Structural basis for heterogeneous kinetics: Reengineering the hairpin ribozyme

The RNA cleavage reaction catalyzed by the hairpin ribozyme shows biphasic kinetics, and chase experiments show that the slow phase of the reaction results from reversible substrate binding to an inactive conformational isomer. To investigate the structural basis for the heterogeneous kinetics, we have developed an enzymatic RNA modification method that selectively traps substrate bound to the inactive conformer and allows the two forms of the ribozyme-substrate complex to be separated and analyzed by using both physical and kinetic strategies. The inactive form of the complex was trapped by the addition of T4 RNA ligase to a cleavage reaction, resulting in covalent linkage of the 5′ end of the substrate to the 3′ end of the ribozyme and in selective and quantitative ablation of the slow kinetic phase of the reaction. This result indicates that the inactive form of the ribozyme-substrate complex can adopt a conformation in which helices 2 and 3 are coaxially stacked, whereas the active form does not have access to this conformation, because of a sharp bend at the helical junction that presumably is stabilized by inter-domain tertiary contacts required for catalytic activity. These results were used to improve the activity of the hairpin ribozyme by designing new interfaces between the two domains, one containing a non-nucleotidic orthobenzene linkage and the other replacing the two-way junction with a three-way junction. Each of these modified ribozymes preferentially adopts the active conformation and displays improved catalytic efficiency.

Identification of a functionally important sequence in the cytoplasmic tail of integrin beta 3 by using cell-permeable peptide analogs.

Integrins are major two-way signaling receptors responsible for the attachment of cells to the extracellular matrix and for cell-cell interactions that underlie immune responses, tumor metastasis, and progression of atherosclerosis and thrombosis. We report the structure-function analysis of the cytoplasmic tail of integrin beta 3 (glycoprotein IIla) based on the cellular import of synthetic peptide analogs of this region. Among the four overlapping cell-permeable peptides, only the peptide carrying residues 747-762 of the carboxyl-terminal segment of integrin beta 3 inhibited adhesion of human erythroleukemia (HEL) cells and of human endothelial cells (ECV) 304 to immobilized fibrinogen mediated by integrin beta 3 heterodimers, alpha IIb beta 3, and alpha v beta 3, respectively. Inhibition of adhesion was integrin-specific because the cell-permeable beta 3 peptide (residues 747-762) did not inhibit adhesion of human fibroblasts mediated by integrin beta 1 heterodimers. Conversely, a cell-permeable peptide representing homologous portion of the integrin beta 1 cytoplasmic tail (residues 788-803) inhibited adhesion of human fibroblasts, whereas it was without effect on adhesion of HEL or ECV 304 cells. The cell-permeable integrin beta 3 peptide (residues 747-762) carrying a known loss-of-function mutation (Ser752Pro) responsible for the genetic disorder Glanzmann thrombasthenia Paris I did not inhibit cell adhesion of HEL or ECV 304 cells, whereas the beta 3 peptide carrying a Ser752Ala mutation was inhibitory. Although Ser752 is not essential, Tyr747 and Tyr759 form a functionally active tandem because conservative mutations Tyr747Phe or Tyr759Phe resulted in a nonfunctional cell permeable integrin beta 3 peptide. We propose that the carboxyl-terminal segment of the integrin beta 3 cytoplasmic tail spanning residues 747-762 constitutes a major intracellular cell adhesion regulatory domain (CARD) that modulates the interaction of integrin beta 3-expressing cells with immobilized fibrinogen. Import of cell-permeable peptides carrying this domain results in inhibition "from within" of the adhesive function of these integrins.

Mice lacking the gene encoding tissue-type plasminogen activator show a selective interference with late-phase long-term potentiation in both Schaffer collateral and mossy fiber pathways.

