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Les cellules souches embryonnaires (ESCs) sont issues de la masse cellulaire interne (ICM) de blastocystes préimplantatoires. Elles sont pluripotentes c'est-à-dire capables de se différencier dans les trois lignages embryonnaires (ectoderme, mésoderme et endoderme) et de s'autorenouveller, c'est-à-dire de se multiplier indéfiniment en culture. Chez la souris, ces cellules (mESCs) sont à la base des techniques de transgénèse permettant des modifications génétiques ciblées. Chez l'Homme ces cellules (hESCs) représentent un grand espoir en médecine régénérative pour traiter des maladies dégénératives comme les maladies de Parkinson ou de Huntington. Le modèle le plus pertinent de l'espèce humaine est le singe. Cependant l'expérimentation sur cette espèce est soumise à une réglementation très stricte. C'est pourquoi il est nécessaire de développer des modèles alternatifs. C'est dans ce cadre que s'inscrit le lapin, qui est phylogénétiquement plus proche de l'Homme que ne l'est la souris. Mon projet de thèse a eu pour but d'étudier la pluripotence dans les ESCs de lapin (rESCs), afin de pouvoir les utiliser en transgénèse et produire des animaux transgéniques, modèles de maladies humaines. La première partie de ces analyses est regroupée au sein de l'article que notre laboratoire a publié en 2013 dans Biology Open (Osteil et al. 2013). D'autres analyses ont abouti à la dérivation de nouvelles lignées stabilisées dans un état plus proche de celui des cellules de l'ICM. L'ensemble des résultats a permis d'établir des bases solides pour la compréhension de la pluripotence et pour la dérivation d'ESCs dites naïves chez un autre mammifère que la souris / Embryonic stem cells (ESCs) result from cultures of inner cell masses (ICMs) isolated at preimplantation blastocyst stage. ESCs are defined by their self-renewal capacity, characterized by robust proliferation while maintaining plutipotent potential, the ability to give rise to cells from all three germ layers mesoderm, endoderm and ectoderm. Mouse ESCs (mESCs) allow the production of transgenic models by site-specific mutagenesis. Human ESCs (hESCs) represent major hope for regenerative medicine in order to treat degenerative diseases like Parkinson or Huntington. The more relevant model of Human is monkey. However, working on this specie is subjected to extremely strict regulation. Consequently it is very important to develop alternative animal models. Rabbit appears to be a very good candidate, because he is phylogenetically closer to Human than the mouse. My thesis project aimed to study the pluripotency mechanism of rabbit ESCs (rESCs), in order to use these cells for the production of transgenic animal models for human diseases. First part of theses analyses is synthesized in a publication into Biology Open in 2013 (Osteil et al. 2013). Other analyses produced new rESCs lines stabilized in a closer state compared to ICM state. All these results led to obtain solid knowledge on pluripotency and derivation on so-called naïve ESCs in a non-rodent specie

Em nosso trabalho anterior mostramos que dentre as citocinas analisadas, os níveis do mRNA de LIF nas células estromais pediátricas, de SMD e de SMD-LMA foram maiores quando comparados às células estromais de crianças saudáveis. No presente estudo, observamos um aumento tempo dependente nos níveis da proteína LIF após adição de SFB em todas as células analisadas (células estromais de crianças saudáveis, de SMD e de SMD-LMA). O envolvimento de p38, ERK e JNK na expressão LIF nestas células foi determinado pelo uso de inibidores dos membros das proteínas quinase ativadas por mitógenos: ERK (PD98059), p38 (SB302580) e JNK (SP600125) os quais inibiram a produção de LIF nas células estromais de crianças saudáveis, após estas serem estimuladas por SFB. No entanto, os níveis da expressão de LIF-induzido por soro nas células estromais de SMD e de SMD-LMA tratadas com SB302580 (p38) foram significativamente diminuídos, em comparação com a inibição observada no tratamento com PD98059 e SP600125 (p <0001, teste ANOVA). Em adição analisamos as formas fosforiladas de p38 e ERK, após 48hs na ausência ou na presença de soro por diferentes tempos. Níveis