Why Ciao? An overview of the ciao system's design philosophy

Our intention in this note is not to provide a listing of the many features of the Ciao system: this can be found in part for example in the brochures announcing upcoming versions, in the Ciao website, or in more feature-oriented descriptions such as. Instead in this document we would like to describe the objectives and reasoning followed in our design as well as the fundamental characteristics that in our opinion make Ciao quite unique and hopefully really useful to you as a Ciao user.

Characterization of the environmental performance of the insulating materials in the enveloping of the building.

Insulating materials in buildings are one of the main factors that should be taken into account when talking about sustainability since with a correct application it could imply important savings for the citizens. In the course of its life, a building requires a series of supplies to perform the duties it has been built for, generating an impact on the environment. The selection of one material or another will establish partly the global environmental impact of the building. Choosing the right insulating material will determine the building's general degree of sustainability, both in its heating savings (energy consumption) and in the environmental impacts caused by its LCA (greenhouse gas emissions). Therefore, we propose to establish guidelines to characterize the insulating material with a better environmental performance in all the stages of its life cycle, taking into account the construction system, the use of the building and its location.

A "Free-Lunch" tour of the Jovian System

An ED-tether mission to Jupiter is presented. A bare tether carrying cathodic devices at both ends but no power supply, and using no propellant, could move 'freely' among Jupiter's 4 great moons. The tour scheme would have current naturally driven throughout by the motional electric field, the Lorentz force switching direction with current around a 'drag' radius of 160,00 kms, where the speed of the jovian ionosphere equals the speed of a spacecraft in circular orbit. With plasma density and magnetic field decreasing rapidly with distance from Jupiter, drag/thrust would only be operated in the inner plasmasphere, current being near shut off conveniently in orbit by disconnecting cathodes or plugging in a very large resistance; the tether could serve as its own power supply by plugging in an electric load where convenient, with just some reduction in thrust or drag. The periapsis of the spacecraft in a heliocentric transfer orbit from Earth would lie inside the drag sphere; with tether deployed and current on around periapsis, magnetic drag allows Jupiter to capture the spacecraft into an elliptic orbit of high eccentricity. Current would be on at succesive perijove passes and off elsewhere, reducing the eccentricity by lowering the apoapsis progressively to allow visits of the giant moons. In a second phase, current is on around apoapsis outside the drag sphere, rising the periapsis until the full orbit lies outside that sphere. In a third phase, current is on at periapsis, increasing the eccentricity until a last push makes the orbit hyperbolic to escape Jupiter. Dynamical issues such as low gravity-gradient at Jupiter and tether orientation in elliptic orbits of high eccentricity are discussed.

Motivating young students to be part of the global research in nuclear through the Seminar of Nuclear Fusion

Jóvenes Nucleares (Spanish Young Generation in Nuclear, JJNN) is a non-profit organization that depends on the Spanish Nuclear Society (SNE). The Universidad Politécnica de Madrid (Technical University of Madrid, UPM) was chosen to host the Seminar as it is one of the most prestigious technical universities of Spain, and has a very strong curriculum in nuclear engineering training and research. Both, the UPM and the SNE, supported strongly the seminar: the opening session was conducted by the member of to board of directors of the Spanish Nuclear Society and Nuclear Engineering professor of the UPM, Emilio Mínguez and the closing session was conducted by the director of the Nuclear Fusion Institute (UPM).

Guidelines for the design of an environmental flow regime program at the regional scale: Application of the ELOHA framework in the Ebro River basin

