Determinants of malnutrition in two states in Sudan

Nearly one in three children in the developing world is malnourished. Poor nutrition contributes to one out of two deaths (53%) associated with infectious diseases among children aged under five in developing countries. Using data from the 2005 World Food Program’s (WFP) Livelihood Vulnerability and Nutritional Assessment of Rural Kassala and Red Sea State this study examines the impact of female headed households and maternal education on malnutrition in children 6-59 months old. The dependent variable investigated in this study is moderate to severe wasting or less than -2 weight for height Z-score, also known as global acute malnutrition (GAM). ^ The study population consisted of 450 households in Kassala State and Red Sea State, Sudan. A total of 900 children 6-59 months of age were part of the households sampled from these states and one child per household (773 children) was randomly chosen for the analysis along with the child’s mother. Results of the study found that 18 percent of children between 6-59 months of age had GAM/wasting. Maternal education, main source of water, and income were strongly related to wasting. Gender of head of household was not found to have a significant relationship with GAM/wasting. Mothers with at least primary education were much less likely to have malnourished children, even after controlling for income and environmental conditions. Children in households with unsafe sources of water were 2.6 more likely to have wasting than those with piped in/tube wells as their main source of water. For every increase of 100 dinar in a household, the children in the household are approximately two-thirds times (.662) less likely to be wasted. ^ The results of this study support the alternate hypothesis that there is an association between maternal education on wasting of children 6-59 months old. The results do not, however, support the alternate hypothesis that there is an association between gender of head of household on wasting of children 6-59 months old. Better understanding of the association of wasting and other measures of malnutrition with maternal education levels can program managers and other health officials to target important nutritional and non-nutritional interventions. ^

Studies of the expression of DNA repair related genes

This dissertation examines the biological functions and the regulation of expression of DNA ligase I by studying its expression under different conditions.^ The gene expression of DNA ligase I was induced two- to four-fold in S-phase lymphoblastoid cells but was decreased to 15% of control after administration of a DNA damaging agent, 4-nitroquinoline-1-oxide. When cells were induced into differentiation, the expression level of DNA ligase I was decreased to less than 15% of that of the control cells. When the gene of DNA ligase I was examined for tissue specific expression in adult rats, high levels of DNA ligase I mRNA were observed in testis (8-fold), intermediate levels in ovary and brain (4-fold), and low levels were found in intestine, spleen, and liver (1- to 2-fold).^ In confluent cells of normal skin fibroblasts, UV irradiation induced the gene expression of DNA ligase I at 24 and 48 h. The induction of DNA ligase I gene expression requires active p53 protein. Introducing a vector containing the wild type p53 protein in the cells caused an induction of the DNA ligase I protein 24 h after the treatment.^ Our results indicate that, in addition to the regulation by phosphorylation/dephosphorylation, cellular DNA ligase I activity can be regulated at the gene transcription level, and the p53 tumor suppresser is one of the transcription factors for the DNA ligase I gene. Also, our results suggest that DNA ligase I is involved in DNA repair as well as in DNA replication.^ Also, as an early attempt to clone the human homolog of the yeast CDC9 gene which has been shown to be involved in DNA replication, DNA repair, and DNA recombination, we have identified a human gene with mRNA of 1.7 kb. This dissertation studies the gene regulation and the possible biological functions of this new human gene by examining its expression at different stages of the cell cycle, during cell differentiation, and in cellular response to DNA damage.^ The new gene that we recently identified from human cells is highly expressed in brain and reproductive organs (BRE). This BRE gene encodes an mRNA of 1.7-1.9 kb, with an open reading frame of 1,149 bp, and gives rise to a deduced polypeptide of 383 amino acid residues. No extensive homology was found between BRE and sequences from the EMBL-Gene Banks. BRE showed tissue-specific expression in adult rats. The steady state mRNA levels were high in testis (5-6 fold), ovary and brain (3-4 fold) compared to the spleen level, but low in intestine and liver (1-2 fold). The expression of this gene is responsive to DNA damage and/or retinoic acid (RA) treatment. Treatment of fibroblast cells with UV irradiation and 4-nitroquinoline-1-oxide caused more than 90% and 50% decreases in BRE mRNA, respectively. Similar decreases in BRE expression were observed after treatment of the brain glioma cell line U-251 and the promyelocytic cell line HL-60 with retinoic acid. (Abstract shortened by UMI). ^

RESPONSES OF INDICATOR AND PATHOGENIC BACTERIA TO THE PRESENCE OF CARBARYL IN WATER

Biodegradability is a desirable, if not a necessary characteristic of pesticides. Carbaryl, as Sevin, is one of the more widely used insecticides for the control of agricultural pests and has been reported to be readily degraded by microorganisms. Because of its broad application, the concentration of Sevin in surface waters has been reported to reach nearly four parts per million (PPM) in surface waters, where it has been reported to affect the growth and metabolic rates of aquatic bacterial populations. Following these reports, it is of public health importance to determine the effects of this insecticide on the growth and metabolic rates of bacteria used to indicate water pollution, and on pathogenic organisms which are found in polluted water.^ This study was conducted to determine the effect of carbaryl on the growth and metabolic rates of indicator and pathogenic organisms. Escherichia coli and Streptococcus faecalis were used as indicators, while Staphylococcus aureus and Salmonella typhimurium were the pathogens studied. Pure and mixed cultures of these organisms were exposed to two concentrations of carbaryl (Sevin).^ The study demonstrated that the fecal pollution indicator organisms, E. coli and S. faecalis respond differently to the presence of small concentrations of carbaryl in water as do the two pathogens tested, (S. typhimurium and S. aureus). The growth of all test organisms as measured by spread plate counts, was reduced by the presence of either one mg/l or five mg/l carbaryl within a period of eight days. Survival of the organisms in the presence of five mg/l carbaryl varied dependent upon whether the organism was in pure or mixed culture. In the presence of five mg/l carbaryl, both pure and mixed culture of E. coli showed longer survival. S. faecalis survived for more than eight days in pure culture, neither S. typhimurium nor S. aureus survived for eight days in pure culture.^ The metabolic rate of S. faecalis and S. aureus was reduced by both five mg/l and one mg/l Sevin concentrations, contrary to E. coli and S. typhimurium which had reduced metabolic rate with the introduction of five mg/l Sevin but showed an increase in the metabolic rate with one mg/l Sevin. There was no difference between the test and control when mixed populations were exposed to five mg/l Sevin and the metabolic rate tested. A mixture of E. coli and S. typhimurium populations showed a respiration increase over the control when exposed to one mg/l Sevin concentration. If similar effects occur in polluted surface waters, misleading results from bacteriological water quality testing may occur. ^

MOLECULAR AND GENOMIC BASED INSIGHTS INTO THE EVOLUTION OF ENTEROCOCCUS FAECIUM FROM COMMENSAL TO HOSPITAL-ADAPTED PATHOGEN

