Evolution of genome sequencing techniques

The quality and the speed for genome sequencing has advanced at the same time that technology boundaries are stretched. This advancement has been divided so far in three generations. The first-generation methods enabled sequencing of clonal DNA populations. The second-generation massively increased throughput by parallelizing many reactions while the third-generation methods allow direct sequencing of single DNA molecules. The first techniques to sequence DNA were not developed until the mid-1970s, when two distinct sequencing methods were developed almost simultaneously, one by Alan Maxam and Walter Gilbert, and the other one by Frederick Sanger. The first one is a chemical method to cleave DNA at specific points and the second one uses ddNTPs, which synthesizes a copy from the DNA chain template. Nevertheless, both methods generate fragments of varying lengths that are further electrophoresed. Moreover, it is important to say that until the 1990s, the sequencing of DNA was relatively expensive and it was seen as a long process. Besides, using radiolabeled nucleotides also compounded the problem through safety concerns and prevented the automation. Some advancements within the first generation include the replacement of radioactive labels by fluorescent labeled ddNTPs and cycle sequencing with thermostable DNA polymerase, which allows automation and signal amplification, making the process cheaper, safer and faster. Another method is Pyrosequencing, which is based on the “sequencing by synthesis” principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation. By the end of the last millennia, parallelization of this method started the Next Generation Sequencing (NGS) with 454 as the first of many methods that can process multiple samples, calling it the 2º generation sequencing. Here electrophoresis was completely eliminated. One of the methods that is sometimes used is SOLiD, based on sequencing by ligation of fluorescently dye-labeled di-base probes which competes to ligate to the sequencing primer. Specificity of the di-base probe is achieved by interrogating every 1st and 2nd base in each ligation reaction. The widely used Solexa/Illumina method uses modified dNTPs containing so called “reversible terminators” which blocks further polymerization. The terminator also contains a fluorescent label, which can be detected by a camera. Now, the previous step towards the third generation was in charge of Ion Torrent, who developed a technique that is based in a method of “sequencing-by-synthesis”. Its main feature is the detection of hydrogen ions that are released during base incorporation. Likewise, the third generation takes into account nanotechnology advancements for the processing of unique DNA molecules to a real time synthesis sequencing system like PacBio; and finally, the NANOPORE, projected since 1995, also uses Nano-sensors forming channels obtained from bacteria that conducts the sample to a sensor that allows the detection of each nucleotide residue in the DNA strand. The advancements in terms of technology that we have nowadays have been so quick, that it makes wonder: ¿How do we imagine the next generation

Rational vs emotional communication models. Definition parameters of advertising discourses

The new information technology, as well as changes in the consumer, have generated transformations in the strategic advertising focus. Since rational communication focuses on the product, it seeks clarity by articulating benefits and by relying on strategic mechanisms such as Reeves’ Unique Selling Proposition (USP). Through this article, we conducted a theoretical review of the resulting rational and emotional communication models from combining different parameters that result in new advertising discourses. For this, we will combine the theory with examples of awarded cases in different Advertising contests to understand the current thin line between the rational and emotional advertising messages.Las nuevas Tecnologías de la Información, así como los cambios en el consumidor, han generado transformaciones en el enfoque estratégico publicitario. La comunicación racional, al estar centrada en el producto, busca la claridad, articulando beneficios claros y apoyándose en mecanismos estratégicos como la Unique Selling Proposition (USP) de Reeves. Por medio de este artículo, realizamos una revisión teórica sobre los modelos de comunicación racional y emocional resultantes si combinamos diferentes parámetros que dan como resultado nuevos discursos publicitarios. Para ello, combinaremos la parte teórica con ejemplos de casos galardonados en diferentes certámenes de Publicidad para comprender la delgada línea entre lo racional y emocional q

Rational vs emotional communication models. Definition parameters of advertising discourses

The new information technology, as well as changes in the consumer, have generated transformations in the strategic advertising focus. Since rational communication focuses on the product, it seeks clarity by articulating benefits and by relying on strategic mechanisms such as Reeves’ Unique Selling Proposition (USP). Through this article, we conducted a theoretical review of the resulting rational and emotional communication models from combining different parameters that result in new advertising discourses. For this, we will combine the theory with examples of awarded cases in different Advertising contests to understand the current thin line between the rational and emotional advertising messages.Las nuevas Tecnologías de la Información, así como los cambios en el consumidor, han generado transformaciones en el enfoque estratégico publicitario. La comunicación racional, al estar centrada en el producto, busca la claridad, articulando beneficios claros y apoyándose en mecanismos estratégicos como la Unique Selling Proposition (USP) de Reeves. Por medio de este artículo, realizamos una revisión teórica sobre los modelos de comunicación racional y emocional resultantes si combinamos diferentes parámetros que dan como resultado nuevos discursos publicitarios. Para ello, combinaremos la parte teórica con ejemplos de casos galardonados en diferentes certámenes de Publicidad para comprender la delgada línea entre lo racional y emocional q

Bodega y vivienda en Cabanelas

Proxecto fin de carreira (UDC.ETSA). Curso 2013/201

Audiovisual Translation and Subtitling. Analyzing Humor in the Spanish subtitles of the TV Series ‘The Office’.

