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This thesis explores lower secondary students’ meaning-making of epigenetic visual representations within and between biological organization levels. Data obtained from five focus group discussions where students indicated and reasoned about eight epigenetic visual representations were explored. By analyzing students’ interactions with multiple visual representations, and the impact of linking and reasoning patterns on their meaning-making, the research contributes insights to the learning of epigenetics.  Epigenetics, which is gaining rapid importance in emerging biology curricula, is communicated at different biological organization levels, and serves as the meaning-making context explored in the thesis. A compelling biology didactics context, where students are required to reason with multiple representations depicted within and between organizational levels to make meaning about epigenetics. The thesis uncovers three primary findings. First, four linking patterns in students’ meaning-making across and between organizational levels using various visual representations are illuminated. Second, five visual characteristics that influence students’ linking within and between levels were discerned. Third, students’ meaning-making processes were observed to emerge through four phases, which involved form and function attributes of the visual representations, and the transfer of scientific ideas across representations. / <p>Article 1 published in thesis as manuscript, now published.</p>

Weed resistance to control measures, particularly herbicides, is a growing problem in agriculture. In the case of herbicides, resistance is sometimes connected to genetic changes that directly affect the target site of the herbicide. Other cases are less straightforward where resistance arises without such a clear-cut mechanism. Understanding the genetic and gene regulatory mechanisms that may lead to the rapid evolution of resistance in weedy species is critical to securing our food supply. To study this phenomenon, we exposed young Arabidopsis plants to sublethal levels of one of four weed management stressors, glyphosate herbicide, trifloxysulfuron herbicide, mechanical clipping, and shading. To evaluate responses to these stressors we collected data on gene expression and regulation via epigenetic modification (methylation) and small RNA (sRNA). For all of the treatments except shade, the stress was limited in duration, and the plants were allowed to recover until flowering, to identify changes that persist to reproduction. At flowering, DNA for methylation bisulfite sequencing, RNA, and sRNA were extracted from newly formed rosette leaf tissue. Analyzing the individual datasets revealed many differential responses when compared to the untreated control for gene expression, methylation, and sRNA expression. All three measures showed increases in differential abundance that were unique to each stressor, with very little overlap between stressors. Herbicide treatments tended to exhibit the largest number of significant differential responses, with glyphosate treatment most often associated with the greatest differences and contributing to overlap. To evaluate how large datasets from methylation, gene expression, and sRNA analyses could be connected and mined to link regulatory information with changes in gene expression, the information from each dataset and for each gene was united in a single large matrix and mined with classification algorithms. Although our models were able to differentiate patterns in a set of simulated data, the raw datasets were too noisy for the models to consistently identify differentially expressed genes. However, by focusing on responses at a local level, we identified several genes with differential expression, differential sRNA, and differential methylation. While further studies will be needed to determine whether these epigenetic changes truly influence gene expression at these sites, the changes detected at the treatment level could prime the plants for future incidents of stress, including herbicides. / Doctor of Philosophy / Growing resistance to herbicides, particularly glyphosate, is one of the many problems facing agriculture. The rapid rise of resistance across herbicide classes has caused some to wonder if there is a mechanism of adaptation that does not involve mutations. Epigenetics is the study of changes in the phenotype that cannot be attributed to changes in the genotype. Typically, studies revolve around two features of the chromosomes: cytosine methylation and histone modifications. The former can influence how proteins interact with DNA, and the latter can influence protein access to DNA. Both can affect each other in self-reinforcing loops. They can affect gene expression, and DNA methylation can be directed by small RNA (sRNA), which can also influence gene expression through other pathways. To study these processes and their role in abiotic stress response, we aimed to analyze sRNA, RNA, and DNA from Arabidopsis thaliana plants under stress. The stresses applied were sublethal doses of the herbicides, glyphosate and trifloxysulfuron, as well as mechanical clipping and shade to represent other weed management stressors. The focus of the project was to analyze these responses individually and together to find epigenetic responses to stresses routinely encountered by weeds. We tested RNA for gene expression changes under our stress conditions and identified many, including some pertaining to DNA methylation regulation. The herbicide treatments were associated with upregulated defense genes and downregulated growth genes. Shade treated plants had many downregulated defense and other stress response genes. We also detected differential methylation and sRNA responses when compared to the control plants. Changes to methylation and sRNA only accounted for about 20% of the variation in gene expression. While attempting to link the epigenetic process of methylation to gene expression, we connected all the data sets and developed computer programs to try to make correlations. While these methods worked on a simulated dataset, we did not detect broad patterns of changes to epigenetic pathways that correlated strongly with gene expression in our experiment's data. There are many factors that can influence gene expression that could create noise that would hinder the algorithms' abilities to detect differentially expressed genes. This does not, however, rule out the possibility of epigenetic influence on gene expression in local contexts. Through scoring the traits of individual genes, we found several that interest us for future studies.

