Sequence-independent characterization of viruses based on the pattern of viral small RNAs produced by the host

Virus surveillance in vector insects is potentially of great benefit to public health. Large-scale sequencing of small and long RNAs has previously been used to detect viruses, but without any formal comparison of different strategies. Furthermore, the identification of viral sequences largely depends on similarity searches against reference databases. Here, we developed a sequence-independent strategy based on virus-derived small RNAs produced by the host response, such as the RNA interference pathway. In insects, we compared sequences of small and long RNAs, demonstrating that viral sequences are enriched in the small RNA fraction. We also noted that the small RNA size profile is a unique signature for each virus and can be used to identify novel viral sequences without known relatives in reference databases. Using this strategy, we characterized six novel viruses in the viromes of laboratory fruit flies and wild populations of two insect vectors: mosquitoes and sandflies. We also show that the small RNA profile could be used to infer viral tropism for ovaries among other aspects of virus biology. Additionally, our results suggest that virus detection utilizing small RNAs can also be applied to vertebrates, although not as efficiently as to plants and insects.A correction has been published: Nucleic Acids Research, Volume 44, Issue 7, 20 April 2016, Pages 3477–3478, https://doi.org/10.1093/nar/gkw04

Análisis de interacciones de letalidad sintética (SL) en cáncer y predicción de tratamientos

Trabajo fin de máster en Bioinformática y Biología ComputacionalEl tratamiento de tumores de forma dirigida todavía supone un desafío en la investigación contra el cáncer. Con el descubrimiento del fenómeno conocido como adicción oncogénica, basado en el concepto de que existen tumores cuya supervivencia depende de (o son adictos a) ciertos genes o rutas biológicas, se produjo un cambio de paradigma en el campo del desarrollo de fármacos antineoplásicos, ahora más dirigido hacia lo que conocemos como medicina personalizada. La idea de medicina personalizada surge de la dependencia que existe entre la eficacia de una terapia y la presencia de determinadas alteraciones genéticas, y busca desarrollar fármacos que actúen sobre dianas específicas del tumor. Pero pese a ser una de las líneas de investigación más importantes para el tratamiento de la enfermedad, de momento sólo ha demostrado ser efectiva en tumores muy determinados y la mayoría de pacientes tienen que recurrir a terapias tradicionales. Una de las estrategias más prometedoras para la mejora y el desarrollo de terapias anticáncer está basada en la explotación de dependencias secundarias, no necesariamente oncogénicas, conocidas como interacciones de letalidad sintética (SL). Se sabe que las células cancerígenas tienen mecanismos de compensación que les ayudan a sobrevivir en el caso de acumular mutaciones en genes críticos, desarrollando dependencias para con dichos genes en muchos de estos casos. El concepto de letalidad sintética surge con la finalidad de aprovechar estas compensaciones y así afectar la viabilidad de la célula tumoral. Utilizando pruebas de detección masivas a lo largo de paneles de líneas celulares tumorales, algunos grupos de investigación han explorado los efectos del silenciamiento de genes y su relación con el comportamiento fenotípico de la célula, detectando dependencias en células cancerígenas. En este proyecto, hemos integrado la información recogida en diversas bases de datos dedicadas a la recolección de interacciones de letalidad sintética, con información de mutaciones puntuales, alteraciones en número de copia y silenciamiento génico. 9 A partir de este análisis se han podido identificar nuevas dianas terapéuticas y biomarcadores predictivos de respuesta en cáncer. En concreto, hemos partido de más de 16 mil interacciones de letalidad sintética y analizado aquellas con opciones terapéuticas disponibles (un 34% del total). La exploración de estas interacciones nos ha llevado a la obtención de nuevas estrategias terapéuticas que involucran a oncogenes sin disponibilidad de tratamientos dirigidos como KRAS o genes supresores de tumores como BRCA. Además, nos ha ayudado a profundizar en los mecanismos biológicos que se encuentran detrás de estos eventos. Para completar ese análisis será necesario integrar más fuentes de información como datos de expresión, metilación, así como explorar los efectos de la inhibición farmacológica de estas dianas. Añadiendo la estrategia de letalidad sintética a las ya existentes, podemos avanzar en la definición de los subtipos de cáncer y su tratamiento mediante terapias dirigida

Bioinformatics Analysis of Chronic Stress Using Targeted Oxford Nanopore Sequencing

