A sedimentological, faunal, and isotopic record of the middle-to-late Pliocene transition at DSDP Hole 80-548

Hydraulic piston coring at DSDP Site 548, on the upper continental slope southwest of Ireland, recovered a nearly complete Pliocene section spanning 103 m of sediment. The sediments are greenish gray carbonate-rich hemipelagites containing abundant nannofossils and foraminifers. Grain-size analysis demonstrates that the texture of the section is fairly constant, with most of the variation occurring in 63- to 32-µm and < 2-µm fractions. Previous research has shown that the middle-to-late Pliocene transition in the North Atlantic was marked by the appearance of the planktonic foraminiferal species Globorotalia inflata and by the first occurrence of significant quantities of ice-rafted sediment grains in deep-sea sediments. The latter is taken to represent the first important development of Northern Hemisphere glaciation. The first appearance of G. inflata is carefully documented for Site 548 and is demonstrated to be an evolutionary datum at this site, rather than an ecologically controlled first appearance. Surface ocean conditions represented in the sediment section spanning the appearance of G. inflata were strongly cyclic, resulting in large periodic changes in the abundances of Globorotalia puncticulata and N. acostaensis. The benthic foraminiferal population was studied in detail over the middle-to-upper Pliocene transition to establish the nature and behavior of the intermediate-depth water mass in the northeastern Atlantic at the time of ice-sheet growth in the Northern Hemisphere. This water mass is presently warm and saline, having its source in the Mediterranean Sea. The benthic data show that the intermediate-depth water mass was undergoing a series of progressive changes over the interval including the first appearance of G. inflata. These changes are particularly reflected in the relative abundances of Globocassidulina subglobosa (Brady), Uvigerina, and Ehrenbergina. Also, the mean size of individuals in the G. subglobosa populations shows systematic variation, indicating changing intermediate-depth water properties. Oxygen-isotope analyses show that the intermediate-depth water mass was cold during the middle-to-late Pliocene transition. This interpretation is supported by the relative abundances of benthic foraminiferal species. Hence, the intermediate-depth northeastern Atlantic water mass of the middle to late Pliocene was considerably different from the intermediate-depth water mass of the present.

Optimal and Long-Term Dynamic Transport Policy Design: Seeking Maximum Social Welfare through a Pricing Scheme.

This article presents an alternative approach to the decision-making process in transport strategy design. The study explores the possibility of integrating forecasting, assessment and optimization procedures in support of a decision-making process designed to reach the best achievable scenario through mobility policies. Long-term evaluation, as required by a dynamic system such as a city, is provided by a strategic Land-Use and Transport Interaction (LUTI) model. The social welfare achieved by implementing mobility LUTI model policies is measured through a cost-benefit analysis and maximized through an optimization process throughout the evaluation period. The method is tested by optimizing a pricing policy scheme in Madrid on a cordon toll in a context requiring system efficiency, social equity and environmental quality. The optimized scheme yields an appreciable increase in social surplus through a relatively low rate compared to other similar pricing toll schemes. The results highlight the different considerations regarding mobility impacts on the case study area, as well as the major contributors to social welfare surplus. This leads the authors to reconsider the cost-analysis approach, as defined in the study, as the best option for formulating sustainability measures.

Integration of congestion pricing and intertemporal preference rate in social welfare function

Assessing social benefits in transport policy implementation has been studied by many researchers using theoretical or empirical measures. However, few of them measure social benefit using different discount rates including the inter-temporal preferences rate of users, the private investment discount rate and the inter-temporal preferences rate of the government. In general, the social discount rate used is the same for all social actors. Therefore, this paper aims to assess a new method by integrating different types of discount rate belonging to different social actors in order to measure the real benefits of each actor in the short, medium and long term. A dynamic simulation is provided by a strategic Land-Use and Transport Interaction (LUTI) model. The method is tested by optimizing a cordon toll scheme in Madrid considering socio- economic efficiency and environmental criteria. Based on the modified social welfare function (WF), the effects on the measure of social benefits are estimated and compared with the classical WF results as well. The results of this research could be a key issue to understanding the relationship between transport system policies and social actors' benefits distribution in a metropolitan context. The results show that the use of more suitable discount rates for each social actor had an effect on the selection and definition of optimal strategy of congestion pricing. The usefulness of the measure of congestion toll declines more quickly overtime.