La gestión de los recursos hídricos se convierte en un reto del presente y del futuro frente a un panorama de continuo incremento de la demanda de agua debido al crecimiento de la población, el crecimiento del desarrollo económico y los posibles efectos del calentamiento global. La política hidráulica desde los años 60 en España se ha centrado en la construcción de infraestructuras que han producido graves alteraciones en el régimen natural de los ríos. Estas alteraciones han provocado y acrecentado los impactos sobre los ecosistemas fluviales y ribereños. Desde los años 90, sin embargo, ha aumentado el interés de la sociedad para conservar estos ecosistemas. El concepto de caudales ambientales consiste en un régimen de caudales que simula las características principales del régimen natural. Los caudales ambientales están diseñados para conservar la estructura y funcionalidad de los ecosistemas asociados al régimen fluvial, bajo la hipótesis de que los elementos que conforman estos ecosistemas están profundamente adaptados al régimen natural de caudales, y que cualquier alteración del régimen natural puede provocar graves daños a todo el sistema. El método ELOHA (Ecological Limits of Hydrological Alteration) tiene como finalidad identificar las componentes del régimen natural de caudales que son clave para mantener el equilibrio de los ecosistemas asociados, y estimar los límites máximos de alteración de estas componentes para garantizar su buen estado. Esta tesis presenta la aplicación del método ELOHA en la cuenca del Ebro. La cuenca del Ebro está profundamente regulada e intervenida por el hombre, y sólo las cabeceras de los principales afluentes del Ebro gozan todavía de un régimen total o cuasi natural. La tesis se estructura en seis capítulos que desarrollan las diferentes partes del método. El primer capítulo explica cómo se originó el concepto “caudales ambientales” y en qué consiste el método ELOHA. El segundo capítulo describe el área de estudio. El tercer capítulo realiza una clasificación de los regímenes naturales de la cuenca (RNC) del Ebro, basada en series de datos de caudal mínimamente alterado y usando exclusivamente parámetros hidrológicos. Se identificaron seis tipos diferentes de régimen natural: pluvial mediterráneo, nivo-pluvial, pluvial mediterréaneo con una fuerte componente del caudal base, pluvial oceánico, pluvio-nival oceánico y Mediterráneo. En el cuarto capítulo se realiza una regionalización a toda la cuenca del Ebro de los seis RNC encontrados en la cueca. Mediante parámetros climáticos y fisiográficos se extrapola la información del tipo de RNC a puntos donde no existen datos de caudal inalterado. El patrón geográfico de los tipos de régimen fluvial obtenido con la regionalización resultó ser coincidente con el patrón obtenido a través de la clasificación hidrológica. El quinto capítulo presenta la validación biológica de los procesos de clasificación anteriores: clasificación hidrológica y regionalización. La validación biológica de los tipos de regímenes fluviales es imprescindible, puesto que los diferentes tipos de régimen fluvial van a servir de unidades de gestión para favorecer el mantenimiento de los ecosistemas fluviales. Se encontraron diferencias significativas entre comunidades biológicas en cinco de los seis tipos de RNC encontrados en la cuenca. Finalmente, en el sexto capítulo se estudian las relaciones hidro-ecológicas existentes en tres de los seis tipos de régimen fluvial encontrados en la cuenca del Ebro. Mediante la construcción de curvas hidro-ecológicas a lo largo de un gradiente de alteración hidrológica, se pueden sugerir los límites de alteración hidrológica (ELOHAs) para garantizar el buen estado ecológico en cada uno de los tipos fluviales estudiados. Se establecieron ELOHAs en tres de los seis tipos de RNC de la cuenca del Ebro Esta tesis, además, pone en evidencia la falta de datos biológicos asociados a registros de caudal. Para llevar a cabo la implantación de un régimen de caudales ambientales en la cuenca, la ubicación de los puntos de muestreo biológico cercanos a estaciones de aforo es imprescindible para poder extraer relaciones causa-efecto de la gestión hidrológica sobre los ecosistemas