INCREASED EXPRESSION OF ONE OF TWO ADENOSINE DEAMINASE ALLELES IN A HUMAN CHORIOCARCINOMA CELL LINE FOLLOWING SELECTION WITH ADENINE NUCLEOSIDES

The human choriocarcinoma cell line JEG-3 is heterozygous at the adenosine deaminase (ADA) gene locus. Both allelic genes are under strong but incomplete repression causing a very low level expression of the gene locus. Because cytotoxic adenosine analogues such as 9-(beta)-D arabinofuranosyladenine (ara-A) and 9-(beta)-D xylofuranosyladenine (xyl-A) can be specifically detoxified by the action of ADA, these analogues were used to select for JEG-3 derived cells which had increased ADA expression. When JEG-3 cells were subjected to a multi-step, successively increasing dosage of either ara-A or xyl-A, resistant cells with increased ADA expression were generated. This increased ADA expression in the resistant cells was unstable, so that when the selective pressure was removed, cellular ADA expression would decrease. Subclone analysis of xyl-A resistant cells revealed that compared to parental JEG-3 cells, individual resistant cells had either elevated ADA levels or decreased adenosine kinase (ADK) levels or both. This altered ADA and ADK expression in the resistant cells were found to be independent events. Because of high endogenous tissue conversion factor (TCF) expression in the JEG-3 cells, the allelic nature of the increased ADA expression in most of the resistant cells could not be determined. However, several resistant subcloned cells were found to have lost TCF expression. These TCF('-) cells expressed only the ADA*2 allelic gene product. Cell fusion experiments demonstrated that the ADA*1 allelic gene was intact and functional in the A3-1A7 cell line. Chromosomal analysis of the A3-1A7 cells showed that they had no double-minutes or homogeneously staining chromosomal regions, although a pair of new chromosomes were found in these cells. Segregation analysis of the hybrid cells indicated that an ADA*2 allelic gene was probably located on this new chromosome. The analysis of the A3-1A7 cell line suggested that the expression of only ADA 2 in these cells was the result of possibly a cis-deregulation of the ADA gene locus or more probably an amplification of the ADA*2 allelic gene. Two effective positive selection systems for ADA('+) cells were also developed and tested. These selection systems should eventually lead to the isolation of the ADA gene.^

SYNTHETIC PROBES FOR STUDYING TUFTSIN-RECEPTOR INTERACTIONS ON HUMAN POLYMORPHONUCLEAR LEUKOCYTES

Tuftsin is an immunopotentiating tetrapeptide of the sequence L-Thr-L-Lys-L-Pro-L-Arg with anti-microbial and anti-tumor enhancing capabilities. These enhancing functions are manifested through the host's granulocytes and monocytes. In delineating tuftsin's mechanism of action, both radiolabeled and fluorescent probes were synthesized. The radiolabeled probe of tuftsin, L-proly-3,4-('3)H(N) -tuftsin, was obtained through the synthesis and subsequent catalytic hydrogenation of L-3,4-dehydroprolyl ('3)-tuftsin using tritium gas. This procedure yielded a probe with a specific activity of 44.9 Ci/mmole. This radiolabeled probe of tuftsin was used in competitive inhibition studies with tuftsin, the tuftsin analogues Lys-Pro-Arg, Thr-Lys-Pro-Arg(NO(,2)) and (DELTA)('3)-pro('3) -tuftsin as well as with the chemotactic peptide f-Met-Leu-Phe. From the competitive binding curves, the K(,D) for tuftsin was estimated to be 80 nM, a value that approaches the concentration of tuftsin that evokes a half maximal biological response. The approximate Ki's for the tuftsin analogues (33 nM) approached that of tuftsin itself (40 nM). On the other hand, approximately a two log difference in the Ki was seen with the chemotactic tripeptide, indicating that tuftsin may indeed be acting through the chemotactic peptide receptor. This conclusion is further strengthened by studies using an N-terminal derivitized mono-fluoresceinated tuftsin probe and image intensification microscopy. These studies showed that like the chemotactic peptide, tuftsin initially binds to diffusely distributed receptors on the surface of human granulocytes. The tuftsin-receptor complexes then rapidly redistribute to form patches (5 min @ 37(DEGREES)C) which are then internalized. Whether redistribution and internalization of tuftsin-receptor complexes is crucial in effecting a biological response, or simply an intermediary point leading ultimately to degradation, is still not clear. This process, however, may provide the target cell with an early time point in modulating the biological effects of tuftsin through down-regulation of cell surface receptor sites. ^

Regulation of vascular endothelial growth factor expression in human pancreatic cancer

