Contribution to integration and coordination mechanisms for mobile distributed services in publish/subscribe networks

Esta tesis doctoral está enmarcada en dos diferentes pero complementarias áreas de investigación: las redes de Publicación/Subscripción y los servicios móviles distribuidos. Con el paso de los años las redes de Publicación/Subscripción han ido ofreciendo el soporte de comunicaciones desacopladas y ligeras que a su vez, han mejorado la distribución de la información en muchos escenarios de aplicación como lo son la ejecución de servicios distribuidos en entornos fijos. Los servicios móviles distribuidos han de ser desplegados en ambientes inalámbricos en donde los dispositivos móviles deben confiar en las mismas características que las redes de Publicación/Subscripción han estado ofreciendo a sus contrapartes fijos. En este contexto, una de las líneas de investigación pendientes consiste en cómo tomar ventaja de estas características, y cómo avanzar hacia nuevas soluciones no existentes con el fin de mejorar la integración entre los dispositivos fijos y móviles, y la ejecución de los servicios móviles distribuidos. En esta tesis doctoral se pretende avanzar en los mecanismos de integración y coordinación de los servicios móviles distribuidos en el contexto de las redes de Publicación/Subscripción. Los objetivos específicos de esta disertación están enfocados en lograr la integración de los sistemas de Publicación/Suscripción fijos y móviles, y la pro-visión de una versión de red de Publicación/Subscripción específica y uniforme que cuente con mecanismos de coordinación que mejoren la ejecución de los servicios móviles distribuidos. Los resultados de esta tesis doctoral están enmarcados en una versión específica de una red de Publicación/Subscripción que integra brokers fijos y móviles, y permite una coordinación totalmente desacoplada y mejorada entre dispositivos móviles que ejecutan fragmentos de servicios. Las contribuciones específicas son las siguientes: una nueva arquitectura de broker móvil que he llamado Rendezvous Mobile broker, un modelo abstracto de servicios móviles distribuidos coordinados sobre una red de Publicación/Subscripción, mejoras en los mecanismos de enrutamiento epidémicos para diseminar eventos de control producidos por fragmentos de servicios, una solución para soportar servicios altamente fragmentados y geográficamente dispersos, y finalmente una solución de interconexión entre dos dominios de red basados en Publicación/Subscripción: una red basada en el protocolo PubSubHubbub y otro en una red basada en el Publish/Subscribe Internet Routing Paradigm (PSIRP). Los experimentos llevados a cabo confirman que la versión específica de red de Pu-blicación/Subscripción propuesta incrementa el rendimiento de la red en términos de tiempo de espera entre nodos finales, permite una coordinación de los servicios móviles distribuidos más resistente a interrupciones y un mejor uso de los recursos de red, y finalmente logra exitosamente, con variaciones mínimas en el rendimiento de las comunicaciones, la interconexión entre estos dominios de Publicación/Subscripción diferentes. ABSTRACT This dissertation is made up of two different but complementary research areas: Publish/Subscribe networks and mobile distributed services. Over the years, Publish/Subscribe networks have been offering the lightweight and decoupled communication characteristics to improve the information distribution in several application domains such as the execution of distributed services. Mobile distributed services are set to be deployed in wireless environments where mobile devices must rely on the same features Publish/Subscribe networks can offer; so one of the pending research directions consists of how to take advantage of these features and further advance to-wards new un-existing solutions that enhance the integration between mobile and fixed systems and the execution of mobile distributed services. This dissertation seeks to advance the integration and coordination mechanisms of mobile distributed services in the context of Publish/Subscribe networks. The specific objectives aim to enable the integration of mobile and fixed Publish/Subscribe systems and provide a uniform and specific version of a Publish/Subscribe network with new coordination mechanisms that improve the execution of mobile distributed services. The results of this dissertation are enclosed in one specific version of a Publish/Subscribe network that integrates mobile and fixed brokers and coordinates the execution of mobile distributed services. These specific contributions are: a new architecture of a mobile broker I called Rendezvous Mobile Broker, an abstract model for coordinating mobile distributed services executions using a Publish/Subscribe net-work, new gossip routing solutions to disseminate events of services, mechanisms to support highly partitioned and geographically dispersed services and finally, an inter-networking solution between two Publish/Subscribe domains: a PubSubHubbub-based network and the Publish/Subscribe Internet Routing Paradigm (PSIRP)-based network. The experimental efforts confirm that the specific version of the Publish/Subscribe proposed in this dissertation improves the performance of the overall network in terms of end-to-end delay, enables a more resilience execution of mobile distributed services, a better usage of the existing network resources, and finally successfully achieves, with minor variations in the network performance, the internetworking between two different Publish/Subscribe domains.