Integration of congestion pricing and intertemporal preferences rate integration in social welfare function

Many researchers have used theoretical or empirical measures to assess social benefits in transport policy implementation. However, few have measured social benefits by using discount rates, including the intertemporal preference rate of users, the private investment discount rate, and the intertemporal preference rate of the government. In general, the social discount rate used is the same for all social actors. This paper aims to assess a new method by integrating different types of discount rates belonging to different social actors to measure the real benefits of each actor in the short term, medium term, and long term. A dynamic simulation is provided by a strategic land use and transport interaction model. The method was tested by optimizing a cordon toll scheme in Madrid, Spain. Socioeconomic efficiency and environmental criteria were considered. On the basis of the modified social welfare function, the effects on the measure of social benefits were estimated and compared with the classical welfare function measures. The results show that the use of more suitable discount rates for each social actor had an effect on the selection and definition of optimal strategy of congestion pricing. The usefulness of the measure of congestion toll declines more quickly over time. This result could be the key to understanding the relationship between transport system policies and the distribution of social actors? benefits in a metropolitan context.

Assessing the impact of adaptive accessibility on the optimal transport policy implementation by using an integrated land-use/transport model for Madrid

Accessibility is an essential concept widely used to evaluate the impact of land-use and transport strategies in transport and urban planning. Accessibility is typically evaluated by using a transport model or a land-use model independently or successively without a feedback loop, thus neglecting the interaction effects between the two systems and the induced competition effects among opportunities due to accessibility improvements. More than a mere methodological curiosity, failure to account for land- use/transport interactions and the competition effect may result in large underestimation of the policy effects. With the recent development of land-use and transport interaction (LUTI) models, there is a growing interest in using these models to adequately measure accessibility and evaluate its impact. The current study joins this research stream by embedding an accessibility measure in a LUTI model with two main aims. The first aim is to account for adaptive accessibility, namely the adjustment of the potential accessibility due to the effect of competition among opportunities (e.g., workplaces) as a result of improved accessibility. LUTI models are particularly suitable for assessing adaptive accessibility because the competition factor is a function of the number of jobs, which is related to land-use attractiveness and the number of workers which is related, among other factors, to the transport demand. The second aim is to identify the optimal implementation scenario of policy measures on the basis of the potential and adaptive accessibility and analyse the results in terms of social welfare and accessibility. The metropolitan area of Madrid is used as a case-study and two transport policy instruments, namely a cordon toll and bus frequency increase, have been chosen for the simulation study in order to present the usefulness of the approach to urban planners and policy makers. The MARS model (Metropolitan Activity Relocation Simulator) calibrated for Madrid was employed as the analysis tool. The impact of accessibility is embedded in the model through a social welfare function that includes not only costs and benefits to both road users and transport operators, but also costs and benefits for the government and society in general (external costs). An optimisation procedure is performed by the MARS model for maximizing the value of objective function in order to find the best (optimal) policy imp lementations intensity (i.e., price, frequency). Last, the two policy strategies are evaluated in terms of their accessibility. Results show that the accessibility with competition factor influences the optimal policy implementation level and also generates different results in terms of social welfare. In addition, mapping the difference between the potential and the adaptive accessibility indicators shows that the main changes occur in areas where there is a strong competition among land-use opportunities.

