149,148 research outputs found

    Genetic and epigenetic characterization of the geminivirus-host interaction

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    Fecha de lectura de Tesis Doctoral: 29 de abril de 2019Los geminivirus constituyen una familia de virus de plantas transmitidos por insectos, contienen una o dos mol茅culas de DNA de cadena sencilla empaquetadas en dos part铆culas gemelas fusionadas. Es uno de los pat贸genos de plantas m谩s devastadores en el mundo, causando importantes p茅rdidas de cosechas en plantas de tomate, ma铆z, algod贸n, cassava, etc. Durante la infecci贸n, los geminivirus interfieren con la maquinaria celular de la planta. Adem谩s deben confrontar mecanismos de defensa tales como el silenciamiento g茅nico. Como primer objetivo, se han generado y analizado de forma integrada los mapas transcript贸micos (mediante RNA-Seq y small RNA-Seq) y epigen茅tico (mediante Bisulfite-Seq) del geminivirus TYLCV durante una infecci贸n en tomate. Esto nos ha permitido describir, a resoluci贸n de nucle贸tido, la relaci贸n existente entre la expresi贸n g茅nica, el patr贸n de metilaci贸n del genoma y los sRNAs derivados (vsRNAs) de TYLCV. Como segundo objetivo, se ha determinado la relevancia de la maquinaria de metilaci贸n del DNA de la planta en una infecci贸n por geminivirus en Arabidopsis y N. benthamiana. Nuestros resultados indican que la inactivaci贸n de la maquinaria de metilaci贸n de mantenimiento no interfiere en la acumulaci贸n de TYLCV en Arabidopsis. Adem谩s, los niveles de DNA de TYLCV no son significativamente diferentes en hojas o tejido floral de plantas silvestres y mutantes. Sin embargo, los niveles de DNA del geminivirus BCTV est谩n incrementados en plantas mutantes en la maquinaria de metilaci贸n, tanto en tejido floral como en hojas apicales. Por otro lado, el silenciamiento de los genes involucrados en metilaci贸n del DNA en N. benthamiana no tuvo efecto en la acumulaci贸n de TYLCV o TYLCSV. Como tercer objetivo, se determin贸 la transmisi贸n de CaLCuV y BCTV a trav茅s de semilla, concluy茅ndose que no existe evidencia de dicha transmisi贸n

    Study on the concordance between different SNP鈥恎enotyping platforms in sheep

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    .Different SNP genotyping technologies are commonly used in multiple studies to perform QTL detection, genotype imputation, and genomic predictions. Therefore, genotyping errors cannot be ignored, as they can reduce the accuracy of different procedures applied in genomic selection, such as genomic imputation, genomic predictions, and false-positive results in genome-wide association studies. Currently, whole-genome resequencing (WGR) also offers the potential for variant calling analysis and high-throughput genotyping. WGR might overshadow array-based genotyping technologies due to the larger amount and precision of the genomic information provided; however, its comparatively higher price per individual still limits its use in larger populations. Thus, the objective of this work was to evaluate the accuracy of the two most popular SNP-chip technologies, namely, Affymetrix and Illumina, for high-throughput genotyping in sheep considering high-coverage WGR datasets as references. Analyses were performed using two reference sheep genome assemblies, the popular Oar_v3.1 reference genome and the latest available version Oar_rambouillet_v1.0. Our results demonstrate that the genotypes from both platforms are suggested to have high concordance rates with the genotypes determined from reference WGR datasets (96.59% and 99.51% for Affymetrix and Illumina technologies, respectively). The concordance results provided in the current study can pinpoint low reproducible markers across multiple platforms used for sheep genotyping data. Comparing results using two reference genome assemblies also informs how genome assembly quality can influence genotype concordance rates among different genotyping platforms. Moreover, we describe an efficient pipeline to test the reliability of markers included in sheep SNP-chip panels against WGR datasets available on public databases. This pipeline may be helpful for discarding low-reliability markers before exploiting genomic information for gene mapping analyses or genomic predictionS

    What is the importance of sperm subpopulations?

