Statistical deconvolution of enthalpic energetic contributions to MHC-peptide binding affinity

Background - MHC Class I molecules present antigenic peptides to cytotoxic T cells, which forms an integral part of the adaptive immune response. Peptides are bound within a groove formed by the MHC heavy chain. Previous approaches to MHC Class I-peptide binding prediction have largely concentrated on the peptide anchor residues located at the P2 and C-terminus positions. Results - A large dataset comprising MHC-peptide structural complexes was created by re-modelling pre-determined x-ray crystallographic structures. Static energetic analysis, following energy minimisation, was performed on the dataset in order to characterise interactions between bound peptides and the MHC Class I molecule, partitioning the interactions within the groove into van der Waals, electrostatic and total non-bonded energy contributions. Conclusion - The QSAR techniques of Genetic Function Approximation (GFA) and Genetic Partial Least Squares (G/PLS) algorithms were used to identify key interactions between the two molecules by comparing the calculated energy values with experimentally-determined BL50 data. Although the peptide termini binding interactions help ensure the stability of the MHC Class I-peptide complex, the central region of the peptide is also important in defining the specificity of the interaction. As thermodynamic studies indicate that peptide association and dissociation may be driven entropically, it may be necessary to incorporate entropic contributions into future calculations.

The production of a framework of competences for pharmacy practice in the European Union:the PHAR-QA project

Background and objectives: The goal of the PHAR-QA (Qualityassurance in European pharmacy education and training) project isthe production of a European framework of competences for pharmacypractice. This PHAR-QA framework (www.phar-qa.eu) will beEuropean and consultative i.e. it will be used for harmonization—butwill not to replace existing national QA systems.Methods: Using the proposals for competences produced by the previousPHARMINE(Pharmacy education in Europe; www.pharmine.eu) project, together with those of other sources, the authors produced a listof 68 personal and patient care competencies. Using internet surveytools the stakeholders—European pharmacy community (universitydepartment staff and students, community, hospital and industrialpharmacists, as well as pharmacists working in clinical biology andother branches, together with representatives of chambers and associations)—were invited to rank the proposals and add comments.Results and conclusions: Pharmacology and pharmacotherapy togetherwith competences such as ‘‘supply of appropriate medicinestaking into account dose, correct formulation, concentration, administrationroute and timing’’ ranked high. Other topics such as ‘‘currentknowledge of design, synthesis, isolation, characterisation and biologicalevaluation of active substances’’ ranked lower.Implications for practice: In the short term, it is anticipated that thissurvey will stimulate a productive discussion on pharmacy educationand practice by the various stakeholders. In the long term, thisframework could serve as a European model framework of competencesfor pharmacy practice.Acknowledgements: With the support of the Lifelong Learningprogramme of the European Union: 527194-LLP-1-2012-1-BEERASMUS-EMCR. This publication reflects the views only of theauthors; the Commission cannot be held responsible for any usewhich may be made of the information contained therein.

A planning model for needs analysis for subacute care

The purpose of this study is to produce a model to be used by state regulating agencies to assess demand for subacute care. In accomplishing this goal, the study refines the definition of subacute care, demonstrates a method for bed need assessment, and measures the effectiveness of this new level of care. This was the largest study of subacute care to date. Research focused on 19 subacute units in 16 states, each of which provides high-intensity rehabilitative and/or restorative care carried out in a high-tech unit. Each of the facilities was based in a nursing home, but utilized separate staff, equipment, and services. Because these facilities are under local control, it was possible to study regional differences in subacute care demand.^ Using this data, a model for predicting demand for subacute care services was created, building on earlier models submitted by John Whitman for the American Hospital Association and Robin E. MacStravic. The Broderick model uses the "bootstrapping" method and takes advantage of high technology: computers and software, databases in business and government, publicly available databases from providers or commercial vendors, professional organizations, and other information sources. Using newly available sources of information, this new model addresses the problems and needs of health care planners as they approach the challenges of the 21st century. ^

