Service Capacity Reserve under Uncertainty by Hospital’s ER Analogies: A Practical Model for Car Services

We define a capacity reserve model to dimension passenger car service installations according to the demographic distribution of the area to be serviced by using hospital?s emergency room analogies. Usually, service facilities are designed applying empirical methods, but customers arrive under uncertain conditions not included in the original estimations, and there is a gap between customer?s real demand and the service?s capacity. Our research establishes a valid methodology and covers the absence of recent researches and the lack of statistical techniques implementation, integrating demand uncertainty in a unique model built in stages by implementing ARIMA forecasting, queuing theory, and Monte Carlo simulation to optimize the service capacity and occupancy, minimizing the implicit cost of the capacity that must be reserved to service unexpected customers. Our model has proved to be a useful tool for optimal decision making under uncertainty integrating the prediction of the cost implicit in the reserve capacity to serve unexpected demand and defining a set of new process indicators, such us capacity, occupancy, and cost of capacity reserve never studied before. The new indicators are intended to optimize the service operation. This set of new indicators could be implemented in the information systems used in the passenger car services.

Analysis of SnS2 hyperdoped with V proposed as efficient absorber material

Intermediate-band materials can improve the photovoltaic efficiency of solar cells through the absorption of two subband-gap photons that allow extra electron-hole pair formations. Previous theoretical and experimental findings support the proposal that the layered SnS2 compound, with a band-gap of around 2 eV, is a candidate for an intermediate-band material when it is doped with a specific transition-metal. In this work we characterize vanadium doped SnS2 using density functional theory at the dilution level experimentally found and including a dispersion correction combined with the site-occupancy-disorder method. In order to analyze the electronic characteristics that depend on geometry, two SnS2 polytypes partially substituted with vanadium in symmetry-adapted non-equivalent configurations were studied. In addition the magnetic configurations of vanadium in a SnS2 2H-polytype and its comparison with a 4H-polytype were also characterized. We demonstrate that a narrow intermediate-band is formed, when these dopant atoms are located in different layers. Our theoretical predictions confirm the recent experimental findings in which a paramagnetic intermediate-band material in a SnS2 2H-polytype with 10% vanadium concentration is obtained.

Service capacity reserve under uncertainty by hospital's ER's analogies: a practical model for car services

El objetivo de esta investigación consiste en definir un modelo de reserva de capacidad, por analogías con emergencias hospitalarias, que pueda ser implementado en el sector de servicios. Este está específicamente enfocado a su aplicación en talleres de servicio de automóviles. Nuestra investigación incorpora la incertidumbre de la demanda en un modelo singular diseñado en etapas que agrupa técnicas ARIMA, teoría de colas y simulación Monte Carlo para definir los conceptos de capacidad y ocupación de servicio, que serán utilizados para minimizar el coste implícito de la reserva capacidad necesaria para atender a clientes que carecen de cita previa. Habitualmente, las compañías automovilísticas estiman la capacidad de sus instalaciones de servicio empíricamente, pero los clientes pueden llegar bajo condiciones de incertidumbre que no se tienen en cuenta en dichas estimaciones, por lo que existe una diferencia entre lo que el cliente realmente demanda y la capacidad que ofrece el servicio. Nuestro enfoque define una metodología válida para el sector automovilístico que cubre la ausencia genérica de investigaciones recientes y la habitual falta de aplicación de técnicas estadísticas en el sector. La equivalencia con la gestión de urgencias hospitalarias se ha validado a lo largo de la investigación en la se definen nuevos indicadores de proceso (KPIs) Tal y como hacen los hospitales, aplicamos modelos estocásticos para dimensionar las instalaciones de servicio de acuerdo con la distribución demográfica del área de influencia. El modelo final propuesto integra la predicción del coste implícito en la reserva de capacidad para atender la demanda no prevista. Asimismo, se ha desarrollado un código en Matlab que puede integrarse como un módulo adicional a los sistemas de información (DMS) que se usan actualmente en el sector, con el fin de emplear los nuevos indicadores de proceso definidos en el modelo. Los resultados principales del modelo son nuevos indicadores de servicio, tales como la capacidad, ocupación y coste de reserva de capacidad, que nunca antes han sido objeto de estudio en la industria automovilística, y que están orientados a gestionar la operativa del servicio. ABSTRACT Our aim is to define a Capacity Reserve model to be implemented in the service sector by hospital's emergency room (ER) analogies, with a practical approach to passenger car services. A stochastic model has been implemented using R and a Monte Carlo simulation code written in Matlab and has proved a very useful tool for optimal decision making under uncertainty. The research integrates demand uncertainty in a unique model which is built in stages by implementing ARIMA forecasting, Queuing Theory and a Monte Carlo simulation to define the concepts of service capacity and occupancy, minimizing the implicit cost of the capacity that must be reserved to service unexpected customers. Usually, passenger car companies estimate their service facilities capacity using empirical methods, but customers arrive under uncertain conditions not included in the estimations. Thus, there is a gap between customer’s real demand and the dealer’s capacity. This research sets a valid methodology for the passenger car industry to cover the generic absence of recent researches and the generic lack of statistical techniques implementation. The hospital’s emergency room (ER) equalization has been confirmed to be valid for the passenger car industry and new process indicators have been defined to support the study. As hospitals do, we aim to apply stochastic models to dimension installations according to the demographic distribution of the area to be serviced. The proposed model integrates the prediction of the cost implicit in the reserve capacity to serve unexpected demand. The Matlab code could be implemented as part of the existing information technology systems (ITs) to support the existing service management tools, creating a set of new process indicators. Main model outputs are new indicators, such us Capacity, Occupancy and Cost of Capacity Reserve, never studied in the passenger car service industry before, and intended to manage the service operation.

