An Agent Architecture to fulfill Real-Time Requirements

In this paper we present AMSIA, an agent architecture that combines the possibility of using di erent reasoning methods with a mechanism to control the resources needed by the agent to ful ll its high level objectives. The architecture is based on the blackboard paradigm which o ers the possibility of combining di erent reasoning techniques and opportunistic behavior. The AMSIA architecture adds a representation of plans of objectives allowing di erent reasoning activities to create plans to guide the future behavior of the agent. The opportunism is in the acquisition of high-level objectives and in the modi cation of the predicted activity when something doesn't happen as expected. A control mechanism is responsible for the translation of plans of objectives to concrete activities, considering resource-boundedness. To do so, all the activity in the agent (including control) is explicitly scheduled, but allowing the necessary exibility to make changes in the face of contingencies that are expected in dynamic environments. Experimental work is also presented.

Multi-agent location system in wireless networks

In this paper we propose a flexible Multi-Agent Architecture together with a methodology for indoor location which allows us to locate any mobile station (MS) such as a Laptop, Smartphone, Tablet or a robotic system in an indoor environment using wireless technology. Our technology is complementary to the GPS location finder as it allows us to locate a mobile system in a specific room on a specific floor using the Wi-Fi networks. The idea is that any MS will have an agent known at a Fuzzy Location Software Agent (FLSA) with a minimum capacity processing at its disposal which collects the power received at different Access Points distributed around the floor and establish its location on a plan of the floor of the building. In order to do so it will have to communicate with the Fuzzy Location Manager Software Agent (FLMSA). The FLMSAs are local agents that form part of the management infrastructure of the Wi-Fi network of the Organization. The FLMSA implements a location estimation methodology divided into three phases (measurement, calibration and estimation) for locating mobile stations (MS). Our solution is a fingerprint-based positioning system that overcomes the problem of the relative effect of doors and walls on signal strength and is independent of the network device manufacturer. In the measurement phase, our system collects received signal strength indicator (RSSI) measurements from multiple access points. In the calibration phase, our system uses these measurements in a normalization process to create a radio map, a database of RSS patterns. Unlike traditional radio map-based methods, our methodology normalizes RSS measurements collected at different locations on a floor. In the third phase, we use Fuzzy Controllers to locate an MS on the plan of the floor of a building. Experimental results demonstrate the accuracy of the proposed method. From these results it is clear that the system is highly likely to be able to locate an MS in a room or adjacent room.

Application of the agency theory for the analysis of performance-based mechanisms in road management

El WCTR es un congreso de reconocido prestigio internacional en el ámbito de la investigación del transporte, y aunque las actas publicadas están en formato digital y sin ISSN ni ISBN, lo consideramos lo suficientemente importante como para que se considere en los indicadores. This paper develops a model based on agency theory to analyze road management systems (under the different contract forms available today) that employ a mechanism of performance indicators to establish the payment of the agent. The base assumption is that of asymmetric information between the principal (Public Authorities) and the agent (contractor) and the risk aversion of this latter. It is assumed that the principal may only measure the agent?s performance indirectly and by means of certain performance indicators that may be verified by the authorities. In this model there is presumed to be a relation between the efforts made by the agent and the performance level measured by the corresponding indicators, though it is also considered that there may be dispersion between both variables that gives rise to a certain degree of randomness in the contract. An analysis of the optimal contract has been made on the basis of this model and in accordance with a series of parameters that characterize the economic environment and the particular conditions of road infrastructure. As a result of the analysis made, it is considered that an optimal contract should generally combine a fixed component and a payment in accordance with the performance level obtained. The higher the risk aversion of the agent and the greater the marginal cost of public funds, the lower the impact of this performance-based payment. By way of conclusion, the system of performance indicators should be as broad as possible but should not overweight those indicators that encompass greater randomness in their results.

