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Das Schicksal einer differenzierten Zelle wird durch epigenetische Grenzen bestimmt und mittels Schutzmechanismen bewahrt, wodurch die Reprogrammierung in andere Zelltypen verhindert wird. In dieser Studie haben wir ein Chromatin-regulierendes Protein, das konservierte MORF4-Verwandte-Gen (MRG) Protein MRG-1, als Barriere für die Reprogrammierung von Zellen in Caenorhabditis elegans (C. elegans) identifiziert. RNAi gegen MRG-1 ermöglicht es uns Keimzellen mittels Überexpression des Neuronen-induzierenden Transkriptionsfaktors CHE-1 in neuronenartige Zellen umzuwandeln. Mittels ChIP-seq fanden wir heraus, dass MRG-1 unterschiedliche DNA Bindungsstellen in den Keimbahnen und somatischen Geweben von C. elegans aufweist. Wir konnten zeigen, dass MRG-1 besonders stark am Genkörper angereichert ist und sich hauptsächlich auf Genen befindet, welche die aktive Histonmarkierung H3K36me3 tragen. Die Charakterisierung der Protein-Protein-Interaktionspartner von MRG-1 mittels Co-IP/MS ergab, dass MRG-1 mit der Histon-H3K9-Methyltransferase SET-26 und der b-gebundenen N-Acetylglucosamin Transferase OGT-1 zusammenarbeitet, um die Umwandlung von Keimzellen in Neuronen zu verhindern. Basierend auf RNA-Seq Experimenten in mrg-1-Mutanten und Wildtyp konnten wir weitreichende Veränderungen der Genexpression mit Auswirkung auf Signalwege wie den Notch Signalweg enthüllen, welcher bekanntermaßen die Zelltyp-Reprogrammierung fördern. Mittels Long-Read basiertem RNA-seq in mrg-1-Mutanten und der Integration entsprechender ChIP-seq Daten habe ich die Beteiligung von MRG-1 am prä-mRNA-Spleißen in C. elegans gezeigt, analog zum Säugetierortholog MRG15. Diese Ergebnisse weisen darauf hin, dass MRG-1 durch die Regulierung des Chromatins und die Sicherstellung des korrekten Spleißens die Expressionsniveaus kritischer Gene und Signalwege aufrechterhält, um eine ordnungsgemäße Keimbahnentwicklung zu gewährleisten und das Schicksal der Keimzellen zu schützen. / Das Schicksal einer differenzierten Zelle wird durch epigenetische Grenzen bestimmt und mittels Schutzmechanismen bewahrt, wodurch die Reprogrammierung in andere Zelltypen verhindert wird. In dieser Studie haben wir ein Chromatin-regulierendes Protein, das konservierte MORF4-Verwandte-Gen (MRG) Protein MRG-1, als Barriere für die Reprogrammierung von Zellen in Caenorhabditis elegans (C. elegans) identifiziert. RNAi gegen MRG-1 ermöglicht es uns Keimzellen mittels Überexpression des Neuronen-induzierenden Transkriptionsfaktors CHE-1 in neuronenartige Zellen umzuwandeln. Mittels ChIP-seq fanden wir heraus, dass MRG-1 unterschiedliche DNA Bindungsstellen in den Keimbahnen und somatischen Geweben von C. elegans aufweist. Wir konnten zeigen, dass MRG-1 besonders stark am Genkörper angereichert ist und sich hauptsächlich auf Genen befindet, welche die aktive Histonmarkierung H3K36me3 tragen. Die Charakterisierung der Protein-Protein-Interaktionspartner von MRG-1 mittels Co-IP/MS ergab, dass MRG-1 mit der Histon-H3K9-Methyltransferase SET-26 und der b-gebundenen N-Acetylglucosamin Transferase OGT-1 zusammenarbeitet, um die Umwandlung von Keimzellen in Neuronen zu verhindern. Basierend auf RNA-Seq Experimenten in mrg-1-Mutanten und Wildtyp konnten wir weitreichende Veränderungen der Genexpression mit Auswirkung auf Signalwege wie den Notch Signalweg enthüllen, welcher bekanntermaßen die Zelltyp-Reprogrammierung fördern. Mittels Long-Read basiertem RNA-seq in mrg-1-Mutanten und der Integration entsprechender ChIP-seq Daten habe ich die Beteiligung von MRG-1 am prä-mRNA-Spleißen in C. elegans gezeigt, analog zum Säugetierortholog MRG15. Diese Ergebnisse weisen darauf hin, dass MRG-1 durch die Regulierung des Chromatins und die Sicherstellung des korrekten Spleißens die Expressionsniveaus kritischer Gene und Signalwege aufrechterhält, um eine ordnungsgemäße Keimbahnentwicklung zu gewährleisten und das Schicksal der Keimzellen zu schützen.

