Detecção do vírus Epstein-Barr em carcinoma espinocelular oral e mucosa oral na região de Araçatuba – SP, Brasil

Pós-graduação em Odontologia - FOA

Detecção do HPV e EBV por nPCR em líquen plano bucal e tecido normal de cavidade bucal

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genotipagem de HLA de classe I e II para seleção de células precursoras dendríticas para estudo da imunobiologia do vírus associado ao Sarcoma de Kaposi (KSHV)

The Kaposi-associated Herpesvirus (KSHV) also known as Human Herpesvirus 8 (HHV-8) is associated with the development of Kaposi’s sarcoma (KS) and others limphoprolipheratives diseases such as Primary Effusion Lymphoma (PEL) and Multicentric Castleman Disease (MCD). Even though the virus is considered lymphotropic, it is able to infect others cell types such as macrophages, dendritic cells, endothelial cells, monocytes and fibroblasts. After infection, KSHV be latent expressing essential viral genes to its maintenance in a infected cell. However, in some circumstances may occur the reactivation of lytic cycle producing new viral particles. K1 protein of KSHV interferes in the cellular signaling inducing proliferation and supporting cellular transformation. K1 is encoded by viral ORF-K1, which shows high variability between different genotypes of KSHV. So far, it is not clear whether different isoforms of K1 have specific immunobiological features. The KSHV latency is maintained under strict control by the immune system supported by an adequate antigen presentation involving Human Leucocyte Antigen (HLA) class I and II. Polymorphisms of HLA class I and II genes confer an enormous variability in molecules that recognize a large amount of antigens, but also can increase the susceptibility to autoimmune diseases. Therefore, the present study aims to genotype HLA class I (A and B) and class II (DR and DQ) from volunteers to identify haplotypes that can provide better response to K1 epitopes of different KSHV genotypes. First of all, 20 volunteers were selected to genotype HLA genes. In our results we observed prevalence of certain HLA class I haplotypes as HLAA1, HLA-A2, HLA-A24, HLA-A26, HLA-B8, HLA-B18 e HLA-B44. After the in silico analysis using BIMAS and SYFPEITHI databases, we observed high scores for epitopes from the B genotype of KSHV, indicating...(Complete abstract click electronic access below)

Produção de vetores recombinantes para expressão de diferentes formas da proteína K1 do herpesvírus associado ao Sarcoma de Kaposi (KSHV)

Kaposi´s sarcoma associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8) is a gammaherpesvirus essential for the development of all forms of Kaposi´s sarcoma (KS). The KSHV’s life cycle is basically divided into latent and lytic phases, which have distinct viral gene expression profiles. Some important oncogenic products of KSHV are expressed during the lytic phase, including the viral K1 protein. As an effect of interfer-ence with intracellular signaling, K1 expression increases proliferation and survival of KSHV-infected cells. Due to its high level of genetic variability compared to other re-gions of the viral genome, the K1-encoding ORF (ORF-K1) is commonly evaluated for KSHV genotyping. It remains unclear whether different viral genotypes have particular biological effects that might modify the KSHV oncogenicity. The present study aimed to contribute to the establishment of an experimental in vitro model for evaluation of the K1 protein from common KSHV genotypes. Recombinant expression vectors with the ORF-K1 from KSHV genotypes A, B and C were prepared by genetic cloning. The recombi-nant vectors pKSHVOK1 obtained by cloning were sequenced for structural validation. After that, HEK293 cell line was transfected with the recombinant vectors, and proteins were extracted for expression analysis by Western blot technique, for K1 functional vali-dation. Results showed that ORF-K1 vectors containing KSHV ORF-K1 from the A, B and C genotypes were produced and structurally validated by DNA sequencing. The K1 expression at the protein level was also confirmed by immunoblots using an antibody for FLAG detection, an epitope from the vector that binds to K1. Based on presented re-sults, it´s possible to conclude that the recombinant vectors will be able to be used in future studies of K1 protein biological properties from distinct KSHV genotypes

Interazioni tra le glicoproteine D, B, H ed L critiche per la fusione indotta dal virus Herpes simplex

Four glycoproteins (gD, gB, gH, and gL) are required for herpes simplex virus (HSV) entry into the cell and for cell-cell fusion in transfected cells. gD serves as the receptor-binding glycoprotein and as the trigger of fusion; the other three glycoproteins execute fusion between the viral envelope and the plasma or endocytic membranes. Little is known on the interaction of gD with gB, gH, and gL. Here, the interactions between herpes simplex virus gD and its nectin1 receptor or between gD, gB, and gH were analyzed by complementation of the N and C portions of split enhanced green fluorescent protein (EGFP) fused to the glycoproteins. Split EGFP complementation was detected between proteins designated gDN + gHC, gDN + gBC, and gHN + gBC + wtgD, both in cells transfected with two or tree glycoproteins and in cells transfected with the four glycoproteins, commited to form syncytia. The in situ assay provides evidence that gD interacts with gH and gB independently one of the other. We further document the interaction between gH and gB. To elucidate which portions of the glycoproteins interact with each other we generated mutants of gD and gB. gD triggers fusion through a specialised domain, named pro-fusion domain (PFD), located C-terminally in the ectodomain. Here, we show that PFD is made of subdomains 1 and 2 (amino acids 260–285 and 285–310) and that each one partially contributed to herpes simplex virus infectivity. Chimeric gB molecules composed of HSV and human herpesvirus 8 (HHV8) sequences failed to reach the cell surface and to complement a gB defective virus. By means of pull down experiments we analyzed the interactions of HSV-HHV8 gB chimeras with gH or gD fused to the strep-tag. The gB sequence between aa residues 219-360 was identified as putative region of interaction with gH or critical to the interaction.

