N2 fixation in marine heterotrophic bacteria: dynamics of environmental and molecular regulation.

Molecular and immunological techniques were used to examine N2 fixation in a ubiquitous heterotrophic marine bacterium, the facultative anaerobic Vibrio natriegens. When batch cultures were shifted from aerobic N-replete to anaerobic N-deplete conditions, transcriptional and post-translational regulation of N2 fixation was observed. Levels of nifHDK mRNA encoding the nitrogenase enzyme were highest at 140 min postshift and undetectable between 6 and 9 h later. Immunologically determined levels of nitrogenase enzyme (Fe protein) were highest between 6 and 15 h postshift, and nitrogenase activity peaked between 6 and 9 h postshift, declining by a factor of 2 after 12-15 h. Unlike their regulation in cyanobacteria, Fe protein and nitrogenase activity were present when nifHDK mRNA was absent in V. natriegens, indicating that nitrogenase is stored and stable under anaerobic conditions. Both nifHDK mRNA and Fe protein disappeared within 40 min after cultures were shifted from N2-fixing conditions (anaerobic, N-deplete) to non- N2-fixing conditions (aerobic, N-enriched) but reappeared when shifted to conditions favoring N2 fixation. Thus, unlike other N2-fixing heterotrophic bacteria, nitrogenase must be resynthesized after aerobic exposure in V. natriegens. Immunological detection based on immunoblot (Western) analysis and immunogold labeling correlated positively with nitrogenase activity; no localization of nitrogenase was observed. Because V. natriegens continues to fix N2 for many hours after anaerobic induction, this species may play an important role in providing "new" nitrogen in marine ecosystems.

Reconstitution of repair-gap UV mutagenesis with purified proteins from Escherichia coli: a role for DNA polymerases III and II.

Using a cell-free system for UV mutagenesis, we have previously demonstrated the existence of a mutagenic pathway associated with nucleotide-excision repair gaps. Here, we report that this pathway can be reconstituted by using six purified proteins: UvrA, UvrB, UvrC, DNA helicase II, DNA polymerase III core, and DNA ligase. This establishes the minimal requirements for repair-gap UV mutagenesis. DNA polymerase II could replace DNA polymerase III, although less effectively, whereas DNA polymerase I, the major repair polymerase, could not. DNA sequence analysis of mutations generated in the in vitro reaction revealed a spectrum typical of mutations targeted to UV lesions. These observations suggest that repair-gap UV mutagenesis is performed by DNA polymerase III, and to a lesser extent by DNA polymerase II, by filling-in of a rare class of excision gaps that contain UV lesions.

X-ray structure of clotting factor IXa: active site and module structure related to Xase activity and hemophilia B.

Hereditary deficiency of factor IXa (fIXa), a key enzyme in blood coagulation, causes hemophilia B, a severe X chromosome-linked bleeding disorder afflicting 1 in 30,000 males; clinical studies have identified nearly 500 deleterious variants. The x-ray structure of porcine fIXa described here shows the atomic origins of the disease, while the spatial distribution of mutation sites suggests a structural model for factor X activation by phospholipid-bound fIXa and cofactor VIIIa. The 3.0-A-resolution diffraction data clearly show the structures of the serine proteinase module and the two preceding epidermal growth factor (EGF)-like modules; the N-terminal Gla module is partially disordered. The catalytic module, with covalent inhibitor D-Phe-1I-Pro-2I-Arg-3I chloromethyl ketone, most closely resembles fXa but differs significantly at several positions. Particularly noteworthy is the strained conformation of Glu-388, a residue strictly conserved in known fIXa sequences but conserved as Gly among other trypsin-like serine proteinases. Flexibility apparent in electron density together with modeling studies suggests that this may cause incomplete active site formation, even after zymogen, and hence the low catalytic activity of fIXa. The principal axes of the oblong EGF-like domains define an angle of 110 degrees, stabilized by a strictly conserved and fIX-specific interdomain salt bridge. The disorder of the Gla module, whose hydrophobic helix is apparent in electron density, can be attributed to the absence of calcium in the crystals; we have modeled the Gla module in its calcium form by using prothrombin fragment 1. The arched module arrangement agrees with fluorescence energy transfer experiments. Most hemophilic mutation sites of surface fIX residues occur on the concave surface of the bent molecule and suggest a plausible model for the membrane-bound ternary fIXa-FVIIIa-fX complex structure: fIXa and an equivalently arranged fX arch across an underlying fVIIIa subdomain from opposite sides; the stabilizing fVIIIa interactions force the catalytic modules together, completing fIXa active site formation and catalytic enhancement.