The gene encoding tissue-type plasminogen activator (t-PA) is an immediate response gene, downstream from CREB-1 and other constitutively expressed transcription factors, which is induced in the hippocampus during the late phase of long-term potentiation (L-LTP). Mice in which the t-PA gene has been ablated (t-PA-/-) showed no gross anatomical, electrophysiological, sensory, or motor abnormalities but manifest a selective reduction in L-LTP in hippocampal slices in both the Schaffer collateral-CA1 and mossy fiber-CA3 pathways. t-PA-/- mice also exhibit reduced potentiation by cAMP analogs and D1/D5 agonists. By contrast, hippocampal-dependent learning and memory were not affected in these mice, whereas performance was impaired on two-way active avoidance, a striatum-dependent task. These results provide genetic evidence that t-PA is a downstream effector gene important for L-LTP and show that modest impairment of L-LTP in CA1 and CA3 does not result in hippocampus-dependent behavioral phenotypes.

Efeito do treinamento físico associado a um programa de perda de peso no controle clínico e nos fatores de saúde relacionados à qualidade de vida em asmáticos obesos

Introdução: O fenótipo asma-obeso caracteriza-se por uma asma mais grave, no qual o controle clínico é mais difícil de ser alcançado, mesmo sob terapia medicamentosa otimizada. A cirurgia bariátrica tem sido recomendada para perda de peso e melhora dos sintomas, porém os benefícios de intervenções não-cirúrgicas têm sido pouco estudados. Objetivo: Avaliar o efeito do treinamento físico associado a um programa de perda de peso no controle clínico da asma, qualidade de vida e sintomas psicossociais em asmáticos obesos. Métodos: 55 pacientes com asma moderada a grave e obesidade grau II (IMC >= 35 e < 39.9 kg/m2) foram alocados em 2 grupos: programa de perda de peso + placebo (PP+P) ou programa de perda de peso + exercícios (PP+E), sendo que o programa de perda de peso incluiu terapia nutricional e psicológica (12 sessões semanais de 60 minutos cada). O grupo PP+E associou exercícios aeróbios e resistidos programa de perda de peso, enquanto o grupo PP+P associou exercícios placebo (respiratórios e alongamentos), 2xvezes/semana, 60 minutos/sessão durante 3 meses. Antes e após as intervenções, foram avaliados o controle clínico da asma, os fatores de saúde relacionados a qualidade de vida (FSRQV), a capacidade física, a composição corporal, os sintomas de ansiedade e depressão, a qualidade do sono, a função pulmonar e as inflamações das vias aéreas e sistêmica. A comparação dos dados contínuos entre os grupos foi realizada por ANOVA de dois fatores com medidas repetidas e das variáveis categóricas pelo teste qui-quadrado. A correlação linear e a regressão linear múltipla foram utilizadas para avaliar associações entre as variáveis avaliadas. Resultados: Foram analisados os resultados de 51 pacientes que foram reavaliados. Comparado com o grupo PP+P, os pacientes que realizaram exercício apresentaram melhora no controle clínico da asma (- 0,7 [-1,3 - -0,3] vs. -0,3 [-0,9 - 0,4] escore ACQ; p=0,01) e nos FSRQV (0,8 [0,3 -2,0] vs. 0,4 [-0,3 - 0,9] escore AQLQ; p=0,02), respectivamente. Essa melhora parece ter sido consequência do aumento do condicionamento físico (3,0 [2,4-4,0] vs. 0,9 [-0,3-1,3] mL.O2/Kg/min; p < 0,001) e da perda de peso (6,8±3,5% vs. 3,1±2,6% do peso corpóreo; p < 0,001) nos pacientes do grupo PP+E, que também apresentaram uma melhora dos sintomas de depressão, da qualidade do sono (ronco, latência e eficiência) e dos níveis séricos de vitamina D. Houve também melhora da função pulmonar (capacidade vital forçada e volume de reserva expiratório) e das inflamações das vias aéreas (FeNO) e sistêmica (CCL2, CXCL9, IL-4, IL-6, TNF-alfa, IL-10 e leptina/adiponectina), que parecem ser possíveis mecanismos associados à melhora do controle clinico da asma nos pacientes do grupo PP+E (p < 0,05 para todas variáveis apresentadas). Conclusão: A inclusão do treinamento físico em um programa de perda de peso a curto prazo deve ser considerada como uma intervenção eficiente para associar à terapia medicamentosa da asma na melhora do controle clínico em asmáticos obesos

