No description available.

Les particules fines atmosphériques (PF) sont capables de pénétrer dans les poumons où certains composés transportés peuvent interagir avec les cellules pulmonaires et atteindre la circulation sanguine. L'exposition aux PF affecte particulièrement les populations sensibles telles que les personnes agées. Cette thèse s'inscrit dans une démarche d'identification des effets des PF sur les lymphocytes T humains (LT) tout en visant à déterminer des biomarqueurs liés à l'exposition et à évaluer la variation de la réponse cellulaire en fonction de l'âge. Des LT ont été isolés de prélèvements sanguins de 91 volontaires appartenant à trois classes d'age (20-30, 45-55, 70-85 ans) puis exposés ex vivo pendant 72h à 45 µg/µl de PF collectées à Dunkerque. Les étapes d'isolement, purification et activation des LT ont d'abord été optimisées. Suite à la caractérisation de la population échantillonnée, une population d'étude homogène a été sélectionnée ( 10 sujets / classe d'âge). Nous avons mis en évidence une induction génique d'enzymes impliquées dans l'activation métabolique des HAP identifiés dans l'échantillon de PF. La caractérisation du profil des Lt a permis de proposer un profil mixte Th1/Th2 causé par l'exposition. L'étude transcriptomique des miARN a mis en évidence une surexpression de miR-124-3p impliqué dans la régulation de plusieurs fonctions au niveau du système immunitaire et de miR-1290 impliqué dans plusieurs types de cancer. Quant à l'influence de l'âge, une surexpression des gènes codant pour les enzymes antioxydantes (NQO1 et HMOX1), une augmentation de la concentration des cytokines (IL-4 et IL-13) ainsi qu'une modification du profil d'expression de certains miARN ont été notées chez les sujets les plus âgés. / Atmospheric fine particulate matter (FP) are able to enter the lungs where some compounds can interact with lung cells and reach the bloodstream . Exposure to FP affects in particular susceptible populations such as the elderly. This thesis is part of a project aiming to identify the effects of FP on human T lymphocytes (LT) while attempting to determine biomarkers related to exposure and to evaluate the variation of the cellular response as a function of age. LT were isolated from blood samples of 91 volunteers belonging to three age groups (20-30, 45-55, 70-85 years) then exposed ex vivo for 72h to 45 µg/µl of FP collected in Dunkirk. The steps of isolation, purification and activation of LT were first optimized. Following the characterization of the sampled population, a homogeneous study population was selected (10 subjects/age class). We have demonstrated an induction of the genes coding for the enzymes involved in the metabolic activation of PAH identified in the PF sample. Characterization of the LT profile made it possible to propose a mixed th1/th2 profile cause by the exposure. Teh transcriptomic study of miRNAs revealed an overexpression of miR-124-3p involved in the regulation of several functions in the immune system and miR-1290 involved in several types of cancer. As for the influence of age, overexpression of the genes coding for the antioxidant enzymes (NQO1 and HMOX1), an increase in the concentration of cytokines (IL-4 and IL-13) as well as a modification of the expression profile of some miRNAs were noted on the elderly.

No description available.

Der Systemische Lupus Erythematodes (SLE) ist eine meist schwer verlaufende Autoimmunerkrankung, die jedes Organ betreffen kann. Trotz zahlreicher und intensiver Anstrengungen die Pathophysiologie des SLE aufzuklären, wird sie aktuell nur in ihren Grundzügen verstanden. Eine Vielzahl zellulärer und molekularer Auffälligkeiten wurden in verschiedenen Immunzellen von Patienten mit SLE beschrieben, wobei die gesteigerte Aktivierung von T und B Zellen ist ein Schlüsselmerkmal ist. Verschiedene Auffälligkeiten der T Zell Funktion wurden in den vergangenen Jahren berichtet, unter anderem die gesteigerte Expression und Aktivierung verschiedener Transkriptionsfaktoren, darunter cAMP Responsive Element Modulator (CREM)α und Signal Transducer and Activator of Transcription (Stat)3. Eine Rolle von CREMα bei der Entstehung von Effektor T Zell Phänotypen bei Patienten mit SLE wurde in Studien belegt. Die gesteigerte Expression von CREMα ist (zumindest teilweise) für die gesteigerte Expression von IL-17A und die reduzierte Expression von IL-2 verantwortlich, welche für die Pathogenese und die Entstehung von Gewebeschäden mitverantwortlich sind. Neben gut charakterisierten CD4+ Effektor T Zellen, spielen TCR+CD3+CD4-CD8-, sogenannte „doppelt negative“ (DN) T Zellen, eine Rolle in der Pathophysiologie des SLE. Die Zahl DN T Zellen ist im peripheren Blut von SLE Patienten gesteigert. DN T Zellen