Estudio de los telómeros en Dictyostelium discoideum. Desarrollo de un modelo celular de disqueratosis congénita

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 8-06-2017Los telómeros son estructuras formadas por ADN y proteínas que protegen los extremos de los cromosomas. Son estructuras esenciales para el mantenimiento de la integridad genómica. Debido a que la maquinaria de replicación no puede copiar completamente los extremos del ADN, los telómeros son extendidos por mecanismos complementarios incluyendo la actividad del complejo telomerasa y el mecanismo ALT (del inglés, Alternative Lengthening of Telomeres). En Dictyostelium discoideum los extremos de los cromosomas no se han podido secuenciar completamente pero se sabe que presentan copias parciales del elemento extracromosomal de ADN ribosomal (ADNr). Este elemento presenta heterogeneidad en su extremos debido a la presencia de una región con la fórmula nucleotídica (CnT)m. En esta tesis hemos completado y ordenado la secuencia presente en este elemento demostrando la existencia de una hebra G y una hebra C y la presencia de un saliente monocatenario 3´, elementos comunes en los telómeros de eucariotas. Nuestra investigación muestra que D. discoideum no presenta un complejo telomerasa funcional por lo que el mantenimiento de los telómeros puede darse por fenómenos de recombinación homóloga entre los extremos de los cromosomas y los de los elementos extracromosomales de ADNr. En apoyo de esta hipótesis, hemos observado que una serie de proteínas implicadas en recombinación que forman corpúsculos nucleares similares a los APBs (del inglés, ALT-associated Promyelocitic Leukaemia (PML) Bodies) presentes en células de mamífero que utilizan el mecanismo ALT también están localizadas en corpúsculos nucleares en D. Discoideum. Estos cuerpos parecen estar asociados con los nucléolos. Posteriormente hemos desarrollasdo un modelo para el estudio de una enfermedad relacionada con el mantenimiento de los telómeros, la disqueratosis congénita. Hemos sobreexpresado en D. discoideum cuatro mutaciones patogénicas del gen que codifica disquerina. También hemos generado cepas de este organismo en los que el gen endógeno dkc1 porta dos de estas mutaciones. Ninguna de las dos funciones principales asociadas a disquerina: el mantenimiento de los telómeros y la pseudouridilación del ARN, parecen afectadas por estas mutaciones. En cambio, la sobreexpresión de la proteína no mutada y de los mutantes producen menor proliferación celular y un aumento de la respuesta al daño del ADN, tanto en situación basal como en presencia del agente citotóxico bleomicina. Dos mutaciones del gen endógeno también producen aumento de la respuesta al daño del ADN en presencia de bleomicina sugiriendo una implicación directa de las mutaciones en disquerina con este proceso biológico.Telomeres are structures composed by DNA and proteins that protect chromosome ends. They are essential to maintain genomic stability. DNA ends cannot be completely replicated by DNA polymerases and, therefore, telomeres have to be extended by complementary mechanism, including telomerase activity as well as the ALT (Alternative Lengthening of Telomeres) mechanism. The chromosome ends of Dictyostelium discoideum have not been completely sequenced but are known to contain partial copies of a extra-chromosomal element that contains rDNA genes. This element is heterogeneous at its ends due to the presence of a region with variable number of copies of the (CnT)m sequence. In this Thesis we have completed and arranged this sequence showing the presence of G-rich and C-rich strands and a 3’- overhanging single strand region. These elements are common in most eukaryotic telomeres. The data reported in this Thesis show that D. discoideum does not present a functional telomerase complex and that telomere elongation could depend on homologous recombination between chromosome ends and the end of extrachromosomal rDNA elements. In support for this hypothesis, we have shown that proteins involved in DNA recombination, components of APB (ALT-associated Promyelocitic Leukemia (PML) Bodies) in mammalian cells that extend telomeres through the ALT mechanism, are also located in nuclear bodies in D. discoideum. These bodies seem to be associated with the nucleolus. We have next developed a model for the study of the dyskeratosis congenita human disease in D. discoideum. Four pathogenic mutants of the dyskerin (DKC1) gene found in dyskeratosis patients were over-expressed in D. discoideum. In addition, mutant strains where two of these mutations were introduced in the endogenous gene were generated. Neither of the two main functions of dyskerin, telomere maintenance and RNA pseudouridylation, where affected by these mutations. Over-expression of the D. discoideum protein, either wild-type or mutated, resulted in decreased cell proliferation and increased response to DNA damage, both in basal conditions and upon treatment with the cytotoxic drug bleomycin. Two of the mutations introduced in the endogenous gene also produced increased DNA-damage response in the presence of bleomycin that makes us suggest a direct implication of dyskerin mutation in this biological process