Enhanced skip -list search algorithm in 3-layer mediator framework

The Three-Layer distributed mediation architecture, designed by Secure System Architecture laboratory, employed a layered framework of presence, integration, and homogenization mediators. The architecture does not have any central component that may affect the system reliability. A distributed search technique was adapted in the system to increase its reliability. An Enhanced Chord-like algorithm (E-Chord) was designed and deployed in the integration layer. The E-Chord is a skip-list algorithm based on Distributed Hash Table (DHT) which is a distributed but structured architecture. DHT is distributed in the sense that no central unit is required to maintain indexes, and it is structured in the sense that indexes are distributed over the nodes in a systematic manner. Each node maintains three kind of routing information: a frequency list, a successor/predecessor list, and a finger table. None of the nodes in the system maintains all indexes, and each node knows about some other nodes in the system. These nodes, also called composer mediators, were connected in a P2P fashion. ^ A special composer mediator called a global mediator initiates the keyword-based matching decomposition of the request using the E-Chord. It generates an Integrated Data Structure Graph (IDSG) on the fly, creates association and dependency relations between nodes in the IDSG, and then generates a Global IDSG (GIDSG). The GIDSG graph is a plan which guides the global mediator how to integrate data. It is also used to stream data from the mediators in the homogenization layer which connected to the data sources. The connectors start sending the data to the global mediator just after the global mediator creates the GIDSG and just before the global mediator sends the answer to the presence mediator. Using the E-Chord and GIDSG made the mediation system more scalable than using a central global schema repository since all the composers in the integration layer are capable of handling and routing requests. Also, when a composer fails, it would only minimally affect the entire mediation system. ^

A planning model for needs analysis for subacute care

The purpose of this study is to produce a model to be used by state regulating agencies to assess demand for subacute care. In accomplishing this goal, the study refines the definition of subacute care, demonstrates a method for bed need assessment, and measures the effectiveness of this new level of care. This was the largest study of subacute care to date. Research focused on 19 subacute units in 16 states, each of which provides high-intensity rehabilitative and/or restorative care carried out in a high-tech unit. Each of the facilities was based in a nursing home, but utilized separate staff, equipment, and services. Because these facilities are under local control, it was possible to study regional differences in subacute care demand. Using this data, a model for predicting demand for subacute care services was created, building on earlier models submitted by John Whitman for the American Hospital Association and Robin E. MacStravic. The Broderick model uses the "bootstrapping" method and takes advantage of high technology: computers and software, databases in business and government, publicly available databases from providers or commercial vendors, professional organizations, and other information sources. Using newly available sources of information, this new model addresses the problems and needs of health care planners as they approach the challenges of the 21st century.

Gestão escolar democrática, participação e autonomia: realidade ou utopia?

This paper analyzed the relation between participation and school autonomy, observing Director actions and School Council actions and if school has pedagogic, administrative and financial autonomy. This is a descriptive search, with quantitative approach, realized in 47 state schools in Rio Grande do Norte, and composed by 292 participants, divided in 3 groups (Direction, Coordination and School Council). The instrument to catch data was a questionnaire and the data treatment realized by way descriptive statistic and inferences using X 2 test. The results show that school administration is composed in great part by women with specialization grade. All schools have School Council and Financial Council, but have not Parents and Masters Association and just one of them has Student Guild. The direction choose process is, in 83% of the schools, by elective way. The indicators show that the relation involving family, society and school stays weak, but Directors, Coordinators and School Council members develop their functions with coherence. About autonomy, it is limited and presents correlation with participation, but the educational system stays rigid and centralizer

Mortalidade em idosos longevos e "mais jovens" no Brasil

The aim of the present study was to trace the mortality profile of the elderly in Brazil using two neighboring age groups: 60 to 69 years (young-old) and 80 years or more (oldest-old). To do this, we sought to characterize the trend and distinctions of different mortality profiles, as well as the quality of the data and associations with socioeconomic and sanitary conditions in the micro-regions of Brazil. Data was collected from the Mortality Information System (SIM) and the Brazilian Institute of Geography and Statistics (IBGE). Based on these data, the coefficients of mortality were calculated for the chapters of the International Disease Classification (ICD-10). A polynomial regression model was used to ascertain the trend of the main chapters. Non-hierarchical cluster analysis (K-Means) was used to obtain the profiles for different Brazilian micro-regions. Factorial analysis of the contextual variables was used to obtain the socio-economic and sanitary deprivation indices (IPSS). The trend of the CMId and of the ratio of its values in the two age groups confirmed a decrease in most of the indicators, particularly for badly-defined causes among the oldest-old. Among the young-old, the following profiles emerged: the Development Profile; the Modernity Profile; the Epidemiological Paradox Profile and the Ignorance Profile. Among the oldest-old, the latter three profiles were confirmed, in addition to the Low Mortality Rates Profile. When comparing the mean IPSS values in global terms, all of the groups were different in both of the age groups. The Ignorance Profile was compared with the other profiles using orthogonal contrasts. This profile differed from all of the others in isolation and in clusters. However, the mean IPSS was similar for the Low Mortality Rates Profile among the oldest-old. Furthermore, associations were found between the data quality indicators, the CMId for badly-defined causes, the general coefficient of mortality for each age group (CGMId) and the IPSS of the micro-regions. The worst rates were recorded in areas with the greatest socioeconomic and sanitary deprivation. The findings of the present study show that, despite the decrease in the mortality coefficients, there are notable differences in the profiles related to contextual conditions, including regional differences in data quality. These differences increase the vulnerability of the age groups studied and the health iniquities that are already present.

