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Orientador: Henrique Marques Barbosa de Souza / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T10:31:42Z (GMT). No. of bitstreams: 1 Rosa_VivianedeSouza_M.pdf: 2727894 bytes, checksum: d0d5ab88ca9670f3109f39586f01a78c (MD5) Previous issue date: 2015 / Resumo: Células-tronco embrionárias (CTE) são células indiferenciadas que possuem a capacidade de (1) se proliferarem indefinidamente (auto-renovação) e, quando induzidas, (2) darem origem a qualquer tipo celular presente no embrião (pluripotência). Uma das abordagens mais comumente utilizadas para o estudo de diferenciação de CTE é através da formação de agregados multicelulares esféricos denominados corpos embrióides (CE). CE passam por um processo de morfogênese semelhante ao observado em embriões, originando derivados dos três folhetos germinativos. Durante o desenvolvimento embrionário, a formação e o posicionamento dos três folhetos ocorre por um processo altamente coordenado que culmina na formação de um embrião polarizado no eixo anteroposterior. Entretanto, um dos grandes desafios de pesquisas que envolvem o uso da diferenciação de CTE em CE é encontrar indícios de que esses processos são recapitulados in vitro e se entender como que células derivadas dos folhetos germinativos, que no embrião ocorrem de forma altamente organizada, são originadas em estruturas celulares sem nenhuma organização global evidente, como visto em CE. Coup-TFII (Chicken ovalbumin upstream promoter-transcription factor II) é um fator de transcrição o qual possui um papel fundamental na regulação do desenvolvimento embrionário e na aquisição de destinos celulares específicos durante a diferenciação de CTE. Utilizando CE como um modelo de estudo, caracterizamos a expressão de Coup-TFII e seu possível envolvimento durante a determinação de destinos celulares. Nossos resultados identificaram uma expressão hemisférica de Coup-TFII em CE em etapas inicias do processo de diferenciação. Esta observação nos levou a caracterizar a distribuição espacial de marcadores moleculares tecido-específicos nos CE em relação à expressão hemisférica de Coup-TFII. Interessantemente, praticamente todas as células identificadas como precursores mesodérmicos e precursores neuroectodérmicos, através da expressão de Brachyury-T e Nestin, respectivamente, estão contidas nas população de células Coup-TFII-positivas. Estes resultados sugerem a existência de um mecanismo de organização global intrínseco nas CTE, onde a expressão de Coup-TFII parece segregar os CE em dois hemisférios e, provavelmente de forma antagônica com Oct4, determinaria diferentes destinos celulares ainda em fases iniciais da diferenciação / Abstract: Embryonic stem cells (ESC) are undifferentiated cells that have the ability to (1) proliferate indefinitely (self-renewal) and when induced, (2) give rise to any cell type present in the embryo (pluripotency). One of the most commonly used approaches for the study of ESC differentiation is through the formation of spherical multicellular aggregates called embryoid bodies (EB). EB undergo a process similar to that observed in morphogenesis embryos, giving derivatives of three germ layers. During embryonic development, formation and placement of the three germ layers is a highly coordinated process by which culminates in the formation of a polarized embryo in the antero-posterior axis. However, one of the great challenges of research involving the use of ESC differentiation in EB is to find evidence that these processes are recapitulated in vitro and in understanding how to cells derived from the germ layers that occurs in the embryo highly organized manner originate on cellular structures with no apparent global organization, as seen in the EB. COUP-TFII (chicken ovalbumin promoter-upstream transcription factor II) is a transcription factor which plays a key role in the regulation of embryonic development and determination of specific cell fates during differentiation ESC. Using EB as a model system, we characterized the expression of Coup-TFII and its possible involvement in the determination of cell fates. Our results identified a hemispheric expression of Coup-TFII in EB at the onset of differentiation. This observation led us to characterize the spatial distribution of tissue-specific molecular markers in EB in relation the hemispheric expression of Coup-TFII. Interestingly, practically all cells identified as mesodermal and neuroectodermal precursors by the expression of Brachyury-T and Nestin, respectively, are contained in the COUP-TFII-positive cell population. These results suggest the existence of a mechanism of global organization intrinsic to ESC, where the expression of Coup-TFII segregates the EB into two hemispheres and probably antagonistically with Oct4, determine different cell fates still in early stages of differentiation / Mestrado / Biologia Tecidual / Mestra em Biologia Celular e Estrutural

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.

