Development of replication-defective lymphocytic choriomeningitis virus vectors for the induction of potent CD8+ T cell immunity

Lymphocytic choriomeningitis virus (LCMV) exhibits natural tropism for dendritic cells and represents the prototypic infection that elicits protective CD8(+) T cell (cytotoxic T lymphocyte (CTL)) immunity. Here we have harnessed the immunobiology of this arenavirus for vaccine delivery. By using producer cells constitutively synthesizing the viral glycoprotein (GP), it was possible to replace the gene encoding LCMV GP with vaccine antigens to create replication-defective vaccine vectors. These rLCMV vaccines elicited CTL responses that were equivalent to or greater than those elicited by recombinant adenovirus 5 or recombinant vaccinia virus in their magnitude and cytokine profiles, and they exhibited more effective protection in several models. In contrast to recombinant adenovirus 5, rLCMV failed to elicit vector-specific antibody immunity, which facilitated re-administration of the same vector for booster vaccination. In addition, rLCMV elicited T helper type 1 CD4+ T cell responses and protective neutralizing antibodies to vaccine antigens. These features, together with low seroprevalence in humans, suggest that rLCMV may show utility as a vaccine platform against infectious diseases and cancer.

Small hive beetles, Aethina tumida, are vectors of Paenibacillus larvae

The transmission of honeybee pathogens by free-flying pests, such as small hive beetles (=SHB), would be independent of bees and beekeepers and thereby constitute a new challenge for pathogen control measures. Here we show that larval and adult SHB become contaminated with Paenibacillus larvae spores when exposed to honeybee brood combs with clinical American foulbrood (=AFB) symptoms in the laboratory. This contamination persists in pupae and newly emerged adults. After exposure to contaminated adult SHB, honeybee field colonies showed higher numbers of P. larvae spores in worker and honey samples after five weeks. Despite these results, the rather low number of P. larvae spores on adult SHB suggests that clinical AFB outbreaks are not likely. However, even small spore numbers can be sufficient to spread P. larvae. Therefore, our data clearly show that SHB are vectors of P. larvae. We suggest considering the role of SHB in AFB control in areas where both pests are established.

Water-soluble fullerene (C60) derivatives as nonviral gene-delivery vectors.

A new class of water-soluble C60 transfecting agents has been prepared using Hirsch-Bingel chemistry and assessed for their ability to act as gene-delivery vectors in vitro. In an effort to elucidate the relationship between the hydrophobicity of the fullerene core, the hydrophilicity of the water-solubilizing groups, and the overall charge state of the C60 vectors in gene delivery and expression, several different C60 derivatives were synthesized to yield either positively charged, negatively charged, or neutral chemical functionalities under physiological conditions. These fullerene derivatives were then tested for their ability to transfect cells grown in culture with DNA carrying the green fluorescent protein (GFP) reporter gene. Statistically significant expression of GFP was observed for all forms of the C60 derivatives when used as DNA vectors and compared to the ability of naked DNA alone to transfect cells. However, efficient in vitro transfection was only achieved with the two positively charged C60 derivatives, namely, an octa-amino derivatized C60 and a dodeca-amino derivatized C60 vector. All C60 vectors showed an increase in toxicity in a dose-dependent manner. Increased levels of cellular toxicity were observed for positively charged C60 vectors relative to the negatively charged and neutral vectors. Structural analyses using dynamic light scattering and optical microscopy offered further insights into possible correlations between the various derivatized C60 compounds, the C60 vector/DNA complexes, their physical attributes (aggregation, charge) and their transfection efficiencies. Recently, similar Gd@C60-based compounds have demonstrated potential as advanced contrast agents for magnetic resonance imaging (MRI). Thus, the successful demonstration of intracellular DNA uptake, intracellular transport, and gene expression from DNA using C60 vectors suggests the possibility of developing analogous Gd@C60-based vectors to serve simultaneously as both therapeutic and diagnostic agents.