The cinema as model for Spanish popular music in the 80´s

Uno de los períodos musicales más brillantes de la historia de la música popular española se produjo en la década de 1980. Una nueva generación surgida en la Transición, influenciada por los movimientos culturales en general, y musicales en particular, que se estaban produciendo en Estados Unidos y Reino Unido, propició la aparición de numerosas formaciones musicales que tuvieron en el cine un punto de referencia y una fuente de inspiración. Este trabajo se centra en analizar dichas influencias.One of the most outstanding periods in Spanish popular music occurred in the eighties.   A younger generation emerged from the Spanish transition to democracy, influenced by other cultural movements in general, especially in music, which were taking place in the USA and the UK. This led to the birth of a countless number of musical bands, for which the cinema brought  a significant point of reference and a never-ending source of inspiration. The aim of this research is to analyse these influences

Effects of autochthonous Kluyveromyces lactis and commercial Enterococcus faecium adjunct cultures on the volatile profile and the sensory characteristics of short-ripened acid-curd Cebreiro cheese

Elaboráronse catro lotes de queixo tipo Cebreiro por duplicado a partir de leite pasterizado. Un lote control foi fabricado só cun cultivo iniciador comercial tipo O. Os outros tres lotes preparáronse co mesmo cultivo iniciador xunto con: (i) un cultivo comercial de Enterococcus faecium; (ii) un cultivo adxunto seleccionado de Kluyveromyces lactis, usado nunha etapa de premaduración do leite de elaboración; e (iii) unha combinación de ambos cultivos adxuntos. Os queixos elaborados co adxunto de lévedos caracterizáronse por valores máis altos de índices de proteolise xeral, pH e actividade da auga, e amosaron recontos de mesófilos totais e de bacterias lácticas polo menos 2 unidades logarítmicas maiores que os correspondentes aos lotes elaborados só con bacterias lácticas. Os perfís de compostos volátiles dos queixos elaborados con K. lactis distinguíronse polo seu alto contido en ésteres, alcohois de cadea ramificada, ácidos graxos, acetoína e 2-feniletanol. Estes lotes tiveron unha textura máis friable e adherente, e amosaron aromas diferenciados picante, a lévedo, alcohólico, acético e afroitado. Ademáis, a adición de enterococos semellou contribuir a acadar características sensoriais máis desexables. Os lotes fabricados con ambos cultivos adxuntos recibiron puntuacións máis altas nos parámetros preferencia de textura, intensidade de sabor, preferencia de aroma e sabor, e preferencia sensorial xeral. Os perfís sensoriais dos queixos elaborados con lévedos engadidos foron moi similares aos do queixo Cebreiro tradicional de leite cru de boa calidade sensorial.Xunta de Galici

Generación automática de objetivos de prueba a partir de casos de uso mediante partición de categorías y variables operacionales

Este trabajo complementa y amplia nuestros trabajos anteriores sobre generación de pruebas a partir de casos de uso presentando un proceso que, de manera sistemática y automática, permite generar objetivos de prueba a partir de casos de uso especificados en un lenguaje no formal. Este proceso aplica el método de categoría-partición y el patrón Use Case Test Pattern, el cual usa variables operacionales. Además se presenta los algoritmos necesarios para la automatización del proceso propuesto, acompañados de un caso práctico

A Case Study for Generating Test Cases from Use Cases

The verification of the correct implementation of use eases is a vital task in software development and quality assurance. Although there are many works describing how to generate test eases from use cases, there are very few ease studies and empirical results of their application and effectiveness. This paper introduces a first ease study that test the correct implementation of use cases in a web system and a command line system, analyses the results and exposes that generation of use cases has a successful about 80%.Ministerio de Educación y Ciencia QSimTest TIN2007-67843-C06 03Ministerio de Educación y Ciencia REPRIS TIN2005-24792-

Genetic Introgression and Morphological Variation in Naked-Back Bats (Chiroptera: Mormoopidae: Pteronotus Species) along Their Contact Zone in Central America