Optimization of the Integrated Guidance and Control for a Dual Aerodynamic Control Missile

Las futuras misiones para misiles aire-aire operando dentro de la atmósfera requieren la interceptación de blancos a mayores velocidades y más maniobrables, incluyendo los esperados vehículos aéreos de combate no tripulados. La intercepción tiene que lograrse desde cualquier ángulo de lanzamiento. Una de las principales discusiones en la tecnología de misiles en la actualidad es cómo satisfacer estos nuevos requisitos incrementando la capacidad de maniobra del misil y en paralelo, a través de mejoras en los métodos de guiado y control modernos. Esta Tesis aborda estos dos objetivos simultáneamente, al proponer un diseño integrando el guiado y el control de vuelo (autopiloto) y aplicarlo a misiles con control aerodinámico simultáneo en canard y cola. Un primer avance de los resultados obtenidos ha sido publicado recientemente en el Journal of Aerospace Engineering, en Abril de 2015, [Ibarrondo y Sanz-Aranguez, 2015]. El valor del diseño integrado obtenido es que permite al misil cumplir con los requisitos operacionales mencionados empleando únicamente control aerodinámico. El diseño propuesto se compara favorablemente con esquemas más tradicionales, consiguiendo menores distancias de paso al blanco y necesitando de menores esfuerzos de control incluso en presencia de ruidos. En esta Tesis se demostrará cómo la introducción del doble mando, donde tanto el canard como las aletas de cola son móviles, puede mejorar las actuaciones de un misil existente. Comparado con un misil con control en cola, el doble control requiere sólo introducir dos servos adicionales para accionar los canards también en guiñada y cabeceo. La sección de cola será responsable de controlar el misil en balanceo mediante deflexiones diferenciales de los controles. En el caso del doble mando, la complicación añadida es que los vórtices desprendidos de los canards se propagan corriente abajo y pueden incidir sobre las superficies de cola, alterando sus características de control. Como un primer aporte, se ha desarrollado un modelo analítico completo para la aerodinámica no lineal de un misil con doble control, incluyendo la caracterización de este efecto de acoplamiento aerodinámico. Hay dos modos de funcionamiento en picado y guiñada para un misil de doble mando: ”desviación” y ”opuesto”. En modo ”desviación”, los controles actúan en la misma dirección, generando un cambio inmediato en la sustentación y produciendo un movimiento de translación en el misil. La respuesta es rápida, pero en el modo ”desviación” los misiles con doble control pueden tener dificultades para alcanzar grandes ángulos de ataque y altas aceleraciones laterales. Cuando los controles actúan en direcciones opuestas, el misil rota y el ángulo de ataque del fuselaje se incrementa para generar mayores aceleraciones en estado estacionario, aunque el tiempo de respuesta es mayor. Con el modelo aerodinámico completo, es posible obtener una parametrización dependiente de los estados de la dinámica de corto periodo del misil. Debido al efecto de acoplamiento entre los controles, la respuesta en bucle abierto no depende linealmente de los controles. El autopiloto se optimiza para obtener la maniobra requerida por la ley de guiado sin exceder ninguno de los límites aerodinámicos o mecánicos del misil. Una segunda contribución de la tesis es el desarrollo de un autopiloto con múltiples entradas de control y que integra la aerodinámica no lineal, controlando los tres canales de picado, guiñada y cabeceo de forma simultánea. Las ganancias del autopiloto dependen de los estados del misil y se calculan a cada paso de integración mediante la resolución de una ecuación de Riccati de orden 21x21. Las ganancias obtenidas son sub-óptimas, debido a que una solución completa de la ecuación de Hamilton-Jacobi-Bellman no puede obtenerse de manera práctica, y se asumen ciertas simplificaciones. Se incorpora asimismo un mecanismo que permite acelerar la respuesta en caso necesario. Como parte del autopiloto, se define una estrategia para repartir el esfuerzo de control entre el canard y la cola. Esto se consigue mediante un controlador aumentado situado antes del bucle de optimización, que minimiza el esfuerzo total de control para maniobrar. Esta ley de alimentación directa mantiene al misil cerca de sus condiciones de equilibrio, garantizando una respuesta transitoria adecuada. El controlador no lineal elimina la respuesta de fase no-mínima característica de la cola. En esta Tesis se consideran dos diseños para el guiado y control, el control en Doble-Lazo y el control Integrado. En la aproximación de Doble-Lazo, el autopiloto se sitúa dentro de un bucle interior y se diseña independientemente del guiado, que conforma el bucle más exterior del control. Esta estructura asume que existe separación espectral entre los dos, esto es, que los tiempos de respuesta del autopiloto son mucho mayores que los tiempos característicos del guiado. En el estudio se combina el autopiloto desarrollado con una ley de guiado óptimo. Los resultados obtenidos demuestran que se consiguen aumentos muy importantes en las actuaciones frente a misiles con control canard o control en cola, y que la interceptación, cuando se lanza cerca del curso de colisión, se consigue desde cualquier ángulo alrededor del blanco. Para el misil de doble mando, la estrategia óptima resulta en utilizar el modo de control opuesto en la aproximación al blanco y utilizar el modo de desviación justo antes del impacto. Sin embargo la lógica de doble bucle no consigue el impacto cuando hay desviaciones importantes con respecto al curso de colisión. Una de las razones es que parte de la demanda de guiado se pierde, ya que el misil solo es capaz de modificar su aceleración lateral, y no tiene control sobre su aceleración axial, a no ser que incorpore un motor de empuje regulable. La hipótesis de separación mencionada, y que constituye la base del Doble-Bucle, puede no ser aplicable cuando la dinámica del misil es muy alta en las proximidades del blanco. Si se combinan el guiado y el autopiloto en un único bucle, la información de los estados del misil está disponible para el cálculo de la ley de guiado, y puede calcularse la estrategia optima de guiado considerando las capacidades y la actitud del misil. Una tercera contribución de la Tesis es la resolución de este segundo diseño, la integración no lineal del guiado y del autopiloto (IGA) para el misil de doble control. Aproximaciones anteriores en la literatura han planteado este sistema en ejes cuerpo, resultando en un sistema muy inestable debido al bajo amortiguamiento del misil en cabeceo y guiñada. Las simplificaciones que se tomaron también causan que el misil se deslice alrededor del blanco y no consiga la intercepción. En nuestra aproximación el problema se plantea