The basis for the recent transition of Enterococcus faecium from a primarily commensal organism to one of the leading causes of hospital-acquired infections in the United States is not yet understood. To address this, the first part of my project assessed isolates from early outbreaks in the USA and South America using sequence analysis, colony hybridizations, and minimal inhibitory concentrations (MICs) which showed clinical isolates possess virulence and antibiotic resistance determinants that are less abundant or lacking in community isolates. I also revealed that the level of ampicillin resistance increased over time in clinical strains. By sequencing the pbp5 gene, I demonstrated an ~5% difference in the pbp5 gene between strains with MICs <4ug/ml and those with MICs >4µg/ml, but no specific sequence changes correlated with increases in MICs within the latter group. A 3-10% nucleotide difference was also seen in three other genes analyzed, which suggested the existence of two distinct subpopulations of E. faecium. This led to the second part of my project analyzing concatenated core gene sequences, SNPs, the 16S rRNA, and phylogenetics of 21 E. faecium genomes confirming two distinct clades; a community-associated (CA) clade and hospital-associated (HA) clade. Molecular clock calculations indicate that these two clades likely diverged ~ 300,000 to > 1 million years ago, long before the modern antibiotic era. Genomic analysis also showed that, in addition to core genomic differences, HA E. faecium harbor specific accessory genetic elements that may confer selection advantages over CA E. faecium. The third part of my project discovered 6 E. faecium genes with the newly identified “WxL” domain. My analyses, using RT-PCR, western blots, patient sera, whole-cell ELISA, and immunogold electron microscopy, indicated that E. faecium WxL genes exist in operons, encode bacterial cell surface localized proteins, that WxL proteins are antigenic in humans, and are more exposed on the surface of clinical isolates versus community isolates (even though they are ubiquitous in both clades). ELISAs and BIAcore analyses also showed that proteins encoded by these operons bind several different host extracellular matrix proteins, as well as to each other, suggesting a novel cell-surface complex. In summary, my studies provide new insights into the evolution of E. faecium by showing that there are two distantly related clades; one being more successful in the hospital setting. My studies also identified operons encoding WxL proteins whose characteristics could also contribute to colonization and virulence within this species.

Modelos de demanda potencial de viajeros en redes de transporte público : aplicaciones en el Metro de Madrid

Este trabajo propone una metodología basada en Sistemas de Información Geográfica para estimar la demanda de viajes en estaciones de redes de transporte público, tomando como ejemplo la red de metro de Madrid. Primero se emplea una serie de datos descriptivos para caracterizar la red, clasificar las estaciones y obtener una tipología de las mismas. Luego, con el objetivo de explicar y predecir los viajes (entradas a la red) se generan dos modelos: uno sencillo a partir de las tasas de penetración de uso del metro en función de la distancia (distance decay), y otro más complejo basado en un modelo de regresión lineal múltiple (MRLM) que incorpora variables relativas a la estación y su entorno (densidad, mezcla de usos, diseño urbano, presencia de modos competidores). Su aplicación muestra resultados alentadores, y se plantea como una alternativa a los clásicos modelos de cuatro etapas, más complejos y con un mayor coste económico.

(Table 1) Gas tie points used to correlate the ice cores NGRIP and EDML

Water isotope records from the EPICA Dronning Maud Land (EDML) and the NorthGRIP ice cores have revealed a one to one coupling between Antarctic Isotope Maxima (AIM) and Greenland Dansgaard-Oeschger (DO) events back to 50 kyr. In order to explore if this north-south coupling is persistent over Marine Isotopic Stage 5 (MIS 5), a common timescale must first be constructed. Here, we present new records of d18O of O2 (d18Oatm) and methane (CH4) measured in the air trapped in ice from the EDML (68-147 kyr) and NorthGRIP (70-123 kyr) ice cores. We demonstrate that, through the period of interest, CH4 records alone are not sufficient to construct a common gas timescale between the two cores. Millennial-scale variations of d18Oatm are evidenced over MIS 5 both on the Antarctic and Greenland ice cores and are coupled to CH4 profiles to synchronise the NorthGRIP and EDML records. They are shown to be a precious tool for ice core synchronisation. With this new dating strategy, we produce the first continuous and accurate sequence of the north-south climatic dynamics on a common ice timescale for the last glacial inception and the first DO events of MIS 5, reducing relative dating uncertainties to an accuracy of a few centuries at the onset of DO events 24 to 20. This EDML-NorthGRIP synchronisation provides new firm evidence that the bipolar seesaw is a pervasive pattern from the beginning of the glacial period. The relationship between Antarctic warming amplitudes and their concurrent Greenland stadial duration highlights the particularity of DO event 21 and its Antarctic counterpart. Our results suggest a smaller Southern Ocean warming rate for this long DO event compared to DO events of MIS 3.

(Table 1) Physical oceanography from several POLARSTERN cruises to the Arctic Ocean, 1993 - 2012

Freshwater in the Arctic Ocean plays an important role in the regional ocean circulation, sea ice, and global climate. From salinity observed by a variety of platforms, we are able, for the first time, to estimate a statistically reliable liquid freshwater trend from monthly gridded fields over all upper Arctic Ocean basins. From 1992 to 2012 this trend was 600±300 km**3/yr. A numerical model agrees very well with the observed freshwater changes. A decrease in salinity made up about two thirds of the freshwater trend and a thickening of the upper layer up to one third. The Arctic Ocean Oscillation index, a measure for the regional wind stress curl, correlated well with our freshwater time series. No clear relation to Arctic Oscillation or Arctic Dipole indices could be found. Following other observational studies, an increased Bering Strait freshwater import to the Arctic Ocean, a decreased Davis Strait export, and enhanced net sea ice melt could have played an important role in the freshwater trend we observed.

A regular perturbation approach to surface tension driven flows

Surface tension induced convection in a liquid bridge held between two parallel, coaxial, solid disks is considered. The surface tension gradient is produced by a small temperature gradient parallel Co the undisturbed surface. The study is performed by using a mathematical regular perturbation approach based on a small parameter, e, which measures the deviation of the imposed temperature field from its mean value. The first order velocity field is given by a Stokes-type problem (viscous terms are dominant) with relatively simple boundary conditions. The first order temperature field is that imposed from the end disks on a liquid bridge immersed in a non-conductive fluid. Radiative effects are supposed to be negligible. The second order temperature field, which accounts for convective effects, is split into three components, one due to the bulk motion, and the other two to the distortion of the free surface. The relative importance of these components in terms of the heat transfer to or from the end disks is assessed