Two sibling bare-backed bat species (Pteronotus fulvus and P. gymnonotus) have been traditionally differentiated by their size. However, intermediate specimens between the two species have been found in sympatric populations along southern Mexico and it has been suggested that they may be the outcome of a hybridization process between the two species. We used one mitochondrial (COI), three nuclear markers (PRKCL, STAT5A and RAG2) and 13 microsatellites to explore the evolutionary relationships between these two species and elucidate whether the intermediate morphotypes correspond to hybrid individuals. These markers have been analyzed in sympatric and allopatric populations of the two species plus the closely related species Pteronotus davyi. We confirmed the species-level differentiation of the three lineages (P. fulvus, P. davyi and P. gymnonotus), but the phylogenetic hypotheses suggested by the nuclear and mitochondrial markers were discordant. We confirm that the discordance between markers is due to genetic introgression through the mitochondrial capture of P. fulvus in P. gymnonotus populations. Such introgression was found in all P. gymnonotus specimens across its sympatric distribution range (Mexico to Costa Rica) and is related to expansion/retraction species distribution pulses associated with changes in forest distribution during the Quaternary climate cycles. Microsatellite analyses showed contemporary genetic contact between the two sympatric species and 3.0% of the samples studied were identified as hybrids. In conclusion, we found a historical and asymmetric genetic introgression (through mitochondrial capture) of P. fulvus into P. gymnonotus in Mexico and Central America and a limited contemporary gene exchange between the two species. However, no relationship was found between hybridization and the intermediate-sized specimens from southern Mexico, which might likely result from a clinal variation with latitude. These results confirm the need for caution when using forearm size to identify these species in the field and when differentiating them in the laboratory based on mitochondrial DNA alone

COVID‑19 vaccine literacy in patients with systemic autoimmune diseases

Funding for open access charge: Universidad de Granada/CBUA.COVID-19 related infodemic is a threat to the successful COVID-19 vaccination campaigns. This might be especially apparent for patients with autoimmune diseases since there is no data available about the balance between benefits and risks of the newly developed COVID-19 vaccines in this population. We aim (i) to evaluate vaccine literacy skills in a population of patients with systemic autoimmune diseases, (ii) to examine the potential associations between vaccine literacy skills and sociodemographic characteristics and (iii) to analyze the relationships between attitudes, perceptions and beliefs about current vaccinations and vaccine literacy skills and sociodemographic characteristics. A cross-sectional study was conducted among 319 patients with systemic autoimmune diseases (92% females; 49.5% of patients in the 31–50 years age category). The vaccine literacy levels were determined using the Health Literacy about Vaccination in adulthood in Italian (HLVa-IT). Sociodemographic characteristics including gender, age, country and area of residence, civil status, socioeconomic status, educational attainment and occupational status were evaluated. The mean vaccine literacy functional and interactive-critical scores were 2.59 ± 0.74 and 3.07 ± 0.60, respectively. The vaccine literacy interactive-critical score was higher in females than in males (p = 0.048). Interactive-critical scores were associated with the area of residence, civil status and socioeconomic status, with the highest score in urban area of ≥ 100.000 inhabitants (p = 0.045), in widow patients (p = 0.023) and in patients with high socioeconomic status (p = 0.018). Significant differences were observed between the different education levels, for both the functional and the interactive-critical scores (p = 0.002 and p < 0.001, respectively), the highest score was observed in patients who completed a university degree. The level of vaccine literacy for functional and interactive-critical scales were medium. Area of residence, civil status and socioeconomic status represented determinants of vaccine literacy interactive-critical scale. Educational attainment also contributes to vaccine literacy functional scale. Insight into these factors is required to ensure an optimal vaccine literacy level in patients with autoimmune diseases.Universidad de Granada/CBU

Towards optimal cut-off trough levels of adalimumab and etanercept for a good therapeutic response in rheumatoid arthritis. Results of the INMUNOREMAR study

Article en format correspondènciaWe read with great interest the paper by Chen et al1 analysing the relationship between therapeutic response to adalimumab and etanercept and serum drug trough levels in 70 patients with rheumatoid arthritis (RA). ..

Prosthetic rehabilitation of an edentulous cleft palate using a denture with a palatal obturator: a clinical report

A cleft palate is a genetic disorder that occurs when an oro-nasal communication is present between the palate and the base of the nose. During pregnancy, the maxillary is not completely merged, and the defect is only seen at birth. Possible causes are hormonal imbalances, nutritional deficiencies, infections, radiation during pregnancy, alcohol or cigarette consumption, the ingestion of other teratogenic substances by the mother, and heredity. The resulting defect requires corrective surgery. In the case of a cleft palate, surgery is postponed until after the first year of life to avoid disturbing the normal development of speech and the risk of aspiration of food, which causes infections such as otitis and pneumonia. Ear infections can harm the development of speech in cases where surgery is not possible or the defect has reappeared. This article describes the prosthetic rehabilitation of a edentulous patient: a woman of 53 years old with a cleft palate who was treated surgically. In a clinical examination, a residual palatal defect was identified. The prosthetic rehabilitation involved the emplacement of a complete adapted prosthesis, using a palatal obturator, with a view to sealing the defect and allowing the patient to acquire better speech quality, and improve her nutrition and well-being