Alterazioni dell'immunità umorale nei pazienti con sarcoma di Kaposi classico: caratterizzazione dei linfociti B circolanti e delle loro sottopopolazioni

Objectives: Human Herpesvirus 8 (HHV-8) is the etiological agent of Kaposi’s Sarcoma (KS) and it is also associated with two B cell lymphoproliferative diseases: primary effusion lymphoma (PEL), and the plasmablastic form of multicentric Castelman’s disease (MCD). HHV-8 establishes persistent infection in the host with tropism for multiple cell types. In KS patients, the virus is found in tumor-spindle cells, peripheral blood monocytes, endothelial progenitor circulating cells, T and B lymphocytes. Peripheral B cells represent one of the major virus reservoir, but the consequences of HHV-8 infection of these cells have been poorly characterized. Therefore, in this study the frequency, the immunophenotypic profile and the functional activity of different peripheral B cell subsets in patients with classic KS (cKS) was analysed in order to identify potential alterations of these cells. The classic variant of KS is ideal to perform such studies, as it lacks confounding factors such as HIV or EBV infection and immunosuppression. Methods: Whole-blood samples from patients with the classical form of KS (cKS) (n=62) and healthy age and sex-matched seronegative controls (HSN) (n=43) were analyzed by multiparametric flow-cytometry to determine the frequency of B cells and their subpopulations, as well as their surface expression of immunoglobulins and activation markers. Results: The frequency of circulating B cells was significantly higher in cKS patients than in controls. In particular, the analysis of the B cell subsets revealed a higher frequency of naïve B cells (CD19+CD27-), among which transitional CD19+CD38highCD5+ and pre-naïve (CD27-CD38intCD5+ ) B cells demonstrated an expansion. Memory B cells (CD19+CD27+) did not differ between the two study groups, except from a higher frequency of CD19+CD27+IgM+IgD+ B cells, the typical phenotype of marginal zone (MZ) B cells, in cKS patients. The characterization of membrane surface activation markers showed lower levels of the activation marker HLA-DR only on CD27- B cells, while CD80 and CD86 were less represented in all the the B cells from cKS patients. Moreover, B cells from cKS patients were smaller and with less granules than the ones from controls. Conclusion: Taken together, these results clearly indicate that circulating B cells are altered in patients with cKS, showing an expansion of the immature phenotypes. These B cell alterations may be due to an indirect viral effect rather than to a direct one: the cytokines expressed in the microenvironment typical of cKS may cause a faster release of immature cells from the bone marrow and a lower grade of peripheral differentiation, as already suggested for other chronic viral infections such as HIV and HCV. Further studies will be necessary to understand how these alterations contribute to the pathogenesis of KS and, eventually, to the different clinical evolution of the disease.

Habitat-specific demography and source-sink dynamics in a population of Siberian jays

P>1. There are a number of models describing population structure, many of which have the capacity to incorporate spatial habitat effects. One such model is the source-sink model, that describes a system where some habitats have a natality that is higher than mortality (source) and others have a mortality that exceeds natality (sink). A source can be maintained in the absence of migration, whereas a sink will go extinct. 2. However, the interaction between population dynamics and habitat quality is complex, and concerns have been raised about the validity of published empirical studies addressing source-sink dynamics. In particular, some of these studies fail to provide data on survival, a significant component in disentangling a sink from a low quality source. Moreover, failing to account for a density-dependent increase in mortality, or decrease in fecundity, can result in a territory being falsely assigned as a sink, when in fact, this density-dependent suppression only decreases the population size to a lower level, hence indicating a 'pseudo-sink'. 3. In this study, we investigate a long-term data set for key components of territory-specific demography (mortality and reproduction) and their relationship to habitat characteristics in the territorial, group-living Siberian jay (Perisoreus infaustus). We also assess territory-specific population growth rates (r), to test whether spatial population dynamics are consistent with the ideas of source-sink dynamics. 4. Although average mortality did not differ between sexes, habitat-specific mortality did. Female mortality was higher in older forests, a pattern not observed in males. Male mortality only increased with an increasing amount of open areas. Moreover, reproductive success was higher further away from human settlement, indicating a strong effect of human-associated nest predators. 5. Averaged over all years, 76% of the territories were sources. These territories generally consisted of less open areas, and were located further away from human settlement. 6. The source-sink model provides a tool for modelling demography in distinct habitat patches of different quality, which can aid in identifying key habitats within the landscape, and thus, reduce the risk of implementing unsound management decisions.

