ZNF292 is a complex protein of ≈3000 amino acids with 16 zinc finger DNA binding domains. First discovered as a Growth Hormone (GH) transcription activator, ZNF292 was then found to play a role in the initiation of certain cancers, in specific autism spectrum disorder types, and to produce high levels of unique circular RNAs (circRNAs). In the rat, we have found that ZNF292 circRNAs are present in multiple related structures containing differing combinations of exons 1-7, thus increasing in sizes like “babushka” or nested Russian dolls. To begin to understand the functions of these ZNF292 circRNAs, we assayed expression in four rat MtT cell lines that model stages of pituitary somatotroph development. These cell lines have specific GH levels: MtT/E are empty: MtT/Se have estrogen stimulated GH expression; MtT/SM somatomammotrophs have both GH and PRL; and MtT/S somatotrophs produce only GH. Rat GH3 somatomammotroph cells were also assayed for comparison. Thus, the defined stages of pituitary development in these cells are an excellent model for evaluating linear or circular ZNF292 RNA changes. All cell lines were grown according to standard conditions, then harvested and total RNA was extracted (RNeasy kit, Qiagen) and used in qRT-PCR assays where both RT and PCR were performed in each well (Luna kit, NEB). Primers were designed in MacVector: RPL19 as a normalization control, and GH, linear, and circular ZNF292 primers targeted selected exons. Standards for quantitation used purified total rat RNA (ThermoFisher). After qRT-PCR, all data was normalized to the amount of RPL19 in each sample, then average RNA levels calculated for each cell line using Excel and GraphPad Prizm. We found that GH RNA rose from 0% in MtT/E and MtT/Se cells to 157% in MtT/SM and 275% in MtT/S cells. Linear ZNF292 was increased to 219% in MtT/Se cells, then declined to 110% in MtT/S and 37% in MtT/SM cells. Circular ZNF292 was expressed in MtT/E cells at 26%, increased to 184% in MtT/Se cells and fell to ≈100% in MtT/SM and MtT/S cells. GH3 cell expression was similar in pattern to MtT/SM cells but reduced in relative abundance. Statistical analysis with two-way ANOVA indicated significant differences. First, these data confirmed that GH RNA levels correlate with stages of somatotroph differentiation in these cell lines. Second, ZNF292 linear RNA expression increases prior to the onset of GH expression, agreeing with the function of linear ZNF292 in initiating GH transcription. Third, ZNF292 circRNAs have a developmental pattern of expression that is higher in somatomammotroph cells. Expression of other RNAs central to pituitary development will be used to evaluate whether ZNF292 circRNA levels specifically relate to GH status or another criterion of cellular differentiation.

abstract: Circular RNAs (circRNAs) are a class of endogenous, non-coding RNAs that are formed when exons back-splice to each other and represent a new area of transcriptomics research. Numerous RNA sequencing (RNAseq) studies since 2012 have revealed that circRNAs are pervasively expressed in eukaryotes, especially in the mammalian brain. While their functional role and impact remains to be clarified, circRNAs have been found to regulate micro-RNAs (miRNAs) as well as parental gene transcription and may thus have key roles in transcriptional regulation. Although circRNAs have continued to gain attention, our understanding of their expression in a cell-, tissue- , and brain region-specific context remains limited. Further, computational algorithms produce varied results in terms of what circRNAs are detected. This thesis aims to advance current knowledge of circRNA expression in a region specific context focusing on the human brain, as well as address computational challenges. The overarching goal of my research unfolds over three aims: (i) evaluating circRNAs and their predicted impact on transcriptional regulatory networks in cell-specific RNAseq data; (ii) developing a novel solution for de novo detection of full length circRNAs as well as in silico validation of selected circRNA junctions using assembly; and (iii) application of these assembly based detection and validation workflows, and integrating existing tools, to systematically identify and characterize circRNAs in functionally distinct human brain regions. To this end, I have developed novel bioinformatics workflows that are applicable to non-polyA selected RNAseq datasets and can be used to characterize circRNA expression across various sample types and diseases. Further, I establish a reference dataset of circRNA expression profiles and regulatory networks in a brain region-specific manner. This resource along with existing databases such as circBase will be invaluable in advancing circRNA research as well as improving our understanding of their role in transcriptional regulation and various neurological conditions. / Dissertation/Thesis / Appendix file containing list of enriched pathways and functions identified in Chapter 4 / Doctoral Dissertation Biomedical Informatics 2018

No description available.