Optimal and Long-Term Dynamic Transport Policy Design: Seeking Maximum Social Welfare through a Pricing Scheme

This article presents an alternative approach to the decision-making process in transport strategy design. The study explores the possibility of integrating forecasting, assessment and optimization procedures in support of a decision-making process designed to reach the best achievable scenario through mobility policies. Long-term evaluation, as required by a dynamic system such as a city, is provided by a strategic Land-Use and Transport Interaction (LUTI) model. The social welfare achieved by implementing mobility LUTI model policies is measured through a cost-benefit analysis and maximized through an optimization process throughout the evaluation period. The method is tested by optimizing a pricing policy scheme in Madrid on a cordon toll in a context requiring system efficiency, social equity and environmental quality. The optimized scheme yields an appreciable increase in social surplus through a relatively low rate compared to other similar pricing toll schemes. The results highlight the different considerations regarding mobility impacts on the case study area, as well as the major contributors to social welfare surplus. This leads the authors to reconsider the cost-analysis approach, as defined in the study, as the best option for formulating sustainability measures.

Regret theory-based scenario building using an interacttive process

This paper is an introduction of the regret theory-based scenario building approach combining with a modified Delphi method that uses an interactive process to design and assess four different TDM measures (i.e., cordon toll, parking charge, increased bus frequency and decreased bus fare). The case study of Madrid is used to present the analysis and provide policy recommendations. The new scenario building approach incorporates expert judgement and transport models in an interactive process. It consists of a two-round modified Delphi survey, which was answeared by a group of Spanish transport experts who were the participants of the Transport Engineering Congress (CIT 2012), and an integrated land-use and transport model (LUTI) for Madrid that is called MARS (Metropolitan Activity Relocation Simulator).

Estudio de los efectos en el viñedo de la modificación del déficit hídrico continuo en los periodos anterior y posterior al envero (cv. Cabernet Sauvignon; Vitis vinifera L.)