MAIA: an event-based modular architecture for intelligent agents

Online services are no longer isolated. The release of public APIs and technologies such as web hooks are allowing users and developers to access their information easily. Intelligent agents could use this information to provide a better user experience across services, connecting services with smart automatic. behaviours or actions. However, agent platforms are not prepared to easily add external sources such as web services, which hinders the usage of agents in the so-called Evented or Live Web. As a solution, this paper introduces an event-based architecture for agent systems, in accordance with the new tendencies in web programming. In particular, it is focused on personal agents that interact with several web services. With this architecture, called MAIA, connecting to new web services does not involve any modification in the platform.

Thermoplastic Polymer Nanocomposites Based on Inorganic Fullerene-like Nanoparticles and Inorganic Nanotubes

Using inorganic fullerene-like (IF) nanoparticles and inorganic nanotubes (INT) in organic-inorganic hybrid composite, materials provide the potential for improving thermal, mechanical, and tribological properties of conventional composites. The processing of such high-performance hybrid thermoplastic polymer nanocomposites is achieved via melt-blending without the aid of any modifier or compatibilizing agent. The incorporation of small quantities (0.1-4 wt.%) of IF/INTs (tungsten disulfide, IF-WS2 or molybdenum disulfide, MoS2) generates notable performance enhancements through reinforcement effects and excellent lubricating ability in comparison with promising carbon nanotubes or other inorganic nanoscale fillers. It was shown that these IF/INT nanocomposites can provide an effective balance between performance, cost effectiveness, and processability, which is of significant importance for extending the practical applications of diverse hierarchical thermoplastic-based composites.

Application of Agent Technology for Fault Diagnosis of Telecommunication Networks

La presente tesis doctoral contribuye al problema del diagnóstico autonómico de fallos en redes de telecomunicación. En las redes de telecomunicación actuales, las operadoras realizan tareas de diagnóstico de forma manual. Dichas operaciones deben ser llevadas a cabo por ingenieros altamente cualificados que cada vez tienen más dificultades a la hora de gestionar debidamente el crecimiento exponencial de la red tanto en tamaño, complejidad y heterogeneidad. Además, el advenimiento del Internet del Futuro hace que la demanda de sistemas que simplifiquen y automaticen la gestión de las redes de telecomunicación se haya incrementado en los últimos años. Para extraer el conocimiento necesario para desarrollar las soluciones propuestas y facilitar su adopción por los operadores de red, se propone una metodología de pruebas de aceptación para sistemas multi-agente enfocada en simplificar la comunicación entre los diferentes grupos de trabajo involucrados en todo proyecto de desarrollo software: clientes y desarrolladores. Para contribuir a la solución del problema del diagnóstico autonómico de fallos, se propone una arquitectura de agente capaz de diagnosticar fallos en redes de telecomunicación de manera autónoma. Dicha arquitectura extiende el modelo de agente Belief-Desire- Intention (BDI) con diferentes modelos de diagnóstico que gestionan las diferentes sub-tareas del proceso. La arquitectura propuesta combina diferentes técnicas de razonamiento para alcanzar su propósito gracias a un modelo estructural de la red, que usa razonamiento basado en ontologías, y un modelo causal de fallos, que usa razonamiento Bayesiano para gestionar debidamente la incertidumbre del proceso de diagnóstico. Para asegurar la adecuación de la arquitectura propuesta en situaciones de gran complejidad y heterogeneidad, se propone un marco de argumentación que permite diagnosticar a agentes que estén ejecutando en dominios federados. Para la aplicación de este marco en un sistema multi-agente, se propone un protocolo de coordinación en el que los agentes dialogan hasta alcanzar una conclusión para un caso de diagnóstico concreto. Como trabajos futuros, se consideran la extensión de la arquitectura para abordar otros problemas de gestión como el auto-descubrimiento o la auto-optimización, el uso de técnicas de reputación dentro del marco de argumentación para mejorar la extensibilidad del sistema de diagnóstico en entornos federados y la aplicación de las arquitecturas propuestas en las arquitecturas de red emergentes, como SDN, que ofrecen mayor capacidad de interacción con la red. ABSTRACT This PhD thesis contributes to the problem of autonomic fault diagnosis of telecommunication networks. Nowadays, in telecommunication networks, operators perform manual diagnosis tasks. Those operations must be carried out by high skilled network engineers which have increasing difficulties to properly manage the growing of those networks, both in size, complexity and heterogeneity. Moreover, the advent of the Future Internet makes the demand of solutions which simplifies and automates the telecommunication network management has been increased in recent years. To collect the domain knowledge required to developed the proposed solutions and to simplify its adoption by the operators, an agile testing methodology is defined for multiagent systems. This methodology is focused on the communication gap between the different work groups involved in any software development project, stakeholders and developers. To contribute to overcoming the problem of autonomic fault diagnosis, an agent architecture for fault diagnosis of telecommunication networks is defined. That architecture extends the Belief-Desire-Intention (BDI) agent model with different diagnostic models which handle the different subtasks of the process. The proposed architecture combines different reasoning techniques to achieve its objective using a structural model of the network, which uses ontology-based reasoning, and a causal model, which uses Bayesian reasoning to properly handle the uncertainty of the diagnosis process. To ensure the suitability of the proposed architecture in complex and heterogeneous environments, an argumentation framework is defined. This framework allows agents to perform fault diagnosis in federated domains. To apply this framework in a multi-agent system, a coordination protocol is defined. This protocol is used by agents to dialogue until a reliable conclusion for a specific diagnosis case is reached. Future work comprises the further extension of the agent architecture to approach other managements problems, such as self-discovery or self-optimisation; the application of reputation techniques in the argumentation framework to improve the extensibility of the diagnostic system in federated domains; and the application of the proposed agent architecture in emergent networking architectures, such as SDN, which offers new capabilities of control for the network.

