Transglutaminase-catalyzed crosslinking of the Aα and γ constituent chains in fibrinogen

Studies on transglutaminases usually focus on the polymerization of protein substrates by intermolecular Nɛ(γ-glutamyl)lysine bridges, without considering the possibility that the monomeric protein units, themselves, could also become crosslinked internally. Both types of crosslinks are produced in the reaction of fibrinogen with red cell transglutaminase. We isolated the transglutaminase-modified, mostly monomeric form (92–96%) of fibrinogen with a Nɛ(γ-glutamyl)lysine content of ≈1.6 moles/mole of fibrinogen. The preparation was fully clottable by thrombin, but the rates of release of fibrinopeptides and clotting times were delayed compared with control. Hybrid Aα⋅γ type of crosslinking, the hallmark of the reaction of the transglutaminase with fibrinogen, occurred by bridging the Aα(408–421) chain segment of the protein to that of γ(392–406). Rotary shadowed electron microscope images showed many monomers to be bent, and the crosslinks seemed to bind the otherwise flexible αC domain closer to the backbone of fibrinogen.

14-3-3 protein is a regulator of the mitochondrial and chloroplast ATP synthase

Mitochondrial and chloroplast ATP synthases are key enzymes in plant metabolism, providing cells with ATP, the universal energy currency. ATP synthases use a transmembrane electrochemical proton gradient to drive synthesis of ATP. The enzyme complexes function as miniature rotary engines, ensuring energy coupling with very high efficiency. Although our understanding of the structure and functioning of the synthase has made enormous progress in recent years, our understanding of regulatory mechanisms is still rather preliminary. Here we report a role for 14-3-3 proteins in the regulation of ATP synthases. These 14-3-3 proteins are highly conserved phosphoserine/phosphothreonine-binding proteins that regulate a wide range of enzymes in plants, animals, and yeast. Recently, the presence of 14-3-3 proteins in chloroplasts was illustrated, and we show here that plant mitochondria harbor 14-3-3s within the inner mitochondrial-membrane compartment. There, the 14-3-3 proteins were found to be associated with the ATP synthases, in a phosphorylation-dependent manner, through direct interaction with the F1 β-subunit. The activity of the ATP synthases in both organelles is drastically reduced by recombinant 14-3-3. The rapid reduction in chloroplast ATPase activity during dark adaptation was prevented by a phosphopeptide containing the 14-3-3 interaction motif, demonstrating a role for endogenous 14-3-3 in the down-regulation of the CFoF1 activity. We conclude that regulation of the ATP synthases by 14-3-3 represents a mechanism for plant adaptation to environmental changes such as light/dark transitions, anoxia in roots, and fluctuations in nutrient supply.

Symmetries in bacterial motility

Descriptions are given of three kinds of symmetries encountered in studies of bacterial locomotion, and of the ways in which they are circumvented or broken. A bacterium swims at very low Reynolds number: it cannot propel itself using reciprocal motion (by moving through a sequence of shapes, first forward and then in reverse); cyclic motion is required. A common solution is rotation of a helical filament, either right- or left-handed. The flagellar rotary motor that drives each filament generates the same torque whether spinning clockwise or counterclockwise. This symmetry is broken by coupling to the filament. Finally, bacterial populations, grown in a nutrient medium from an inoculum placed at a single point, usually move outward in symmetric circular rings. Under certain conditions, the cells excrete a chemoattractant, and the rings break up into discrete aggregates that can display remarkable geometric order.

Cell Adhesion Molecule L1 in Folded (Horseshoe) and Extended Conformations

We have investigated the structure of the cell adhesion molecule L1 by electron microscopy. We were particularly interested in the conformation of the four N-terminal immunoglobulin domains, because x-ray diffraction showed that these domains are bent into a horseshoe shape in the related molecules hemolin and axonin-1. Surprisingly, rotary-shadowed specimens showed the molecules to be elongated, with no indication of the horseshoe shape. However, sedimentation data suggested that these domains of L1 were folded into a compact shape in solution; therefore, this prompted us to look at the molecules by an alternative technique, negative stain. The negative stain images showed a compact shape consistent with the expected horseshoe conformation. We speculate that in rotary shadowing the contact with the mica caused a distortion of the protein, weakening the bonds forming the horseshoe and permitting the molecule to extend. We have thus confirmed that the L1 molecule is primarily in the horseshoe conformation in solution, and we have visualized for the first time its opening into an extended conformation. Our study resolves conflicting interpretations from previous electron microscopy studies of L1.