en ejes inerciales y se recurre a la dinámica de los cuaterniones, eliminado estos inconvenientes. No se limita a la dinámica de corto periodo del misil, porque se construye incluyendo de modo explícito la velocidad dentro del bucle de optimización. La formulación resultante en el IGA es independiente de la maniobra del blanco, que sin embargo se ha de incluir en el cálculo del modelo en Doble-bucle. Un típico inconveniente de los sistemas integrados con controlador proporcional, es el problema de las escalas. Los errores de guiado dominan sobre los errores de posición del misil y saturan el controlador, provocando la pérdida del misil. Este problema se ha tratado aquí con un controlador aumentado previo al bucle de optimización, que define un estado de equilibrio local para el sistema integrado, que pasa a actuar como un regulador. Los criterios de actuaciones para el IGA son los mismos que para el sistema de Doble-Bucle. Sin embargo el problema matemático resultante es muy complejo. El problema óptimo para tiempo finito resulta en una ecuación diferencial de Riccati con condiciones terminales, que no puede resolverse. Mediante un cambio de variable y la introducción de una matriz de transición, este problema se transforma en una ecuación diferencial de Lyapunov que puede resolverse mediante métodos numéricos. La solución resultante solo es aplicable en un entorno cercano del blanco. Cuando la distancia entre misil y blanco es mayor, se desarrolla una solución aproximada basada en la solución de una ecuación algebraica de Riccati para cada paso de integración. Los resultados que se han obtenido demuestran, a través de análisis numéricos en distintos escenarios, que la solución integrada es mejor que el sistema de Doble-Bucle. Las trayectorias resultantes son muy distintas. El IGA preserva el guiado del misil y consigue maximizar el uso de la propulsión, consiguiendo la interceptación del blanco en menores tiempos de vuelo. El sistema es capaz de lograr el impacto donde el Doble-Bucle falla, y además requiere un orden menos de magnitud en la cantidad de cálculos necesarios. El efecto de los ruidos radar, datos discretos y errores del radomo se investigan. El IGA es más robusto, resultando menos afectado por perturbaciones que el Doble- Bucle, especialmente porque el núcleo de optimización en el IGA es independiente de la maniobra del blanco. La estimación de la maniobra del blanco es siempre imprecisa y contaminada por ruido, y degrada la precisión de la solución de Doble-Bucle. Finalmente, como una cuarta contribución, se demuestra que el misil con guiado IGA es capaz de realizar una maniobra de defensa contra un blanco que ataque por su cola, sólo con control aerodinámico. Las trayectorias estudiadas consideran una fase pre-programada de alta velocidad de giro, manteniendo siempre el misil dentro de su envuelta de vuelo. Este procedimiento no necesita recurrir a soluciones técnicamente más complejas como el control vectorial del empuje o control por chorro para ejecutar esta maniobra. En todas las demostraciones matemáticas se utiliza el producto de Kronecker como una herramienta practica para manejar las parametrizaciones dependientes de variables, que resultan en matrices de grandes dimensiones. ABSTRACT Future missions for air to air endo-atmospheric missiles require the interception of targets with higher speeds and more maneuverable, including forthcoming unmanned supersonic combat vehicles. The interception will need to be achieved from any angle and off-boresight launch conditions. One of the most significant discussions in missile technology today is how to satisfy these new operational requirements by increasing missile maneuvering capabilities and in parallel, through the development of more advanced guidance and control methods. This Thesis addresses these two objectives by proposing a novel optimal integrated guidance and autopilot design scheme, applicable to more maneuverable missiles with forward and rearward aerodynamic controls. A first insight of these results have been recently published in the Journal of Aerospace Engineering in April 2015, [Ibarrondo and Sanz-Aránguez, 2015]. The value of this integrated solution is that it allows the missile to comply with the aforementioned requirements only by applying aerodynamic control. The proposed design is compared against more traditional guidance and control approaches with positive results, achieving reduced control efforts and lower miss distances with the integrated logic even in the presence of noises. In this Thesis it will be demonstrated how the dual control missile, where canard and tail fins are both movable, can enhance the capabilities of an existing missile airframe. Compared to a tail missile, dual control only requires two additional servos to actuate the canards in pitch and yaw. The tail section will be responsible to maintain the missile stabilized in roll, like in a classic tail missile. The additional complexity is that the vortices shed from the canard propagate downstream where they interact with the tail surfaces, altering the tail expected control characteristics. These aerodynamic phenomena must be properly described, as a preliminary step, with high enough precision for advanced guidance and control studies. As a first contribution we have developed a full analytical model of the nonlinear aerodynamics of a missile with dual control, including the characterization of this cross-control coupling effect. This development has been produced from a theoretical model validated with reliable practical data obtained from wind tunnel experiments available in the scientific literature, complement with computer fluid dynamics and semi-experimental methods. There are two modes of operating a missile with forward and rear controls, ”divert” and ”opposite” modes. In divert mode, controls are deflected in the same direction, generating an increment in direct lift and missile translation. Response is fast, but in this mode, dual control missiles may have difficulties in achieving large angles of attack and high level of lateral accelerations. When controls are deflected in opposite directions (opposite mode) the missile airframe rotates and the body angle of attack is increased to generate greater accelerations in steady-state, although the response time is larger. With the aero-model, a state dependent parametrization of the dual control missile short term dynamics can be obtained. Due to the cross-coupling effect, the open loop dynamics for the dual control missile is not linearly dependent of the fin positions. The short term missile dynamics are blended with the servo system to obtain an extended autopilot model, where the response is linear with the control fins turning rates, that will be the control variables. The flight control loop is optimized to achieve the maneuver required by the