infiltrieren entzündete Gewebe, insbesondere die Nieren, wo sie IL-17A exprimieren und zu Gewebeschäden beitragen können. Da DN T Zellen durch den Verlust der Oberflächenexpression des CD8 Co-Rezeptors aus CD8+ T Lymphozyten hervorgehen können, stellte wir die Frage, ob CREMα an diesem Prozess beteiligt ist. In den vorliegenden Studien konnten wir zeigen, dass CREMα an hochkonservierte nichtkodierende Sequenzen des CD8 Gen Clusters bindet und den CD8B Promoter trans-reprimiert. CREMα stellt damit den ersten berichteten Transkriptionsfaktor dar, der zu trans-Repression von CD8 führt. Zudem co-rekrutiert CREMα die DNA Methyltransferase DNMT3a und die Histon Methyltransferase G9a an hochkonservierte nichtkodierende Elemente des CD8 Gen Clusters in CD8+ T Zellen. Hierdurch trägt CREMα zur Chromatinkondensation, folglich reduzierter CD8 Expression und letztendlich der Generierung von DN T Zellen bei. Da die Expression von CREMα sowohl in T Zellen von SLE Patienten als auch in T Zellen von MRL.lrp Mäusen gesteigert ist, könnten die beschriebenen Effekte auf die CD8 Expression eine Rolle für eine Reihe von Autoimmunerkrankungen spielen, die mit einer erhöhten Zahl von DN T Zellen einhergehen (z.B. Patienten mit Autoimmune Lymphoproliferative Syndrome; ALPS). Proteine der Stat Transkriptionsfaktor Familie spielen eine Rolle während der Differenzierung und Aktivierung von Effektor T Zellen. Speziell die Transkriptionsfaktoren Stat3 und Stat5 scheinen für das Gleichgewicht zwischen Th17 Effektor Phänotypen (Stat3) und regulatorischen T Zellen (Stat5) von Bedeutung zu sein. Stat3 spielt eine wichtige Rolle bei der Differenzierung von IL-17A produzierenden CD4+ Th17 Helferzellen, welche eine pathophysiologische Rolle beim SLE spielen. Durch die Aktivierung der Expression weiterer Zytokine (z.B. IL-6, IL-10, und IL-21) in verschiedenen T Lymphozytenpopulationen, sind die Effekte von Stat3 nicht auf die genannten T Helferzellpopulationen beschränkt. Interleukin-10 ist ein immunregulatorisches Zytokin, welches neben seinen anti-inflammatorischen Effekten auch zur Aktivierung von B Lymphozyten und Antikörperproduktion beiträgt. Eine mögliche Pathophysiologische Rolle von IL-10 beim SLE ergibt sich aus gesteigerten IL-10 Serumspiegeln in SLE Patienten und nicht zuletzt aus einer kleinen Kohorte von SLE Patienten, die klinische Besserung nach therapeutischer Blockade von IL-10 erfahren hatte. Wie IL17A, wird auch IL10 durch Transkriptionsfaktoren der Stat Familie kontrolliert. Da die Expression und Aktivierung von Stat3 in T Zellen von SLE Patienten gesteigert ist, untersuchten wir am Beispiel des IL10 Gens Effekte von fehlregulierter Stat Aktivierung. Wir konnten zeigen, dass Stat3 und Stat5 das IL10 Gen durch trans-Aktivierung und die Induktion von epigenetischen Remodeling durch die Co-Rekrutierung von p300 regulieren. Der transkriptionelle Co-Aktivator p300 besitzt Histon Azetyltransferase Aktivität und induziert die „Öffnung“ des IL10 Gens. In T Zellen von SLE Patienten ist die Rekrutierung von Stat3 durch reduzierte DNA Methylierung am proximalen Promoter und einem intronischen Enhancer (I-SRE) erleichtert. Zudem verdrängt Stat3 den Transkriptionsfaktor Stat5 von einem Bindungselement im 4. Intron (I-SRE) des IL10 Gens. Zusammen führen diese Ereignisse zu gesteigerter Expression von IL-10 in T Zellen von SLE Patienten. Da die Aktivierung von Stat3 zu gesteigerter Expression einer Reihe von Zytokinen beträgt und die Stat3 Aktivierung sowohl beim SLE als auch bei anderen Autoimmunerkrankungen gesteigert ist, könnten die beschriebenen Effekte nicht nur auf die Expression von IL-10 in T Zellen von SLE Patienten beschränkt sein. Unsere Beobachtungen unterstreichen das Potenzial fehregulierte Transkriptionsfaktoren, speziell CREMα und Stat3, als Biomarker und/oder therapeutische Ziele beim SLE zu nutzen. Es bleibt jedoch an dieser Stelle noch zu klären, ob CREMα und/oder Stat3 auch Chromatin Remodeling während der physiologischen Generierung von DN oder CD4+ T Helfer Zellen kontrollieren oder ob sie ausschließlich oder zumindest in gesteigertem Maße an der pathologischen Generierung von Effektor T Zellen bei Autoimmunerkrankungen beteiligt sein. Die translationale Bedeutung unserer Beobachtungen wird durch den neuerdings begonnenen Einsatz von JAK/Stat Inhibitoren in der Therapie verschiedener Autoimmunerkrankungen unterstrichen. / Systemic lupus erythematosus (SLE) is a severe autoimmune disease that can affect any organ of the human body. Despite intense efforts towards a better understanding, the pathophysiology of SLE remains largely unknown. A number of cellular and molecular anomalies have been reported in immune cells from patients with SLE, and increased activation of B and T lymphocytes are considered