de atividade de ERK e do p38 foram inicialmente elevados na ausência de soro. A atividade de p38 foi sustentada após tratamento com SFB, entretanto, ERK apresentou uma variação de atividade durante o tratamento. Sugerimos que a sinalização das MAPKs (p38, ERK e JNK), em resposta a fatores de crescimento presentes no soro, parece desempenhar um papel importante na expressão da LIF em células estromais de crianças saudáveis, mas a sinalização do p38 parece ser funcionalmente mais importante nas mielodisplasias ou naquelas associadas à LMA / Our previous report showed that among the cytokines analysed, LIF mRNA levels in stromal cells from pediatric MDS and MDS-AML were higher as compared to those found in healthy stromal cells. In the present study, we have observed an increased protein LIF levels in a time dependent manner after FCS stimulation in all stromal cells analysed (MDS, MDS-AML and healthy children) and the involvement of p38, ERK and JNK pathways in the LIF expression in these cells was determined. In stromal cells from two healthy children, LIF production was equally inhibited in a dose dependent manner after FCS stimulation by mitogen-activated protein kinase (MAPKs) members inhibitors: ERK (PD98059), p38 (SB302580) and JNK (SP600125). However, in MDS and MDS-AML stromal cells, the levels of LIF-induced by serum, were significantly decreased by SB302580, as compared with the inhibition observed by treatment with PD98059 and SP600125 (p <0,001, ANOVA test). In addition we have analysed the presence of p38 and ERK phosphorylated forms in stromal cells, after 48hs of serum starvation or in the presence of FCS for different times. Activated ERK and p38MAPK levels were initially elevated in the absence of serum. p38MAPK activation was sustained after treatment with FCS, whereas ERK presented a variation of the activated forms during treatment. We suggest that the signalling of the MAPKs (p38, ERK and JNK) in response to growth factors present in the serum, seems to play an important role in the LIF expression by stromal cells of healthy children, but p38 MAPK signalling appears to be functionally more important in MDS and MDS-AML

E consenso que o crescente consumo de combustíveis fósseis na geração de potencia tem provocado uma maior degradação do meio ambiente. Para mitigar os efeitos adversos desse consumo, novas fontes e usos sustentáveis de energia são necessários. O uso de etanol na atual geração de motores de combustão interna demanda informações detalhadas sobre os processos de nebulização, evaporação, mistura e combustão das gotas desse combustível. Adicionalmente, novas estratégias de injeção de combustível em MCIs com injeção direta estão em desenvolvimento, como a injeção estraticada. Essa estratégia consiste na injeção de combustível em elevadas frequências durante as etapas de admissão e combustão do ciclo motor. Neste trabalho, técnicas avançadas de diagnostico a laser são utilizadas para analisar a inuência da frequência de injeção de combustível no processo de combustão de sprays pulsados de etanol, em que as chamas resultantes são estabilizadas por um swirler. A técnica de interferometria por efeito Doppler (PDI) foi utilizada para medir o diâmetro e a componente axial da velocidade das gotas. O campo de velocidades do escoamento de ar foi avaliado pela técnica de velocimetria por imagens de partículas (PIV) em duas taxas de aquisição (7,4 Hz e 2.000 Hz) para avaliar tanto valores médios quanto rastrear estruturas coerentes no escoamento. O mesmo sistema PIV foi utilizado para identificar a posição de aglomerados de gotas pela técnica de espalhamento Mie ao longo de eventos individuais de injeção. Já a técnica de uorescência induzida por laser do radical hidroxila (LIF-OH) foi utilizada com taxa de aquisição de 4.700 Hz para rastrear a região com presença deste radical próximo ao queimador utilizado. Imagens da luminescência química espontânea foram obtidas para observar características globais das chamas. O queimador, posicionado em um ambiente aberto, e composto por um dispositivo swirler e um injetor automotivo. As frequências de injeção de 100 Hz, 250 Hz e 400 Hz foram escolhidas para as análises por produzirem chamas com características que