Flexibilidad Polivalencia: modelos de libertad para la vivienda social en España

Este trabajo estudia la validez de la flexibilidad y la polivalencia, en tanto que estrategias de diseño doméstico, para superar los problemas derivados de la coincidencia de dos circunstancias opuestas en el campo de la vivienda social. Por un lado, la existencia en España de un rígido conjunto de normativas de diseño, originado a mediados del siglo pasado bajo unos supuestos culturales e ideológicos distintos a los actuales, que fija una idea muy precisa de hogar. Por otro lado, la creciente variedad de modos de habitar derivada del progreso social, económico y tecnológico de las últimas décadas. La tesis presenta dos partes ligadas entre sí. La primera es una investigación bibliográfica en la que se analizan los diferentes conceptos asociados a la flexibilidad y la polivalencia manejados hasta hoy, se estudian las formas en las que estas estrategias se presentan a través de ejemplos, algunos de ellos muy poco conocidos, y se exponen las oportunidades y los obstáculos para su desarrollo en nuestro país. La segunda parte es una investigación experimental que incluye los estudios de evaluación de dos edificios madrileños de referencia, las viviendas flexibles de Aranguren y Gallegos en Carabanchel y las viviendas polivalentes de la Casa de las Flores, de Secundino Zuazo. En ellos se analizan las ventajas y desventajas de los distintos dispositivos flexibles y polivalentes existentes en dichas viviendas; cómo influyen en la organización de la vida doméstica; cuáles de sus características merecen ser implementadas en nuevos proyectos y bajo qué condiciones. La investigación revela que la flexibilidad ofrece ventajas funcionales objetivas, pero que su generalización en nuevas promociones de vivienda ocasionaría problemas de índole cultural. Por otro lado, la polivalencia es eficaz para dar cabida a nuevos modelos de convivencia, pero choca con las preferencias de los grupos familiares tradicionales. En ambos casos, se proponen soluciones para mejorar su eficacia. ABSTRACT The aim of this thesis is to assess the validity of flexibility and polyvalence as architectural responses to address some of the issues derived from two concurrent –yet opposing– circumstances in the field of Spanish social housing. On the one hand, the existence of a thick set of rules that strictly defines the form and use of the domestic space. These design norms were originated in the mid-20th century, according to an institutionalised idea of nuclear family and family home that does not match the current social trends in the country. On the other hand, the growing variety of lifestyles and family groups derived from the social, economic and technological progress of the last decades. The thesis is divided into two interrelated parts: The first one is a bibliographic research and an analysis of the diverse concepts attached to flexibility and polyvalence existing in the literature, a study of the ways in which these strategies are practiced in relevant housing buildings, and an explanation of the benefits and difficulties they pose with respect to their development in Spain. The second part is an experimental research including two post-occupancy evaluations in two housing buildings in Madrid: the flexible apartments in Carabanchel by Aranguren & Gallegos, and the polyvalent apartments in la Casa de las Flores by Secundino Zuazo. These studies are aimed at determining the extent to which the flexible and polyvalent elements in these projects are successful, which users take advantage of them and, over all, which of these features deserve to be applied to future developments, and under which conditions. The research indicates that, despite offering objective functional advantages, flexibility cannot be proposed as a general strategy in future subsidised housing developments due to the cultural problems it provokes. Besides, polyvalence is useful to host new cohabitations models, but it clashes with the preferences of traditional family groups. In both cases, several solutions to improve their efficiency are presented.

