Academic literature on the topic 'Self-signalling'

Self-incompatibility (SI) is a genetic mechanism which prevents self-fertilisation via the recognition and rejection of ‘self’ pollen. In the self-incompatible species Papaver rhoeas L., rejection of incompatible pollen is achieved through interaction of the female and male S-determinants, PrsS and PrpS, respectively. This interaction results in a Ca\(^{2+}\)-dependent signalling cascade in the ‘self’ pollen, which mediates programmed cell death (PCD). To date, many downstream effects of SI signalling cascade have been identified, including actin depolymerization, the formation of actin foci, and the activation of caspase-like activities. Work presented in this thesis identified the involvement of Reactive Oxygen Species (ROS) and Nitric Oxide (NO) in the SI response, and the temporal and spatial patterns were characterized. Other studies identified SI-induced cytosolic acidification as a key step in SI. Moreover, investigation of the role of ROS, NO and H\(^+\) revealed that they all play a role in triggering key features of SI: actin foci formation and caspase-3-like activity. Other studies also provided the first evidence for vacuolar breakdown in SI in this species. Data presented also show the first documentation of SI-induced alterations in phospholipids. Together these data further our understanding of mechanisms involved in the complex SI signalling network.

Canonical Wnt signalling influences the 'sternness' of both embryonic stem (ES) cells and haemopoietic stem cells (RSC). In this study we investigated how modulation of the canonical Wnt pathway affected ES cell self-renewal and haemopoietic differentiation. We observed that Wnt activation suppressed differentiation whereas inhibiting Wnt signalling enhanced differentiation. Relative Quantification using TaqMan Pluripotency Array cards ™ indicated that genes correlating to 'sternness' such as AFP, Tert, Nanog, Zgp 42, Tcf were up-regulated along with characteristic mesodermal markers (Brachyury, Pecam J, Cdh 5, Nodal, Myo DJ) whereas differentiation markers (Myf 5, Foxa 2, Pax 6, Actc, Neuro DJ) were down regulated following activation of the pathway using the GSK3 inhibitor BIO or expression of dominant positive ~-catenin (DP-~C). Chromatin Immunoprecipitation technology demonstrated a complex interplay occurring between ~-cateninlTCF/LEFlBrachyury and Nanog expression following Wnt signalling. In addition during haemopoietic differentiation we demonstrate that at the early hemangioblast stage, the primitive erythroid genes (Pecaml , LM02 and Tie2) , genes related to globin switching (Hbz, Hbb-BHJ, Hba-al , Hba-a2, Hbb-bJ Hbb-b2) and the definitive erythroid genes and haematopoietic regulators PUJ, GATA J, 3 and 4, EPO-R and KLF were all up- regulated following Wnt activation. A higher percentage of cells at this stage expressed Scal , c-Kit, CD44 and CD45 compared to control cells. Furthermore when directed to form multipotent progenitors, Wnt activation resulted in cells having a megakaryotype erythroid progenitor phenotype characterised by high CD4l, CD7l, CD45 and CD24 and Ter1l91ow expression. In colony assays following myeloid differentiation Wnt activation suppressed granulocyte-macrophage colony formation, resulting in a significant increase in erythroid and mixed colonies. Overall this study demonstrates that activation of the canonical Wnt pathway not only maintains 'sternness' during mesodermal differentiation, but also enhances hematopoietic differentiation towards the erythroid lineage, thus establishing an important role for this pathway in the onset of primitive and definitive erythropoiesis.

Many flowering plants are hermaphrodite, which poses the problem of self-fertilisation and the subsequent loss of genetic fitness in the offspring. To prevent this, plants have developed a genetically controlled mechanism called self-incompatibility (SI) which allows self (incompatible) pollen to be recognised and rejected before fertilisation can occur. The SI response of Papaver rhoeas (field poppy) has been extensively studied at the molecular and cellular level. Rejection of incompatible pollen occurs on the stigma surface when the pollen S-determinant PrpS, a transmembrane protein, interacts with the stigmatic S-determinant, secreted S-proteins. This triggers a calcium-mediated signalling cascade that targets the cytoskeleton and results in programmed cell death (PCD) of incompatible pollen. Work presented in this thesis investigated the localisation of PrpS and S-proteins. Other studies investigated the role of the cytoskeleton in SI. These demonstrated the involvement of the microtubule cytoskeleton for the first time. Microtubules were rapidly depolymerised and this was implicated in signalling to PCD. The actin cytoskeleton has previously been shown to exhibit biphasic alterations during SI involving depolymerisation, followed by formation of F-actin foci. Studies described here represent the first steps toward characterisation of the F-actin foci. Their potential involvement in PCD and signalling is discussed.