Cabecera de televisión por cable

En el presente proyecto se realiza un estudio para la construcción de una cabecera de televisión por cable. Se trata de un proyecto puramente teórico en el que se especifican cada una de las partes que forman una cabecera de televisión y cómo funciona cada una de ellas. En un principio, se sitúa la cabecera de televisión dentro de una plataforma general de transmisión, para indicar sus funciones. Posteriormente, se analizan las distintas tecnologías que implementan esta transmisión y los estándares DVB que las rigen, como son DVB-C y DVB-C2 para las transmisiones por cable propiamente dichas y DVB-IPTV para las transmisiones por IP, para elegir cuál de las opciones es la más acertada y adaptar la cabecera de televisión a la misma. En cuanto al desarrollo teórico de la cabecera, se estudia el proceso que sigue la señal dentro de la misma, desde la recepción de los canales hasta el envío de los mismos hacia los hogares de los distintos usuarios, pasando previamente por las etapas de codificación y multiplexación. Además, se especifican los equipos necesarios para el correcto funcionamiento de cada una de las etapas. En la recepción, se reciben los canales por cada uno de los medios posibles (satélite, cable, TDT y estudio), que son demodulados y decodificados por el receptor. A continuación, son codificados (en este proyecto en MPEG-2 o H.264) para posteriormente ser multiplexados. En la etapa de multiplexación, se forma una trama Transport Stream por cada canal, compuesta por su flujo de video, audio y datos. Estos datos se trata de una serie de tablas (SI y PSI) que guían al set-topbox del usuario en la decodificación de los programas (tablas PSI) y que proporcionan información de cada uno de los mismos y del sistema (tablas SI). Con estas últimas el decodificador forma la EPG. Posteriormente, se realiza una segunda multiplexación, de forma que se incluyen múltiples programas en una sola trama Transport Stream (MPTS). Estos MPTS son los flujos que les son enviados a cada uno de los usuarios. El mecanismo de transmisión es de dos tipos en función del contenido y los destinatarios: multicast o unicast. Por último, se especifica el funcionamiento básico de un sistema de acceso condicional, así como su estructura, el cual es imprescindible en todas las cabeceras para asegurar que cada usuario solo visualiza los contenidos contratados. In this project, a study is realized for the cable television head-end construction . It is a theoretical project in which there are specified each of the parts that form a television headend and how their works each of them. At first, the television head-end places inside a general platform of transmission, to indicate its functions. Later, the different technologies that implement this transmission and the standards DVB that govern them are analyzed, since the standards that govern the cable transmissions (DVB-C and DVB-C2) to the standard that govern the IP transmissions (DVB-IPTV), to choose which of the options is the most guessed right and to adapt the television head-end to the same one. The theoretical development of the head-end, there is studied the process that follows the sign inside the same one, from the receipt of the channels up to the sending of the same ones towards the homes of the different users, happening before for the stages of codification and multiplexación. In addition, there are specified the equipments necessary for the correct functioning of each one of the stages. In the reception, the channels are receiving for each of the possible systems(satellite, cable, TDT and study), and they are demodulated and decoded by the receiver. Later, they are codified (in this project in MPEG-2 or H.264). The next stage is the stage of multiplexing. In the multiplexing stage, the channels are packetized in Transport Stream, composed by his video flow, audio and information. The information are composed by many tables(SI and PSI). The PSI tables guide the set-top-box of the user in the programs decoding and the SI tables provide information about the programs and system. With the information mentioned the decoder forms the EPG. Later, a second multiplexación is realized, so that there includes multiple programs in an alone Transport Stream (MPTS). These MPTS are the flows that are sent to each of the users. Two types of transmission are possible: unicast (VoD channels) and multicast (live channels). Finally, the basic functioning of a conditional access system is specified and his structure too, which is indispensable in all the head-end to assure that every users visualizes the contracted contents only.

Strengthening and rehabilitation of two damaged bridges in the motorway M-410 in Madrid (Spain)

The new highway M-410 in Madrid was constructed in the year 2007. This motorway near to Parla city crosses the road from Madrid to Toledo. To solve this crossing it was needed to constructed three bridges, the central with two spans over the existing motorway and the other two with one span at each side of the previous one.

A model of amacrine and ganglion cells for optical information processing

Based on a previously reported logic cell structure (see SPIE, vol. 2038, p. 67-77, 1993), the two types of cells present at the inner and ganglion cell layers of the vertebrate retina and their intracellular response, as well as their connections with each other, have been simulated. These cells are amacrines and ganglion cells. The main scheme of the authors' configuration is shown in a figure. These two types of cells, as well as some of their possible interconnections, have been implemented with the authors' previously reported optical-processing element. As it has been shown, the authors' logic structure is able to process two optical input binary signals, being the output two logical functions. Moreover, if a delayed feedback from one of the two possible outputs to one or both of the inputs is introduced, a very different behaviour is obtained. Depending on the value of the time delay, an oscillatory output can be obtained from a constant optical input signal. Period and length pulses are dependent on delay values, both external and internal, as well as on other control signals. Moreover, a chaotic behaviour can be obtained too under certain conditions

Aplicación de la física estadística a los medios granulares

Esta tesis se centra en el estudio de medios granulares blandos y atascados mediante la aplicación de la física estadística. Esta aproximación se sitúa entre los tradicionales enfoques macro y micromecánicos: trata de establecer cuáles son las propiedades macroscópicas esperables de un sistema granular en base a un análisis de las propiedades de las partículas y las interacciones que se producen entre ellas y a una consideración de las restricciones macroscópicas del sistema. Para ello se utiliza la teoría estadística junto con algunos principios, conceptos y definiciones de la teoría de los medios continuos (campo de tensiones y deformaciones, energía potencial elástica, etc) y algunas técnicas de homogeneización. La interacción entre las partículas es analizada mediante las aportaciones de la teoría del contacto y de las fuerzas capilares (producidas por eventuales meniscos de líquido cuando el medio está húmedo). La idea básica de la mecánica estadística es que entre todas soluciones de un problema físico (como puede ser el ensamblaje en equilibrio estático de partículas de un medio granular) existe un conjunto que es compatible con el conocimiento macroscópico que tenemos del sistema (por ejemplo, su volumen, la tensión a la que está sometido, la energía potencial elástica que almacena, etc.). Este conjunto todavía contiene un número enorme de soluciones. Pues bien, si no hay ninguna información adicional es razonable pensar que no existe ningún motivo para que alguna de estas soluciones sea más probable que las demás. Entonces parece natural asignarles a todas ellas el mismo peso estadístico y construir una función matemática compatible. Actuando de este modo se obtiene cuál es la función de distribución más probable de algunas cantidades asociadas a las soluciones, para lo cual es muy importante asegurarse de que todas ellas son igualmente accesibles por el procedimiento de ensamblaje o protocolo. Este enfoque se desarrolló en sus orígenes para el estudio de los gases ideales pero se puede extender para sistemas no térmicos como los analizados en esta tesis. En este sentido el primer intento se produjo hace poco más de veinte años y es la colectividad de volumen. Desde entonces esta ha sido empleada y mejorada por muchos investigadores en todo el mundo, mientras que han surgido otras, como la de la energía o la del fuerza-momento (tensión multiplicada por volumen). Cada colectividad describe, en definitiva, conjuntos de soluciones caracterizados por diferentes restricciones macroscópicas, pero de todos ellos resultan distribuciones estadísticas de tipo Maxwell-Boltzmann y controladas por dichas restricciones. En base a estos trabajos previos, en esta tesis se ha adaptado el enfoque clásico de la física estadística para el caso de medios granulares blandos. Se ha propuesto un marco general para estudiar estas colectividades que se basa en la comparación de todas las posibles soluciones en un espacio matemático definido por las componentes del fuerza-momento y en unas funciones de densidad de estados. Este desarrollo teórico se complementa con resultados obtenidos mediante simulación de la compresión cíclica de sistemas granulares bidimensionales. Se utilizó para ello un método de dinámica molecular, MD (o DEM). Las simulaciones consideran una interacción mecánica elástica, lineal y amortiguada a la que se ha añadido, en algunos casos, la fuerza cohesiva producida por meniscos de agua. Se realizaron cálculos en serie y en paralelo. Los resultados no solo prueban que las funciones de distribución de las componentes de fuerza-momento del sistema sometido a un protocolo específico parecen ser universales, sino que también revelan que existen muchos aspectos computacionales que pueden determinar cuáles son las soluciones accesibles. This thesis focuses on the application of statistical mechanics for the study of static and jammed packings of soft granular media. Such approach lies between micro and macromechanics: it tries to establish what the expected macroscopic properties of a granular system are, by starting from a micromechanical analysis of the features of the particles, and the interactions between them, and by considering the macroscopic constraints of the system. To do that, statistics together with some principles, concepts and definitions of continuum mechanics (e.g. stress and strain fields, elastic potential energy, etc.) as well as some homogenization techniques are used. The interaction between the particles of a granular system is examined too and theories on contact and capillary forces (when the media are wet) are revisited. The basic idea of statistical mechanics is that among the solutions of a physical problem (e.g. the static arrangement of particles in mechanical equilibrium) there is a class that is compatible with our macroscopic knowledge of the system (volume, stress, elastic potential energy,...). This class still contains an enormous number of solutions. In the absence of further information there is not any a priori reason for favoring one of these more than any other. Hence we shall naturally construct the equilibrium function by assigning equal statistical weights to all the functions compatible with our requirements. This procedure leads to the most probable statistical distribution of some quantities, but it is necessary to guarantee that all the solutions are likely accessed. This approach was originally set up for the study of ideal gases, but it can be extended to non-thermal systems too. In this connection, the first attempt for granular systems was the volume ensemble, developed about 20 years ago. Since then, this model has been followed and improved upon by many researchers around the world, while other two approaches have also been set up: energy and force-moment (i.e. stress multiplied by volume) ensembles. Each ensemble is described by different macroscopic constraints but all of them result on a Maxwell-Boltzmann statistical distribution, which is precisely controlled by the respective constraints. According to this previous work, in this thesis the classical statistical mechanics approach is introduced and adapted to the case of soft granular media. A general framework, which includes these three ensembles and uses a force-moment phase space and a density of states function, is proposed. This theoretical development is complemented by molecular dynamics (or DEM) simulations of the cyclic compression of 2D granular systems. Simulations were carried out by considering spring-dashpot mechanical interactions and attractive capillary forces in some cases. They were run on single and parallel processors. Results not only prove that the statistical distributions of the force-moment components obtained with a specific protocol seem to be universal, but also that there are many computational issues that can determine what the attained packings or solutions are.