Contribution to the improvement of communication services in nanosatellite constellations using delay and disruption tolerant networking based architectures

Esta tesis se desarrolla dentro del marco de las comunicaciones satelitales en el innovador campo de los pequeños satélites también llamados nanosatélites o cubesats, llamados así por su forma cubica. Estos nanosatélites se caracterizan por su bajo costo debido a que usan componentes comerciales llamados COTS (commercial off-the-shelf) y su pequeño tamaño como los Cubesats 1U (10cm*10 cm*10 cm) con masa aproximada a 1 kg. Este trabajo de tesis tiene como base una iniciativa propuesta por el autor de la tesis para poner en órbita el primer satélite peruano en mi país llamado chasqui I, actualmente puesto en órbita desde la Estación Espacial Internacional. La experiencia de este trabajo de investigación me llevo a proponer una constelación de pequeños satélites llamada Waposat para dar servicio de monitoreo de sensores de calidad de agua a nivel global, escenario que es usado en esta tesis. Es ente entorno y dadas las características limitadas de los pequeños satélites, tanto en potencia como en velocidad de datos, es que propongo investigar una nueva arquitectura de comunicaciones que permita resolver en forma óptima la problemática planteada por los nanosatélites en órbita LEO debido a su carácter disruptivo en sus comunicaciones poniendo énfasis en las capas de enlace y aplicación. Esta tesis presenta y evalúa una nueva arquitectura de comunicaciones para proveer servicio a una red de sensores terrestres usando una solución basada en DTN (Delay/Disruption Tolerant Networking) para comunicaciones espaciales. Adicionalmente, propongo un nuevo protocolo de acceso múltiple que usa una extensión del protocolo ALOHA no ranurado, el cual toma en cuenta la prioridad del trafico del Gateway (ALOHAGP) con un mecanismo de contienda adaptativo. Utiliza la realimentación del satélite para implementar el control de la congestión y adapta dinámicamente el rendimiento efectivo del canal de una manera óptima. Asumimos un modelo de población de sensores finito y una condición de tráfico saturado en el que cada sensor tiene siempre tramas que transmitir. El desempeño de la red se evaluó en términos de rendimiento efectivo, retardo y la equidad del sistema. Además, se ha definido una capa de convergencia DTN (ALOHAGP-CL) como un subconjunto del estándar TCP-CL (Transmission Control Protocol-Convergency Layer). Esta tesis muestra que ALOHAGP/CL soporta adecuadamente el escenario DTN propuesto, sobre todo cuando se utiliza la fragmentación reactiva. Finalmente, esta tesis investiga una transferencia óptima de mensajes DTN (Bundles) utilizando estrategias de fragmentación proactivas para dar servicio a una red de sensores terrestres utilizando un enlace de comunicaciones satelitales que utiliza el mecanismo de acceso múltiple con prioridad en el tráfico de enlace descendente (ALOHAGP). El rendimiento efectivo ha sido optimizado mediante la adaptación de los parámetros del protocolo como una función del número actual de los sensores activos recibidos desde el satélite. También, actualmente no existe un método para advertir o negociar el tamaño máximo de un “bundle” que puede ser aceptado por un agente DTN “bundle” en las comunicaciones por satélite tanto para el almacenamiento y la entrega, por lo que los “bundles” que son demasiado grandes son eliminados o demasiado pequeños son ineficientes. He caracterizado este tipo de escenario obteniendo una distribución de probabilidad de la llegada de tramas al nanosatélite así como una distribución de probabilidad del tiempo de visibilidad del nanosatélite, los cuales proveen una fragmentación proactiva óptima de los DTN “bundles”. He encontrado que el rendimiento efectivo (goodput) de la fragmentación proactiva alcanza un valor ligeramente inferior al de la fragmentación reactiva. Esta contribución permite utilizar la fragmentación activa de forma óptima con todas sus ventajas tales como permitir implantar el modelo de seguridad de DTN y la simplicidad al implementarlo en