L'encéphalomyélite myalgique (EM), communément appelée syndrome de fatigue chronique (SFC), est une maladie chronique complexe, impliquant divers facteurs biochimiques, métaboliques, génétiques et épigénétiques dans sa pathogenèse. L'EM se caractérise par une fatigue persistante et débilitante, un malaise post-effort (PEM), des douleurs, des troubles du sommeil, des troubles cognitifs et d'autres symptômes. L'EM constitue un problème de santé important, compte tenu de sa prévalence dans la population générale. Cependant, notre compréhension des origines de l’EM reste limitée; son diagnostic est difficile et il n’existe aucun traitement curatif définitif ni de biomarqueur officiellement approuvé. Les approches de prise en charge actuelles visent principalement à soulager les symptômes, soulignant le besoin urgent de mieux comprendre ses causes sous-jacentes. Le développement de l’EM est reconnu comme une confluence de prédispositions génétiques et d’expositions environnementales, compte tenu de l’hétérogénéité clinique de cette pathologie. Ces dernières années, de nombreuses voies étiologiques ont été suggérées, notamment un développement post-infectieux de l’EM, qui reste l’une des principales hypothèses. L'implication de mécanismes épigénétiques, principalement la méthylation de l'ADN, un régulateur bien documenté de l'expression des gènes, est également apparu comme un acteur essentiel dans la pathogenèse de l'EM. Il est intéressant de noter, et avec une note d’inquiétude, que près de deux ans après la pandémie de SRAS-CoV-2 à l’origine du COVID-19, une cohorte croissante d’individus auparavant en bonne santé souffre du Long COVID (LC), une manifestation de symptômes persistants provenant de l’infection initiale. Il est remarquable de constater qu’il existe un chevauchement évident entre les présentations cliniques de l’EM et de la LC. Cependant, les mécanismes qui sous-tendent ce chevauchement restent encore à élucider. Dans le cadre de cette thèse, je me suis intéressée dans un premier temps à identifier les changements épigénétiques associés à l'EM à partir de la salive. Le choix d’utiliser la salive représente une approche non-invasive et découle également du fait que des études précédentes ont mis en évidence que les profils de méthylation de l’ADN obtenu des cellules buccales présentes dans la salive étaient assez similaires aux profiles observés au niveau du cerveau et des muscles (des tissus difficilement accessibles mais pourtant atteints dans l’EM) comparativement aux cellules mononuclées du sang périphériques (PBMCs). Cette approche pourrait aider à établir une signature épigénétique susceptible de combler le fossé conceptuel entre la génétique et l'influence environnementale dans la pathogenèse de l'EM. En effet, les sites CpG/gènes identifiés avec une méthylation différentielle pourraient être impliqués dans des mécanismes physiopathologiques associés au développement et/ou à la gravité de l'EM. Après correction de Bonferroni, mes travaux ont révélé un seul site CpG, "cg19803194", différentiellement méthylé chez les patients atteints d'EM par rapport aux sujets sains appariés pour l’âge. Ce site CpG est situé dans le corps du gène PTPRN2 codant pour la tyrosine phosphatase receptor type N2. De plus, le gène PTPRN2 contient un microARN intronique, le miR-153-3p (miR-153-2), qui est corégulé avec son gène hôte. En raison des défis éthiques et logistiques liés à l'accès aux tissus cérébraux ou musculaires, nous avons mesuré les niveaux circulants en miR-153-3p dans le plasma comme mesure de substitution pour évaluer les fluctuations de l'expression de PTPRN2. Nous avons donc analysé ce miARN comme un proxy prometteur pour mieux comprendre la dynamique d’expression de PTPRN2. Notre stratification des patients atteints d'EM basée sur les niveaux de méthylation de l'ADN cg19803194 (hypo vs hyper) a révélé qu'une réduction des niveaux circulants du miR-153-3p a été observée dans le groupe hypo-méthylé, ce qui concorde avec de précédentes études montrant que l'hypométhylation au niveau du corps des gènes, entraine habituellement une diminution de leur transcription. Cependant, l’intrigue a été amplifiée par un schéma contre-intuitif observé chez un sous-ensemble de patients. Cette découverte inattendue, nous a incité à découvrir un mécanisme alternatif régissant la régulation des niveaux de miR-153-3p en circulation, qui implique de manière complexe une interaction dynamique des niveaux nucléaires de PHB2 (Prohibitine 2) dans le contrôle de la maturation de certains miARN dont le miR-153-3p. La LC est un trouble complexe avec des symptômes prolongés et hétérogènes qui s’apparentent avec l’EM. Dans le cadre de ma seconde étude, nous avons utilisé une manoeuvre de provocation standardisée pour induire un malaise post-effort (PEM), ce qui nous a permis de stratifier les patients LC en deux groupes : LC1 (sévère) et LC2 (léger à modéré) en fonction de la gravité du PEM. Les patients LC1 présentaient des scores de gravité de la maladie significativement plus élevés et des scores neurocognitifs plus mauvais que les patients classés dans le groupe LC2. Ceci était étroitement lié aux scores PEM élevés chez les patients LC1 et aux scores PEM réduits chez les patients LC2. Nous avons également utilisé le profilage de la méthylation de l'ADN des cellules mononucléées du sang périphérique (PBMC) pour étudier les changements dans la méthylation de l'ADN associés aux symptômes de la LC (n = 24) par rapport aux participants ayant eu la COVID-19, suivie d’une rémission complète (SC pour Short COVID en anglais) (n = 4). Il est intéressant de noter que nous avons identifié des altérations de plusieurs gènes impliqués dans