Predicting the evolution of Kaposi sarcoma, in the highly active antiretroviral therapy era

BACKGROUND: The outcome of Kaposi sarcoma varies. While many patients do well on highly active antiretroviral therapy, others have progressive disease and need chemotherapy. In order to predict which patients are at risk of unfavorable evolution, we established a prognostic score. METHOD: The survival analysis (Kaplan-Meier method; Cox proportional hazards models) of 144 patients with Kaposi sarcoma prospectively included in the Swiss HIV Cohort Study, from January 1996 to December 2004, was conducted. OUTCOME ANALYZED: use of chemotherapy or death. VARIABLES ANALYZED: demographics, tumor staging [T0 or T1 (16)], CD4 cell counts and HIV-1 RNA concentration, human herpesvirus 8 (HHV8) DNA in plasma and serological titers to latent and lytic antigens. RESULTS: Of 144 patients, 54 needed chemotherapy or died. In the univariate analysis, tumor stage T1, CD4 cell count below 200 cells/microl, positive HHV8 DNA and absence of antibodies against the HHV8 lytic antigen at the time of diagnosis were significantly associated with a bad outcome.Using multivariate analysis, the following variables were associated with an increased risk of unfavorable outcome: T1 [hazard ratio (HR) 5.22; 95% confidence interval (CI) 2.97-9.18], CD4 cell count below 200 cells/microl (HR 2.33; 95% CI 1.22-4.45) and positive HHV8 DNA (HR 2.14; 95% CI 1.79-2.85).We created a score with these variables ranging from 0 to 4: T1 stage counted for two points, CD4 cell count below 200 cells/microl for one point, and positive HHV8 viral load for one point. Each point increase was associated with a HR of 2.26 (95% CI 1.79-2.85). CONCLUSION: In the multivariate analysis, staging (T1), CD4 cell count (<200 cells/microl), positive HHV8 DNA in plasma, at the time of diagnosis, predict evolution towards death or the need of chemotherapy.

The power of emotional valence – from cognitive to affective processes in reading

The comprehension of stories requires the reader to imagine the cognitive and affective states of the characters. The content of many stories is unpleasant, as they often deal with conflict, disturbance or crisis. Nevertheless, unpleasant stories can be liked and enjoyed. In this fMRI study, we used a parametric approach to examine (1) the capacity of increasing negative valence of story contents to activate the mentalizing network (cognitive and affective theory of mind, ToM), and (2) the neural substrate of liking negatively valenced narratives. A set of 80 short narratives was compiled, ranging from neutral to negative emotional valence. For each story mean rating values on valence and liking were obtained from a group of 32 participants in a prestudy, and later included as parametric regressors in the fMRI analysis. Another group of 24 participants passively read the narratives in a three Tesla MRI scanner. Results revealed a stronger engagement of affective ToM-related brain areas with increasingly negative story valence. Stories that were unpleasant, but simultaneously liked, engaged the medial prefrontal cortex (mPFC), which might reflect the moral exploration of the story content. Further analysis showed that the more the mPFC becomes engaged during the reading of negatively valenced stories, the more coactivation can be observed in other brain areas related to the neural processing of affective ToM and empathy.

Epstein-Barr virus infection of Langerhans cell precursors as a mechanism of oral epithelial entry, persistence, and reactivation.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus associated with many malignant and nonmalignant human diseases. Life-long latent EBV persistence occurs in blood-borne B lymphocytes, while EBV intermittently productively replicates in mucosal epithelia. Although several models have previously been proposed, the mechanism of EBV transition between these two reservoirs of infection has not been determined. In this study, we present the first evidence demonstrating that EBV latently infects a unique subset of blood-borne mononuclear cells that are direct precursors to Langerhans cells and that EBV both latently and productively infects oral epithelium-resident cells that are likely Langerhans cells. These data form the basis of a proposed new model of EBV transition from blood to oral epithelium in which EBV-infected Langerhans cell precursors serve to transport EBV to the oral epithelium as they migrate and differentiate into oral Langerhans cells. This new model contributes fresh insight into the natural history of EBV infection and the pathogenesis of EBV-associated epithelial disease.

Cloning and characterization of the mouse ret finger protein (rfp), a B box zinc finger protein

An important question in biology is to understand the role of specific gene products in regulating embryogenesis and cellular differentiation. Many of the regulatory proteins possess specific motifs, such as the homeodomain, basic helix-loop-helix structure, zinc finger, and leucine zipper. These sequence motifs participate in specific protein-DNA, protein-RNA, and protein-protein interactions, and are important for the function of these regulatory proteins.^ The human rfp (ret finger protein) belongs to a novel zinc finger protein family, the B box zinc finger family. Most of the B box proteins, including rfp, have a conserved tripartite motif, consisting of two novel zinc fingers (the RING finger and the B box) and a coiled-coil domain. Interestingly, a fusion protein between the tripartite motif of rfp and the tyrosine kinase domain of c-ret has transforming activity. In this study, we examined the expression of rfp during mouse development, and characterized the role of the tripartite motif in rfp function.^ We cloned the mouse rfp cDNA, which shares a 98.4% homology with the human sequence at amino acid level. Such strikingly high degree of homology indicates the high evolutionary pressure on the conservation of the sequence, suggesting that rfp may have an important function. Using the somatic cell hybrid system, we assigned the rfp gene to mouse chromosome 13 and human chromosome 6. Rfp transcripts and protein were ubiquitous in day 10.5-13.5 mouse embryos; however, they were restricted in adult mice, with the highest level of expression in the testis. Rfp expression in the testis is detected only in late pachytene spermatocytes and round spermatids. In both embryos and spermatogenic cells, rfp protein was distributed within cell nuclei in a punctate pattern, similar to the PODs (PML oncogenic domains) observed with another B box protein, PML. In cultured mammalian cells, we found that rfp was indeed co-localized to the PODs with PML. Using the yeast two-hybrid system, we showed that the rfp could specifically interact with PML, and that the interaction was dependent on the distal portion of the rfp coiled-coil domain.^ We also showed that rfp could form homodimers, and both the B box and coiled-coil domain were required for proper dimerization. It seems that the proximal portion of the coiled-coil domain provides the interacting interface, while the B box zinc finger orients the coil and maintains the correct structure of the whole molecule. Our data are consistent with the zinc-binding property and structural analysis of the B box. The RING finger seems to be involved in rfp nuclear localization through interaction with other proteins. We believe that homodimerization and interaction with PML are important for the normal interaction of rfp during development and differentiation. In addition, rfp homodimerization may also be essential for the oncogenic activation of the rfp-ret fusion protein. ^

