The potential of embryonic stem (ES) cells to generate any developmental or adult cell type holds much promise for regenerative medicine and in vitro modelling of development and disease. Haematopoietic stem cells (HSCs) regenerate all lineages of the blood throughout adult life and are essential for the treatment of a vast number of haematalogic disorders. Current sources of HSCs for clinical use and research, including adult bone marrow, peripheral blood stem cells and umbilical cord blood, are limited by the number of HSCs they contain and by the availability of a suitable donor. A system that generates a reliable source of HSCs from ES cells would therefore be an ideal alternative. While much progress has been made in the generation of downstream lineages of the haematopoietic system, progress in the derivation of HSCs capable of long-term self-renewal and multilineage reconstitution from ES cells has been limited. Understanding of the developmental steps leading to HSC emergence in the embryo has been advancing in recent years. In particular, precursors of HSCs (preHSCs) have been isolated from the mouse embryo, characterised and matured into HSCs ex vivo using the specialised conditions of aggregate culture systems (Taoudi et al 2008, Rybtsov et al 2011). We hypothesised that application of the aggregate culture system in the differentiation of ES cells could provide a missing link in the in vitro generation of HSCs. Here I have developed a novel ES cell differentiation system that employs the specialised conditions of the aggregate culture system, after an initial stage of mesoderm differentiation. I show that this system creates an environment for efficient haematopoietic and endothelial progenitor formation and generates cells of a preHSC type I (VE-Cadherin+CD45-CD41lo) and preHSC type II (VE-Cadhein+CD45+) surface phenotype. Notably, the system gives rise to cells that achieve low-levels of haematopoietic repopulation in sublethally irradiated NSG mice. The low-level repopulating cells persist for over 4 months in animals and show both myeloid and lymphoid potential. I identify genes that are expressed in cells of a preHSC II surface marker-phenotype from the E11.5 dorsal aorta, but not in cells of this phenotype from the E11.5 Yolk sac or differentiated ES cells. I also show that enforced expression of Notch downstream target Hes1 in Flk1+ mesoderm during ES cell differentiation does not improve levels of ES-derived repopulation.

Colonic stromal cells provide critical structural support but also regulate immunity, tolerance and inflammatory responses in the mucosa. Substantial variability and plasticity of mucosal stromal cells has been reported but a paucity of distinct marker genes exist to identify distinct cell states. Here single-cell RNA-sequencing is used to document heterogeneity and subtype specific markers of individual colonic stromal cells in human and mouse. Marker-free transcriptional clustering of fibroblast-like cells derived from healthy human tissue reveals distinct populations corresponding to myofibroblasts and three transcriptionally and functionally dissimilar populations of fibroblasts. A SOX6 high fibroblast subset occupies a position adjacent to the epithelial basement membrane and expresses multiple epithelial morphogens including WNT5A and BMP2. Additional fibroblast subtypes show specific enrichment for chemokine signalling and prostaglandin E₂ synthesis respectively. In ulcerative colitis, substantial remodelling occurs with depletion of the SOX6 high population and emergence of an immune enriched population expressing genes associated with fibroblastic reticular cells including CCL19, CCL21 and IL33. A large murine dataset comprising over 7,000 colonic mesenchymal cells from an acute colitis model and matched healthy controls reveals strong preservation of the SOX6 high and myofibroblast transcriptional signatures. Unsupervised pseudotemporal ordering is used to relate fibroblast subsets to one another producing a branched developmental hierarchy that includes a potential progenitor population with mesothelial characteristics at its origin. This work provides a molecular basis for re-classification of colonic stromal cells and identifies pathological changes in these cells underpinning inflammation in UC.