Energy-driven subunit rotation at the interface between subunit a and the c oligomer in the FO sector of Escherichia coli ATP synthase

Subunit rotation within the F1 catalytic sector of the ATP synthase has been well documented, identifying the synthase as the smallest known rotary motor. In the membrane-embedded FO sector, it is thought that proton transport occurs at a rotor/stator interface between the oligomeric ring of c subunits (rotor) and the single-copy a subunit (stator). Here we report evidence for an energy-dependent rotation at this interface. FOF1 was expressed with a pair of substituted cysteines positioned to allow an intersubunit disulfide crosslink between subunit a and a c subunit [aN214C/cM65C; Jiang, W. & Fillingame, R. H. (1998) Proc. Natl. Acad. Sci. USA 95, 6607–6612]. Membranes were treated with N,N′-dicyclohexyl-[14C]carbodiimide to radiolabel the D61 residue on less than 20% of the c subunits. After oxidation to form an a–c crosslink, the c subunit properly aligned to crosslink to subunit a was found to contain very little 14C label relative to other members of the c ring. However, exposure to MgATP before oxidation significantly increased the radiolabel in the a–c crosslink, indicating that a different c subunit was now aligned with subunit a. This increase was not induced by exposure to MgADP/Pi. Furthermore, preincubation with MgADP and azide to inhibit F1 or with high concentrations of N,N′-dicyclohexylcarbodiimide to label most c subunits prevented the ATP effect. These results provide evidence for an energy-dependent rotation of the c ring relative to subunit a.

An axoplasmic myosin with a calmodulin-like light chain.

Organelles in the axoplasm from the squid giant axon move along exogenous actin filaments toward their barbed ends. An approximately 235-kDa protein, the only band recognized by a pan-myosin antibody in Western blots of isolated axoplasmic organelles, has been previously proposed to be a motor for these movements. Here, we purify this approximately 235-kDa protein (p235) from axoplasm and demonstrate that it is a myosin, because it is recognized by a pan-myosin antibody and has an actin-activated Mg-ATPase activity per mg of protein 40-fold higher than that of axoplasm. By low-angle rotary shadowing, p235 differs from myosin II and it does not form bipolar filaments in low salt. The amino acid sequence of a 17-kDa protein that copurifies with p235 shows that it is a squid optic lobe calcium-binding protein, which is more similar by amino acid sequence to calmodulin (69% identity) than to the light chains of myosin II (33% identity). A polyclonal antibody to this light chain was raised by using a synthetic peptide representing the calcium binding domain least similar to calmodulin. We then cloned this light chain by reverse transcriptase-PCR and showed that this antibody recognizes the bacterially expressed protein but not brain calmodulin. In Western blots of sucrose gradient fractions, the 17-kDa protein is found in the organelle fraction, suggesting that it is a light chain of the p235 myosin that is also associated with organelles.

Atenuação de vibrações em pás de helicópteros utilizando circuito piezelétrico semi-passivo

O uso de materiais inteligentes em problemas de controle de vibração tem sido investigado em diversas pesquisas ao longo dos últimos anos. Apesar de que diferentes materiais inteligentes estão disponíveis, o piezelétrico tem recebido grande atenção devido à facilidade de uso como sensores, atuadores, ou ambos simultaneamente. As principais técnicas de controle usando materiais piezoelétricos são os ativos e passivos. Circuitos piezelétricos passivos são ajustados para uma frequência específica e, portanto, a largura de banda efetiva é pequena. Embora os sistemas ativos possam apresentar um bom desempenho no controle de vibração, a quantidade de energia externa e hardware adicionado são questões importantes. As técnicas SSD (Synchronized Switch Damping) foram desenvolvidas como uma alternativa aos controladores passivos e controladores ativos de vibração. Elas podem ser técnicas semi-ativas ou semi-passivas que introduzem um tratamento não linear na tensão elétrica proveniente do material piezelétrico e induz um aumento na conversão de energia mecânica para energia elétrica e, consequentemente, um aumento no efeito de amortecimento. Neste trabalho, o controle piezoelétrico semi-passivo de uma pá piezelétrica engastada é apresentado e comparado com outros controladores. O modelo não linear electromecânico de uma pá com piezocerâmicas incorporados é determinado com base no método variacional-assintótico (VAM). O sistema rotativo acoplado não linear é resolvido no domínio do tempo, utilizando um método de integração alfa-generalizado afim de garantir a estabilidade numérica. As simulações são realizadas para uma vasta gama de velocidades de rotação. Em primeiro lugar, um conjunto de resistências (variando desde a condição de curto-circuito para a condição de circuito aberto) é considerada. O efeito da resistência ótima (que resulta em máximo amortecimento) sobre o comportamento do sistema é investigado para o aumento da velocidade de rotação. Mais tarde, a técnica SSDS é utilizada para amortecer as oscilações da pá com o aumento da velocidade de rotação. Os resultados mostram que a técnica SSDS pode ser um método útil para o controle de vibrações de vigas rotativas não lineares, tais como pás de helicóptero.