Vivienda y fondo edificatorio: análisis de las consecuencias del parámetro fondo en el edificio de vivienda colectiva

Este estudio ofrece una herramienta de aproximación al espacio morfológico-métrico en el que se formula la ciudad de alta densidad desde la vivienda colectiva. La vivienda colectiva es la célula básica de la ciudad. El estudio configurativo y dimensional del tejido urbano muestra la importancia del fondo edificatorio como parámetro clave a mitad de camino entre la vivienda y la ciudad. El fondo edificatorio traza el margen de la arquitectura en la ciudad y desde él se equipa y cuantifica el territorio urbano. Sus dinámicas van caracterizando los distintos entornos, mientras en su interior se formula el tipo en un ajuste de continua verificación y adaptación. La forma de la ciudad y sus distintas posibilidades configurativas —en cuanto masa construida y espacio público, pero sin perder de vista la relación entre ambos— depende en gran medida del fondo edificatorio. Se trata, por tanto, de un parámetro importante de relación entre las distintas configuraciones del espacio exterior e interior. Al proyectar, una vez establecido un fondo, algunas propiedades se adaptan con facilidad mientras que otras requieren un cierto grado de interpretación o deben ser descartadas. Dada una superficie, la especificación del fondo fuerza la dimensión del frente en las configuraciones posibles. Ambas dimensiones son vitales en el valor del factor de forma del continuo edificado y en su relación se produce el complejo rango de posibilidades. Partiendo de la ciudad, un gran fondo encierra y mezcla en su interior todo tipo de usos sin distinción, repercute un menor coste por unidad de superficie edificada y comparte su frente reduciendo los intercambios térmicos y lumínicos. Sin embargo la ciudad de fondo reducido ajusta la forma al uso y se desarrolla linealmente con repetitividad a lo largo de sus frentes exteriores. En ella, el fuerte intercambio energético se opone a las grandes posibilidades del espacio libre. En cambio desde la casa las distintas medidas del fondo se producen bajo determinados condicionantes: clima, compacidad, ocupación, hibridación, tamaño de casa, etc., mientras que el tipo se desarrolla en base a una métrica afín. Este trabajo parte de esta dialéctica. Estudia la relación de dependencia entre las condiciones del edificio de viviendas y su métrica. Jerarquiza edificios en base al parámetro “fondo” para constituir una herramienta que como un ábaco sea capaz de visibilizar las dinámicas relacionales entre configuración y métrica bajo la condición de alta densidad. Para ello en una primera fase se gestiona una extensa muestra de edificios representativos de vivienda colectiva principalmente europea, extraída de tres prestigiosos libros en forma de repertorio. Se ordenan y categorizan extrayendo datos conmensurables y temas principales que ligan la profundidad de la huella a la morfología y posteriormente, esta información se estudia en diagramas que ponen de manifiesto convergencias y divergencias, acumulaciones y vacíos, límites, intervalos característicos, márgenes y ejes, parámetros y atributos... cuya relación trata de factorizar el lugar morfológico y métrico de la casa como metavivienda y ciudad. La herramienta se establece así como un complejo marco relacional en el que posicionar casos concretos y trazar nexos transversales, tanto de tipo morfológico como cultural, climático o técnico, normativo o tecnológico. Cada nuevo caso o traza añadida produce consonancias y disonancias en el marco que requieren interpretación y verificación. De este modo este instrumento de análisis comparativo se tempera, se especializa, se completa y se perfecciona con su uso. La forma de la residencia en la ciudad densa se muestra así sobre un subsistema morfológico unitario y su entendimiento se hace más fácilmente alcanzable y acumulable tanto para investigaciones posteriores como para el aprendizaje o el ejercicio profesional. ABSTRACT This research study offers a tool to approach the morphometric space in which (multi-family) housing defines high-density cities. Multi-family housing is the basic cell of the city. The configuration and dimension studies of the urban fabric render the importance of building depth as a key parameter half way between the dwelling and the city. The building depth traces de limit of architecture in the city. It qualifies and quantifies the urban territory. Its dynamics characterize the different environments while in its essence, an adjustment process of continuous verification and adaption defines type. The shape of the city and its different configuration possibilities —in terms of built fabric and public space, always keeping an eye on the relationship between them— depend majorly on the building depth. Therefore, it is a relevant parameter that relates the diverse configurations between interior and exterior space. When designing, once the depth is established, some properties are easily adpated. However, others require a certain degree of interpretation or have to be left out of the study. Given a ceratin surface, the establishment of the depth forces the dimensions of the facade in the different configurations. Both depth and facade dimensions are crucial for the form factor of the built mass. Its relationship produces a complex range of possibilities. From an urban point of view, great depth means multiple uses (making no distinction whatsoever,) it presents a lower cost per unit of built area and shares its facade optimizing temperature and light exchange. On the contrary, the city of reduced depth adjusts its shape to the use, and develops linearly and repetitively along its facades. The strong energy exchange opposes to the great possibilities of free space. From the perspective of the dwelling, the different dimensions of depth are produced under certain determinants: climate, compactness, occupancy, hybridization, dwelling size, etc. Meanwhile, the type is developed based on a related meter (as in poetry). This work starts from the previous premise. It studies the dependency relation bewteen the conditions of the dwellings and their meter (dimensions). It organizes buildings hierarchically based on the parameter “depth” to create a tool that, as an abacus, is able to visibilise the relational dynamics between configuration and dimension in high density conditions. For this, in the first stage a large group of representative multi-family housing buildings is managed, mostly from Europe, picked from three prestigious books as a repertoir. They are categorized and ordered drawing commensurable data and key issues that link the depth of the fooprint to its morphology. Later, this information is studied deeply with diagrams that bring out connections and discrepancies, voids and accumulations, limits, charasteristic intervals, margins and axii, parameters, attributes, etc. These relationships try to create factors from a morphological and metrical point of view of the house as a metadwelling. This tool is established as a complex relation frame in which case studies are postitioned and cross-cutting nexii are traced. These can deal with morphology, climate, technique, law or technology. Each new case or nexus produces affinities and discrepancies that require interpretation and verification. Thus, this instrument of comparative analysis is fine-tuned, especialized and completed as its use is improved. The way housing is understood in high density cities is shown as a unitary metric subsystem and its understanding is easy to reach and accumulate for future researchers, students or practicing architects.