Stress impacts us on a molecular level by altering our DNA methylation patterns. Uncovering these changes methylation is vital to understanding molecular mechanisms that underlie stress-related diseases and conditions. Our goal was to evaluate Oxford Nanopore sequencing with CRISPR-Cas9 enrichment as a way to examine DNA methylation landscapes across stress-responsive genes. We enhanced the wet lab protocols from Gilpatrick et al. by using additional probes during digestion and wide bore tips during extraction. We improved the sequencing depth to generate the number of reads necessary for highly accurate DNA methylation calls. After analyzing the reads, we were able to accurately capture the underlying epigenetic landscape such as hypomethylated regions at CpG islands, and predicted changes between young and old cells. In addition, by visualizing DNA methylation levels with heat maps, we uncovered co-regulated methylation regions spanning long stretches of the GILZ gene. This technique may soon enable us to determine differential methylation due to chronic stress and to uncover the molecular mechanisms of stress-related diseases.Bachelor of Scienc

The regulatory role of Malat1 on the alternative splicing factor SRSF1 during CD4+ T cell differentiation

The proper activation and subsequent differentiation of naive CD4+ T cells into effector T helper and regulatory T cells is vital for directing an appropriate adaptive immune response to specific infections. Recent evidence has identified long non-coding RNAs as novel regulators of CD4+ T cell activation and differentiation. Work by the Lagos group and others has identified the long non-coding RNA Metastasis associated lung adenocarcinoma transcript 1 (Malat1) as a critical regulator of Th cell function and immune response to chronic infection in mice. However, the mechanism behind this regulation by Malat1 is not yet fully understood. This project aimed to investigate the RNA binding protein and splicing factor SRSF1, a known Malat1 binding partner and prominent regulator of gene expression and alternative splicing in the immune system, as a mediator for Malat1 regulation of Th cell function. Through analysis of individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP), we have shown that SRSF1 displays alternative RNA binding behaviour in Th2 cells upon Malat1 loss. This alternative binding is directed towards RNA transcripts involved in T cell activation and differentiation, including Il2ra and Runx3. Following this, we found that Runx3 abundance is reduced and isoform usage is altered upon Malat1 loss in Th2 cells. To complement studies within Malat1-/- CD4+ T cells we attempted to develop Srsf1-/- CD4+ T cell models. Attempts to develop Srsf1-/- EL4 cell lines using CRISPRCas9- editing caused just a transient knockdown of SRSF1 expression, suggesting SRSF1 is essential for viability in this mouse T cell lymphoma cell line. Initial studies for establishing CRISPR-Cas9 RNP transfection into in vitro primary CD4+ T cell activation assays were unsuccessful at producing an SRSF1 knockout but have laid a foundation for further optimisation. Overall, our results identified Malat1 regulation of SRSF1 mediated RNA interaction during the Th2 cell differentiation, which serves as a promising mechanism for further investigation to better characterise Malat1 regulation of Th cell phenotype and cytokine expression

Gestión de la calidad educativa en la educación virtual de la I.E.I. 479 distrito de Callería-Ucayali 2022

La búsqueda de la calidad educativa, aún es una brecha que las políticas educativas tienen como reto agudo. El objetivo de la investigación fue conocer la influencia de la gestión de la calidad educativa en la educación virtual en la I.E.I. 479 distrito de Callería-Ucayali 2022. Dada la naturaleza cualitativa de la investigación, el nivel fue descriptivo con diseño fenomenológico. Los datos se recogieron a través de un Focus Group. Se contó con la participación de 6 miembros de la comunidad educativa: directivos, docentes y padres de familia. El contar con los elementos de calidad educativa implementadas en la gestión, influyen significativamente en la educación virtual y, por ende, en la calidad del servicio que se brinda a través de ello. Siendo un indicador de fuerte influencia para lograr o no con el perfil de egreso de los estudiantes. La presente investigación concluye que: La gestión de calidad educativa tiene influencia en una buena práctica de la educación virtual. Si se realiza una buena gestión de la calidad estarían cumpliendo con los cuatro factores determinantes de una buena calidad educativa impartida a los estudiantes y población que recibe el servicio educativo, así como los que lo conforma