Contribuição ao estudo de instabilidade em túneis não revestidos da estrada de ferro Vitória-Minas através da teoria dos blocos-chave e caracterização da rocha através de ensaios laboratoriais e de campo.

No país existem inúmeras estruturas e obras civis que estão em operação a dezenas de anos e necessitam de monitoramento periódico devido a sua importância. Por este motivo, a dissertação apresenta um caso de um túnel antigo com problema de queda de bloco e visa instigar novas pesquisas e aumentar o conhecimento sobre o tema. Foram realizadas inspeções em campo em alguns túneis não revestidos da Estrada de Ferro Vitória-Minas (EFVM), bem como os ensaios em laboratório e in situ realizados nas amostras e no maciço rochoso para caracterizar o problema. Para o estudo foi escolhido o túnel Monte Seco Linha 1 e Linha 2 nos quais foram realizadas sondagens rotativas inclinadas e orientadas próximas ao eixo para investigação dos planos de descontinuidade. Os conceitos da Teoria dos Blocos-Chave foram aplicados às famílias de descontinuidades encontradas nos Túneis Monte Seco L1 e L2 para identificar os possíveis blocos instáveis formados pelas escavações. Para obtenção dos parâmetros geotécnicos de resistência e deformabilidade foram realizados ensaios de compressão uniaxial instrumentados com strain gages. A resistência a tração foi obtida através de Ensaio de Compressão Diametral (ECD). No ensaio de campo foi utilizado o Martelo de Schmidt para avaliação da rocha in situ. Através da análise dos dados foi possível distinguir setores cuja ocorrência de queda de blocos são maiores e a classe do maciço rochoso de acordo com a proposta de Bieniawski.

Inventario y descripción de los hornos de cal en el agrosistema tradicional de la Finca Buixcarró (P.N. Sierra de Mariola)

Se ha realizado un inventario de los hornos de cal en la Finca Buixcarró, término municipal de Bocairente (Valencia), situada en el Parque Natural de la Sierra de Mariola. Para localizar los hornos, se ha contado con la colaboración del responsable técnico y también se fotointerpretarán mediante la aplicación Google Earth, con tal de encontrar los hornos. Para ello se elaborará una capa de puntos con formato *.kml, posteriormente introduciremos en al GPS, mediante el software (MapSource). Se muestreó la finca en búsqueda de hornos y se georreferenció cada uno de ellos mediante la utilización de un dispositivo GPS, generando un waypoint con su X-Y correspondiente. Una vez georreferenciados los hornos de la finca con formato *.gpx, se elaboró la cartografía correspondiente. Se utilizará GPSBabel y QuantumGis para elaborar la cartografía de los hornos de cal. Se han registrado un total de 24 hornos de cal, de los cuales el 60% presentan un estado de degradación bastante elevado. Se han identificado 20 categorías de usos del suelo, siendo el bosque mixto (Pinus halepensis – Quercus ilex) y el pinar carrasco (Pinus halepensis) las coberturas que mayor superficie ocupan. El inventariado de estas infraestructuras contribuye a la conservación del patrimonio material y cultural de este espacio natural protegido.