Die Reprogrammierung somatischer Zellen hat sich kürlich als leistungsfähige Technik für die Herstellung von induzierten pluripotenten Stammzellen (iPS Zellen) aus terminal differenzierten Zellen bewährt. Trotz der großen Hoffnung, die sie speziell im Bezug auf patientenspezifische Stammzelltherapie darstellt, gibt es viele Hindernisse auf dem Weg zur Anwendung in der Humanmedizin, die sich von niedrigen Effizienzen bei der technischen Umsetzung bis hin zur unerwünschten Integration von Onkogenen in das menschliche Genom erstrecken. Aus diesem Grund ist es unabdingbar, unser Verständnis der zugrundeliegenden Prozesse und Mechanismen zu vertiefen. Durch neue Datengewinnungsmethoden und stetig wachsende biologische Komplexität hat sich der Denkansatz der Systembiologie in den letzten Jahrzehnten stark etabliert und erfährt eine fortwährende Entwicklung seiner Anwendbarkeit auf komplexe biologische und biochemische Zusammenhänge. Verschiedene mathematische Modellierungsmethoden werden auf den Reprogrammierungsprozess angewendet um Engpässe und mögliche Effizienz-Optimierungen zu erforschen. Es werden topologische Merkmale eines Pluripotenznetzwerkes untersucht, um Unterschiede zu zufällig generierten Netzen und so topologische Einschränkungen des biologisch relevanten Netzwerkes zu finden. Die Optimierung eines Booleschen Modells aus einem selbst kuratierten Netzwerk in Bezug auf Genexpressionsdaten aus Reprogrammierungsexperimenten gewährt tiefgreifende Einblicke in die ersten Schritte und wichtigsten Faktoren des Prozesses. Der Transkriptionsfaktor SP1 spielt hierbei eine wichtige Rolle zur Induktion eines intermediären, transkriptionell inaktiven Zustands. Ein probabilistisches Boole''sches Modell verdeutlicht das Zusammenspiel epigenetischer und transkriptioneller Kontrollprozesse zusammen, um Pluripotenz- und Zelllinien-Entscheidungen in Reprogrammierung und Differenzierung zu treffen. Erklärungen für die geringe Effizienz werden versucht. / Somatic Cell Reprogramming has emerged as a powerful technique for the generation of induced pluripotent stem cells (iPSCs) from terminally differentiated cells in recent years. Although holding great promises for future clinical development, especially in patient specific stem cell therapy, the barriers on the way to a human application are manifold ranging from low technical efficiencies to undesirable integration of oncogenes into the genome. It is thus indispensable to further our understanding of the underlying processes involved in this technique. With the advent of new data acquisition technologies and an ever-growing complexity of biological knowledge, the Systems Biology approach has seen an evolution of its applicability to the elaborate questions and problems of researchers. Using different mathematical modeling approaches the process of somatic cell reprogramming is examined to find out bottlenecks and possible enhancements of its efficiency. I analyze the topological characteristics of a pluripotency network in order to find differences to randomly generated networks and thus deduce constraints of the biologically relevant network. The optimization of a Boolean model from a curated network against early reprogramming gene expression profiles reveals profound insights into the first steps and most important factors of the process. The transcription factor SP1 emerges to play an important role in the induction of an intermediate, transcriptionally inactive state. A probabilistic Boolean network (PBN) illustrates the interplay of transcriptional and epigenetic regulatory processes in order to explain pluripotency and cell lineage decisions in reprogramming and differentiation. Explanations for the low reprogramming efficiencies are tried.