Indiana University-Purdue University Indianapolis (IUPUI) / The differentiation of naïve CD4+ T cells into subsets of T helper cells (Th) is an essential process that impacts host defense and the pathogenesis of immunemediated diseases. Signal transducers and activators of transcription (STAT) proteins, activated downstream of instructive cytokines, dictate and perpetuate the lineage decision of Th cells through both positive and negative effects. This is accomplished by regulating transcription factors, surface receptors and promoting epigenetic changes in gene expression through chromatin remodeling. Transforming growth factor-β1 (TGF-β1) can induce Foxp3 in developing Th cells and these Foxp3-expressing adaptive T regulatory cells (aTregs) are able to suppress inflammation in vitro and in vivo. To define the mechanism by which STAT proteins regulate Th cell pro- and anti-inflammatory phenotypes, we examined T cells deficient in Stat3, Stat4, and Stat6 as well as T cells expressing two STAT4 isoforms after being cultured in the presence or absence of TGF-β1 and cytokines known to be instructive in Th cell development. The negative effects of STAT proteins are demonstrated by our results indicating STAT3, STAT4 and STAT6 proteins activated downstream of the instructive cytokines IL- 6, IL-12 and IL-4, respectively, negatively regulate the development of TGF-β induced Foxp3 and aTreg development. STAT3, STAT4, and STAT6 utilize a vi Mark H. Kaplan, Ph.D., Chair common mechanism to inhibit aTreg generation by inhibiting STAT5, a positive regulator of Foxp3 expression, from binding to the Foxp3 gene. STAT proteins positively effecting inflammatory immunity are demonstrated by our analysis of STAT4 isoforms and their ability to regulate the production of proinflammatory cytokines downstream of IL-12. STAT4β, a STAT4 splice isoform that lacks a Cterminal domain, and STAT4α, a full-length isoform are both capable of mediating inflammatory cell development. However, STAT4β promotes greater inflammation in vivo than STAT4α independent of its ability to repress Foxp3. Instead, the inflammation correlates with STAT4 isoform-dependent expression of inflammatory cytokines. Thus, cytokine-stimulated STAT proteins orchestrate T helper cell pro- and anti-inflammatory cell phenotypes.

Ligament/tendon tissue engineering has the potential to provide therapies that overcome the limitations of incomplete natural healing responses and inadequate graft materials. While ligament/tendon fibroblasts are an obvious choice of cell type for these applications, difficulties associated with finding a suitable cell source have limited their utility. Mesenchymal stem cells/marrow stromal cells (MSCs) are seen as a viable alternative since they can be harvested through routine medical procedures and can be differentiated toward a ligament/tendon fibroblast lineage. Further study is needed to create an optimal biomaterial/biomechanical environment for ligament/tendon fibroblastic differentiation of MSCs. The overall goal of this dissertation was to improve the understanding of the role that biomechanical stimulation and the biomaterial environment play, both independently and combined, on human MSC (hMSC) differentiation toward a ligament/tendon fibroblast phenotype. Specifically, the effects of cyclic tensile stimuli were studied in a biomaterial environment that provided controlled presentation of biological moieties. The influence of an enzymatically-degradable biomaterial environment on hMSC differentiation was investigated by creating biomaterials containing enzymatically-cleavable moieties. The role that preculture may play in tensile responses of hMSCs was also explored. Together, these studies provided insights into the contributions of the biomaterial and biomechanical environment to hMSC differentiation toward a ligament/tendon fibroblast phenotype.