Gene transfer by viral vectors

To initiate our clinical trial for chemotherapy protection, I established the retroviral vector system for human MDR1 cDNA gene transfer. The human MDR1 cDNA continued to be expressed in the transduced bone marrow cells after four cohorts of serial transplants, 17 months after the initial transduction and transplant. In addition, we used this retroviral vector pVMDR1 to transduce human bone marrow and peripheral blood CD34$\sp+$ cells on stromal monolayer in the presence of hematopoietic growth factors. These data suggest that the retroviral vector pVMDR1 could modify hematopoietic precursor cells with a capacity for long-term self renewal. Thus, it may be possible to use the MDR1 retroviruses to confer chemotherapeutic protection on human normal hematopoietic precursor cells of ovarian and breast cancer patients in whom high doses of MDR drugs may be required to control the diseases.^ Another promising vector system is recombinant adeno-associated virus (rAAV) vector. An impediment to use rAAV vectors is that production of rAAV vectors for clinical use is extremely cumbersome and labor intensive. First I set up the rAAV vector system in our laboratory and then, I focused on studies related to the production of rAAV vectors for clinical use. By using a self-inactivating retroviral vector carrying a selection marker under the control of the CMV immediate early promoter and an AAV genome with the deletion of both ITRs, I have developed either a transient or a stable method to produce rAAV vectors. These methods involve infection only and can generate high-titer rAAV vectors (up to 2 x 10$\sp5$ cfu/ml of CVL) with much less work.^ Although recombinant adenoviral vectors hardly infect early hematopoietic precursor cells lacking $\alpha\sb v\beta\sb5$ or $\alpha\sb v\beta\sb3$ integrin on their surface, but efficiently infect other cells, we can use these properties of adenoviral vectors for bone marrow purging as well as for development of new viral vectors such as pseudotyped retroviral vectors and rAAV vectors. Replacement of self-inactivating retroviral vectors by recombinant adenoviral vectors will facilitate the above strategies for production of new viral vectors. In order to accomplish these goals, I developed a new method which is much more efficient than the current methods to construct adenoviral vectors. This method involves a cosmid vector system which is utilized to construct the full-length recombinant adenoviral vectors in vitro.^ First, I developed an efficient and flexible method for in vitro construction of the full-length recombinant adenoviral vectors in the cosmid vector system by use of a three-DNA fragment ligation. Then, this system was improved by use of a two-DNA fragment ligation. The cloning capacity of recombinant adenoviral vectors constructed by this method to develop recombinant adenoviral vectors depends on the efficiency of transfection only. No homologous recombination is required for development of infectious adenoviral vectors. Thus, the efficiency of generating the recombinant adenoviral vectors by the cosmid method reported here was much higher than that by the in vitro direct ligation method or the in vivo homologous recombination method reported before. This method of the in vitro construction of recombinant adenoviral vectors in the cosmid vector system may facilitate the development of adenoviral vector for human gene therapy. (Abstract shortened by UMI.) ^

New Shuttle Vector-Based Expression System To Generate Polyhistidine-Tagged Fusion Proteins in Staphylococcus aureus and Escherichia coli.

Four Staphylococcus aureus-Escherichia coli shuttle vectors were constructed for gene expression and production of tagged fusion proteins. Vectors pBUS1-HC and pTSSCm have no promoter upstream of the multiple cloning site (MCS), and this allows study of genes under the control of their native promoters, and pBUS1-Pcap-HC and pTSSCm-Pcap contain the strong constitutive promoter of S. aureus type 1 capsule gene 1A (Pcap) upstream of a novel MCS harboring codons for the peptide tag Arg-Gly-Ser-hexa-His (rgs-his6). All plasmids contained the backbone derived from pBUS1, including the E. coli origin ColE1, five copies of terminator rrnB T1, and tetracycline resistance marker tet(L) for S. aureus and E. coli. The minimum pAMα1 replicon from pBUS1 was improved through either complementation with the single-strand origin oriL from pUB110 (pBUS1-HC and pBUS1-Pcap-HC) or substitution with a pT181-family replicon (pTSSCm and pTSSCm-Pcap). The new constructs displayed increased plasmid yield and segregational stability in S. aureus. Furthermore, pBUS1-Pcap-HC and pTSSCm-Pcap offer the potential to generate C-terminal RGS-His6 translational fusions of cloned genes using simple molecular manipulation. BcgI-induced DNA excision followed by religation converts the TGA stop codon of the MCS into a TGC codon and links the rgs-his6 codons to the 3' end of the target gene. The generation of the rgs-his6 codon-fusion, gene expression, and protein purification were demonstrated in both S. aureus and E. coli using the macrolide-lincosamide-streptogramin B resistance gene erm(44) inserted downstream of Pcap. The new His tag expression system represents a helpful tool for the direct analysis of target gene function in staphylococcal cells.