Borboletas no semiárido: sazonalidade e padrões de diversidade de borboletas frugívoras em um ambiente extremo

The spatial and temporal distribution of the population reflects the adjustment of their biological characteristics to environmental conditions and biotic interactions as adaptive and phylogenetic precursors elements. The habitat’s heterogeneity and alternating seasons tend to cause patterns of activity of organisms and species diversity. However, these seasonal and spatial patterns in butterfly communities in dry environments are not yet clear. We studied a community of frugivorous butterflies in ESEC Seridó, in northeastern Brazil, aiming to characterize the guild in semiarid and check the relative contribution of climate and vegetation variables on its composition, diversity and phenofaunistic. The butterflies were sampled monthly during one year, and the distribution of species was associated with structural characteristics of three vegetation types (eg. richness and abundance of tree and shrub species, canopy cover, herbaceous cover, litter) and climatological data (temperature, rainfall and humidity). We captured 9580 individuals of 16 species of butterflies belonging to four subfamilies (Biblidinae, Charaxinae, Nymphalinae and Satyrinae). The richness, abundance and diversity varied in different scales, especially in time, being higher in the rainy season, while the β-diversity and turnover was higher in the dry. The distribution of species mainly followed the changes in humidity, rainfall and vegetation phenology, with no defined boundaries between habitats. The flight period was shared within subfamilies, which should have distinct response to environmental stimuli, as well as respond to the phenology of host plants and have different reproductive strategies. There is even evidence of physiological and behavioral adaptations as seasonal reproduction and aestivation. So there was environmental control over the distribution and diversity of species, with the key role climate Association and vegetation structure in the community of differentiation in the seasons, and the availability and quality of resources on the variation of species abundance in small scales. These results may support the biomonitoring and conservation preserved areas, particularly in environments under human pressure and extreme environmental conditions such as semi-arid.

Functional Evaluation of Causal Mutations Identified in Human Genetic Studies

Human genetics has been experiencing a wave of genetic discoveries thanks to the development of several technologies, such as genome-wide association studies (GWAS), whole-exome sequencing, and whole genome sequencing. Despite the massive genetic discoveries of new variants associated with human diseases, several key challenges emerge following the genetic discovery. GWAS is known to be good at identifying the locus associated with the patient phenotype. However, the actually causal variants responsible for the phenotype are often elusive. Another challenge in human genetics is that even the causal mutations are already known, the underlying biological effect might remain largely ambiguous. Functional evaluation plays a key role to solve these key challenges in human genetics both to identify causal variants responsible for the phenotype, and to further develop the biological insights from the disease-causing mutations.

We adopted various methods to characterize the effects of variants identified in human genetic studies, including patient genetic and phenotypic data, RNA chemistry, molecular biology, virology, and multi-electrode array and primary neuronal culture systems. Chapter 1 is a broader introduction for the motivation and challenges for functional evaluation in human genetic studies, and the background of several genetics discoveries, such as hepatitis C treatment response, in which we performed functional characterization.

Chapter 2 focuses on the characterization of causal variants following the GWAS study for hepatitis C treatment response. We characterized a non-coding SNP (rs4803217) of IL28B (IFNL3) in high linkage disequilibrium (LD) with the discovery SNP identified in the GWAS. In this chapter, we used inter-disciplinary approaches to characterize rs4803217 on RNA structure, disease association, and protein translation.