Orientadores: Carmen Verissima Ferreira, Hiroshi Aoyama / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T13:30:15Z (GMT). No. of bitstreams: 1 Cavagis_AlexandreDonizetiMartins_D.pdf: 4238254 bytes, checksum: af81f06637a0dca49e3ab3fabe00d10d (MD5) Previous issue date: 2005 / Resumo: O objetivo central deste trabalho foi avaliar a modulação de MAPKs em células de leucemia humana induzidas a apoptose e diferenciação. No capítulo 1, estudou-se o efeito da tetrahidroxiquinona (THQ), uma molécula que pode participar de um ciclo redox formando radicais semiquinona e, conseqüentemente, espécies reativas de oxigênio (EROs). A citotoxicidade da THQ, mediada pela formação de EROs, foi revertida por tratamento das células com glutationa e N-acetil-L-cisteína. A produção de EROs ocorreu concomitan-temente com a apoptose das células HL60 através da via mitocondrial e com redução na atividade de várias moléculas anti-apoptóticas da via de sobrevivência celular, inclusive da proteína quinase B (PKB). A transfecção das células HL60 com a PKB levou à diminuição da citotoxicidade dependente de EROs gerada pela THQ. No capítulo 2, demonstrou-se que a análise de um arranjo simultâneo de 1176 substratos com seqüências de consenso de diferentes quinases permitiu identi-ficar a via de sinalização das MAPKs como principal alvo da violaceína, um pigmento com propriedades antitumorais in vitro produzido pela Chromobacterium violaceum do rio Amazonas e que induz diferenciação de células HL60 em monócitos e granulócitos. Os resultados também indicaram que a análise do quinoma empregando arranjos metabólicos é uma ferramenta promissora na elucidação do modo de ação de fármacos em nível molecular. O capítulo 3 corresponde a um artigo de revisão em português sobre a riboflavi-na, componente do complexo vitamínico B2 que participa de importantes eventos bioquímicos, como reações redox envolvendo transferência de um ou dois elétrons, e também agindo como fotossensibilizador. Este artigo mostra que o comportamento peculiar e multifuncional da riboflavina permite que ela atue como nucleófilo ou eletrófilo e também descreve a associação deste composto com diferentes doenças como o câncer e doenças cardiovasculares / Abstract: The principal aim of this work was to assess the MAPKs family status in human leukaemia cells induced to undergo apoptosis and differentiation. In chapter one, the effect of tetrahydroxyquinone (THQ), a highly redox active molecule which can participate of a redox cycle with semiquinone radicals leading to the formation of reactive oxygen species (ROS), was investigated in HL60 cells. THQ caused substantial ROS formation followed by cytotoxicity that was sensitive to glutathione and N-acetyl-L-cysteine. Furthermore, ROS production coincided with HL60 cell apoptosis through the mitochondrial pathway and was followed by reduced activity of various anti-apoptotic survival molecules, including the protein kinase B pathway. Importantly, transfection of protein kinase B into HL60 cells leading to an artificial increase in protein kinase B activity inhibited ROS-dependent cytotoxicity. In chapter two, peptide arrays of 1176 different kinase consensus substrates unambiguously identified the MAP kinase pathway as a major target for violacein, the anti-leukemic purple-colored pigment produced by Chromobacterium violaceum from the Amazon River that stimulates HL60 cells to differentiate into monocytes and granulocytes. The results also indicate that kinome profiling using metabolic arrays is a promising and suitable tool for studying the molecular mechanisms of drug action. Chapter three is a review article in Portuguese about riboflavin, a component of the vitamin B2 complex, that has important roles in biochemistry, especially in redox reactions due to its ability to participate in both one- and two-electron transfers as well as acting as photosensitizer. This article describes the peculiar and multifunctional behavior of riboflavin which allows it to take part in several biochemical pathways both as nucleophile and electrophile. Moreover, the association of this vitamin with different diseases, including cancer and cardiovascular diseases, is also discussed. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Mitochondria are essential for the viability of mammalian cells and provide a compartment for specific chemical reactions. Cellular respiration -- the main mitochondrial function -- is tightly connected with ROS production: the mitochondrial electron transport chain complexes I and III are the main ROS sources in mammalian cells. It has been reported that complex I and complex III activities are essential for cell cycle, apoptosis and stem cell differentiation (Spitkovsky et al., 2004; Varum et al., 2009; Lee et al., 2011; Ma et al., 2011; Tormos et al., 2012). In our work, we aimed to investigate the role of mitochondrial electron transport chain activity in the regulation of the differentiation potential and to unravel signaling pathways that could participate in this regulation. As a model, we used the P19 pluripotent stem cell line that can be easily differentiated into trophoblasts, expressing intermediate filaments cytokeratin 8/18, and neurons, which express cytoskeleton protein beta-III-tubulin. We first showed that both trophoblast and neural differentiation of P19 cells were accompanied by activation of cellular respiration. The analysis of respiratory chain complexes and supercomplexes, however, showed that undifferentiated P19 cells, as well as their differentiated derivatives did not differ in their respiratory machinery, including functional respirasomes. While undifferentiated cells did not use respiration as the main energy source, cellular respiration was activated during differentiation, indicating that oxidative metabolism was important for efficient differentiation. To investigate the potential role of mitochondrial electron transport chain activity we monitored the influence of a disrupted electron flow on the differentiation of P19 cells. We found that the activity of complex I and complex III influenced the differentiation potential of the pluripotent P19 cell line: the presence of complex I and complex III inhibitors rotenone, antimycin A, or myxothiazol increased the amount of cytokeratin 8/18+ cells during trophoblast differentiation, but almost completely prevented the formation of neuron-like beta-III-tubulin+ cells during neuron differentiation. Moreover, a low oxygen level (1 % O2 vs 21 % O2 in atmosphere) - the final electron acceptor - had the same effect on differentiation. These data suggest that mitochondrial electron transport chain activity contributes to the regulation of differentiation. The presence of complex I and complex III inhibitors, as well as oxygen scarcity, increase ROS production. We suggested that increased ROS level could explain the observed effects. By visualizing mitochondrial superoxide production with a specific dye – MitoSox - we confirmed that rotenone, antimycin A, myxothiazol, as well as low oxygen conditions, increased the superoxide level. These results suggest that the observed changes of the differentiation potential of P19 cells are associated with ROS production. To prove this idea, we differentiated P19 cells in presence of paraquat – a known ROS inducer. In line with our hypothesis paraquat promoted trophoblast differentiation. The received results suggest that the mitochondrial electron transport chain activity regulates differentiation through the ROS level. ROS are secondary messengers that participate in numerous processes including cell proliferation and differentiation. We aimed to predict the signal pathway that connects ROS level in stem cells and their differentiation potential. For this purpose, we performed a microarray analysis and compared the gene expression profiles of cells grown under hypoxia or in the presence of the complex III inhibitor myxothiazol with untreated control cells. The expression analysis revealed p53 as a transcriptional factor that impacts the differentiation potential in treated cells. p53 is a known redox-sensing molecule (Bigarella et al., 2014) that influences the differentiation potential through cell cycle control (Maimets et al., 2008). This observation is in line with our results and suggests that p53 may regulate the differentiation potential of P19 cells. We are planning to investigate the role of p53 signaling in the regulation of cell cycle and differentiation potential of P19 cell line.