Análisis de la cadena de valor, canales cortos de comercialización y horticultura multifuncional como opciones de puesta en valor: Caso de la Huerta de Aranjuez

La Vega de Aranjuez ha sido desde hace siglos conocida por la notoriedad de sus jardines y huertas históricas y también, en épocas más recientes, por ser la despensa de Madrid. A mediados del siglo XX, sin embargo, con las transformaciones que el sector agrario ha experimentado no sólo a nivel local, sino a escala global, empezó un proceso de declive que se ha acentuado en los últimos años. Paralelamente, en el año 2001, la Unesco declara el Paisaje Cultural de Aranjuez, como Patrimonio de la Humanidad con un valor universal excepcional. Parte de lo que sustenta este reconocimiento a nivel internacional radica en las peculiaridades del paisaje que conforma la actividad agraria. El cambio de orientación, eminentemente hortofrutícola hacia cultivos extensivos y superficies en barbecho, y la pérdida de importancia del sector, no han pasado desapercibidos para la administración local y han sido varios los intentos de recuperación y dinamización del sector en los últimos años. La investigación de la que es objeto esta Tesis Doctoral surgió por iniciativa del Ayuntamiento de Aranjuez, que en al año 2010 deseó conocer la situación del sector hortícola en el municipio, para lo cual, encargó un estudio a la UPM-ETSIA. Para conocer la realidad desde una perspectiva integral, se planteó abordar el análisis desde la perspectiva de Cadena de Valor. Las implicaciones de este concepto, su evolución y su papel como instrumento de análisis de la cadena alimentaria, han sido revisadas y presentadas en el epígrafe del Marco Conceptual. Por cadena de valor se entiende el conjunto de procesos desde el consumidor, a través de los proveedores que proporcionan productos, servicios e información que añaden valor hacia los clientes (Lambert & Cooper, 2000; Chen, 2004). Para el análisis de la situación del sector hortícola en Aranjuez, se procedió en varios pasos, los cuales constituyeron la primera fase de la investigación y contemplaron lo siguiente: 1. La identificación de los problemas y de las demandas de los actores de la cadena, realizando cortes transversales en el mercado a distintos niveles para conocer los puntos de vista de los operadores de la cadena (Gunasekaran & Patel ,2004; Rojas, 2009; Schiefer, 2007). Para ello se diseñaron cuestionarios y se dirigieron a representantes de cinco eslabones de la cadena presentes en el caso de estudio: Agricultores (15), Mayoristas (11), Detallistas (55), Consumidores (85) y Restaurantes (36), tanto en Aranjuez como en Madrid (un total de 202). 2. Análisis D.A.F.O de cada uno de los eslabones y de la cadena completa, como herramienta para identificar y sintetizar la problemática y las potencialidades del sector. 3. Análisis global de la Cadena de Valor mediante el Método de Organización Sectorial, según Briz, de Felipe y Briz (2010), el cual permite estudiar aspectos de la estructura, la conducta y el funcionamiento de la cadena. 4. Jornada de análisis por parte de un Panel de Expertos en la sede de la Fundación Foro Agrario, en la cual se presentaron las conclusiones del análisis de la cadena de valor y se plantearon estrategias para la dinamización del sector. Con los resultados del análisis en esta primera fase de la investigación, se obtuvo una panorámica de la cadena de valor. Algunos de los aspectos más destacados son los siguientes: • El eslabón productivo en Aranjuez está muy atomizado y trabaja por lo general de forma independiente. Cultiva hasta veinte productos hortícolas diferentes, entre los cuales los frutales están casi en desaparición y hay poca presencia de producto ecológico. Le preocupan los precios poco estables y bajos y por lo general no trabaja con contratos. •El eslabón mayorista prácticamente carece de presencia en Aranjuez y está representado en su mayoría por operadores de Mercamadrid, los cuales demandan mayores volúmenes y mejor normalización de los productos hortícolas procedentes de Aranjuez. • El eslabón detallista consultado es diverso (con representación de comercios minoristas, supermercados e hipermercados) y también demanda mayores volúmenes de producción y mejoras en la normalización. Un 80% conoce los productos de Aranjuez, especialmente el espárrago y la fresa-fresón citados en un 74% y 63% de los casos respectivamente. • En el eslabón de consumidores, el 79% de los encuestados da importancia a la procedencia de los productos hortícolas y un 82% conoce los productos de la Huerta de Aranjuez, siendo los más consumidos el espárrago y el fresón. Un 42% de los encuestados compra producto ecológico por razones de salud, sabor y buscando la ausencia de químicos. • El eslabón de restaurantes es un gran consumidor de hortalizas, la gran mayoría de los productos incluidos en el análisis se oferta en más del 75% de los casos. Las más presentes son tomate y espárrago. La fresa y el fresón (en un 83% de los casos), y el espárrago (72%) seguido de la alcachofa (44%) son los productos de Aranjuez que más se conocen en el conjunto de los restaurantes entrevistados. El 75% de los restaurantes prefiere el producto fresco frente al procesado o congelado. La mayoría de los restaurantes locales (92%) estaría dispuesto a incluir en su carta una diferenciación para los productos de Aranjuez. Sin embargo, en el resto de restaurantes el porcentaje es mucho menor (21%). Entre las conclusiones del análisis se evidenció que un 52% de las ventas de los agricultores se realizaba sin intermediarios y un 69% de los consumidores se manifestaba interesado en adquirir productos de Aranjuez directamente