A second nitrogenase in vegetative cells of a heterocyst-forming cyanobacterium.

In many filamentous cyanobacteria nitrogen fixation occurs in differentiated cells called heterocysts. Filamentous strains that do not form heterocysts may fix nitrogen in vegetative cells, primarily under anaerobic conditions. We describe here two functional Mo-dependent nitrogenases in a single organism, the cyanobacterium Anabaena variabilis. Using a lacZ reporter with a fluorescent beta-galactoside substrate for in situ localization of gene expression, we have shown that the two clusters of nif genes are expressed independently. One nitrogenase functions only in heterocysts under either aerobic or anaerobic growth conditions, whereas the second nitrogenase functions only under anaerobic conditions in vegetative cells and heterocysts. Differences between the two nif clusters suggest that the nitrogenase that is expressed in heterocysts is developmentally regulated while the other is regulated by environmental factors.

Chloroplast gene sequence data suggest a single origin of the predisposition for symbiotic nitrogen fixation in angiosperms.

Of the approximately 380 families of angiosperms, representatives of only 10 are known to form symbiotic associations with nitrogen-fixing bacteria in root nodules. The morphologically based classification schemes proposed by taxonomists suggest that many of these 10 families of plants are only distantly related, engendering the hypothesis that the capacity to fix nitrogen evolved independently several, if not many, times. This has in turn influenced attitudes toward the likelihood of transferring genes responsible for symbiotic nitrogen fixation to crop species lacking this ability. Phylogenetic analysis of DNA sequences for the chloroplast gene rbcL indicates, however, that representatives of all 10 families with nitrogen-fixing symbioses occur together, with several families lacking this association, in a single clade. This study therefore indicates that only one lineage of closely related taxa achieved the underlying genetic architecture necessary for symbiotic nitrogen fixation in root nodules.

Estudo morfológico e eletrofisiológico dos efeitos da injeção intravítrea de ácido micofenólico em coelhos utilizando um modelo de uveíte crônica experimental

Uveítes são inflamações intra-oculares geralmente crônicas e constituem uma das principais causas de cegueira no mundo. Os corticosteroides são a droga de primeira escolha para o tratamento das uveítes não infecciosas, mas muitas vezes há necessidade do uso de outras drogas imunossupressoras. O micofenolato de mofetila (MMF) é um potente imunossupressor administrado por via oral que vem sendo utilizado com sucesso no tratamento das uveítes, mas cujos efeitos colaterais muitas vezes tornam necessária sua suspensão. O MMF é uma pró-droga, que é transformada no fígado em ácido micofenólico (MPA), o imunossupressor ativo. Para minimizar os efeitos colaterais do uso do MPA e permitir que o olho receba uma dose maior da droga, testamos os efeitos da injeção intravítrea do MPA em um modelo de uveíte crônica experimental (UCE) em olhos de coelhos. Os objetivos deste estudo foram: 1) reproduzir um modelo de UCE em coelhos através da injeção intravítrea de M. tuberculosis; 2) estabelecer uma dose segura de MPA a ser injetada no vítreo; e 3) analisar os efeitos morfológicos, clínicos e eletrofisiológicos da injeção intravítrea de MPA em coelhos utilizados como modelo de UCE. O modelo de UCE reproduzido apresentou uma inflamação autolimitada, possuindo um pico de inflamação no 17° dia após a indução da uveíte. As doses de MPA testadas (0,1 e 1mg) não foram toxicas para a retina do coelho. O modelo de UCE recebeu uma injeção intravítrea de 0,1mg de MPA e as análises clinicas demonstraram uma redução na inflamação. As análises realizadas com o eletrorretinograma (ERG) também apontaram uma melhora na inflamação através da recuperação da latência das ondas-a e b (fotópicas e escotópica) e recuperação da amplitude da onda-a (fotópica). As análises morfológicas com HE não apresentaram alterações na estrutura retinia, porem a imunohistoquimica para proteína GFAP evidenciou gliose das células de Müller, sinalizando um processo inflamatório. Concluímos que o modelo de UCE reproduziu uma uveíte anterior semelhante à uveíte causada em humanos e a dose de MPA utilizada apresentou efeitos terapêuticos durante o pico de inflamação, mostrando uma diminuição da inflamação e promovendo a recuperação de fotorreceptores e células bipolares-ON. Este resultado faz das injeções intravítreas de MPA um recurso promissor no tratamento de uveítes. Porém, novos experimentos são necessários para padronizar os resultados encontrados

Sensoriamento automático e participativo em cidades.