guidance law without exceeding any of the missile aerodynamic or mechanical limitations. The specific aero-limitations and relevant performance indicators for the dual control are set as part of the analysis. A second contribution of this Thesis is the development of a step-tracking multi-input autopilot that integrates non-linear aerodynamics. The designed dual control missile autopilot is a full three dimensional autopilot, where roll, pitch and yaw are integrated, calculating command inputs simultaneously. The autopilot control gains are state dependent, and calculated at each integration step solving a matrix Riccati equation of order 21x21. The resulting gains are sub-optimal as a full solution for the Hamilton-Jacobi-Bellman equation cannot be resolved in practical terms and some simplifications are taken. Acceleration mechanisms with an λ-shift is incorporated in the design. As part of the autopilot, a strategy is defined for proper allocation of control effort between canard and tail channels. This is achieved with an augmented feed forward controller that minimizes the total control effort of the missile to maneuver. The feedforward law also maintains the missile near trim conditions, obtaining a well manner response of the missile. The nonlinear controller proves to eliminate the non-minimum phase effect of the tail. Two guidance and control designs have been considered in this Thesis: the Two- Loop and the Integrated approaches. In the Two-Loop approach, the autopilot is placed in an inner loop and designed separately from an outer guidance loop. This structure assumes that spectral separation holds, meaning that the autopilot response times are much higher than the guidance command updates. The developed nonlinear autopilot is linked in the study to an optimal guidance law. Simulations are carried on launching close to collision course against supersonic and highly maneuver targets. Results demonstrate a large boost in performance provided by the dual control versus more traditional canard and tail missiles, where interception with the dual control close to collision course is achieved form 365deg all around the target. It is shown that for the dual control missile the optimal flight strategy results in using opposite control in its approach to target and quick corrections with divert just before impact. However the Two-Loop logic fails to achieve target interception when there are large deviations initially from collision course. One of the reasons is that part of the guidance command is not followed, because the missile is not able to control its axial acceleration without a throttleable engine. Also the separation hypothesis may not be applicable for a high dynamic vehicle like a dual control missile approaching a maneuvering target. If the guidance and autopilot are combined into a single loop, the guidance law will have information of the missile states and could calculate the most optimal approach to the target considering the actual capabilities and attitude of the missile. A third contribution of this Thesis is the resolution of the mentioned second design, the non-linear integrated guidance and autopilot (IGA) problem for the dual control missile. Previous approaches in the literature have posed the problem in body axes, resulting in high unstable behavior due to the low damping of the missile, and have also caused the missile to slide around the target and not actually hitting it. The IGA system is posed here in inertial axes and quaternion dynamics, eliminating these inconveniences. It is not restricted to the missile short term dynamic, and we have explicitly included the missile speed as a state variable. The IGA formulation is also independent of the target maneuver model that is explicitly included in the Two-loop optimal guidance law model. A typical problem of the integrated systems with a proportional control law is the problem of scales. The guidance errors are larger than missile state errors during most of the flight and result in high gains, control saturation and loss of control. It has been addressed here with an integrated feedforward controller that defines a local equilibrium state at each flight point and the controller acts as a regulator to minimize the IGA states excursions versus the defined feedforward state. The performance criteria for the IGA are the same as in the Two-Loop case. However the resulting optimization problem is mathematically very complex. The optimal problem in a finite-time horizon results in an irresoluble state dependent differential Riccati equation with terminal conditions. With a change of variable and the introduction of a transition matrix, the equation is transformed into a time differential Lyapunov equation that can be solved with known numerical methods in real time. This solution results range limited, and applicable when the missile is in a close neighborhood of the target. For larger ranges, an approximate solution is used, obtained from solution of an algebraic matrix Riccati equation at each integration step. The results obtained show, by mean of several comparative numerical tests in diverse homing scenarios, than the integrated approach is a better solution that the Two- Loop scheme. Trajectories obtained are very different in the two cases. The IGA fully preserves the guidance command and it is able to maximize the utilization of the missile propulsion system, achieving interception with lower miss distances and in lower flight times. The IGA can achieve interception against off-boresight targets where the Two- Loop was not able to success. As an additional advantage, the IGA also requires one order of magnitude less calculations than the Two-Loop solution. The effects of radar noises, discrete radar data and radome errors are investigated. IGA solution is robust, and less affected by radar than the Two-Loop, especially because the target maneuvers are not part of the IGA core optimization loop. Estimation of target acceleration is always imprecise and noisy and degrade the performance of the two-Loop solution. The IGA trajectories are such that minimize the impact of radome errors in the guidance loop. Finally, as a fourth contribution, it is demonstrated that the missile with IGA guidance is capable of performing a defense against attacks from its rear hemisphere, as a tail attack, only with aerodynamic control. The studied trajectories have a preprogrammed high rate turn maneuver, maintaining the missile within its controllable envelope. This solution does not recur to more complex features in service today, like vector control of the missile thrust or side thrusters. In all the mathematical treatments and demonstrations, the Kronecker product has been introduced as a practical tool to handle the state dependent parametrizations that have resulted in very high order matrix equations.