New geometries for black hole horizons

We construct several classes of worldvolume effective actions for black holes by integrating out spatial sections of the worldvolume geometry of asymptotically flat black branes. This provides a generalisation of the blackfold approach for higher-dimensional black holes and yields a map between different effective theories, which we exploit by obtaining new hydrodynamic and elastic transport coefficients via simple integrations. Using Euclidean minimal surfaces in order to decouple the fluid dynamics on different sections of the worldvolume, we obtain local effective theories for ultraspinning Myers-Perry branes and helicoidal black branes, described in terms of a stress-energy tensor, particle currents and non-trivial boost vectors. We then study in detail and present novel compact and non-compact geometries for black hole horizons in higher-dimensional asymptotically flat space-time. These include doubly-spinning black rings, black helicoids and helicoidal p-branes as well as helicoidal black rings and helicoidal black tori in D ≥ 6.

Cellular Trafficking of Single and Multistage Vectors

Nanomedicine is an innovative field of science which has recently generated many drug delivery platforms with exciting results. The great potential of these strategies rely on the unique characteristics of the devices at the nano-scale in terms of long time circulation in the blood stream, selective accumulation at the lesions sites, increased solubility in aqueous solutions, etc. Herein we report on a new drug delivery system known as a multistage system which is comprised of non-spherical, mesoporous silicon particles loaded with second stage nanoparticles. The rationally designed particle shape, the possibility to modulate the surface properties and the degree of porosity allow these carriers to be optimized for vascular targeting and to overcome the numerous biological barriers found in drug delivery. In this study we investigated the intra and inter cellular trafficking of the multistage system in endothelial cells bringing evidence of its bio-compatibility as well as its ability to perform multiple intra and inter cellular tasks. Once internalized in cells, the multi-particle construct is able to dissociate, localizing in different subcellular compartments which can be targeted for exocytosis. In particular the second stage nanoparticles were found to be secreted in microvesicles which can act as mediators of transfer of particles across the endothelium and between different endothelial and cancer cells.

Seagrass biofilm communities at a naturally CO2-rich vent at Papua New Guinea

Seagrass meadows are a crucial component of tropical marine reef ecosystems. The seagrass plants are colonized by a multitude of epiphytic organisms that contribute to determining the ecological role of seagrasses. To better understand how environmental changes like ocean acidification might affect epiphytic assemblages, the microbial community composition of the epiphytic biofilm of Enhalus acroides was investigated at a natural CO2 vent in Papua New Guinea using molecular fingerprinting and next generation sequencing of 16S and 18S rRNA genes. Both bacterial and eukaryotic epiphytes formed distinct communities at the CO2-impacted site compared to the control site. This site-related CO2 effect was also visible in the succession pattern of microbial epiphytes. We further found an increased abundance of bacterial types associated with coral diseases at the CO2-impacted site (Fusobacteria, Thalassomonas) whereas eukaryotes such as certain crustose coralline algae commonly related to healthy reefs were less diverse. These trends in the epiphytic community of E. acroides suggest a potential role of seagrasses as vectors of coral pathogens and may support previous predictions of a decrease in reef health and prevalence of diseases under future ocean acidification scenarios.