[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<br>[CAT] 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<br>[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.<br>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.<br>Mnatsakanyan Movsesyan, H. (2019). ROLE OF IONS IN STEM CELLS SIGNALLING [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/123063<br>TESIS

Supramolecular chemistry is the chemistry of the non-covalent bond, i.e. all those chemical processes relying on non-covalent interactions between molecules. In the last decades, chemists have learned to exploit non-covalent interactions for the construction of sensors, catalysts and materials. In particular self-assembly, defined as the spontaneous organization of small molecules into a well-defined structure has emerged as the most attractive way to prepare highly complex molecular architectures of nanosized dimensions. In this Thesis, the self-assembly of small molecules on the surface of monolayer protected gold nanoparticles (Au NPs) is introduced as an approach toward the realization of complex chemical systems with minimal synthetic efforts. An essential role is played by Au NPs as they provide a robust multivalent surface for the binding of external molecules. The unique features of Au NPs such as stability, biocompatibility, ease of preparation and optoelectronic properties have led to their wide-spread use in a variety of functional supramolecular systems. The Au NPs used in this Thesis have a monolayer composed of thiols terminating with a 1,4,7-triazacyclononane (TACN)•Zn2+ complex. The resulting multivalent cationic surface provides a scaffold for the binding of small anionic molecules (e.g. peptides and nucleotides). In the first part, the attention is focused on understanding the interactions that drive the self-assembly of small oliogoanions on the monolayer surface. This process was studied by means of fluorescence spectroscopy, taking advantage of the well-known ability of gold clusters to quench the fluorescence of bound fluorophores. It emerged that the high affinity is determined by i) the number of negative charges of the oligoanion, ii) the chemical nature of the anionic groups and iii) the presence of hydrophobic moieties able to penetrate into the apolar part of the monolayer. These studies also showed that the metal ions in the monolayer acts as regulatory elements of the self-assembly process. It was shown that the valency of the self-assembled system, i.e. the number of bound molecules , can be precisely controlled in a reversible manner by the addition and removal of Zn2+ from the monolayer. The ability to control the self-assembly process was demonstrated by showing that a mixture of two different oligoanions, with the same number of negative charges but with different anionic groups (phosphate vs carboxylate), spontaneously self-sorted on two different monolayer surfaces, thus creating two discrete topological domains in a homogeneous system. In the second part of the thesis, the obtained knowledge was used for the development of supramolecular sensors for the detection of small molecules and enzymes. The self-assembly of multiple fluorescence indicators on the monolayer surface resulted in a dynamic responsive surface able to generate finger-print patterns upon the addition of eight different di- and tri-nucleotides. The ability of the system to discriminate between ATP and ADP was then used for the development of a protein kinase assay relying on the monitoring of ATP→ADP conversion. With the aim to improve the sensitivity of this assay, an alternative system was subsequently developed in which ATP→ADP conversion led to activation of the catalytic activity of Au NPs. Also in this case a crucial role was played by the Zn2+ metal ions in the monolayer, by the formation of catalytic pockets able to catalyze a chromogenic reaction generating the output signal. In the final chapter all concepts were used to set up a dissipative system able to generate a transient signal, whose duration is determined by the amount of ATP added to the system. This last methodology provides for a straightforward way to gain temporal control over supramolecular processes, showing important analogies with the behaviours of natural systems.<br>La chimica supramolecolare è definita come la chimica del legame non covalente, ovvero la chimica di tutti quei processi chimici che dipendono da interazioni non-covalenti tra molecole. Negli ultimi decenni, i chimici hanno imparato a sfruttare le interazioni non covalenti nello sviluppo di sensori, catalizzatori e materiali. In particolare, l'autoassemblaggio, definito come l'organizzazione spontanea di piccole molecole in una struttura ben definita, si è dimostrato essere il modo più conveniente per preparare architetture molecolari molto complesse e di dimensioni nanometriche. In questa Tesi, l'auto-assemblaggio di piccole molecole sulla superficie di nanoparticelle d'oro funzionalizzate (Au NPs) è usato come approccio per la realizzazione di sistemi chimici complessi, senza l'ausilio di complicate vie di sintesi. Le Au NPs svolgono un ruolo fondamentale, in quanto forniscono una robusta superficie polivalente per il legame con molecole esterne. Le caratteristiche uniche delle Au NPs, quali la stabilità, la biocompatibilità, la facilità di preparazione e le proprietà optoelettroniche, hanno portato ad una loro larga diffusione. Le Au NPs utilizzate in questa Tesi sono ricoperte da un monostrato organico composto da tioli la cui unità periferica è un legante (1,4,7-triazaciclononano) in grado di complessare ioni metallici come lo Zn2+. La superficie cationica multivalente che ne deriva fornisce un'ottima piattaforma per il legame di piccole molecole anioniche (per esempio peptidi e nucleotidi). Nella prima parte di questa Tesi, l'attenzione è stata focalizzata sulla comprensione delle interazioni che guidano l'auto-assemblaggio di piccoli oliogoanioni sulla superficie del monostrato. Questo processo è stato studiato mediante spettroscopia di fluorescenza, sfruttando le note capacità dei cluster d'oro di spegnere la fluorescenza di fluorofori ad essi legati. Da questi studi è emerso che l'alta affinità è determinata i) dal numero di cariche negative dell'oligoanione, ii) dalla natura chimica dei gruppi anionici e iii) dalla presenza di porzioni idrofobiche in grado di penetrare nella parte apolare del monostrato. Questi studi hanno inoltre dimostrato che gli ioni metallici nel monostrato sono in grado di regolare il processo di auto-assemblaggio. È stato dimostrato che la valenza del sistema auto-assemblato, ovvero il numero di molecole legate, può essere controllata con precisione in modo reversibile attraverso l'aggiunta e la rimozione di Zn2+. La capacità di controllare il processo di auto-assemblaggio è stato confermato dimostrando come due oligoanioni differenti, aventi lo stesso numero di cariche negative ma diversi gruppi anionici (fosfato vs carbossilato), siano capaci di auto-organizzarsi spontaneamente sulla superficie di due monostrati differenti, creando due domini topologici in un sistema omogeneo. Nella seconda parte della Tesi, la conoscenza acquisita è stata utilizzata per lo sviluppo di sensori supramolecolari atti alla rivelazione di piccole molecole ed enzimi. Attraverso l'auto-assemblaggio di più indicatori di fluorescenza sulla superficie di un unico monostrato è stato possibile realizzare una superficie in grado di generare segnali specifici in risposta all'aggiunta di otto diversi nucleotidi. La capacità del sistema di discriminare tra ATP e ADP è stata poi fruttata per lo sviluppo di un saggio per protein-chinasi, basato sul monitoraggio della conversione ATP→ADP. Al fine di migliorare la sensibilità di questo saggio, è stato successivamente sviluppato un sistema alternativo, in cui la conversione ATP→ADP porta all'attivazione dell'attività catalitica delle Au NPs. Anche in questo caso, gli ioni Zn2+ presenti nel monostrato giocano un ruolo cruciale, formando dei siti catalitici in grado di catalizzare una reazione cromogena e quindi di generare un segnale rilevabile. Nel capitolo finale, questi concetti sono stati usati per sviluppare un sistema dissipativo in grado di generare un segnale transiente, la cui durata dipende dalla quantità di ATP aggiunto al sistema. Quest'ultima metodologia fornisce un modo semplice per controllare processi supramolecolari a livello temporale, mostrando importanti analogie proprie dei sistemi naturali.