Improvement of root architecture under abiotic stress through control of auxin homeostasis in Arabidopsis and Brassica crops

Auxin plays an important role in many aspects of plant development including stress responses. Here we briefly summarize how auxin is involved in salt stress, drought (i.e. mostly osmotic stress), waterlogging and nutrient deficiency in Brassica plants. In addition, some mechanisms to control auxin levels and signaling in relation to root formation (under stress) will be reviewed. Molecular studies are mainly described for the model plant Arabidopsis thaliana, but we also like to demonstrate how this knowledge can be transferred to agriculturally important Brassica species, such as Brassica rapa, Brassica napus and Brassica campestris. Moreover, beneficial fungi could play a role in the adaptation response of Brassica roots to abiotic stresses. Therefore, the possible influence of Piriformospora indica will also be covered since the growth promoting response of plants colonized by P. indica is also linked to plant hormones, among them auxin.

Control of fertilization-independent endosperm development by the MEDEA polycomb gene in Arabidopsis

Higher plant reproduction is unique because two cells are fertilized in the haploid female gametophyte. Egg and sperm nuclei fuse to form the embryo. A second sperm nucleus fuses with the central cell nucleus that replicates to generate the endosperm, a tissue that supports embryo development. To understand mechanisms that initiate reproduction, we isolated a mutation in Arabidopsis, f644, that allows for replication of the central cell and subsequent endosperm development without fertilization. When mutant f644 egg and central cells are fertilized by wild-type sperm, embryo development is inhibited, and endosperm is overproduced. By using a map-based strategy, we cloned and sequenced the F644 gene and showed that it encodes a SET-domain polycomb protein. Subsequently, we found that F644 is identical to MEDEA (MEA), a gene whose maternal-derived allele is required for embryogenesis [Grossniklaus, U., Vielle-Calzada, J.-P., Hoeppner, M. A. & Gagliano, W. B. (1998) Science 280, 446–450]. Together, these results reveal functions for plant polycomb proteins in the suppression of central cell proliferation and endosperm development. We discuss models to explain how polycomb proteins function to suppress endosperm and promote embryo development.