En regiones semiáridas, con veranos cálidos, evapotranspiraciones elevadas, humedades relativas bajas, además de precipitaciones intermitentes y escasas (400 mm al año) que reducen la recarga del perfil en invierno, se puede ver afectada la productividad y sostenibilidad de los viñedos. Por eso en estas zonas puede ser necesario utilizar el riego suplementario para mantener la calidad y aumentar la productividad. En este marco de circunstancias es en el que se desarrolla la mayoría de la viticultura española, y en concreto en la zona centro donde se realizó el estudio. Con este trabajo se pretende estudiar la influencia del agua del riego, con distintas dosis y aplicada en diferentes fases de crecimiento y maduración de la baya, en la producción y calidad de la uva. Se estudió la influencia del déficit moderado continuo aplicado antes y después de envero, en el aprovechamiento de los recursos ambientales y en la respuesta del cv. Cabernet Sauvignon. El ensayo se desarrolló en 2010 y 2011 en la finca “La Alcoholera”, perteneciente a bodegas LICINIA S.L., ubicada en la localidad de Chinchón, Comunidad de Madrid, España. Durante 2010 y 2011 se realizaron estudios en cepas del cv. Cabernet Sauvignon (clon 15), injertadas sobre 41B y plantadas en 2005. Con poda corta en Cordón Royat unilateral de 80 cm de altura, y cuya vegetación alcanzó una altura de 97 cm. Las cepas del ensayo fueron conducidas verticalmente en espaldera (VSP), con 10 yemas por metro lineal. Las plantas dentro de la fila fueron separadas 1 m y la distancia de la calle fue de 3 m. La orientación de las filas norte – sur. Se plantearon cuatro tratamientos experimentales con diferentes dosis y momento de aplicación del riego. - T: Testigo. Déficit ligero, con un aporte continuado de agua desde floración (420 mm). - DMc: Déficit moderado continuo. Manejo del riego convencional, empezando en pre-envero (154 mm) - DM1: Déficit moderado a partir de pre-envero. Aporte de riego continuado desde floración reduciéndose la dosis de riego en pre-envero (312 mm). - DM2: Déficit moderado hasta pre-envero. Se empezó a regar a partir de preenvero (230 mm). Se observó que bajo condiciones de estrés hídrico leve, el crecimiento se reduce disminuyendo la migración de fotoasimilados hacia los órganos vegetativos. Generando menor área foliar en las plantas sometidas a déficit hídrico moderado entre floración y cuajado. Disminuyendo su consumo. En maduración la humedad del suelo dependió principalmente del riego aplicado y del consumo de la planta; dicho consumo dependió a la vez del desarrollo foliar del canopy y de la necesidad hídrica de la vid. El aumento del déficit hídrico disminuyó el contenido de agua en el suelo, lo que provocó diferencias en el estado hídrico y en el intercambio gaseoso de las hojas. En condiciones de déficit moderado, la fotosíntesis se encontró altamente correlacionada con el potencial hídrico foliar medido a mediodía solar. Además en condiciones de déficit moderado continuo la disponibilidad hídrica, la demanda atmosférica y el nivel de hidratación de las hojas, interaccionaron de forma compleja en la regulación estomática de las hojas, condicionando el intercambio gaseoso y la eficiencia en el uso del agua. Al aumentar el volumen de agua aplicado el peso de madera de poda por metro de fila fue superior. Las diferencias encontradas en el rendimiento fueron debidas a las diferencias en el peso de baya. El cual estuvo condicionado por la estrategia de riego, ya que, el déficit hídrico moderado antes de envero fue más crítico para el rendimiento que el déficit impuesto durante la madurez. Por otro lado, la biomasa dependió de la actividad fisiológica de la planta, la cual fue altamente dependiente de la disponibilidad hídrica. Déficit hídrico moderado aplicado de cuajado a envero, generó bayas más pequeñas y aumentó la relación hollejo:pulpa. El déficit moderado aplicado después de envero favoreció la acumulación de IPT y antocianos extraíbles, mejorando la calidad de la uva, pero disminuyó la acidez de la baya. El riego aplicado de envero a vendimia desaceleró la concentración de azúcares en bayas que fueron sometidas a déficit antes de pre-envero. ABSTRACT Mediterranean climate is characterized by hot summers, high evapotranspiration rates, and scarce precipitations (400 mm per year) during grapevine cycle. These extremely dry conditions affect vineyard productivity and sustainability. Supplementary irrigation is needed practice in order to maintain yield and quality. Almost all Spanish grape growing regions are characterized by these conditions, especially in the center region, where this trial was performed. The main objective of this work is to study the influence of water irrigation on yield and quality. For this aim, different levels of irrigation (mm of water applied) were applied during different stages of growth and berry maturity of Caberent Sauvignon grapevines. The work was conducted from 2010 to 2011 and located in Licinia (40º 12´ N, 3º 28´ W), Madrid, Spain. The cultivar utilized was Cabernet Sauvignon, clone 15 grafted onto rootstock 41B, planted in 2005. The vineyard was oriented north-south, with spacing on 3 meters between rows and 1 meter between plants. Vines were spurpruned to 10 buds per meter and trained in unilateral cordon with a height of 80 cm. Shoots were positioned vertically (VSP). Considering the amount of water and the moment of the application, four experimental treatments were applied: - T: Control sample. Slight deficit (420 mm) applied from bloom to maturity. - DMc: Continuous moderate deficit. Traditional irrigation: application of 154 mm of water from pre-veraison to maturity. - DM1: Deficit moderate from pre-veraison. Irrigation of 312 mm of water from bloom to pre-veraison. - DM2: Deficit moderate to pre-veraison. Irrigation of 230 mm of water from preveraison to maturity Under moderated water stress conditions it was seen than leaf growth decreases due to the reduction of migration of photo-assimilates to vegetative organs. Vines with moderate water deficit between flowering and ripening develop less leaf area and decrease its water consumption. During maturation of berries, soil moisture depends on irrigation and plant consumption. This consumption further depends on vegetation development and on the vine needs for water. By increasing water deficit, the water content in the soil decreases. This causes differences in leaf water status and in the gases exchange. Under moderate deficit conditions, photosynthesis was found highly correlated with midday leaf water potential. Further, atmospheric demand and the level of leaves hydration interact in complex ways in the stomatal regulation, which affects leaf gas exchange and the efficiency of water use. The amount of water applied is directly proportional to pruning weight. Changes in berry weight cause differences in yield ratios. The differences in berry weights are conditioned by the irrigation strategy, the moderate water deficit before veraison influences more than the deficit applied from veraison. Biomass generated for the plant depends on its physiological activity, which is highly related to the water availability. Moderate water deficit applied from fruit set to ripening generates smaller berries, increasing the pulp/skin ratio. Moderate deficit applied after veraison promotes the accumulation of extractables anthocyans and TPI. Despite this treatment improves color parameters of the grapes, it decreases its total acidity. Irrigation applied from veraison to harvest slows down sugar accumulation in berries compared to those under deficit before veraison conditions.