Chapter 3 describes another avenue of functional characterization following GWAS focusing on the novel transcripts and proteins identified near the IL28B (IFNL3) locus. It has been recently speculated that this novel protein, which was named IFNL4, may affect the HCV treatment response and clearance. In this chapter, we used molecular biology, virology, and patient genetic and phenotypic data to further characterize and understand the biology of IFNL4. The efforts in chapter 2 and 3 provided new insights to the candidate causal variant(s) responsible for the GWAS for HCV treatment response, however, more evidence is still required to make claims for the exact causal roles of these variants for the GWAS association.

Chapter 4 aims to characterize a mutation already known to cause a disease (seizure) in a mouse model. We demonstrate the potential use of multi-electrode array (MEA) system for the functional characterization and drug testing on mutations found in neurological diseases, such as seizure. Functional characterization in neurological diseases is relatively challenging and available systematic tools are relatively limited. This chapter shows an exploratory research and example to establish a system for the broader use for functional characterization and translational opportunities for mutations found in neurological diseases.

Overall, this dissertation spans a range of challenges of functional evaluations in human genetics. It is expected that the functional characterization to understand human mutations will become more central in human genetics, because there are still many biological questions remaining to be answered after the explosion of human genetic discoveries. The recent advance in several technologies, including genome editing and pluripotent stem cells, is also expected to make new tools available for functional studies in human diseases.

The end of the shock of the new

‘Shock’ advertising is the new black and the subject of the reflection in which this article engages. We do this in particular through consideration of the (largely) British high-street fashion house French Connection’s seemingly endless ‘FCUK’ campaign. The obvious resonance between this abbreviation and perhaps the most popular word in the English language was at the heart of the campaign’s appeal and it continues today through various extensions on both slogans and logos on French Connection’s own goods and indeed those who seek to piggy back upon and/or subvert its market power. It is far from the only example of such ‘shock’ tactics. Whether discussing reproduction in graphic detail with children, joyously dismantling chastity, or merely fucking with fuck, it seems that traditional mores can no longer remain virgin territory, unsullied by rapacious marketing. Our mediated experiences of reaching ‘extremes’, it now appears, are not paralysing, mesmerising, fascinating or inspiring but simply a further prod down the path leading to (gleeful) purchase. In this paper we explore how, via a series of semiotic reversals, the new, the strange, the unfamiliar and the would-be shocking are rendered banal, and thus thoroughly comprehensible through brand association and the endless re-iteration of existing works.

Étude des mécanismes d’extraction lipidique par le peptide mélittine et la protéine BSP1