Pancreatic adenocarcinoma is currently the fifth-leading cause of cancer-related death in the United States. Like with other solid tumors, the growth and metastasis of pancreatic adenocarcinoma are dependent on angiogenesis. Vascular endothelial growth factor (VEGF) is a key angiogenic molecule that plays an important role in angiogenesis, growth and metastasis of many types of human cancer, including pancreatic adenocarcinoma. However, the expression and regulation of VEGF in human pancreatic cancer cells are mostly unknown. ^ To examine the hypothesis that VEGF is constitutively expressed in human pancreatic cancer cells, and can be further induced by tumor environment factors such as nitric oxide, a panel of human pancreatic cancer cell lines were studied for constitutive and inducible VEGF expression. All the cell lines examined were shown to constitutively express various levels of VEGF. To identify the mechanisms responsible for the elevated expression of VEGF, its rates of turnover and transcription were then investigated. While the half-live of VEGF was unaffected, higher transcription rates and increased VEGF promoter activity were observed in tumor cells that constitutively expressed elevated levels of VEGF. Detailed VEGF promoter analyses revealed that the region from −267 to +50, which contains five putative Sp1 binding sites, was responsible for this VEGF promoter activity. Further deletion and point mutation analyses indicated that deletion of any of the four proximal Sp1 binding sites significantly diminished VEGF promoter activity and when all four binding sites were mutated, it was completely abrogated. Consistent with these observations, high levels of constitutive Sp1 expression and DNA binding activities were detected in pancreatic cancer cells expressing high levels of VEGF. Collectively, our data indicates that constitutively expressed Sp1 leads to the constitutive expression of VEGF, and implicates that both molecules involve in the aggressive pathogenesis of human pancreatic cancer. ^ Although constitutively expressed in pancreatic cancer cells, VEGF can be further induced. In human pancreatic cancer specimens, we found that in addition to VEGF, both inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were overexpressed, suggesting that nitric oxide might upregulate VEGF expression. Indeed, a nitric oxide donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) significantly induced VEGF mRNA expression and protein secretion in pancreatic adenocarcinoma cells in a time- and dose-dependant manner. Using a luciferase reporter containing both the VEGF promoter and the 3′ -UTR, we showed that SNAP significantly increased luciferase activity in human pancreatic cancer cells. Notwithstanding its ability to induce VEGF in vitro, pancreatic cancer cells genetically engineered to produce NO did not exhibit increased tumor growth. This inability of NO to promote tumor growth appears to be related to NO-mediated cytotoxicity. The balance between NO mediated effects on pro-angiogenesis and cytotoxicity would determine the biological outcome of NO action on tumor cells. ^ In summary, we have demonstrated that VEGF is constitutively expressed in human pancreatic cancer cells, and that overexpression of transcription factor Sp1 is primarily responsible. Although constitutively expressed in these cells, VEGF can be further induced by NO. However, using a mouse model, we have shown that NO inhibited tumor growth by promoting cytotoxicity. These studies suggest that both Sp1 and NO may be important targets for designing potentially effective therapies of human pancreatic cancer and warrant further investigation. ^

New aspects of estrogen action in the endometrium: Reciprocal interplay with retinoic acid pathway and a novel estrogen-regulated gene

The uterine endometrium is a major target for the estrogen. However, the molecular basis of estrogen action in the endometrium is largely unknown. I have used two approaches to study the effects of estrogen on the endometrium. One approach involved the study of the interaction between estrogen and retinoic acid (RA) pathways in the endometrium. I have demonstrated that estrogen administration to rodents and estrogen replacement therapy (ERT) in postmenopausal women selectively induced the endometrial expression of retinaldehyde dehydrogenase II (RALDH2), a critical enzyme of RA biosynthesis. RALDH2 was expressed exclusively in the stromal cells, especially in the stroma adjacent to the luminal and glandular epithelia. The induction of RALDH2 by estrogen required estrogen receptor and occurred via a direct increase in RALDH2 transcription. Among the three RA receptors, estrogen selectively induced the expression of RARα. In parallel, estrogen also increased the utilization of all-trans retinol (the substrate for RA biosynthesis) and the expression of two RA-regulated marker genes, cellular retinoic acid binding protein II (CRABP2) and tissue transglutaminase (tTG) in the endometrium. Thus estrogen coordinately upregulated both the production and signaling of RA in both the rodent and human endometrium. This coordinate upregulation of RA system appeared to play a role in counterbalancing the stimulatory effects of estrogen on the endometrium, since the depletion of endogenous RA in mice led to an increase in estrogen-stimulated stromal proliferation and endometrial Akt phosphorylation. In addition, I have also used a systematic approach (DNA microarray) to categorize genes and pathways affected by the ERT in the endometrium of postmenopausal women and identified a novel estrogen-regulated gene EIG121. EIG121 was exclusively expressed in the glandular epithelial cells of the endometrium and induced by estrogen in vivo and in cultured cell lines. Compared with the normal endometrium, EIG121 was highly overexpressed in type 1 endometrial cancer, but profoundly suppressed in type 2 endometrial tumors. Taken together, these studies suggested that estrogen regulates the expression of many genes of both the pro-proliferative and anti-proliferative pathways and the abnormality of these pathways may increase the risks for estrogen-dependent endometrial hyperplasia and endometrial cancer. ^