Chez la souris, le Leukemia Inhibitory Factor (LIF) joue un rôle clé dans le maintien des cellules souches embryonnaires (ES) à l’état pluripotent. Le LIF agit en activant le facteur de transcription STAT3 via les kinases Jak. Cette activation est nécessaire et suffisante au maintien des cellules ES en autorenouvellement en présence de sérum. Une étude du transcriptome de STAT3 réalisée au laboratoire a permis d’identifier plusieurs gènes cibles de ce facteur, parmi lesquels plusieurs gènes inconnus. L’un d’eux, le gène 1456160_at, est fortement exprimé dans les cellules ES de souris et son expression diminue après induction de la différenciation. Ce gène a été appelé Asgard pour Another Self-renewal GuARDian. La caractérisation et le séquençage de ce gène ont permis de mettre en évidence qu'Asgard code pour un microARN. De nombreux microARNs jouent un rôle clé dans le maintien de l'autorenouvellement des cellules ES et dans le contrôle de la différenciation. Des expériences d’inhibition et de surexpression ont permis de montrer que Asgard est impliqué dans la régulation de la différenciation endoderme versus mésoderme. Des analyses préliminaires ont permis d’identifier Pbx3, FoxA2 et Sox17 comme cibles potentielles. Bien que les mécanismes d’action du microARN Asgard restent à confirmer, ce travail a permis d’identifier un nouveau gène clé de l'autorenouvellement des cellules ES de souris<br>The Leukemia Inhibitory Factor (LIF) activates the transcription factor STAT3, which results in the maintenance of mouse embryonic stem cells in the undifferentiated state by inhibiting mesodermal and endodermal differentiation. We identified several target genes of STAT3 by transcriptomic analysis. Among them, we focused on an unknown gene referred as 1456160_at on Affymetrix array. This gene is highly expressed in embryonic stem cells and its expression level decreases during differentiation. We named this gene Asgard for Another Self-renewal GuARDian. Its characterization and sequencing revealed that Asgard encodes for a microRNA sequence. Several microRNAs have been shown to play key role in the maintenance of self-renewal of mouse ES cells and in the control of differentiation. Inhibition and overexpression assays showed that Asgard inhibits endodermal differentiation in order to maintain self-renewal. Through preliminary analysis, we identified Pbx3, FoxA2 and Sox17 as potential targets of the microRNA Asgard. Our work enables us to identify a new key gene of self-renewal of mouse ES cells

