramas. Na primeira parte descrevem-se os métodos de análise espectral, bem como a representação tempo-frequência. A decomposição de sinais para detectar e isolar os batimentos cardíacos do electrocardiograma é implementada. Descrevem-se algoritmos de isolamento para detecção dos eventos interessantes e pontos fiduciais que os constituem. Na segunda parte estima-se a variação da frequência cardíaca e estabelece-se a sua relação com as variações temporais e espectrais das características encontradas pelos métodos implementados na primeira parte do trabalho.

Aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is a member of the xanthine oxidase (XO) family of mononuclear Mo-enzymes that catalyzes the oxidation of aldehydes to carboxylic acids. The molybdenum site in the enzymes of the XO family 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. We report here steady-state kinetic studies of DgAOR with the inhibitors cyanide, ethylene glycol, glycerol, and arsenite, together with crystallographic and EPR studies of the enzyme after reaction with the two alcohols. In contrast to what has been observed in other members of the XO family, cyanide, ethylene glycol, and glycerol are reversible inhibitors of DgAOR. Kinetic data with both cyanide and samples prepared from single crystals confirm that DgAOR does not need a sulfido ligand for catalysis and confirm the absence of this ligand in the coordination sphere of the molybdenum atom in the active enzyme. Addition of ethylene glycol and glycerol to dithionite-reduced DgAOR yields rhombic Mo(V) EPR signals, suggesting that the nearly square pyramidal coordination of the active enzyme is distorted upon alcohol inhibition. This is in agreement with the X-ray structure of the ethylene glycol and glycerol-inhibited enzyme, where the catalytically labile OH/OH(2) ligand is lost and both alcohols coordinate the Mo site in a eta(2) fashion. The two adducts present a direct interaction between the molybdenum and one of the carbon atoms of the alcohol moiety, which constitutes the first structural evidence for such a bond in a biological system.

The orange-coloured protein (ORP) from Desulfovibrio gigas is a 12 kDa protein that contains a novel mixed-metal sulfide cluster of the type [S(2)MoS(2)CuS(2)MoS(2)]. Diffracting crystals of the apo form of ORP have been obtained. Data have been collected for the apo form of ORP to 2.25 A resolution in-house and to beyond 2.0 A resolution at ESRF, Grenoble. The crystals belonged to a trigonal space group, with unit-cell parameters a = 43, b = 43, c = 106 A.

A conductometric biosensor for nitrite detection was developed using cytochrome c nitrite reductase (ccNiR) extracted from Desulfovibrio desulfuricans ATCC 27774 cells immobilized on a planar interdigitated electrode by cross-linking with saturated glutaraldehyde (GA) vapour in the presence of bovine serum albumin, methyl viologen (MV), Nafion, and glycerol. The configuration parameters for this biosensor, including the enzyme concentration, ccNiR/BSA ratio, MV concentration, and Nafion concentration, were optimized. Various experimental parameters, such as sodium dithionite added, working buffer solution, and temperature, were investigated with regard to their effect on the conductance response of the biosensor to nitrite. Under the optimum conditions at room temperature (about 25 degrees C), the conductometric biosensor showed a fast response to nitrite (about 10s) with a linear range of 0.2-120 microM, a sensitivity of 0.194 microS/microM [NO(2)(-)], and a detection limit of 0.05 microM. The biosensor also showed satisfactory reproducibility (relative standard deviation of 6%, n=5). The apparent Michaelis-Menten constant (K(M,app)) was 338 microM. When stored in potassium phosphate buffer (100mM, pH 7.6) at 4 degrees C, the biosensor showed good stability over 1 month. No obvious interference from other ionic species familiar in natural waters was detected. The application experiments show that the biosensor is suitable for use in real water samples.

The characterization of a novel Mo-Fe protein (MorP) associated with a system that responds to Mo in Desulfovibrio alaskensis is reported. Biochemical characterization shows that MorP is a periplasmic homomultimer of high molecular weight (260 +/- 13 kDa) consisting of 16-18 monomers of 15321.1 +/- 0.5 Da. The UV/visible absorption spectrum of the as-isolated protein shows absorption peaks around 280, 320, and 570 nm with extinction coefficients of 18700, 12800, and 5000 M(-1) cm(-1), respectively. Metal content, EXAFS data and DFT calculations support the presence of a Mo-2S-[2Fe-2S]-2S-Mo cluster never reported before. Analysis of the available genomes from Desulfovibrio species shows that the MorP encoding gene is located downstream of a sensor and a regulator gene. This type of gene arrangement, called two component system, is used by the cell to regulate diverse physiological processes in response to changes in environmental conditions. Increase of both gene expression and protein production was observed when cells were cultured in the presence of 45 microM molybdenum. Involvement of this system in Mo tolerance of sulfate reducing bacteria is proposed.