Chemopreventive function of retinoid X receptors in human squamous cell carcinoma of the skin

Retinoid therapy has been successful for the treatment of skin squamous cell carcinoma (SCC). A suppression of the predominant retinoid X receptor expressed in skin, retinoid X receptor α (RXRα), has been reported in skin SCC. These observations have led to the hypothesis that retinoid receptor loss contributes to the tumorigenic phenotype of epithelial cancers. To test this hypothesis, the RXRα gene was mapped in order to generate a targeting construct. Additionally the transcriptional regulation of the human RXRα a gene in keratinocytes was characterized after identifying the transcription initiation sites, the promoter, and enhancer regions of this gene. The structure is highly conserved between human and mouse. A nontumorigenic human skin-derived cell line called near diploid immortalized keratinocytes (NIKS) has the advantage of growing as organotypic raft cultures, under physiological conditions closely resembling in-vivo squamous stratification. We have exploited the raft culture technique to develop an in-vitro model for skin SCC progression that includes the NIKS cells, HaCaT cells, a premalignant cell line, and SRB 12-p9 cells, a tumorigenic SCC skin cell line. The differentiation, proliferation and nuclear receptor ligand response characteristics of this system were studied and significant and novel results were obtained. RXRs are obligate heterodimerization partners with many of the nuclear hormone receptors, including retinoic acid receptors (RARs), vitamin D3 receptors (VDR), thyroid hormone receptors (T3 R) and peroxisome proliferator activate receptors (PPARs), which are all known to be active in skin. Treatment of the three cell lines in raft culture with the RXR specific ligand BMS649, BMS961 (RARγ-specific), vitamin D3 (VDR ligand), thryoid hormone (T3R ligand) and clofibrate (PPARa ligand), and the combination of BMS649 with each of the 4 receptor partner ligands, resulted in distinct effects on differentiation, proliferation and apoptosis. The effects of activation of RXRs in each of the four-receptor pathways; in the context of skin SCC progression, with an emphasis on the VDR/RXR pathway, are discussed. These studies will lead to a better understanding of RXRα action in human skin and will help determine its role in SCC tumorigenesis, as well as its potential as a target for the prevention, treatment, and control of skin cancer. ^

Bortezomib modulates TRAIL sensitivity in human bladder and prostate cancer

Tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is a member of the TNF superfamily of cytokines that can induce cell death through engagement of cognate death receptors. Unlike other death receptor ligands, it selectively kills tumor cells while sparing normal cells. Preclinical studies in non-human primates have generated much enthusiasm regarding its therapeutic potential. However, many human cancer cell lines exhibit significant resistance to TRAIL-induced apoptosis, and the molecular mechanisms underling this are controversial. Possible explanations are typically cell-type dependent, but include alterations of receptor expression, enhancement of pro-apoptotic intracellular signaling molecules, and reductions in anti-apoptotic proteins. We show here that the proteasome inhibitor bortezomib (Velcade, PS-341) produces synergistic apoptosis in both bladder and prostate cancer cell lines within 4-6 hours when co-treated with recombinant human TRAIL which is associated with accumulation of p21 and cdk1/2 inhibition. Our data suggest that bortezomib's mechanism of action involves a p21-dependent enhancement of caspase maturation. Furthermore, we found enhanced tumor cell death in in vivo models using athymic nude mice. This is associated with increases in caspase-8 and caspase-3 cleavage as well as significant reductions in microvessel density (MVD) and proliferation. Although TRAIL alone had less of an effect, its biological significance as a single agent requires further investigations. Toxicity studies reveal that the combination of bortezomib and rhTRAIL has fatal consequences that can be circumvented by altering treatment schedules. Based on our findings, we conclude that this strategy has significant therapeutic potential as an anti-cancer agent. ^

Regulation of squamous cell differentiation in human head and neck carcinoma cells by retinoids