Low-resource language recognition using a fusion of phoneme posteriorgram counts, acoustic and glottal-based i-vectors

This paper presents a description of our system for the Albayzin 2012 LRE competition. One of the main characteristics of this evaluation was the reduced number of available files for training the system, especially for the empty condition where no training data set was provided but only a development set. In addition, the whole database was created from online videos and around one third of the training data was labeled as noisy files. Our primary system was the fusion of three different i-vector based systems: one acoustic system based on MFCCs, a phonotactic system using trigrams of phone-posteriorgram counts, and another acoustic system based on RPLPs that improved robustness against noise. A contrastive system that included new features based on the glottal source was also presented. Official and postevaluation results for all the conditions using the proposed metrics for the evaluation and the Cavg metric are presented in the paper.

New methodology for the integration of biometric features in speaker recognition systems applied to security environments

La cuestión principal abordada en esta tesis doctoral es la mejora de los sistemas biométricos de reconocimiento de personas a partir de la voz, proponiendo el uso de una nueva parametrización, que hemos denominado parametrización biométrica extendida dependiente de género (GDEBP en sus siglas en inglés). No se propone una ruptura completa respecto a los parámetros clásicos sino una nueva forma de utilizarlos y complementarlos. En concreto, proponemos el uso de parámetros diferentes dependiendo del género del locutor, ya que como es bien sabido, la voz masculina y femenina presentan características diferentes que deberán modelarse, por tanto, de diferente manera. Además complementamos los parámetros clásicos utilizados (MFFC extraídos de la señal de voz), con un nuevo conjunto de parámetros extraídos a partir de la deconstrucción de la señal de voz en sus componentes de fuente glótica (más relacionada con el proceso y órganos de fonación y por tanto con características físicas del locutor) y de tracto vocal (más relacionada con la articulación acústica y por tanto con el mensaje emitido). Para verificar la validez de esta propuesta se plantean diversos escenarios, utilizando diferentes bases de datos, para validar que la GDEBP permite generar una descripción más precisa de los locutores que los parámetros MFCC clásicos independientes del género. En concreto se plantean diferentes escenarios de identificación sobre texto restringido y texto independiente utilizando las bases de datos de HESPERIA y ALBAYZIN. El trabajo también se completa con la participación en dos competiciones internacionales de reconocimiento de locutor, NIST SRE (2010 y 2012) y MOBIO 2013. En el primer caso debido a la naturaleza de las bases de datos utilizadas se obtuvieron resultados cercanos al estado del arte, mientras que en el segundo de los casos el sistema presentado obtuvo la mejor tasa de reconocimiento para locutores femeninos. A pesar de que el objetivo principal de esta tesis no es el estudio de sistemas de clasificación, sí ha sido necesario analizar el rendimiento de diferentes sistemas de clasificación, para ver el rendimiento de la parametrización propuesta. En concreto, se ha abordado el uso de sistemas de reconocimiento basados en el paradigma GMM-UBM, supervectores e i-vectors. Los resultados que se presentan confirman que la utilización de características que permitan describir los locutores de manera más precisa es en cierto modo más importante que la elección del sistema de clasificación utilizado por el sistema. En este sentido la parametrización propuesta supone un paso adelante en la mejora de los sistemas de reconocimiento biométrico de personas por la voz, ya que incluso con sistemas de clasificación relativamente simples se consiguen tasas de reconocimiento realmente competitivas. ABSTRACT The main question addressed in this thesis is the improvement of automatic speaker recognition systems, by the introduction of a new front-end module that we have called Gender Dependent Extended Biometric Parameterisation (GDEBP). This front-end do not constitute a complete break with respect to classical parameterisation techniques used in speaker recognition but a new way to obtain these parameters while introducing some complementary ones. Specifically, we propose a gender-dependent parameterisation, since as it is well known male and female voices have different characteristic, and therefore the use of different parameters to model these distinguishing characteristics should provide a better characterisation of speakers. Additionally, we propose the introduction of a new set of biometric parameters extracted from the components which result from the deconstruction of the voice into its glottal source estimate (close related to the phonation process and the involved organs, and therefore the physical characteristics of the speaker) and vocal tract estimate (close related to acoustic articulation and therefore to the spoken message). These biometric parameters constitute a complement to the classical MFCC extracted from the power spectral density of speech as a whole. In order to check the validity of this proposal we establish different practical scenarios, using different databases, so we can conclude that a GDEBP generates a more accurate description of speakers than classical approaches based on gender-independent MFCC. Specifically, we propose scenarios based on text-constrain and text-independent test using HESPERIA and ALBAYZIN databases. This work is also completed with the participation in two international speaker recognition evaluations: NIST SRE (2010 and 2012) and MOBIO 2013, with diverse results. In the first case, due to the nature of the NIST databases, we obtain results closed to state-of-the-art although confirming our hypothesis, whereas in the MOBIO SRE we obtain the best simple system performance for female speakers. Although the study of classification systems is beyond the scope of this thesis, we found it necessary to analise the performance of different classification systems, in order to verify the effect of them on the propose parameterisation. In particular, we have addressed the use of speaker recognition systems based on the GMM-UBM paradigm, supervectors and i-vectors. The presented results confirm that the selection of a set of parameters that allows for a more accurate description of the speakers is as important as the selection of the classification method used by the biometric system. In this sense, the proposed parameterisation constitutes a step forward in improving speaker recognition systems, since even when using relatively simple classification systems, really competitive recognition rates are achieved.