Die vorliegende Arbeit befasst sich mit Aspekten der frühen Entwicklung der Phoronida („Hufeisenwürmer“). An drei Arten wird der Furchungsprozess untersucht (Phoronis pallida, Phoronis muelleri, Phoronis vancouverensis). Dies erfolgt sowohl mithilfe der 4D-Mikroskopie als auch anhand von immunocytochemischen Markierungen der Mitosespindeln und konfokaler Laser-Scanning-Mikroskopie. Verschiedene morphologische Merkmale des Furchungsprozesses werden quantitativ erfasst und innerhalb sowie zwischen den Arten verglichen. Die Ergebnisse zeigen eine weitgehend übereinstimmende Furchung bei P. pallida und P. muelleri Embryonen: Ab dem dritten Zellzyklus teilen sich die Blastomeren meist schräg – und alternierend dextral und sinistral – zur animal-vegetativ Achse. Dieses Muster zeigt überraschende Übereinstimmungen mit dem Muster der Spiralfurchung. Dies kann als morphologische Unterstützung molekular-phylogenetischer Befunde einer Stellung der Phoronida innerhalb der Spiralia/Lophotrochozoa interpretiert werden. Die Furchung bei P. vancouverensis unterscheidet sich von der Furchung der anderen beiden Arten; sie weist jedoch auch Unterschiede zu einer Radiärfurchung auf. Generell zeigt die Furchung aller drei Arten einen gewissen Grad an Variabilität. Anhand von in-vivo Einzelzellmarkierungen untersucht die Studie darüber hinaus das Schicksal der Blastomeren früher P. pallida Embryonen bis zu späten Gastrulationsstadien. Diese Analysen zeigen, dass die ersten beiden Furchungsteilungen durch die spätere Achse Blastoporus-Apikalplatte, jedoch in keinem konstanten Orientierungsverhältnis zur Ebene der Bilateralsymmetrie der Gastrula verlaufen. Dies unterscheidet sich von der Situation, wie sie von spiralfurchenden Tieren bekannt ist. Die Unterschiede und die beobachtete Variabilität des Furchungsprozesses werden im Licht unterschiedlicher Mechanismen der Spezifizierung von Zellschicksalen und Körperachsen bei verschiedenen Taxa der Spiralia und den Phoronida diskutiert. / This study addresses aspects of the early development of Phoronida (“horseshoe worms”). The cleavage process is analyzed for three species (Phoronis pallida, Phoronis muelleri, Phoronis vancouverensis). These investigations are performed using 4D-microscopy as well as immunocytochemical stainings of the mitotic spindle apparatuses in combination with confocal laser-scanning microscopy. Different morphological features of the cleavage process are quantified and compared within as well as between the species. The results reveal a highly consistent cleavage of P. pallida and P. muelleri embryos: from the third cell cycle onward, the blastomeres divide mostly obliquely – and alternatingly dextral and sinistral – with respect to the animal-vegetal axis. This cleavage pattern shows surprising correspondences to the pattern of spiral cleavage. The finding can be interpreted as morphological support for recent molecule-based phylogenies, which indicate a position of Phoronida within the Spiralia/Lophotrochozoa clade. The cleavage of P. vancouverensis differs from the cleavage in the other two species; however, it also shows differences to a radial cleavage pattern. In all three species, the cleavage process also involves some degree of variability. Furthermore, the study traces the cell fates of early P. pallida embryos up to the state of late gastrulation, by the use of fluorescent in-vivo single cell markings. These analyses reveal that the first two cleavage divisions both pass through the later axis blastopore-apical plate of the gastrula, yet they do not pass in a constant relationship with respect to the later plane of bilateral symmetry. This differs from the situation known from spiral cleaving animals. The differences and the encountered variability of the cleavage process are discussed with respect to different mechanisms of the specification of cell fates and body axes in different taxa of the Spiralia and the Phoronida.