dependientes. ABSTRACT In view of a growing freshwater demand because of population raising, improvement of economies and the potential effects of climate change, water resources management has become a challenge for present and future societies. Water policies in Spain have been focused from the 60’s on constructing hydraulic infrastructures, in order to dampen flow variability and granting water availability along the year. Consequently, natural flow regimes have been deeply altered and so the depending habitats and its ecosystems. However, an increasing acknowledgment of societies for preserving healthy freshwater ecosystems started in the 90’s and agreed that to maintain healthy freshwater ecosystems, it was necessary to set environmental flow regimes based on the natural flow variability. The Natural Flow Regime paradigm (Richter et al. 1996, Poff et al. 1997) bases on the hypothesis that freshwater ecosystems are made up by elements adapted to natural flow conditions, and any change on these conditions can provoke deep impacts on the whole system. Environmental flow regime concept consists in designing a flow regime that emulates natural flow characteristics, so that ecosystem structure, functions and services are maintained. ELOHA framework (Ecological Limits of Hydrological Alteration) aims to identify key features of the natural flow regime (NFR) that are needed to maintain and preserve healthy freshwater and riparian ecosystems. Moreover, ELOHA framework aims to quantify thresholds of alteration of these flow features according to ecological impacts. This thesis describes the application of the ELOHA framework in the Ebro River Basin. The Ebro River basin is the second largest basin in Spain and it is highly regulated for human demands. Only the Ebro headwaters tributaries still have completely unimpaired flow regime. The thesis has six chapters and the process is described step by step. The first chapter makes an introduction to the origin of the environmental flow concept and the necessity to come up. The second chapter shows a description of the study area. The third chapter develops a classification of NFRs in the basin based on natural flow data and using exclusively hydrological parameters. Six NFRs were found in the basin: continental Mediterranean-pluvial, nivo-pluvial, continental Mediterranean pluvial (with groundwater-dominated flow pattern), pluvio-oceanic, pluvio-nival-oceanic and Mediterranean. The fourth chapter develops a regionalization of the six NFR types across the basin by using climatic and physiographic variables. The geographical pattern obtained from the regionalization process was consistent with the pattern obtained with the hydrologic classification. The fifth chapter performs a biological validation of both classifications, obtained from the hydrologic classification and the posterior extrapolation. When the aim of flow classification is managing water resources according to ecosystem requirements, a validation based on biological data is compulsory. We found significant differences in reference macroinvertebrate communities between five over the six NFR types identified in the Ebro River basin. Finally, in the sixth chapter we explored the existence of significant and explicative flow alteration-ecological response relationships (FA-E curves) within NFR types in the Ebro River basin. The aim of these curves is to find out thresholds of hydrological alteration (ELOHAs), in order to preserve healthy freshwater ecosystem. We set ELOHA values in three NFR types identified in the Ebro River basin. During the development of this thesis, an inadequate biological monitoring in the Ebro River basin was identified. The design and establishment of appropriate monitoring arrangements is a critical final step in the assessment and implementation of environmental flows. Cause-effect relationships between hydrology and macroinvertebrate community condition are the principal data that sustain FA-E curves. Therefore, both data sites must be closely located, so that the effects of external factors are minimized. The scarce hydro-biological pairs of data available in the basin prevented us to apply the ELOHA method at all NFR types.