Sintetizador analógico en Arduino

Este proyecto consiste en el diseño y construcción de un sintetizador basado en el chip 6581 Sound Interface Device (SID). Este chip era el encargado de la generación de sonido en el Commodore 64, ordenador personal comercializado en 1982, y fue el primer sintetizador complejo construido para ordenador. El chip en cuestión es un sintetizador de tres voces, cada una de ellas capaz de generar cuatro diferentes formas de onda. Cada voz tiene control independiente de varios parámetros, permitiendo una relativamente amplia variedad de sonidos y efectos, muy útil para su uso en videojuegos. Además está dotado de un filtro programable para conseguir distintos timbres mediante síntesis sustractiva. El sintetizador se ha construido sobre Arduino, una plataforma de electrónica abierta concebida para la creación de prototipos, consistente en una placa de circuito impreso con un microcontrolador, programable desde un PC para que realice múltiples funciones (desde encender LEDs hasta controlar servomecanismos en robótica, procesado y transmisión de datos, etc.). El sintetizador es controlable vía MIDI, por ejemplo, desde un teclado de piano. A través de MIDI recibe información tal como qué notas debe tocar, o los valores de los parámetros del SID que modifican las propiedades del sonido. Además, toda esa información también la puede recibir de un PC mediante una conexión USB. Se han construido dos versiones del sintetizador: una versión “hardware”, que utiliza el SID para la generación de sonido, y otra “software”, que reemplaza el SID por un emulador, es decir, un programa que se comporta (en la medida de lo posible) de la misma manera que el SID. El emulador se ha implementado en un microcontrolador Atmega 168 de Atmel, el mismo que utiliza Arduino. ABSTRACT. This project consists on design and construction of a synthesizer which is based on chip 6581 Sound Interface Device (SID). This chip was used for sound generation on the Commodore 64, a home computer presented in 1982, and it was the first complex synthesizer built for computers. The chip is a three-voice synthesizer, each voice capable of generating four different waveforms. Each voice has independent control of several parameters, allowing a relatively wide variety of sounds and effects, very useful for its use on videogames. It also includes a programmable filter, allowing more timbre control via subtractive synthesis. The synthesizer has been built on Arduino, an open-source electronics prototyping platform that consists on a printed circuit board with a microcontroller, which is programmable with a computer to do several functions (lighting LEDs, controlling servomechanisms on robotics, data processing or transmission, etc.). The synthesizer is controlled via MIDI, in example, from a piano-type keyboard. It receives from MIDI information such as the notes that should be played or SID’s parameter values that modify the sound. It also can receive that information from a PC via USB connection. Two versions of the synthesizer have been built: a hardware one that uses the SID chip for sound generation, and a software one that replaces SID by an emulator, it is, a program that behaves (as far as possible) in the same way the SID would. The emulator is implemented on an Atmel’s Atmega 168 microcontroller, the same one that is used on Arduino.