One of the effects of globalization is the ease with which any TV series or movie, regardless of its origin, can reach an almost infinite audience. This factor, coupled with the increasing demand for visual entertainment, makes audiovisual translation and subtitling the order of the day. Despite this, not everything is so simple, as cultural barriers are still present. This is why humor, despite being a universal language, has difficulties when it comes to translation. In this project, taking the American sitcom “The Office” as a reference, different aspects of humor translation will be analyzed, taking into account cultural references.Uno de los efectos de la globalización es la facilidad que tiene cualquier serie o película de televisión, independientemente de su origen, para llegar a un público casi infinito. Este factor, sumado al aumento de demanda de entretenimiento visual, hacen que la traducción audiovisual y el subtitulado estén a al orden del día. A pesar de esto, no todo es tan sencillo, puesto que las barreras culturales siguen estando presentes. Es por esto, que el humor, a pesar de ser un lenguaje universal, tiene dificultades a la hora de ser traducido. En este trabajo de fin de grado, tomando como referencia la serie estadounidense “The Office”, se analizarándiferentes aspectos de la traducción del humor, teniendo en cuenta las referencias culturales.Departamento de Filología InglesaGrado en Estudios Inglese

Efectos de la fijación simbiótica del N sobre la eficiencia en el uso de N y en el uso del agua en plantas de judía: caracterización fisiológica, molecular y bioquímica