le dysfonctionnement neurocognitif et le changement de la réponse immunitaire liés à la persistance des symptômes de la LC. En outre, dans le cadre de ma troisième étude, nous avons effectué une analyse complète de la méthylation de l'ADN à l'échelle du génome, à partir d'échantillons de salive provenant du même groupe de patients LC (n=24) LC, de 4 participants SC et de 13 témoins sains (HC) pré-pandémiques. Nous avons étudié les signatures épigénétiques associées à la LC. Nous avons constaté que les patients LC présentaient une hypométhylation significative de trois gènes communs, MEST, DDR1 et LRP1, par rapport aux participants SC et HC. Ces gènes sont impliqués dans la cascade de l’inflammation, l’altération de la réponse immunitaire et des maladies neurologiques. Nous avons également signalé des altérations de la méthylation de LGR6, ZFAND2A et TDG dans les PBMCs et la salive chez les mêmes individus associés à la sévérité de la LC. Plus précisément, nous avons constaté une hypométhylation au niveau des gènes LGR6 et ZFAND2A et une hyperméthylation au niveau du gène TDG dans les cas graves (groupe LC1) par rapport aux cas légers à modérés (groupe LC2). Cette convergence de profils entre la salive et les PBMCs souligne l'importance de LGR6, ZFAND2A et TDG dans la gravité des LC. L’ensemble des résultats obtenus dans le cadre de cette thèse apporte un éclairage nouveau et pertinent sur les mécanismes épigénétiques qui sous-tendent l’EM et la COVID longue. Les nouvelles connaissances présentées dans les trois manuscrits ci-joints devraient nous permettre de mieux comprendre l’EM et la COVID longue, de développer de futures stratégies diagnostiques, thérapeutiques et préventives, mais également de mieux gérer les effets à long terme de ces conditions. En effet, les altération épigénétiques identifiées dans ces deux contextes pathologiques, plus précisément au niveau de la méthylation de l’ADN, pourraient être impliquées dans la sévérité et la persistance des symptômes de ces pathologies complexes, distinctes et présentant tout de même des similitudes. / Myalgic encephalomyelitis (ME), also known as chronic fatigue syndrome (CFS), is a complex chronic disease involving various biochemical, metabolic, genetic, and epigenetic factors. Patients with ME experience persistent and debilitating fatigue, post-exertional malaise (PEM), pain, sleep disturbances, cognitive impairment, and other symptoms. ME is a significant health problem, with an estimated prevalence of 0.2% to 0.5% in the general population. However, our understanding of the origins of ME remains limited; its diagnosis is complex, and there is no definitive cure or officially approved biomarker. Current management approaches focus primarily on relieving symptoms, highlighting the urgent need to understand its underlying causes better. The development of ME is thought to be a confluence of genetic predispositions and environmental exposures. In recent years, many etiological pathways have been suggested, including a post-infectious development of ME, which remains one of the main hypotheses. The involvement of epigenetic mechanisms, primarily DNA methylation, a well-documented regulator of gene expression, has also emerged as a critical player in the pathogenesis of ME. Interestingly, and with a note of concern, nearly two years after the SARS-CoV-2 pandemic that caused COVID-19, a growing cohort of previously healthy individuals suffer from Long COVID (LC), a manifestation of persistent symptoms from the initial infection. Remarkably, there is a clear overlap between the clinical presentations of ME and LC. However, the mechanisms underlying this overlap remain to be elucidated. This thesis investigated the epigenetic mechanisms underlying ME and LC. As part of this thesis, I was initially interested in identifying the epigenetic changes associated with ME from saliva. Saliva is a non-invasive sample that can be easily collected, and previous studies have shown that the DNA methylation profiles obtained from buccal cells present in saliva are similar to those observed in the brain and muscles. This suggests that saliva could be used as a proxy for these tissues, which are difficult to access but are thought to be involved in the pathogenesis of ME. I used Bonferroni's correction to identify a single CpG site, "cg19803194", differentially methylated in ME patients compared to age-matched healthy subjects. This CpG site is located in the body of the PTPRN2 gene, which encodes the tyrosine phosphatase receptor type N2. The PTPRN2 gene also contains an intronic microRNA, miR-153-3p (miR-153-2), which is co-regulated with its host gene. Due to ethical and logistical challenges, I measured circulating levels of miR-153-3p in plasma as a surrogate measure to assess fluctuations in PTPRN2 expression. This miRNA is a promising proxy for understanding the expression dynamics of PTPRN2 because it is located in the same gene and is co-regulated with it. My stratification of ME patients based on cg19803194 DNA methylation levels (hypo vs hyper) revealed that a reduction in circulating levels of miR-153-3p was observed in the hypo-methylated group. These findings agree with previous studies showing that hypomethylation in the body of genes usually decreases their transcription. However, a counterintuitive pattern was observed in a subset of patients. This unexpected finding prompted me to discover an alternative mechanism governing the regulation of circulating miR-153-3p levels. This mechanism involves a dynamic interaction of nuclear PHB2 (Prohibitin 2) levels in controlling the maturation of certain miRNAs, including miR-153-3p. LC is a complex disorder with prolonged