Desarrollo de tecnologías de telemanipulación con alto grado de escalado orientadas a la interacción hombre-robot en entornos nucleares

Esta tesis se centra en desarrollo de tecnologías para la interacción hombre-robot en entornos nucleares de fusión. La problemática principal del sector de fusión nuclear radica en las condiciones ambientales tan extremas que hay en el interior del reactor, y la necesidad de que los equipos cumplan requisitos muy restrictivos para poder aguantar esos niveles de radiación, magnetismo, ultravacío, temperatura... Como no es viable la ejecución de tareas directamente por parte de humanos, habrá que utilizar dispositivos de manipulación remota para llevar a cabo los procesos de operación y mantenimiento. En las instalaciones de ITER es obligatorio tener un entorno controlado de extrema seguridad, que necesita de estándares validados. La definición y uso de protocolos es indispensable para regir su buen funcionamiento. Si nos centramos en la telemanipulación con algo grado de escalado, surge la necesidad de definir protocolos para sistemas abiertos que permitan la interacción entre equipos y dispositivos de diversa índole. En este contexto se plantea la definición del Protocolo de Teleoperación que permita la interconexión entre dispositivos maestros y esclavos de distinta tipología, pudiéndose comunicar bilateralmente entre sí y utilizar distintos algoritmos de control según la tarea a desempeñar. Este protocolo y su interconectividad se han puesto a prueba en la Plataforma Abierta de Teleoperación (P.A.T.) que se ha desarrollado e integrado en la ETSII UPM como una herramienta que permita probar, validar y realizar experimentos de telerrobótica. Actualmente, este Protocolo de Teleoperación se ha propuesto a través de AENOR al grupo ISO de Telerobotics como una solución válida al problema existente y se encuentra bajo revisión. Con el diseño de dicho protocolo se ha conseguido enlazar maestro y esclavo, sin embargo con los niveles de radiación tan altos que hay en ITER la electrónica del controlador no puede entrar dentro del tokamak. Por ello se propone que a través de una mínima electrónica convenientemente protegida se puedan multiplexar las señales de control que van a través del cableado umbilical desde el controlador hasta la base del robot. En este ejercicio teórico se demuestra la utilidad y viabilidad de utilizar este tipo de solución para reducir el volumen y peso del cableado umbilical en cifras aproximadas de un 90%, para ello hay que desarrollar una electrónica específica y con certificación RadHard para soportar los enormes niveles de radiación de ITER. Para este manipulador de tipo genérico y con ayuda de la Plataforma Abierta de Teleoperación, se ha desarrollado un algoritmo que mediante un sensor de fuerza/par y una IMU colocados en la muñeca del robot, y convenientemente protegidos ante la radiación, permiten calcular las fuerzas e inercias que produce la carga, esto es necesario para poder transmitirle al operador unas fuerzas escaladas, y que pueda sentir la carga que manipula, y no otras fuerzas que puedan influir en el esclavo remoto, como ocurre con otras técnicas de estimación de fuerzas. Como el blindaje de los sensores no debe ser grande ni pesado, habrá que destinar este tipo de tecnología a las tareas de mantenimiento de las paradas programadas de ITER, que es cuando los niveles de radiación están en sus valores mínimos. Por otro lado para que el operador sienta lo más fielmente posible la fuerza de carga se ha desarrollado una electrónica que mediante el control en corriente de los motores permita realizar un control en fuerza a partir de la caracterización de los motores del maestro. Además para aumentar la percepción del operador se han realizado unos experimentos que demuestran que al aplicar estímulos multimodales (visuales, auditivos y hápticos) aumenta su inmersión y el rendimiento en la consecución de la tarea puesto que influyen directamente en su capacidad de respuesta. Finalmente, y en referencia a la realimentación visual del operador, en ITER se trabaja con cámaras situadas en localizaciones estratégicas, si bien el humano cuando manipula objetos hace uso de su visión binocular cambiando constantemente el punto de vista adecuándose a las necesidades visuales de cada momento durante el desarrollo de la tarea. Por ello, se ha realizado una reconstrucción tridimensional del espacio de la tarea a partir de una cámara-sensor RGB-D, lo cual nos permite obtener un punto de vista binocular virtual móvil a partir de una cámara situada en un punto fijo que se puede proyectar en un dispositivo de visualización 3D para que el operador pueda variar el punto de vista estereoscópico según sus