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    .The study of sperm subpopulations spans three decades. The origin, meaning, and practical significance, however, are less clear. Current technology for assessing sperm morphology (CASA-Morph) and motility (CASA-Mot) has enabled the accurate evaluation of these features, and there are many options for data classification. Subpopulations could occur as a result of the stage of development of each spermatozoon in the subpopulation. Spermatogenesis might contribute to the production of these subpopulations. Insights from evolutionary biology and recent molecular research are indicative of the diversity among male gametes that could occur from unequal sharing of transcripts and other elements through cytoplasmic bridges between spermatids. Sperm cohorts exiting the gonads would contain different RNA and protein contents, affecting the spermatozoon physiology and associations with the surrounding environmental milieu. Subsequently, these differences could affect how spermatozoa interact with the environmental milieu (maturation, mixing with seminal plasma, and interacting with the environmental milieu, or female genital tract and female gamete). The emergence of sperm subpopulations as an outcome of evolution, related to the reproductive strategies of the species, genital tract structures, and copulatory and fertilization processes. This kind of approach in determining the importance of sperm subpopulations in fertilization capacity should have a practical impact for conducting reproductive technologies, inspiring and enabling new ways for the more efficient use of spermatozoa in the medical, animal breeding, and conservation fields. This manuscript is a contribution to the Special Issue in memory of Dr. Duane GarnerS

    Bioinformatic characterization of a triacylglycerol lipase produced by Aspergillus flavus isolated from the decaying seed of Cucumeropsis mannii

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    Lipases are enzymes of industrial importance responsible for the hydrolysis of ester bonds of triglycerides. A lipolytic fungus was isolated and subsequently identified based on the ITS sequence analysis as putative Aspergillus flavus with accession number LC424503. The gene coding for extracellular triacylglycerol lipase was isolated from Aspergillus flavus species, sequenced, and characterised using bioinformatics tools. An open reading frame of 420 amino acid sequence was obtained and designated as Aspergillus flavus lipase (AFL) sequence. Alignment of the amino acid sequence with other lipases revealed the presence GHSLG sequence which is the lipase consensus sequence Gly-X1-Ser-X2-Gly indicating that it a classical lipase. A catalytic active site lid domain composed of TYITDTIIDLS amino acids sequence was also revealed. This lid protects the active site, control the catalytic activity and substrate selectivity in lipases. The 3-Dimensional structural model shared 34.08% sequence identity with a lipase from Yarrowia lipolytica covering 272 amino acid residues of the template model. A search of the lipase engineering database using AFL sequence revealed that it belongs to the class GX-lipase, superfamily abH23 and homologous family abH23.02, molecular weight and isoelectric point values of 46.95鈥塊Da and 5.7, respectively. N-glycosylation sites were predicted at residues 164, 236 and 333, with potentials of 0.7250, 0.7037 and 0.7048, respectively. O-glycosylation sites were predicted at residues 355, 358, 360 and 366. A signal sequence of 37 amino acids was revealed at the N-terminal of the polypeptide. This is a short peptide sequence that marks a protein for transport across the cell membrane and indicates that AFL is an extracellular lipase. The findings on the structural and molecular properties of Aspergillus flavus lipase in this work will be crucial in future studies aiming at engineering the enzyme for biotechnology applications

    Medicina personalizada en lactantes con c谩ncer: Estudio farmacogen茅tico de polimorfismos relacionados con toxicidad y respuesta a la quimioterapia