Managing Layoff Survivors

‰Û÷Survivor syndrome' or negative consequences of a layoff on survivors is the key reason why layoffs fail. This Capstone Project draws on downsizing-related research and proposes a customizable plan to revitalize the surviving workforce after a downsizing initiative. The plan divides downsizing-related interventions into three phases: pre-downsizing, day of the announcement, and post-downsizing. Key elements of this plan include linking the plan to a revised vision, detailed planning of the process, open, two-way communication systems, employee involvement in all stages of the process, respectable treatment of departing employees, plans to support survivors, training frontline managers, and implementing a top talent retention strategy. The Capstone also addresses roles and responsibilities of leaders, HR professionals, and managers in the survivor management process.

Transmedialidad: series de ficción y redes sociales. El caso de Pulseras Rojas en el grupo oficial de Facebook (Antena 3. España)

Analizamos el Facebook de la teleserie Pulseras Rojas emitida en España por Antena 3, en tanto recurso transmedia y fuente de oportunidades para estimular el engagement del espectador. Estudiamos el tráfico generado durante la emisión de la segunda temporada, entre julio y agosto de 2013. Siguiendo la propuesta de Askwith (2007), se pueden identificar siete modalidades de touchpoints, es decir, puntos de contacto con la narración, adicionales a la propia emisión televisiva: acceso expandido, contenido adaptado, contenido expandido, productos de marca, actividades relacionadas, interacción social e interactividad. El estudio exploratorio se basa en el análisis del contenido del grupo de la serie en Facebook e identifica las modalidades de touchpoints y sus resultados. Facebook se revela como un potente instrumento para involucrar al espectador, aunque la estrategia comunicativa de la cadena de televisión presenta una gestión incipiente, poco evolucionada, que desaprovecha el carácter interactivo y bidireccional de la red social.

An introduction to alysidal algebra (IV)

Purpose – Deontical impure systems are systems whose object set is formed by an s-impure set, whose elements are perceptuales significances (relative beings) of material and/or energetic objects (absolute beings) and whose relational set is freeways of relations, formed by sheaves of relations going in two-way directions and at least one of its relations has deontical properties such as permission, prohibition, obligation and faculty. The paper aims to discuss these issues. Design/methodology/approach – Mathematical and logical development of human society ethical and normative structure. Findings – Existence of relations with positive imperative modality (obligation) would constitute the skeleton of the system. Negative imperative modality (prohibition) would be the immunological system of protection of the system. Modality permission the muscular system, that gives the necessary flexibility. Four theorems have been formulated based on Gödel's theorem demonstrating the inconsistency or incompleteness of DISs. For each constructed systemic conception can happen to it one of the two following things: either some allowed responses are not produced or else some forbidden responses are produced. Responses prohibited by the system are defined as nonwished effects. Originality/value – This paper is a continuation of the four previous papers and is developed the theory of deontical impure systems.

Experiencia de transferencia de conocimiento en el proyecto de investigación ITSAL (Inmigración, Trabajo y SALud) con representantes de la población diana

Se describe una experiencia de transferencia de conocimiento entre investigadores del proyecto ITSAL (Inmigración, Trabajo y SALud) y representantes de asociaciones que trabajan con población inmigrante para discutir los resultados obtenidos en esta investigación y las líneas de futuro. Para ello se llevó a cabo una reunión en la que participaron tres investigadoras y 18 representantes de 11 instituciones. Tras una exposición de la metodología y los resultados del proyecto ITSAL, todos los asistentes comentaron los resultados presentados y líneas de investigación de interés. Se pusieron de manifiesto coincidencias entre ambas partes y también se obtuvieron propuestas de interés para el proyecto ITSAL. Entendemos el proceso descrito como una aproximación a la validación social de algunos resultados principales del proyecto. Esta experiencia nos ha permitido abrir un canal de comunicación con la población diana del estudio, en línea con la necesaria interacción de investigadores y población.