equipos con muchas limitaciones de CPU y memoria. La implementación de estas contribuciones se han contemplado inicialmente como parte de la carga útil del nanosatélite QBito, que forma parte de la constelación de 50 nanosatélites que se está llevando a cabo dentro del proyecto QB50. ABSTRACT This thesis is developed within the framework of satellite communications in the innovative field of small satellites also known as nanosatellites (<10 kg) or CubeSats, so called from their cubic form. These nanosatellites are characterized by their low cost because they use commercial components called COTS (commercial off-the-shelf), and their small size and mass, such as 1U Cubesats (10cm * 10cm * 10cm) with approximately 1 kg mass. This thesis is based on a proposal made by the author of the thesis to put into orbit the first Peruvian satellite in his country called Chasqui I, which was successfully launched into orbit from the International Space Station in 2014. The experience of this research work led me to propose a constellation of small satellites named Waposat to provide water quality monitoring sensors worldwide, scenario that is used in this thesis. In this scenario and given the limited features of nanosatellites, both power and data rate, I propose to investigate a new communications architecture that allows solving in an optimal manner the problems of nanosatellites in orbit LEO due to the disruptive nature of their communications by putting emphasis on the link and application layers. This thesis presents and evaluates a new communications architecture to provide services to terrestrial sensor networks using a space Delay/Disruption Tolerant Networking (DTN) based solution. In addition, I propose a new multiple access mechanism protocol based on extended unslotted ALOHA that takes into account the priority of gateway traffic, which we call ALOHA multiple access with gateway priority (ALOHAGP) with an adaptive contention mechanism. It uses satellite feedback to implement the congestion control, and to dynamically adapt the channel effective throughput in an optimal way. We assume a finite sensor population model and a saturated traffic condition where every sensor always has frames to transmit. The performance was evaluated in terms of effective throughput, delay and system fairness. In addition, a DTN convergence layer (ALOHAGP-CL) has been defined as a subset of the standard TCP-CL (Transmission Control Protocol-Convergence Layer). This thesis reveals that ALOHAGP/CL adequately supports the proposed DTN scenario, mainly when reactive fragmentation is used. Finally, this thesis investigates an optimal DTN message (bundles) transfer using proactive fragmentation strategies to give service to a ground sensor network using a nanosatellite communications link which uses a multi-access mechanism with priority in downlink traffic (ALOHAGP). The effective throughput has been optimized by adapting the protocol parameters as a function of the current number of active sensors received from satellite. Also, there is currently no method for advertising or negotiating the maximum size of a bundle which can be accepted by a bundle agent in satellite communications for storage and delivery, so that bundles which are too large can be dropped or which are too small are inefficient. We have characterized this kind of scenario obtaining a probability distribution for frame arrivals to nanosatellite and visibility time distribution that provide an optimal proactive fragmentation of DTN bundles. We have found that the proactive effective throughput (goodput) reaches a value slightly lower than reactive fragmentation approach. This contribution allows to use the proactive fragmentation optimally with all its advantages such as the incorporation of the security model of DTN and simplicity in protocol implementation for computers with many CPU and memory limitations. The implementation of these contributions was initially contemplated as part of the payload of the nanosatellite QBito, which is part of the constellation of 50 nanosatellites envisaged under the QB50 project.