A negative regulator mediates quorum-sensing control of exopolysaccharide production in Pantoea stewartii subsp. stewartii

Classical quorum-sensing (autoinduction) regulation, as exemplified by the lux system of Vibrio fischeri, requires N-acyl homoserine lactone (AHL) signals to stimulate cognate transcriptional activators for the cell density-dependent expression of specific target gene systems. For Pantoea stewartii subsp. stewartii, a bacterial pathogen of sweet corn and maize, the extracellular polysaccharide (EPS) stewartan is a major virulence factor, and its production is controlled by quorum sensing in a population density-dependent manner. Two genes, esaI and esaR, encode essential regulatory proteins for quorum sensing. EsaI is the AHL signal synthase, and EsaR is the cognate gene regulator. esaI, ΔesaR, and ΔesaI-esaR mutations were constructed to establish the regulatory role of EsaR. We report here that strains containing an esaR mutation produce high levels of EPS independently of cell density and in the absence of the AHL signal. Our data indicate that quorum-sensing regulation in P. s. subsp. stewartii, in contrast to most other described systems, uses EsaR to repress EPS synthesis at low cell density, and that derepression requires micromolar amounts of AHL. In addition, derepressed esaR strains, which synthesize EPS constitutively at low cell densities, were significantly less virulent than the wild-type parent. This finding suggests that quorum sensing in P. s. subsp. stewartii may be a mechanism to delay the expression of EPS during the early stages of infection so that it does not interfere with other mechanisms of pathogenesis.

The role of the synthetic enzyme GAD65 in the control of neuronal γ-aminobutyric acid release

We have studied GABAergic synaptic transmission in retinal ganglion cells and hippocampal pyramidal cells to determine, at a cellular level, what is the effect of the targeted disruption of the gene encoding the synthetic enzyme GAD65 on the synaptic release of γ-aminobutyric acid (GABA). Neither the size nor the frequency of GABA-mediated spontaneous inhibitory postsynaptic currents (IPSCs) were reduced in retina or hippocampus in GAD65−/− mice. However, the release of GABA during sustained synaptic activation was substantially reduced. In the retina both electrical- and K+-induced increases in IPSC frequency were depressed without a change in IPSC amplitude. In the hippocampus the transient increase in the probability of inhibitory transmitter release associated with posttetanic potentiation was absent in the GAD65−/− mice. These results indicate that during and immediately after sustained stimulation the increase in the probability of transmitter release is not maintained in GAD65−/− mice. Such a finding suggests a decrease in the size or refilling kinetics of the releasable pool of vesicles, and various mechanisms are discussed that could account for such a defect.

Mechanical stretch induces vascular permeability factor in human mesangial cells: Mechanisms of signal transduction

Hemodynamic abnormalities have been implicated in the pathogenesis of the increased glomerular permeability to protein of diabetic and other glomerulopathies. Vascular permeability factor (VPF) is one of the most powerful promoters of vascular permeability. We studied the effect of stretch on VPF production by human mesangial cells and the intracellular signaling pathways involved. The application of mechanical stretch (elongation 10%) for 6 h induced a 2.4-fold increase over control in the VPF mRNA level (P < 0.05). There was a corresponding 3-fold increase in VPF protein level by 12 h (P < 0.001), returning to the baseline by 24 h. Stretch-induced VPF secretion was partially prevented both by the protein kinase C (PKC) inhibitor H7 (50 μM: 72% inhibition, P < 0.05) and by pretreatment with phorbol ester (phorbol-12-myristate-13 acetate 10−7 M: 77% inhibition, P < 0.05). A variety of protein tyrosine kinase (PTK) inhibitors, genistein (20 μg/ml), herbimycin A (3.4 μM), and a specific pp60src peptide inhibitor (21 μM) also significantly reduced, but did not entirely prevent, stretch-induced VPF protein secretion (respectively 63%, 80%, and 75% inhibition; P < 0.05 for all). The combination of both PKC and PTK inhibition completely abolished the VPF response to mechanical stretch (100% inhibition, P < 0.05). Stretch induces VPF gene expression and protein secretion in human mesangial cells via PKC- and PTK-dependent mechanisms.

Torpor in mice is induced by both leptin-dependent and -independent mechanisms

We tested the effect of chronic leptin treatment on fasting-induced torpor in leptin-deficient A-ZIP/F-1 and ob/ob mice. A-ZIP/F-1 mice have virtually no white adipose tissue and low leptin levels, whereas ob/ob mice have an abundance of fat but no leptin. These two models allowed us to examine the roles of adipose tissue and leptin in the regulation of entry into torpor. Torpor is a short-term hibernation-like state that allows conservation of metabolic fuels. We first characterized the A-ZIP/F-1 animals, which have a 10-fold reduction in total body triglyceride stores. Upon fasting, A-ZIP/F-1 mice develop a lower metabolic rate and decreased plasma glucose, insulin, and triglyceride levels, with no increase in free fatty acids or β-hydroxybutyrate. Unlike control mice, by 24 hr of fasting, they have nearly exhausted their triglycerides and are catabolizing protein. To conserve energy supplies during fasting, A-ZIP/F-1 (but not control) mice entered deep torpor, with a minimum core body temperature of 24°C, 2°C above ambient. In ob/ob mice, fasting-induced torpor was completely reversed by leptin treatment. In contrast, neither leptin nor thyroid hormone prevented torpor in A-ZIP/F-1 mice. These data suggest that there are at least two signals for entry into torpor in mice, a low leptin level and another signal that is independent of leptin and thyroid hormone levels. Studying rodent torpor provides insight into human torpor-like states such as near drowning in cold water and induced hypothermia for surgery.