del campo a su casa. Se identificó de esta manera que el acortamiento de la cadena se está utilizando como una forma de crear valor. Para profundizar en este aspecto, se planteó una segunda fase en la investigación. Además de estudiar diversos aspectos de los canales cortos de comercialización, se planteó estudiar el enfoque multifuncional de la horticultura, como estrategia para poner en valor la Huerta ribereña. Para dar una coherencia con la fase anterior, el planteamiento se realizó también desde la perspectiva de la cadena de valor, orientando la consulta hacia el eslabón consumidor. De esta manera, se realizó una consulta mediante cuestionario a 221 consumidores sobre diferentes aspectos relativos a los Canales Cortos de Comercialización (CCC) y la Horticultura Multifuncional (HM)1, lo cual permitió realizar un análisis cuantitativo de diferentes variables. Para completar la visión integral de la cadena corta, se seleccionaron 6 proyectos significativos que son ejemplos de diversas tipologías de canales cortos de comercialización en Aranjuez y Madrid, como representantes del denominado eslabón de “Promotores de CCC” y se consultó también a la Asociación de Productores de la Huerta de Aranjuez, constituida formalmente en el año 2014, como representante del eslabón Productor. Para la obtención de información, la cual tiene un carácter cualitativo en el caso de estos dos eslabones de la cadena, se realizaron cuestionarios y Estudios de Caso de cada proyecto. Los cuestionarios de esta segunda fase de consulta a los eslabones de la cadena corta contuvieron cuestiones relativas a los canales cortos de comercialización, tanto a nivel general (concepto de canal corto, puntos fuertes y ventajas que aportan, dificultades para su desarrollo, factores clave para su éxito) como para el caso concreto de la Huerta de Aranjuez (perfil del consumidor, factores implicados en la compra de verduras, utilización de diversas modalidades de canal corto, relación con las tecnologías de la información y comunicación (TICs) y la producción ecológica o la importancia del factor confianza). También aspectos relacionados con la Horticultura Multifuncional, como son, la valoración de diferentes actividades o proyectos de carácter agro-turístico, educativo, social o terapéutico, o la percepción del consumidor de las actividades de huerta como fuente de bienestar y como agente satisfactor de diversas necesidades humanas. Para completar la visión sobre estos dos temas, CCC y HM, se realizó una consulta mediante cuestionario a Expertos en el campo de los canales cortos de comercialización, procedentes del ámbito académico, y a Profesionales en activo trabajando en proyectos de horticultura social y terapéutica. La información aportada, aunque tiene carácter cualitativo, complementa el estudio ofreciendo la perspectiva académica en el caso de los canales cortos y amplía la información sobre la horticultura multifuncional, tratando cuestiones relativas al desempeño profesional o a la formación existente en España en el campo de la horticultura social y terapéutica. Los resultados de esta segunda fase de la investigación, entre otras cuestiones, evidenciaron que: • Los canales cortos de comercialización implican mucho más que la simple reducción de intermediarios y comprenden una gran diversidad de tipologías. • Los casos estudiados, están enfocados en su mayoría al producto ecológico y su funcionamiento está muy fundamentado en el uso de TICs y en el factor confianza. • En relación a la compra de verduras en el ámbito de los canales cortos, son aspectos muy valorados por el consumidor la calidad del producto, la rapidez y frescura con la que llega del campo a la mesa y que el Producto sea recogido en su punto óptimo de maduración. • Las actividades en el ámbito de la horticultura multifuncional son valoradas positivamente por los consumidores, siendo las más puntuadas las de huerto educativo, huerto terapéutico, seguidas de visitas guiadas y degustaciones de productos de huerta en restaurantes. • Por lo general existe una valoración muy alta de la huerta como fuente de bienestar y de satisfacción de necesidades humanas básicas, especialmente las de una alimentación saludable y de conexión con la naturaleza. Para terminar esta síntesis de la investigación realizada, se presentan las principales conclusiones a nivel global de la Tesis, que son las siguientes: 1. La metodología de cadena de valor ha resultado adecuada para conocer la complejidad y el funcionamiento del sector hortícola arancetano desde una perspectiva integral. 2. La Huerta de Aranjuez cuenta con importantes fortalezas, sustentadas en la calidad de sus suelos y en la fama que mantienen sus productos, pero también debilidades. Esto supone poco volumen de producción, que dificulta la relación con mayoristas y grandes detallistas. 3. El acortamiento de la cadena, mediante canales cortos de comercialización se ha identificado como una forma de creación de valor en la cadena. Existen oportunidades de abastecimiento a consumidores de Madrid mediante canales cortos, sin embargo, las modalidades que requieren mayor organización o requisitos de producción ecológica todavía no están desarrolladas. 4. La producción ecológica podría ser una estrategia para crear valor pero todavía es un método de cultivo muy minoritario en la cadena productiva arancetana. 5. Las peculiaridades de la Huerta de Aranjuez propician la puesta en práctica del enfoque multifuncional de la horticultura como vía de desarrollo económico. Los resultados apuntan a una posible demanda de servicios que contemplen actividades de horticultura de carácter educativo, terapéutico y agro-turístico, conducidas por profesionales. Existe una percepción positiva sobre el potencial de la huerta como fuente de bienestar y de satisfacción de necesidades humanas básicas. 