Retinoids have been found to be effective in the prevention of premalignant lesions and second primary cancers in the upper aerodigestive tract. Further development of retinoids for prevention and therapy of head and neck squamous cell carcinoma (HNSCC) requires a better understanding of their mechanism of action on the growth and differentiation of such cells. I have chosen to employ cultured HNSCC cell lines as a model system for investigating the mechanism underlying the effects of retinoids. These cells are useful because all-trans retinoic acid (ATRA) inhibits their proliferation. Furthermore, two HNSCC cell lines were found to express three squamous differentiation (SqD) markers characteristic of normal keratinocytes and ATRA suppressed the expression of these markers as reported for normal keratinocytes. It is thought that nuclear retinoic acid receptors (RARs and RXRs), which act as DNA-binding transcription modulating factors, mediate the effects of retinoids on the growth and differentiation of normal and tumor cells. I found that all four cell lines examined expressed RAR-$\alpha ,$ RAR-$\tau ,$ and RXR-$\alpha$ and three of four expressed RAR-$\beta .$ ATRA treatment increased the level of RAR-$\alpha ,$ -$\beta ,$ and -$\tau$ in four cell lines. Two HNSCC cell lines that exhibited a progressive increase in the expression of SqD markers during growth in culture also showed a concurrent decrease in RAR-$\beta$ level. Moreover, increasing concentrations of RA suppressed the SqD marker while inducing RAR-$\beta$ mRNA. Several synthetic retinoids which exhibit a preference for binding to specific nuclear RARs showed a differential ability to inhibit cell proliferation, transactivate transcription of the reporter genes (CAT and luciferase) from the RA response element (RARE) of the RAR-$\beta$ gene, and induce RAR-$\beta$ expression. Those retinoids that were effective inducers of RAR-$\beta$ also suppressed SqD effectively, indicating an inverse relationship exists between the expression of RAR-$\beta$ and SqD. This inverse relationship suggests a role for RAR-$\beta$ in the suppression of SqD. ^

Survey and brain storming studies about machines, constructions, human and environmental risk consideration in the careers of the Universidad Politécnica of Madrid

The Universidad Politécnica of Madrid (UPM) includes schools and faculties that were for engineering degrees, architecture and computer science, that are now in a quick EEES Bolonia Plan metamorphosis getting into degrees, masters and doctorate structures. They are focused towards action in machines, constructions, enterprises, that are subjected to machines, human and environment created risks. These are present in actions such as use loads, wind, snow, waves, flows, earthquakes, forces and effects in machines, vehicles behavior, chemical effects, and other environmental factors including effects of crops, cattle and beasts, forests, and varied essential economic and social disturbances. Emphasis is for authors in this session more about risks of natural origin, such as for hail, winds, snow or waves that are not exactly known a priori, but that are often considered with statistical expected distributions giving extreme values for convenient return periods. These distributions are known from measures in time, statistic of extremes and models about hazard scenarios and about responses of man made constructions or devices. In each engineering field theories were built about hazards scenarios and how to cover for important risks. Engineers must get that the systems they handle, such as vehicles, machines, firms or agro lands or forests, obtain production with enough safety for persons and with decent economic results in spite of risks. For that risks must be considered in planning, in realization and in operation, and safety margins must be taken but at a reasonable cost. That is a small level of risks will often remain, due to limitations in costs or because of due to strange hazards, and maybe they will be covered by insurance in cases such as in transport with cars, ships or aircrafts, in agro for hail, or for fire in houses or in forests. These and other decisions about quality, security for men or about business financial risks are sometimes considered with Decision Theories models, using often tools from Statistics or operational Research. The authors have done and are following field surveys about risk consideration in the careers in UPM, making deep analysis of curricula taking into account the new structures of degrees in the EEES Bolonia Plan, and they have considered the risk structures offered by diverse schools of Decision theories. That gives an aspect of the needs and uses, and recommendations about improving in the teaching about risk, that may include special subjects especially oriented for each career, school or faculty, so as to be recommended to be included into the curricula, including an elaboration and presentation format using a multi-criteria decision model.

Contributions to the contextualization of human-machine spoken interaction systems

