The stochastic nature of intracellular chemistry guarantees that even genetically identical cells sharing an environment will differ in composition. The question of whether this chemical diversity translates into significant phenotypic individuality is tied to the relative timescales of the processes involved. In order for cells in a population to have distinct functional identities, they must maintain their states for an appreciable period of time. Quantification of these timescales requires accurate time-lapse measurements covering tens or even hundreds of generations, a technical hurdle that has left these questions largely underexplored. In this thesis I present three pieces of work that aim to provide a foundation for the study of fluctuation timescales in bacteria. In the first part, I describe modifications to a recently developed microfluidic platform for continuous culture of cells under constant conditions. These revised devices enable the high-throughput, long-term measurement of gene expression dynamics while eliminating several confounding experimental factors that interfere with timescale measurements. In the second part, I employ one of these devices to survey fluctuation timescales in ~50 reporters for Eshcerichia coli gene expression. Under rich conditions, all reporters exhibited nearly identical, rapid fluctuation dynamics that were captured by a simple model of gene expression. In contrast, under poor nutritional conditions gene expression states became correlated over several cell divisions. However, accounting for instantaneous growth rate fluctuations eliminated these slow timescales, revealing an exceedingly simple behavior. In the third part, I describe our work to dissect the stochastic transition between the solitary motile state and sessile multicellular state in exponentially growing Bacillus subtilis</italic.. By enforcing static environmental conditions, we uncover the cell's internal strategies for state switching. The transition to the multicellular state occurs without regard to the cell's state history, whereas commitment to the multicellular state is tightly timed. By manipulating the genetic circuit responsible for the switch, we also expose surprising functional modularity in the commitment. I believe that the striking range of gene expression timescales we observe--from the fast fluctuations in E. coli gene expression to the feedback-amplified noise in B. subtilis--will serve as a useful starting point for future studies.

The cell division cycles of differentiating cells are coordinated so as to generate sufficient numbers of mature cells. The cell cycle may also regulate the process of differentiation, in ways that are not well understood. We previously discovered that during erythropoiesis, the cell cycle is synchronized with a specific developmental switch, where erythroid progenitors known as colony-forming-unit-erythroid (CFU-e) transition from a self-renewal state to a state of erythroid terminal differentiation (ETD). This switch takes place during a single cell cycle S phase and is dependent on S-phase progression. My work shows that this S phase is unusual, in that it is shorter than S phase in preceding cycles, as a result of a global increase in replication fork speed. I found that the CDK inhibitor, p57KIP2, negatively regulates replication fork speed in self-renewing CFU-e, and its down-regulation at the switch to ETD results in S-phase shortening. p57KIP2-mediated inhibition of CDK2 is essential for CFU-e self-renewal. It exerts this effect by reducing replication stress and also reducing the probability of transition from CFU-e to ETD, promoting CFU-e self-renewal instead. CDK2 inhibiting drugs that mimic the action of p57KIP2 stimulate erythropoiesis both in vitro and in vivo, through expansion of the CFU-e pool. In addition to p57KIP2, E2f4 also regulates S-phase shortening and the efficiency of the CFU-e to ETD transition. Overall, my work shows that S-phase speed regulates a key erythroid cell fate decision, and suggests a possible translational application of CDK2 inhibiting drugs in the stimulation of erythropoiesis.