La gastronomía como Industria Creativa en un contexto digital. Análisis de webs y redes sociales de los restaurantes españoles con estrella Michelin

En los últimos años es evidente el éxito creciente de la gastronomía, no sólo como mercado económico sino también desde una perspectiva comunicativa. Entendida como una Industria Creativa, la gastronomía está adquiriendo cada vez más peso en el mercado de la comunicación y se están desarrollando planes estratégicos que acercan progresivamente el sector gastronómico a todos los públicos, democratizando el arte de la cocina y haciendo de éste un talento accesible y disponible para todos los públicos. En pleno contexto de crecimiento, las estrategias y recursos comunicativos que utilizan tanto los mass media como los propios restaurantes y profesionales de la cocina, se han de convertir en objeto de estudio necesario para entender, por ejemplo, de qué manera se explotan los recursos comunicacionales y cuál es el alcance de los mismos así como sus oportunidades. El presente artículo se centra en un análisis de contenido de las páginas webs y las redes sociales utilizadas por los ocho restaurantes españoles galardonados con tres estrellas por la Guía Michelin España-Portugal (2014), sometidos a estudio en la misma semana (del 15 al 21 de septiembre). El objetivo principal es tratar de conocer estrategia de comunicación online llevada a cabo por estos restaurantes y establecer la importancia que se les atribuye a los recursos web a la hora de contribuir tanto a la consolidación de sus propias marcas como a la propia Industria Creativa gastronómica. Los resultados muestran cierta disparidad en el uso tanto de los recursos web como de los social media y avanzan que el sector gastronómico y de la restauración española tiene un reto: seguir comunicando implicándose en mayor medida con la bidireccionalidad e invitación a la participación de sus públicos, con el objetivo de captar nuevos contactos, fidelizar a los clientes actuales y convertir a todos ellos en prescriptores de sus servicios.

Estudio de caso de los sMOOC y su pedagogía en el contexto online

En un contexto dominado por las nuevas tecnologías, los MOOCs parecen posicionarse como una alternativa o complemento a la formación tradicional. Se observa sin embargo, que la metodología empleada por este tipo de cursos masivos, no difiere de la comúnmente utilizada en el aula. Este artículo evalúa y describe una nueva tipología de MOOC, denominada Social MOOC, la cual se presenta como disruptiva en cuanto a pedagogía se refiere. A través de la plataforma ECO Learning, se analiza de forma exhaustiva mediante un estudio de caso, la pedagogía empleada y el rol que asume el estudiante en el primer sMOOC llevado a cabo que tuvo lugar durante los meses de marzo y abril de 2015. Se concluye, que la pedagogía empleada en esta nueva versión social de los ya citados MOOC, es innovadora tanto dentro como fuera del contexto digital. Los estudiantes asumen un papel muy activo y la comunicación se torna de un carácter bidireccional entre el facilitador y los mismos.

Testing for Subsidiarity. Bruges European Economic Policy (BEEP) Briefing 13/2006

In the EU circuit (especially the European Parliament, the Council and Coreper) as well as in national parliaments of the EU Member States, one observes a powerful tendency to regard 'subsidiarity' as a 'political' issue. Moreover, subsidiarity is frequently seen as a one-way street : powers going 'back to' Member States. Both interpretations are at least partly flawed and less than helpful when looking for practical ways to deal with subsidiarity at both EU and Member states' levels. The present paper shows that subsidiarity as a principle is profoundly 'functional' in nature and, hence, is and must be a two-way principle. A functional subsidiarity test is developed and its application is illustrated for a range of policy issues in the internal market in its widest sense, for equity and for macro-economic stabilisation questions in European integration. Misapplications of 'subsidiarity' are also demonstrated. For a good understanding, subsidiarity being a functional, two-way principle neither means that elected politicians should not have the final (political!) say (for which they are accountable), nor that subsidiarity tests, even if properly conducted, cannot and will not be politicised once the results enter the policy debate. Such politicisation forms a natural run-up to the decision-making by those elected for it. But the quality and reasoning of the test as well as structuring the information in a logical sequence ( in accordance with the current protocol and with the one in the constitutional treaty) is likely to be directly helpful for decisionmakers, confronted with complicated and often specialised proposals. EU debates and decision-making is therefore best served by separating the functional subsidiarity test (prepared by independent professionals) from the final political decision itself. If the test were accepted Union-wide, it would also assist national parliaments in conducting comparable tests in a relatively short period, as the basis for possible joint action (as suggested by the constitutional treaty). The core of the paper explains how the test is formulated and applied. A functional approach to subsidiarity in the framework of European representative democracy seeks to find the optimal assignment of regulatory or policy competences to the various tiers of government. In the final analysis, this is about structures facilitating the highest possible welfare in the Union, in the fundamental sense that preferences and needs are best satisfied. What is required for such an analysis is no less than a systematic cost/benefit framework to assess the (de)merits of (de)centralisation in the EU.