hallmarks of the disease. Several alterations to T cell function and phenotypes have been reported, including the increased expression of the transcription factors cAMP response element modulatorα (CREM α) and signal transducer and activator of transcription 3 (Stat3). A role of CREMα in the generation of effector T cells has been demonstrated. Enhanced expression of CREMα is (at least partially) responsible for increased expression of IL-17A and reduced expression of IL-2 from effector T cells in SLE patients; and altered cytokine expression contributes to the pathophysiology and tissue damage. In addition to well-characterized effector CD4+ T cells, TCR+CD3+CD4-CD8-, so-called “double negative” (DN) T cells, also play a role in the pathophysiology of SLE. Increased numbers of DN T cells in the peripheral blood of SLE patients invade inflamed tissues, including the kidneys, where they produce IL-17A and contribute to tissue damage. Double negative T cells can derive from CD8+ T cells through the down-regulation of CD8 co-receptor expression. Thus, we asked whether CREMα may be involved in this process. In the studies presented here, we demonstrate that CREMα recruits to highly conserved non-coding sequences of the CD8 gene cluster and trans-represses the CD8B promoter. Thus, CREMα is the first reported transcription factor that negatively regulates CD8 expression. Furthermore, CREMα co-recruits DNA methyltransferase (DNMT)3a and histone methyltransferase G9a to highly conserved regions within the CD8 cluster in CD8+ T cells. Through these interactions, CREMα induces chromatin condensation, reduced CD8 expression, and the generation of DN T cells. Since CREMα expression is greater in T cells from SLE patients and lupus-prone MRL.lpr mice, the reported effects may play a role in several autoimmune disorders that are characterized by increased numbers of DN T cells (such as autoimmune lymphoproliferative syndrome; ALPS). Stat family transcription factors play a role during the differentiation and activation of T cells. Particularly Stat3 and Stat5 appear to be of central importance to the balance between effector Th17 phenotypes (Stat3) and regulatory T cells (Stat5). Stat3 is involved in the generation of IL-17A producing CD4+ Th17 cells, which contribute to tissue damage. Through the induction of cytokines other than IL-17A (e.g. IL-6, IL-10, IL-21), effects of Stat3 are not limited to individual T helper cell populations. Interleukin-10 is an immune-regulatory cytokine. In addition to anti-inflammatory effects, IL-10 is involved in the activation of B lymphocytes and induces immunoglobulin production. Increased IL-10 serum levels in SLE patients and a cohort of SLE patients that clinically responded to therapeutic blockade of IL-10 suggest a pathophysiological role for IL-10 in the disease. As with IL17A, the IL10 gene is regulated by Stat transcription factors. Since expression and activation of Stat3 are increased in T cells from patients with SLE, we investigated effects of dysbalanced Stat activation on the IL10 gene. In the presented study, we demonstrate that Stat3 and Stat5 trans-activate IL10 and induce epigenetic remodeling through co-recruitment of p300. The transcriptional co-activator p300 functions as histone acetyltransferase and induced epigenetic “opening” of the IL10 gene. In T cells from SLE patients, recruitment of Stat3 is enhanced by reduced levels of DNA methylation of the proximal promoter and an intronic enhancer, harboring a Stat responsive element (I-SRE). Stat3 replaces the transcription factor Stat5 at I-SRE in a potentially competitive manner. Altogether, these effects result in increased expression of IL-10 in T cells from patients with SLE. Activation of Stat3 induces the expression of a number of cytokines. Since Stat3 activation is enhanced in several autoimmune/inflammatory disorders, including SLE, we concluded that Stat3-mediated effects on gene expression are most likely not limited to just IL-10 expression in SLE. The herewith reported observations suggest high potential for the application of dysregulated transcription factor networks, particularly CREMα and Stat3, as biomarkers and/or molecular targets for future therapeutic interventions in SLE. However, it remains to be investigated whether and to what extent CREMα and/or Stat3 are involved in chromatin remodeling during the physiological generation of DN and CD4+ T helper cell subsets, or whether they contribute exclusively to the generation of effector T cell phenotypes in SLE and other autoimmune/inflammatory disorders. The translational importance of our observations is underscored by the recently initiated application of JAK/Stat inhibitors in the treatment of autoimmune/inflammatory conditions.