DNS-Methylierung ist ein zentraler epigenetischer Prozess, der essentiell für die Differenzierung embryonaler Stammzellen ist, über dessen Funktion in somatischen Zellen allerdings wenig bekannt ist. In der vorliegenden Doktorarbeit wurden zwei Mausmodelle analysiert, um die Rolle der durch DNS Methyltransferase 1 (DNMT1) hergestellten DNS-Methylierung im adulten hämatopoetischen System zu untersuchen. Als erstes wurde ein „Knockout“-Modell gewählt, um DNMT1 im hämatopoetischen System zu eliminieren. Des Weiteren wurde eine Mausmutante mit reduzierter DNMT1 Expression analysiert. Die vollständige Entfernung von DNMT1 aus dem hämatopoetischen System adulter Mäuse resultierte in Zytopenie und Anämie, gefolgt vom raschen Tod aller Tiere. Die Analyse des Knochenmarks dieser Mäuse zeigte einen fast vollständigen Verlust von hämatopoetischen Stamm- sowie Vorläuferzellen. Dies zeigt, dass die durch DNMT1 erzeugte DNS-Methylierung essentiell für Homöostase und Differenzierung von hämatopoetischen Stammzellen ist. Mäuse mit reduzierter DNMT1 Expression hingegen sind lebensfähig und zeigen einen niedrigen Grad an DNS-Methylierung in verschiedensten Geweben, einschließlich des hämatopoetischen Systems. Durch eine detaillierte phänotypische und funktionelle Analyse der hämatopoetischen Stammzellen zeigte sich, dass der veränderte DNS-Methylierungsgrad ein vermindertes Selbsterneuerungspotenzial zur Folge hat. Interessanterweise fehlen DNMT1 hypomorphen Mäusen lymphoide Vorläuferzellen sowie reife lymphoide Zellen, wohingegen myeloide und erythroide Zellpopulationen keine Veränderungen zeigten. Genomweite Expressionsanalysen von Stammzellen sowie myeloiden Vorläuferzellen zeigten, dass hypomethylierte Stammzellen eine verfrühte myeloerythroide Entwicklung vollziehen und liefern damit eine Erklärung für den Verlust des Selbsterneurungspotenzials und der lymphoiden Entwicklung. Diese Resultate identifizieren eine bis hierhin unbekannte Funktion von spezifischen DNS-Methylierungsgraden für die Steuerung von funktionellen Programmen wie Selbsterneuerung und Differenzierung in hämatopoetischen Stammzellen. / DNA methylation is one of the major epigenetic mechanisms which is known to play a role in embryonic stem cell fate, but its function in somatic stem cells is not well understood. In this thesis two different genetic mouse models were chosen to address the role of DNA methyltransferase 1 (DNMT1) controlled DNA methylation in adult hematopoiesis. First, a conditional knockout approach was used to delete DNMT1 in the adult hematopoietic system. Second, DNMT1 hypomorphic mice with reduced DNMT1 expression were analyzed. Complete DNMT1 deletion in hematopoietic cells led to severe cytopenia and anemia causing rapid lethality of all animals. Bone marrow analysis revealed an almost complete absence of hematopoietic stem and progenitor cells in DNMT1 ablated primary mice as well as in secondary chimeric mice. These results indicated that DNMT1 controlled maintenance of DNA methylation is indispensable for HSCs preservation and differentiation. In contrast to complete DNMT1 deletion, mice with hypomorphic DNMT1 expression were viable, but showed low methylation levels in multiple tissues including the hematopoietic system. Detailed phenotypical and functional analysis of the hypomethylated hematopoietic stem cell (HSCs) compartment revealed an impaired homeostasis and self-renewal capacity. Intriguingly, mutant animals had profoundly reduced lymphoid cell compartments, whereas myeloid and erythroid compartments were unchanged. Expression profiling of stem and myeloid progenitor cells unexpectedly demonstrated that reduced DNA methylation forces the HSC to adopt a myeloid lineage identity. These results, showing the inability of hypomethylated HSCs to maintain an undifferentiated state, provided an explanation for their disturbed capability to self-renew and produce lymphocytes. Taken together, these findings suggest that distinct levels of DNA methylation are required to control different functional programs such as self-renewal and alternative lineage choices in HSCs, thus uncovering a previously unrecognized function for DNMT1 activity.