Extended phone log-likelihood ratio features and acoustic-based I-vectors for language recognition

This paper presents new techniques with relevant improvements added to the primary system presented by our group to the Albayzin 2012 LRE competition, where the use of any additional corpora for training or optimizing the models was forbidden. In this work, we present the incorporation of an additional phonotactic subsystem based on the use of phone log-likelihood ratio features (PLLR) extracted from different phonotactic recognizers that contributes to improve the accuracy of the system in a 21.4% in terms of Cavg (we also present results for the official metric during the evaluation, Fact). We will present how using these features at the phone state level provides significant improvements, when used together with dimensionality reduction techniques, especially PCA. We have also experimented with applying alternative SDC-like configurations on these PLLR features with additional improvements. Also, we will describe some modifications to the MFCC-based acoustic i-vector system which have also contributed to additional improvements. The final fused system outperformed the baseline in 27.4% in Cavg.

Influence of new integrated pest management strategies on aphidophagous predators in horticultural crops

El 1 de enero de 2014 entró en vigor la Directiva Europea 2009/128/CE sobre uso sostenible de plaguicidas y el Real Decreto 1311/2012 por el cual se traspone dicha normativa comunitaria al ámbito nacional. Estos reglamentos establecen el marco legal por el que las explotaciones agrícolas deben cumplir los principios generales de la Gestión Integrada de Plagas (GIP). Los principios de la GIP dan preferencia a aquellos métodos de control que sean sostenibles y respetuosos con el medio ambiente, dando prioridad al control biológico, al físico y a otros de carácter no químico. Sin embargo, el uso de insecticidas selectivos con los enemigos naturales es necesario en ocasiones para el adecuado manejo de las plagas en cultivos hortícolas. Por ello, el objetivo general de esta Tesis ha sido aportar conocimientos para la mejora del control de plagas en cultivos hortícolas, mediante la integración de estrategias de lucha biológica, física y química. La primera de las líneas de investigación de esta Tesis se centró en el estudio del efecto de la presencia dos depredadores, larvas Chrysoperla carnea y adultos de Adalia bipunctata, en la dispersión del virus de transmisión no persistente Cucumber mosaic virus (CMV) y del virus de transmisión persistente Cucurbit aphid-borne yellows virus (CABYV), transmitidos por el pulgón Aphis gosypii en cultivo de pepino. La tasa de transmisión de CMV fue baja para los dos tiempos de evaluación ensayados (1 y 5 días), debido al limitado movimiento de su vector A. gossypii. Las plantas que resultaron infectadas se localizaron próximas a la fuente de inóculo central y la presencia de ambos enemigos naturales no incrementó significativamente el porcentaje de plantas ocupadas por pulgones ni la tasa de transmisión de CMV. Los patrones de distribución de A. gossypii y de CMV tan solo fueron coincidentes en las proximidades de la planta central infectada en la que se liberaron los insectos. En los ensayos con CABYV, la presencia de C. carnea y de A. bipunctata respectivamente provocó un incremento significativo de la dispersión de A. gossypii tras 14 días, pero no tras 7 días desde la liberación de los insectos. La reducción en el número inicial de pulgones en la planta central infectada con CABYV fue siempre mayor tras la liberación de C. carnea en comparación con A. bipunctata. Sin embargo, la tasa de transmisión de CABYV y su distribución espacial no se vieron significativamente modificadas por la presencia de ninguno de los depredadores, ni tras 7 días ni tras 14 días desde el inicio de los ensayos. Al igual que se estudió el efecto de la presencia de enemigos naturales en el comportamiento de las plagas y en la epidemiología