As cidades estão a seu tempo e a seu modo, modernizando os serviços prestados à população. Entre os diversos fatores que estão contribuindo para esta evolução estão a diversificação e proliferação de sensores, nos diversos domínios de serviços das cidades, e os novos canais de comunicação com os munícipes, entre eles, as redes sociais e mais recentemente os sistemas crowdsensing, motivados pelos anseios sociais, por melhores serviços públicos e pela popularização dos dispositivos móveis. Nesta direção, a eficiência administrativa é um fator essencial, uma vez que as cidades estão se mostrando mais complexas na medida em que cresce a população nas áreas urbanas. A utilização de técnicas de sistemas distribuídos para que múltiplos domínios de serviços usufruam da mesma infraestrutura computacional, pode auxiliar na eficiência das cidades, evitando gastos administrativos duplicados e até mesmo, possibilitando a correlação de eventos entre os serviços, favorecendo a identificação de fatores de causalidades e assim, a tomada de decisões administrativas mais objetivas e precisas. Neste contexto, este trabalho concentra-se na análise de um middleware direcionado à gestão de cidades para coleta, integração e interpretação dos dados de sensores, pertencentes aos serviços disponíveis da própria cidade, junto com os dados do sensoriamento colaborado pelos cidadãos. Para avaliação do conceito foi investigado o cenário de monitoração da conservação de vias públicas. Após 3 meses de coletas de dados por um sistema de sensoriamento automático, totalizando mais de 360 mil pontos e também mais de 90 relatórios pelo sensoriamento participativo, verificou-se que um sistema distribuído pode realizar a interpretação de séries históricas, engajar os munícipes apoiar a manutenção dos serviços da cidade e também indicar objetivamente aos gestores públicos os pontos que devem ser prioritariamente atendidos. Aliar ferramentas pelas quais o cidadão pode, de acordo com sua necessidade, convicção e altruísmo, exercer influência nos gestores públicos com o suporte de informação contínua e critérios objetivos das redes de sensores, pode estimular a continua excelência dos serviços públicos.

Dibujando la arquitectura olvidada: las defensas militares de la guerra de 1936-39 [PPT]

A decade before there was getting up the 'Atlantic Wall', there was executed a system of defenses along the Mediterranean coast in Spain (1936-39). The recovery of the same constructions (both of his graphical documents and of the constructed works that stay in foot) and his putting in value it can help to consolidate an own memory of the 20th century. This work considers to inventory, to measure and to draw the planes of these architectures to fix the memory that is diluted by the erosion of the time. The military pieces place in many borders: are these properly architecture? These are walking between two epochs: the one that perpetuates the epic acts in opposite to the one that shows the disasters in order that they do not forget. They are the most modern ruins of our history. In this process of reconstruction of the memory, there turns out to be crucial the graphical restitution. The drawing is a source of knowledge and demonstrates facts that were constructed.

Dibujando la arquitectura olvidada: las defensas militares de la guerra de 1936-39

Comunicación presentada en el XI Congreso Internacional de Expresión Gráfica Aplicada a la Edificación, APEGA 2012, Valencia, 29 noviemnre-1 diciembre 2012.

Muro Mediterráneo versus movimiento moderno

Una década antes de que fuera construido el Muro Atlántico fue ejecutado un sistema de defensas a lo largo de la costa del Mediterráneo español (1936-39). La recuperación de estas construcciones (de sus documentos gráficos) y su puesta en valor pueden ayudar a consolidar una memoria propia del siglo XX. Este trabajo consiste en inventariar, medir y dibujar los planos de estas arquitecturas a fin de fijar la memoria que se diluye por la erosión del tiempo. Estas defensas militares se sitúan en muchas fronteras: ¿son propiamente arquitectura o piezas industriales? ¿Son arquitectura moderna? Estas transitan entre dos mundos: uno que proyecta arquitecturas ligeras, flexibles y con caducidad frente a otro que construye obras compactas, rígidas y eternas. También se mueven por dos épocas: una que perpetúa las hazañas épicas frente a otras que muestra los desastres. Espacio, tiempo y materia. Son las ruinas de hormigón más modernas de nuestra historia que se encuentran camufladas en la topografía: templos y tumbas a la vez. En esta reconstrucción de la memoria, resulta crucial la restitución gráfica que es donde comienza el conocimiento.

