Apesar dos avanços dos últimos anos, a reposição de cardiomiócitos permanece como um dos maiores desafios da medicina regenerativa. A comprovação da existência de mecanismos endógenos de proliferação cardíaca nos impulsionou a buscar o entendimento dos eventos moleculares envolvidos na proliferação de cardiomiócitos na vida pós-natal. Neste trabalho foi testada a hipótese da influência do metabolismo energético mitocondrial na cardiomiogênese, e seu impacto na regeneração cardíaca. No primeiro momento, foi descrito pela primeira vez o modelo de ressecção apical cardíaca em ratos neonatos. Demonstrou-se que há um período restrito as primeiras 24 horas de vida em que o animal é capaz de regenerar o tecido cardíaco, formando novos cardiomiócitos e permitindo a manutenção da função cardíaca na vida adulta. Esta capacidade é perdida 7 dias após o nascimento, havendo apenas reparo com tecido fibroso e prejuízo à função cardíaca. De maneira interessante, os dados apontaram para hipoperfusão da região apical em ambos os animais ressectados. Isso possivelmente acarretou em dano mitocondrial na vida adulta, sem influenciar a função cardíaca. De maneira a investigar os eventos moleculares da regeneração cardíaca neonatal foi realizado o sequenciamento de RNA dos corações de ratos neonatos de 1 e 7 dias de vida, ressectados e sham, pela técnica de RNASeq, que apontou a relevância da idade nas diferenças de expressão de genes relacionados ao metabolismo, sendo que a intervenção da ressecção pouco influenciou o perfil de expressão gênica. Os resultados mostraram a troca de expressão de isoformas da via glicolítica com a maturação pós-natal, e a hiper-regulação da expressão de genes das vias da ?-oxidação, fosforilação oxidativa e ciclo do ácido tricarboxílico durante o mesmo período. Entretanto, os dados funcionais da atividade metabólica do tecido cardíaco e cultura de cardiomiócitos neonatais mostraram que tanto a glicólise anaeróbia quanto o consumo de oxigênio relacionado à oxidação mitocondrial estiveram elevados no neonato de 1 dia, e foram reduzidos com o desenvolvimento cardíaco. As elevadas taxas de consumo de oxigênio nas culturas de cardiomiócitos de 1 dia de vida foram relacionadas principalmente à produção de ATP. Esses cardiomiócitos foram capazes de proliferar em cultura na presença de soro como estimulador. Assim sendo, as análises de expressão gênica sozinhas pareceram ser indicadores parciais do estado funcional do metabolismo. A inibição não letal da fosforilação oxidativa evidenciou a importância do metabolismo mitocondrial na capacidade proliferativa dos cardiomiócitos na vida pós-natal. Os dados sugerem que o primeiro dia após o nascimento abrange uma alta demanda energética tanto para a diferenciação terminal quanto para a última fase robusta de proliferação de cardiomiócitos na vida pós-natal, e assim evidenciam a importância do metabolismo mitocondrial no processo regenerativo / Despite advances in recent years, the replacement of cardiomyocytes remains one of the biggest challenges in regenerative medicine. The existence of endogenous mechanisms of cardiac proliferation prompted us to seek the understanding of molecular events involved in cardiomyocyte proliferation in postnatal life. In this study, we investigated the influence of mitochondrial energy metabolism in cardiomyogenesis, and its impact on cardiac regeneration. At first, it was described for the first time the model of heart apical resection in neonatal rats, where there is a limited period the first 24 hours of life that animal is able to regenerate cardiac tissue, forming new cardiomyocytes and allowing the maintenance cardiac function in adulthood. This ability is lost seven days after birth, when repair is basically by fibrotic tissue and consequent impairment for heart function. Interestingly, data showed hypoperfusion of the apical region in both resected animals, which possibly resulted in mitochondrial damage in adulthood without affecting heart function. In order to investigate the molecular events of neonatal cardiac regeneration was performed RNA sequencing of hearts from newborn rats with 1 and 7 days of life, resected and sham, which pointed out the importance of age in the different expression of genes related to metabolism, and the intervention of resection had little influence on this. The results showed exchange of expression of enzymes isoforms from glycolytic pathway and hyperregulation of genes from beta-oxidation, oxidative phosphorylation and tricarboxylic acid cycle pathways, during postnatal maturation. However, the functional data of the metabolic activity of cardiac tissue and culture of neonatal cardiomyocytes showed that both anaerobic glycolysis and oxygen consumption related to mitochondrial oxidation were higher in 1-day-old newborns, and were reduced with cardiac development. The high rates of oxygen consumption in 1-day-old cardiomyocytes were related mainly to ATP production. These 1-day-old cardiomyocytes were able to proliferate in culture by serum stimulation. Therefore, the analysis of gene expression alone appeared to be a partial indicator of functional state of metabolism. The non-lethal inhibition of oxidative phosphorylation highlighted the importance of mitochondrial metabolism in the proliferative capacity of cardiomyocytes in postnatal life. Data suggest that the first day after birth covers a high energy demand for both terminal differentiation of cardiac cells and last robust phase of cardiomyocyte proliferation in postnatal life, and show the importance of mitochondrial metabolism in the regenerative process