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    Introducci贸n La farmacogen茅tica es una herramienta de la 鈥淢edicina personalizada鈥 que contribuye a optimizar los tratamientos antineopl谩sicos, adapt谩ndolos a las caracter铆sticas gen茅ticas particulares de cada individuo, maximizando su eficacia y minimizando su toxicidad. El lactante con c谩ncer es un paciente de particular vulnerabilidad y sus comorbilidades tiene una especial repercusi贸n vital. El estudio farmacogen茅tico en esta poblaci贸n resulta pionero y novedoso en nuestro medio. La identificaci贸n de marcadores predictivos espec铆ficos permitir谩 individualizar m谩s la terap茅utica en esta poblaci贸n tan fr谩gil y mejorar en consecuencia su calidad de vida y su pron贸stico vital. Material y m茅todos Estudio de cohortes ambispectivo de pacientes oncol贸gicos entre 1 y 18 meses de edad, receptores de quimioterapia en el Hospital La Fe de Valencia, en el periodo comprendido entre enero 2007 y agosto 2019. La parte retrospectiva comprende hasta diciembre 2015, el estudio prospectivo desde enero 2016 hasta agosto 2019. En primer lugar, se realiza un an谩lisis descriptivo de variables epidemiol贸gicas, cl铆nico/biol贸gicas, terap茅uticas y pron贸sticas de 72 pacientes con dichas caracter铆sticas. Se describe la toxicidad derivada de sus 578 ciclos de quimioterapia (37 variables cl铆nicas), el tiempo de seguimiento y su supervivencia (meses). En segundo lugar, se realiza un estudio anal铆tico cuyo objetivo es correlacionar los polimorfismos gen茅ticos relacionados con la quimioterapia de 64 de los pacientes, la toxicidad grave secundaria al tratamiento (鈮 grado 3 seg煤n CTAE 4.0) y su supervivencia. El genotipado se realiza en el Centro Nacional de Genotipado (CEGEN) mediante la tecnolog铆a MassArray (AgenaBioscience), previa configuraci贸n de un panel farmacogen茅tico pedi谩trico en base a las evidencias recogidas en la base de datos PharmGKB, fichas t茅cnicas de los medicamentos y consorcios internacionales expertos. El an谩lisis estad铆stico descriptivo se realiza con los programas Excel 2016 y R: las variables cualitativas con el recuento num茅rico (porcentaje) y las variables cuantitativas como mediana +/- rango intercuart铆lico ante ausencia de normalidad en la distribuci贸n de los datos (p <0,05, prueba de Kolmogorov-Smirnov). En el an谩lisis de supervivencia se utiliza el estimador Kaplan-Meier. El an谩lisis estad铆stico anal铆tico de correlaci贸n entre polimorfismos y toxicidad se realiza mediante regresi贸n log铆stica penalizada por Elastic Net empleando R. El an谩lisis estad铆stico anal铆tico de correlaci贸n entre polimorfismos y reca铆da/muerte se realiza mediante regresi贸n de Cox penalizada por Elastic Net. Resultados Las variables epidemiol贸gicas, cl铆nico/biol贸gicas y terap茅uticas de los pacientes de la muestra son consecuentes con las descritas en la literatura del lactante con c谩ncer. Las neoplasias con mayor impacto negativo en la supervivencia son la leucemia mielobl谩stica aguda y los tumores del sistema nervioso central. La toxicidad m谩s prevalente es hematol贸gica, digestiva e infecciosa. Existe correlaci贸n entre la toxicidad grave secundaria a los quimioter谩picos en forma de anemia, neutropenia y/o trombopenia y 46 polimorfismos gen茅ticos diferentes. As铆 mismo se encuentra asociaci贸n estad铆sticamente significativa entre la supervivencia global y la supervivencia libre de enfermedad y ciertos polimorfismos gen茅ticos (26 y 13 respectivamente). Los polimorfismos obtenidos pertenecen a genes encargados del transporte (6 genes) y metabolismo (17 genes) de los f谩rmacos, de la reparaci贸n del material gen茅tico y la supresi贸n tumoral (5 genes) y de otras funciones biol贸gicas (4 genes). Conclusi贸n Los resultados del presente estudio muestran correlaci贸n estad铆stica entre 46 polimorfismos de genes implicados en la cin茅tica farmacol贸gica y la reparaci贸n del material gen茅tico y la variabilidad en la toxicidad quimioter谩pica y supervivencia de pacientes lactantes con c谩ncer. En definitiva, aportaciones de la farmacogen茅tica que pueden contribuir a la optimizaci贸n del tratamiento antineopl谩sico en esta poblaci贸n particular y a la predicci贸n de sus riesgos, de especial impacto en los supervivientes del c谩ncer infantil

    Uso de las histonas circulantes y sus modificaciones post-traduccionales como biomarcadores en sepsis y shock s茅ptico