O desenvolvimento de um pacote de software para lidar facilmente com electrocardiogramas de alta resolução tornou-se importante para pesquisa na área de electrocardiografia. O desenvolvimento de novas técnicas para detecção de potenciais tardios e outros problemas associados a arritmias cardíacas têm sido objecto de estudo ao longo dos anos. No entanto, ainda existe a lacuna de um pacote de software que facilmente permita implementar algumas destas inovadoras técnicas de uma forma integrada, possibilitando avaliar técnicas clássicas como o protocolo de Simson para a detecção de sinais não estacionários (potenciais tardios). Algumas destas inovadoras técnicas envolvem a detecção tempo-frequência usando escalogramas ou a análise espectral usando metodologias wavelet-packet, sendo implementadas no software desenvolvido com flexibilidade e versatilidade suficientes para que futuramente sirva de plataforma de pesquisa para o refinamento destas mesmas técnicas no que toca ao processamento de sinais de electrocardiogramas de alta resolução. O software aqui desenvolvido foi também desenhado de forma a suportar dois tipos de ficheiros diferentes provenientes de outros tantos sistemas de aquisição. Os sistemas suportados são o ActiveTwo da Biosemi e o USBamp da g.tec.

Nanotechnology is a multidisciplinary field that brings together diverse fields of research and development such as engineering, biology, physics and chemistry. Formal definitions of nanotechnology refer to man-made devices, components and structures in the 1-100 nm range in at least one dimension. Advances in nanoscience are having a significant impact on many scientific fields, boosting the development of a variety of important technologies. Nanotechnology offers an unprecedented opportunity to interact with cancer cells in real time at the molecular and cellular scale. Because of their small size, nanoscale devices can readily interact with biomolecules on both the surface of cells and inside of cells. The concerted development of nanoscale devices, structures and components have provided essential breakthroughs in monitoring and fighting cancer at the earliest stages of the cancer process. Nanotechnology offers a wealth of tools that may provide researchers with new and innovative ways to diagnose and treat cancer - new imaging agents; systems for real-time assessments of therapeutic and surgical efficacy; multifunctional, targeted devices capable of bypassing biological barriers to deliver multiple therapeutic agents directly to cancer cells and tissues that play a critical role in cancer growth and metastasis; agents that can monitor predictive molecular changes allowing for preventive action against precancerous cells becoming malignant; minimizing costs for multiplex analysis. Nanotechnology, if properly integrated with conventional cancer research, may provide extraordinary prospects towards better diagnosis and effective therapy.

FRET can be used as a strategy to assign different simultaneous events in the same sample but {"}cross-talk{"} problems are a limitation. Here we present a contribution for the better understanding of the {"}cross-talk{"} in FRET experiments that include several pairs in the same sample. Using oligonucleotide probes labeled with fluorescent dyes which can be selectively excited at a specific wavelength, and using target oligonucleotides tagged with a fluorescent dye with specific characteristics that allow only it to emit light upon selective excitation of a specific probe by energy transfer (FRET), we aim to identify the exact probe-target hybridized pair. When using three donors to probe the presence of complementary targets, only 20% of possible donor/acceptor combinations give straightforward signals readily identifiable with the sample composition, while in the remaining cases severe cross-excitation prevents the direct identification of the sample composition. To correctly resolve the samples identity, we developed a theoretical model that enables the unequivocal attribution of a sample composition to a given set of fluorescence signals, in the presence of three donors.

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Flavylium salts contain the basic structure and show a pH-dependent sequence of reactions identical to natural anthocyanins, which are responsible for most of the red and blue colors of flowers and fruits. In this work we investigated the effect of the water-soluble molecular clips C1 and C2 substituted by hydrogen phosphate or sulfate groups on the stability and reactions of the flavylium salts 1-4 by the use of UV-vis absorption, fluorescence, and NMR spectroscopy as well as of the time-resolved pH jump and flash photolysis methods. Clip C1 forms highly stable host-guest complexes with the flavylium salts 1 and 2 and the quinoidal base 3A in methanol. The binding constants were determined by fluorometric titration to be log K = 4.1, 4.7, and 5.6, respectively. Large complexation-induced (1)H NMR shifts of guest signals, Delta delta(max), indicate that in the case of the flavylium salts 1 and 2 the pyrylium ring and in the case of the quinoidal base 3A the o-hydroxyquinone ring are preferentially bound inside the clip cavity. Due to the poor solubility of these host-guest complexes in water, the association constants could be only determined in highly diluted aqueous solution by UV-vis titration experiments for the complex formation of clip C1 with the flavylium salt 3AH(+) at pH = 2 and the quinoidal base 3A at pH = 5.3 to be log K = 4.9 for both complexes. Similar results were obtained for the formation of the complexes of the sulfate-substituted clip C2 with flavylium salt 4AH(+) and its quinoidal base 4A which are slightly better soluble in water (log K = 4.3 and 4.0, respectively). According to the kinetic analysis (performed by using the methods mentioned above) the thermally induced trans-cis chalcone isomerization (4Ct -> 4Cc) and the H(2)O addition to flavylium cation 4AH(+) followed by H(+) elimination leading to hemiketal 4B are both retarded in the presence of clip C2, whereas the photochemically induced trans-cis isomerization (4Ct -> 4Cc) is not affected by clip C2. The results presented here are explained with dominating hydrophobic interactions between the molecular clips and the flavylium guest molecules. The other potential interactions (ion-ion, cation-pi, pi-pi, and CH-pi), which certainly determine the structures of these host-guest complexes to a large extent, seem to be of minor importance for their stability.