Die Entstehung von Tumoren und damit einhergehenden Veränderungen wurden insbesondere im letzten Jahrzehnt kontrovers diskutiert. Bisher standen nur wenige Datensätze mit ausreichender Datendichte zur Verfügung um eine umfassende Untersuchung der Regulation des Stoffwechsels durchzuführen. Die in dieser Arbeit zusammengefassten Projekte adressieren verschiedene Aspekte der Stoffwechselregulation und beschreiben die Verknüpfung von Zellkulturexperimenten mit innovativen Hochdurchsatz-Technologien, komplexer Datenanalyse und Computer-basierter Modellierung zur Bestimmung der Stoffwechselflüsse in eukaryotischen Zellen. Die Kombination von GC-MS und LC-MS basierten Technologien ermöglicht die quantitative Analyse des zentralen Kohlenstoffwechsels. Markierungsexperimente mit stabilen Isotopen (pSIRM) erlauben die dynamische Analyse der Stoffwechselaktivität. In verschiedenen Projekten wurden das Proteom und Metabolom von Krebszellen, humanen Stammzellen (hESCs), induzierten pluripotenten Stammzellen (iPS) und deren dazugehörigen differenzierten Vorläufer- oder Nachfolgerzellen bestimmt. Die multivariate, statistische Analyse der Daten ermöglichte die Differenzierung verschiedener Zelltypen basierend auf der Kombination aller quantitativ bestimmten Daten. Quantitative Bestimmungen der Poolgrössen, Isotopeninkorporationen, sowie der extrazellulären Raten in neuronalen, pluripotenten Vorläuferzellen (Luhmes d0) und Neuronen (Luhmes d6) ermöglichte die Bestimmung der Stoffwechselflusskarte beider Zelltypen unter Verwendung der instationären metabolischenen Flussanalyse (INST-MFA). Die Etablierung einer Qualitätskontrolle für GC-MS basierte Daten (MTXQC), sowie die Zuordnung der GC-MS Fragmente zur Molekülstruktur, ermöglichten den Ausbau des Netzwerkes des zentralen Kohlenstoffwechsels und die Implementierung der Daten für die metabolische Flussanalyse. / Metabolic reprogramming of the central carbon metabolism (CCM) is highly debated during the last decade. It describes the rearrangement of nutrient consumption for providing energy and building blocks for cellular proliferation and maintenance. So far, only sparse data are available for an in-depth analysis of metabolic reprogramming events. The herein summarised projects address metabolic programming from different perspectives and show the implementation of cell culture experiments, cutting-edge high-throughput technologies, bioinformatics, and computational modelling into one workflow providing the determination of metabolic flux maps of mammalian cells. The combination of GC-MS and LC-MS-based methodologies enable the quantitative analysis of proteins and metabolites of the CCM. Pulsed stable isotope-resolved metabolomics (pSIRM) experiments allow monitoring the fate of nutrients within the network of the CCM. The time-dependent and position-specific incorporation of carbon-13 leads to an indirect measurement of the metabolic flux, the only one functional readout of a cell. High-throughput technologies were applied in four projects to gain insights in metabolic reprogramming in cancer cell lines, human embryonic stem cells (hESCs), induced pluripotent stem (iPS) cells and their derived fibroblasts. A global principal component analysis demonstrated the discrimination of phenotypes by different classes of quantitative data. The comparison of metabolic and protein levels confirms the presence of the Warburg effect in both cell types. Though, the executing enzymes vary regarding their isoenzyme identity and expression levels. Methodological improvements provided the implementation of GC-MS derived data for INST-MFA. The mapping of GC-MS derived fragments to the molecule structure enables an extension of the CCM network. Robustness of the input data has been improved by the development of a R-scripting based quality control tool (MTXQC).