[ES] Los procesos de comunicación celular permiten a las células desarrollar una acción coordinada durante la embriogénesis y asimilar de forma coherente las señales recibidas a través del entorno. Algunas de las moléculas señalizadoras más usadas en la clínica y la investigación son las citoquinas. Sin embargo, existe una tendencia creciente en el uso de otro tipo de moléculas, como los iones metálicos. Algunos iones como el calcio y el zinc actúan como segundos mensajeros intracelulares. Otros como el litio son capaces de inactivar proteínas quinasa alterando rutas de señalización. En el desarrollo de esta tesis doctoral, se ha estudiado el efecto del zinc en células musculares de ratón, el papel del zinc en la auto-renovación de células madre embrionarias (CMEs), y el papel del litio en la diferenciación de CMEs. El estudio del efecto del zinc sobre los mioblastos demostró que el zinc es capaz de estimular la diferenciación de los mioblastos. El análisis del zinc intracelular, en los diferentes estadios de diferenciación de las células musculares, demostró que los miotubos eran capaces de albergar mayor cantidad de zinc en su interior. Los resultados mostraron que la adición de zinc extracelular estimula la fosforilación y activación de la proteína quinasa Akt. También se ha visto que el transportador de zinc, Zip7, es crítico en el proceso de diferenciación celular mediado por el zinc, además, su activación incrementa la fosforilación de Akt. La inhibición de Zip7 mediante ARN interferente redujo la fosforilación de Akt y consecuentemente origino unos niveles menores de diferenciación de los mioblastos expuestos a zinc extracelular. Nuestros resultados demuestran que altas concentraciones de zinc extracelular producen un incremento en la diferenciación de los mioblastos debido a la activación de Akt mediada por Zip7. Para el segundo estudio, se analizó el efecto del zinc sobre las CMEs. Como control de mantenimiento de la pluripotencia se usó medio suplementado con factor inhibidor de leucemia (LIF). Se ha observado que la adición externa de concentraciones de zinc superiores a 100 µM produce un incremento inmediato de la concentración de zinc intracelular activando Akt. Los resultados demuestran que las células tratadas con altas concentraciones de zinc mantienen su capacidad de auto-renovación. Para demostrar que el efecto del zinc en CMEs está asociado a la activación de Akt mediada por Zip7, se inhibió la fosforilación de Akt y se silenció Zip7. Ambos abordajes dieron como resultado un incremento en la diferenciación de las células tratadas con zinc. Por otro lado, CMEs cultivadas durante 30 días en presencia de zinc fueron capaces de retener su pluripotencia, mientras que el control sin zinc presentaba rasgos claros de diferenciación celular. Por último, la combinación de LIF con zinc produjo un incremento importante del efecto del LIF en cuanto al mantenimiento de la capacidad de auto-renovación celular. Por último, se ha estudiado el efecto del litio en la diferenciación de las CMEs. El litio es un inhibidor de la glucógeno sintasa quinasa 3ß (GSK3ß). En términos de CMEs, GSK3ß activa los mecanismos de diferenciación. Los resultados obtenidos indican que altas concentraciones de litio (10 mM) son capaces de fosforilar e inhibir la proteína GSK3ß. Sin embargo, en lugar de mantener la pluripotencia, las células madre se diferenciaron hacia el linaje del mesodermo tras 3 días de cultivo. Después de un total de 6 días, las células tratadas con 10 mM de litio presentaron características de endotelio hemogénico. La inhibición de GSK3ß dio como resultado la activación de la proteína ß-catenina, cuya actividad transcripcional es necesaria para la hematogénesis embrionaria. La capacidad de las células endoteliales con potencial hemogénico obtenidas de derivar en células madre hematopoyéticas fue confirmada tras su maduración durante 11 día / [CA] Els processos de comunicació cel·lular permeten a les cèl·lules desenvolupar una acció coordinada durant la embriogènesis y assimilar de forma coherent als senyals rebudes a través de l'entorn. Algunes de les molècules senyalitzadores més usades en la clínica i la investigació són les citocines. No obstant, hi ha una tendència creixent en l'ús d'un altre tipus de molècules, com els ions metàl·lics. Alguns ions com el calci i el zinc són capaços de dur a terme funcions de missatger secundari. Altres com el liti són capaços d'inactivar proteïnes quinasa alterant rutes de senyalització. Durant el desenvolupament d'aquest treball de tesi doctoral, s'ha estudiat l'efecte del zinc sobre mioblasts de ratolí, el paper del zinc en l'auto-renovació de les cèl·lules mare embrionàries (CMEs), i el paper del liti sobre la diferenciació de les CMEs. L'estudi de l'efecte del zinc sobre els mioblasts ha demostrat que el zinc és capaç d'incrementar la diferenciació dels mioblasts. L'anàlisi del zinc intracel·lular ha demostrat que els mioblasts diferenciats eren capaços d'albergar major quantitat de zinc intracel·lular. Els resultats han mostrat que suplementar les cèl·lules amb zinc extracel·lular produïx una major fosforilació