and heterogeneous symptoms that resemble myalgic encephalomyelitis (ME). In my second study, we used a standardized provocative maneuver to induce post-exertional sickness (PEM), which allowed us to stratify LC patients into two groups: LC1 (severe) and LC2 (mild to moderate), depending on the severity of the PEM. LC1 patients had significantly higher disease severity scores and worse neurocognitive scores than patients classified in the LC2 group. This was closely related to high PEM scores in LC1 patients and reduced PEM scores in LC2 patients. We also used peripheral blood mononuclear cell (PBMC) DNA methylation profiling to investigate changes in DNA methylation associated with LC symptoms (n=24) compared to participants who had COVID followed by complete remission Short COVID (SC) (n = 4). Interestingly, we identified alterations in several genes implicated in neurocognitive dysfunction and changes in immune response related to the persistence of LC symptoms. In my third study, we also performed a comprehensive genome-wide DNA methylation analysis of saliva samples from the same group of LC patients (n=24), 4 SC participants, and 13 pre-pandemic healthy controls (HC). We studied the epigenetic signatures associated with LC. We found that LC patients showed significant hypomethylation of three common genes, MEST, DDR1, and LRP1, compared to SC and HC participants. These genes are involved in the cascade of inflammation, impaired immune response, and neurological disease. We also reported LGR6, ZFAND2A, and TDG methylation alterations in PBMCs and saliva in the same individuals associated with CL severity. Specifically, we found hypomethylation at the LGR6 and ZFAND2A genes and hypermethylation at the TDG gene in severe cases (LC1 group) compared to mild to moderate cases (LC2 group). This convergence of profiles between saliva and PBMCs highlights the importance of LGR6, ZFAND2A, and TDG in the severity of CL. All the results obtained in this thesis shed new and relevant light on the epigenetic mechanisms underlying ME and LC. The new knowledge in the three attached manuscripts allowed us to understand ME and LC better, develop future diagnostic, therapeutic, and preventive strategies, and better manage these conditions' long-term effects. Epigenetic alterations, more precisely at the level of DNA methylation, have been identified in both ME and long-term COVID. These alterations could be involved in the severity and persistence of the symptoms of these complex pathologies, which are distinct but share some similarities. The findings of this thesis suggest that epigenetics could be a promising target for developing new treatments for ME and long-term COVID. Further research is needed to confirm these findings and to develop effective therapeutic interventions.

Tesis por compendio / [ES] Resumen Como pez de gran valor económico, procedente de una de las líneas de teleósteos más antiguas, con un ciclo de vida misterioso, un potencial de acuicultura excepcional, y con importancia cultural y actividades de pesca en casi todos los países de Europa, la anguila europea posee un enorme valor socioeconómico. Este valor se suma a la desgraciada situación actual en peligro crítico de población natural de anguilas europeas. Como el ciclo de vida de la anguila aún no se ha conseguido cerrar en cautiverio, si la especie se extingue en la naturaleza, no seremos capaces de recuperarla. El cierre del ciclo de vida de la anguila europea ha sido, por lo tanto, el objetivo final de varios estudios. Sin embargo, a pesar de una investigación científica sustancial, desde la década de 1930, varios aspectos de la maduración de la anguila, como el mecanismo que bloquea la maduración de la anguila en la etapa prepúber en cautiverio, aún no se conocen bien. Por lo tanto, es necesario ampliar nuestro conocimiento sobre la reproducción de la anguila para inducir mejores hipótesis y lograr un progreso sustancial. Para profundizar en este campo, esta tesis se realizó con el objetivo específico de desarrollar métodos innovadores para la inducción de la maduración de la anguila y aumentar el conjunto de conocimientos sobre los procesos europeos de maduración de la anguila. Los procedimientos hormonales utilizados actualmente para la maduración sexual de la anguila artificial probablemente no induzcan el proceso natural de maduración. Por lo tanto, esta tesis ha evaluado el potencial de las hormonas recombinantes específicas de la anguila para inducir un proceso de maduración más natural. Este estudio específico mostró que la espermatogénesis completa y la espermiación se pueden inducir con gonadotropinas específicas de anguila recombinante; sin embargo, la calidad del gameto resultante es aún inferior a los resultados de los protocolos establecidos. Sin embargo, la utilización de hormonas recombinantes tiene un gran potencial para futuras implementaciones. Además, el experimento de gonadotropina recombinante ha generado nuevos detalles sobre el efecto de las gonadotropinas homólogas en el eje BPG de las anguilas europeas. Trabajos previos han llevado a la hipótesis de que un tratamiento térmico adecuado puede reducir o reemplazar parcialmente los tratamientos hormonales estándar para la maduración sexual de la anguila europea, o puede mejorar la calidad y / o cantidad de gametos. En esta tesis, se probó el efecto de varios regímenes térmicos en el eje BPG de machos de anguila europeos prepúberes, sin administración de hormonas. Los resultados muestran claramente que un tratamiento de agua de mar fría durante 2 semanas (10 ° C) afecta el eje BPG de los machos de anguila europeas. Los resultados específicos incluyeron un aumento en la