A Personal Agent Architecture for Task Automation in the Web of Data. Bringing intelligence to everyday tasks

Internet está evolucionando hacia la conocida como Live Web. En esta nueva etapa en la evolución de Internet, se pone al servicio de los usuarios multitud de streams de datos sociales. Gracias a estas fuentes de datos, los usuarios han pasado de navegar por páginas web estáticas a interacturar con aplicaciones que ofrecen contenido personalizado, basada en sus preferencias. Cada usuario interactúa a diario con multiples aplicaciones que ofrecen notificaciones y alertas, en este sentido cada usuario es una fuente de eventos, y a menudo los usuarios se sienten desbordados y no son capaces de procesar toda esa información a la carta. Para lidiar con esta sobresaturación, han aparecido múltiples herramientas que automatizan las tareas más habituales, desde gestores de bandeja de entrada, gestores de alertas en redes sociales, a complejos CRMs o smart-home hubs. La contrapartida es que aunque ofrecen una solución a problemas comunes, no pueden adaptarse a las necesidades de cada usuario ofreciendo una solucion personalizada. Los Servicios de Automatización de Tareas (TAS de sus siglas en inglés) entraron en escena a partir de 2012 para dar solución a esta liminación. Dada su semejanza, estos servicios también son considerados como un nuevo enfoque en la tecnología de mash-ups pero centra en el usuarios. Los usuarios de estas plataformas tienen la capacidad de interconectar servicios, sensores y otros aparatos con connexión a internet diseñando las automatizaciones que se ajustan a sus necesidades. La propuesta ha sido ámpliamante aceptada por los usuarios. Este hecho ha propiciado multitud de plataformas que ofrecen servicios TAS entren en escena. Al ser un nuevo campo de investigación, esta tesis presenta las principales características de los TAS, describe sus componentes, e identifica las dimensiones fundamentales que los defines y permiten su clasificación. En este trabajo se acuña el termino Servicio de Automatización de Tareas (TAS) dando una descripción formal para estos servicios y sus componentes (llamados canales), y proporciona una arquitectura de referencia. De igual forma, existe una falta de herramientas para describir servicios de automatización, y las reglas de automatización. A este respecto, esta tesis propone un modelo común que se concreta en la ontología EWE (Evented WEb Ontology). Este modelo permite com parar y equiparar canales y automatizaciones de distintos TASs, constituyendo un aporte considerable paraa la portabilidad de automatizaciones de usuarios entre plataformas. De igual manera, dado el carácter semántico del modelo, permite incluir en las automatizaciones elementos de fuentes externas sobre los que razonar, como es el caso de Linked Open Data. Utilizando este modelo, se ha generado un dataset de canales y automatizaciones, con los datos obtenidos de algunos de los TAS existentes en el mercado. Como último paso hacia el lograr un modelo común para describir TAS, se ha desarrollado un algoritmo para aprender ontologías de forma automática a partir de los datos del dataset. De esta forma, se favorece el descubrimiento de nuevos canales, y se reduce el coste de mantenimiento del modelo, el cual se actualiza de forma semi-automática. En conclusión, las principales contribuciones de esta tesis son: i) describir el estado del arte en automatización de tareas y acuñar el término Servicio de Automatización de Tareas, ii) desarrollar una ontología para el modelado de los componentes de TASs y automatizaciones, iii) poblar un dataset de datos de canales y automatizaciones, usado para desarrollar un algoritmo de aprendizaje automatico de ontologías, y iv) diseñar una arquitectura de agentes para la asistencia a usuarios en la creación de automatizaciones. ABSTRACT The new stage in the evolution of the Web (the Live Web or Evented Web) puts lots of social data-streams at the service of users, who no longer browse static web pages but interact with applications that present them contextual and relevant experiences. Given that each user is a potential source of events, a typical user often gets overwhelmed. To deal with that huge amount of data, multiple automation tools have emerged, covering from simple social media managers or notification aggregators to complex CRMs or smart-home Hub/Apps. As a downside, they cannot tailor to the needs of every single user. As a natural response to this downside, Task Automation Services broke in the Internet. They may be seen as a new model of mash-up technology for combining social streams, services and connected devices from an end-user perspective: end-users are empowered to connect those stream however they want, designing the automations they need. The numbers of those platforms that appeared early on shot up, and as a consequence the amount of platforms following this approach is growing fast. Being a novel field, this thesis aims to shed light on it, presenting and exemplifying the main characteristics of Task Automation Services, describing their components, and identifying several dimensions to classify them. This thesis coins the term Task Automation Services (TAS) by providing a formal definition of them, their components (called channels), as well a TAS reference architecture. There is also a lack of tools for describing automation services and automations rules. In this regard, this thesis proposes a theoretical common model of TAS and formalizes it as the EWE ontology This model enables to compare channels and automations from different TASs, which has a high impact in interoperability; and enhances automations providing a mechanism to reason over external sources such as Linked Open Data. Based on this model, a dataset of components of TAS was built, harvesting data from the web sites of actual TASs. Going a step further towards this common model, an algorithm for categorizing them was designed, enabling their discovery across different TAS. Thus, the main contributions of the thesis are: i) surveying the state of the art on task automation and coining the term Task Automation Service; ii) providing a semantic common model for describing TAS components and automations; iii) populating a categorized dataset of TAS components, used to learn ontologies of particular domains from the TAS perspective; and iv) designing an agent architecture for assisting users in setting up automations, that is aware of their context and acts in consequence.