Environmental stress has shown to affect both the exposed individuals and the development of their offspring. Generally, it is thought that the stressed organism responds to stress by trying to adapt to it. This thesis investigates possible evolutionary consequences of cross-generational transmissions of stress, where the parent has been stressed but the offspring has not. In two studies we have exposed chicken parents of different breeds to an unpredictable circadian light rhythm, to investigate the influence of genetic background on the transmission of behaviour and patterns of genome-wide gene expression across generations. In Paper I, we can show that the domesticated chicken, by means of epigenetic factors, transmit their behaviours as well as their gene expression profiles to their offspring to a higher extent than their wild ancestor, the red junglefowl. Furthermore, in Paper II, even though the offspring never experienced the stress or had any contact with their stressed parents, they seemed to have adapted to it, which suggests that the parents might have prepared (or pre-adapted) them for living in the unpredictable environment. Additionally, eggs of stressed hens showed increased levels of estradiol that might have affected gene expression of specific immune genes, which were up-regulated in the offspring of stressed parents. It is possible that the traditional distinction between stress responses and evolutionary adaptation may be reevaluated, since our results indicate that they could be parts of the same evolutionary event.

Chemoresistance is a major obstacle in successful chemotherapy. This research explored several genetic and epigenetic factors involved in chemoresistance, angiogenesis and metastasis in the human non-small cell lung cancer cell line A549 and its paclitaxel resistant subclone A549-T24. We characterized various morphological and biochemical differences (with a special focus on the Bcl-2 family of apoptotic regulators) between the two cell lines. Although paclitaxel induced apoptosis in both the cell lines, the subclone was 10 fold more resistant to this drug. Our immunocytochemistry data indicated that VEGF (a potent inducer of angiogenesis) and VEGF receptor-2 mRNA expression levels were higher in A549-T24 cells compared to those in A549 cells. We also observed a higher angiogenic potential in A549-T24 cells as determined by the effect of these cells on endothelial cell growth and cell sprouting using EA.hy926 human umbilical vein endothelial cells and rat aortic ring models, respectively. Our data suggested that tumor cell-induced angiogenesis may involve activation of nitric oxide, calcium and PI3K signaling pathways. Intracellular calcium [Ca 2+ ] i plays a critical role in cellular growth and apoptosis. We characterized alterations in the regulatory pathways of [Ca 2+ ] i handling in our cell lines. While the endoplasmic reticulum calcium store ([Ca 2+ ] er ) was significantly lower, calcium influx pathways were considerably inhibited in A549-T24 cells compared to A549 cells. We investigated the actions of 2-aminoethoxydiphenyl borate to release [Ca 2+ ] er and to block store operated Ca 2+ channels. In addition, we studied the role of Ca 2+ in thapsigargin-induced apoptosis in A549 cells. Integrins, a family of cell adhesion proteins, inhibit apoptosis via activation of survival signals. Integrin (mainly β 3 and α 5 ) gene expression patterns and functions differed between A549 and A549-T24 cell lines, suggesting that adhesion to matrix elements may modulate the response to paclitaxel. Indeed, adhesion to extracellular matrix proteins via integrins resulted in a further decrease in chemosensitivity in A549-T24 cells with simultaneous inactivation of BAD (a pro-apoptotic protein). Cell adhesion mediated drug resistance was successfully reversed using integrin blockers (GRGDS and LM609). In summary, our data suggested that chemoresistance is multifactorial. Understanding the molecular mechanisms of chemoresistance will enable the design of better anticancer agents.