Our earlier work has shown that pre-B cells can be converted into macrophages by the transcription factor C/EBPα at very high frequencies and also that a clonal pre-B cell line with an inducible form of C/EBPα can be converted into macrophage-like cells. Using these systems we have performed a systematic analysis of the questions whether during transdifferentiation the cells retrodifferentiate to a precursor cell state and whether cell cycle is required for reprogramming. As for the first question, a transcriptome analysis of transdifferentiating cells showed that most genes are continuously up or downregulated, acquiring a macrophage phenotype within 5 days. In addition, we observed the transient reactivation of a subset of immature myeloid markers, as well as low levels of the progenitor markers Kit and Flt3 and a few lineage inappropriate genes. Importantly, we were unable to observe the re-expression of cell surface marker combinations that characterize hematopoietic stem and progenitor cells (HSPCs), including c-Kit and Flt3. This was the case even when C/EBPα was activated in pre-B cells under culture conditions that favor HSPC growth or when the transcription factor was activated in a time limited fashion. As for the second question, using the C11-inducible pre-B cell line, time-lapse experiments showed that a subpopulation of about 8% of the pre-B cells did not divide before acquiring macrophage properties, with the majority of cells dividing once and a few percent dividing twice. In agreement with these results we found that 8% of the induced cells did not incorporate BrdU during reprogramming. Importantly, the non-dividing cell subset expressed the highest levels of C/EBPα and was the fastest in acquiring a macrophage phenotype. Inhibition of DNA synthesis by aphidicolin led to an impairment of transdifferentiation in >70% of the cells, suggesting a requirement for traversing the cell cycle. However, sorting pre-B cells into G0/G1 and G2/M fractions followed by induction showed no significant differences in the reprogramming kinetics. Finally, we showed that knocking down p53 in the inducible pre-B cells does not alter their conversion into macrophages, suggesting that an acceleration of the cell cycle has no effect. Together, our findings show that the conversion of pre-B cells to macrophages does not involve overt retrodifferentiation and that high concentrations of C/EBPα bypass the cell cycle-dependency of immune cell transdifferentiation / Recientemente, nuestro grupo ha demostrado que las células pre-B se pueden reprogramar a macrófagos mediante la sobreexpresión del factor de transcripción C/EBP, con una eficiencia elevada. Así mismo, mediante la expresión de la forma inducible de C/EBP en una línea de células pre-B (C11), éstas también se puede convertir en células similares a macrófagos. Usando este sistema hemos estudiado si durante el proceso de trans-diferenciacion las células requieren volver a un estadio de célula precursora, y si el ciclo celular es necesario para este proceso. En cuanto a la primera cuestión, el análisis del transcriptoma de células trans-diferenciadas mostró que la expresión de la mayoría de los genes están regulados durante todo el proceso bien aumentando o disminuyendo, y que adquieren el fenotipo de macrófago a los 5 días después de iniciar el proceso. Así mismo, se observó la reactivación transitoria de un grupo de genes que codifican para marcadores de células mieloides inmaduras; también cabe destacar que observamos una disminución en la expresión de los genes expresados en células progenitoras Kit y Flt3, así como de genes de linajes impropios. Es importante destacar que nunca hemos llegado a observar la expresión de combinaciones de marcadores de superficie característicos de las células madre hematopoyéticas y las células progenitoras (HSPCs), incluyendo c-Kit y Flt3, mediante el análisis por citometría de flujo. Estos resultados se reprodujeron incluso cuando C/EBP se sobreexpresó en células pre-B que fueron cultivadas en condiciones que favorecen el crecimiento de las HSPC o cuando el factor de transcripción se activó de forma limitada en el tiempo. En cuanto a la segunda pregunta, usando la línea de células inducibles pre-B C11, el análisis mediante microscopia a diferentes tiempos después de la inducción de la reprogramación mostraron que una subpoblación de aproximadamente el 8% de las células pre-B no se dividen antes de adquirir las propiedades de macrófago, mientras que la mayoría de las células se dividen sólo una vez y un pequeño porcentaje dos veces antes de que se reprogramen totalmente a macrófagos. De acuerdo con estos resultados se encontró que un 8% de las células inducidas no incorporan BrdU durante la reprogramación. Es importante destacar que el subconjunto de células que no se dividen expresan los niveles más altos de C/EBP, con lo que cabe pensar que la adquisición del fenotipo de macrófago es más rápida en estas células. La inhibición de la síntesis de ADN por afidicolina bloqueó la transdiferenciación en mas de un 70% de las células, lo que sugiere que la correcta progresión del ciclo celular es un requisito para la transdiferenciación. Sin embargo, al separar la linea de células pre-B C11 en fracciones G0/G1 y G2/M seguido de la inducción, la cinética de la reprogramación no mostró diferencias significativas. Por último, también demostramos que la reducción en la expresión de p53 en las células pre-B inducibles no altera el proceso de conversión a macrófago, lo que sugiere que la aceleración del ciclo celular no tiene ningún efecto. En conjunto, nuestros resultados muestran que la conversión de células pre-B a macrófagos no requiere retro-diferenciación y que las células con una expresión mayor de C/EBP pueden llegar a prescindir de la dependencia del ciclo celular para la trans-diferenciación de las células inmunitarias.