La judía común (Phaseolus vulgaris) es uno de los cultivos con mayor importancia, no solo por su alto valor nutricional, sino también medioambiental, ya que, como leguminosa, la judía común tiene la capacidad de fijar el N atmosférico (N2) en sus nódulos radiculares, disminuyendo así el uso de fertilizantes y su consecuente emisión de gases de efecto invernadero. Sin embargo, la fijación de N2 es particularmente sensible a diferentes condiciones adversas como la sequía, cuyos efectos se están agravando por el cambio climático. Por tanto, estudiar los efectos de la sequía en los cultivos de estas leguminosas es de especial relevancia en regiones áridas como Andalucía (España), donde la disponibilidad de agua es cada vez más escasa y siendo este uno de los factores que más limitan su productividad. El presente trabajo de tesis está organizado en 4 capítulos en los cuales se estudia la regulación a nivel molecular, bioquímico y fisiológico de los mecanismos de defensa de las plantas de P. vulgaris frente al estrés hídrico. Recientemente, el desarrollo de tecnologías -ómicas, que permiten evaluar cambios moleculares a gran escala, se ha convertido en una herramienta de gran utilidad para la evaluación de la respuesta al estrés en plantas. Por tanto, en el capítulo 1 se analizaron los cambios transcriptómicos (RNAseq) en respuesta al estrés hídrico en una variedad local de P. vulgaris (PHA-0683) resistente a la sequía. Para ello se utilizó tejido foliar de plantas control y plantas sometidas a 10 días de sequía, cultivadas en condiciones de fijación de N2. El análisis mostró la disminución de la expresión de genes asociados con la inhibición de la respuesta del ácido abscísico (ABA), la principal hormona de respuesta al estrés en la planta. Además, la prevalencia los genes expresados diferencialmente (DEG) relacionados con la respuesta a la carencia de fosfato (P) y el análisis fisiológico de plantas PHA-0683 en respuesta a sequía con diferentes concentraciones de P, demostraron que, entre los mecanismos de tolerancia de esta variedad está, precisamente, la capacidad de movilizar fosfato en condiciones de estrés. La domesticación, la expansión global y las prácticas agrícolas locales han implicado el desarrollo de una amplia variedad de líneas o genotipos de judía común con diferentes grados de resistencia al estrés hídrico. Por lo que en el capítulo 2 se llevó a cabo la comparación de la respuesta molecular y fisiológica al estrés hídrico de dos variedades de judía. Para ello se utilizaron la variedad local de P. vulgaris (PHA-0683), con un alto grado de tolerancia a los periodos de sequía, y una línea comercial (PMB-0220) de tolerancia intermedia a la sequía, pero mayor productividad. La comparación mostró que mientras la variedad local, PHA-0683, mantenía relativamente estables sus parámetros fisiológicos en respuesta a diez días de falta de riego, la variedad PMB-0220 disminuía su contenido hídrico foliar, su biomasa aérea y el contenido en clorofilas en las hojas más viejas. El análisis de los niveles de expresión de genes relacionados con la respuesta al ABA y de varios factores de transcripción que regulan la senescencia en la planta, demostró que había claras diferencias cualitativas y cuantitativas en los patrones de expresión de varios de estos reguladores en respuesta a la sequía entre las dos variedades. Con el fin de incrementar la tolerancia a la sequía en cultivos de judía más productivos, en el capítulo 3 se analizó de forma más completa la respuesta al estrés hídrico en plantas de la línea comercial PMB-0220 que es menos tolerante a la sequía que PHA-0683, pero tiene mayor productividad. A pesar de las ventajas que supone cultivar leguminosas como la judía común en condiciones de fijación de N2, muchos agricultores prefieren el uso de fertilizantes nitrogenados que aseguren una buena cosecha. En este capítulo se estudió la respuesta a la sequía en plantas de judía PMB-02202 cultivadas con nitrógeno inorgánico procedente de la fertilización, o en condiciones de simbiosis y fijación de nitrógeno. Para ello, en este capítulo se combinaron análisis transcriptómicos y metabolómicos con los análisis fisiológicos. El estudio de la expresión diferencial de genes mostró que las plantas cultivadas con fijación de N2 tenían mayor expresión de genes asociados con la síntesis de ABA y de respuesta al estrés hídrico, incluso en ausencia de estrés, comparadas con las plantas fertilizadas con NO3-. Así mismo, también se observó la inducción de genes relacionados con el metabolismo de los azúcares solubles y el mantenimiento del balance osmótico en plantas fijadoras de N2 y sometidas a sequía. El análisis metabolómico confirmó que las plantas en simbiosis y sometidas a 10 días de sequía también acumulaban mayor concentración de metabolitos de defensa a la sequía, como la propia hormona ABA y ureidos (alantoína), azúcares solubles, y otros metabolitos que mantienen el balance osmótico en la planta como rafinosa y prolina, además de diversos lípidos que ayudan en el mantenimiento de la fluidez de las membranas. Por último, el estudio fisiológico confirmó que las plantas fijadoras de N2 mantenían mejor el contenido hídrico foliar y estaban mejor adaptadas que las plantas fertilizadas con NO3 -. En este capítulo 3, también se realizó un experimento de recuperación tras el tratamiento de estrés hídrico, en el que las plantas volvieron a regarse después de los 10 días de sequía y se cultivaron hasta el momento de la recogida de frutos. Tras la rehidratación, las plantas en fijación de N2 recuperaron el intercambio de gases, tasa de fotosíntesis y la producción de frutos, mientras que las plantas fertilizadas con NO3 - no consiguieron recuperarse de la sequía, mantuvieron valores reducidos de intercambio de gases y fotosíntesis a pesar de la rehidratación y disminuyeron la productividad de su cosecha. Varios estudios han indicado el papel positivo de los ureidos en diferentes situaciones de estrés. Los nucleótidos de purina, precursores de los ureidos, pueden sintetizarse mediante la síntesis de novo u obtenerse mediante vías de reciclaje de las bases de purina. Dos enzimas son las responsables del reciclaje de purinas, la adenina fosforribosiltransferasa (APRT) que cataliza el reciclaje de adenina y la hipoxantina guanina fosforribosiltransferasa (HGPRT) que cataliza el reciclaje de guanina e hipoxantina. En el capítulo 4 se aborda la caracterización molecular, bioquímica y funcional de la primera de estas dos enzimas. La adenina fosforribosiltransferasa está codificada por cuatro genes en P. vulgaris. El análisis filogenético de las secuencias, así como también la sobreexpresión de las cuatro proteínas fusionadas a -YFP (Yellow fluorescent protein) en plantas de Nicotiana benthamiana, reveló que las APRTs de judía se agrupaban en dos “clusters”, dos de las cuatro proteínas tienen localización cloroplástica, mientras que las otras dos se localizan en el citosol. La caracterización bioquímica, el patrón de expresión génica y los niveles de actividad en los diferentes tejidos de P. vulgaris mostraron que PvAPRT1 y PvAPRT5 eran las proteínas predominantes y que, curiosamente, tenían altos niveles de expresión en raíz y nódulos. Con el fin de dilucidar el papel específico que llevan a cabo estas isoformas, se generaron raíces mutadas en cada uno de los dos genes mediante edición génica usando la tecnología CRISPR/Cas9. Finalmente, el análisis fisiológico y metabolómico de estas raíces transgénicas reveló que, si bien el gen PvARPT1 actúa principalmente en la vía de reciclaje de la adenina, el gen PvAPRT5 participa en la regulación de los niveles de citoquininas, modificando el crecimiento de la raíz y los nódulos de la