Mechanisms of β cell death in diabetes: A minor role for CD95

Insulin-dependent diabetes mellitus is an autoimmune disease, under polygenic control, manifested only when >90% of the insulin-producing β cells are destroyed. Although the disease is T cell mediated, the demise of the β cell results from a number of different insults from the immune system. It has been proposed that foremost amongst these effector mechanisms is CD95 ligand-induced β cell death. Using the nonobese diabetic lpr mouse as a model system, we have found, to the contrary, that CD95 plays only a minor role in the death of β cells. Islet grafts from nonobese diabetic mice that carry the lpr mutation and therefore lack CD95 were protected only marginally from immune attack when grafted into diabetic mice. An explanation to reconcile these differing results is provided.

Mechanisms Governing the Activation and Trafficking of Yeast G Protein-coupled Receptors

We have addressed the mechanisms governing the activation and trafficking of G protein-coupled receptors (GPCRs) by analyzing constitutively active mating pheromone receptors (Ste2p and Ste3p) of the yeast Saccharomyces cerevisiae. Substitution of the highly conserved proline residue in transmembrane segment VI of these receptors causes constitutive signaling. This proline residue may facilitate folding of GPCRs into native, inactive conformations, and/or mediate agonist-induced structural changes leading to G protein activation. Constitutive signaling by mutant receptors is suppressed upon coexpression with wild-type, but not G protein coupling-defective, receptors. Wild-type receptors may therefore sequester a limiting pool of G proteins; this apparent “precoupling” of receptors and G proteins could facilitate signal production at sites where cell surface projections form during mating partner discrimination. Finally, rather than being expressed mainly at the cell surface, constitutively active pheromone receptors accumulate in post-endoplasmic reticulum compartments. This is in contrast to other defective membrane proteins, which apparently are targeted by default to the vacuole. We suggest that the quality-control mechanism that retains receptors in post-endoplasmic reticulum compartments may normally allow wild-type receptors to fold into their native, fully inactive conformations before reaching the cell surface. This may ensure that receptors do not trigger a response in the absence of agonist.

Mechanisms of Human Mitochondrial DNA Maintenance: The Determining Role of Primary Sequence and Length over Function

Although the regulation of mitochondrial DNA (mtDNA) copy number is performed by nuclear-coded factors, very little is known about the mechanisms controlling this process. We attempted to introduce nonhuman ape mtDNA into human cells harboring either no mtDNA or mutated mtDNAs (partial deletion and tRNA gene point mutation). Unexpectedly, only cells containing no mtDNA could be repopulated with nonhuman ape mtDNA. Cells containing a defective human mtDNA did not incorporate or maintain ape mtDNA and therefore died under selection for oxidative phosphorylation function. On the other hand, foreign human mtDNA was readily incorporated and maintained in these cells. The suicidal preference for self-mtDNA showed that functional parameters associated with oxidative phosphorylation are less relevant to mtDNA maintenance and copy number control than recognition of mtDNA self-determinants. Non–self-mtDNA could not be maintained into cells with mtDNA even if no selection for oxidative phosphorylation was applied. The repopulation kinetics of several mtDNA forms after severe depletion by ethidium bromide treatment showed that replication and maintenance of mtDNA in human cells are highly dependent on molecular features, because partially deleted mtDNA molecules repopulated cells significantly faster than full-length mtDNA. Taken together, our results suggest that mtDNA copy number may be controlled by competition for limiting levels of trans-acting factors that recognize primarily mtDNA molecular features. In agreement with this hypothesis, marked variations in mtDNA levels did not affect the transcription of nuclear-coded factors involved in mtDNA replication.

Quality Control in the Secretory Pathway: The Role of Calreticulin, Calnexin and BiP in the Retention of Glycoproteins with C-Terminal Truncations

Unlike properly folded and assembled proteins, most misfolded and incompletely assembled proteins are retained in the endoplasmic reticulum of mammalian cells and degraded without transport to the Golgi complex. To analyze the mechanisms underlying this unique sorting process and its fidelity, the fate of C-terminally truncated fragments of influenza hemagglutinin was determined. An assortment of different fragments was generated by adding puromycin at low concentrations to influenza virus-infected tissue culture cells. Of the fragments generated, <2% was secreted, indicating that the system for detecting defects in newly synthesized proteins is quite stringent. The majority of secreted species corresponded to folding domains within the viral spike glycoprotein. The retained fragments acquired a partially folded structure with intrachain disulfide bonds and conformation-dependent antigenic epitopes. They associated with two lectin-like endoplasmic reticulum chaperones (calnexin and calreticulin) but not BiP/GRP78. Inhibition of the association with calnexin and calreticulin by the addition of castanospermine significantly increased fragment secretion. However, it also caused association with BiP/GRP78. These results indicated that the association with calnexin and calreticulin was involved in retaining the fragments. They also suggested that BiP/GRP78 could serve as a backup for calnexin and calreticulin in retaining the fragments. In summary, the results showed that the quality control system in the secretory pathway was efficient and sensitive to folding defects, and that it involved multiple interactions with endoplasmic reticulum chaperones.