Les peptides et protéines extracteurs de lipides (PEL) se lient aux membranes lipidiques puis en extraient des lipides en formant de plus petits auto-assemblages, un phénomène qui peut aller jusqu'à la fragmentation des membranes. Dans la nature, cette extraction se produit sur une gamme de cellules et entraîne des conséquences variées, comme la modification de la composition de la membrane et la mort de la cellule. Cette thèse se penche sur l’extraction lipidique, ou fragmentation, induite par le peptide mélittine et la protéine Binder-of-SPerm 1 (BSP1) sur des membranes lipidiques modèles. Pour ce faire, des liposomes de différentes compositions sont préparés et incubés avec la mélittine ou la BSP1. L'association aux membranes est déterminée par la fluorescence intrinsèque des PEL, tandis que l'extraction est caractérisée par une plateforme analytique combinant des tests colorimétriques et des analyses en chromatographie en phase liquide et spectrométrie de masse (LCMS). La mélittine fait partie des peptides antimicrobiens cationiques, un groupe de PEL très répandu chez les organismes vivants. Ces peptides sont intéressants du point du vue médical étant donné leur mode d’action qui vise directement les lipides des membranes. Plusieurs de ceux-ci agissent sur les membranes des bactéries selon le mécanisme dit « en tapis », par lequel ils s’adsorbent à leur surface, forment des pores et ultimement causent leur fragmentation. Dans cette thèse, la mélittine est utilisée comme peptide modèle afin d’étudier le mécanisme par lequel les peptides antimicrobiens cationiques fragmentent les membranes. Les résultats montrent que la fragmentation des membranes de phosphatidylcholines (PC) est réduite par une déméthylation graduelle de leur groupement ammonium. L'analyse du matériel fragmenté révèle que les PC sont préférentiellement extraites des membranes, dû à un enrichissement local en PC autour de la mélittine à l'intérieur de la membrane. De plus, un analogue de la mélittine, dont la majorité des résidus cationiques sont neutralisés, est utilisé pour évaluer le rôle du caractère cationique de la mélittine native. La neutralisation augmente l'affinité du peptide pour les membranes neutres et anioniques, réduit la fragmentation des membranes neutres et augmente la fragmentation des membranes anioniques. Malgré les interactions électrostatiques entre le peptide cationique et les lipides anioniques, aucune spécificité lipidique n'est observée dans l'extraction. La BSP1 est la protéine la plus abondante du liquide séminal bovin et constitue un autre exemple de PEL naturel important. Elle se mélange aux spermatozoïdes lors de l’éjaculation et extrait des lipides de leur membrane, notamment le cholestérol et les phosphatidylcholines. Cette étape cruciale modifie la composition lipidique de la membrane du spermatozoïde, ce qui faciliterait par la suite la fécondation de l’ovule. Cependant, le contact prolongé de la protéine avec les spermatozoïdes endommagerait la semence. Cette thèse cherche donc à approfondir notre compréhension de ce délicat phénomène en étudiant le mécanisme moléculaire par lequel la protéine fragmente les membranes lipidiques. Les résultats des présents travaux permettent de proposer un mécanisme d’extraction lipidique en 3 étapes : 1) L'association à l’interface des membranes; 2) La relocalisation de l’interface vers le cœur lipidique; 3) La fragmentation des membranes. La BSP1 se lie directement à deux PC à l'interface; une quantité suffisante de PC dans les membranes est nécessaire pour permettre l'association et la fragmentation. Cette liaison spécifique ne mène généralement pas à une extraction lipidique sélective. L'impact des insaturations des chaînes lipidiques, de la présence de lysophosphatidylcholines, de phosphatidyléthanolamine, de cholestérol et de lipides anioniques est également évalué. Les présentes observations soulignent la complexe relation entre l'affinité d'un PEL pour une membrane et le niveau de fragmentation qu'il induit. L'importance de la relocalisation des PEL de l'interface vers le cœur hydrophobe des membranes pour permettre leur fragmentation est réitérée. Cette fragmentation semble s'accompagner d'une extraction lipidique préférentielle seulement lorsqu'une séparation de phase est induite au niveau de la membrane, nonobstant les interactions spécifiques PEL-lipide. Les prévalences des structures amphiphiles chez certains PEL, ainsi que de la fragmentation en auto-assemblages discoïdaux sont discutées. Finalement, le rôle des interactions électrostatiques entre les peptides antimicrobiens cationiques et les membranes bactériennes anioniques est nuancé : les résidus chargés diminueraient l'association des peptides aux membranes neutres suite à l'augmentation de leur énergie de solvatation.

Continuidades y rupturas en el proceso de centralización de la administración sanitaria argentina (1880-1945)

El objetivo del trabajo es presentar el proceso de centralización administrativa de la repartición sanitaria nacional. Para ello se analizan, en primer lugar y con la intención de situar el problema en un plazo más largo, los límites que debió afrontar el Departamento Nacional de Higiene desde el momento de su creación, en 1880, para avanzar en sus intenciones centralizadoras. Entre ellos se encuentran su escasa autonomía administrativa, las superposiciones jurisdiccionales con otras dependencias del Estado, las indefiniciones respecto de su supremacía jerárquica, la resistencia de las provincias, los municipios fuertes y las asociaciones benéficas y los conflictos de proyectos al interior mismo de la repartición. En segundo lugar se muestra cómo los sucesivos presidentes del Departamento Nacional de Higiene asumieron ese límite para su gestión y apostaron a la organización interna de la repartición antes que al desafío de la centralización de la asistencia sanitaria. Su estrategia fue el fortalecimiento de nuevas áreas de incumbencia que constituyeron una agenda que sirvió como base de la definitiva centralización de la administración sanitaria. En esta tarea contaron con el apoyo parlamentario, fundamentalmente de la bancada socialista, que logró convertir en ley durante los años 30 a una serie de nuevas atribuciones del Departamento en aspectos que ligaban la salud con la asistencia social. Por último se analiza un momento clave de este proceso, el primer ensayo de centralización sanitaria a través de la creación, en 1943, de la Dirección Nacional de Salud Pública y Asistencia Social.