Caracterización de la ventilación en la edificación residencial existente. Conciliación entre calidad del aire interior y eficiencia en la rehabilitación energética

La edificación residencial existente en España y en Europa se encuentra abocada a una rehabilitación profunda para cumplir los objetivos marcados en la estrategia europea para el año 2050. Estos, para el sector de la edificación, se proponen una reducción del 90% de emisiones de gases de efecto invernadero (GEI) respecto a niveles del año 1990. Este plan a largo plazo establece hitos intermedios de control, con objetivos parciales para el año 2020 y 2030. El objetivo último es aprovechar el potencial de reducción de demanda energética del sector de la edificación, del cual la edificación residencial supone el 85% en España. Dentro de estos requerimientos, de reducción de demanda energética en la edificación, la ventilación en la edificación residencial se convierte en uno de los retos a resolver por su vinculación directa a la salud y el confort de los ocupantes de la misma, y al mismo tiempo su relación proporcional con la demanda energética que presenta el edificio asociada al acondicionamiento térmico. Gran parte de las pérdidas térmicas de la edificación residencial se producen por el aire de renovación y la infiltración de aire a través de la envolvente. La directiva europea de eficiencia energética de la edificación (EPBD), que establece las directrices necesarias para alcanzar los objetivos de este sector en cuanto a emisiones de CO2 y gases de efecto invernadero (GEI), contempla la ventilación con aire limpio como un requisito fundamental a tener en cuenta de cara a las nuevas construcciones y a la rehabilitación energética de los edificios existentes. El síndrome del edificio enfermo, un conjunto de molestias y síntomas asociados a la baja calidad del aire de edificios no residenciales que surgió a raíz de la crisis del petróleo de 1973, tuvo su origen en una ventilación deficiente y una renovación del aire interior insuficiente de estos edificios, producto del intento de ahorro en la factura energética. Teniendo en cuenta que, de media, pasamos un 58% de nuestro tiempo en las viviendas, es fundamental cuidar la calidad del aire interior y no empeorarla aplicando medidas de “eficiencia energética” con efectos no esperados. Para conseguir esto es fundamental conocer en profundidad cómo se produce la ventilación en la edificación en bloque en España en sus aspectos de calidad del aire interior y demanda energética asociada a la ventilación. El objetivo de esta tesis es establecer una metodología de caracterización y de optimización de las necesidades de ventilación para los espacios residenciales existentes en España que aúne el doble objetivo de garantizar la calidad ambiental y reducir la demanda energética de los mismos. La caracterización del parque edificatorio residencial español en cuanto a ventilación es concluyente: La vivienda en España se distribuye principalmente en tres periodos en los que se encuentran más del 80% del total de las viviendas construidas. El periodo anterior a las normas básicas de la edificación (NBE), de 1960 a 1980, el periodo desde 1980 al año 2005, con el mayor número total de viviendas construidas, guiado por la NTE ISV 75, y el periodo correspondiente a la edificación construida a partir del Código Técnico de la Edificación, en 2006, cuyo documento básico de condiciones de salubridad (DB HS3) es la primera norma de obligado cumplimiento en diseño y dimensionamiento de ventilación residencial en España. La selección de un modelo de bloque de viviendas de referencia, un valor medio y representativo, seleccionado de entre estos periodos, pero con cualidades que se extienden más allá de uno de ellos, nos permite realizar un intensivo análisis comparativo de las condiciones de calidad de aire interior y la demanda energética del mismo, aplicando las distintas configuraciones que presenta la ventilación en viviendas dependiendo del escenario o época constructiva (o normativa) en que esta fuera construida. Este análisis se lleva a cabo apoyándose en un doble enfoque: el modelado numérico de simulaciones y el análisis de datos experimentales, para comprobar y afinar los modelos y observar la situación real de las viviendas en estos dos aspectos. Gracias a las conclusiones del análisis previo, se define una estrategia de optimización de la ventilación basada fundamentalmente en dos medidas: 1) La introducción de un sistema de extracción mecánica y recuperación de calor que permita reducir la demanda energética debida a la renovación del aire y a la vez diluir los contaminantes interiores más eficazmente para mejorar, de esta forma, la calidad del ambiente interior. 