Development of a Mechanism and Process to Continuously Produce Spherical, Torrefied Biomass

Thesis (Master, Mechanical and Materials Engineering) -- Queen's University, 2016-06-17 02:15:25.215

Acompanhamento da construção de uma captação de água subterrânea

Tese de mestrado, Geologia Aplicada (Hidrogeologia) Universidade de Lisboa, Faculdade de Ciências, 2016

Mobbing nos enfermeiros

Introdução: As pressões constantes do mundo laboral e, a especificidade do seu trabalho expõe os enfermeiros a actos de violência como o mobbing ou assédio moral. Este é um fenómeno dissimulado e psicossocial que afecta o indivíduo, o grupo de trabalho e a organização e, que importa aprender a combater. Objectivos: Determinar a prevalência de mobbing nos enfermeiros; caracterizar o mobbing; relacionar a influência das características sociodemográficas e profissionais na percepção de mobbing dos enfermeiros. Metodologia: Estudo quantitativo, de carácter descritivo-correlacional, transversal. A amostra não probabilística por conveniência foi constituída por 143 enfermeiros do Hospital Sousa Martins, Guarda. Maioritariamente são mulheres (71,3%) e, com média de idades de 37 anos. Possuem formação base 71,3%, 69,9% pratica horário rotativo, 69,2% tem vínculo estável e, tempo médio de exercício profissional de 14 anos. Os dados foram colhidos através de questionário que integrou a escala LIPT-60. Resultados: Os enfermeiros em estudo experienciaram baixos níveis de mobbing no seu contexto laboral. Em média, referem sentir oito condutas de assédio moral com efeito (0,20) e intensidade reduzida (1,37). As condutas mais experimentadas visam o bloqueio à comunicação e a difamação. Cerca de 42,0% dos enfermeiros admitem já ter sido vítima de mobbing e 24,1% referem que aconteceu por um período de seis meses. Os principais agressores identificados foram os médicos (40,0%) e os superiores hierárquicos (37,1%). Contudo, a percepção de mobbing é maior à medida que se progride na carreira, bem como nos enfermeiros que praticam regime de horário fixo e, que trabalham no mesmo serviço há 5 – 10 anos. Conclusões: Apesar dos baixos índices de percepção de mobbing, este está presente no contexto laboral dos enfermeiros tornando-os vulneráveis e afectando a prestação de cuidados. Os ataques sentidos acontecem sobretudo, de forma dissimulada fazendo denotar a gravidade deste fenómeno, sobre o qual impera prevenir e intervir. Palavras-Chave: Mobbing; Assédio moral; Abuso psicológico; Violência psicológica; Agressão psicológica no trabalho.

Enriching plant diversity in grasslands by large-scale experimental sward disturbance and seed addition along gradients of land-use intensity

Aims The relationship between biodiversity and ecosystem functioning is among the most active areas of ecological research. Furthermore, enhancing the diversity of degraded ecosystems is a major goal in applied restoration ecology. In grasslands, many species may be locally absent due to dispersal or microsite limitation and may therefore profit from mechanical disturbance of the resident vegetation. We established a seed addition and disturbance experiment across several grassland sites of different land use to test whether plant diversity can be increased in these grasslands. Additionally, the experiment will allow us testing the consequences of increased plant diversity for ecosystem processes and for the diversity of other taxa in real-world ecosystems. Here we present details of the experimental design and report results from the first vegetation survey one year after disturbance and seed addition. Moreover, we tested whether the effects of seed addition and disturbance varied among grassland depending on their land use or pre-disturbance plant diversity. Methods A full-factorial experiment was installed in 73 grasslands in three regions across Germany. Grasslands were under regular agricultural use, but varied in the type and the intensity of management, thereby representing the range of management typical for large parts of Central Europe. The disturbance treatment consisted of disturbing the top 10 cm of the sward using a rotavator or rotary harrow. Seed addition consisted of sowing a high-diversity seed mixture of regional plant species. These species were all regionally present, but often locally absent, depending on the resident vegetation composition and richness of each grassland. Important findings One year after sward disturbance it had significantly increased cover of bare soil, seedling species richness and numbers of seedlings. Seed addition had increased plant species richness, but only in combination with sward disturbance. The increase in species richness, when both seed addition and disturbance was applied, was higher at high land-use intensity and low resident diversity. Thus, we show that at least the early recruitment of many species is possible also at high land-use intensity, indicating the potential to restore and enhance biodiversity of species-poor agricultural grasslands. Our newly established experiment provides a unique platform for broad-scale research on the land-use dependence of future trajectories of vegetation diversity and composition and their effects on ecosystem functioning.