The design and development of spoken interaction systems has been a thoroughly studied research scope for the last decades. The aim is to obtain systems with the ability to interact with human agents with a high degree of naturalness and efficiency, allowing them to carry out the actions they desire using speech, as it is the most natural means of communication between humans. To achieve that degree of naturalness, it is not enough to endow systems with the ability to accurately understand the user’s utterances and to properly react to them, even considering the information provided by the user in his or her previous interactions. The system has also to be aware of the evolution of the conditions under which the interaction takes place, in order to act the most coherent way as possible at each moment. Consequently, one of the most important features of the system is that it has to be context-aware. This context awareness of the system can be reflected in the modification of the behaviour of the system taking into account the current situation of the interaction. For instance, the system should decide which action it has to carry out, or the way to perform it, depending on the user that requests it, on the way that the user addresses the system, on the characteristics of the environment in which the interaction takes place, and so on. In other words, the system has to adapt its behaviour to these evolving elements of the interaction. Moreover that adaptation has to be carried out, if possible, in such a way that the user: i) does not perceive that the system has to make any additional effort, or to devote interaction time to perform tasks other than carrying out the requested actions, and ii) does not have to provide the system with any additional information to carry out the adaptation, which could imply a lesser efficiency of the interaction, since users should devote several interactions only to allow the system to become adapted. In the state-of-the-art spoken dialogue systems, researchers have proposed several disparate strategies to adapt the elements of the system to different conditions of the interaction (such as the acoustic characteristics of a specific user’s speech, the actions previously requested, and so on). Nevertheless, to our knowledge there is not any consensus on the procedures to carry out these adaptation. The approaches are to an extent unrelated from one another, in the sense that each one considers different pieces of information, and the treatment of that information is different taking into account the adaptation carried out. In this regard, the main contributions of this Thesis are the following ones: Definition of a contextualization framework. We propose a unified approach that can cover any strategy to adapt the behaviour of a dialogue system to the conditions of the interaction (i.e. the context). In our theoretical definition of the contextualization framework we consider the system’s context as all the sources of variability present at any time of the interaction, either those ones related to the environment in which the interaction takes place, or to the human agent that addresses the system at each moment. Our proposal relies on three aspects that any contextualization approach should fulfill: plasticity (i.e. the system has to be able to modify its behaviour in the most proactive way taking into account the conditions under which the interaction takes place), adaptivity (i.e. the system has also to be able to consider the most appropriate sources of information at each moment, both environmental and user- and dialogue-dependent, to effectively adapt to the conditions aforementioned), and transparency (i.e. the system has to carry out the contextualizaton-related tasks in such a way that the user neither perceives them nor has to do any effort in providing the system with any information that it needs to perform that contextualization). Additionally, we could include a generality aspect to our proposed framework: the main features of the framework should be easy to adopt in any dialogue system, regardless of the solution proposed to manage the dialogue. Once we define the theoretical basis of our contextualization framework, we propose two cases of study on its application in a spoken dialogue system. We focus on two aspects of the interaction: the contextualization of the speech recognition models, and the incorporation of user-specific information into the dialogue flow. One of the modules of a dialogue system that is more prone to be contextualized is the speech recognition system. This module makes use of several models to emit a recognition hypothesis from the user’s speech signal. Generally speaking, a recognition system considers two types of models: an acoustic one (that models each of the phonemes that the recognition system has to consider) and a linguistic one (that models the sequences of words that make sense for the system). In this work we contextualize the language model of the recognition system in such a way that it takes into account the information provided by the user in both his or her current utterance and in the previous ones. These utterances convey information useful to help the system in the recognition of the next utterance. The contextualization approach that we propose consists of a dynamic adaptation of the language model that is used by the recognition system. We carry out this adaptation by means of a linear interpolation between several models. Instead of training the best interpolation weights, we make them dependent on the conditions of the dialogue. In our approach, the system itself will obtain these weights as a function of the reliability of the different elements of information available, such as the semantic