In der vorliegenden Arbeit werden eine sichere FPGA-Stellwerksplattform und ein Transformationsverfahren entwickelt, mit dem die Schaltungen bestehender Relaisstellwerke in eine FPGA-Logik überführt werden können. Die FPGA-Stellwerksplattform ersetzt die Innenanlage eines Relaisstellwerks. Ihre Schnittstellen entsprechen den bisherigen Schnittstellen am Kabelabschlussgestell und zur Bedien- und Meldeeinrichtung. Damit ist eine einfache Migration bestehender Stellwerke möglich. Das Sicherheitskonzept basiert auf einer zweikanaligen Struktur mit sicherem Vergleicher und zusätzlichen Selbsttests zur schnellen, datenflussunabhängigen Ausfalloffenbarung. Die erreichbare Gefährdungsrate liegt im Bereich von SIL 4 und entspricht damit dem Sicherheitsziel für Stellwerke der Deutschen Bahn. Die Transformation sieht eine Trennung der Stellwerkslogik in Logik- und Leistungsteil vor. Der Logikteil wird auf dem FPGA realisiert. Die im Leistungsteil verbliebenen Kontakte und Überwacherrelais werden durch sichere Stellteile ersetzt. Die logischen Ansteuerbedingungen der Relais werden in Schaltnetze überführt. Die gesteuerten Relais werden durch Instanzen generischer Zustandsmodelle ersetzt. Für jeden verwendeten Relaistyp wurde ein entsprechendes Modell entwickelt, das bei der Transformation als Baustein eingesetzt werden kann. Die generischen Zustandsmodelle berücksichtigen auch die sicherheitsrelevanten konstruktiven Eigenschaften der Relais. So wird bei der Auftrennung einer Schaltung in Logik- und Leistungsteil sichergestellt, dass die in getrennte Schaltungsteile überführten Öffner und Schließer eines Relais nie gleichzeitig geschlossen sein können (Zwangsführung der Kontakte). Dies ist eine Voraussetzung für die Beibehaltung der sicherheitsrelevanten Funktionsbedingungen der Originalschaltung. Das Transformationsverfahren und die implementierten Mechanismen zur Ausfalloffenbarung sind unabhängig von der Anwenderlogik und vom gewählten Schaltkreistyp. Damit kann der generierte VHDL-Code bei Obsoleszenz eines Schaltkreises auch auf andere FPGA-Typen portiert werden. In einer Ressourcenabschätzung wird gezeigt, dass der gewählte Lösungsansatz geeignet ist, die Schaltungen kleinerer Relaisstellwerke vollständig auf einem FPGA zu realisieren. Die Anwendung des vorgestellten Verfahrens wird am Beispiel der Weichengruppe des Stellwerkstyps GS II DR demonstriert. Das Transformationsverfahren ist aber auch für andere Stellwerksbauformen geeignet. Dabei ist es unerheblich, ob diese nach dem tabellarischen Verschlussplanprinzip oder dem Spurplanprinzip arbeiten.