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    La sepsis es una afecci贸n potencialmente mortal causada por una respuesta anormal del hu茅sped a una infecci贸n, produciendo respuestas fisiol贸gicas alteradas que da帽an los propios tejidos del paciente y pueden provocar disfunci贸n org谩nica e incluso la muerte. Asimismo, algunos pacientes s茅pticos progresan a shock s茅ptico, caracterizado por alteraciones circulatorias, celulares y metab贸licas sustanciales que aumentan el riesgo de mortalidad. A pesar de que la sepsis se caracteriza por un mal funcionamiento del sistema inmunol贸gico, lo que a su vez conduce a una respuesta inmune alterada e inmunosupresi贸n, la alta complejidad de la fisiopatolog铆a de la sepsis requiere una mayor investigaci贸n para comprender las respuestas inmunes que ocurren durante la sepsis. Asimismo, las histonas extracelulares circulantes han ganado relevancia como mediadores citot贸xicos en la sepsis, ya que act煤an como patrones moleculares asociados a da帽o, que inducen estr茅s oxidativo y activan el inflamasoma NLRP3. Estos mecanismos median la activaci贸n de la piroptosis, un mecanismo de muerte celular programada que produce inflamaci贸n mediante la expresi贸n de IL-18, IL-1尾 and IL-1伪. Sin embargo, a pesar de la evidencia de activaci贸n del inflamasoma en las c茅lulas inmunes durante la sepsis, se desconoce si las histonas extracelulares son capaces de activar los inflamasomas endoteliales y sus consecuencias. En este trabajo destacamos el papel previamente desconocido de las histonas extracelulares, mediando la activaci贸n del inflamasoma NLRP3 y la piroptosis en las c茅lulas endoteliales, contribuyendo a la disfunci贸n endotelial y la desregulaci贸n de la respuesta inmune mediada por el endotelio. Asimismo, tambi茅n demostramos c贸mo la acetilaci贸n de histonas disminuye la activaci贸n de la piroptosis. Adem谩s, demostramos que la piroptosis se produce en pacientes con shock s茅ptico y los niveles de histonas circulantes se correlacionan con la expresi贸n de citoquinas proinflamatorias y citoquinas piropt贸ticas, la liberaci贸n de factores de adhesi贸n endotelial y la gravedad de la enfermedad. Proponemos la piroptosis mediada por histonas como un nuevo objetivo para desarrollar intervenciones cl铆nicas. De manera similar, hemos analizado las respuestas inmunorelacionadas que ocurren durante las primeras etapas de la sepsis con el objetivo de proporcionar nuevos datos comparando las cantidades de citoquinas, inmunomoduladores y otros mediadores endoteliales en pacientes cr铆ticamente enfermos no s茅pticos, s茅pticos y de shock s茅ptico. Nuestro enfoque ayudar谩 a caracterizar r谩pidamente las respuestas inmunes alteradas en pacientes s茅pticos y de shock s茅ptico ingresados en la Unidad de Cuidados Intensivos. Finalmente analizamos el papel de la metilaci贸n del ADN en el control del sistema inmune s茅ptico. Nuestros resultados demostraron el papel central de la metilaci贸n del ADN modulando la respuesta molecular en los pacientes de shock s茅ptico y contribuyendo a la inmunosupresi贸n, a trav茅s de la alteraci贸n de los patrones de metilaci贸n de los promotores de IL-10 y TREM-2.Sepsis is a life-threatening condition caused by an abnormal host response to an infection that produce altered physiological responses which damages own tissues of the patient and can result in organ dysfunction and in some cases death. Likewise, a subset of septic patients progresses to septic shock, characterized by substantial circulatory, cellular and metabolic abnormalities, which substantially increase the risk of mortality. Sepsis is characterized by a malfunction of the immune system and it can lead to an altered immune response and immunosuppression. Moreover, the high complexity of the pathophysiology of sepsis requires of further investigation to characterize the immune responses in sepsis and septic shock. Likewise, circulating extracellular histones have gained relevance as cytotoxic mediators in sepsis pathophysiology, since they act as damage-associated molecular patterns, which induce oxidative stress and activate NLRP3 inflammasome. Subsequently, inflammasome mediates pyroptosis activation, a programmed cell death mechanism that produces inflammation through the release of IL-18, IL-1尾 and IL-1伪. However, despite inflammasome activation may occur in immune cells during sepsis, it is unknown if this process also takes place in endothelial cells and particularly whether extracellular histones are capable of activating endothelial inflammasomes and their consequences. In this work we highlight a previously unknown role for extracellular histones, that mediates the activation of NLRP3 inflammasome and pyroptosis in endothelial cells by contributing to endothelial dysfunction and the dysregulation of the immune response mediated by endothelium. Likewise, we demonstrated how histone acetylation decreases pyroptosis activation. Furthermore, we show how pyroptosis occurs in septic shock patients and how circulating histone levels correlate with the expression of pro-inflammatory and pyroptotic cytokines, the release of endothelial adhesion factors and septic shock severity. We propose histone-mediated pyroptosis as a new target to develop clinical interventions. Similarly, we have analyzed the immune-related responses occurring during the early stages of sepsis with the aim of providing new data by comparing the amounts of cytokines, immune modulators and other endothelial mediators in critically-ill non-septic patients, septic and septic shock patients. Our approach will help to rapidly characterize the altered immune responses in septic and septic shock patients admitted in the Intensive Care Unit. Finally, we also analyzed the role of DNA methylation in the control of septic immune system. Our results demonstrated the central role of DNA methylation modulating the molecular response in septic shock patients and contributing to immunosuppression, through the alteration of DNA methylation patterns of IL-10 and TREM2 promoters