de las virosis que transmiten, en una segunda línea de investigación se evaluó el posible efecto del consumo de pulgones portadores de virus por parte de los enemigos naturales. Este trabajo se llevó a cabo en el Laboratorio de Ecotoxicología del Departamento de Entomología de la Universidade Federal de Lavras (UFLA) (Brasil). En él se evaluó la influencia en los parámetros biológicos del enemigo natural Chrysoperla externa al alimentarse de Myzus persicae contaminados con el virus de transmisión persistente Potato leafroll virus (PLRV). El consumo de M. persicae contaminados con PLRV incrementó significativamente la duración de la fase larvaria, reduciendo también la supervivencia en comparación a otras dos dietas a base de M. persicae no contaminados con el virus y huevos del lepidóptero Ephestia kuehniella. La duración de la fase de pupa de C. externa no difirió significativamente entre las dietas a base de pulgones contaminados con PLRV y pulgones no contaminados, pero ambas fueron menores que con la dieta con huevos de E. kuehniella. Sin embargo, ni la supervivencia en la fase de pupa ni los parámetros reproductivos de los adultos emergidos mostraron diferencias significativas entre las dietas evaluadas. Por el contrario, la supervivencia de los adultos durante los 30 primeros días desde su emergencia sí se vio significativamente afectada por la dieta, siendo al término de este periodo del 54% para aquellos adultos de C. externa que durante su fase larvaria consumieron pulgones con PLRV. Dentro de la GIP, una de las estrategias de carácter físico que se emplean para el control de plagas y enfermedades en cultivos hortícolas protegidos es el uso de plásticos con propiedades fotoselectivas de absorción de la radiación ultravioleta (UV). Por ello, la tercera línea de investigación de la Tesis se centró en el estudio de los efectos directos e indirectos (mediados por la planta) de condiciones especiales de baja radiación UV sobre el crecimiento poblacional del pulgón A. gossypii y los parámetros biológicos del enemigo natural C. carnea, así como sobre las plantas de pepino en las que se liberaron los insectos. Los ensayos se realizaron en jaulones dentro de invernadero, utilizándose en el primero de ellos plantas de pepino sanas, mientras que en el segundo las plantas de pepino fueron previamente infectadas con CABYV para estudiar de qué manera afectaba la incidencia del virus en las mismas condiciones. Las condiciones de baja radiación UV (bajo plástico Térmico Antivirus®) ejercieron un efecto directo en las fases iniciales del cultivo de pepino, promoviendo su crecimiento, mientras que en fases más avanzadas del cultivo indujeron un aumento en el contenido en nitrógeno de las plantas. Las plantas de pepino que fueron sometidas a mayor intensidad de radiación UV (bajo plástico Térmico Blanco®) al inicio del cultivo mostraron un engrosamiento significativo de las paredes de las células epidérmicas del haz de las hojas, así como de la cutícula. El uso del plástico Térmico Antivirus®, utilizado como barrera fotoselectiva para crear condiciones de baja radiación UV, no alteró con respecto al plástico Térmico Blanco® (utilizado como control) el desarrollo poblacional del pulgón A. gossypii ni los parámetros biológicos evaluados en el depredador C. carnea. En el segundo experimento, realizado con plantas infectadas con CABYV, la incidencia de la virosis enmascaró las diferencias encontradas en experimento con plantas sanas, reduciendo aparentemente la influencia de las distintas condiciones de radiación UV. Por último, para el desarrollo de las estrategias de GIP es importante estudiar los posibles efectos secundarios que los plaguicidas pueden tener en los enemigos naturales de las plagas. Es por ello que en la Tesis se evaluaron la toxicidad y los efectos subletales (fecundidad y fertilidad) de flonicamida, flubendiamida, metaflumizona, spirotetramat, sulfoxaflor y deltametrina en los enemigos naturales C. carnea y A. bipunctata. Los efectos