A cardiomiopatia arritmogênica do ventrículo direito (CAVD) tem origem genética e é caracterizada pela substituição de células miocárdicas por tecido fibroadiposo. A doença tem uma prevalência aproximada de 1:3500, sendo mais frequentemente diagnosticada em indivíduos jovens, atletas e do sexo masculino. Atualmente, várias alterações genéticas associadas a CAVD foram descritas em 12 genes diferentes. No entanto, existem poucos estudos na literatura que descrevem o espectro mutacional da doença usando um painel abrangente de genes potencialmente causais, em populações diferentes das coortes descendentes de europeus. O sequenciamento de nova geração (NGS) como ferramenta para o diagnóstico molecular da doença, permite um avanço na correlação entre alterações genotípicas e fenotípicas e tem aportado potenciais benefícios que crescem juntamente com os desafios na sua interpretação. Além disso, o uso de hiPSCs como modelo in vitro de determinadas doenças cardíacas, permite avaliar especificamente a relação do genótipo com as diferentes consequências fenotípicas celulares da CAVD. Entretanto, os mecanismos moleculares da doença ainda são pouco esclarecidos e não há na literatura estudos que englobem ao mesmo tempo o perfil mutacional (com um painel extenso de genes) e estudo funcional das alterações encontradas com o uso de hiPSC-CMs. Esta tese teve como objetivo descrever a prevalência de variantes causais em genes associados à CAVD na população brasileira, e caracterizar, do ponto de vista funcional, os cardiomiócitos derivados de hiPSC (hiPSC-CMs) de pacientes com mutações identificadas, a fim de associar o perfil mutacional e a expressão fenotípica celular. Quarenta e sete indivíduos, não aparentados, sendo 38 (80,85%) pacientes do sexo masculino, idade média 40,2 ± 15,56 anos, com diagnóstico clínico de CAVD, foram submetidos ao sequenciamento de um painel genético relacionado à cardiomiopatias hereditárias, compreendendo os 12 genes previamente descritos como causadores de CAVD, utilizando sequenciamento de nova geração (NGS). As variantes foram interpretadas e classificadas de acordo com os critérios da ACMG. Variantes patogênicas ou provavelmente patogênicas foram encontradas em dezoito probandos (38,3%), com maior número de ocorrências no gene PKP2 (38,8%). Entre os 18 casos positivos, treze variantes diferentes foram encontradas, quatro delas novas variantes em genes desmossomais, sem descrição prévia na literatura. Variantes de significado incerto (VSI) foram encontradas em 16 pacientes. A presença de uma variante causal ocorreu em todos os probandos assintomáticos e foi significativamente associada a probandos com histórico familiar de morte súbita cardíaca abaixo de 35 anos. Para a modelagem celular da CAVD, foram geradas hiPSCs de dois pacientes a partir de células progenitoras de urina (UPCs) e fibroblastos, por transfecção episomal. O primeiro paciente possuía alteração missense no gene PKP2 e o segundo, uma inserção no gene DSC2. As hiPSCs foram caracterizadas quanto ao seu potencial de pluripotência e posteriormente diferenciadas em cardiomiócitos (hiPSC-CMs). Nossos resultados demonstraram diferenças fenotípicas significativas entre os CAVD-CMs comparados com os controle-CMs, como: reduções significativas de expressão das proteínas desmossomais e desmossomos estruturalmente alterados; presença de marcadores do acúmulo de gotículas lipídicas e regulação aumentada do fator de transcrição proadipogênico PPAR-gama; aumento de duração do potencial de campo (FPD) e do potencial de ação em 90% de repolarização (APD90); velocidade de condução mais lenta e uma força de contração menor. Em conclusão, este é o primeiro trabalho a caracterizar o perfil genético da CAVD, abrangendo todos os genes descritos até o momento relacionados à doença, na população brasileira. Os dados obtidos neste trabalho sugerem que, pacientes com história familiar de MSC ( < 35 anos) têm maior probabilidade de portar uma variante causal. Além disso, nossos achados sugerem que pacientes com alteração causal no gene PKP2 têm uma maior gravidade da apresentação fenotípica de arritmia. Nosso modelo celular, que contemplou células paciente-específicas com diferentes alterações das estudadas até o presente momento,sugere ser possível o estudo do efeito das alterações genéticas na CAVD e pode ser