2) La racionalización del horario de utilización de estos sistemas, no malgastando la energía en periodos de no ocupación, permitiendo una leve ventilación de fondo, debida a la infiltración, que no incida en pérdidas energéticas cuantiosas. A esta optimización, además de aplicar la metodología de análisis previo, en cuanto a demanda energética y calidad del aire, se aplica una valoración económica integradora y comparativa basada en el reglamento delegado EU244/2012 de coste óptimo (Cost Optimal Methodology). Los resultados principales de esta tesis son: • Un diagnóstico de la calidad del aire interior de la edificación residencial en España y su demanda energética asociada, imprescindible para lograr una rehabilitación energética profunda garantizando la calidad del aire interior. • Un indicador de la relación directa entre calidad de aire y demanda energética, para evaluar la adecuación de los sistemas de ventilación, respecto de las nuevas normativas de eficiencia energética y ventilación. • Una estrategia de optimización, que ofrece una alternativa de intervención, y la aplicación de un método de valoración que permite evaluar la amortización comparada de la instalación de los sistemas. ABSTRACT The housing building stock already built in Spain and Europe faces a deep renovation in the present and near future to accomplish with the objectives agreed in the European strategy for 2050. These objectives, for the building sector, are set in a 90% of Green House Gases (GHG) reduction compared to levels in 1990. This long‐term plan has set milestones to control the correct advance of achievement in 2020 and 2030. The main objective is to take advantage of the great potential to reduce energy demand from the building sector, in which housing represents 85% share in Spain. Among this reduction on building energy demand requirements, ventilation of dwellings becomes one of the challenges to solve as it’s directly connected to the indoor air quality (IAQ) and comfort conditions for the users, as well as proportional to the building energy demand on thermal conditioning. A big share of thermal losses in housing is caused by air renovation and infiltration through the envelope leaks. The European Directive on Building energy performance (EPBD), establishes the roots needed to reach the building sector objectives in terms of CO2 and GHG emissions. This directive sets the ventilation and renovation with clean air of the new and existing buildings as a fundamental requirement. The Sick Building Syndrome (SBS), an aggregation of symptoms and annoys associated to low air quality in non residential buildings, appeared as common after the 1973 oil crisis. It is originated in defective ventilation systems and deficient air renovation rates, as a consequence of trying to lower the energy bill. Accounting that we spend 58% of our time in dwellings, it becomes crucial to look after the indoor air quality and focus in not worsening it by applying “energy efficient” measures, with not expected side effects. To do so, it is primary to research in deep how the ventilation takes place in the housing blocks in Spain, in the aspects related to IAQ and ventilation energy demand. This thesis main objective is to establish a characterization and optimization methodology regarding the ventilation needs for existing housing in Spain, considering the twofold objective of guaranteeing the air quality as reducing the energy demand. The characterization of the existing housing building stock in Spain regarding ventilation is conclusive. More of 80% of the housing stock is distributed in 3 main periods: before the implementation of the firsts regulations on building comfort conditions (Normas Básicas de la Edificación), from 1960 to 1980; the period after the first recommendations on ventilation (NTE ISV 75) for housing were set, around 1980 until 2005 and; the period corresponding to the housing built after the existing mandatory regulation in terms of indoor sanity conditions and ventilation (Spanish Building Code, DB HS3) was set, in 2006. Selecting a representative blueprint of a housing block in Spain, which has medium characteristics not just within the 3 periods mention, but which qualities extent beyond the 3 of them, allows the next step, analyzing. This comparative and intense analyzing phase is focused on the air indoor conditions and the related energy demand, applying different configurations to the ventilation systems according to the different constructive or regulation period in which the building is built. This analysis is also twofold: 1) Numerical modeling with computer simulations and 2) experimental data collection from existing housing in real conditions to check and refine the models to be tested. Thanks to the analyzing phase conclusions, an optimization strategy on the ventilation of the housing stock is set, based on two actions to take: 1) To introduce a mechanical exhaust and intake ventilation system with heat recovery that allows reducing energy demand, as improves the capacity of the system to dilute the pollutant load. This way, the environmental quality is improved. 2) To optimize the schedule of the system use, avoids waste of energy in no occupancy periods, relying ventilation during this time in a light infiltration ventilation, intended not to become large and not causing extra energy losses. Apart from applying the previous analyzing methodology to the optimization strategy, regarding energy demand and air quality, a ROI valorization is performed, based on the cost optimal methodology (delegated regulation EU244/2012). The main results from the thesis are: • To obtain a through diagnose regarding air quality and energy demand for the existing housing stock in Spain, unavoidable to reach a energy deep retrofitting scheme with no air quality worsening. • To obtain a marker to relate air quality and energy demand and evaluate adequateness of ventilation systems, for the new regulations to come. • To establish an optimization strategy to improve both air quality and energy demand, applying a compared valorization methodology to obtain the Return On Investment (ROI).