Das Ziel dieser Arbeit war, die funktionellen Charkteristika von humanen antigenerfahrenen CD4-T-Zellen in Relation zur Expression von IL-7Ra und CCR7 zu untersuchen. Im Rahmen dessen wurden zwei verschiedene experimentelle Ansätze wurden gewählt: Zur Analyse von Populationen, die während einer Primärantwort auftreten, wurden antigenerfahrene CD4-T-Zellen in vitro mit TSST-beladenen dendritischen Zellen stimuliert. Der zweite Ansatz bestand darin, zirkulierende antigenerfahrene CD4-T-Zellen entsprechend ihrer CCR7- und IL7R-Expression zu isolieren, um die heterogenen zirkulierenden T-Zellen zu untersuchen. Die Experimente zeigen, daß IL7RhiCCR7+ T-Zellen Charakteristika zentraler Gedächtniszellen besitzen. IL7RlowCCR7–identifiziert hingegen Zellen mit Effektor-T-Zellmerkmalen. Dementsprechend wiesen IL7RlowCCR7– T-Zellen ein stark verringertes Zellüberleben auf, waren stark beeinträchtigt in darin in Anwesenheit von homöostatischen Zytokinen zu proliferieren und exprimierten nur wenig IL-2. Im Gegenzug überlebten IL7RhiCCR7+ T-Zellen gut, reagierten auf homöostatische Zytokine, sekretierten IL-2 und expandierten nach antigenspezifischer Stimulation. Interessanterweise erkannten ex vivo isolierte IL7Rlow T-Zellen vornehmlich persistierende Antigene. Dies weist darauf hin, daß diese Zellen chronisch aktiviert sind. In vitro konnte demonstriert werden, daß die funktionelle Ausprägung der den zentralen Gedächtniszellen ähnlichen IL7RhiCCR7+ T-Zellen deutlich von der Stärke der Stimulation während der Generierung der IL7RhiCCR7+ T-Zellen abhängt. Dieser Umstand stützt die Hypothese, daß die Quantität der Signale während der primären Stimulation die Richtung der Zelldifferenzierung bestimmt und ein solcher Mechanismus zur Heterogenität der zentralen Gedächtnis-T-Zellen in vivo beiträgt. Zusammenfassend kann man feststellen, daß, sich die Marker CCR7 und IL7R in Kombination als ein wertvoll zur Identifizierung von CD4-Gedächtnis- und –Effektor-T-Zellen erweisen / The aim of this work was to elucidate the functional characteristics of human antigen-experienced CD4 T cells according to the expression of IL7RAlpha and CCR7. Two different approaches were used: in order to analyze the subsets occurring during a primary response, antigen-experienced CD4 cells were analyzed in vitro after priming with the superantigen TSST. The signal strength of TCR-stimulation during the priming was modulated in order to understand how the levels of stimulation might influence the generation of memory-like T cells. The second approach was to isolate circulating CD4 T cells expressing different combinations of CCR7 and IL7R in order to analyze the heterogeneous pool of antigen-experienced cells found under steady state conditions ex vivo. The results revealed that both in vitro and ex vivo the IL7RhiCCR7+ T cell subset corresponds to cells with TCM characteristics whereas IL7RlowCCR7– identifies cells with effector characteristics. Correspondingly, IL7RlowCCR7–CD4 T cells showed low survival rates, impaired proliferation in the presence of homeostatic cytokines and a low IL-2 production, IL7RhiCCR7+ CD4 T cells survived well, responded to homeostatic cytokines, secreted IL-2 and expanded upon antigenic stimulation. Notably, ex vivo isolated IL7Rlow T cells preferentially recognized under steady state conditions persistent antigens, suggesting that these cells are chronically activated. Finally, it was demonstrated that IL7RhiCCR7+ TCM-like cells generated in vitro acquired different functional properties depending on the strength of stimulation during the priming. This fact supports the concept that the amount of signal received during the priming influences the cell fate decision contributing to the heterogeneity of the TCM pool in vivo. In summary, despite some differences observed between in vitro and ex vivo CD4 T cell subsets, the combination of the markers CCR7 and IL7R is useful to distinguish memory- from effector-like CD4 T cells.