um acréscimo às ferramentas disponíveis para estudar o mecanismo desta doença complexa / Arrhythmogenic right ventricular cardiomyopathy (ARVC) has a genetic origin and is mainly characterized by the replacement of myocardial cells with fibroadipose tissue. The disease has a prevalence of approximately 1: 3.500, being more frequently diagnosed in young individuals, athletes and males. Currently, several mutations associated with ARVC have been described in 12 different genes. However, there are few studies in the literature that describe the mutational spectrum of the disease using a comprehensive panel of potentially causal genes in populations other than European-descent cohorts. Next Generation Sequencing (NGS) as a tool for molecular diagnosis of the disease allows an advance in the correlation between genotypic and clinical phenotypic aspects and has potential benefits that grow along with the challenges in its interpretation. In addition, the use of hiPSCs as an in vitro model of certain heart diseases, allows to specifically evaluate the relationship of the genotype with the different cellular phenotypic consequences of ARVC. However, the molecular mechanisms of the disease are still poorly understood and there are no studies in the literature that include both the mutational profile (with an extensive panel of genes) and functional study of different causal variants, with the use of hiPSC-CMs. The aim of this thesis was to describe the prevalence of causal variants in ARVC-associated genes in the Brazilian population, and to characterize, from a functional point of view, cardiomyocytes derived from hiPSC (hiPSC-CMs) from patients with identified mutations, in order to associate the mutational profile and cellular phenotypic expression. Forty-seven unrelated probands, 38 (80.85%) male, mean age 40.2 ± 15.56 years, with clinical diagnosis of ARVC, were submitted to a cardiomyopathy-related gene panel sequencing, comprising 12 genes, using next-generation sequencing (NGS). Variants were interpreted and classified according to the ACMG criteria. Pathogenic or Likely Pathogenic variants were found in eighteen probands (38.3%), with the largest number of occurrences in the PKP2 gene (38.8%). Among the 18 positive cases, thirteen different variants were found, four of them novel mutations in desmosomal genes, without previous description in the literature. Variants of uncertain significance (VUS) were found in 16 patients. The presence of a causal variant was present in all asymptomatic probands and was significantly associated with probands who have a family history of sudden cardiac death under 35 years. For the cellular modeling, from urinary progenitor cells (UPCs) and fibroblasts, hiPSCs from two patients were generated by episomal transfection. The first patient had a missense variant in the PKP2 gene, while the second had an insertion in the DSC2 gene. The hiPSCs were characterized for its pluripotency potential and subsequently differentiated into cardiomyocytes (hiPSC-CMs). Our results demonstrated significant phenotypic differences between the ARVC-CMs compared to the control-CMs, such as: significant reductions in the expression of desmosomal proteins and structurally altered desmosomes; presence of lipid droplet accumulation markers and increased regulation of the proadipogenic transcription factor PPAR-gamma; prolonged field potential duration (FPD) and action potential in 90% repolarization (APD90); slower conduction velocity and a lower active contraction force. In conclusion, this is the first work to characterize the genetic profile of ARVC, covering all genes described to date related to the disease, in the Brazilian population. The data obtained in this study suggests that patients with a family history of sudden cardiac death ( < 35 years) are more likely to carry a causal variant. In addition, our findings suggest that patients with causal variant in the PKP2 gene have a greater severity of the phenotypic presentation of arrhythmia. Our cellular model, which contemplated patient-specific cells with different causal variants of the previous studies, suggests that it is possible to study the effect of the genetic changes in ARVC, and may be an addition to the tools available to study the mechanism of this complex disease

No description available.