6. La puesta en marcha de proyectos empresariales en el ámbito de la horticultura social es una apuesta interesante para crear valor en la huerta que ha sido valorada positivamente por los eslabones de la cadena corta consultados. 7. El campo de la Horticultura Multifuncional que contempla aspectos educativos, sociales y terapéuticos conforma una disciplina con posibilidades de desarrollo que en la investigación se perciben como limitadas por la falta de profesionales y su acceso a una formación adecuada en España. El estudio de los Canales Cortos de Comercialización y de la Horticultura Multifuncional como vías de puesta en valor en el caso de la Huerta de Aranjuez ha tenido un carácter exploratorio y en gran parte cualitativo en esta Tesis Doctoral. Ambos conceptos han desvelado cierta complejidad y requieren de un mayor conocimiento en diversos aspectos para su puesta en práctica con éxito. Se abre, por tanto, un campo para futuras investigaciones que profundicen en estos ámbitos. ABSTRACT La Vega de Aranjuez has been known for centuries for the reputation of its orchards and historic gardens and, more recently, as the pantry of Madrid. However, in the mid-twentieth century, with the transformations in agriculture, not only locally, but globally, began a process of decline that has been accentuated in recent years. Meanwhile, in 2001, Unesco declared Aranjuez Cultural Landscape, as a World Heritage Site with outstanding universal value. Part of what underpins this international recognition lies in the peculiarities of the landscape created by farming. The shift, from an eminently horticultural vocation to extensive field crops and fallow surfaces, and the loss of importance of the sector, have not gone unnoticed for local authorities and have been several attempts at recovery and revitalization of the sector in recent years. The beginning of this research came at the initiative of the municipality of Aranjuez, which in 2010 wanted to know the situation of the horticultural sector, for which he commissioned a study by the UPM-ETSIA. To know reality from an integral perspective, it was proposed to approach the analysis from the perspective of value chain. The implications of this concept, its evolution and its role as an instrument of analysis of the food chain, have been reviewed and presented in Chapter 3.2. The value chain concept refers to all the processes from the consumer, through suppliers who provide products, services and information that add value to customers (Lambert & Cooper, 2000; Chen, 2004). For the analysis of the situation of the horticultural sector in Aranjuez, which constituted the first phase of research, it proceeded in several steps: 1. Identifying the problems and demands of the actors in the chain, making transverse cuts in the market at different levels to meet the views of the chain operators (Gunasekaran & Patel , 2004; Rojas, 2009; Schiefer, 2007). Questionnaires were designed for it and went to representatives of the five links in the chain: Farmers (15), Wholesalers (11), Retailers (55), Consumers (85) and Restaurants (36), both in Aranjuez and Madrid (a total of 202). 2. SWOT analysis of each chain actor and of the whole supply chain, as a tool to identify and synthesize the problems and potential of the sector. 3. Analysis of the whole supply chain by Industrial Organization Method according to Briz et al. (2010), which allows to study aspects of the structure, conduct and performance of the chain. 4. Analysis by a Panel of Experts at Foro Agrario Foundation headquarters, where the conclusions of the analysis were presented and strategies for the revitalization of the sector were raised. The results of the analysis in this first phase of the research, presented an overview of the value chain. Some of the highlights are: - The productive sector in Aranjuez is very fragmented and usually works independently. With a wide variety of horticultural products (up to 20), fruit crops almost disappearing and little presence of organic product. Is concerned about the unstable and low prices and usually does not work with contracts. - The wholesale sector with virtually no presence in Aranjuez is represented mostly by Mercamadrid operators, who demand higher volumes and better standardization of horticultural products from Aranjuez. - The retailer sector is diversified (with representation from retailers, supermarkets and hypermarkets) and also demand higher production volumes and improved standardization. 80% know the products of Aranjuez, especially asparagus and strawberry-strawberry cited by 74% and 63% of cases respectively. - Among the consumers, 79% give importance to the origin of horticultural products and 82% know the products from Aranjuez, the most consumed asparagus and strawberries. 42% buy organic products for health, taste and absence of chemicals. - Restaurants are big consumers of vegetables, most of the products included in the analysis is offered in over 75% of cases. The most: tomato and asparagus. Strawberry (83% of cases), and asparagus (72%) followed by the artichoke (44%) are the products of Aranjuez more known in all the surveyed restaurants. 