preferencias. La correcta integración de estas tecnologías para la interacción hombre-robot en la P.A.T. ha permitido validar mediante pruebas y experimentos para verificar su utilidad en la aplicación práctica de la telemanipulación con alto grado de escalado en entornos nucleares de fusión. Abstract This thesis focuses on developing technologies for human-robot interaction in nuclear fusion environments. The main problem of nuclear fusion sector resides in such extreme environmental conditions existing in the hot-cell, leading to very restrictive requirements for equipment in order to deal with these high levels of radiation, magnetism, ultravacuum, temperature... Since it is not feasible to carry out tasks directly by humans, we must use remote handling devices for accomplishing operation and maintenance processes. In ITER facilities it is mandatory to have a controlled environment of extreme safety and security with validated standards. The definition and use of protocols is essential to govern its operation. Focusing on Remote Handling with some degree of escalation, protocols must be defined for open systems to allow interaction among different kind of equipment and several multifunctional devices. In this context, a Teleoperation Protocol definition enables interconnection between master and slave devices from different typologies, being able to communicate bilaterally one each other and using different control algorithms depending on the task to perform. This protocol and its interconnectivity have been tested in the Teleoperation Open Platform (T.O.P.) that has been developed and integrated in the ETSII UPM as a tool to test, validate and conduct experiments in Telerobotics. Currently, this protocol has been proposed for Teleoperation through AENOR to the ISO Telerobotics group as a valid solution to the existing problem, and it is under review. Master and slave connection has been achieved with this protocol design, however with such high radiation levels in ITER, the controller electronics cannot enter inside the tokamak. Therefore it is proposed a multiplexed electronic board, that through suitable and RadHard protection processes, to transmit control signals through an umbilical cable from the controller to the robot base. In this theoretical exercise the utility and feasibility of using this type of solution reduce the volume and weight of the umbilical wiring approximate 90% less, although it is necessary to develop specific electronic hardware and validate in RadHard qualifications in order to handle huge levels of ITER radiation. Using generic manipulators does not allow to implement regular sensors for force feedback in ITER conditions. In this line of research, an algorithm to calculate the forces and inertia produced by the load has been developed using a force/torque sensor and IMU, both conveniently protected against radiation and placed on the robot wrist. Scaled forces should be transmitted to the operator, feeling load forces but not other undesirable forces in slave system as those resulting from other force estimation techniques. Since shielding of the sensors should not be large and heavy, it will be necessary to allocate this type of technology for programmed maintenance periods of ITER, when radiation levels are at their lowest levels. Moreover, the operator perception needs to feel load forces as accurate as possible, so some current control electronics were developed to perform a force control of master joint motors going through a correct motor characterization. In addition to increase the perception of the operator, some experiments were conducted to demonstrate applying multimodal stimuli (visual, auditory and haptic) increases immersion and performance in achieving the task since it is directly correlated with response time. Finally, referring to the visual feedback to the operator in ITER, it is usual to work with 2D cameras in strategic locations, while humans use binocular vision in direct object manipulation, constantly changing the point of view adapting it to the visual needs for performing manipulation during task procedures. In this line a three-dimensional reconstruction of non-structured scenarios has been developed using RGB-D sensor instead of cameras in the remote environment. Thus a mobile virtual binocular point of view could be generated from a camera at a fixed point, projecting stereoscopic images in 3D display device according to operator preferences. The successful integration of these technologies for human-robot interaction in the T.O.P., and validating them through tests and experiments, verify its usefulness in practical application of high scaling remote handling at nuclear fusion environments.