Model-based Self-awareness Patterns for Autonomy

Los sistemas técnicos son cada vez más complejos, incorporan funciones más avanzadas, están más integrados con otros sistemas y trabajan en entornos menos controlados. Todo esto supone unas condiciones más exigentes y con mayor incertidumbre para los sistemas de control, a los que además se demanda un comportamiento más autónomo y fiable. La adaptabilidad de manera autónoma es un reto para tecnologías de control actualmente. El proyecto de investigación ASys propone abordarlo trasladando la responsabilidad de la capacidad de adaptación del sistema de los ingenieros en tiempo de diseño al propio sistema en operación. Esta tesis pretende avanzar en la formulación y materialización técnica de los principios de ASys de cognición y auto-consciencia basadas en modelos y autogestión de los sistemas en tiempo de operación para una autonomía robusta. Para ello el trabajo se ha centrado en la capacidad de auto-conciencia, inspirada en los sistemas biológicos, y se ha explorado la posibilidad de integrarla en la arquitectura de los sistemas de control. Además de la auto-consciencia, se han explorado otros temas relevantes: modelado funcional, modelado de software, tecnología de los patrones, tecnología de componentes, tolerancia a fallos. Se ha analizado el estado de la técnica en los ámbitos pertinentes para las cuestiones de la auto-consciencia y la adaptabilidad en sistemas técnicos: arquitecturas cognitivas, control tolerante a fallos, y arquitecturas software dinámicas y computación autonómica. El marco teórico de ASys existente de sistemas autónomos cognitivos ha sido adaptado para servir de base para este análisis de autoconsciencia y adaptación y para dar sustento conceptual al posterior desarrollo de la solución. La tesis propone una solución general de diseño para la construcción de sistemas autónomos auto-conscientes. La idea central es la integración de un meta-controlador en la arquitectura de control del sistema autónomo, capaz de percibir la estado funcional del sistema de control y, si es necesario, reconfigurarlo en tiempo de operación. Esta solución de metacontrol se ha formalizado en cuatro patrones de diseño: i) el Patrón Metacontrol, que define la integración de un subsistema de metacontrol, responsable de controlar al propio sistema de control a través de la interfaz proporcionada por su plataforma de componentes, ii) el patrón Bucle de Control Epistémico, que define un bucle de control cognitivo basado en el modelos y que se puede aplicar al diseño del metacontrol, iii) el patrón de Reflexión basada en Modelo Profundo propone una solución para construir el modelo ejecutable utilizado por el meta-controlador mediante una transformación de modelo a modelo a partir del modelo de ingeniería del sistema, y, finalmente, iv) el Patrón Metacontrol Funcional, que estructura el meta-controlador en dos bucles, uno para el control de la configuración de los componentes del sistema de control, y otro sobre éste, controlando las funciones que realiza dicha configuración de componentes; de esta manera las consideraciones funcionales y estructurales se desacoplan. La Arquitectura OM y el metamodelo TOMASys son las piezas centrales del marco arquitectónico desarrollado para materializar la solución compuesta de los patrones anteriores. El metamodelo TOMASys ha sido desarrollado para la representación de la estructura y su relación con los requisitos funcionales de cualquier sistema autónomo. La Arquitectura OM es un patrón de referencia para la construcción de una metacontrolador integrando los patrones de diseño propuestos. Este meta-controlador se puede integrar en la arquitectura de cualquier sistema control basado en componentes. El elemento clave de su funcionamiento es un modelo TOMASys del sistema decontrol, que el meta-controlador usa para monitorizarlo y calcular las acciones de reconfiguración necesarias para adaptarlo a las circunstancias en cada momento. Un proceso de ingeniería, complementado con otros recursos, ha sido elaborado para guiar la aplicación del marco arquitectónico OM. Dicho Proceso de Ingeniería OM define la metodología a seguir para construir el subsistema de metacontrol para un sistema autónomo a partir del modelo funcional del mismo. La librería OMJava proporciona una implementación del meta-controlador OM que se puede integrar en el control de cualquier sistema autónomo, independientemente del dominio de la aplicación o de su tecnología de implementación. Para concluir, la solución completa ha sido validada con el desarrollo de un robot móvil autónomo que incorpora un meta-controlador con la Arquitectura OM. Las propiedades de auto-consciencia y adaptación proporcionadas por el meta-controlador han sido validadas en diferentes escenarios de operación del robot, en los que el sistema era capaz de sobreponerse a fallos en el sistema de control mediante reconfiguraciones orquestadas por el metacontrolador. ABSTRACT Technical systems are becoming more complex, they incorporate more advanced functionalities, they are more integrated with other systems and they are deployed in less controlled environments. All this supposes a more demanding and uncertain scenario for control systems, which are also required to be more autonomous and dependable. Autonomous adaptivity is a current challenge for extant control technologies. The ASys research project proposes to address it by moving the responsibility for adaptivity from the engineers at design time to the system at run-time. This thesis has intended to advance in the formulation and technical reification of ASys principles of model-based self-cognition and having systems self-handle at runtime for robust autonomy. For that it has focused on the biologically inspired capability of self-awareness, and explored the possibilities to embed it into the very architecture of control systems. Besides self-awareness, other themes related to the envisioned solution have been explored: functional modeling, software modeling, patterns technology, components technology, fault tolerance. The state of the art in fields relevant for the issues of self-awareness and adaptivity has been analysed: cognitive architectures, fault-tolerant control, and software architectural reflection and autonomic computing. The extant and evolving ASys Theoretical Framework for cognitive autonomous systems has been adapted to provide a basement for this selfhood-centred analysis and to conceptually support the subsequent development of our solution. The thesis proposes a general design solution for building self-aware autonomous systems. Its central idea is the integration of a metacontroller in the control architecture of the autonomous system, capable of perceiving the functional state of the control system and reconfiguring it if necessary at run-time. This metacontrol solution has been formalised into four design patterns: i) the Metacontrol Pattern, which defines the integration of a metacontrol subsystem, controlling the domain control system through an interface provided by its implementation component platform, ii) the Epistemic Control Loop pattern, which defines a modelbased cognitive control loop that can be applied to the design of such a metacontroller, iii) the Deep Model Reflection pattern proposes a solution to produce the online executable model used by the metacontroller by model-to-model transformation from the engineering model, and, finally, iv) the Functional Metacontrol pattern, which proposes to structure the metacontroller in two loops, one for controlling the configuration of components of the controller, and another one on top of the former, controlling the functions being realised by that configuration; this way the functional and structural concerns become decoupled. The OM Architecture and the TOMASys metamodel are the core pieces of the architectural framework developed to reify this patterned solution. The TOMASys metamodel has been developed for representing the structure and its relation to the functional requirements of any autonomous system. The OM architecture is a blueprint for building a metacontroller according to the patterns. This metacontroller can be integrated on top of any component-based control architecture. At the core of its operation lies a TOMASys model of the control system. An engineering process and accompanying assets have been constructed to complete and exploit the architectural framework. The OM Engineering Process defines the process to follow to develop the metacontrol subsystem from the functional model of the controller of the autonomous system. The OMJava library provides a domain and application-independent implementation of an OM Metacontroller than can be used in the implementation phase of OMEP. Finally, the complete solution has been validated in the development of an autonomous mobile robot that incorporates an OM metacontroller. The functional selfawareness and adaptivity properties achieved thanks to the metacontrol system have been validated in different scenarios. In these scenarios the robot was able to overcome failures in the control system thanks to reconfigurations performed by the metacontroller.

Resistance to malathion and lambda-cyhalothrin in Ceratitis capitata (Wiedemann) (Diptera: Tephritidae)