secundarios fueron evaluados por contacto residual tanto para larvas como para adultos de ambos enemigos naturales en condiciones de laboratorio. Flonicamida, flubendiamida, metaflumizona y spirotetramat fueron inocuos para larvas de último estadio y adultos de C. carnea y A. bipunctata. Por este motivo, estos insecticidas se presentan como buenos candidatos para ser incorporados dentro de programas de GIP en combinación con estos enemigos naturales para el control de plagas de cultivos hortícolas. Sulfoxaflor fue ligeramente tóxico para adultos de C. carnea y altamente tóxico para larvas de último estadio de A. bipunctata. Para A. bipunctata, sulfoxaflor y deltametrina fueron los compuestos más dañinos. Deltametrina fue también el compuesto más tóxico para larvas y adultos de C. carnea. Por tanto, el uso de deltametrina y sulfoxaflor en programas de GIP debería tomarse en consideración cuando se liberasen cualquiera de estos dos enemigos naturales debido al comportamiento tóxico que mostraron en condiciones de laboratorio. ABSTRACT On 1 January 2014 came into effect the Directive 2009/128/EC of the European Parliament about sustainable use of pesticides and the Royal Decree 1311/2012 that transposes the regulation to the Spanish level. These regulations establish the legal framework that agricultural holdings must adhere to in order to accomplish the general principles of Integrated Pest Management (IPM). The guidelines of IPM give priority to sustainable and eco-friendly pest control techniques, such as biological and physical measures. Nevertheless, the use of pesticides that are selective to natural enemies is sometimes a necessary strategy to implement accurate pest management programs in horticultural protected crops. Therefore, the general objective of this Thesis was to contribute to the improvement of pest management strategies in horticultural crops, by means of the integration of biological, physical and chemical techniques. The first research line of this Thesis was focused on the evaluation of the effects of two aphidophagous predators, Chrysoperla carnea larvae and Adalia bipunctata adults, on the spread of the non-persistently transmitted Cucumber mosaic virus (CMV, Cucumovirus) and the persistently transmitted Cucurbit aphid-borne yellows virus (CABYV, Polerovirus), by the aphid vector Aphis gossypii in a cucumber crop under greenhouse conditions. The CMV transmission rate was generally low, both after 1 and 5 days, due to the limited movement of its aphid vector A. gossypii. Infected plants were mainly located around the central virusinfected source plant, and the percentage of aphid occupation and CMV-infected plants did not differ significantly in absence and presence of natural enemies. The distribution patterns of A. gossypii and CMV were only coincident close to the central plant where insects were released. In the CABYV experiments, the presence of C. carnea larvae and A. bipunctata adults induced significant A. gossypii dispersal after 14 days but not after 7 days. The reduction in the initial aphid population established in the central plant was always higher for C. carnea than for A. bipunctata. Nevertheless, CABYV spread was not significantly modified by the presence of each predator either in the short term (7 days) or in the long term (14 days). Furthermore, the percentage of CABYV-infected plants did not significantly differ when each natural enemy was present in any evaluation period. It is important to evaluate the influence that natural enemies have on pest dynamics and on the spread of viral diseases, but it should be also taken into account the possible effect on the performance of natural enemies when they feed on preys that act as vectors of viruses. Thus, in a second research line developed in the Laboratory of Ecotoxicology, Department of Entomology, of the Universidade Federal de Lavras (UFLA) (Brazil), it was evaluated the performance of Chrysoperla externa under the condition of consuming Myzus