Assessing the accessibility impact of transport policy by a land-use and transport interaction model - The case of Madrid

Accessibility is an essential concept widely used to evaluate the impact of transport and land-use strategies in urban planning and policy making. Accessibility is typically evaluated by using separately a transport model or a land-use model. This paper embeds two accessibility indicators (i.e., potential and adaptive accessibility) in a land use and transport interaction (LUTI) model in order to assess transport policies implementation. The first aim is to define the adaptive accessibility, considering the competition factor at territorial level (e.g. workplaces and workers). The second aim is to identify the optimal implementation scenario of policy measures using potential and adaptive accessibility indicators. The analysis of the results in terms of social welfare and accessibility changes closes the paper. Two transport policy measures are applied in Madrid region: a cordon toll and increase bus frequency. They have been simulated through the MARS model (Metropolitan Activity Relocation Simulator, i.e. LUTI model). An optimisation procedure is performed by MARS for maximizing the value of the objective function in order to find the optimal policy implementation (first best). Both policy measures are evaluated in terms of accessibility. Results show that the introduction of the accessibility indicators (potential and adaptive) influence the optimal value of the toll price and bus frequency level, generating different results in terms of social welfare. Mapping the difference between potential and adaptive accessibility indicator shows that the main changes occur in areas where there is a strong competition among different land-use opportunities.

Alloantigen-induced unresponsiveness in cord blood T lymphocytes is associated with defective activation of Ras

Human umbilical cord blood T lymphocytes (CBTL) respond to primary allostimulation but they do not proliferate upon rechallenge with alloantigen. Using PKH-26-labeled cells created a proliferative block that was observed only in CBTL that have divided during primary stimulation (PKH-26dim) but not in unstimulated (PKH-26bright) CBTL. CBTL’s secondary unresponsiveness resembles anergy and can be overcome by treatment with phorbol myristate acetate (PMA) and ionomycin or by high doses (50–100 units/ml) of interleukin 2. Addition of interleukin 2 to the primary cultures does not prevent the induction of secondary unresponsiveness. Defective Ras activation is detected in PKH-26dim CBTL during secondary response to alloantigen or after antibody-mediated T cell receptor stimulation whereas Ras is activated and proliferation is induced in CBTL during primary alloantigenic stimulation. Upon stimulation with PMA plus ionomycin, PMA plus alloantigen, but not alloantigen plus ionomycin, Ras is activated in PKH-26dim CBTL, and the block in proliferation is overcome. Correction of PKH-26dim CBTL’s proliferative defect correlates with PMA-induced Ras activation, suggesting a defect in the signaling pathway leading to Ras. Ras-independent signals, necessary but not sufficient to induce PKH-26dim CBTL proliferation, are provided by alloantigen exposure, as evident by the ability of PMA plus alloantigen but not PMA alone to overcome the proliferative block. Functional signal transduction through CD28 in PKH-26dim CBTL is supported by detectable CD28-mediated PI-3 kinase activation after PKH-26dim CBTL’s exposure to alloantigen or CD28 cross-linking. These results suggest that defective activation of Ras plays a key role in PKH-26dim CBTL’s secondary unresponsiveness and point to a defect along the T cell receptor rather than the CD28 signaling pathway.