Pedestrian Motion Prediction: A Graph Based Approach

A novel pedestrian motion prediction technique is presented in this paper. Its main achievement regards to none previous observation, any knowledge of pedestrian trajectories nor the existence of possible destinations is required; hence making it useful for autonomous surveillance applications. Prediction only requires initial position of the pedestrian and a 2D representation of the scenario as occupancy grid. First, it uses the Fast Marching Method (FMM) to calculate the pedestrian arrival time for each position in the map and then, the likelihood that the pedestrian reaches those positions is estimated. The technique has been tested with synthetic and real scenarios. In all cases, accurate probability maps as well as their representative graphs were obtained with low computational cost.

Urokinase-Type Plasminogen Activator Receptor Is Internalized by Different Mechanisms in Polarized and Nonpolarized Madin–Darby Canine Kidney Epithelial Cells

Accumulated data indicate that endocytosis of the glycosylphosphatidyl-inositol-anchored protein urokinase plasminogen activator receptor (uPAR) depends on binding of the ligand uPA:plasminogen activator inhibitor-1 (PAI-1) and subsequent interaction with internalization receptors of the low-density lipoprotein receptor family, which are internalized through clathrin-coated pits. This interaction is inhibited by receptor-associated protein (RAP). We show that uPAR with bound uPA:PAI-1 is capable of entering cells in a clathrin-independent process. First, HeLaK44A cells expressing mutant dynamin efficiently internalized uPA:PAI-1 under conditions in which transferrin endocytosis was blocked. Second, in polarized Madin–Darby canine kidney (MDCK) cells, which expressed human uPAR apically, the low basal rate of uPAR ligand endocytosis, which could not be inhibited by RAP, was increased by forskolin or phorbol ester (phorbol 12-myristate 13-acetate), which selectively up-regulate clathrin-independent endocytosis from the apical domain of epithelial cells. Third, in subconfluent nonpolarized MDCK cells, endocytosis of uPA:PAI-1 was only decreased marginally by RAP. At the ultrastructural level uPAR was largely excluded from clathrin-coated pits in these cells and localized in invaginated caveolae only in the presence of cross-linking antibodies. Interestingly, a larger fraction of uPAR in nonpolarized relative to polarized MDCK cells was insoluble in Triton X-100 at 0°C, and by surface labeling with biotin we also show that internalized uPAR was mainly detergent insoluble, suggesting a correlation between association with detergent-resistant membrane microdomains and higher degree of clathrin-independent endocytosis. Furthermore, by cryoimmunogold labeling we show that 5–10% of internalized uPAR in nonpolarized, but not polarized, MDCK cells is targeted to lysosomes by a mechanism that is regulated by ligand occupancy.