Introduction: Myocardial infarction is the most common form of acute cardiac injury attributed to heart failure. Despite advancements in prognosis and treatment, acute MI (AMI) still bears a considerable mortality rate within the initial year, with a significant portion of patients succumbing within the initial 30 days post-MI. The overall prognosis hinges on factors such as the extent of heart muscle damage, duration of the inflammatory response, and the efficacy of administered treatments in mitigating myocardial cell death and injury. This underscores the need for deeper mechanistic understanding and development of targeted therapies for cardiovascular diseases. In response to cardiac injury, macrophages are initially recruited to the infarcted myocardium and undergo phenotypic change from pro-inflammatory (M1) macrophages in the early stage to an anti-inflammatory (M2) macrophages phenotype in the later stage, orchestrating the initiation, maintenance, and eventual resolution of the inflammatory response. However, in chronic ischemia or severe infarction, continuous cardiomyocyte death prolongs pro-inflammatory macrophage activation resulting in robust secretion of pro-inflammatory cytokines perpetuating the inflammatory response and resulting in increased myocardial damage. Despite some understanding, further research is needed on the mechanisms and factors influencing macrophage function during injury. Circular RNAs are newly discovered non-coding RNA generated from protein-coding genes ubiquitously expressed in mammalian tissue, highly conserved among species, and recently implicated in the possible regulation of macrophage activation. However, their role in immunomodulation during cardiovascular injury remains unknown. Objective: This study focused on determining the specific role of circ-cdr1as in phenotypic switching between pro-, and anti-inflammatory macrophages and to determine whether functional regulation of circ-cdr1as modulates macrophage function post-cardiac injury. Methods and Results: We performed circular RNA microarray analyses to assess circular RNA transcriptome changes using RNA isolated from bone marrow derived macrophages (BMDM) polarized to a M1 phenotype by INFγ and TNFα or a M2 phenotype by IL-10, IL-4, and TGF-β. Following RNA isolation, samples are treated with RNaseR for enrichment of circular RNA and removal of linear RNA. We identified circRNAs differentially expressed in pro-and anti-inflammatory macrophages, including circRNA cdr1as (circ-cdr1as). RT-qPCR analysis revealed circ-cdr1as was one of the most downregulated in pro-inflammatory macrophages and significantly upregulated in anti-inflammatory macrophages in vitro. We established a circ-cdr1as overexpression system by generating a circ-cdr1as plasmid using pc3.1 plasmid with flanking regions that allows circularization of specified sequence for in vitro studies. For knockdown of circ-cdr1as, we used small hairpin RNA targeting the splicing junction found only in circular RNA. RT-qPCR and fluorescence activated cell sorting (FACS) analyses showed that overexpression of circ-Cdr1as increased transcription of anti-inflammatory markers and percentage of CD206+ (M2 macrophage marker) cells in naïve and pro-inflammatory macrophages in vitro. Meanwhile, knockdown decreased transcription of anti-inflammatory markers and increased the percentage of CD86+ (M1 macrophage marker) cells in naïve and anti-inflammatory macrophages in vitro. Disease enrichment analysis based on IPA system of the diseases associated with circular RNA involved in macrophage polarization indicated that cardiac fibrosis and cardiac enlargement as the top diseases. Therefore, we investigated the expression levels of circ-cdr1as in the heart after myocardial infarction (MI) injury in a mouse model. RT-qPCR analysis revealed downregulation of circ-cdr1as in the heart 3 days post MI, suggesting a possible physiological role for circ-cdr1as in MI pathophysiology. We isolated fibroblast, cardiomyocytes, CD31+ endothelial cells, and F4/80+ macrophages and investigated the transcriptional changes of circ-cdr1as to determine if it is cell-type specific. RT-qPCR analysis showed no significant difference in transcription of circ-cdr1as in fibroblast and endothelial cells. However, in cardiomyocytes and macrophages there was a significant downregulation of circ-cdr1as. To understand the role of circ-cdr1asmodulated macrophages in post-MI cardiac repair in vivo, we overexpressed circ-cdr1as in fluorescently