    Pontus in Antiquity: aspects of identity

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    The purpose of this thesis is the presentation of the interaction between the successive inhabitants of Pontus in antiquity, indigenous Anatolians, Greeks, Persians and Romans. Limited archaeological evidence cannot determine the precise extent of interaction, although the available information substantiates the notion of a slow, but steady amalgamation. Initially, the intermingling was based on mutual trading links. Although the Hellenic cultural element tended to surface, Eastern factors remained visible. The Mithridatic dynasty was established around the vicinity of Pontus, creating the 'Kingdom of Pontus' which reached its height under Mithridates VI. His administrative and military policy appears to have placed the foundations for the later, Roman corresponding structures. His policies-propaganda reflected the GraecoEastern image of a king, which appealed to the Greek and Persian-Eastern inhabitants of his kingdom, Asia Minor and, to a lesser extent, mainland Greece. This GraecoEastern image might have nourished the concept of a shared history among the inhabitants of Pontus. Their interactions appear to have given rise to an unnamed, local culture, which was enriched with the relevant Roman practices. Around the third century A.D., the Roman administrative patterns might have established an externally defined appellation. During Roman times, Christianity started to be established in Pontus. Although it was not yet a socio-political factor, its non-racial nature prevailed in later centuries. The influence of the Roman-Christian elements can still be observed in the modern Ponti an identity. In antiquity, (lack of) evidence indicates that no group defined themselves as 'Pontics' or 'Pontians' and an internally defined Pontic identity is unlikely to have existed. However, people associated themselves with the geographical area of Pont us, cultural and religious concepts were frequently amalgamated, while the notion of a common descent and a shared history might have been unconsciously fostered. These factors can assist in the understanding of the 'Pontians' today

    Metabolic phenotyping of opioid and psychostimulant addiction: A novel approach for biomarker discovery and biochemical understanding of the disorder.

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    Despite the progress in characterising the pharmacological profile of drugs of abuse, their precise biochemical impact remains unclear. The metabolome reflects the multifaceted biochemical processes occurring within a biological system. This includes those encoded in the genome but also those arising from environmental/exogenous exposures and interactions between the two. Using metabolomics, the biochemical derangements associated with substance abuse can be determined as the individual transitions from recreational drug to chronic use (dependence). By understanding the biomolecular perturbations along this time course and how they vary across individuals, metabolomics can elucidate biochemical mechanisms of the addiction cycle (dependence/withdrawal/relapse) and predict prognosis (recovery/relapse). In this review, we summarise human and animal metabolomic studies in the field of opioid and psychostimulant addiction. We highlight the importance of metabolomics as a powerful approach for biomarker discovery and its potential to guide personalised pharmacotherapeutic strategies for addiction targeted towards the individual's metabolome
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