planta. Además, tanto los datos transcriptómicos de los capítulos anteriores, como los perfiles metabolómicos de las raíces con mutaciones en los genes que codifican APRTs indican que estas enzimas tienen también un papel importante en las respuestas a estrés en las plantas de judía.Common bean (Phaseolus vulgaris L) is one of the most important crops, not only for its high nutritional value, but also for its environmental advantages. This is because, as a legume, common bean can fix atmospheric N (N2) in its root nodules, thus reducing the requirement of fertilizers and its consequent emission of greenhouse gases. However, N2 fixation is particularly sensitive to various adverse conditions such as drought, whose effects are being aggravated by climate change. For this reason, farmers opt for the use of fertilizers that ensure a good harvest. Therefore, studying the effects of drought on these legumes is of special relevance in arid regions such as Andalusia (Spain), where the availability of water is increasingly scarce, and drought is one of the factors that most limit crop productivity. This Thesis work is organized into 4 chapters in which the regulation at the molecular, biochemical, and physiological level of the response mechanisms of P. vulgaris plants against water stress is studied. Recently, transcriptomics has become a very useful tool for the evaluation of the environmental responses in different plant species. Therefore, in chapter 1 we analysed the transcriptomic changes (RNA-seq) in response to water stress in a P. vulgaris (PHA-0683) landrace resistant to drought. Leaves from control plants and plants subjected to 10 days of drought, cultivated under N2 fixation conditions, were used. The transcriptomic analysis showed the decrease in the expression of genes associated with the inhibition of the abscisic acid (ABA) response, the main stress response hormone in the plant. In addition, they were a significant number of differentially expressed genes (DEG) related to the response to phosphate (P) deficiency. Relevance of P acquisition under stress was confirmed by drought experiments performed with various concentrations of P, thus revealing that, among the tolerance mechanisms of this resistant landrace is the ability to mobilize phosphate under stress conditions. Domestication, global expansion, and local agricultural practices have involved the development of a wide variety of common bean lines or genotypes with varying degrees of resistance to water stress. Therefore, in chapter 2, the study of the molecular and physiological response to water stress of two common bean genotypes was carried out. For this, the P. vulgaris landrace (PHA-0683), previously described as having a high degree of tolerance to drought, and the market class breeding line Great Northern (PMB- 0220), with intermediate tolerance to drought, but higher productivity, were compared. The comparison showed that while the landrace PHA-068 kept its physiological parameters stable, the market class PMB-0220 decreased its leaf water content, its shoots biomass, and the chlorophyll content in its oldest leaf tissue. The analysis of expresión levels of relevant genes related to the ABA response and transcription factors that regulate senescence showed clear qualitative and quantitative differences in the expresión patterns in response to drought between the two genotypes. To increase tolerance to drought in more productive bean crops, in chapter 3 the response to water stress was analyzed in greater depth in plants of the commercial line PMB-0220, which despite being less tolerant to drought than PHA-0683, has higher productivity. Despite the advantages of growing legumes such as N2 fixation common beans, many farmers prefer the use of nitrogen fertilizers to ensure a good harvest. In this chapter, the response to drought was studied in PMB-0220 bean plants grown with inorganic nitrogen from fertilization, or under symbiosis and nitrogen fixation conditions. To do this, in this chapter we combined transcriptomic and metabolomic analysis, together with physiological analysis. Leaf tissue from plants subjected to 10 days of drought and fertilized with nitrate or in symbiosis with rhizobia was used in the -omic analyses. The study of differential gene expression showed that plants grown with N2 fixation had higher induction of genes associated with ABA synthesis and response to water stress, even in the absence of stress, compared to plants fertilized with NO3 -. Likewise, the induction of genes related to the metabolism of soluble sugars and with the maintenance of osmotic balance in N2-fixing plants subjected to drought was also observed. The metabolomic analysis confirmed that plants in symbiosis and subjected to 10 days of drought accumulated a higher concentration of drought response metabolites, such as the ABA hormone itself, ureides (allantoin), soluble sugars, and other metabolites that maintain the osmotic balance in the plant, as raffinose and proline, as well as various lipids that help maintain membrane fluidity. The physiological study confirmed that N2- fixing plants maintained their leaf water content better and were better adapted than plants fertilized with NO3 -. In this chapter, a recovery experiment was carried out after the wáter stress treatment, in which the plants were irrigated again after 10 days of drought and cultivated until the pod collection. The N2-fixing plants, after rehydration, recovered their gas exchange values, their photosynthesis rate and even maintained pod production, whereas the NO3 - fertilized plants did not manage to recover from the drought, they maintained reduced gas exchange and photosynthesis values despite rehydration and decreased their pod and seeds yields. Several studies have indicated the positive role of ureides in different stress situations. Purine nucleotides, precursors to ureides, can be synthesized by the de novo synthesis or obtained by salvage pathways of purine bases. Two enzymes are responsible for purine salvage, adenine phosphoribosyltransferase (APRT) which catalyzes the adenine salvage and hypoxanthine guanine phosphoribosyltransferase (HGPRT) which catalyzes the salvage of guanine and hypoxanthine. In chapter 4, the molecular, biochemical, and functional characterization of the first of these two enzymes is addressed. Adenine phosphoribosyltransferase is encoded by four genes in P. vulgaris. Phylogenetic analysis, as well as the overexpression of the four coded proteins fused to -YFP (Yellow fluorescent protein) in Nicotiana benthamiana plants, revealed that the common vean APRTs were grouped into two "clusters", two of the four proteins have possible location to chloroplast, while the other two are in the cytosol. The biochemical characterization, gene expression pattern, and the levels of activity in the different tissues of P. vulgaris showed that PvAPRT1 and PvAPRT5 were the predominant proteins and that, interestingly, they had high levels of expression in roots and nodules. To elucidate the specific role played by these isoforms, roots mutated in each of the two genes were generated by gene editing using CRISPR/Cas9 technology. Finally, the physiological and metabolomic analysis of these transgenic roots revealed that, although the PvAPRT1 is mainly involved in the adenine salvage pathway, the PvAPRT5 could function in the regulation of cytokinin levels, modifying root growth and nodulation. In addition, both the transcriptomic data from the previous chapters, and the results of the metabolomic profiles of roots with mutations in the genes that encode APRTs, indicate that these enzymes also have an important role in stress responses in common bean plants