Receptores purinérgicos e canais de cálcio voltagem-dependentes estão envolvidos em diversos processos biológicos como na gastrulação, durante o desenvolvimento embrionário, e na diferenciação neural. Quando ativados, canais de cálcio voltagem-dependentes e receptores purinérgicos do tipo P2, ativados por nucleotídeos, desencadeiam transientes de cálcio intracelulares controlando diversos processos biológicos. Neste trabalho, nós estudamos a participação de canais de cálcio voltagem-dependentes e receptores do tipo P2 na geração de transientes de cálcio espontâneos e sua regulação na expressão de fatores de transcrição relacionados com a neurogênese utilizando como modelo células tronco (CTE) induzidas à diferenciação em células tronco neurais (NSC) com ácido retinóico. Descrevemos que CTE indiferenciadas podem ter a proliferação acelerada pela ativação de receptores P2X7, enquanto que a expressão e a atividade desse receptor precisam ser inibidas para o progresso da diferenciação em neuroblasto. Além disso, ao longo da diferenciação neural, por análise em tempo real dos níveis de cálcio intracelular livre identificamos 3 padrões de oscilações espontâneas de cálcio (onda, pico e unique), e mostramos que ondas e picos tiveram a frequência e amplitude aumentadas conforme o andamento da diferenciação. Células tratadas com o inibidor do receptor de inositol 1,4,5-trifosfato (IP3R), Xestospongin C, apresentaram picos mas não ondas, indicando que ondas dependem exclusivamente de cálcio oriundo do retículo endoplasmático pela ativação de IP3R. NSC de telencéfalo de embrião de camundongos transgênicos ou pré-diferenciadas de CTE tratadas com Bz-ATP, o agonista do receptor P2X7, e com 2SUTP, agonista de P2Y2 e P2Y4, aumentaram a frequência e a amplitude das oscilações espontâneas de cálcio do tipo pico. Dados, obtidos por microscopia de luminescência, da expressão em tempo real de gene repórter luciferase fusionado à Mash1 e Ngn2 revelou que a ativação dos receptores P2Y2/P2Y4 aumentou a expressão estável de Mash1 enquanto que ativação do receptor P2X7 levou ao aumento de Ngn2. Além disso, células na presença do quelante de cálcio extracelular (EGTA) ou do depletor dos estoques intracelulares de cálcio do retículo endoplasmático (thapsigargin) apresentaram redução na expressão de Mash1 e Ngn2, indicando que ambos são regulados pela sinalização de cálcio. A investigação dos canais de cálcio voltagem-dependentes demonstrou que o influxo de cálcio gerado por despolarização da membrana de NSC diferenciadas de CTE é decorrente da ativação de canais de cálcio voltagem-dependentes do tipo L. Além disso, esse influxo pode controlar o destino celular por estabilizar expressão de Mash1 e induzir a diferenciação neuronal por fosforilação e translocação do fator de transcrição CREB. Esses dados sugerem que os receptores P2X7, P2Y2, P2Y4 e canais de cálcio voltagem-dependentes do tipo L podem modular as oscilações espontâneas de cálcio durante a diferenciação neural e consequentemente alteram o padrão de expressão de Mash1 e Ngn2 favorecendo a decisão do destino celular neuronal. / Purinergic receptors and voltage gated Ca2+ channels have been attributed with developmental functions including gastrulation and neural differentiation. Upon activation, nucleotide-activated P2 purinergic receptor and voltage-gated Ca2+ channel subtypes trigger intracellular calcium transients controlling cellular processes. Here, we studied the participation of voltage-gated calcium channels and P2 receptor activity in spontaneous calcium transients and consequent regulation expression of transcription factors related to retinoic acid-induced neurogenesis of mouse neural stem and embryonic stem cells (ESC). In embryonic pluripotent stem cells, proliferation is accelerated by P2X7 receptor activation, while receptor expression / activity needs to be down-regulated for the progress of neuroblast differentiation. Moreover, along neural differentiation time lapse imaging with means of a cytosolic calcium-sensitive fluorescent probe provided different patterns of spontaneous calcium transients (waves and spikes) showing that both, frequency and amplitude increased along differentiation. Cells treated with the inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor Xestospongin C showed spikes but not waves, indicating that waves exclusively depended on calcium release from endoplasmic reticulum by IP3R activation. Cells treated with the P2X7 receptor subtype agonist Bz-ATP and the P2Y2 and P2Y4 receptor 2-S-UTP increased frequency and amplitudes of calcium transients, mainly spikes, in embryonic telencephalon neural stem cells (NSC) and NSC pre-differentiated from ESC. Data obtained by luminescence time lapse imaging of stable transfected cells with Mash1 or Ngn2 promoter-protein fusion to luciferase reporter construct revealed increased Mash1 expression due to activation of P2Y2/P2Y4 receptor subtypes, while increased expression of Ngn2 was observed following P2X7 receptor activation. In addition, cells imaged in presence of the extracellular calcium chelator EGTA or following endoplasmic reticulum calcium store depletion by thapsigargin showed a decrease in Mash1 and Ngn2 expression, indicating that both are regulated by calcium signaling. Investigation of the roles of voltage gated Ca2+ channels in neural differentiation showed that Ca2+ influx in NSC pre-differentiated from ESC is due to membrane depolarization and L-type voltage gated Ca2+ channel activation, thereby controlling cell fate decision, by stabilizing the expression of MASH1 and inducing differentiation, by phosphorylation of the transcription factor CREB. Altogether these data suggest that P2X7, P2Y2, P2Y4 receptors and L-type voltage gated Ca2+ channels can modulate spontaneous calcium oscillations during neural differentiation and consequently change the Mash1 and Ngn2 expression patterns, thus favoring the cell fate decision to the neuronal phenotype.