concepts extracted from the user’s utterance, the actions that he or she wants to carry out, the information provided in the previous interactions, and so on. One of the aspects more frequently addressed in Human-Computer Interaction research is the inclusion of user specific characteristics into the information structures managed by the system. The idea is to take into account the features that make each user different from the others in order to offer to each particular user different services (or the same service, but in a different way). We could consider this approach as a user-dependent contextualization of the system. In our work we propose the definition of a user model that contains all the information of each user that could be potentially useful to the system at a given moment of the interaction. In particular we will analyze the actions that each user carries out throughout his or her interaction. The objective is to determine which of these actions become the preferences of that user. We represent the specific information of each user as a feature vector. Each of the characteristics that the system will take into account has a confidence score associated. With these elements, we propose a probabilistic definition of a user preference, as the action whose likelihood of being addressed by the user is greater than the one for the rest of actions. To include the user dependent information into the dialogue flow, we modify the information structures on which the dialogue manager relies to retrieve information that could be needed to solve the actions addressed by the user. Usage preferences become another source of contextual information that will be considered by the system towards a more efficient interaction (since the new information source will help to decrease the need of the system to ask users for additional information, thus reducing the number of turns needed to carry out a specific action). To test the benefits of the contextualization framework that we propose, we carry out an evaluation of the two strategies aforementioned. We gather several performance metrics, both objective and subjective, that allow us to compare the improvements of a contextualized system against the baseline one. We will also gather the user’s opinions as regards their perceptions on the behaviour of the system, and its degree of adaptation to the specific features of each interaction. Resumen El diseño y el desarrollo de sistemas de interacción hablada ha sido objeto de profundo estudio durante las pasadas décadas. El propósito es la consecución de sistemas con la capacidad de interactuar con agentes humanos con un alto grado de eficiencia y naturalidad. De esta manera, los usuarios pueden desempeñar las tareas que deseen empleando la voz, que es el medio de comunicación más natural para los humanos. A fin de alcanzar el grado de naturalidad deseado, no basta con dotar a los sistemas de la abilidad de comprender las intervenciones de los usuarios y reaccionar a ellas de manera apropiada (teniendo en consideración, incluso, la información proporcionada en previas interacciones). Adicionalmente, el sistema ha de ser consciente de las condiciones bajo las cuales transcurre la interacción, así como de la evolución de las mismas, de tal manera que pueda actuar de la manera más coherente en cada instante de la interacción. En consecuencia, una de las características primordiales del sistema es que debe ser sensible al contexto. Esta capacidad del sistema de conocer y emplear el contexto de la interacción puede verse reflejada en la modificación de su comportamiento debida a las características actuales de la interacción. Por ejemplo, el sistema debería decidir cuál es la acción más apropiada, o la mejor manera de llevarla a término, dependiendo del usuario que la solicita, del modo en el que lo hace, etcétera. En otras palabras, el sistema ha de adaptar su comportamiento a tales elementos mutables (o dinámicos) de la interacción. Dos características adicionales son requeridas a dicha adaptación: i) el usuario no ha de percibir que el sistema dedica recursos (temporales o computacionales) a realizar tareas distintas a las que aquél le solicita, y ii) el usuario no ha de dedicar esfuerzo alguno a proporcionar al sistema información adicional para llevar a cabo la interacción. Esto último implicaría una menor eficiencia de la interacción, puesto que los usuarios deberían dedicar parte de la misma a proporcionar información al sistema para su adaptación, sin ningún beneficio inmediato. En los sistemas de diálogo hablado propuestos en la literatura, se han propuesto diferentes estrategias para llevar a cabo la adaptación de los elementos del sistema a las diferentes condiciones de la interacción (tales como las características acústicas del habla de un usuario particular, o a las acciones a las que se ha referido con anterioridad). Sin embargo, no existe una estrategia fija para proceder a dicha adaptación, sino que las mismas no suelen guardar una relación entre sí. En este sentido, cada una de ellas tiene en cuenta distintas fuentes de información, la cual es tratada de manera diferente en función de las características de la adaptación buscada. Teniendo en cuenta lo anterior, las contribuciones principales de esta Tesis son las siguientes: Definición de un marco de contextualización. Proponemos un criterio unificador que pueda cubrir cualquier estrategia de adaptación del comportamiento de un sistema de diálogo a las condiciones de la interacción (esto es, el contexto de la misma). En nuestra definición teórica del marco de contextualización consideramos el contexto del sistema como todas aquellas fuentes de variabilidad presentes en cualquier instante de la interacción, ya estén relacionadas con el