variam desde uma chama estável e ancorada ao queimador até o comportamento similar a uma chama suspensa e instável. Etanol anidro líquido foi utilizado como combustível e fornecido ao injetor a pressão e vazão constantes para todos os casos. Os resultados indicaram que a variação na frequência de injeção produziu sprays com diferentes densidades. A menor frequência de injeção produziu um spray com maior densidade, em que as gotas formaram um aglomerado pouco sensível ao escoamento de ar na linha de centro do queimador. Esse aglomerado produz uma chama ancorada ao queimador com formato alongado e estreito. Já a maior frequência de injeção produziu um spray com menor densidade, permitindo que a dinâmica das gotas seja mais susceptível ao escoamento de ar. Essa maior inuência do escoamento de ar promoveu uma maior dispersão espacial das gotas e um processo de combustão com maior susceptibilidade a instabilidades locais do escoamento. Essas instabilidades locais foram associadas a passagem periódica de estruturas coerentes do escoamento de ar através da região de reação das chamas, produzindo uma chama com presença intermitente do radical OH e ausência de emissão de luminescência química espontânea próximo a saída do queimador. / The increase in consumption of petroleum-based fuels promotes environmental degradation and, to mitigate the adverse effects of this consumption, new sustainable sources and uses of energy are required. The use of ethanol as an option to conventional fuels on current generation of engines and gas turbines requires detailed information on atomization, evaporation, mixture and combustion processes of ethanol droplets. In addition, new strategies to fuel injection in ICEs are being developed, such as the stratied fuel injection. In this strategy, fuel is injected in higher injection frequencies along the air intake and combustion phases of the engine cycle. In this work, advanced techniques of laser diagnostic are applied to analyze the inuence of fuel injection frequency on the behavior of the combustion process of pulsed ethanol sprays, in which the resulting ames are stabilized by a swirler. The Phase Doppler Interferometry (PDI) technique is applied to measure both diameter and axial velocity of droplets produced by the injector under different conditions of injection frequency. The velocity eld of the airow is evaluated by the Particle Image Velocimetry (PIV) using two repetition rates (7,4 Hz and 2.000 Hz) to evaluated both mean values and to track coherent structures in the ow. The PIV equipment is also used to evaluate the position of droplet groups by Mie scattering during the individual injection events. A high repetition rate (4.700 Hz) Laser Induced Fluorescence of hydroxyl radical (LIF-OH) system is applied to track the region with presence of OH in a vertical plane near the exit of the burner. Images of spontaneous chemical luminescence are acquired to analyze general aspects of the ames. The burner consists on a swirler and an automotive injector and is positioned in an open space with quiescent air. The analyzed injection frequencies are 100 Hz, 250Hz and 400 Hz, presenting characteristics ranging from a stable anchored ame to a lifted-like unstable ame. Liquid anhydrous ethanol is delivered to the injector with constant pressure and ow rate for all cases. Results show different spray densities produced by the change in the injection frequency. The case with lower injection frequency produced higher spray densities, in which the formation of large groups of droplets prevents the inuence of the recirculating airow on droplet dynamics. This case presented an anchored, elongated, narrow and stable ame. The case with higher injection frequency produced lower spray densities, in which droplet dynamics are more susceptible to the velocities of the airow. This inuence of the airow promotes spatial dispersion of droplets, been more prone to instabilities on the local combustion process. These local instabilities are associated to the inuence of periodic coherent structures of the ow passing through the reaction zone, which leads to the intermittent presence of OH and the absence of spontaneous chemical luminescence near the injection plane of the burner.