Construcciones identitarias, interculturalidad y resistencia : los pueblos originarios y migrantes frente al Estado monocultural

Fil: García Vázquez, Cristina B.. Universidad Nacional del Comahu

Cuando el Otro se convierte en Sujeto de estudio : el etnógrafo desde la perspectiva de sus informantes

Fil: García Vázquez, Cristina B.. Universidad Nacional del Comahu

Complementary music therapy for cancer patients in at-home palliative care and their caregivers: protocol for a multicentre randomised controlled trial

Background Patients with advanced cancer, receiving at-home palliative care, are subject to numerous symptoms that are changeable and often require attention, a stressful situation that also impacts on the family caregiver. It has been suggested that music therapy may benefit both the patient and the caregiver. We propose a study to analyse the efficacy and cost utility of a music intervention programme, applied as complementary therapy, for cancer patients in palliative care and for their at-home caregivers, compared to usual treatment. Method A randomised, double-blind, multicentre clinical trial will be performed in cancer patients in at-home palliative care and their family caregivers. The study population will include two samples of 40 patients and two samples of 41 caregivers. Participants will be randomly assigned either to the intervention group or to the control group. The intervention group will receive a seven-day programme including music sessions, while the control group will receive seven sessions of (spoken word) therapeutic education. In this study, the primary outcome measure is the assessment of patients' symptoms, according to the Edmonton Symptom Assessment System, and of the overload experienced by family caregivers, measured by the Caregiver Strain Index. The secondary outcomes considered will be the participants' health-related quality of life, their satisfaction with the intervention, and an economic valuation. Discussion This study is expected to enhance our understanding of the efficacy and cost-utility of music therapy for cancer patients in palliative care and for their family caregivers. The results of this project are expected to be applicable and transferrable to usual clinical practice for patients in home palliative care and for their caregivers. The approach described can be incorporated as an additional therapeutic resource within comprehensive palliative care. To our knowledge, no previous high quality studies, based on a double-blind clinical trial, have been undertaken to evaluate the cost-effectiveness of music therapy. The cost-effectiveness of the project will provide information to support decision making, thereby improving the management of health resources and their use within the health system