sincronización de espermatogonias, niveles elevados de testosterona y 11-ketotestosterona en plasma, agrupamiento de muestras de transcriptomas del eje BPG del grupo tratado con agua de mar fría y posiblemente niveles aumentados de la proteína subunidad ß de la hormona luteinizante de la hipófisis. Los genes transcritos diferencialmente incluyeron varios genes, procesos y vías interesantes, que parecen estar involucrados en la maduración "natural" temprana de la anguila y que pueden ser biomarcadores adecuados para las distintas etapas de este proceso. Para un análisis adecuado de los datos transcriptómicos, se creó un transcriptoma de anguila europea de novo. Se demostró que este transcriptoma de novo posee una superior integridad al genoma de anguila europea disponible y, por lo tanto, es una herramienta útil para el análisis adicional de genes específicos. Un análisis de este transcriptoma reveló un gran número de pares de genes parálogos, que mostraron una baja divergencia entre secuencias sinónimas. Entre las hipótesis potenciales sobre e / [CA] Com a espècie de renom culinari que pertany a un dels llinatges teleostis més antics, amb un cicle vital misteriós, un potencial d'aqüicultura excepcional, i una tradició pesquera a gairebé tots els països d'Europa, l'anguila europea posseeix un enorme valor socioeconòmic. No obstant això, aquest valor només fa que augmentar la preocupació de la seva població, que actualment es troba catalogada com "en perill crític d'extinció". Atès que el cicle de vida de les anguiles encara no ha estat tancat en captivitat, l'espècie no serà salvable en el cas que s'extingeixi en estat natural, per la qual cosa tancar el cicle de vida d'aquesta espècie ha estat l'objectiu final de diversos grups d'investigació durant els últims anys.. No obstant això, i malgrat la investigació científica de qualitat duta a terme des de la dècada de 1930, encara hi han diversos aspectes de la maduració de les anguiles -com el mecanisme que bloqueja la maduració sexual de l'anguila a l'etapa pre-puberal en captivitat- que son poc coneguts en l'actualitat. Per tal d'ampliar els coneixements sobre la reproducció de les anguiles i aconseguir un progrés substancial, aquesta tesi es va dur a terme amb l'objectiu específic de desenvolupar mètodes innovadors per a la inducció de la maduració de l'anguila europea, a més de afegir-hi el coneixement en els processos de maduració bàsics d'aquesta espècie. Els procediments hormonals utilitzats actualment per a la maduració artificial de l'anguila europea no acaben d'induir el procés de maduració natural tal i com probablement es dóna a la natura. Doncs, en primer lloc, aquesta tesi va avaluar el potencial d'hormones recombinants específiques d'anguila europea per induir un procés de maduració molt més natural. Aquest estudi específic va mostrar que mitjançant estes gonadotropines específiques d'anguila europea és possible induir l'espermatogènesi i l'espermiació completes. Tot i que els resultats van mostrar que la qualitat dels gamets va ser inferior als resultats que generen els protocols establerts fins ara amb un altre tipus d'hormones (generalment d'origen humà), la utilització d'hormones recombinants específiques es presenta amb un gran potencial per a la seva implementació futura en la inducció de la maduració sexual de l'anguila europea, ja que l'estudi va generar noves idees sobre l'efecte de les gonadotropines l'eix BPG de l'anguila europea. En segon lloc, i treballant amb la hipòtesi que un tractament tèrmic adequat pot reduir o substituir parcialment els tractaments hormonals estàndards per a la maduració sexual de l'anguila europea, en aquesta tesi es va provar l'efecte de diversos règims tèrmics (sense administració d'hormones) en l'eix BPG dels mascles europeus pre-puberals amb l'objectiu de millorar la qualitat i / o quantitat dels gamets. Els resultats mostraren clarament que un tractament d'aigua de mar de 2 setmanes a baixa temperatura (10 °C) va afectar l'eix BPG dels mascles europeus d'anguila. Resultats més específics van mostrar un augment de la sincronització de les espermatogonies, elevats nivells plasmàtics de testosterona i 11-ketotestosterona, una agrupació de mostres de transcriptoma de l'eix BPG del grup tractat amb aigua de mar freda i, possiblement, un augment dels nivells de la proteïna de la subunitat ß de la hormona luteinitzant de la hipofisi. Els gens transcrits diferencials van al·ludir a diversos gens, processos i vies interessants, que semblen estar implicats en la maduració inicial de l'anguila "natural" i podrien resultar biomarcadors adequats per a les etapes d'aquest procés. No obstant això, es necessiten estudis addicionals per avaluar el potencial dels biomarcadors d'aquests gens i, de manera complementària, comprovar si un pre-tractament d'aigua de mar freda pot millorar la resposta de les anguiles europees a un tractament hormonal artificial, com suggereixen els resultats. Finalment, amb l'objectiu / [EN] As an expensive fish from one of the most ancient teleost lineages, with a mysterious life cycle, exceptional aquaculture potential, and cultural associations and fishing activity in almost every country in Europe, the European eel possess huge socioeconomic value. This value only adds to the misfortune of the current critically endangered state of the wild European eel population. As the eel lifecycle has not yet been closed in captivity, the species will not be salvable if it went extinct in the wild. Closing the life-cycle of the European eel has thus been the ultimate objective of several studies. However, despite the substantial scientific investigation, since the 1930s, several aspects of eel maturation, such