The field of stem cell research has been growing more because of the interest in using stem cells to cure diseases and heal injuries. Human embryonic stem cells, because of the controversy surrounding them—and subsequently the difficulties in acquiring samples of the existing aging cell lines—can only be used in limited capacities. While the development of induced pluripotent stem cells in the last decade has allowed the field to progress closer to medical treatments, the low efficiency of reprogramming a somatic cell to a pluripotent state, and the vast molecular and genomic differences between human embryonic stem cells and human induced pluripotent stem cells is still an issue. Therefore, the goal is to discover methods, chemicals, and factors that can reduce these differences and increase the efficiency of inducing pluripotency. This proposal aims to look at the effects of the protein ECAT1 in inducing pluripotency in human somatic cells. Little is known about ECAT1, otherwise known as Embryonic Stem Cell-Associated Transcript 1, beyond its presence in human embryonic stem cells and oocytes and its absence in differentiated cells. While originally considered by scientists during the development of the reprogramming technique, ECAT1's effects have not been tested in humans. Therefore, a series of experiments will be performed in which ECAT1 will be used in conjunction with OSKM to induce pluripotency in adult human dermal fibroblasts, which will then be differentiated into spinal motor neurons. The three stages of this proposal--inducing pluripotency, comparing pluripotencies in the reprogrammed cells and embryonic stem cells, and differentiating the stem cells--should answer questions about ECAT1 and the reprogramming process. It is predicted that ECAT1 should reduce the genomic and molecular differences between embryonic stem cells and induced pluripotent stem cells. ECAT1's presence should also increase the efficiency of reprogramming as well as successful differentiation to other cell types.

Background: Replacement of dysfunctional β-cells in the islets of Langerhans by transdifferentiation of pancreatic acinar cells has been proposed as a regenerative therapy for diabetes. Adult acinar cells spontaneously revert to a multipotent state upon tissue dissociation in vitro and can be stimulated to redifferentiate into β-cells. Despite accumulating evidence that contact-mediated signals are involved, the mechanisms regulating acinar-to-islet cell transdifferentiation remain poorly understood. Results: In this study, we propose that the crosstalk between two contact-mediated signaling mechanisms, lateral inhibition and lateral stabilization, controls cell fate stability and transdifferentiation of pancreatic cells. Analysis of a mathematical model combining gene regulation with contact-mediated signaling reveals the multistability of acinar and islet cell fates. Inhibition of one or both modes of signaling results in transdifferentiation from the acinar to the islet cell fate, either by dedifferentiation to a multipotent state or by direct lineage switching. Conclusions: This study provides a theoretical framework to understand the role of contact-mediated signaling in pancreatic cell fate control that may help to improve acinar-to-islet cell transdifferentiation strategies for β-cell neogenesis.

No description available.

Background: Replacement of dysfunctional β-cells in the islets of Langerhans by transdifferentiation of pancreatic acinar cells has been proposed as a regenerative therapy for diabetes. Adult acinar cells spontaneously revert to a multipotent state upon tissue dissociation in vitro and can be stimulated to redifferentiate into β-cells. Despite accumulating evidence that contact-mediated signals are involved, the mechanisms regulating acinar-to-islet cell transdifferentiation remain poorly understood. Results: In this study, we propose that the crosstalk between two contact-mediated signaling mechanisms, lateral inhibition and lateral stabilization, controls cell fate stability and transdifferentiation of pancreatic cells. Analysis of a mathematical model combining gene regulation with contact-mediated signaling reveals the multistability of acinar and islet cell fates. Inhibition of one or both modes of signaling results in transdifferentiation from the acinar to the islet cell fate, either by dedifferentiation to a multipotent state or by direct lineage switching. Conclusions: This study provides a theoretical framework to understand the role of contact-mediated signaling in pancreatic cell fate control that may help to improve acinar-to-islet cell transdifferentiation strategies for β-cell neogenesis.