labeled BMDMs and directly injected them into the ischemic myocardium immediately following MI surgery. FACS and immunohistochemistry analyses showed that these macrophages retained their anti-inflammatory phenotype during the initial stages of cardiac injury, and in the later stages improved cardiac left ventricular (LV) functions and reduced infarct size. Since circ-cdr1as was also decreased in cardiomyocytes post-MI, we additionally generated circ-cdr1as adeno associated virus 9 (circ-cdr1as-AAV-9) vectors to overexpress circ-cdr1as in mouse hearts. We performed tail vein injections of circ-cdr1as-AAV9 vectors 14 days prior to MI and conducted physiological and histological studies. Administration of circ-cdr1as-AAV9 significantly improved post-MI LV functions including ejection fraction (%EF) and fractional shortening (%FS) at 21-28D post MI, decreased infarct size, and improved angiogenesis. Interestingly, in the initial stages of cardiac injury, overexpression of circ-cdr1as reduced cardiomyocyte apoptosis and increased percentage of anti-inflammatory macrophages at injury site. Lastly, emerging evidence suggests that some circular RNAs function as microRNA (miR) sponges. Therefore, we investigated this mechanism to assess whether circular cdr1as invokes phenotypic changes in macrophages in both the steady-state and injured heart by acting as a sponge for miRNA to inhibit its function. Circ-cdr1as contains over 70 binding sites for miR-7, a microRNA known to exacerbate inflammation in lung related diseases through inhibition of KLF4. Pull-down assay indicated that circ-cdr1as directly interacts with miR-7. We found a reciprocal relationship between circ-cdr1as and miR-7 in macrophages and in the heart 3 days post-MI. Overexpression of miR-7 by miR-7-5p mimic increased the percentage of pro-inflammatory marker CD86 in naïve, pro-, and anti-inflammatory macrophages and upregulated transcription of pro-inflammatory markers. Meanwhile, inhibition of miR-7 had the opposite effect. We also found that expression of miR-7 target gene, KLF4, was reduced when macrophages were treated with miR-7 and increased when miR-7 was inhibited. Conclusions: In summary, this study suggests that circ-cdr1as plays a key role in regulating anti-inflammatory phenotype of macrophages through the modulation of miR-7 and its targets and exogenous delivery of circ-cdr1as may improve LV function over time. Therefore, circ-cdr1as may potentially be developed as an anti-inflammatory regulator in tissue inflammation after cardiac injury. / Biomedical Sciences

Opioid use disorder (OUD) is chronic relapse neurological disorder, best characterized by profound drug-induced neuroadaptations which drive drug-seeking phenotypes and persist in the absence of drug. In this dissertation we will examine circular RNA (circRNA) dysregulation following a rodent model of heroin self-administration and characterize a novel circRNA pathway regulating opioid induced neuroadaptations. We identified 76 circRNAs regulated by heroin self-administration, of these we selected circGrin2b_011731 (circGrin2b) for further characterization. circGrin2b is derived from exon 3 of linear Grin2b, which encodes the regulatory subunit of the NMDA receptor, GluN2b, a protein essential for opioid learning and memory paradigms. First, using a custom small-interfering RNA (siRNA) targeting the backsplice junction of circGrin2b, we show that knock-down of orbitofrontal cortex (OFC) circGrin2b significantly reduces heroin intake during self-administration, while only non-significantly reducing heroin-seeking phenotypes. This suggests that OFC circGrin2b is functionally relevant to heroin self-administration phenotypes. Next, we utilized an in vitro primary cortical culture morphine exposure model to interrogate putative upstream biogenesis mechanisms and examine expression of potential circGrin2b microRNA (miRNA)-sponge targets. We validated circGrin2b interactions with miR-26b-3p and miR-100-3p utilizing a luciferase binding assay. Sponging of these 2 miRNA targets can have extensive impact of translation of miRNA-repressed genes, as both miRNAs are involved in regulation of messenger RNAs involved in opioid signaling. Taken together, this dissertation characterized the behavioral and molecular role of a novel circRNA species in mediating opioid-induced neuroadaptations and expands upon the current understanding of the role of circRNAs in regulating synaptic plasticity. / Biomedical Sciences

No description available.