entorno en el que tiene lugar la interacción, ya dependan del agente humano que se dirige al sistema en cada momento. Nuestra propuesta se basa en tres aspectos que cualquier estrategia de contextualización debería cumplir: plasticidad (es decir, el sistema ha de ser capaz de modificar su comportamiento de la manera más proactiva posible, teniendo en cuenta las condiciones en las que tiene lugar la interacción), adaptabilidad (esto es, el sistema ha de ser capaz de considerar la información oportuna en cada instante, ya dependa del entorno o del usuario, de tal manera que adecúe su comportamiento de manera eficaz a las condiciones mencionadas), y transparencia (que implica que el sistema ha de desarrollar las tareas relacionadas con la contextualización de tal manera que el usuario no perciba la manera en que dichas tareas se llevan a cabo, ni tampoco deba proporcionar al sistema con información adicional alguna). De manera adicional, incluiremos en el marco propuesto el aspecto de la generalidad: las características del marco de contextualización han de ser portables a cualquier sistema de diálogo, con independencia de la solución propuesta en los mismos para gestionar el diálogo. Una vez hemos definido las características de alto nivel de nuestro marco de contextualización, proponemos dos estrategias de aplicación del mismo a un sistema de diálogo hablado. Nos centraremos en dos aspectos de la interacción a adaptar: los modelos empleados en el reconocimiento de habla, y la incorporación de información específica de cada usuario en el flujo de diálogo. Uno de los módulos de un sistema de diálogo más susceptible de ser contextualizado es el sistema de reconocimiento de habla. Este módulo hace uso de varios modelos para generar una hipótesis de reconocimiento a partir de la señal de habla. En general, un sistema de reconocimiento emplea dos tipos de modelos: uno acústico (que modela cada uno de los fonemas considerados por el reconocedor) y uno lingüístico (que modela las secuencias de palabras que tienen sentido desde el punto de vista de la interacción). En este trabajo contextualizamos el modelo lingüístico del reconocedor de habla, de tal manera que tenga en cuenta la información proporcionada por el usuario, tanto en su intervención actual como en las previas. Estas intervenciones contienen información (semántica y/o discursiva) que puede contribuir a un mejor reconocimiento de las subsiguientes intervenciones del usuario. La estrategia de contextualización propuesta consiste en una adaptación dinámica del modelo de lenguaje empleado en el reconocedor de habla. Dicha adaptación se lleva a cabo mediante una interpolación lineal entre diferentes modelos. En lugar de entrenar los mejores pesos de interpolación, proponemos hacer los mismos dependientes de las condiciones actuales de cada diálogo. El propio sistema obtendrá estos pesos como función de la disponibilidad y relevancia de las diferentes fuentes de información disponibles, tales como los conceptos semánticos extraídos a partir de la intervención del usuario, o las acciones que el mismo desea ejecutar. Uno de los aspectos más comúnmente analizados en la investigación de la Interacción Persona-Máquina es la inclusión de las características específicas de cada usuario en las estructuras de información empleadas por el sistema. El objetivo es tener en cuenta los aspectos que diferencian a cada usuario, de tal manera que el sistema pueda ofrecer a cada uno de ellos el servicio más apropiado (o un mismo servicio, pero de la manera más adecuada a cada usuario). Podemos considerar esta estrategia como una contextualización dependiente del usuario. En este trabajo proponemos la definición de un modelo de usuario que contenga toda la información relativa a cada usuario, que pueda ser potencialmente utilizada por el sistema en un momento determinado de la interacción. En particular, analizaremos aquellas acciones que cada usuario decide ejecutar a lo largo de sus diálogos con el sistema. Nuestro objetivo es determinar cuáles de dichas acciones se convierten en las preferencias de cada usuario. La información de cada usuario quedará representada mediante un vector de características, cada una de las cuales tendrá asociado un valor de confianza. Con ambos elementos proponemos una definición probabilística de una preferencia de uso, como aquella acción cuya verosimilitud es mayor que la del resto de acciones solicitadas por el usuario. A fin de incluir la información dependiente de usuario en el flujo de diálogo, llevamos a cabo una modificación de las estructuras de información en las que se apoya el gestor de diálogo para recuperar información necesaria para resolver ciertos diálogos. En dicha modificación las preferencias de cada usuario pasarán a ser una fuente adicional de información contextual, que será tenida en cuenta por el sistema en aras de una interacción más eficiente (puesto que la nueva fuente de información contribuirá a reducir la necesidad del sistema de solicitar al usuario información adicional, dando lugar en consecuencia a una reducción del número de intervenciones necesarias para llevar a cabo una acción determinada). Para determinar los beneficios de las aplicaciones del marco de contextualización propuesto, llevamos a cabo una evaluación de un sistema de diálogo que incluye las estrategias mencionadas. Hemos recogido diversas métricas, tanto objetivas como subjetivas, que nos permiten determinar las mejoras aportadas por un sistema contextualizado en comparación con el sistema sin contextualizar. De igual manera, hemos recogido las opiniones de los participantes en la evaluación acerca de su percepción del comportamiento del sistema, y de su capacidad de adaptación a las condiciones concretas de cada interacción.