Development of a tool to introduce DNA methylation into a genomic locus in vivo

The Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 (Cas9) system, also called CRISPR-Cas9, has been manipulated for application in a wide variety of biomedical research. The simplicity of the CRISPR system has made it easy for its successful application in introducing both genetic and epigenetic modifications in vitro and in vivo. While essential for development and differentiation in organisms, epigenetic processes (DNA Methylation, Histone modification, and non-coding RNAs) have been found to be influenced by endogenous, biotic, and abiotic factors. Epigenetic alterations, particularly DNA methylation changes that occur due to early-life environmental exposures have been identified as one of the potential mechanisms underlying the developmental origins of health and disease (DOHaD). The Cas9 protein can be made devoid of its endonuclease activity resulting in a nuclease dead Cas9 (dCas9) molecule that cannot cleave target DNA. Transcriptional effectors are proteins that can activate or repress gene transcription. Although a few experiments have been performed using transcriptional effectors in the Crispr-dcas9 system in vitro, an effective and reproducible technique to epigenetically activate/suppress genes using activator and repressor elements in vivo has not been developed. Given that embryos undergo epigenetic reprogramming of parental epigenetic marks during the cleavage stages, it is difficult to determine if epigenome targeting at the one-cell stage can escape genome-wide reprogramming of the embryo occurring at cleavage stages. It is also unknown if de novo-established epigenetic alterations are programmed differently from those inherited from parental gametes. To date, there have been no studies demonstrating the fate of epigenetic edits during epigenetic reprogramming in vivo. This study examines if the epigenome editing performed at zygotic stages will alter expression of a heritable epigenetic trait that can withstand the reprogramming window during the subsequent cleavage stages. To this end, we are using a fish model medaka (Oryzias latipes) which has been established as an excellent biomedical model given its similarity in germ cell development to mouse and humans and applying CRISPR-dCcas9-DNMT3aa repressor at the first few cell stages. This study will help delineate the applicability of the CRISPR system in introducing epigenetic modifications during the epigenetic reprogramming windows

Importância da região 3´UTR do mRNA do deltaC no desenvolvimento de Peixe-Zebra

A somitogénese é um processo morfogenético altamente regulado e conservado entre os vertebrados. Durante este processo ocorre a formação de sómitos de forma rítmica e sequencial ao longo do eixo ântero-posterior do embrião a partir de um tecido progenitor não segmentado denominado de mesoderme pré-somítica (PSM). Os sómitos são aglomerados de células da mesoderme, precursores do esqueleto e da musculatura axial. A periodicidade da formação de sómitos a partir da PSM é imposta por um oscilador molecular denominado de relógio molecular que leva à expressão cíclica de um conjunto de genes, que se propaga ao longo da PSM no sentido caudo-rostral. A comunicação celular e a sincronização das oscilações de expressão na PSM através da sinalização intercelular Notch-Delta são fundamentais no controlo da somitogénese e da diferenciação celular. Esta oscilação da expressão dos genes do relógio é controlada por um mecanismo de regulação de feedback negativo, onde a instabilidade do mRNA desempenha um papel fundamental. A região 3´UTR geralmente contém vários elementos reguladores que governam a expressão espacial e temporal de diferentes mRNAs. Estudos anteriores do laboratório demostraram uma estabilização do mRNA do gene deltaC de peixe-zebra na ausência de porções da região 3´UTR. Para melhor compreensão do fenótipo, neste trabalho foi avaliado o papel da região 3’UTR do gene deltaC na somitogénese de embriões de peixe-zebra. Para isso foram utilizadas diferentes linhas mutantes em que a região 3'UTR do gene deltaC foi editada usando a tecnologia CRISPR/Cas9. Procedeu-se à caracterização do padrão de expressão dos genes her7 e deltaC e caracterização morfológica dos sómitos formados para perceber o impacto destas alterações. Os resultados do presente estudo confirmaram que a região 3´UTR de deltaC é importante para a correta expressão de deltaC e her7, bem como para a segmentação corporal do embrião de peixe-zebra. Embora nos embriões RR2 e RR3 a segmentação não é interrompida, o número total de sómitos é menor e os sómitos anteriores são ligeiramente mais curtos. Usando uma linha transgénica em que o Notch Intracellular Domain (NICD) é expresso constitutivamente após indução térmica observou-se diminuição do tamanho dos sómitos como nos embriões RR3 o que sugere que o fenótipo observado nos embriões mutantes poderá ser devido a um aumento da sinalização Notch durante a somitogénese.Embryonic somitogenesis is a highly regulated and conserved morphogenetic process among vertebrates. During this process, somites are formed rhythmically and sequentially from a non-segmented progenitor tissue called presomitic mesoderm (PSM) along the anterior-posterior axis of the embryo. Somites are clusters of mesoderm cells that are precursors of the axial skeleton and musculature. The periodicity of somite formation from the PSM is due to an oscillator mechanism called the molecular clock that leads to the cyclic expression of a set of genes that propagate along the PSM in a caudal-rostral direction. Cellular communication and synchronization of expression oscillations in the PSM through Notch-Delta intercellular signaling are fundamental in the control of somitogenesis and cell differentiation. The oscillation of clock gene expression is controlled by negative feedback, where mRNA instability plays a key role. The mRNA 3'UTR region generally includes several regulatory elements that govern the spatial and temporal expression of mRNAs. Previous studies in the lab suggest that mRNA stabilization in the absence of portions of the 3'UTR region of zebrafish deltaC gene might be involved in the regulation of the molecular clock. For a better understanding of the phenotype, this work was designed to evaluate the role of the 3’UTR region of the deltaC gene in the somitogenesis of zebrafish embryos. For this study, different mutant lines generated by CRISPR / Cas9 were used, where the 3'UTR region of the deltaC gene was edited. The characterization of expression patterns of her7 and deltaC genes was carried out, as well as the morphological characterization of the somites formed in these mutants. The results of this study confirmed that the deltaC 3'UTR region is importante for proper expression of deltaC and her7, as well as for zebrafish embryo body segmentation. Although in RR2 and RR3 embryos segmentation is not interrupted, the total number of somites is smaller and the anterior somites are slightly shorter. Using a transgenic line where the Notch Intracellular Domain (NICD) is constitutively expressed after heat-shock was observed a decrease in somite size as observed in the RR3 embryos suggesting that the phenotype observed in the mutant embryos could be due to enhanced Notch signaling during somitogenesis