as the mechanism which blocks eel sexual maturation at the pre-pubertal stage in captivity, is still poorly understood. Therefore, it is necessary to broaden our knowledge of eel reproduction to induce better hypotheses and therethrough achieve substantial progress. In order to further this field, this thesis was conducted with the specific objective of developing innovative methods for induction of eel maturation and add to the pool of knowledge of European eel maturation processes. The hormonal procedures currently used for artificial eel sexual maturation are probably not inducing the natural maturation process. Therefore, this thesis has evaluated the potential of eel specific recombinant hormones to induce a more natural maturation process. This specific study showed that full spermatogenesis and spermiation can be induced with recombinant eel specific gonadotropins; however, the resulting gamete quality is still inferior to the results of established protocols. Nevertheless, the utilization of recombinant hormones holds a large potential for future implementation. Furthermore, the recombinant gonadotropin experiment has generated novel insights into the effect of homologous gonadotropins on the BPG axis of European eels. Previous work has led to the hypothesis that the right thermal environmental treatment may reduce or partially replace the standard hormonal treatments for sexual maturation of European eel, or may improve gamete quality and/or quantity. In this thesis, the effect of various thermal regimes was tested on the BPG axis of pre-pubertal European eel males, without administration of hormones. The results clearly show that a 2 week cold (10 °C) seawater treatment effects the BPG-axis of European eel males. Specific results included an increase in the synchronization of spermatogonial cells, elevated testosterone and 11-ketotestosterone plasma levels, clustering of BPG-axis transcriptome samples from the cold seawater treated group and possibly increased levels of pituitary luteinizing hormone ß-subunit protein. Differentially transcribed genes alluded to several interesting genes, processes, and pathways, which appears to be involved in early "natural" eel maturation and may prove to be suitable biomarkers for the stages of this process. In order for proper analysis of the transcriptomic data, a de novo European eel transcriptome was assembled. This de novo transcriptome was proven to have superior completeness to the available European eel genome and is thus a useful tool for further analysis of specific genes. An analysis of this transcriptome revealed a large number of paralog gene pairs, which showed low synonymous sequence divergence. Among the potential hypothesis regarding the origin of these paralog gene pairs, the hypothesis of a 4R whole genome duplication is among the most parsimonious. Several of these duplicated genes were involved in reproduction and the onset of puberty. Regardless of the origin, further analysis of these genes may reveal eel specific adaptations, which could help to better understand the exceptional reproductive system of eels. / Rozenfeld, C. (2019). Development of innovative methods for induction of European eel (Anguilla anguilla) spermatogenesis [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/125697 / Compendio

No / Human aging involves a gradual decrease in cellular integrity that contributes to multiple complex disorders such as neurodegenerative disorders, cancer, diabetes, and cardiovascular diseases. Genome-wide association studies (GWAS) play a key role in discovering genetic variations that may contribute towards disease vulnerability. However, mostly disease-associated SNPs lie within non-coding part of the genome; majority of the variants are also present in linkage disequilibrium (LD) with the genome-wide significant SNPs (GWAS lead SNPs). Overall 600 SNPs were analyzed, out of which 291 returned RegulomeDB scores of 1-6. It was observed that just 4 out of those 291 SNPs show strong evidence of regulatory effects (RegulomeDB score < 3), while none of them includes any GWAS lead SNP. Nevertheless, this study demonstrates that by combining ENCODE project data along with GWAS reported information will provide important insights on the impact of a genetic variant-moving from GWAS towards understanding disease pathways. It is noteworthy that both genome-wide significant SNPs as well as the SNPs in LD must be considered for future studies; this may prove to be crucial in deciphering the potential regulatory elements involved in complex disorders and aging in particular.

Neueste Arbeiten zeigen, dass die Selbsterneuerung und Pluripotenz von Stammzellen durch hochkonservierte Signalwege kontrolliert werden. Störungen dieser Prozesse verursachen Tumoren, die von Zellen mit stammzellähnlichen Eigenschaften ausgehen, den sog. Krebsstammzellen. Diese Arbeit zeigt, dass erhöhte Wnt/beta-Catenin- und erniedrigte Bmp-Signalgebung eine wichtige Rolle in der Entstehung humaner Plattenepithel-Karzinome der Speicheldrüsen spielen. Es wurde ein Mausmodell hergestellt, bei dem die Aktivierung der Wnt/beta-Catenin und der Verlust der Bmp-Signalgebung ebenfalls Speicheldrüsenkarzinome verursachen. Die Speicheldrüsen-Tumoren der Maus enthielten eine erhöhte Zahl von CD24+CD29+-Stammzellen, von denen bereits 500 Zellen transplantierbare Tumoren in NOD/SCID-Mäusen induzierten, d. h. dass sie Krebsstammzellen darstellen. Die Veränderung nur eines Signalweges, Wnt/beta-Catenin oder Bmp allein, war nicht tumorigen, resultierte aber in einer erhöhten Anzahl von CD24+CD29+-Stammzellen in der Speicheldrüse und einer fünffach schnelleren Geweberegeneration nach Verwundung. Die