<p dir="ltr">Neurotrauma encompasses a broad category of injuries affecting the central nervous system (CNS), which includes both the traumatic brain injury (TBI) and spinal cord injury (SCI). These injuries can result from various causes, including accidents, falls, sports-related incidents, and other traumatic events, affecting millions of individuals annually. Traumatic injuries are the leading cause of disability, and moreover are associated with elevated risk of developing cognitive impairments and neurodegenerative diseases (ND) such as Alzheimer’s Disease (AD) and Parkinson’s Disease (PD). The elevated ND risk arising from neurotrauma poses significant burdens on healthcare systems and affect life quality of affected individuals, emphasizing the critical need for research aimed at understanding the underlying mechanisms conferring ND risk from the lesion center to CNS. The goal of my thesis is to understand persistent molecular changes post SCI associated with ND using a combination of a rat animal model and neuronal cultures derived from human induced pluripotent stem cells.</p><p dir="ltr">I started with Sprague-Dawley rats with T10 spinal cord contusive injury; and assessed immediate and persistent changes in transcriptomic and epigenetic markers via next generation sequencing (NGS) at primary lesion site and distal spinal cord tissue. Along with global changes in chromatin arrangements and DNA methylation, we observed significant transcriptomic changes enriched for pathways of inflammatory responses, and synaptogenesis. These changes were further verified using immunohistochemistry and super resolution microscopy. To further understand the long-term brain abnormality linked to SCI, we investigated persistent alterations in the composition and molecular profiles of both the male and female motor cortex 30 days after injury. Immunohistochemistry revealed that SCI leads to neuronal loss and changes in synaptic density and morphology; and significant alterations in the neuron-astrocyte ratio and astrocyte morphology, in male motor cortex supporting our hypothesis that SCI may increase the risk of neurodegeneration by affecting the motor cortex. Comparison of transcriptomic data collected at a sub-acute stage in male rats, namely 7 days post injury, with 30 days post injury, identified persistent and de novo changes that occur primarily after recovery of spinal cord injury, which are enriched for neuronal and synaptic function related pathways. Interestingly, neuroendocrine-related pathways were prominently implicated at the chronic stage of SCI, with Esr1 identified as a major upstream regulator offering protective effects in females that did not exhibit significant alterations in cellular composition or morphology after SCI. Collectively, our study paved the way towards understanding sexual dimorphism in brains after spinal cord injury and provides a plausible connection between spinal cord injury and neurodegeneration later in life that were further investigated using a humanized culture model.</p><p dir="ltr">We established the feasibility of using hiPSC derived neurons to examine long term neurotoxic mechanism using lead (Pb) as a model chemical with strong associations with elevated AD risks later in life. A similar culture system was then used to assess persistent neurotoxicity of acrolein, a chemical that is known to emerge in brains post traumatic injury. We found that acrolein induced alterations in neuronal network morphology, synaptic density, and excitability. Furthermore, acrolein exposure negatively impacted mitochondrial function and persistently altered neuronal resilience towards a secondary stressor of mitochondria, namely MPP+. Acrolein exposure also alters the expression of tau and tau phosphorylation which collectively result in increased cellular vulnerability toward paired helical filament (PHF-tau) seeding, a known neurotoxin associated with ND. These findings collectively provide molecular insights as to how acrolein can partake alterations in neural function and resilience to stressors; and relay ND risks in neurotrauma patients later in life.</p><p dir="ltr">In conclusion, our comprehensive investigation employing both rat and hiPSC models uncovers plausible molecular pathways connecting SCI to neurodegenerative diseases, providing insights into the enduring consequences of these injuries on affected patients.</p>