La mosca mediterránea de la fruta, Ceratitis capitata (Wiedemann, 1824) (Diptera: Tephritidae), es una de las plagas de mayor incidencia económica en cítricos y otros frutales a nivel mundial. En España las medidas de control de esta plaga en cítricos, desde mediados de los 90 hasta 2009, se basaron principalmente en el monitoreo de las poblaciones y en la aplicación de tratamientos aéreos y terrestres con malatión cebo. Sin embargo, desde la retirada en la Unión Europea en 2009 de los productos fitosanitarios que contienen malatión, los insecticidas más utilizados para el control de esta plaga han sido lambda-cihalotrina y spinosad. En 2004-2005 se detectaron poblaciones españolas de C. capitata resistentes a malatión. Esta resistencia se ha asociado a una mutación (G328A) en la acetilcolinesterasa (AChE), a una duplicación del gen de la AChE (Ccace2) (una de las copias lleva la mutación G328A) y a resistencia metabólica mediada por esterasas (posiblemente aliesterasas). Sin embargo, cuando se secuenció la aliesterasa CcE7 en individuos de una línea resistente a malatión, no se encontró ninguna de las mutaciones (G137D y/o W251L/S/G) asociadas a resistencia en otras especies, si bien se encontraron otras mutaciones al compararlos con individuos de una línea susceptible. Asimismo, mediante la selección en laboratorio de una línea resistente a malatión (W-4Km) con lambda-cihalotrina, se ha podido obtener una línea resistente a lambda-cihalotrina (W-1K). Finalmente, se ha demostrado la capacidad de esta especie para desarrollar resistencia a spinosad mediante selección en laboratorio. Los múltiples mecanismos de resistencia identificados evidencian el potencial de esta especie para desarrollar resistencia a insecticidas con diferentes modos de acción. Los objetivos de esta tesis doctoral son: 1) evaluar la susceptibilidad de poblaciones españolas de campo de C. capitata a lambda-cihalotrina y dilucidar los mecanismos de resistencia en la línea W-1Kλ; 2) comparar la herencia, el coste biológico y la estabilidad de la resistencia a malatión mediada por la mutación G328A y la duplicación del gen Ccace2 (una de las copias lleva la mutación G328A); y 3) investigar el papel de las mutaciones identificadas en la aliesterasa CcαE7 en la resistencia a malatión. Estos estudios son de utilidad para el desarrollo de estrategias de manejo de la resistencia que puedan prevenir o retrasar la aparición de resistencia y aumentar la sostenibilidad de los insecticidas disponibles para el control de esta plaga. Nuestros resultados indican que las poblaciones españolas de C. capitata analizadas han desarrollado resistencia a lambda-cihalotrina. Los valores de CL50 estimados para las poblaciones recogidas en la Comunidad Valenciana, Cataluña y Andalucía oscilaron entre 129 ppm y 287 ppm, igualando o sobrepasando la concentración recomendada para los tratamientos de campo (125 ppm). Estos resultados contrastan con los obtenidos con tres poblaciones de campo recogidas en Túnez, cuya susceptibilidad fue similar a la de la línea control (C). La línea resistente a lambda-cihalotrina W-1K se continuó seleccionando en el laboratorio alcanzándose unos niveles de resistencia de 205 veces con respecto a la línea C, siendo su CL50 (4224 ppm) más de 30 veces superior a la concentración recomendada para los tratamientos de campo. Esta línea resistente mostró altos niveles de resistencia cruzada a deltametrina (150 veces) y a etofenprox (240 veces), lo que sugiere que el desarrollo de resistencia a lambda-cihalotrina podría comprometer la eficacia de otros piretroides para el control de esta plaga. Hemos demostrado que la resistencia de la línea W-1K a lambda-cihalotrina fue casi completamente suprimida por el sinergista PBO, lo que indica que las enzimas P450 desempeñan un papel muy importante en la resistencia a este insecticida. Sin embargo, tanto las moscas de la línea susceptible C como las de la línea resistente W-1K perdieron inmediatamente la capacidad de caminar (efecto “knock-down”) al ser tratadas tópicamente con lambda-cihalotrina, lo que sugiere que la resistencia no está mediada por alteraciones en la molécula diana (resistencia tipo “kdr”). La resistencia metabólica mediada por P450 fue analizada comparando la expresión de 53 genes CYP (codifican enzimas P450) de las familias CYP4, CYP6, CYP9 y CYP12 en adultos de la línea resistente W-1K y de la línea susceptible C. Nuestros resultados muestran que el gen CYP6A51 (número de acceso GenBank XM_004534804) fue sobreexpresado (13-18 veces) en la línea W-1K. Por otra parte, la expresión del gen CYP6A51 fue inducida tanto en adultos de la línea W-1K como de la línea C al ser tratados con lambda-cihalotrina. Sin embargo, no se obtuvieron diferencias significativas entre la línea susceptible C y la línea resistente W-1K al comparar la cantidad de P450 y la actividad NADPH-citocromo c reductasa presente en fracciones microsomales obtenidas a partir de abdómenes. Asimismo, no hemos podido correlacionar el metabolismo de deltametrina, estimado in vitro mediante la incubación de este insecticida con fracciones microsomales, con el nivel de resistencia a este piretroide observado en los bioensayos con la línea W-1K. Por otro lado, no se encontró ninguna alteración en la región promotora 5'UTR del gen CYP6A51 (-500 pb desde el inicio de la traducción) que pudiera explicar su sobreexpresión en la línea W-1K. Los datos obtenidos sugieren que la resistencia a lambda-cihalotrina en la línea W-1K está mediada por P450 y que la sobreexpresión de CYP6A51 puede desempeñar un papel importante, aunque se necesitan más evidencias para establecer una asociación directa de la resistencia con este gen. Hemos estudiado la herencia, el coste biológico y la estabilidad de la resistencia a malatión mediada por la mutación G328A y la duplicación del gen Ccace2 (una de las copias lleva la mutación G328A). La línea susceptible C, donde no se encuentra la mutación G328A (genotipo S/S), se cruzó con dos isolíneas establecidas para representar genotipos únicos correspondientes a los dos mecanismos de resistencia asociados a la molécula diana: 1) la isolínea 267Y (genotipo R/R) establecida a partir de una pareja que portaba la mutación G328A en homocigosis; 2) la isolínea 306TY (genotipo RS/RS) establecida a partir de una pareja que portaba en homocigosis la duplicación del gen Ccace2. No se realizaron cruces recíprocos, ya que mediante experimentos de hibridación in situ en cromosomas politénicos se pudo comprobar que el locus de la AChE y la duplicación (probablemente en tándem) se localizan en el cromosoma autosómico 2L. La susceptibilidad al malatión de los parentales resistentes (R/R o RS/RS) y susceptibles (S/S), los cruces F1 (S/R, S/RS y R/RS) y los retrocruzamientos indican que la resistencia a malatión es semi-dominante en ambos casos. Sin embargo, nuestros resultados no fueron concluyentes con respecto a la naturaleza monogénica de la resistencia a malatión en estas isolíneas. Por lo tanto, no podemos descartar que otros genes que contribuyan a la resistencia, además de la mutación G328A (isolínea 267Y) y de la duplicación del gen Ccace2 (isolínea 306TY), puedan haber sido seleccionados durante el proceso de selección de 267Y y 306TY. Varios parámetros biológicos fueron evaluados para determinar si estos dos mecanismos de resistencia a malatión suponen un coste biológico para los genotipos resistentes. Individuos con genotipo R/R mostraron un retraso en el tiempo de desarrollo de huevo a pupa, un peso de pupa reducido y una menor longevidad de los adultos, en comparación con los individuos con genotipo S/S. Sin embargo, el peso de pupa de los individuos con genotipo RS/RS fue similar al de los individuos S/S, y su desarrollo de huevo a pupa intermedio entre S/S y R/R. Estas diferencias en el coste biológico pueden estar relacionadas con la reducción de la eficiencia catalítica de la AChE mutada en los individuos R/R, y al efecto compensatorio que la copia no mutada del gen tiene en los individuos RS/RS que portan la duplicación. La estabilidad de la resistencia a malatión mediada por la mutación G328A y la duplicación se analizó mediante el seguimiento de los caracteres de resistencia en la progenie de retrocruzamientos S/R x R/R y S/RS x RS/RS a lo largo de varias generaciones en ausencia de presión de selección con insecticidas. Nuestros resultados muestran que la frecuencia del alelo que porta la mutación G328A disminuyó desde 67,5% en la primera generación del retrocruzamiento S/R x R/R (75% esperado, asumiendo segregación mendeliana y que sólo hay dos alelos: uno mutado y otro no mutado) a 12% después de 10 generaciones. Por el contrario, la frecuencia de la duplicación sólo disminuyó desde 75% en en la primera generación del retrocruzamiento S/RS x RS/RS (75% esperado, asumiendo