Balancing transcriptional interference and initiation on the GAL7 promoter of Saccharomyces cerevisiae

Transcriptional termination of the GAL10 gene in Saccharomyces cerevisiae depends on the efficiency of polyadenylation. Either cis mutations in the poly(A) signal or trans mutations of mRNA 3′ end cleavage factors result in GAL10 read-through transcripts into the adjacent GAL7 gene and inactivation (occlusion) of the GAL7 promoter. Herein, we present a molecular explanation of this transcriptional interference phenomenon. In vivo footprinting data reveal that GAL7 promoter occlusion is associated with the displacement of Gal4p transcription factors from the promoter. Interestingly, overexpression of Gal4p restores promoter occupancy, activates GAL7 expression, and rescues growth on the otherwise toxic galactose substrate. Our data therefore demonstrate a precise balance between transcriptional interference and initiation.

The structure of the ultraspiracle ligand-binding domain reveals a nuclear receptor locked in an inactive conformation

Ultraspiracle (USP) is the invertebrate homologue of the mammalian retinoid X receptor (RXR). RXR plays a uniquely important role in differentiation, development, and homeostasis through its ability to serve as a heterodimeric partner to many other nuclear receptors. RXR is able to influence the activity of its partner receptors through the action of the ligand 9-cis retinoic acid. In contrast to RXR, USP has no known high-affinity ligand and is thought to be a silent component in the heterodimeric complex with partner receptors such as the ecdysone receptor. Here we report the 2.4-Å crystal structure of the USP ligand-binding domain. The structure shows that a conserved sequence motif found in dipteran and lepidopteran USPs, but not in mammalian RXRs, serves to lock USP in an inactive conformation. It also shows that USP has a large hydrophobic cavity, implying that there is almost certainly a natural ligand for USP. This cavity is larger than that seen previously for most other nuclear receptors. Intriguingly, this cavity has partial occupancy by a bound lipid, which is likely to resemble the natural ligand for USP.

Detection of alkali metal ions in DNA crystals using state-of-the-art X-ray diffraction experiments

The observation of light metal ions in nucleic acids crystals is generally a fortuitous event. Sodium ions in particular are notoriously difficult to detect because their X-ray scattering contributions are virtually identical to those of water and Na+…O distances are only slightly shorter than strong hydrogen bonds between well-ordered water molecules. We demonstrate here that replacement of Na+ by K+, Rb+ or Cs+ and precise measurements of anomalous differences in intensities provide a particularly sensitive method for detecting alkali metal ion-binding sites in nucleic acid crystals. Not only can alkali metal ions be readily located in such structures, but the presence of Rb+ or Cs+ also allows structure determination by the single wavelength anomalous diffraction technique. Besides allowing identification of high occupancy binding sites, the combination of high resolution and anomalous diffraction data established here can also pinpoint binding sites that feature only partial occupancy. Conversely, high resolution of the data alone does not necessarily allow differentiation between water and partially ordered metal ions, as demonstrated with the crystal structure of a DNA duplex determined to a resolution of 0.6 Å.

Switch from Myc/Max to Mad1/Max binding and decrease in histone acetylation at the telomerase reverse transcriptase promoter during differentiation of HL60 cells

Recent evidence suggests that the Myc and Mad1 proteins are implicated in the regulation of the gene encoding the human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase. We have analyzed the in vivo interaction between endogenous c-Myc and Mad1 proteins and the hTERT promoter in HL60 cells with the use of the chromatin immunoprecipitation assay. The E-boxes at the hTERT proximal promoter were occupied in vivo by c-Myc in exponentially proliferating HL60 cells but not in cells induced to differentiate by DMSO. In contrast, Mad1 protein was induced and bound to the hTERT promoter in differentiated HL60 cells. Concomitantly, the acetylation of the histones at the promoter was significantly reduced. These data suggest that the reciprocal E-box occupancy by c-Myc and Mad1 is responsible for activation and repression of the hTERT gene in proliferating and differentiated HL60 cells, respectively. Furthermore, the histone deacetylase inhibitor trichostatin A inhibited deacetylation of histones at the hTERT promoter and attenuated the repression of hTERT transcription during HL60 cell differentiation. In addition, trichostatin A treatment activated hTERT transcription in resting human lymphocytes and fibroblasts. Taken together, these results indicate that acetylation/deacetylation of histones is operative in the regulation of hTERT expression.