Malgré les progrès réalisés dans le séquençage de l’ADN, nous n’avons pas encore compris comment le phénotype d’un organisme se rapporte au contenu de son génome. Cependant, il est devenu clair que l'impact des gènes dépend du contexte. La simple présence d'un gène dans un génome ne nous informe pas du moment où il est exprimé et des autres gènes qui y sont exprimés. Comprendre comment l'expression des gènes est régulée est un élément nécessaire pour comprendre comment les phénotypes émergent d'un génotype donné. Les facteurs de transcription, qui peuvent activer ou réprimer l'expression d'un gène, forment un réseau complexe d'interactions entre eux et leurs gènes ciblés. Ce réseau consiste en une hiérarchie de groupes de facteurs de transcription fortement liés, chacun lié à des processus cellulaires distincts. La structure de ce réseau de régulation transcriptionnelle est-elle significative pour la réponse transcriptionnelle d'une cellule? Ici, nous utilisons une protéine de liaison à l'ADN programmable appelée CRISPR (répétitions courtes palindromiques groupées régulièrement) pour perturber l'expression génique des régulateurs globaux au sein du réseau de régulation transcriptionnelle. Ces régulateurs mondiaux régulent de nombreux processus cellulaires distincts et ont de nombreuses cibles génétiques. Le système CRISPR nous permet de perturber ces régulateurs dans toutes les combinaisons possibles, y compris les perturbations d'ordre supérieur avec tous les régulateurs mondiaux potentiellement ciblés perturbés en même temps. Nous enregistrons ensuite à la fois le modèle d'expression du transciptome en utilisant le séquençage de l'ARN et l'adéquation de chaque souche. Nous trouvons que la structure du réseau de régulation augmente la dimensionnalité de la réponse transcriptionnelle plutôt que de la réduire. Cela se traduit par une épistasie importante au-delà des interactions par paires. Cela a des implications sur la façon dont ces réseaux évoluent. L'épistasie par paires que nous trouvons entre les facteurs de transcription globaux repose sur la présence ou l'absence d'autres perturbations. Cela implique que d'autres perturbations pourraient agir comme des mutations de potentialisation. Le nombre de voies d'évolution potentielles augmente avec les épistasies d'ordre élevé, même si cela ne nous dit rien sur la qualité de ces voies. Fait important, les répliques de cette thèse sont toujours en cours et les données présentées ici n’ont pas encore exclu les artefacts expérimentaux. / Despite advances in DNA sequencing, we have yet to understand how an organism’s phenotype relates to the contents of their genome. However it has become clear that the impact of genes are context dependant. The mere presence of a gene within a genome does not inform us of when it is expressed, and which other genes are expressed along with it. Understanding how gene expression is regulated is a necessary piece of understanding how phenotypes emerge from a given genotype. Transcription factors, which can activate or repress the expression of a gene, form a complex network of interactions between themselves and their targeted genes. This network consists of a hierarchy of groups of strongly connected transcription factors, each relating to distinct cellular processes. Is the structure of this transcriptional regulatory network significant to the transcriptional response of a cell? Here we use a programmable DNA binding protein called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) to perturb gene expression of global regulators within the transcriptional regulatory network. These global regulators are regulating many distinct cellular processes and have many genetic targets. The CRISPR system allows us to perturb these regulators in all possible combinations, including higher order perturbations with potentially all targeted global regulators perturbed at the same time. We then record both the expression pattern of the transciptome using RNA sequencing, and the fitness of each strain. We find that the structure of the regulatory network increases the dimensionality of the transcriptional response rather than reducing it. This results in significant high order epistasis beyond pair-wise interactions. This has implications for how these networks evolve. The pair-wise epistasis we find between global transcription factors rely on the presence or absence of other perturbations. This implies that other perturbations could act as potentiating mutations. The number of potential evolutionary paths increases with high order epistasis, although this alone tells us nothing about the quality of those paths. Importantly, the replicates for this thesis are still on-going and the data presented here has not yet excluded experimental artefacts.