Receptores purinérgicos e canais de cálcio voltagem-dependentes estão envolvidos em diversos processos biológicos como na gastrulação, durante o desenvolvimento embrionário, e na diferenciação neural. Quando ativados, canais de cálcio voltagem-dependentes e receptores purinérgicos do tipo P2, ativados por nucleotídeos, desencadeiam transientes de cálcio intracelulares controlando diversos processos biológicos. Neste trabalho, nós estudamos a participação de canais de cálcio voltagem-dependentes e receptores do tipo P2 na geração de transientes de cálcio espontâneos e sua regulação na expressão de fatores de transcrição relacionados com a neurogênese utilizando como modelo células tronco (CTE) induzidas à diferenciação em células tronco neurais (NSC) com ácido retinóico. Descrevemos que CTE indiferenciadas podem ter a proliferação acelerada pela ativação de receptores P2X7, enquanto que a expressão e a atividade desse receptor precisam ser inibidas para o progresso da diferenciação em neuroblasto. Além disso, ao longo da diferenciação neural, por análise em tempo real dos níveis de cálcio intracelular livre identificamos 3 padrões de oscilações espontâneas de cálcio (onda, pico e unique), e mostramos que ondas e picos tiveram a frequência e amplitude aumentadas conforme o andamento da diferenciação. Células tratadas com o inibidor do receptor de inositol 1,4,5-trifosfato (IP3R), Xestospongin C, apresentaram picos mas não ondas, indicando que ondas dependem exclusivamente de cálcio oriundo do retículo endoplasmático pela ativação de IP3R. NSC de telencéfalo de embrião de camundongos transgênicos ou pré-diferenciadas de CTE tratadas com Bz-ATP, o agonista do receptor P2X7, e com 2SUTP, agonista de P2Y2 e P2Y4, aumentaram a frequência e a amplitude das oscilações espontâneas de cálcio do tipo pico. Dados, obtidos por microscopia de luminescência, da expressão em tempo real de gene repórter luciferase fusionado à Mash1 e Ngn2 revelou que a ativação dos receptores P2Y2/P2Y4 aumentou a expressão estável de Mash1 enquanto que ativação do receptor P2X7 levou ao aumento de Ngn2. Além disso, células na presença do quelante de cálcio extracelular (EGTA) ou do depletor dos estoques intracelulares de cálcio do retículo endoplasmático (thapsigargin) apresentaram redução na expressão de Mash1 e Ngn2, indicando que ambos são regulados pela sinalização de cálcio. A investigação dos canais de cálcio voltagem-dependentes demonstrou que o influxo de cálcio gerado por despolarização da membrana de NSC diferenciadas de CTE é decorrente da ativação de canais de cálcio voltagem-dependentes do tipo L. Além disso, esse influxo pode controlar o destino celular por estabilizar expressão de Mash1 e induzir a diferenciação neuronal por fosforilação e translocação do fator de transcrição CREB. Esses dados sugerem que os receptores P2X7, P2Y2, P2Y4 e canais de cálcio voltagem-dependentes do tipo L podem modular as oscilações espontâneas de cálcio durante a diferenciação neural e consequentemente alteram o padrão de expressão de Mash1 e Ngn2 favorecendo a decisão do destino celular neuronal. / Purinergic receptors and voltage gated Ca2+ channels have been attributed with developmental functions including gastrulation and neural differentiation. Upon activation, nucleotide-activated P2 purinergic receptor and voltage-gated Ca2+ channel subtypes trigger intracellular calcium transients controlling cellular processes. Here, we studied the participation of voltage-gated calcium channels and P2 receptor activity in spontaneous calcium transients and consequent regulation expression of transcription factors related to retinoic acid-induced neurogenesis of mouse neural stem and embryonic stem cells (ESC). In embryonic pluripotent stem cells, proliferation is accelerated by P2X7 receptor activation, while receptor expression / activity needs to be down-regulated for the progress of neuroblast differentiation. Moreover, along neural differentiation time lapse imaging with means of a cytosolic calcium-sensitive fluorescent probe provided different patterns of spontaneous calcium transients (waves and spikes) showing that both, frequency and amplitude increased along differentiation. Cells treated with the inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor Xestospongin C showed spikes but not waves, indicating that