Bulge-helix-bulge (BHB) elements guide the enzymatic splicing machinery that in Archaea excises introns from tRNAs, rRNAs from their primary precursor, and accounts for the assembly of piece-wise encoded tRNAs. This processing pathway renders the intronic sequences as circularized RNA species. Although archaeal transcriptomes harbor a large number of circular small RNAs, it remains unknown whether most or all of them are produced through BHB-dependent splicing. We therefore conduct a genome-wide survey of BHB elements of a phylogenetically diverse set of archaeal species and complement this approach by searching for BHB-like structures in the vicinity of circularized transcripts. We find that besides tRNA introns, the majority of box C/D snoRNAs is associated with BHB elements. Not all circularized sRNAs, however, can be explained by BHB elements, suggesting that there is at least one other mechanism of RNA circularization at work in Archaea. Pattern search methods were unable, however, to identify common sequence and/or secondary structure features that could be characteristic for such a mechanism.

Les translocations chromosomiques sont associées à un grand nombre de cancers. Les translocations chromosomiques sont impliquées dans la tumorigenèse par différents mécanismes : elles conduisent soit à une dérégulation d’un oncogène, soit à la formation d’un nouvel oncogène de fusion. Cependant, le lien direct entre l'apparition d'une translocation chromosomique et la formation d'une tumeur n'est pas totalement établi. Par exemple, plusieurs translocations associées au cancer ont été détectées dans le sang d’individus sains voire dans le sang de cordon des bébés avec une prévalence bien supérieure à celle de la maladie. Ceci suggère que la seule formation de la translocation ne suffit pas toujours à induire l’oncogenèse. La plupart des travaux de recherche antérieurs reposaient sur la surexpression de la protéine de fusion, oncogène supposé. Ces approches présentent de nombreuses limites, la translocation chromosomique est alors absente de même que le contexte chromosomique natif du gène de fusion (promoteur endogène, statut de la chromatine, etc.) ou les éventuels effets d’haplo-insuffisance qui ne sont pas récapitulés. La molécule d’ADN étant organisée de manière non aléatoire dans le noyau, les réarrangements chromosomiques sont également susceptibles d’affecter le statut épigénétique, la réplication et la transcription du chromosome dérivatif entier, en plus des segments d’ADN nouvellement juxtaposés. Or la technologie CRISPR/Cas9, permet de reproduire la translocation chromosomique in situ, après avoir induit deux cassures double-brin simultanées. Ce travail de thèse a porté spécifiquement sur la translocation t(2,5) (p23, q35) qui induit l’expression de la protéine de fusion NPM1-ALK fréquemment rencontrée dans le lymphome anaplasique à grandes cellules (ALCL). Nous avons reproduit la t(2,5) à la fois dans des lignées cellulaires mais aussi dans des cellules T primaires à la fin de ma thèse. Nous avons pu montrer des modifications significatives du timing de réplication des cellules qui portent la translocation en comparaison des cellules isogéniques de départ (par la méthode du Répli-seq) pouvant avoir un impact sur l’homéostasie des cellules tumorales. En parallèle, nous avons mis en évidence la formation d'ARN circulaires de fusion spécifiques, exprimés à partir du gène de fusion, spécifiques des lignées tumorales. Ces ARN circulaires pourraient donner naissance à de nouveaux biomarqueurs diagnostic/pronostic dans le futur. Ces travaux permettront de mieux comprendre les conséquences des translocations chromosomiques oncogéniques dans les cellules humaines et pourraient mener vers de nouvelles orientations thérapeutiques à l’avenir. / Chromosomal translocations are associated with a wide range of cancers. These chromosomal rearrangements are implicated in tumorigenesis by different mechanisms: either they lead to oncogene upregulation or tumor suppressor downregulation. However, the direct link between the appearance of one chromosomal translocation and tumor formation is not always clear. For example, several cancer translocations have been found in PBMCs or in cord blood cells from healthy individuals, suggesting that translocation formation alone is not always sufficient to drive oncogenesis. Most of previous research works on cancer translocation relied on studies using overexpression of the fusion protein. These approaches do not reproduce the chromosome arm translocation nor the chromosomal context of the fusion gene (endogenous promotor, chromatin status etc…) or do not recapitulate a potential haplo-insufficiency of the translocated cells. Because the DNA molecule is organized non-randomly in the nucleus, chromosomal rearrangements are also likely to impact the epigenetic, replication and transcriptional status of the whole rearranged chromosome in addition to the newly juxtaposed gene segments. Using CRISPR/Cas9 technology, we can recapitulate chromosomal translocation in situ, after inducing 2 concurrent double-strand breaks. In this work, we focus on t(2,5)(p23,q35) leading to NPM1-ALK fusion protein frequently found in Anaplasic Large Cell Lymphoma (ALCL). We could recapitulate t(2;5) in cell lines but more importantly in human primary T cells from healthy donors. We showed significant modifications on Replication Timing in model cell lines compare to isogenic non-translocated cells (using Repli-seq analysis). Importantly, these changes might have a direct impact on tumor cell homeostasis. In parallel, we also highlighted the formation of specific fusion circular RNAs expressed from the fusion gene also found in tumor cells. These circular RNAs could give rise to new diagnostic/prognostic biomarkers in the future. This work will lead to a better understanding of the consequences of cancer translocation in human cells and could give new directions for therapeutics in future.