Ein neuartiger Ansatz für die Immunphänotypisierung wird vorgestellt. Die Durchflusszytometrie (FACS) ist die übliche Methode für die Charaketrisierung des Immunsystems. Jedoch ist die Verfügbarkeit von frischem Vollblut, sowie ein schnelle Probenlogistik Vorraussetzung für die Analyse. Als potentialle Alternative werden epigentische qPCR Assays vorgestellt. Für die Quantifizierung von B- und NK-Zellen wurden epigenetische qPCR Systeme etabliert. Anhand eines erweiterten epigenetischen Markerpanels wurde die klinische Anwendung in drei Kohorten getestet: a) 41 Patienten mit primären Immundefizienzen (PID); b) 19 Neugeborene mit und ohne PID und c) 28 Patienten nach einer Stammzelltransplantation (SZT). In Kohorte a) und c) konnte die Äquivalenz der Ergebnisse mit FACS bestätigt werden. Diskrepanzen bei der regulatorischen T-Zell Quantifizierung in einzelnen PID Patienten wurde festgestellt, welche durch Mutationen verursacht wurden, die die Integrität der analysierten Proteine beeinflussen. Zudem konnte die Anwendung der epigentischen Quantifizierung in Trockenblutkarten von Neugeborenen gezeigt werden. Dies würde die Anwendung auch im Neugeborenen-Screening für die Erkennung von PIDs ermöglichen. Für die Anwendung in der SZT konnte gezeigt werden, dass das epigenetische System eine frühe Analyse der Immunrekonstitution ermöglicht, welche eine prädiktive Aussage über das Überleben der Patienten erlaubt. Patienten, welche eine Immunantwort der Lymphozyten gegen eine Virusinfektion bereits am Tag 26 nach Transplantation aufwiesen, hatten eine signifikant höhere Überlebenschance als Patienten ohne Immunantwort. Zusammengefassend zeigen die Daten, dass die epigenetische Systeme für klinische Anwendungen eine zuverlässige Methode darstellt. Die Aussagekraft der Daten ist aufgrund der Studiengröße noch limitiert, und komplizierte klinische Szenarien erschweren die Evaluierung. Deshalb sind weitere Studien erforderlich, um das gezeigte Potenzial zu validieren. / A novel approach for immunophenotyping for clinical applications is presented here. Flow cytometry is currently a common method to characterize the immune system but requiring fresh whole blood and good sample logistics which is not always available. To overcome this limitations, epigenetic qPCR assays are introduced as potential alternative. New epigenetic qPCR systems to quantiy B and NK cells have been established. Using an extended epigenetic marker panel, clinical applications were tested in three patient cohorts: a) 41 patients with different primary immunodeficiencies (PID); b) 19 newborns with and without PID and c) 28 patients after stem cell transplantation (SCT). In cohort a) and c) the equivalence of the epigenetic quantification with flow cytometry was confirmed. However, discrepancies between both methods for regulatory T-cell quantification were found in individual PID patients caused by disease-associated mutations affecting the integrity of the respective protein. Furthermore, the epigenetic quantification using dried blood spots from newborns was demonstrated. This would allow the implementation of epigenetic immunophenotyping in neonatal screening for the detection of congenital immunodeficiencies. For the application in SCT, it was shown that the epigenetic system allows an early analysis of immune reconstitution, which may allow a prediction of the patients' overall survival. Patients who showed an immune response of lymphocytes against viral infections at day 26 after transplantation had a significantly higher survival rate. In summary, the available data show that epigenetic immunophenotyping is a reliable analytical method for various clinical applications. The significance of the data is still limited due to the size of the study and complicated clinical scenarios make the evaluation of individual measurements difficult. Therefore, further extensive investigations are needed to clinically validate the demonstrated potential.

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

[ES] Los síndromes mielodisplásicos (SMD) constituyen un grupo heterogéneo de enfermedades de naturaleza clonal caracterizadas por presentar una hematopoyesis ineficaz, citopenias y riesgo variable de evolución a leucemia mieloide aguda (LMA) secundaria. En la última década, las nuevas tecnologías de secuenciación masiva han revelado que más del 80 % de pacientes con SMD presenta mutaciones somáticas y que éstas pueden agruparse en diversas categorías en función de las rutas biológicas que se vean alteradas. Además, se ha visto que existen patrones de concurrencia y exclusión entre estas categorías de mutaciones. La adquisición secuencial y la concurrencia entre estas mutaciones desencadenan, en parte, el desarrollo de la enfermedad y genera la heterogeneidad clínica característica de los SMD. Las mutaciones en factores de splicing aparecen a menudo simultáneamente con mutaciones en reguladores epigenéticos como es el caso de los genes U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) y Ten-eleven translocation 2 (TET2) que se encuentran co-mutados en un 13 % de los casos. A pesar de su prevalencia, los efectos de la concurrencia en las mutaciones en U2AF1 y TET2 no han sido estudiados. Por ello, en esta tesis nos propusimos estudiar esta cooperación cruzando, en primer lugar, dos líneas mutantes de ratón generadas mediante el sistema de edición genética CRISPR/Cas9. El efecto de estas alteraciones sobre la hematopoyesis