Continuidades y rupturas en el proceso de centralización de la administración sanitaria argentina (1880-1945)

El objetivo del trabajo es presentar el proceso de centralización administrativa de la repartición sanitaria nacional. Para ello se analizan, en primer lugar y con la intención de situar el problema en un plazo más largo, los límites que debió afrontar el Departamento Nacional de Higiene desde el momento de su creación, en 1880, para avanzar en sus intenciones centralizadoras. Entre ellos se encuentran su escasa autonomía administrativa, las superposiciones jurisdiccionales con otras dependencias del Estado, las indefiniciones respecto de su supremacía jerárquica, la resistencia de las provincias, los municipios fuertes y las asociaciones benéficas y los conflictos de proyectos al interior mismo de la repartición. En segundo lugar se muestra cómo los sucesivos presidentes del Departamento Nacional de Higiene asumieron ese límite para su gestión y apostaron a la organización interna de la repartición antes que al desafío de la centralización de la asistencia sanitaria. Su estrategia fue el fortalecimiento de nuevas áreas de incumbencia que constituyeron una agenda que sirvió como base de la definitiva centralización de la administración sanitaria. En esta tarea contaron con el apoyo parlamentario, fundamentalmente de la bancada socialista, que logró convertir en ley durante los años 30 a una serie de nuevas atribuciones del Departamento en aspectos que ligaban la salud con la asistencia social. Por último se analiza un momento clave de este proceso, el primer ensayo de centralización sanitaria a través de la creación, en 1943, de la Dirección Nacional de Salud Pública y Asistencia Social.

Continuidades y rupturas en el proceso de centralización de la administración sanitaria argentina (1880-1945)

El objetivo del trabajo es presentar el proceso de centralización administrativa de la repartición sanitaria nacional. Para ello se analizan, en primer lugar y con la intención de situar el problema en un plazo más largo, los límites que debió afrontar el Departamento Nacional de Higiene desde el momento de su creación, en 1880, para avanzar en sus intenciones centralizadoras. Entre ellos se encuentran su escasa autonomía administrativa, las superposiciones jurisdiccionales con otras dependencias del Estado, las indefiniciones respecto de su supremacía jerárquica, la resistencia de las provincias, los municipios fuertes y las asociaciones benéficas y los conflictos de proyectos al interior mismo de la repartición. En segundo lugar se muestra cómo los sucesivos presidentes del Departamento Nacional de Higiene asumieron ese límite para su gestión y apostaron a la organización interna de la repartición antes que al desafío de la centralización de la asistencia sanitaria. Su estrategia fue el fortalecimiento de nuevas áreas de incumbencia que constituyeron una agenda que sirvió como base de la definitiva centralización de la administración sanitaria. En esta tarea contaron con el apoyo parlamentario, fundamentalmente de la bancada socialista, que logró convertir en ley durante los años 30 a una serie de nuevas atribuciones del Departamento en aspectos que ligaban la salud con la asistencia social. Por último se analiza un momento clave de este proceso, el primer ensayo de centralización sanitaria a través de la creación, en 1943, de la Dirección Nacional de Salud Pública y Asistencia Social.

La creación de valor en el museo y la sociomuseología

Los museos tienen normalmente mucha dificultad en justificar su existencia y en demostrar su valor real y su contribución al desarrollo de las comunidades a las que sirven. El coste del mantenimiento de un museo, después de inaugurado, nunca parece estar justificado para las tutelas y financiadores que lo promovieron. El museo, grande o pequeño, puede ser un elemento esencial en el desarrollo cultural del conjunto social, pero necesita tener una actividad que lo justifique y que su labor sea reconocida. Para contribuir con eficacia debe compartir con la comunidad su misión y sus actividades y para probar su valor debe tener sistemas de evaluación que permitan verificar si su labor de investigación y acción sobre el patrimonio, de promoción de la cultura y la ciudadanía y de colaboración con escuelas y asociaciones, reduce el fracaso escolar, aumenta la participación en la vida y cultura o contribuye a la cohesión social y a la formación de ciudadanos más responsables y solidarios. La Sociomuseología estudia estos campos de la actividad del museo, acompaña experiencias y propone soluciones a estas dificultades.