[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 / [CAT] 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 no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/125697 / TESIS

Seasonal allergic rhinitis (SAR) is a disease caused by allergens from both environmental and genetic factors. Some researchers have studied the SAR based on traditional genetic methodologies. As technology develops, a new technique called single-cell RNA sequencing (scRNA-seq) is developed, which can generate high-dimension data. We apply two machine learning (ML) algorithms, random forest (RF) and partial least squares discriminant analysis (PLS-DA), for cell source classification and gene selection based on the SAR scRNA-seq time-series data from three allergic patients and four healthy controls denoised by single-cell variational inference (scVI). We additionally propose a new fitting method consisting of bootstrap and cubic smoothing splines to fit the averaged gene expressions per cell from different populations. To sum up, we find that both RF and PLS-DA could provide high classification accuracy, and RF is more preferable, considering its stable performance and strong gene-selection ability. Based on our analysis, there are 10 genes having discriminatory power to classify cells of allergic patients and healthy controls at any timepoints. Although there is no literature founded to show the direct connections between such 10 genes and SAR, the potential associations are indirectly confirmed by some studies. It shows a possibility that we can alarm allergic patients before a disease outbreak based on their genetic information. Meanwhile, our experiment results indicate that ML algorithms may discover something between genes and SAR compared with traditional techniques, which needs to be analyzed in genetics in the future.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第22593号 / 生博第426号 / 新制||生||56(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 荒木 崇, 教授 河内 孝之, 教授 上村 匡 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Vitiligo is an autoimmune disease in which CD8+ T cells selectively destroy melanocytes, leading to a patchy, disfiguring depigmentation of the skin. Our group and others have highlighted the central role of IFN-γ-dependent chemokines in the progression of disease; however, IFN-γ is also reported to have pleiotropic effects on melanocyte biology. We examined whether IFN-γ has a direct role in melanocyte killing. We tested the T-cell effector functions IFN-γ, Fas ligand and perforin by deleting them from autoreactive T cells used to induce vitiligo in mice. We found that disease incidence, disease severity and T cell accumulation in the skin was reduced in mice receiving adoptive transfer of either IFN-γ deficient or Fas ligand deficient gp100-specific T cells; however, perforin was dispensable and led to increased disease scores and T cell accumulation. To determine how melanocytes are affected by IFN-γ signaling during vitiligo, we performed single-cell RNA-sequencing on suction blister biopsies obtained from vitiligo and healthy subjects. We discovered that integrin expression and TGFb2 signaling was decreased only in lesional melanocyte transcriptomes. Moreover, melanocytes appear to participate in their own demise by increasing HLA expression and recruiting effector cells through the chemotactic ligand CCL18. The loss of melanocyte retention factors may explain their clean disappearance from the skin during keratinocyte turnover. Taken together, we believe IFN-γ production by autoreactive T cells in the skin leads to clean loss of melanocytes by downregulation of melanocyte retention factors and by increasing their potential to be detected by effector cells during vitiligo.

Panarthropoda is a monophyletic group comprised of arthropods and lobopods, molting animals with a segmented body, paired appendages, dorsal brain, and ventral nerve cords. Evolutionary Developmental Biology (EvoDevo) is an interdisciplinary field that seeks to understand how changes in development form the basis for variations in morphology and phenotypic evolution, including the genetic network underlying these processes. To study the evolution of panarthropods from such an EvoDevo perspective, one typically uses standard molecular techniques. A first step here is to investigate the expression of a gene of interest in order to find out where and when it is transcribed during development. A hallmark of EvoDevo studies is its comparative character, often with respect to model organisms such as the fruit fly Drosophila melanogaster. Recently developed single-cell RNA sequencing technologies allow the profiling of a plethora of gene expression on the level of individual cells, and thus provide a much more detailed insight into gene expression. In Paper I, I applied standard molecular techniques used in EvoDevo research such as PCR, gene cloning, probe synthesis and whole mount in situ hybridization, to investigate the embryonic expression patterns of the tiptop/teashirt (tio/tsh) and spalt (sal) genes in a range of arthropods representing all main groups of this phylum, and an onychophoran. In the arthropod model Drosophila, these genes act as trunk-specifiers, and the objective of my work was to find out if this is conserved in Arthropoda or even Panarthropoda as a whole. I provide comprehensive data on arthropod tio/tsh and sal expression, including the first data from an onychophoran. The results support the idea that tio/tsh genes are involved in the development of ‘trunk’ segments by regulating limb development. In addition, my data suggest that the function of Sal is unlikely to be conserved in trunk vs head development. Early expression of sal, however, is in line with a potential homeotic function of this gene, at least in Arthropoda. In Paper II, I provide an embryonic tissue dissociation protocol for embryos of the common house spider Parasteatoda tepidariorum that I developed and that I successfully applied for single-cell RNA sequencing. In addition, I report on the progress of this experiment, and provide and discuss preliminary results.

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