de las tres líneas mutantes, U2af1mut/+, Tet2-/- y U2af1mut/+ Tet2-/-, fue examinado mediante el hemograma, citometría de flujo (CF), análisis morfológicos, ensayos de Unidades Formadoras de Colonias (CFU) y estudios funcionales como el trasplante hematopoyético. Para finalizar, se realizó un análisis transcriptómico mediante secuenciación de ARN (ARN-seq) para detectar los posibles cambios en el patrón de splicing entre las líneas mutantes y los controles. La línea mutante U2af1mut/+ no presentó ninguna alteración destacable de la hematopoyesis ni en ratones jóvenes (12-13 semanas) ni envejecidos (2 años). Sin embargo, sus células madre y progenitoras hematopoyéticas (HSPC) fueron incapaces de injertar en la médula ósea de ratones trasplantados. En el caso de los ratones mutantes Tet2-/-, observamos un incremento de células mieloides, esplenomegalia, aumento del compartimento LSK (HSPC con inmunofenotipo Linaje- Sca-1+ c-kit+) y, en los experimentos de trasplante, una capacidad de reconstitución hematopoyética superior a la de los controles. Por último, la cooperación de ambas alteraciones en la línea doble mutante U2af1mut/+ Tet2-/-, no mostró un efecto sinérgico entre ellas. Así pues, se detectaron variaciones en los progenitores mieloeritroides y un aumento significativo de células mieloides y LSK. No obstante, igual que ocurría con la línea U2af1mut/+, las HSPC no producían prendimiento en los ratones trasplantados. A pesar de las alteraciones observadas, ninguna de las tres líneas mutantes desarrollaba SMD ni fallecía antes que los controles. Respecto al análisis transcriptómico, el salto de exón fue el evento de splicing alternativo observado con mayor frecuencia en las líneas U2af1mut/+, Tet2-/- y U2af1mut/+ Tet2-/-. Únicamente un 6.6 % del total de genes que presentaba eventos de splicing alternativo fueron coincidentes en las tres líneas mutantes. A pesar de que en el análisis bioinformático se detectaron alteraciones en las rutas biológicas relacionadas con el ciclo celular, en los ratones U2af1mut/+, y el daño al ADN, en las líneas U2af1mut/+ y U2af1mut/+ Tet2-/-, en la validación mediante CF no se encontraron variaciones respecto a los controles. Para concluir, nuestros datos sugieren que, a pesar de producirse alteraciones en la hematopoyesis, la cooperación entre la mutación en U2af1 y la pérdida de Tet2 es insuficiente para iniciar SMD en ratón. / [CA] Les síndromes mielodisplàstiques (SMD) constituïxen un grup heterogeni de malalties de naturalesa clonal caracteritzades per presentar una hematopoesi ineficaç, citopènies i risc variable d'evolució a leucèmia mieloide aguda (LMA) secundària. En l'última dècada, les noves tecnologies de seqüenciació massiva han revelat que més del 80 % de pacients amb SMD presenta mutacions somàtiques i que aquestes poden agrupar-se en diverses categories en funció de les rutes biològiques que es vegen alterades. A més, s'ha vist que hi ha patrons de concurrència i exclusió entre aquestes categories de mutacions. L'adquisició seqüencial i la concurrència entre aquestes mutacions desencadenen, en part, el desenvolupament de la malaltia i genera l'heterogeneïtat clínica característica de les SMD. Les mutacions en factors de splicing apareixen sovint simultàniament amb mutacions en reguladors epigenètics com és el cas dels gens U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) i Ten-eleven translocation 2 (TET2) que es troben co-mutats en un 13 % dels casos. A pesar de la seua prevalença, els efectes de la concurrència en les mutacions en U2AF1 i TET2 no han sigut estudiats. Per això, en aquesta tesi ens vam proposar estudiar aquesta cooperació creuant, en primer lloc, dos línies mutants de ratolí generades per mitjà del sistema d'edició genètica CRISPR/Cas9. L'efecte d'aquestes alteracions sobre l'hematopoesi de les tres línies mutants, U2af1mut/+, Tet2-/- i U2af1mut/+ Tet2-/-, va ser examinat per mitjà de l'hemograma, citometría de flux (CF), anàlisis morfològiques, assajos d'Unitats Formadores de Colònies (CFU) i estudis funcionals com el trasplantament hematopoètic. Per últim, es va realitzar l'anàlisi transcriptòmic per mitjà de seqüenciació d'ARN (ARN-seq) per a detectar els possibles canvis en el patró de splicing entre les línies mutants i els controls. La línia mutant U2af1mut/+ no va presentar cap alteració destacable de l'hematopoesi ni en ratolins jóvens (12-13 setmanes) ni envellits (2 anys). No obstant això, les seues cèl·lules mare i progenitores hematopoetiques (HSPC) van ser incapaços d'empeltar en la medul·la òssia de ratolins trasplantats. En el cas dels ratolins mutants Tet2-/-, observarem un increment de cèl·lules mieloides, esplenomegàlia, augment del compartiment LSK (cèl·lules mare amb inmunofenotip Llinatge- Sca-1+ c-kit+) i, en els experiments de trasplantament, una capacitat de reconstitució hematopoética superior a la dels controls. Finalment, la cooperació d'ambdues alteracions en la línia