A Vision-based Quadrotor Multi-robot Solution for the Indoor Autonomy Challenge of the 2013 International Micro Air Vehicle Competition

This paper presents a completely autonomous solution to participate in the Indoor Challenge of the 2013 International Micro Air Vehicle Competition (IMAV 2013). Our proposal is a multi-robot system with no centralized coordination whose robotic agents share their position estimates. The capability of each agent to navigate avoiding collisions is a consequence of the resulting emergent behavior. Each agent consists of a ground station running an instance of the proposed architecture that communicates over WiFi with an AR Drone 2.0 quadrotor. Visual markers are employed to sense and map obstacles and to improve the pose estimation based on Inertial Measurement Unit (IMU) and ground optical flow data. Based on our architecture, each robotic agent can navigate avoiding obstacles and other members of the multi-robot system. The solution is demonstrated and the achieved navigation performance is evaluated by means of experimental flights. This work also analyzes the capabilities of the presented solution in simulated flights of the IMAV 2013 Indoor Challenge. The performance of the CVG UPM team was awarded with the First Prize in the Indoor Autonomy Challenge of the IMAV 2013 competition.

α1-Antitrypsin Portland, a bioengineered serpin highly selective for furin: Application as an antipathogenic agent

The important role of furin in the proteolytic activation of many pathogenic molecules has made this endoprotease a target for the development of potent and selective antiproteolytic agents. Here, we demonstrate the utility of the protein-based inhibitor α1-antitrypsin Portland (α1-PDX) as an antipathogenic agent that can be used prophylactically to block furin-dependent cell killing by Pseudomonas exotoxin A. Biochemical analysis of the specificity of a bacterially expressed His- and FLAG-tagged α1-PDX (α1-PDX/hf) revealed the selectivity of the α1-PDX/hf reactive site loop for furin (Ki, 600 pM) but not for other proprotein convertase family members or other unrelated endoproteases. Kinetic studies show that α1-PDX/hf inhibits furin by a slow tight-binding mechanism characteristic of serpin molecules and functions as a suicide substrate inhibitor. Once bound to furin’s active site, α1-PDX/hf partitions with equal probability to undergo proteolysis by furin at the C-terminal side of the reactive center -Arg355-Ile-Pro-Arg358-↓ or to form a kinetically trapped SDS-stable complex with the enzyme. This partitioning between the complex-forming and proteolytic pathways contributes to the ability of α1-PDX/hf to differentially inhibit members of the proprotein convertase family. Finally, we propose a structural model of the α1-PDX-reactive site loop that explains the high degree of enzyme selectivity of this serpin and which can be used to generate small molecule furin inhibitors.