segregación Mendeliana y que la duplicación segrega como un único alelo) a 50% en el mismo período, lo que indica que la duplicación es más estable que la mutación. Asimismo, se analizó la presencia de la mutación y de la duplicación en poblaciones de campo recogidas en seis localidades en 2004-2007, cuando todavía se usaba el malatión, y se comparó con poblaciones recogidas en los mismos campos en 2010, un año después de la prohibición del malatión en la Unión Europea. La frecuencia media del genotipo susceptible (S/S) aumentó del 55,9% en el período 2004-2007 a 70,8% en 2010, mientras que la frecuencia de los genotipos portadores de la mutación en homocigosis o heterocigosis (R/R y S/R) disminuyó del 30,4 al 9,2%, los que llevan la duplicación en homocigosis o heterocigosis (RS/RS y S/RS) aumentaron levemente desde 12,8 hasta 13,3%, y los que llevan a la vez la mutación y la duplicación (R/RS) también aumentaron del 1 al 6,7%. Estos resultados son consistentes con que la duplicación del gen Ccace2 (con una copia con la mutación G328A y la otra copia no mutada) es más ventajosa que la mutación G328A por si sola, ya que la duplicación mantiene los niveles de resistencia a la vez que limita el coste biológico. Para investigar la asociación entre la resistencia a malatión y las mutaciones encontradas previamente en CcE7, hemos generado isolíneas con mutaciones específicas seleccionadas por su ubicación próxima a la entrada al centro activo de la enzima. La isolínea Sm2 (procedente de una hembra heterocigota para la mutación V96L y un macho homocigoto para el alelo no mutado) mantuvo altos niveles de resistencia a malatión, incluso después de 30 generaciones sin presión de selección. Por el contrario, la isolínea 267Y (compuesta por individuos homocigotos para la mutación L267Y) y la línea 306TY (compuesta por individuos homocigotos para la doble mutación R306T-N307Y) mostraron una reducción significativa en los niveles de resistencia. También hemos encontrado que la resistencia a malatión de la línea Sm2 fue parcialmente revertida por DEF y TPP, y que Sm2 mostró una reducción significativa en la actividad MTB, como se ha descrito en otras especies que muestran resistencia específica a malatión mediada por aliesterases. Además, fue posible asociar la presencia de la mutación V96L en individuos de la línea Sm2 con supervivencia a una concentración discriminante de malatión (5,000 ppm) y con una baja actividad MTB. Estos resultados sugieren una posible relación entre la mutación V96L en la aliesterasa CcE7 y la resistencia a malatión, aunque todavía no se puede concluir que la resistencia es causada por esta mutación, siendo necesarios más estudios para comprobar su contribución a la resistencia. En conclusión, se ha encontrado por primera vez resistencia a lambda-cihalotrina en poblaciones de campo de C. capitata, y nuestros resultados indican que las P450 son el principal mecanismo de resistencia en la línea W-1K. Esta situación se suma al caso previamente descrito de resistencia en campo a malatión asociada a la mutación G328A, a la duplicación del gen Ccace2 (una de las copias lleva la mutación G328A) y a resistencia metabólica mediada por esterasas. Nuestros resultados también indican que la alteración de la molécula diana AChE parece ser responsable de un cierto nivel de resistencia a malatión en C. capitata, que puede ser estimada como aproximadamente 25-40 veces para la mutación G328A y 40-60 veces para la duplicación; mientras que la resistencia mediada por esterasas y que ha sido asociada en este estudio con la mutación V96L en CcE7 puede conferir un efecto multiplicativo (por un factor de 5 a 10) aumentando la resistencia a malatión a 200-400 veces. Por otra parte, hemos demostrado que los insectos resistentes que llevan la duplicación tienen un coste biológico menor y muestran una estabilidad mayor que aquellos con la mutación G328A en ausencia de presión de selección con insecticidas. Esto representa un escenario en el que los genotipos con la duplicación permanecerán en el campo en frecuencias bajas a moderadas, pero podrían ser seleccionados rápidamente si se utilizan malatión u otros insecticidas que muestren resistencia cruzada. Estos resultados tienen importantes implicaciones para los programas de manejo de la resistencia, ya que el repertorio de insecticidas eficaces para el control de C. capitata es cada vez más limitado. Además, la coexistencia de múltiples mecanismos de resistencia en poblaciones de campo ofrece el potencial para desarrollar resistencia frente a otros insecticidas disponibles para el control de esta plaga. Estrategias para de manejo de la resistencia basadas en la alternancia de insecticidas con diferentes modos de acción, y su combinación con otros métodos de control, deben ser implementadas para evitar el desarrollo de resistencia en campo. ABSTRACT The Mediterranean fruit fly (Medfly), Ceratitis capitata (Wiedemann, 1824) (Diptera: Tephritidae), is one of the most economically damaging pests of citrus and other fruit crops worldwide. Control measures in citrus crops in Spain from the mid 90's to 2009 were mainly based on field monitoring of population levels and aerial and ground treatments with malathion bait sprays. However, since the withdrawal of phytosanitary products containing malathion in the European Union in 2009, lambda-cyhalothrin and spinosad have become the most widely used insecticides for the control of this pest. Resistance to malathion was found in Spanish field populations of C. capitata in 2004-2005. This resistance has been associated with a mutation G328A in the acetylcholinesterase (AChE), a duplication of the AChE gene (Ccace2) (one of the copies bearing the mutation G328A), and metabolic resistance mediated by esterases (probably aliesterases). However, when the gene of the aliesterase CcE7 was sequenced in individuals from a malathion resistant strain of C. capitata, none of the known G137D and/or W251L/S/G mutations associated to resistance in other species were found, though other mutations were detected when compared with individuals from a susceptible strain. Noteworthy, a lambda-cyhalothrin resistant strain (W-1K) was obtained by selecting a field-derived malathion resistant strain (W-4Km) with lambda-cyhalothrin. Moreover, it has also been demonstrated the capacity of this species to develop resistance to spinosad by laboratory selection. The multiple resistance mechanisms identified highlight the potential of this species to develop resistance to insecticides with different modes of action. The objectives of this PhD Thesis are: 1) to assess the susceptibility of Spanish field populations of C. capitata to lambda-cyhalothrin and to elucidate the resistance mechanisms in the W-1Kλ strain; 2) to compare the inheritance, fitness cost and stability of the malathion resistance mediated by the G328A mutation and the duplication of the Ccace2 gene (with one of the copies bearing the mutation G328A); and 3) to investigate the role of the aliesterase CcαE7 mutations in malathion resistance. All these studies will be of use for devising proactive resistance management strategies that could prevent or delay resistance development and would increase the sustainability of the insecticides available for Medfly control. Our results indicate that Spanish field populations of C. capitata have developed resistance to lambda-cyhalothrin. The LC50 values estimated for populations collected at Comunidad Valenciana, Cataluña and Andalucía ranged from 129 ppm to 287 ppm, equaling or overpassing the recommended concentration for field treatments (125 ppm). These results contrast with those obtained with three different Tunisian field populations, whose susceptibility was similar to that of the control (C) strain. The lambda-cyhalothrin resistant W-1K strain has been further selected to achieve a 205-fold resistance compared to the C strain, being its LC50 (4,224 ppm) more than 30 times higher than the recommended concentration for field applications. This resistant strain showed high levels of cross-resistance to deltamethrin (150-fold) and etofenprox (240-fold), suggesting that the development of resistance to lambda-cyhalothrin may compromise the effectiveness of other pyrethroids for the control of this species. We have shown that the resistance of the W-1K strain to lambda-cyhalothrin was almost completely suppressed by the synergist PBO, indicating that P450 enzymes play a very important role in resistance to this insecticide. However, both susceptible C and resistant W-1K flies were knocked down after topical treatment with lambda-cyhalothrin, suggesting that kdr resistance mediated by alterations of the target site is not playing a major role. Metabolic resistance mediated by P450 was further analyzed by comparing the expression of 53 