persicae acting as vector of Potato leafroll virus (PLRV). The diet composed of PLRV-infected M. persicae significantly increased the length and reduced the survival rate, of the larval period in regard to the other two diets, composed of non-infected M. persicae and Ephestia kuehniella eggs. The lengths of the pupal stage were not significantly different between the aphid diets, but both were significantly shorter than that of E. kuehniella eggs. Neither pupal survival nor reproductive parameters revealed significant differences among the diets. Nevertheless, the adult survival curves during the first 30 days after emergence showed significant differences, reaching at the end of this interval a value of 54% for those C. externa adults fed on PLRVinfected aphids during their larval period. According to the IPM guidelines, one of the physical strategies for the control of pests and diseases in horticultural protected crops is the use of plastic films with photoselective properties that act as ultraviolet (UV) radiation blocking barriers. In this sense, the third research line of the Thesis dealt with the study of the direct and plant-mediated influence of low UV radiation conditions on the performance of the aphid A. gossypii and on the biological parameters of the natural enemy C. carnea, as well as on the cucumber plants where insects were released. The experiments were conducted inside cages under greenhouse conditions, using for the first one healthy cucumber plants, while for the second experiment the cucumber plants were previously infected with CABYV in order to assess the influence of the virus in the same conditions. The low UV radiation conditions (under Térmico Antivirus® plastic film) seemed to exert a direct effect in the early stages of cucumber plants, enhancing their growth, and in an increasing nitrogen content at further developmental stages. The higher UV radiation exposure (under Térmico Blanco® plastic film) in the early stages of the cucumber crop induced the thickening of the adaxial epidermal cell walls and the cuticle of leaves. The use of Térmico Antivirus® plastic film as a photoselective barrier to induce low UV radiation conditions did not modify, in regard to Térmico Blanco® plastic film (used as control), neither the population development of A. gossypii nor the studied biological parameters of the predator C. carnea. In the second experiment, done with CABYV-infected cucumber plants, the incidence of the virus seemed to mask the direct and plant-mediated influence of the different UV radiation conditions. In last term, for the development of IPM strategies it is important to study the potential side effects that pesticides might have on natural enemies. For this reason, in the Thesis were tested the toxicity and sublethal effects (fecundity and fertility) of flonicamid, flubendiamide, metaflumizone, spirotetramat, sulfoxaflor and deltamethrin on the natural enemies C. carnea and A. bipunctata. The side effects of the active ingredients of the insecticides were evaluated with residual contact tests for the larvae and adults of these predators under laboratory conditions. Flonicamid, flubendiamide, metaflumizone and spirotetramat were innocuous to last instar larvae and adults of C. carnea and A. bipunctata. Therefore, these pesticides are promising candidates for being incorporated into IPM programs in combination with these natural enemies for the control of particular greenhouse pests. In contrast, sulfoxaflor was slightly toxic to adults of C. carnea and was highly toxic to last instar larvae of A. bipunctata. For A. bipunctata, sulfoxaflor and deltamethrin were the most damaging compounds. Deltamethrin was also the most toxic compound to larvae and adults of C. carnea. In accordance with this fact, the use of sulfoxaflor and deltamethrin in IPM strategies should be taken into consideration when releasing either of these biological control agents, due to the toxic behavior observed under laboratory conditions.