Systematic analysis of the decoding delay in multiview video

We present a framework for the analysis of the decoding delay in multiview video coding (MVC). We show that in real-time applications, an accurate estimation of the decoding delay is essential to achieve a minimum communication latency. As opposed to single-view codecs, the complexity of the multiview prediction structure and the parallel decoding of several views requires a systematic analysis of this decoding delay, which we solve using graph theory and a model of the decoder hardware architecture. Our framework assumes a decoder implementation in general purpose multi-core processors with multi-threading capabilities. For this hardware model, we show that frame processing times depend on the computational load of the decoder and we provide an iterative algorithm to compute jointly frame processing times and decoding delay. Finally, we show that decoding delay analysis can be applied to design decoders with the objective of minimizing the communication latency of the MVC system.

A real case of a cognitive multiplayer game: design and development of the iOS client in Swift and its cloud architecture

as tecnologías emergentes como el cloud computing y los dispositivos móviles están creando una oportunidad sin precedentes para mejorar el sistema educativo, permitiendo tanto a los educadores personalizar y mejorar la experiencia de aprendizaje, como facilitar a los estudiantes que adquieran conocimientos sin importar dónde estén. Por otra parte, a través de técnicas de gamificacion será posible promover y motivar a los estudiantes a que aprendan materias arduas haciendo que la experiencia sea más motivadora. Los juegos móviles pueden ser el camino correcto para dar soporte a esta experiencia de aprendizaje mejorada. Este proyecto integra el diseño y desarrollo de una arquitectura en la nube altamente escalable y con alto rendimiento, así como el propio cliente de iOS, para dar soporte a una nueva version de Temporis, un juego móvil multijugador orientado a reordenar eventos históricos en una línea temporal (e.j. historia, arte, deportes, entretenimiento y literatura). Temporis actualmente está disponible en Google Play. Esta memoria describe el desarrollo de la nueva versión de Temporis (Temporis v.2.0) proporcionando detalles acerca de la mejora y adaptación basados en el Temporis original. En particular se describe el nuevo backend hecho en Go sobre Google App Engine creado para soportar miles de usuarios, asó como otras características por ejemplo como conseguir enviar noticaciones push desde la propia plataforma. Por último, el cliente de iOS en Temporis v.2.0 se ha desarrollado utilizando las últimas y más relevantes tecnologías, prestando especial atención a Swift (el lenguaje de programación nuevo de Apple, que es seguro y rápido), el Paradigma Funcional Reactivo (que ayuda a construir aplicaciones altamente interactivas además de a minimizar errores) y la arquitectura VIPER (una arquitectura que sigue los principios SOLID, se centra en la separación de asuntos y favorece la reutilización de código en otras plataformas). ABSTRACT Emerging technologies such as cloud computing and mobile devices are creating an unprecedented opportunity for enhancing the educational system, letting both educators customize and improve the learning experience, and students acquire knowledge regardless of where they are. Moreover, through gamification techniques it would be possible to encourage and motivate students to learn arduous subjects by making the experience more motivating. Mobile games can be a perfect vehicle to support this enhanced learning experience. This project integrates the design and development of a highly scalable and performant cloud architecture, as well as the iOS client that uses it, in order to provide support to a new version of Temporis, a mobile multiplayer game focused on ordering time-based (e.g. history, art, sports, entertainment and literature) in a timeline that currently is available on Google Play. This work describes the development of the new Temporis version (Temporis v.2.0), providing details about improvements and details on the adaptation of the original Temporis. In particular, the new Google App Engine backend is described, which was created to support thousand of users developed in Go language are provided, in addition to other features like how to achieve push notications in this platform. Finally, the mobile iOS client developed using the latest and more relevant technologies is explained paying special attention to Swift (Apple's new programming language, that is safe and fast), the Functional Reactive Paradigm (that helps building highly interactive apps while minimizing bugs) and the VIPER architecture (a SOLID architecture that enforces separation of concerns and makes it easy to reuse code for other platforms).

Proceso metodológico para optimizar la toma de decisiones en el proyecto de envolventes arquitectónicas multicapa = Methodological process for the optimization of the decision making in multilayer building envelope projects