Análise da precisão e reprodutibilidade de um scanner intraoral: estudo clínico

Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz

Avaliação in vitro da resistência adesiva à microtracção e caracterização morfológica com SEM de um novo sistema adesivo

Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz

Exploring complementarities between goods and services trade in trade agreements

More than three decades of research on trade costs and goods trade have unveiled fundamental insights into the determinants, the nature and the consequences of goods trade agreements. A cottage literature has also evolved studying similar issues from a services trade perspective, but the two-way interaction between goods and services trade has not been explored formally. We bridge this gap by providing a formal treatment of the inter-linkages between goods and services trade. The model provides insights into how trade agreements impact goods and services trade. We also explore the impact of the complementarities of goods and services agreements on goods and services trade empirically using bilateral goods and services trade data for OECD and BRICS trading partners over 1995-2010.

Chemistry and boron isotope ratios of Izu arc front volcanic rocks

The process of fluid release from the subducting slab beneath the Izu arc volcanic front (Izu VF) was examined by measuring B concentrations and B isotope ratios in the Neogene fallout tephra (ODP Site 782A). Both were measured by secondary ion mass spectrometry, in a subset of matrix glasses and glassy plagioclase-hosted melt inclusions selected from material previously analyzed for major and trace elements (glasses) and radiogenic isotopes (Sr, Nd, Pb; bulk tephra). These tephra glasses have high B abundances (~10-60 ppm) and heavy delta11B values (+4.5? to +12.0?), extending the previously reported range for Izu VF rocks (delta11B, +7.0? to +7.3?). The glasses show striking negative correlations of delta11B with large ion lithophile element (LILE)/Nb ratios. These correlations cannot be explained by mixing two separate slab fluids, originating from the subducting sediment and the subducting basaltic crust, respectively (model A). Two alternative models (models B and C) are proposed. Model B proposes that the inverse correlations are inherited from altered oceanic crust (AOC), which shows a systematic decrease of B and LILE with increasing depth (from basaltic layer 2A to layer 3), paralleled by an increase in delta11B (from ~ +1? to +10? to +24?). In this model, the contribution of sedimentary B is insignificant (<4% of B in the Izu VF rocks). Model C explains the correlation as a mixture of a low-delta11B (~ +1?) 'composite' slab fluid (a mixture of metasediment- and metabasalt-derived fluids) with a metasomatized mantle wedge containing elevated B (~1-2 ppm) and heavy delta11B (~ +14?). The mantle wedge was likely metasomatized by 11B-rich fluids beneath the outer forearc, and subsequently down dragged to arc front depths by the descending slab. Pb-B isotope systematics indicate that, at arc front depths, ~ 53% of the B in the Izu VF is derived from the wedge. This implies that the heavy delta11B values of Izu VF rocks are largely a result of fluid fractionation, and do not reflect variations in slab source provenance (i.e. subducting sediment vs. basaltic crust). Since the B content of the peridotite at the outer forearc (7-58 ppm B, mean 24 +/- 16 ppm) is much higher than beneath the arc front (~1-2 ppm B), the hydrated mantle wedge must have released a B-rich fluid on its downward path. This 'wedge flux' can explain (1) the across-arc decrease in B and delta11B (e.g. Izu, Kuriles), without requiring a progressive decrease in fluid flux from the subducting slab, and (2) the thermal structure of volcanic arcs, as reflected in the B and delta11B variations of volcanic arc rocks.