Bulge-helix-bulge (BHB) elements guide the enzymatic splicing machinery that in Archaea excises introns from tRNAs, rRNAs from their primary precursor, and accounts for the assembly of piece-wise encoded tRNAs. This processing pathway renders the intronic sequences as circularized RNA species. Although archaeal transcriptomes harbor a large number of circular small RNAs, it remains unknown whether most or all of them are produced through BHB-dependent splicing. We therefore conduct a genome-wide survey of BHB elements of a phylogenetically diverse set of archaeal species and complement this approach by searching for BHB-like structures in the vicinity of circularized transcripts. We find that besides tRNA introns, the majority of box C/D snoRNAs is associated with BHB elements. Not all circularized sRNAs, however, can be explained by BHB elements, suggesting that there is at least one other mechanism of RNA circularization at work in Archaea. Pattern search methods were unable, however, to identify common sequence and/or secondary structure features that could be characteristic for such a mechanism.

Tesis por compendio / [ES] Los viroides son los patógenos con replicación autónoma más simples y sólo se han encontrado de forma natural infectando plantas superiores. Desde que se descubrieron en los años setenta, se ha adquirido un conocimiento considerable sobre su naturaleza y mecanismos de replicación en las plantas huésped. Sin embargo, aún quedan por descubrir muchos aspectos de la biología de los viroides. Por lo tanto, un conocimiento más profundo de la naturaleza y el modo de acción de los viroides han sido los objetivos principales que engloban esta tesis. Para ello, es esencial contar con procedimientos sencillos y eficientes para la obtención de clones de ADNc infecciosos. Se desarrolló un nuevo método eficiente para construir clones de viroides infecciosos y se probó con un viroide de cada familia: El viroide latente de la berenjena (ELVd, Avsunviroidae) y el viroide del lúpulo (HSVd, Pospiviroidae). Esta aproximación se basó en enzimas de restricción de tipo IIS que cortan fuera del sitio de reconocimiento y supone un procedimiento universal para obtener clones infecciosos de un viroide independientemente de su secuencia, con una alta eficiencia. A pesar de que los viroides han sido considerados como ARN no codificantes desde su descubrimiento, nuestro análisis computacional predijo pequeños marcos de lectura abiertos en cada uno de los genomas de HSVd y ELVd. No se encontraron similitudes significativas con las proteínas de la base de datos de plantas superiores, pero algunos de estos péptidos predichos estaban altamente conservados entre todas las variantes de HSVd y ELVd. Curiosamente, la fusión de estas secuencias conservadas con una proteína fluorescente reveló una localización subcelular específica en el correspondiente orgánulo donde tiene lugar la replicación/acumulación para cada viroide: nucleolo y cloroplasto para HSVd y ELVd, respectivamente. Las mutaciones que truncan el dominio nucleolar de HSVd fueron perjudiciales para el viroide, mientras que el truncamiento de cualquiera de los dos ORF de ELVd que contiene una señal de localización al cloroplasto también disminuyó (pero en menor medida) la eficiencia biológica del viroide, tal vez debido a la redundancia funcional. Se encontraron formas circulares de los ARN de HSVd y ELVd en fracciones polisómicas, lo que revela su interacción física con la maquinaria de traducción de la célula vegetal. En conjunto, estas observaciones experimentales indican que no se puede descartar la capacidad de codificación de los viroides, aunque la prueba definitiva (la detección de los péptidos codificados por los circRNAs) es un reto tecnológico que deberá abordarse en futuras líneas de investigación. Finalmente, para estudiar qué cambios se producen en el huésped durante la infección con un viroide sintomático, se realizó un análisis integrador de las alteraciones genómicas de plantas de pepino infectadas con HSVd. Se integraron los transcriptomas, el sRNAnomas y el metilomas para determinar la respuesta temporal a la infección por el viroide. Nuestros resultados apoyan que el HSVd promueve el rediseño de las vías