doble mutant U2af1mut/+ Tet2-/-, no va mostrar un efecte sinèrgic entre elles. Així, doncs, es van detectar variacions en els progenitors mieloeritroids i un augment significatiu de cèl·lules mieloides i LSK. No obstant això, igual que ocorria amb la línia U2af1mut/+, les HSPC no produïen empelt en els ratolins trasplantats. A pesar de les alteracions observades, cap de les tres línies mutants desenvolupava SMD ni moria abans que els controls. Respecte a l'anàlisi transcriptòmic, el salt d'exó va ser l'esdeveniment de splicing alternatiu observat amb major freqüència en les línies U2af1mut/+, Tet2-/- i U2af1mut/+ Tet2-/-. Únicament un 6.6 % del total de gens que presentava esdeveniments de splicing alternatiu van ser coincidents en les tres línies mutants. Encara que en l'anàlisi bioinformàtica es van detectar alteracions en les rutes biològiques relacionades amb el cicle cel·lular, en els ratolins U2af1mut/+, i el dany a l'ADN, en les línies U2af1mut/+ i U2af1mut/+ Tet2-/-, en la validació per mitjà de CF no es van trobar variacions respecte als controls. Per a concloure, les nostres dades suggerixen que, a pesar de produir-se alteracions en l'hematopoesi, la cooperació entre la mutació en U2af1 i la pèrdua de Tet2 és insuficient per a iniciar SMD en ratolí. / [EN] Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal malignancies characterized by ineffective hematopoiesis, cytopenia and a variable risk of progression to secondary acute myeloid leukemia (AML). In the last decade, next-generation sequencing technologies have deciphered that more than 80 % of MDS patients have somatic mutations and that those can be grouped into several categories depending on which biological routes have been altered. Furthermore, it has been observed that there are concurrency and exclusion patterns among these mutation categories. The sequential acquisition and the concurrency between these driver mutations trigger, in part, the development of the disease and generate the clinical heterogeneity characteristic of MDS. The splicing factor mutations often occur simultaneously with mutations in epigenetic regulators such as the U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) and Ten-eleven translocation 2 (TET2) genes, which are found co-mutated in 13 % of cases. Despite their prevalence, the effects of concurrence in mutations in U2AF1 and TET2 have not been studied. Consequently, in this thesis we aim to study this cooperation. Firstly, we crossed two mutant mouse lines that were previously generated using the CRISPR/Cas9 gene editing system. The effects of these alterations on hematopoiesis in the three mutant lines, U2af1mut/+, Tet2-/- y U2af1mut/+ Tet2-/-, was examinated by the blood counts, flow cytometry (FC), morphological analysis, Colony Forming Units assays (CFU) and functional studies such as the hematopoietic transplantation. Finally, transcriptomic analysis was peformed by RNA sequencing (RNA-seq) to detect possible splicing pattern changes between mutant lines and control samples. U2af1mut/+ mutant line did not present any remarkable alteration of hematopoiesis in either in young (12-13 weeks) or aged (2 years) mice. However, their hematopoietic stem and progenitor cells (HSPC) were unable to engraft into the bone marrow of transplanted mice. In the case of Tet2-/- mutant mice, we observed an increase of myeloid cells, splenomegaly, an increased LSK compartment (HSPC: Lineage- Sca-1+ c-kit+) and an enhanced ability, relative to wild-type, to reconstitute hematopoiesis in transplantation assays. Finally, the cooperation of both alterations in U2af1mut/+ Tet2-/- double mutant line did not show a synergistic effect between them. Nonetheless, the myeloerythroid progenitors were altered and also myeloid and LSK cells were increased. However, as in the U2af1mut/+ line, HSPC did not produce any engraftment in transplanted mice. Despite the observed alterations, none of the three mutant lines developed MDS or die earlier than control mice. Regarding the transcriptomic analysis, exon skipping was the most frequently observed alternative splicing event in the U2af1mut/+, Tet2-/- y U2af1mut/+ Tet2-/- lines. Only 6.6 % of the total number of genes showing alternative splicing events were coincident in the three mutant lines. Although the bioinformatic analysis revealed alterations in biological pathways related to the cell cycle in the U2af1mut/+ mice and DNA damage in the U2af1mut/+ and U2af1mut/+ Tet2-/- lines, the validation by CF found no variations with respect to the controls. In conclusion, our data suggest that, despite alterations in hematopoiesis, the cooperation between U2af1 mutation and Tet2 loss is insufficient to initiate MDS in mice. / Martínez Valiente, C. (2022). Desarrollo y caracterización de un modelo de ratón doble mutante en U2af1 y Tet2 para el estudio de los Síndromes Mielodisplásicos [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/187749