The structures of anthranilate synthase of Serratia marcescens crystallized in the presence of (i) its substrates, chorismate and glutamine, and a product, glutamate, and (ii) its end-product inhibitor, l-tryptophan

The crystal structure of anthranilate synthase (AS) from Serratia marcescens, a mesophilic bacterium, has been solved in the presence of its substrates, chorismate and glutamine, and one product, glutamate, at 1.95 Å, and with its bound feedback inhibitor, tryptophan, at 2.4 Å. In comparison with the AS structure from the hyperthermophile Sulfolobus solfataricus, the S. marcescens structure shows similar subunit structures but a markedly different oligomeric organization. One crystal form of the S. marcescens enzyme displays a bound pyruvate as well as a putative anthranilate (the nitrogen group is ambiguous) in the TrpE subunit. It also confirms the presence of a covalently bound glutamyl thioester intermediate in the TrpG subunit. The tryptophan-bound form reveals that the inhibitor binds at a site distinct from that of the substrate, chorismate. Bound tryptophan appears to prevent chorismate binding by a demonstrable conformational effect, and the structure reveals how occupancy of only one of the two feedback inhibition sites can immobilize the catalytic activity of both TrpE subunits. The presence of effectors in the structure provides a view of the locations of some of the amino acid residues in the active sites. Our findings are discussed in terms of the previously described AS structure of S. solfataricus, mutational data obtained from enteric bacteria, and the enzyme's mechanism of action.

Repression of TFII-I-dependent transcription by nuclear exclusion

TFII-I is an unusual transcription factor possessing both basal and signal-induced transcriptional functions. Here we report the characterization of a TFII-I-related factor (MusTRD1/BEN) that regulates transcriptional functions of TFII-I by controlling its nuclear residency. MusTRD1/BEN has five or six direct repeats, each containing helix–loop–helix motifs, and, thus, belongs to the TFII-I family of transcription factors. TFII-I and MusTRD1/BEN, when expressed individually, show predominant nuclear localization. However, when the two proteins are coexpressed ectopically, MusTRD1/BEN locates almost exclusively to the nucleus, whereas TFII-I is largely excluded from the nucleus, resulting in a loss of TFII-I-dependent transcriptional activation of the c-fos promoter. Mutation of a consensus nuclear localization signal in MusTRD1/BEN results in a reversal of nuclear residency of the two proteins and a concomitant gain of c-fos promoter activity. These data suggest a means of transcriptional repression by competition at the level of nuclear occupancy.

The "allosteric three-site model" of elongation cannot be confirmed in a well-defined ribosome system from Escherichia coli.

For the functional role of the ribosomal tRNA exit (E) site, two different models have been proposed. It has been suggested that transient E-site binding of the tRNA leaving the peptidyl (P) site promotes elongation factor G (EF-G)-dependent translocation by lowering the energetic barrier of tRNA release [Lill, R., Robertson, J. M. & Wintermeyer, W. (1989) EMBO J. 8, 3933-3938]. The alternative "allosteric three-site model" [Nierhaus, K.H. (1990) Biochemistry 29, 4997-5008] features stable, codon-dependent tRNA binding to the E site and postulates a coupling between E and aminoacyl (A) sites that regulates the tRNA binding affinity of the two sites in an anticooperative manner. Extending our testing of the two conflicting models, we have performed translocation experiments with fully active ribosomes programmed with heteropolymeric mRNA. The results confirm that the deacylated tRNA released from the P site is bound to the E site in a kinetically labile fashion, and that the affinity of binding, i.e., the occupancy of the E site, is increased by Mg2+ or polyamines. At conditions of high E-site occupancy in the posttranslocation complex, filling the A site with aminoacyl-tRNA had no influence on the E site, i.e., there was no detectable anticooperative coupling between the two sites, provided that second-round translocation was avoided by removing EF-G. On the basis of these results, which are entirely consistent with our previous results, we consider the allosteric three-site model of elongation untenable. Rather, as proposed earlier, the E site-bound state of the leaving tRNA is a transient intermediate and, as such, is a mechanistic feature of the classic two-state model of the elongating ribosome.