Magnetic particles {(MNPs)} offer attractive possibilities in biotechnology. {MNPs} can get close to a target biological entity, as their controllable sizes range from a few nanometres up to tens of nanometres, and their surface can be modified to add affinity and specificity towards desired molecules. Additionally, they can be manipulated by an external magnetic field gradient. In this work, the study of ferric oxide {(Fe3O4)} {MNPs} with different coating agents was conducted, particularly in terms of strategies for antibody attachment at the surfaces (covalent and physical adsorption) and the effects of blocking buffer composition and incubation times on the specific and non-specific interactions observed. The considered biological model system consisted of a coating antibody (goat {IgG)}, bovine serum albumin {(BSA)} as blocking agent, and a complementary antibody labelled with {FITC} (anti-goat {IgG).} The detection of antibody binding was followed by fluorescence microscopy and the intensity of the signals quantified. The ratio between the mean grey values of negative and positive controls, as well as the maximum intensity attainable in positive controls, were considered in the evaluation of the assays efficiency. The covalent immobilization of the coating antibody was more successful as opposed to protein adsorption. For covalent immobilization, silica-coated {MNPs}, a 5% (w/v) concentration of {BSA} in the blocking buffer and incubation times of 1 h produced the best results in terms of assay sensitivity. However, when conducting the assay for incubation periods of 10 min, the fluorescence signal was reduced by 44% but the assay specificity was maintained.

The surface modification of iron oxide magnetic nanoparticles {(MNPs)} with gum Arabic {(GA)} via adsorption and covalent coupling was studied. The adsorption of {GA} was assessed during {MNP} chemical synthesis by the co-precipitation method {(MNP\_GA)}, and after {MNP} synthesis on both bare magnetite and {MNP\_GA.} The covalent immobilization of {GA} at the surface of aldehyde-activated {(MNP\_GAAPTES)} or aminated {MNPs} {(MNP\_GAEDC)} was achieved through free terminal amino and carboxylate groups from {GA.} The presence of {GA} at the surface of the {MNPs} was confirmed by {FTIR} and by the quantification of {GA} by the bicinchoninic acid test. Results indicated that the maximum of {GA} coating was obtained for the covalent coupling of {GA} through its free carboxylate groups {(MNP\_GAEDC)}, yielding a maximum of 1.8&\#xa0;g of {GA} bound/g of dried particles. The hydrodynamic diameter of {MNPs} modified with {GA} after synthesis resulted in the lowest values, in opposition to the {MNPs} co-precipitated with {GA} which presented the tendency to form larger aggregates of up to 1&\#xa0;μm. The zeta potentials indicate the existence of negatively charged surfaces before and after {GA} coating. The potential of the {GA} coated {MNPs} for further biomolecule attachment was assessed through anchorage of a model antibody to aldehyde-functionalized {MNP\_GA} and its subsequent detection with an {FITC} labeled anti-antibody.

Wound healing is a complex process involving an integrated response by many different cell types and growth factors in order to achieve rapid restoration of skin architecture and function. The present study evaluated the applicability of a chitosan hydrogel (CH) as a wound dressing. Scanning electron microscopy analysis was used to characterize CH morphology. Fibroblast cells isolated from rat skin were used to assess the cytotoxicity of the hydrogel. CH was able to promote cell adhesion and proliferation. Cell viability studies showed that the hydrogel and its degradation by-products are noncytotoxic. The evaluation of the applicability of CH in the treatment of dermal burns in Wistar rats was performed by induction of full-thickness transcutaneous dermal wounds. Wound healing was monitored through macroscopic and histological analysis. From macroscopic analysis, the wound beds of the animals treated with CH were considerably smaller than those of the controls. Histological analysis revealed lack of a reactive or a granulomatous inflammatory reaction in skin lesions with CH and the absence of pathological abnormalities in the organs obtained by necropsy, which supported the local and systemic histocompatibility of the biomaterial. The present results suggest that this biomaterial may aid the re-establishment of skin architecture.

Electrospun cellulose-based nano and microfibers and a nematic liquid crystal are used to assemble an electro-optical (EO) light-scattering device that shows enhanced characteristics when compared to similar devices. Based on the controlled scattering of light in the composite system, the device can achieve light transmission coefficients tunable from 1% up to around 89%. Simulation of the EO behavior indicates that the roughness of the polymer-liquid crystal interface is crucial for the optical performance of the device.

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