Modeling the role of fixational eye movements in real-world scenes

Our eyes never remain still. Even when we stare at a fixed point, small involuntary movements take place in our eyes in an imperceptible manner. Researchers agree on the presence of three main contributions to eye movements when we fix the gaze: microsaccades, drifts and tremor. These small movements carry the image across the retina stimulating the photoreceptors and thus avoiding fading. Nowadays it is commonly accepted that these movements can improve the discrimination performance of the retina. In this paper, several retina models with and without fixational eye movements were implemented by mean of RetinaStudio tool to test the feasibility of these models to be incorporated in future neuroprostheses. For this purpose each retina model has been stimulated with natural scene images in two experiments. Results are discussed from the point of view of a neuroprosthesis development.

An adaptive algorithm for clustering cumulative probability distribution functions using the Kolmogorov–Smirnov two-sample test

This paper proposes an adaptive algorithm for clustering cumulative probability distribution functions (c.p.d.f.) of a continuous random variable, observed in different populations, into the minimum homogeneous clusters, making no parametric assumptions about the c.p.d.f.’s. The distance function for clustering c.p.d.f.’s that is proposed is based on the Kolmogorov–Smirnov two sample statistic. This test is able to detect differences in position, dispersion or shape of the c.p.d.f.’s. In our context, this statistic allows us to cluster the recorded data with a homogeneity criterion based on the whole distribution of each data set, and to decide whether it is necessary to add more clusters or not. In this sense, the proposed algorithm is adaptive as it automatically increases the number of clusters only as necessary; therefore, there is no need to fix in advance the number of clusters. The output of the algorithm are the common c.p.d.f. of all observed data in the cluster (the centroid) and, for each cluster, the Kolmogorov–Smirnov statistic between the centroid and the most distant c.p.d.f. The proposed algorithm has been used for a large data set of solar global irradiation spectra distributions. The results obtained enable to reduce all the information of more than 270,000 c.p.d.f.’s in only 6 different clusters that correspond to 6 different c.p.d.f.’s.

La aplicación de stop-motion en arquitectura: materia y luz

Se inicia un análisis de los procesos de trabajo de stop-motion porque ayudan a comprender las diferentes escalas en arquitectura donde las maquetas se convierten en futuros prototipos de infraestructuras de edificios o de paisaje. Stop motion es una técnica de animación fotograma a fotograma de objetos estáticos mediante la manipulación de figuras de plastilina en entornos fijos con cambios de luz, color y sonido. Igual que dicha técnica reúne lo mejor del rodaje tradicional -story board, escenografía, fotografía, personajes, iluminación- la animación de maquetas de interiores sintetiza micro-procesos de mayor repercusión -habitaciones con cambios de humedad, de temperatura, de ventilación y de iluminación- incorporando efectos especiales que son procesados digitalmente en post-producción. Se construyen varios prototipos de habitación con parámetros fijos como el tamaño y la posición de la cámara y otros variables como los materiales, los personajes y la iluminación. Representan un mundo en miniatura que intenta aportar un acercamiento sensorial y atmosférico analizando la magia y la fantasía que Junichirô Tanizaki describe en la penumbra de las construcciones tradicionales japonesas y estudiando las imperfecciones de los escenarios que Tim Burton manipula en su películas de animación con una textura que las tecnologías digitales no pueden igualar. El objetivo es utilizar una escala micro para realizar unos modelos interiores donde las condiciones atmosféricas están controladas y reducidas, y tomar datos que se podrían aplicar a un proceso de modelado a escala intermedia para testar prototipos de edificios como el túnel de viento; o, finalmente, a una escala macro con maquetas de un sector de la costa o de un río donde los fenómenos meteorológicos son los protagonistas para simular inundaciones y diseñar futuras medidas de prevención y seguridad.

In Search of a Feasible EU-Mercosul Free Trade Agreement. CEPS Working Document No. 378, 25 February 2013

This paper aims at identifying ways to pursue the EU–Mercosul negotiations leading to a free trade agreement (FTA). After reviewing their already long history, it outlines a basic framework, in goods, services and other themes, judged possible. The main point is that, given the prevailing conditions on both sides, an agreement to be signed within a reasonable time must be modest, i.e. along the described lines. It then clearly sets up the decisions confronting the negotiators: either to pursue the modest, feasible option or to terminate negotiations under the FTA heading. The latter, however, does not imply an end to the dialogue. Many actions and measures may be taken – which are easier to discuss and fix – that could pave the way for, in due time, a closer-to-ideal FTA to be considered again. These are the subjects of a last section.