Combinatorial roles for pRB, p107, and p130 in E2F-mediated cell cycle control

Numerous studies have implicated the pRB family of nuclear proteins in the control of cell cycle progression. Although over-expression experiments have revealed that each of these proteins, pRB, p107, and p130, can induce a G1 cell cycle arrest, mouse knockouts demonstrated distinct developmental requirements for these proteins, as well as partial functional redundancy between family members. To study the mechanism by which the closely related pRB family proteins contribute to cell cycle progression, we generated 3T3 fibroblasts derived from embryos that lack one or more of these proteins (pRB−/−, p107−/−, p130−/−, pRB−/−/p107−/−, pRB−/−/p130−/−, and p107−/−/p130−/−). By comparing the growth and cell cycle characteristics of these cells, we have observed clear differences in the manner in which they transit through the G1 and S phases as well as exit from the cell cycle. Deletion of Rb, or more than one of the family members, results in a shortening of G1 and a lengthening of S phase, as well as a reduction in growth factor requirements. In addition, the individual cell lines showed differential regulation of a subset of E2F-dependent gene promoters, as well as differences in cell cycle-dependent kinase activity. Taken together, these observations suggest that the closely related pRB family proteins affect cell cycle progression through distinct biochemical mechanisms and that their coordinated action may contribute to their diverse functions in various physiological settings.

Mechanism in the Sequential Control of Cell Morphology and S Phase Entry by Epidermal Growth Factor Involves Distinct MEK/ERK Activations

Cell shape plays a role in cell growth, differentiation, and death. Herein, we used the hepatocyte, a normal, highly differentiated cell characterized by a long G1 phase, to understand the mechanisms that link cell shape to growth. First, evidence was provided that the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) cascade is a key transduction pathway controlling the hepatocyte morphology. MEK2/ERK2 activation in early G1 phase did not lead to cell proliferation but induced cell shape spreading and demonstration was provided that this MAPK-dependent spreading was required for reaching G1/S transition and DNA replication. Moreover, epidermal growth factor (EGF) was found to control this morphogenic signal in addition to its mitogenic effect. Thus, blockade of cell spreading by cytochalasin D or PD98059 treatment resulted in inhibition of EGF-dependent DNA replication. Our data led us to assess the first third of G1, is exclusively devoted to the growth factor-dependent morphogenic events, whereas the mitogenic signal occured at only approximately mid-G1 phase. Moreover, these two growth factor-related sequential signaling events involved successively activation of MEK2-ERK2 and then MEK1/2-ERK1/2 isoforms. In addition, we demonstrated that inhibition of extracellular matrix receptor, such as integrin β1 subunit, leads to cell arrest in G1, whereas EGF was found to up-regulated integrin β1 and fibronectin in a MEK-ERK–dependent manner. This process in relation to cytoskeletal reorganization could induce hepatocyte spreading, making them permissive for DNA replication. Our results provide new insight into the mechanisms by which a growth factor can temporally control dual morphogenic and mitogenic signals during the G1 phase.

Forebrain mechanisms of nociception and pain: Analysis through imaging

Pain is a unified experience composed of interacting discriminative, affective-motivational, and cognitive components, each of which is mediated and modulated through forebrain mechanisms acting at spinal, brainstem, and cerebral levels. The size of the human forebrain in relation to the spinal cord gives anatomical emphasis to forebrain control over nociceptive processing. Human forebrain pathology can cause pain without the activation of nociceptors. Functional imaging of the normal human brain with positron emission tomography (PET) shows synaptically induced increases in regional cerebral blood flow (rCBF) in several regions specifically during pain. We have examined the variables of gender, type of noxious stimulus, and the origin of nociceptive input as potential determinants of the pattern and intensity of rCBF responses. The structures most consistently activated across genders and during contact heat pain, cold pain, cutaneous laser pain or intramuscular pain were the contralateral insula and anterior cingulate cortex, the bilateral thalamus and premotor cortex, and the cerebellar vermis. These regions are commonly activated in PET studies of pain conducted by other investigators, and the intensity of the brain rCBF response correlates parametrically with perceived pain intensity. To complement the human studies, we developed an animal model for investigating stimulus-induced rCBF responses in the rat. In accord with behavioral measures and the results of human PET, there is a progressive and selective activation of somatosensory and limbic system structures in the brain and brainstem following the subcutaneous injection of formalin. The animal model and human PET studies should be mutually reinforcing and thus facilitate progress in understanding forebrain mechanisms of normal and pathological pain.