reguladoras del pepino afectando predominantemente a capas reguladoras específicas en diferentes fases de la infección. La respuesta inicial se caracterizó por una reconfiguración del transcriptoma del hospedador mediante el uso diferencial de exones, seguido de una predominante regulación a la baja de la actividad transcripcional modulada por los cambios epigenéticos del hospedador asociados a la infección y caracterizada por un aumento de la hipermetilación. Las alteraciones en el metabolismo de los ARN pequeños y microARNs del huésped fueron marginales y se produjeron principalmente en la fase tardía. En general, estos datos constituyen el primer mapa exhaustivo de las respuestas de la planta a la infección de un viroide. / [CA] Els viroids són els patògenes més simples amb replicació autònoma i només s'han identificat de forma natural infectant a plantes superiors. Des que es descobriren als anys setanta, s'ha adquirit un coneixement considerable sobre la seua natura i els mecanismes de replicació en plantes hoste. No obstant, encara queden per descobrir molts aspectes de la biologia dels viroids. Per tant, un coneixement més profund de la natura i el mode d'acció dels viroids han sigut els objectius principals que engloben aquesta tesi. Per a això, és essencial la disponibilitat de procediments senzills i eficients per a l'obtenció de clones infecciosos. Es va desenvolupar un nou mètode eficient per a construir clones infecciosos y es fa provar amb un viroid de cada família: el viroide latent de la albergínia (ELVd, Avsunviroidae) y el viroid del llúpol (HSVd, Pospiviroidae). Aquesta aproximació es basà en enzims de restricció de tipus IIS que tallen fora del lloc de reconeixement i suposa un procediment universal per obtenir clones infecciosos de un viroid independentment de la seua seqüencia amb una elevada eficiència. Tot i que els viroids s'han considerat com ARNs no codificants des del seu descobriment, el nostre anàlisi computacional va predir xicotets ORF als genomes de HSVd y ELVd. No es trobaren similituds significatives amb proteïnes depositades a les bases de dades, però alguns d'aquest pèptids estaven altament conservats a les variants de HSVd y ELVd. Curiosament, la fusió d'aquestes seqüencies conservades amb una proteïna fluorescent revelà una localització subcel·lular especifica al orgànul on te lloc la replicació/acumulació de cada viroid: nuclèol i cloroplast per a HSVd i ELVd, respectivament. Les mutacions que trunquen el domini nucleolar de HSVd foren perjudicials per al viroid, mentre que el truncament de qualsevol de les dos ORF de ELVd que contenen una senyal de localització al cloroplast també va disminuir (però en menor mesura) l'eficiència biològica del viroid, el que pot ser degut a una redundància funcional. Es detectaren formes d'ARN circular de HSVd i ELVd a les fraccions polisòmiques, el que revela la seua interacció física amb la maquinaria de traducció cel·lular. En conjunt, aquestes observacions experimentals indiquen que no es pot descartar la capacitat codificants dels viroids, encara que la evidencia definitiva (la detecció del pèptids codificats per ARN circulars) es un repte tecnològic que s'haurà d'adreçar en línies d'investigació futures. Finalment, per tal d'estudiar que canvis es produeixen a l'hoste durant la infecció amb un viroid simptomàtic, es va realitzar un anàlisi integrador de les alteracions genòmiques de les plantes de cogombre infectades amb HSVd. S'integraren els transcriptomes, sARNomes i metilomes per determinar la resposta temporal a la infecció per viroid. Els resultats obtinguts suporten que HSVd promou un redisseny de les vies reguladores de cogombre afectant predominantment a nivells reguladors específics a les diferents etapes de la infecció. La resposta inicial es caracteritzà per una reconfiguració del transcriptoma de l'hoste mitjançant l'ús diferencial d'exons, seguit d'una repressió transcripticional modulada per canvis epigenètics de l'hoste caracteritzats per una major hipermetilació. Les alteracions al metabolisme de ARN xicotets i microARNs de l'hoste van ser marginals i es produïren principalment al final de la infecció. En general, aquestes dades constitueixen el primer mapa exhaustiu de les