Krebsstammzellen der Speicheldrüsen zeigten eine erhöhte Expression von Pluripotenzgenen und globale Veränderungen von trimethyliertem Lysin 4 und 27 des Histons 3. Diese Veränderungen korrelieren häufig mit einer Zunahme aktiven und einer Abnahme repressiven Chromatins. Die Krebsstammzellen wuchsen in vitro als undifferenzierte Salisphären und konnten durch Inhibierung des Wnt/beta-Catenin-Signalweges in drüsenartige Strukturen differenziert werden. Dieser Prozess war von repressivem Chromatin abhängig, da DNA-Methylierungs- oder Histon-Deazetylase-Inhibitoren den ursprünglichen Krebsstammzell-Status wieder reaktivieren konnten. Diese Arbeit zeigt, dass ein aktiver Wnt/beta-Catenin-Signalweg die Transformation von normalen Stammzellen zu Krebsstammzellen durch einen epigenetischen Mechanismus fördert. Die Ergebnisse eröffnen neue Strategien für die Tumortherapie beim Menschen. / Little is known about the processes by which cancer stem cells arise in the different tissues. Our analysis of aggressive squamous cell carcinomas (SCCs) of the salivary gland in human patients suggested a link to the Wnt/beta-catenin and Bmp signaling systems. Using a genetically modified mouse strain in which Wnt signaling is up-regulated and Bmp is suppressed, we found that Wnt/beta-catenin promotes the transformation of normal stem cells into cancer stem cells through an epigenetic mechanism. Mouse SCCs of the salivary gland contained high numbers of CD24+CD29+ cancer stem cells. As few as 500 of these cells sufficed to cause tumors when transplanted into NOD/SCID mice. Mice in which only one of the signaling systems was altered had higher numbers of stem cells in the salivary gland, more efficient tissue regeneration, and no apparent tumors. We discovered that the difference of normal compared to cancer stem cells in the salivary gland is an up-regulation of specific pluripotency genes, e.g. Dppa5, as well as global changes in trimethylated Lysine 4 and 27 of histone 3. This indicates an increase of active chromatin and a decrease in the repressive form, which suggests a mechanistic explanation for the change of cell fate. Cancer stem cells of the salivary gland grew as non-adherent spheres and retained the capacity for differentiation if beta-catenin is inhibited. This depended on repressive chromatin, as shown by the fact that 5-azacytidine or HDAC inhibitors restored stemness. Our data opens new strategies for future cancer therapies in humans.

Next-generation sequencing (NGS) ermöglicht das molekulare Profiling von Zellen mit beispiellos hohem Durchsatz. Allerdings ist der Fokus oftmals auf proteinkodierende Proteine beschränkt, wodurch die vollständige Diversität des Transkriptoms übersehen wird. Nicht-kodierende RNA-Moleküle variieren stark in ihrer Biogenese, Struktur und Funktion, wodurch ihre unverzerrte Inklusion in die Analyse erschwert wird. Diese Promotion fokussiert sich auf das Verständnis nicht-kodierender RNA und navigiert durch drei aufeinander aufbauende Säulen in der Analyse, um Beobachtungen in Wissen zu verwandeln: Generierung von Daten, Quantifizierung und Interpretation. Diese drei Säulen werden in den drei Kapiteln der Dissertation aus der bioinformatischen Perspektive adressiert, indem Schlüsselherausforderungen beschrieben und neue Lösungen vorgestellt werden, um die Analyse des gesamten Transkriptoms mit NGS-Techniken zu verbessern. Zunächst wird ein vollautomatischer Algorithmus vorgestellt, welcher die verschiedenen Quellen von aus der Vorberei- tung von Bibliotheken resultierenden Artefakten mittels unüberwachtes Lernen erkennt, was anschließend zur Optimierung der Protokolle zur Vorbereitung von total-RNA-seq-Bibliotheken genutzt werden kann. Zudem werden die primären Herausforderungen der Quantifizierung von total-RNA-seq behandelt: die Prozessierung von Reads, die mehreren, möglicherweise überlappenden Loci zugeordnet werden können, wie auch die Tatsache, dass manche Loci mehrfach im Genom vorkommen und ein Read zu all diesen Loci passen kann. Diese beiden Fälle können auch gleichzeitig vorkommen, was die Analyse von nicht-kodierender RNA mit üblichen Methoden erschwert. Um diese Problematik anzugehen, wird eine neue Software namens Multi-Graph count (MGcount) vorgestellt. Diese ordnet hierarchisch Reads Transkripten zu, um unter anderem eine Diskrepanz zwischen der Loci-Länge von small und long RNA zu berücksichtigen. Wenn Reads konsistent mehrfach alignieren, fasst MGcount Loci in Communitys zusammen. Es wird gezeigt, dass die Beurteilung der Expression auf der Community-Ebene eine genauere Quantifizierung von biologisch bedeutsamen RNA-Einheiten (Einfachtranskript oder Locusfamilien) ermöglicht. Schließlich wird MGcount angewandt, um nicht-kodierende RNA während der Differenzierung von induzierten pluripotenten Stammzellen in die Keimblätter Mesoderm, Endoderm und Ektoderm zu analysieren. In dieser Dissertation wird eine Multi-Omics-Analyse erfolgreich angewandt, um sowohl die Expressionsverläufe von verschiedenen RNA-Biotypen während der Determination zu charakterisieren als auch einen Zusammenhang bezüglich Chromatin-Remodellierung (“chromatin remodeling“) und DNA-Methylierung an den jeweiligen Loci herzustellen. Schlussendlich dient diese Dissertation als Ratgeber für alle Forschenden, die neue Einsichten in das nicht-kodierende Transkriptom gewinnen wollen. / Next-generation sequencing (NGS) techniques enable the molecular profiling of cells with unprecedented high throughput. Yet, in transcriptome analysis, the focus is often restricted to protein-coding RNA, overlooking the transcriptome in its entire diversity. Non-coding RNA molecules largely vary in biogenesis, structure and function