A model-based approach for assessing in vivo combination therapy interactions

We present an approach for evaluating the efficacy of combination antitumor agent schedules that accounts for order and timing of drug administration. Our model-based approach compares in vivo tumor volume data over a time course and offers a quantitative definition for additivity of drug effects, relative to which synergism and antagonism are interpreted. We begin by fitting data from individual mice receiving at most one drug to a differential equation tumor growth/drug effect model and combine individual parameter estimates to obtain population statistics. Using two null hypotheses: (i) combination therapy is consistent with additivity or (ii) combination therapy is equivalent to treating with the more effective single agent alone, we compute predicted tumor growth trajectories and their distribution for combination treated animals. We illustrate this approach by comparing entire observed and expected tumor volume trajectories for a data set in which HER-2/neu-overexpressing MCF-7 human breast cancer xenografts are treated with a humanized, anti-HER-2 monoclonal antibody (rhuMAb HER-2), doxorubicin, or one of five proposed combination therapy schedules.

The design of an agent to bend DNA.

An artificial DNA bending agent has been designed to assess helix flexibility over regions as small as a protein binding site. Bending was obtained by linking a pair of 15-base-long triple helix forming oligonucleotides (TFOs) by an adjustable polymeric linker. By design, DNA bending was introduced into the double helix within a 10-bp spacer region positioned between the two sites of 15-base triple helix formation. The existence of this bend has been confirmed by circular permutation and phase-sensitive electrophoresis, and the directionality of the bend has been determined as a compression of the minor helix groove. The magnitude of the resulting duplex bend was found to be dependent on the length of the polymeric linker in a fashion consistent with a simple geometric model. Data suggested that a 50-70 degrees bend was achieved by binding of the TFO chimera with the shortest linker span (18 rotatable bonds). Equilibrium analysis showed that, relative to a chimera which did not bend the duplex, the stability of the triple helix possessing a 50-70 degrees bend was reduced by less than 1 kcal/mol of that of the unbent complex. Based upon this similarity, it is proposed that duplex DNA may be much more flexible with respect to minor groove compression than previously assumed. It is shown that this unusual flexibility is consistent with recent quantitation of protein-induced minor groove bending.

Leukotriene A4 hydrolase: protection from mechanism-based inactivation by mutation of tyrosine-378.

Leukotriene A4 (LTA4) hydrolase [(7E,9E,11Z,14Z)-(5S,6S)-5,6-epoxyicosa-7, 9,11,14-tetraenoate hydrolase; EC 3.3.2.6] is a bifunctional zinc metalloenzyme that catalyzes the final step in the biosynthesis of the potent chemotactic agent leukotriene B4 (LTB4). LTA4 hydrolase/aminopeptidase is suicide inactivated during catalysis via an apparently mechanism-based irreversible binding of LTA4 to the protein in a 1:1 stoichiometry. Previously, we have identified a henicosapeptide, encompassing residues Leu-365 to Lys-385 in human LTA4 hydrolase, which contains a site involved in the covalent binding of LTA4 to the native enzyme. To investigate the role of Tyr-378, a potential candidate for this binding site, we exchanged Tyr for Phe or Gln in two separate mutants. In addition, each of two adjacent and potentially reactive residues, Ser-379 and Ser-380, were exchanged for Ala. The mutated enzymes were expressed as (His)6-tagged fusion proteins in Escherichia coli, purified to apparent homogeneity, and characterized. Enzyme activity determinations and differential peptide mapping, before and after repeated exposure to LTA4, revealed that wild-type enzyme and the mutants [S379A] and [S380A]LTA4hydrolase were equally susceptible to suicide inactivation whereas the mutants in position 378 were no longer inactivated or covalently modified by LTA4. Furthermore, in [Y378F]LTA4 hydrolase, the value of kcat for epoxide hydrolysis was increased 2.5-fold over that of the wild-type enzyme. Thus, by a single-point mutation in LTA4 hydrolase, catalysis and covalent modification/inactivation have been dissociated, yielding an enzyme with increased turnover and resistance to mechanism-based inactivation.