La presente tesis doctoral se enmarca dentro del concepto de la sistematización del conocimiento en arquitectura, más concretamente en el campo de las construcciones arquitectónicas y la toma de decisiones en la fase de proyecto de envolventes arquitectónicas multicapa. Por tanto, el objetivo principal es el establecimiento de las bases para una toma de decisiones informadas durante el proyecto de una envolvente multicapa con el fin de colaborar en su optimización. Del mismo modo que la historia de la arquitectura está relacionada con la historia de la innovación en construcción, la construcción está sujeta a cambios como respuesta a los fracasos anteriores. En base a esto, se identifica la toma de decisiones en la fase de proyecto como el estadio inicial para establecer un punto estratégico de reflexión y de control sobre los procesos constructivos. La presente investigación, conceptualmente, define los parámetros intervinientes en el proyecto de envolventes arquitectónicas multicapa a partir de una clasificación y sistematización de todos los componentes (elementos, unidades y sistemas constructivos) utilizados en las fachadas multicapa. Dicha sistematización se materializa en una hoja matriz de datos en la que, dentro de una organización a modo de árbol, se puede acceder a la consulta de cada componente y de su caracterización. Dicha matriz permite la incorporación futura de cualquier componente o sistema nuevo que aparezca en el mercado, relacionándolo con aquellos con los que comparta ubicación, tipo de material, etc. Con base en esa matriz de datos, se diseña la sistematización de la toma de decisiones en la fase de proyecto de una envolvente arquitectónica, en concreto, en el caso de una fachada. Operativamente, el resultado se presenta como una herramienta que permite al arquitecto o proyectista reflexionar y seleccionar el sistema constructivo más adecuado, al enfrentarse con las distintas decisiones o elecciones posibles. La herramienta se basa en las elecciones iniciales tomadas por el proyectista y se estructura, a continuación y sucesivamente, en distintas aproximaciones, criterios, subcriterios y posibilidades que responden a los distintos avances en la definición del sistema constructivo. Se proponen una serie de fichas operativas de comprobación que informan sobre el estadio de decisión y de definición de proyecto alcanzados en cada caso. Asimismo, el sistema permite la conexión con otros sistemas de revisión de proyectos para fomentar la reflexión sobre la normalización de los riesgos asociados tanto al proprio sistema como a su proceso constructivo y comportamiento futuros. La herramienta proporciona un sistema de ayuda para ser utilizado en el proceso de toma de decisiones en la fase de diseño de una fachada multicapa, minimizando la arbitrariedad y ofreciendo una cualificación previa a la cuantificación que supondrá la elaboración del detalle constructivo y de su medición en las sucesivas fases del proyecto. Al mismo tiempo, la sistematización de dicha toma de decisiones en la fase del proyecto puede constituirse como un sistema de comprobación en las diferentes fases del proceso de decisión proyectual y de definición de la envolvente de un edificio. ABSTRACT The central issue of this doctoral Thesis is founded on the framework of the concept of the systematization of knowledge in architecture, in particular, in respect of the field of building construction and the decision making in the design stage of multilayer building envelope projects. Therefore, the main objective is to establish the bases for knowledgeable decision making during a multilayer building envelope design process, in order to collaborate with its optimization. Just as the history of architecture is connected to the history of innovation in construction, construction itself is subject to changes as a response to previous failures. On this basis, the decisions made during the project design phase are identified as the initial state to establish an strategic point for reflection and control, referred to the constructive processes. Conceptually, this research defines the parameters involving the multilayer building envelope projects, on the basis of a classification and systematization for all the components (elements, constructive units and constructive systems) used in multilayer façades. The mentioned systematization is materialized into a data matrix sheet in which, following a tree‐like organization, the access to every single component and its characterization is possible. The above data matrix allows the future inclusion of any new component or system that may appear in the construction market. That new component or system can be put into a relationship with another, which it shares location, type of material,… with. Based on the data matrix, the systematization of the decision making process for a building envelope design stage is designed, more particularly in the case of a façade. Putting this into practice, it is represented as a tool which allows the architect or the designer, to reflect and to select the appropriate building system when facing the different elections or the different options. The tool is based on the initial elections taken by the designer. Then and successively, it is shaped on the form of different operative steps, criteria, sub‐criteria and possibilities which respond to a different progress in the definition of the building construction system. In order to inform about the stage of the decision and the definition reached by the project in every particular case, a range of operative sheets are proposed. Additionally, the system allows the connection with other reviewing methods for building projects. The aim of this last possibility is to encourage the reflection on standardization of the associated risks to the building system itself and its future performance. The tool provides a helping system to be used during the decision making process for a multilayer façade design. It minimizes the arbitrariness and offers a qualification previous to the quantification that will be done with the development of the construction details and their bill of quantities, that in subsequent project stages will be executed. At the same time, the systematization of the mentioned decision making during the design phase, can be found as a checking system in the different stages of the decision making design process and in the different stages of the building envelope definition.

Characterization of the transposition pattern of the Ac element in Arabidopsis thaliana using endonuclease I-SceI

We have investigated physical distances and directions of transposition of the maize transposable element Ac in Arabidopsis thaliana. We prepared a transferred DNA (T-DNA) construct that carried a non-autonomous derivative of Ac with a site for cleavage by endonuclease I-SceI (designated dAc-I-RS element). Another cleavage site was also introduced into the T-DNA region outside dAc-I-RS. Three transgenic Arabidopsis plants were generated, each of which had a single copy of the T-DNA at a different chromosomal location. These transgenic plants were crossed with the Arabidopsis that carried the gene for Ac transposase and progeny in which dAc-I-RS had been transposed were isolated. After digestion of the genomic DNA of these progeny with endonuclease I-SceI, sizes of segment of DNA were determined by pulse-field gel electrophoresis. We also performed linkage analysis for the transposed elements and sites of mutations near the elements. Our results showed that 50% of all transposition events had occurred within 1,700 kb on the same chromosome, with 35% within 200 kb, and that the elements transposed in both directions on the chromosome with roughly equal probability. The data thus indicate that the Ac–Ds system is most useful for tagging of genes that are present within 200 kb of the chromosomal site of Ac in Arabidopsis. In addition, determination of the precise localization of the transposed dAc-I-RS element should definitely assist in map-based cloning of genes around insertion sites.