Immunotargeting of liposomes to activated vascular endothelial cells: A strategy for site-selective delivery in the cardiovascular system

Endothelial-selective delivery of therapeutic agents, such as drugs or genes, would provide a useful tool for modifying vascular function in various disease states. A potential molecular target for such delivery is E-selectin, an endothelial-specific cell surface molecule expressed at sites of activation in vivo and inducible in cultured human umbilical vein endothelial cells (HUVEC) by treatment with cytokines such as recombinant human interleukin 1β (IL-1β). Liposomes of various types (classical, sterically stabilized, cationic, pH-sensitive), each conjugated with mAb H18/7, a murine monoclonal antibody that recognizes the extracellular domain of E-selectin, bound selectively and specifically to IL-1β-activated HUVEC at levels up to 275-fold higher than to unactivated HUVEC. E-selectin-targeted immunoliposomes appeared in acidic, perinuclear vesicles 2–4 hr after binding to the cell surface, consistent with internalization via the endosome/lysosome pathway. Activated HUVEC incubated with E-selectin-targeted immunoliposomes, loaded with the cytotoxic agent doxorubicin, exhibited significantly decreased cell survival, whereas unactivated HUVEC were unaffected by such treatment. These results demonstrate the feasibility of exploiting cell surface activation markers for the endothelial-selective delivery of biologically active agents via immunoliposomes. Application of this targeting approach in vivo may lead to novel therapeutic strategies in the treatment of cardiovascular disease.

Down-regulation of the expression of endothelial NO synthase is likely to contribute to glucocorticoid-mediated hypertension

Hypertension is a side effect of systemically administered glucocorticoids, but the underlying molecular mechanism remains poorly understood. Ingestion of dexamethasone by rats telemetrically instrumented increased blood pressure progressively over 7 days. Plasma concentrations of Na+ and K+ and urinary Na+ and K+ excretion remained constant, excluding a mineralocorticoid-mediated mechanism. Plasma NO2−/NO3− (the oxidation products of NO) decreased to 40%, and the expression of endothelial NO synthase (NOS III) was found down-regulated in the aorta and several other tissues of glucocorticoid-treated rats. The vasodilator response of resistance arterioles was tested by intravital microscopy in the mouse dorsal skinfold chamber model. Dexamethasone treatment significantly attenuated the relaxation to the endothelium-dependent vasodilator acetylcholine, but not to the endothelium-independent vasodilator S-nitroso-N-acetyl-d,l-penicillamine. Incubation of human umbilical vein endothelial cells, EA.hy 926 cells, or bovine aortic endothelial cells with several glucocorticoids reduced NOS III mRNA and protein expression to 60–70% of control, an effect that was prevented by the glucocorticoid receptor antagonist mifepristone. Glucocorticoids decreased NOS III mRNA stability and reduced the activity of the human NOS III promoter (3.5 kilobases) to ≈70% by decreasing the binding activity of the essential transcription factor GATA. The expressional down-regulation of endothelial NOS III may contribute to the hypertension caused by glucocorticoids.

Tumor necrosis factor-α induces adhesion molecule expression through the sphingosine kinase pathway

The signaling pathways that couple tumor necrosis factor-α (TNFα) receptors to functional, especially inflammatory, responses have remained elusive. We report here that TNFα induces endothelial cell activation, as measured by the expression of adhesion protein E-selectin and vascular adhesion molecule-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with TNFα resulted in a rapid SKase activation and sphingosine 1-phosphate (S1P) generation. S1P, but not ceramide or sphingosine, was a potent dose-dependent stimulator of adhesion protein expression. S1P was able to mimic the effect of TNFα on endothelial cells leading to extracellular signal-regulated kinases and NF-κB activation, whereas ceramide or sphingosine was not. Furthermore, N,N-dimethylsphingosine, an inhibitor of SKase, profoundly inhibited TNFα-induced extracellular signal-regulated kinases and NF-κB activation and adhesion protein expression. Thus we demonstrate that the SKase pathway through the generation of S1P is critically involved in mediating TNFα-induced endothelial cell activation.