A general genetic approach in Escherichia coli for determining the mechanism(s) of action of tumoricidal agents: application to DMP 840, a tumoricidal agent.

We describe here a simple and easily manipulatable Escherichia coli-based genetic system that permits us to identify bacterial gene products that modulate the sensitivity of bacteria to tumoricidal agents, such as DMP 840, a bisnaphthalimide drug. To the extent that the action of these agents is conserved, these studies may expand our understanding agents is conserved, these studies may expand our understanding of how the agents work in mammalian cells. The approach briefly is to use a library of E. coli genes that are overexpressed in a high copy number vector to select bacterial clones that are resistant to the cytotoxic effects of drugs. AtolC bacterial mutant is used to maximize permeability of cells to hydrophobic organic molecules. By using DMP 840 to model the system, we have identified two genes, designated mdaA and mdaB, that impart resistance to DMP 840 when they are expressed at elevated levels. mdaB maps to E. coli map coordinate 66, is located between the parE and parC genes, and encodes a protein of 22 kDa. mdaA maps to E. coli map coordinate 18, is located adjacent to the glutaredoxin (grx) gene, and encodes a protein of 24 kDa. Specific and regulatable overproduction of both of these proteins correlates with DMP 840 resistance. Overproduction of the MdaB protein also imparts resistance to two mammalian topoisomerase inhibitors, Adriamycin and etoposide. In contrast, overproduction of the MdaA protein produces resistance only to Adriamycin. Based on its drug-resistance properties and its location between genes that encode the two subunits of the bacterial topoisomerase IV, we suggest that mdaB acts by modulating topoisomerase IV activity. The location of the mdaA gene adjacent to grx suggests it acts by a drug detoxification mechanism.

Leukotriene A4 hydrolase: mapping of a henicosapeptide involved in mechanism-based inactivation.

Leukotriene A4 (LTA4) hydrolase [7E,9E,11Z,14Z)-(5S,6S)-5,6-epoxyicosa-7,9 ,11,14-tetraenoate hydrolase; EC 3.3.2.6] is a bifunctional zinc metalloenzyme which converts LTA4 into the chemotactic agent leukotriene B4 (LTB4). Suicide inactivation, a typical feature of LTA4 hydrolase/aminopeptidase, occurs via an irreversible, apparently mechanism-based, covalent binding of LTA4 to the protein in a 1:1 stoichiometry. Differential lysine-specific peptide mapping of unmodified and suicide-inactivated LTA4 hydrolase has been used to identify a henicosapeptide, encompassing the amino acid residues 365-385 of human LTA4 hydrolase, which is involved in the binding of LTA4, LTA4 methyl ester, and LTA4 ethyl ester to the native enzyme. A modified form of this peptide, generated by lysine-specific digestion of LTA4 hydrolase inactivated by LTA4 ethyl ester, could be isolated for complete Edman degradation. The sequence analysis revealed a gap at position 14, which shows that binding of the leukotriene epoxide had occurred via Tyr-378 in LTA4 hydrolase. Inactivation of the epoxide hydrolase and the aminopeptidase activity was accompanied by a proportionate modification of the peptide. Furthermore, both enzyme inactivation and peptide modification could be prevented by preincubation of LTA4 hydrolase with the competitive inhibitor bestatin, which demonstrates that the henicosapeptide contains functional elements of the active site(s). It may now be possible to clarify the molecular mechanisms underlying suicide inactivation and epoxide hydrolysis by site-directed mutagenesis combined with structural analysis of the lipid molecule, covalently bound to the peptide.