Prediction of Secondary Protein Structure

Αυτό το έργο στοχεύει να δείξει στους αναγνώστες του μια προσπάθεια για την επίλυση του προβλήματος πρόβλεψης της δευτερογενούς δομής πρωτεΐνης χρησιμοποιώντας βαθιά υπολειμματικά νευρωνικά δίκτυα και άλλες μεθόδους. Οι πρωτεΐνες είναι ένα από τα πιο ζωτικά συστατικά κάθε ζωντανού όντος. Παίζουν πολύ σημαντικό ρόλο καθώς καθορίζουν τις λειτουργίες ενός οργανισμού. Επομένως, η γνώση της δομής της πρωτεΐνης είναι μεγάλης σημασίας. Συγκεκριμένα, η δομή της πρωτεΐνης αποτελείται από τέσσερα επίπεδα. πρωτοταγής, δευτεροταγής, τριτοταγής και τεταρτοταγής πρωτεϊνική δομή. Η πιο σημαντική είναι η δομή στον τρισδιάστατο χώρο, η τριτοταγής δομή , γιατί αυτή καθορίζει τον βιολογικό ρόλο της πρωτεΐνης. Ως αποτέλεσμα, η γνώση των πρωτεϊνικών λειτουργιών μπορεί να βοηθήσει στη θεραπεία πολλών ασθενειών. Δυστυχώς, οι μεθοδολογίες εξαγωγών που έχουν αναπτυχθεί μέχρι τώρα, είναι πολύ περίπλοκες και χρονοβόρες διαδικασίες. Ο ορισμός της δευτεροταγής δομής είναι απαραίτητος για την εξαγωγή της τριτοταγής δομής και αυτός είναι ο λόγος που μελετάται. Η δευτεροταγής δομή εξάγεται από την πρωτοταγή δομή, η οποία περιλαμβάνει μια αλληλουχία αμινοξέων. Σε αυτό το έργο θα αναλυθούν κυρίως τα βαθιά υπολειμματικά δίκτυα και ο τρόπος που μπορούν να βοηθήσουν στην πρόβλεψη της δευτεροταγούς δομής της πρωτεΐνης. Τέτοια δίκτυα ανήκουν στην κατηγορία των βαθιών νευρωνικών δικτύων, τα οποία ουσιαστικά αποτελούνται από συγκλίνοντα επίπεδα με προσθετικές συνδέσεις μεταξύ τους.This project aims to show its readers an effort for the solution of the prediction problem of the protein secondary structure using deep residual neural networks and other methods. Proteins are one of the most vital components of every living being. They play a quite important role as they define the functions of an organism. Therefore, knowing the protein structure is of great importance. Specifically, protein structure consists of four levels; primary, secondary, tertiary and quaternary protein structure. The most significant is the structure in the three-dimensional space, the tertiary structure because this one defines the biological role of the protein. As a result, knowing the protein functions may help the treatment of many diseases. Unfortunately, the export methodologies that are developed so far, are very complicated and time-wasting procedures. The definition of the secondary structure is needed to export the tertiary structure and that is the reason it is studied. The secondary structure is exported by the primary structure, which includes an amino acid sequence. In this project the deep residual networks and the way they can help for the prediction of the protein secondary structure will mainly be analyzed. Such networks belong to the category of deep residual neural ones, which essentially consist of convergent levels with additive connections among them