waves exclusively depended on calcium release from endoplasmic reticulum by IP3R activation. Cells treated with the P2X7 receptor subtype agonist Bz-ATP and the P2Y2 and P2Y4 receptor 2-S-UTP increased frequency and amplitudes of calcium transients, mainly spikes, in embryonic telencephalon neural stem cells (NSC) and NSC pre-differentiated from ESC. Data obtained by luminescence time lapse imaging of stable transfected cells with Mash1 or Ngn2 promoter-protein fusion to luciferase reporter construct revealed increased Mash1 expression due to activation of P2Y2/P2Y4 receptor subtypes, while increased expression of Ngn2 was observed following P2X7 receptor activation. In addition, cells imaged in presence of the extracellular calcium chelator EGTA or following endoplasmic reticulum calcium store depletion by thapsigargin showed a decrease in Mash1 and Ngn2 expression, indicating that both are regulated by calcium signaling. Investigation of the roles of voltage gated Ca2+ channels in neural differentiation showed that Ca2+ influx in NSC pre-differentiated from ESC is due to membrane depolarization and L-type voltage gated Ca2+ channel activation, thereby controlling cell fate decision, by stabilizing the expression of MASH1 and inducing differentiation, by phosphorylation of the transcription factor CREB. Altogether these data suggest that P2X7, P2Y2, P2Y4 receptors and L-type voltage gated Ca2+ channels can modulate spontaneous calcium oscillations during neural differentiation and consequently change the Mash1 and Ngn2 expression patterns, thus favoring the cell fate decision to the neuronal phenotype.

On admet communément que les tumeurs proviennent de cellules échappant aux contrôles homéostatiques qui sous-tendent les structures histologiques saines et que le phénotype d’une cellule n’est pas le résultat de processus génétiques et biochimiques déterministes mais la conséquence stochastique de réseaux de régulation intra- et intercellulaires. Ce doctorat vise à étudier quantitativement l’homéostasie phénotypique de populations cellulaires et à présenter une approche à la question fondamentale, mais jusqu’alors jamais étudiée, concernant l’autonomie versus le contrôle collectif du devenir des cellules. Nous avons étudié sur le long terme, par cytométrie de flux et dans des conditions 2D puis 3D, le niveau d’expression de CD24 et CD44 de deux lignées cellulaires de cancer du sein (SUM149-PT et SUM159-PT). Trois phénotypes ont été isolés (CD24-/CD44+, CD24+/CD44+, CD24-/CD44-), ce dernier n’avait pour le moment pas été documenté dans la littérature. Le comportement phénotypique des sous-populations CD44-low et CD44-high a été caractérisé en évaluant leur proportion et en analysant leur spectre de fluorescence. Ainsi nous avons observé des comportements périodiques d’apparition et de disparition de pool de cellules caractéristiques des lignées et une re-diversification des phénotypes pour chacune des sous-population. Seule la population issue de CD24-/CD44- re-diversifiée présente le même équilibre que la population initiale non triée. En 3D, le processus de re-diversification a été observé dans les tumorsphères issues de CD24-/CD44+ et CD24+/CD44+. Les cellules CD24-/CD44- n’ont pas ce potentiel mais survivent néanmoins à l’anoïkis. Ces comportements laissent penser qu’il existe une coordination intercellulaire régulant l’équilibre des proportions phénotypiques. Pour découvrir les règles sociales régissant l’organisation spatiale inter-phénotypique, nous avons mis en place un rapporteur des variations du niveau d’expression endogène des marqueurs d’intérêt et élaboré un modèle théorique d’interactions cellulaires. Ce travail a conforté notre hypothèse selon laquelle il s’établit des règles sociales inter-cellulaires déterminant l’expression phénotypique à l’échelle uni- et pluricellulaire. / It is commonly accepted that tumors arise from cells that escape the homeostatic controls which underlie the healthy histological structure and that cell phenotype is not the result of deterministic biochemical and genetic processes, but rather the stochastic and dynamic outcome of multiple intra- and intercellular regulation networks. This PhD aims to quantitatively study the phenotypic homeostasis of the cell populations and to present an approach to the fundamental question, never heretofore