and this challenges their unbiased inclusion into the analyses. This doctoral research places non-coding RNA understanding at the focus spot and navigates through the three workflow pillars that must align effectively to turn observations into knowledge: data generation, quantification, and interpretation. Throughout three chapters, this Thesis addresses these pillars from a Bioinformatics perspective, by outlining key challenges and introducing novel solutions to improve whole-transcriptome analysis through NGS techniques. First, we introduce a fully automatic algorithm that identifies sources of library preparation artifacts in an unsupervised manner and we demonstrate its utility within the development and optimization of total-RNA-seq library preparation protocols. Secondly, we address a major challenge in total-RNA-seq quantification; processing reads that align to multiple loci that overlap within the same genomic region or/and multiple loci that are present in high copy numbers. Such ambiguous alignments commonly arise due to the inherent characteristics of non-coding RNA. To tackle this, we introduce a novel software, named Multi-Graph count (MGcount), that hierarchically assigns reads to transcripts to account for loci length disparity between small-RNA and long-RNA and subsequently collapses loci where reads consistently multi-map into communities defined in a data-driven fashion. We show that these cohesive communities allow the quantification of biologically meaningful RNA entities (single-transcripts or locus-families) and estimate their abundance more accurately. Finally, we apply the developed method to investigate non-coding RNA in early development, specifically during the differentiation of Induced Pluripotent Stem Cells into the three germ-layer lineages, namely, mesoderm, endoderm, and ectoderm. In this study, we leverage a multi-omics analysis to characterize the expression trajectories of diverse RNA biotypes along cell-commitment and the interplay with chromatin remodeling and DNA methylation patterns at the locus surroundings. Ultimately, this work is intended to serve as a guide for all those who want to gain new insights from the non-coding transcriptome.

Bei Säugerkeimzellen, Zygoten und Embryos in frühen Stadien kommt der epigenetischen Neuprogammierung eine außergewöhnlich wichtige Rolle in der Regulation der Genomfunktionen in entscheidenden Entwicklungsstadien zu. Die epigenetische Neuprogrammierung in Keimzellen löscht zuerst die Imprinting-Markierungen und Epi-Mutationen und stellt dann geschlechtsspezifische Markierungen (genomische Prägung) wieder her. Die vorliegende Arbeit bezieht sich auf das Löschen epigenetischer Modifikationen in primordialen Mauskeimzellen (primordial germ cells (PGCs)) zwischen dem 10.5 bis 13.5 Tag nach der Befruchtung. Entgegen früheren Annahmen zeigen unsere Ergebnisse, daß primordiale Mauskeimzellen (PGCs) beim Eintritt in die embryonalen Keimdrüsen noch immer DNS Methylierungsmarker besitzen, die ähnlich dem Marker in somatischen Zellen sind. Kurz nach dem Eintritt in die Keimdrüsen werden die DNS Methylierungsmarker, die in Verbindung mit geprägten und nicht geprägten Genen stehen, gelöscht. Für die Mehrzahl der Gene beginnt die Löschung der Marker in männlichen und weiblichen Embryos gleichzeitig und ist innerhalb eines Entwicklungstages abgeschlossen. Diese Kinetik deutet auf einen aktiven Demethylierungsprozess hin, initiiert durch ein somatisches Signal, ausgehend von der embryonalen Keimdrüse. Der Zeitpunkt der Neuprogrammierung in den primordialen Keimzellen ist entscheidend, da er sicherstellt, daß Keimzellen beiden Geschlechts einen epigenetisch äquivalenten Status erhalten, bevor sie geschlechtsspezifisch ausdifferenzieren und anschließend neu elterlich geprägt werden. Vollständiges Verständnis des Prozesses der Neuprogrammierung der Keimzellen ist nicht nur im Hinblick auf genomisches Imprinting wichtig, sondern auch für die Erforschung von Mechanismen für die Wiederherstellung von omnipotenten Zellen bei Klonierung und Stammzellenerhaltung. / Epigenetic reprogramming in mammalian germ cells, zygote and early embryos, plays a crucial role in regulating genome functions at critical stages of development. Germ line epigenetic reprogramming assures erasure of all the imprinting marks and epi-mutations and establishment of new sex-specific gametic imprints. The presented work focuses on the erasure of epigenetic modifications that occur in mouse primordial germ cells (PGCs) between day 10.5 to 13.5 post coitum (dpc). Contrary to previous assumptions, our results show that as they enter the genital ridge the PGCs still possess DNA methylation marks comparable to those found in somatic cells. Shortly after the entry of PGCs into the gonadal anlagen the DNA methylation marks associated with imprinted and non-imprinted genes are erased. For most genes the erasure commences simultaneously in PGCs of both male and female embryos and is completed within only one day of development. The kinetics of this process indicates that is an active demethylation process initiated by a somatic signal emanating from the stroma of the genital ridge. The timing of reprogramming in PGCs is crucial since it ensures that germ cells of both sexes acquire an equivalent epigenetic state prior to the differentiation of the definitive male and female germ cells in which, new parental imprints are established subsequently. Complete understanding of the germline reprogramming processes is important not only in the light of genomic imprinting but also for resolving other mechanisms connected with restoring cellular totipotency, such as cloning and stem cell derivation.