Immediate and simultaneous sensory reorganization at cortical and subcortical levels of the somatosensory system

The occurrence of cortical plasticity during adulthood has been demonstrated using many experimental paradigms. Whether this phenomenon is generated exclusively by changes in intrinsic cortical circuitry, or whether it involves concomitant cortical and subcortical reorganization, remains controversial. Here, we addressed this issue by simultaneously recording the extracellular activity of up to 135 neurons in the primary somatosensory cortex, ventral posterior medial nucleus of the thalamus, and trigeminal brainstem complex of adult rats, before and after a reversible sensory deactivation was produced by subcutaneous injections of lidocaine. Following the onset of the deactivation, immediate and simultaneous sensory reorganization was observed at all levels of the somatosensory system. No statistical difference was observed when the overall spatial extent of the cortical (9.1 ± 1.2 whiskers, mean ± SE) and the thalamic (6.1 ± 1.6 whiskers) reorganization was compared. Likewise, no significant difference was found in the percentage of cortical (71.1 ± 5.2%) and thalamic (66.4 ± 10.7%) neurons exhibiting unmasked sensory responses. Although unmasked cortical responses occurred at significantly higher latencies (19.6 ± 0.3 ms, mean ± SE) than thalamic responses (13.1 ± 0.6 ms), variations in neuronal latency induced by the sensory deafferentation occurred as often in the thalamus as in the cortex. These data clearly demonstrate that peripheral sensory deafferentation triggers a system-wide reorganization, and strongly suggest that the spatiotemporal attributes of cortical plasticity are paralleled by subcortical reorganization.

Molecular switch in signal transduction: Reaction paths of the conformational changes in ras p21

Conformational changes in ras p21 triggered by the hydrolysis of GTP play an essential role in the signal transduction pathway. The path for the conformational change is determined by molecular dynamics simulation with a holonomic constraint directing the system from the known GTP-bound structure (with the γ-phosphate removed) to the GDP-bound structure. The simulation is done with a shell of water molecules surrounding the protein. In the switch I region, the side chain of Tyr-32, which undergoes a large displacement, moves through the space between loop 2 and the rest of the protein, rather than on the outside of the protein. As a result, the charged residues Glu-31 and Asp-33, which interact with Raf in the homologous RafRBD–Raps complex, remain exposed during the transition. In the switch II region, the conformational changes of α2 and loop 4 are strongly coupled. A transient hydrogen bonding complex between Arg-68 and Tyr-71 in the switch II region and Glu-37 in switch I region stabilizes the intermediate conformation of α2 and facilitates the unwinding of a helical turn of α2 (residues 66–69), which in turn permits the larger scale motion of loop 4. Hydrogen bond exchange between the protein and solvent molecules is found to be important in the transition. Possible functional implications of the results are discussed.

Molecular determinants of the modulation of cyclic nucleotide-activated channels by calmodulin

The action of calmodulin (CaM) on target proteins is important for a variety of cellular functions. We demonstrate here, however, that the presence of a CaM-binding site on a protein does not necessarily imply a functional effect. The α-subunit of the cGMP-gated cation channel of human retinal cones has a CaM-binding site on its cytoplasmic N-terminal region, but the homomeric channel that it forms is not functionally modulated by CaM. Mutational analysis based on comparison to the highly homologous olfactory cyclic nucleotide-gated channel α-subunit, which does form a CaM-modulated channel, indicates that residues downstream of the CaM-binding domain on these channels are also important for CaM to have an effect. These findings suggest that a CaM-binding site and complementary structural features in a protein probably evolve independently, and an effect caused by CaM occurs only in the presence of both elements. More generally, the same may be true for other recognized binding sites on proteins for modulators or activators, so that a demonstrated physical interaction does not necessarily imply functional consequence.