studied, regarding the autonomy versus collective control of cell fate. We studied in the long run, using flow cytometry and in 2D and 3D conditions, the level of expression of CD24 and CD44 of two breast cancer cell lines (SUM149-PT and SUM159-PT). Three phenotypes were isolated (CD24-/CD44+, CD24+/CD44+, CD24-/CD44-), the latter had not previously been documented in the literature. The phenotypic behavior of CD44-low and CD44-high subpopulations has been characterized by assessing their proportion and analyzing the fluorescence map. Thereby, we observed both a periodic behavior of appearance and disappearance of pool of cells characteristics of each cell lines and a phenotypic re-diversification for each subpopulation. Only the resulting population derived from CD24-/CD44- provided the same balance as the original unsorted population. 3D re-diversification process was observed in tumorspheres from CD24-/CD44+ and CD24+/CD44+. The cells CD24-/CD44did not have that potential but nonetheless outlived anoikis. These behaviors suggest that there is an inter-cell coordination regulating the balance of phenotypic proportions. To discover the social rules regulating inter-phenotypic spatial organization, we have set up a reporter of the endogenous variations of CD24 and CD44 and developed a theoretical model of cell interactions. This work has confirmed our hypothesis that inter-cellular social rules are determining the phenotypic expression at both the uni- and multicellular scales.

Zelluläre Entscheidungen, wie z. B. die Differenzierung von T-Helferzellen (Th-Zellen) in spezialisierte Effektorlinien, haben großen Einfluss auf die Spezifität von Immunreaktionen. Solche Reaktionen sind das Ergebnis eines komplexen Zusammenspiels einzelner Zellen, die über kleine Signalmoleküle, so genannte Zytokine, kommunizieren. Die hohe Anzahl der Komponenten, sowie deren komplizierte und oft nichtlineare Interaktionen erschweren dabei die Vorhersage, wie bestimmte zelluläre Reaktionen erzeugt werden. Aus diesem Grund sind die globalen Auswirkungen der gezielten Beeinflussung einzelner Zellen oder spezifischer Signalwege nur unzureichend verstanden. So wirken beispielsweise etablierte Behandlungen von Autoimmunkrankheiten oft nur bei einem Teil der Patienten. Durch Einzelzellmethoden wie Live-Cell-Imaging, Massenzytometrie und Einzelzellsequenzierung, können Immunzellen heutzutage quantitativ auf mehreren Ebenen charakterisiert werden. Diese Ansammlung quantitativer Daten erlaubt die Formulierung datengetriebener Modelle zur Vorhersage von zellulären Entscheidungen, allerdings fehlen in vielen Fällen Methoden, um die verschiedenen Daten auf geeignete Weise zu integrieren und zu annotieren. Die vorliegende Arbeit befasst sich mit quantitativen Modellformulierungen für die Entscheidungsfindung von Zellen im Immunsystem mit dem Schwerpunkt auf Lymphozytenproliferation, -differenzierung und -tod. / Cellular decisions, such as the differentiation of T helper (Th) cells into specialized effector lineages, largely impact the direction of immune responses. Such population-level responses are the result of a complex interplay of individual cells which communicate via small signaling molecules called cytokines. The system's complexity, stemming not only from the number of components but also from their intricate and oftentimes non-linear interactions, makes it difficult to develop intuition for how cellular responses are actually generated. Not surprisingly, the global effects of targeting individual cells or specific signaling pathways through perturbations are poorly understood. For instance, common treatments of autoimmune diseases often work for some patients, but not for others. Recently developed methods such as live-cell imaging, mass cytometry and single-cell sequencing now enable quantitative characterization of individual immune cells. This accumulating wealth of quantitative data has laid the basis to derive predictive, data-driven models of immune cell behavior, but in many cases, methods to integrate and annotate the data in a way suitable for model formulation are missing. In this thesis, quantitative workflows and methods are introduced that allow to formulate data-driven models of immune cell decision-making with a particular focus on lymphocyte proliferation, differentiation and death.