Perspectives on climate change and human development in the Mediterranean agriculture

The Mediterranean region is one of the world's climate change hotspots. Future climate projections envisage dramatic implications for the agricultural and water sectors that will endanger economic development and lead to natural resources degradation and social instability.

Post-fracture behavior of laminated plates after human impact test

For safety barriers the load bearing capacity of the glass when subjected to the soft body impact should be verified. The soft body pendulum test became a testing standard to classify safety glass plates. The classification of the safety glass do not consider the structural behavior when one sheet of a laminated glass is broken; in situations when the replacement of the plate could not be very urgent, structural behavior should be evaluated. The main objective of this paper is to present the structural behavior o laminated glass plates, though modal test and human impact test, including the post fracture behavior for the laminated cases. A god reproducibility and repeatability is obtained. Two main aspects of the structural behavior can be observed: the increment of the rupture load for laminated plates after the failure of the first sheet, and some similarities with a tempered monolithic behavior of equivalent thickness.

Analysis of human cytochrome P450 3A4 cooperativity: Construction and characterization of a site-directed mutant that displays hyperbolic steroid hydroxylation kinetics

Cytochrome P450 3A4 is generally considered to be the most important human drug-metabolizing enzyme and is known to catalyze the oxidation of a number of substrates in a cooperative manner. An allosteric mechanism is usually invoked to explain the cooperativity. Based on a structure–activity study from another laboratory using various effector–substrate combinations and on our own studies using site-directed mutagenesis and computer modeling of P450 3A4, the most likely location of effector binding is in the active site along with the substrate. Our study was designed to test this hypothesis by replacing residues Leu-211 and Asp-214 with the larger Phe and Glu, respectively. These residues were predicted to constitute a portion of the effector binding site, and the substitutions were designed to mimic the action of the effector by reducing the size of the active site. The L211F/D214E double mutant displayed an increased rate of testosterone and progesterone 6β-hydroxylation at low substrate concentrations and a decreased level of heterotropic stimulation elicited by α-naphthoflavone. Kinetic analyses of the double mutant revealed the absence of homotropic cooperativity with either steroid substrate. At low substrate concentrations the steroid 6β-hydroxylase activity of the wild-type enzyme was stimulated by a second steroid, whereas L211F/D214E displayed simple substrate inhibition. To analyze L211F/D214E at a more mechanistic level, spectral binding studies were carried out. Testosterone binding by the wild-type enzyme displayed homotropic cooperativity, whereas substrate binding by L211F/D214E displayed hyperbolic behavior.

Interaction of the human androgen receptor transactivation function with the general transcription factor TFIIF

The human androgen receptor (AR) is a ligand-activated transcription factor that regulates genes important for male sexual differentiation and development. To better understand the role of the receptor as a transcription factor we have studied the mechanism of action of the N-terminal transactivation function. In a protein–protein interaction assay the AR N terminus (amino acids 142–485) selectively bound to the basal transcription factors TFIIF and the TATA-box-binding protein (TBP). Reconstitution of the transactivation activity in vitro revealed that AR142–485 fused to the LexA protein DNA-binding domain was competent to activate a reporter gene in the presence of a competing DNA template lacking LexA binding sites. Furthermore, consistent with direct interaction with basal transcription factors, addition of recombinant TFIIF relieved squelching of basal transcription by AR142–485. Taken together these results suggest that one mechanism of transcriptional activation by the AR involves binding to TFIIF and recruitment of the transcriptional machinery.

Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil

Increasing resistance of Plasmodium falciparum malaria parasites to chloroquine and the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and cycloguanil have sparked renewed interest in the antimalarial drugs WR99210 and proguanil, the cycloguanil precursor. To investigate suggestions that WR99210 and proguanil act against a target other than the reductase moiety of the P. falciparum bifunctional DHFR–thymidylate synthase enzyme, we have transformed P. falciparum with a variant form of human DHFR selectable by methotrexate. Human DHFR was found to fully negate the antiparasitic effect of WR99210, thus demonstrating that the only significant action of WR99210 is against parasite DHFR. Although the human enzyme also resulted in greater resistance to cycloguanil, no decrease was found in the level of susceptibility of transformed parasites to proguanil, thus providing evidence of intrinsic activity of this parent compound against a target other than DHFR. The transformation system described here has the advantage that P. falciparum drug-resistant lines are uniformly sensitive to methotrexate and will complement transformation with existing pyrimethamine-resistance markers in functional studies of P. falciparum genes. This system also provides an approach for screening and identifying novel DHFR inhibitors that will be important in combined chemotherapeutic formulations against malaria.

Characterization of human cardiac Na+ channel mutations in the congenital long QT syndrome

The congenital long QT syndrome (LQTS) is an inherited disorder characterized by a prolonged cardiac action potential. This delay in cellular repolarization can lead to potentially fatal arrhythmias. One form of LQTS (LQT3) has been linked to the human cardiac voltage-gated sodium channel gene (SCN5A). Three distinct mutations have been identified in the sodium channel gene. The biophysical and functional characteristics of each of these mutant channels were determined by heterologous expression of a recombinant human heart sodium channel in a mammalian cell line. Each mutation caused a sustained, non-inactivating sodium current amounting to a few percent of the peak inward sodium current, observable during long (>50 msec) depolarizations. The voltage dependence and rate of inactivation were altered, and the rate of recovery from inactivation was changed compared with wild-type channels. These mutations in diverse regions of the ion channel protein, all produced a common defect in channel gating that can cause the long QT phenotype. The sustained inward current caused by these mutations will prolong the action potential. Furthermore, they may create conditions that promote arrhythmias due to prolonged depolarization and the altered recovery from inactivation. These results provide insights for successful intervention in the disease.