Ligation of intestinal epithelial CD1d induces bioactive IL-10: Critical role of the cytoplasmic tail in autocrine signaling

The intestinal epithelium is anatomically positioned to serve as the critical interface between the lumen and the mucosal immune system. In addition to MHC class I and II antigens, intestinal epithelia constitutively express the nonclassical MHC molecule CD1d, a transmembrane molecule with a short cytoplasmic tail expressed as a β2-microglobulin-associated 48-kDa glycoprotein and novel β2-microglobulin-independent 37-kDa nonglycosylated protein on intestinal epithelia. At present, it is not known whether extracellular ligands can signal intestinal epithelial CD1d. To define signaling of CD1d cytoplasmic tail, retrovirus-mediated gene transfer was used to generate stable cell lines expressing wild-type CD1d or a chimeric molecule (extracellular CD1d and cytoplasmic CD1a), and surface CD1d was triggered by antibody crosslinking. Although wild-type CD1d was readily activated (tyrosine phosphorylation), no demonstrable signal was evident in cell lines expressing the chimeric molecule. Subsequent studies revealed that anti-CD1d crosslinking specifically induces epithelial IL-10 mRNA and protein and is blocked by the tyrosine kinase inhibitor genistein. Further studies addressing epithelial-derived IL-10 revealed that anti-CD1d crosslinking attenuates IFN-γ signaling and that such attenuation is reversed by addition of functionally inhibitory IL-10 antibodies. These results define signaling through surface CD1d, and, importantly, they demonstrate that this pathway may serve to dampen epithelial proinflammatory signals.

Rho1p-Bni1p-Spa2p Interactions: Implication in Localization of Bni1p at the Bud Site and Regulation of the Actin Cytoskeleton in Saccharomyces cerevisiae

Rho1p is a yeast homolog of mammalian RhoA small GTP-binding protein. Rho1p is localized at the growth sites and required for bud formation. We have recently shown that Bni1p is a potential target of Rho1p and that Bni1p regulates reorganization of the actin cytoskeleton through interactions with profilin, an actin monomer-binding protein. Using the yeast two-hybrid screening system, we cloned a gene encoding a protein that interacted with Bni1p. This protein, Spa2p, was known to be localized at the bud tip and to be implicated in the establishment of cell polarity. The C-terminal 254 amino acid region of Spa2p, Spa2p(1213–1466), directly bound to a 162-amino acid region of Bni1p, Bni1p(826–987). Genetic analyses revealed that both the bni1 and spa2 mutations showed synthetic lethal interactions with mutations in the genes encoding components of the Pkc1p-mitogen-activated protein kinase pathway, in which Pkc1p is another target of Rho1p. Immunofluorescence microscopic analysis showed that Bni1p was localized at the bud tip in wild-type cells. However, in the spa2 mutant, Bni1p was not localized at the bud tip and instead localized diffusely in the cytoplasm. A mutant Bni1p, which lacked the Rho1p-binding region, also failed to be localized at the bud tip. These results indicate that both Rho1p and Spa2p are involved in the localization of Bni1p at the growth sites where Rho1p regulates reorganization of the actin cytoskeleton through Bni1p.

Activation and Cellular Localization of the Cyclosporine A-sensitive Transcription Factor NF-AT in Skeletal Muscle Cells

The widely used immunosuppressant cyclosporine A (CSA) blocks nuclear translocation of the transcription factor, NF-AT (nuclear factor of activated T cells), preventing its activity. mRNA for several NF-AT isoforms has been shown to exist in cells outside of the immune system, suggesting a possible mechanism for side effects associated with CSA treatment. In this study, we demonstrate that CSA inhibits biochemical and morphological differentiation of skeletal muscle cells while having a minimal effect on proliferation. Furthermore, in vivo treatment with CSA inhibits muscle regeneration after induced trauma in mice. These results suggest a role for NF-AT–mediated transcription outside of the immune system. In subsequent experiments, we examined the activation and cellular localization of NF-AT in skeletal muscle cells in vitro. Known pharmacological inducers of NF-AT in lymphoid cells also stimulate transcription from an NF-AT–responsive reporter gene in muscle cells. Three isoforms of NF-AT (NF-ATp, c, and 4/x/c3) are present in the cytoplasm of muscle cells at all stages of myogenesis tested. However, each isoform undergoes calcium-induced nuclear translocation from the cytoplasm at specific stages of muscle differentiation, suggesting specificity among NF-AT isoforms in gene regulation. Strikingly, one isoform (NF-ATc) can preferentially translocate to a subset of nuclei within a single multinucleated myotube. These results demonstrate that skeletal muscle cells express functionally active NF-AT proteins and that the nuclear translocation of individual NF-AT isoforms, which is essential for the ability to coordinate gene expression, is influenced markedly by the differentiation state of the muscle cell.