75% of the restaurants prefer fresh product against processed or frozen. Most local restaurants (92%) would be willing to include in their menu a differentiation for products of Aranjuez. However, for those restaurants from Madrid the percentage is much lower (21%). Among the conclusions of the analysis it showed that 52% of sales were realized from farmers without intermediaries and 69% of consumers expressed interest in acquiring products directly from field to table. It has been identified that the shortening of the chain is being used as a way to create value. To deepen this aspect, a second phase investigation arose. Besides studying various aspects of the short supply chains, it was also proposed to study the functional approach of horticulture as a strategy to add value. To provide consistency with the previous phase, the focus was also conducted from the perspective of the value chain, directing the query to consumers. Thus, again it was used the questionnaire as a methodological tool, and 221 consumers were asked about different aspects of the Short Suppy Chains (SSC) and Multifunctional Horticulture (MH)2, which allowed a quantitative analysis of several variables. To complete the comprehensive view of the short chain, 6 significant projects were selected as examples of different types of short supply chains in Aranjuez and Madrid, representing "SSC Promoters" and also the “Asociación de Productores de la Huerta de Aranjuez”, formally constituted in 2014, was asked representing the productive sector. The Study Case and again the questionnaire were elected as methodological tools in a qualitative analysis. The questionnaires of this second phase of research contained short supply chain issues, as a general topic (short supply chain concept, strengths and advantages they bring difficulties for its development, key factors success) and also refered to the case of Aranjuez (consumer profile, factors involved in the purchase of vegetables, use of several types of short supply chains, relation with information and communication technologies (ICTs) and organic production or the importance of trust in short supply chains). It also contemplated aspects of multifunctional horticulture, such as the valuation of different activities (agro-tourism, educational, social or therapeutic horticulture) and consumer perception about horticultural activities as a source of welfare and satisfactor of human needs. To complete the vision of these two issues, SSC and MH, experts in the field of short supply chains and professionals working in the field of social and therapeutic horticulture were asked. The qualitative information provided, complements the study offering a new perspective in the value chain analysis, such as those relating to job performance, the difficulties encountered or training existing in our country in the field of social and therapeutic horticulture. The results of this second phase of research showed that: Short supply chains involve much more than simply reducing intermediaries and cover a wide range of types. The cases studied are mostly focused on ecological product and its operation is heavily based on the use of ICTs and the trust factor. In connection with the purchase of vegetables in the field of short supply chains, product quality, speed and freshness with which comes from the field to the table and products picked at its peak maturation, are aspects highly valued by the consumer. Activities in the field of multifunctional horticulture are positively valued by consumers, the most scored: the educational garden, therapeutic garden, followed by guided tours and tastings of vegetables from Aranjuez in restaurants. Horticultural activities were highly valuated as a source of welfare and satisfaction of human needs, especially those of healthy eating and connection with nature. To complete this summary, the main conclusions of the research are presented as follows: 1. The value chain approach has been adequate to meet the complexity and operation of the horticultural sector in Aranjuez from a holistic perspective. 2. La Huerta de Aranjuez has important strengths, underpinned by the quality of its soils and fame that keep their products, but also weaknesses. This implies low volume of production, which makes difficult the link with wholesalers and large retailers. 3. The shortening of the chain by short supply chains has been identified as a way of creating value in the chain. Opportunities exist to supply consumers from Madrid by short supply chains, however, methods that require greater organization or requirements of organic production are not yet developed. 4. Organic production could be a strategy to create value but is not generally being implemented in the production chain. 5. The peculiarity of the Huerta de Aranjuez favours the implementation of the multifunctional approach as a means of economic development. The results point to a possible demand for multifunctional horticulture that include educational, therapeutic and agro-tourism activities. There is a positive perception of the potential of horticultural activities as a source of welfare and satisfaction of basic human needs. 6. The implementation of business projects in the field of social horticulture are an interesting way to create value that has been highly valued in the short supply chain. 7. The field of Multifunctional Horticulture which includes educational, social and therapeutic aspects, forms a discipline with possibilities of development, which in research are seen as limited by the lack of professionals and access to adequate training in our country. The study of Short Supply Chains and Multifunctional Horticulture as strategies to create value in the case of the Huerta de Aranjuez has an exploratory character and largely qualitative in this research. Both concepts have revealed some complexity and require greater knowledge in various aspects for successful implementation. It opens therefore a field for future research to deepen in these areas.