i activació de la proteïna quinasa Akt. D'altra banda, s'ha observat que el transportador de zinc Zip7 es crític per a la diferenciació cel·lular mediada pel zinc. S'ha demostrat que l'activació d'aquest transportador mitjançant zinc extracel·lular és capaç d'incrementar la fosforilació d'Akt. La inhibició d'aquest transportador mitjançant ARN interferent ha donat com a resultat una menor fosforilació d'Akt i una menor diferenciació dels mioblasts exposats a zinc. Aquests resultats demostren que altes concentracions de zinc extracel·lular produeixen un incrementar la diferenciació dels mioblasts a causa de l'activació d'Akt per mitja de Zip7. Per al segon estudi, s'ha analitzat l'efecte del zinc sobre les CMEs. Com a control de manteniment de la pluripotència es va usar medi suplementat amb factor inhibidor de leucèmia (LIF). S'ha observat que les concentracions extracel·lulars de zinc a partir de 100 µM produïxen un increment immediat de la concentració intracel·lular, produint l'activació d'Akt per mitja de Zip7. Les CMEs tractades amb altes concentracions de zinc mantenen l'auto-renovació. Per demostrar que aquest efecte està associat a l'activació d'Akt mediada per Zip7, es va inhibir la fosforilació d'Akt i es va silenciar el transportador Zip7. Tots dos abordatges han donat com a resultat un increment en la diferenciació de les CMEs tractades amb zinc. D'altra banda, les CMEs van ser capaços de retenir la seva pluripotència després de ser cultivades durant 30 dies en presència de zinc, mentre que el control sense zinc presentava trets clars de diferenciació cel·lular. Finalment, la combinació de LIF amb zinc ha produit un increment sinèrgic de l'efecte del LIF. Finalment, també s'ha estudiat l'efecte del liti en la diferenciació de les CMEs. El liti és un inhibidor de la glicogen sintasa quinasa 3 beta (GSK3ß). En termes de CMEs, aquesta proteïna activa els mecanismes de diferenciació. Els resultats obtinguts indiquen que altes concentracions de liti (10 mM) tenen la capacitat de fosforilar i inhibir la proteïna GSK3ß. No obstant això, en lloc de mantenir la pluripotència, les CMEs es van diferenciar cap al llinatge del mesoderma després de 3 dies. Després d'un total de 6 dies, les cèl·lules tractades amb 10 mM de liti presentaven característiques d'endoteli hemogénic. La fosforilació de GSK3ß va donar com a resultat l'activació de la proteïna ß-catenina, l'activitat trasncripcional d'aquesta proteïna és necessària per a la hematogénesis embrionària. La capacitat de les cèl·lules endotelials amb potencial hemogénic obtingudes de derivar en cèl·lules mare hematopoètiques va ser confirmada després de la / [EN] The cell signalling process allows cells to develop a coordinated action during embryogenesis and assimilate coherently the signals received through the environment. Some of the most currently used signalling molecules in clinics and research are growth factors and cytokines. However, there is a growing trend in the use of other types of molecules, such as metal ions. Some ions such as calcium and zinc are able to carry out secondary messenger functions, transmitting signals in cascade. Others ions, such as lithium, are capable to inactivate protein kinases altering signalling pathways. During the development of this doctoral thesis, we investigated the effect of zinc on mouse muscle cells (myoblasts), the role of zinc in embryonic stem cells (ESCs) self-renewal, and the role of lithium in the differentiation of ESCs. In the first chapter, we showed that zinc is able to increase the differentiation of myoblasts. The analysis of intracellular zinc indicated that the differentiated myoblasts were capable to harbour higher concentration of intracellular zinc than undifferentiated ones. Addition of high concentration of extracellular zinc increased protein kinase Akt phosphorylation and activation. Akt activity is critical for myoblasts differentiation and has been well studied by other authors. Our results indicated that zinc transporter Zip7 was critical for zinc-mediated cell differentiation. It was prior demonstrated that the activation of this transporter by extracellular zinc increased the phosphorylation of Akt. The inhibition of Zip7 by interfering RNA resulted in a lower phosphorylation of Akt and reduced differentiation of the myoblasts exposed to extracellular zinc. These results demonstrated that high concentration of extracellular zinc enhances the differentiation of myoblasts through activation of Akt mediated by Zip7. In the second chapter, we have analysed