respostes de la planta a la infecció per un viroid. / [EN] Viroids are the simplest pathogens with autonomous replication and have only been found naturally infecting higher plants. Since viroids were discovered in the seventies, we have gained considerable knowledge about their nature and replication mechanisms in host plants. However, many aspects of viroid biology are yet to be discovered. Therefore, a deeper understanding of the nature and mode of action of viroids have been the encompassing main goals of this thesis. For this purpose, simple and efficient procedures for obtaining infectious cDNA clones are essential. A new efficient method for constructing infectious viroid clones was developed and tested with one viroid of each family: eggplant latent viroid (ELVd, Avsunviroidae) and hop stunt viroid (HSVd, Pospiviroidae). This procedure was based on type IIS restrictions enzymes that cut outside of the recognition site and supposes a universal procedure for obtaining infectious clones of a viroid independently of its sequence, with a high efficiency. Despite viroids have been considered as plant-pathogenic non-coding RNAs since their discovery, our computational analysis predicted small open reading frames in each of the HSVd and ELVd genomes. No significant similarities with proteins in the database of higher plants were found, but some of these predicted peptides were highly conserved among all HSVd and ELVd variants. Interestingly, the fusion of these conserved sequences to a fluorescent protein revealed a specific subcellular localization in the corresponding organelle where replication/accumulation takes place for each viroid: nucleolus and chloroplast for HSVd and ELVd, respectively. Mutations that truncate the nucleolar domain of HSVd were detrimental for the viroid while truncating any of the two ELVd ORF that contains a chloroplast transit signal also diminished (but to a lesser extent) viroid biological efficiency, maybe because of functional redundancy. Circular forms of both, HSVd and ELVd RNAs were found in polysome fractions, revealing their physical interaction with the translational machinery of the plant cell. Altogether, these experimental observations indicate that the coding capacity of viroids cannot be ruled out, although the definitive evidence (detection of the circRNA-encoded peptides) is a technological challenge to be addressed in future research lines. Finally, to study the host changes that are produced during a symptomatic viroid infection, an integrative analysis of the timing and intensity of the genome-wide alterations in cucumber plants infected with HSVd was performed. Differential host transcriptome, sRNAnome and methylome were integrated to determine the temporal response to viroid-infection. Our results support that HSVd promotes the redesign of the cucumber regulatory-pathways predominantly affecting specific regulatory layers at different infection-phases. The initial response was characterized by a reconfiguration of the host-transcriptome by differential exon usage, followed by a predominant down-regulation of the transcriptional activity modulated by the host epigenetic changes associated to infection and characterized by increased hypermethylation. The alterations in host sRNA and microRNA metabolism were marginal and mainly occurred at the late stage. Overall, these data constitute the first comprehensive map of the plant responses to a viroid infection. / La Conselleria d’Educació, Investigació, Cultura i Esports (Generalitat Valenciana) y el Fondo Social Europeo (FSECV 2014-2020) han cofinanciado la contratación del doctorando como personal investigador de carácter predoctoral (ACIF/2017/114) y unas estancias predoctorales fuera de la Comunitat Valenciana (BEFPI/2020). La realización de esta tesis doctoral también se ha realizado en el marco de dos proyectos de investigación del Ministerio de Ciencia, Innovación y Universidades, con cofinanciación de fondos FEDER [BIO2017-88321-R y AGL2016-79825-R] . / Márquez Molins, J. (2022). Novel Insights of Viroid Biology and Host Responses to Their Infection [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/183479 / Premios Extraordinarios de tesis doctorales / Compendio