CLIMA ORGANIZACIONAL E BURNOUT: UM ESTUDO COM SERVIDORES PÚBLICOS FEDERAIS

As condições inadequadas vivenciadas nas organizações afligem não só os trabalhadores da iniciativa privada, pois são igualmente encontradas no segmento estatal, contrariando a expectativa de que o aparato governamental eliminaria as condições insalubres e criaria outras melhores nas quais prevalecesse à promoção de saúde. Diante desse panorama questionou-se porque, uma vez que, pelo menos do ponto de vista da sociedade leiga, esses servidores estão submetidos a condições privilegiadas de trabalho. O presente estudo objetivou identificar e descrever possíveis relações entre o clima organizacional e o burnout em servidores públicos de uma instituição federal de ensino. Objetivou-se ainda descrever o clima organizacional predominante. A pesquisa realizada teve cunho quantitativo, tipo estudo de caso e exploratória. A coleta de dados deu-se por meio das escalas ECO (escala de clima organizacional), ECB (escala de caracterização do burnout) e um questionário sociodemográfico, todos os instrumentos autoaplicáveis eletronicamente disponíveis à instituição. Participaram do estudo 201 servidores públicos federais, com idade média de 37 anos, majoritariamente de nível superior e casados. Os resultados revelaram que cerca de um quarto dos participantes raramente experimentaram burnout, no entanto outra quarta parte deles frequentemente experimentaram altos níveis de burnout, resultado bastante expressivo. Os servidores perceberam clima organizacional mediano, destacando-se a boa coesão entre os colegas de trabalho e a percepção de baixa recompensa. Merece destaque a grande dispersão entre as percepções de clima, o que permite inferir haver subclimas não identificados nesta investigação, possivelmente ocasionados por uma força de clima fraca e pela participação dos servidores de unidades de ensino geograficamente distintas, geridas por gestores locais com relativa autonomia. Os resultados dos cálculos de correlação revelaram que, quanto menos os participantes percebem apoio da chefia e da organização, coesão entre colegas, e mais controle/pressão, mais exaustos se sentem, mais desumanizam as pessoas com quem tratam e mais se decepcionam no trabalho e vice-versa. Conforto físico menor está associado a maior desumanização e a mais decepção no trabalho e vice-versa; e que controle/pressão, relaciona-se positiva e fracamente com desumanização e vice-versa. Desta forma, a hipótese de que existe associação entre burnout e clima organizacional foi confirmada. Os resultados também revelaram que os servidores com burnout, perceberam pior clima organizacional que os seus pares sem burnout, confirmando a segunda hipótese. Esses servidores também se mostraram neutros quanto à percepção de apoio da chefia e conforto físico; não percebem controle pressão, nem recompensa; todavia percebem coesão entre os colegas. Esses resultados sugerem que os participantes têm se apoiado nessas relações para suportar a indiferença e ausência de estímulos experimentados no trabalho. Os resultados obtidos nesse estudo permitiram concluir que o clima organizacional é fraco, provavelmente influenciado por uma cultura organizacional fraca, explicando a heterogeneidade da percepção do clima organizacional pelos servidores. Além disso, embora haja burnout entre poucos participantes, há que se atentar que cerca de um quarto deles, encontra-se acometido desta síndrome e isto poderá contagiar os demais.

Corticotropin-releasing factor-binding protein ligand inhibitor blunts excessive weight gain in genetically obese Zucker rats and rats during nicotine withdrawal

Elevation of the neuropeptide corticotropin-releasing factor (CRF) in the brain is associated with a reduction of food intake and body weight gain in normal and obese animals. A protein that binds CRF and the related peptide, urocortin, with high affinity, CRF-binding protein (CRF-BP), may play a role in energy homeostasis by inactivating members of this peptide family in ingestive and metabolic regulatory brain regions. Intracerebroventricular administration in rats of the high-affinity CRF-BP ligand inhibitor, rat/human CRF (6-33), which dissociates CRF or urocortin from CRF-BP and increases endogenous brain levels of “free” CRF or urocortin significantly blunted exaggerated weight gain in Zucker obese subjects and in animals withdrawn from chronic nicotine. Chronic administration of CRF suppressed weight gain nonselectively by 60% in both Zucker obese and lean control rats, whereas CRF-BP ligand inhibitor treatment significantly reduced weight gain in obese subjects, without altering weight gain in lean control subjects. Nicotine abstinent subjects, but not nicotine-naive controls, experienced a 35% appetite suppression and a 25% weight gain reduction following acute and chronic administration, respectively, of CRF-BP ligand inhibitor. In marked contrast to the effects of a CRF-receptor agonist, the CRF-BP ligand inhibitor did not stimulate adrenocorticotropic hormone secretion or elevate heart rate and blood pressure. These results provide support for the hypothesis that the CRF-BP may function within the brain to limit selected actions of CRF and/or urocortin. Furthermore, CRF-BP may represent a novel and functionally selective target for the symptomatic treatment of excessive weight gain associated with obesity of multiple etiology.

Epstein-Barr viral latency is disrupted by the immediate-early BRLF1 protein through a cell-specific mechanism.

Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, is a human herpesvirus associated with epithelial cell malignancies (nasopharyngeal carcinoma) as well as B-cell malignancies. Understanding how viral latency is disrupted is a central issue in herpesvirus biology. Epithelial cells are the major site of lytic EBV replication within the human host, and viral reactivation occurs in EBV-associated nasopharyngeal carcinomas. It is known that expression of a single viral immediate-early protein, BZLF1, is sufficient to initiate the switch from latent to lytic infection in B cells. Cellular regulation of BZLF1 transcription is therefore thought to play a key role in regulating the stringency of viral latency. Here we show that, unexpectedly, expression of another viral immediate-early protein, BRLF1, can disrupt viral latency in an epithelial cell-specific fashion. Therefore, the mechanisms leading to disruption of EBV latency appear to be cell-type specific.