the effect of zinc on ESCs. Leukaemia inhibitory factor (LIF) was used as pluripotency sustaining factor. We observed that extracellular supplementation of 100 ¿M zinc produced an immediate increase of the intracellular concentration, which resulted in the activation of Akt mediated by Zip7 transporter. ESCs treated with high concentrations of zinc maintained self-renewal. The role of Akt on ESCs self-renewal has been well established in the literature. To demonstrate that this effect is associated with the activation of Akt mediated by Zip7, we inhibited Akt phosphorylation and silenced the expression of Zip7. Both approaches resulted in an increase in the differentiation levels of the ESCs treated with zinc. We further demonstrated that ESCs treated with zinc during 30 days were able to retain their pluripotency, while the control condition cultured 30 days without zinc presented evident traits of spontaneous cellular differentiation. Finally, the combination of LIF with zinc produced a synergistic-like increase in the effect of LIF on ESCs self-renewal. Finally, we addressed the effect of lithium on the differentiation of ESCs. Lithium is an inhibitor of glycogen synthase kinase 3 beta (GSK3ß). In terms of ESCs, GSK3ß activates differentiation mechanisms. Our results indicated that high concentration of lithium (10 mM) was able to phosphorylate and strongly inhibit the activity of GSK3ß. However, instead of maintaining pluripotency, ESCs differentiated into the mesoderm lineage after 3 days of culture. After a total of 6 days, ESCs treated with 10 mM lithium showed haemogenic endothelium characteristics, expressing CD31, Sca-1 and CD31/Sca-1 positive cells. The phosphorylation of GSK3ß resulted in the activation of the ß-catenin protein, whose transcriptional activity is necessary for embryonic hematogenesis. The ability of endothelial cells with hemogenic potential obtained from lithium-treated ESCs to derive into hematopoietic stem cells was confirmed after maturation of these cells, resulting in rounded cell aggregates positive for Sox17. / Ministerio de Ciencia, Innovación y Universidades a través de la beca BES-2013-064052 y los proyectos MAT2012-38359-C03-01 y MAT2015-69315-C3-1-R. / Mnatsakanyan Movsesyan, H. (2019). ROLE OF IONS IN STEM CELLS SIGNALLING [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/123063

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第25201号 / 医科博第157号 / 新制||医科||10(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 遊佐 宏介, 教授 近藤 玄, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Phospholipase C enzymes are important cell signalling enzymes that catalyse the cleavage of phosphatidylinositol 4,5-bisphophate PI(4,5)P₂ into two biologically active second messenger molecules. These are the inositol 1,4,5-trisphosphate which initiates Ca²⁺ release from the endoplasmic reticulum and the diacylglycerol that activates protein kinase C. Although this basic function is shared between the different isoforms, the PLC family encompasses a diverse collection of proteins with various domain structures in addition to the PLC-specific domains. The neuron-specific “6th family” of these enzymes, PLCηs have most recently been identified with two members, PLCη1 and PLCη2. The aim of the thesis is to characterise the PLCη2 variant from several aspects. Firstly, it describes that PLCη2 possesses a high sensitivity towards Ca²⁺. Secondly, it investigates how the Ca²⁺-induced enzymatic activity of PLCη2 is controlled by its different domains. Also it provides evidence that the pleckstrin homology domain targets PLCη2 to membranes by recognising PI(3,4,5)P₃. Moreover, the uniquely structured EF-hand is responsible for the Ca²⁺-sensitivity of the enzyme. Finally, it is demonstrated that the C2 domain is important for activity. The initial biochemical characterisation is followed by the description of a physiological role for PLCη2. It is shown using a neuroblast model that PLCη2 is crucial for neuronal differentiation and neurite growth. Further efforts were made to assess how PLCη2 is responsible for this effect. It was revealed that it might be involved in regulating intracellular Ca²⁺ dynamics, transcriptional activity and actin reorganisation in differentiating neurons. As the functions of PLCη2 are just beginning to come to light, more aspects for future research are also suggested in the thesis. Hopefully, this and the data presented within the thesis will stimulate even greater interest in this fascinating new field of research.

Abstract not available