genes of the families CYP4, CYP6, CYP9 and CYP12 in adults flies from the resistant W-1K and the susceptible C strains. We found that the gene CYP6A51 (GenBank accession number XM_004534804) was overexpressed (13-18-fold) in the W-1K strain. Moreover, the expression of the CYP6A51 gene was induced when adults of the W-1K and C strains were treated with lambda-cyhalothrin. However, no significant differences were obtained between susceptible C and resistant W-1K strains for the quantity of P450 and for the activity of NADPH- cytochrome c reductase measured in microsomal fractions obtained from abdomens. Moreover, we failed to correlate the metabolism of deltamethrin, analyzed in vitro by incubating this insecticide with microsomal fractions, with the resistance level against this pyrethroid observed in bioassays with W-1K. The sequencing of the 5´UTR region of the CYP6A51 gene failed in finding an alteration in the promoter region (-500 bp from translation start site) that could explain overexpression in the W-1K strain. All data obtained suggest that resistance to lambda-cyhalothrin in the W- 1K strain is mediated by P450 and that overexpression of CYP6A51 may play a major role, although further evidences are needed to establish a direct association of resistance with this gene. We have studied the inheritance, fitness cost and stability of the malathion resistance mediated by the G328A mutation and the duplication of the Ccace2 gene (with one of the copies bearing the mutation G328A). The malathion-susceptible C strain where the G328A mutation is not found (S/S genotype) was crossed with two isolines established to represent unique genotypes corresponding to the two target-site resistance mechanisms: 1) the 267Y isoline (genotype R/R) was established from a couple bearing the mutation G328A in homozygosis; and 2) the 306TY isoline (genotype RS/RS) was established from a couple being homozygous for the duplication of the Ccace2 gene. Reciprocal crosses have not been performed, since in situ hybridization on polythene chromosomes showed that the AChE locus and the duplication (most probably in tandem) are placed at the autosomal chromosome 2L. Mortality responses to malathion of resistant isolines (R/R or RS/RS) and susceptible (S/S) genotypes, F1 crosses (S/R, S/RS, and R/RS), and the back-crosses indicated that resistance to malathion is inherited as a semi-dominant trait in both cases. However, our results were not conclusive about the monogenic nature of the resistance to malathion in these isolines. Thus, we can not discard that other genes contributing to resistance, in addition to the mutation G328A (isoline 267Y) and the duplication of the Ccace2 gene (isoline 306TY), may have been selected during the selection process of 267Y and 306TY. Several biological parameters were evaluated to determine if these two malathion resistance mechanisms impose a fitness cost for resistant genotypes. Individuals with genotype R/R have a reduced fitness in terms of developmental time from egg to pupa, pupal weight and adult longevity, when compared to susceptible individuals (genotype S/S). Interestingly, the fitness cost was substantially diminished in individuals with genotype RS/RS. These differences in fitness may be related to the reduction of the catalytic efficiency of mutated AChE in individuals R/R, and the compensatory effect that the non-mutated copy of the gene has on individuals RS/RS bearing the duplication. The stability of malathion reistance associated with the mutation G328A or the duplication was analyzed by following these resistant traits in the progeny of the back-crosses S/RS x RS/RS and S/R x R/R over consecutive generations in the absence of insecticide selection pressure. Our results show that the frequency of the allele bearing the mutation G328A decreased from 67.5% at the first generation of the back-cross S/R x R/R (75% expected, assuming Mendelian segregation and that there are only two alleles: one mutated and the other non-mutated) to 12% after 10 generations. By contrast, the frequency of the duplication only declined from 75% at the first generation of the back-cross S/RS x RS/RS (75% expected, assuming Mendelian segregation and that the duplication segregates as an unique allele) to 50% in the same period, indicating that the duplication is more stable than the mutation. The presence of the mutation and the duplication was analyzed in field populations collected in six localities in 2004-2007, when malathion was still used, and compared to populations collected in the same fields in 2010, one year after the prohibition of malathion in the European Union. The average frequency of the susceptible genotype (S/S) increased from 55.9% in the period 2004-2007 to 70.8% in 2010, whereas the frequency of those genotypes carrying the mutation in homozygosis or heterozygosis (R/R and S/R) declined from 30.4 to 9.2%, those carrying the duplication in homozygosis or heterozygosis (RS/RS and S/RS) increased slightly from 12.8 to 13.3%, and those carrying both the mutation and the duplication (R/RS) also increased from 1 to 6.7%. These results are consistent with the duplication of the Ccace2 gene (with one of the copies bearing the mutation G328A and the other copy non-mutated) being more advantageous than the G328A mutation alone by maintaining resistance while restoring part of the fitness. In order to investigate the association of malathion resistance with mutations previously found in the aliesterase CcE7, we have generated isolines bearing specific mutations selected by their putative location near the upper part of the active site gorge of the enzyme. The isoline Sm2 (originating from a female heterozygous for the mutation V96L and a male homozygous for the non-mutated allele) kept high levels of resistance to malathion, even after 30 generations without selection pressure. On the contrary, the isoline 267Y (composed by individuals homozygous for the mutation L267Y) and the strain 306TY (composed by homozygous for the double mutation R306T-N307Y) showed a significant reduction in the levels of resistance. We have found also that resistance to malathion in the Sm2 isoline was partially reverted by DEF and TPP, and that Sm2 showed a significant reduction in MTB activity, as reported for other species showing malathion-specific resistance mediated by aliesterases. Besides, it was possible to associate the presence of the mutation V96L in individuals from the Sm2 isoline with both survival to a discriminating concentration of malathion (5,000 ppm) and low MTB activity. Our results point out to a possible connection betwen the mutation V96L in the aliesterase CcE7 and resistance to malathion, though we can not yet conclude that the resistance is caused by the mutation, being needed further work to understand its contribution to resistance. In conclusion, resistance to lambda-cyhalothrin has been found for the first time in field populations of C. capitata, and metabolic resistance mediated by P450 appears to be the main resistance mechanism in the resistant strain W-1K. These findings add to the previously reported case of field resistance to malathion, associated to the G328A mutation and the duplication of the Ccace2 gene (with one of the copies bearing the mutation G328A) and to metabolic resistance mediated by esterases. Our results also indicate that altered target site AChE appears to be responsible for a certain level of resistance to malathion in C. capitata, that can be estimated as about 25-40-fold for the mutation G328A and 40-60-fold for the duplication; whereas metabolic resistance mediated by esterases and associated in this study with the mutation V96L in CcE7 may confer a multiplicative effect (by a factor of 5 to10) increasing malathion resistance to 200-400-fold. Moreover, we have shown that resistant insects carrying the duplication have better fitness and exhibit a higher stability than those with the mutation G328A in the absence of insecticide pressure. This represents a scenario where genotypes with the duplication will remain in the field at low to moderate frequencies, but could be rapidly selected if malathion or other insecticides showing cross-resistance are used. These findings have important implications for resistance management programs, as the repertoire of effective insecticides for C. capitata control is becoming very limited. Besides, multiple resistance mechanisms coexisting in field populations provide the potential to develop resistance to other available insecticides for the control of this pest. Appropriate resistance management strategies based on the alternation of insecticides with different modes of action, and their combination with other control methods, must then be implemented to avoid the evolution of resistance in the field.