E consenso que o crescente consumo de combustíveis fósseis na geração de potencia tem provocado uma maior degradação do meio ambiente. Para mitigar os efeitos adversos desse consumo, novas fontes e usos sustentáveis de energia são necessários. O uso de etanol na atual geração de motores de combustão interna demanda informações detalhadas sobre os processos de nebulização, evaporação, mistura e combustão das gotas desse combustível. Adicionalmente, novas estratégias de injeção de combustível em MCIs com injeção direta estão em desenvolvimento, como a injeção estraticada. Essa estratégia consiste na injeção de combustível em elevadas frequências durante as etapas de admissão e combustão do ciclo motor. Neste trabalho, técnicas avançadas de diagnostico a laser são utilizadas para analisar a inuência da frequência de injeção de combustível no processo de combustão de sprays pulsados de etanol, em que as chamas resultantes são estabilizadas por um swirler. A técnica de interferometria por efeito Doppler (PDI) foi utilizada para medir o diâmetro e a componente axial da velocidade das gotas. O campo de velocidades do escoamento de ar foi avaliado pela técnica de velocimetria por imagens de partículas (PIV) em duas taxas de aquisição (7,4 Hz e 2.000 Hz) para avaliar tanto valores médios quanto rastrear estruturas coerentes no escoamento. O mesmo sistema PIV foi utilizado para identificar a posição de aglomerados de gotas pela técnica de espalhamento Mie ao longo de eventos individuais de injeção. Já a técnica de uorescência induzida por laser do radical hidroxila (LIF-OH) foi utilizada com taxa de aquisição de 4.700 Hz para rastrear a região com presença deste radical próximo ao queimador utilizado. Imagens da luminescência química espontânea foram obtidas para observar características globais das chamas. O queimador, posicionado em um ambiente aberto, e composto por um dispositivo swirler e um injetor automotivo. As frequências de injeção de 100 Hz, 250 Hz e 400 Hz foram escolhidas para as análises por produzirem chamas com características que variam desde uma chama estável e ancorada ao queimador até o comportamento similar a uma chama suspensa e instável. Etanol anidro líquido foi utilizado como combustível e fornecido ao injetor a pressão e vazão constantes para todos os casos. Os resultados indicaram que a variação na frequência de injeção produziu sprays com diferentes densidades. A menor frequência de injeção produziu um spray com maior densidade, em que as gotas formaram um aglomerado pouco sensível ao escoamento de ar na linha de centro do queimador. Esse aglomerado produz uma chama ancorada ao queimador com formato alongado e estreito. Já a maior frequência de injeção produziu um spray com menor densidade, permitindo que a dinâmica das gotas seja mais susceptível ao escoamento de ar. Essa maior inuência do escoamento de ar promoveu uma maior dispersão espacial das gotas e um processo de combustão com maior susceptibilidade a instabilidades locais do escoamento. Essas instabilidades locais foram associadas a passagem periódica de estruturas coerentes do escoamento de ar através da região de reação das chamas, produzindo uma chama com presença intermitente do radical OH e ausência de emissão de luminescência química espontânea próximo a saída do queimador. / The increase in consumption of petroleum-based fuels promotes environmental degradation and, to mitigate the adverse effects of this consumption, new sustainable sources and uses of energy are required. The use of ethanol as an option to conventional fuels on current generation of engines and gas turbines requires detailed information on atomization, evaporation, mixture and combustion processes of ethanol droplets. In addition, new strategies to fuel injection in ICEs are being developed, such as the stratied fuel injection. In this strategy, fuel is injected in higher injection frequencies along the air intake and combustion phases of the engine cycle. In this work, advanced techniques of laser diagnostic are applied to analyze the inuence of fuel injection frequency on the behavior of the combustion process of pulsed ethanol sprays, in which the resulting ames are stabilized by a swirler. The Phase Doppler Interferometry (PDI) technique is applied to measure both diameter and axial velocity of droplets produced by the injector under different conditions of injection frequency. The velocity eld of the airow is evaluated by the Particle Image Velocimetry (PIV) using two repetition rates (7,4 Hz and 2.000 Hz) to evaluated both mean values and to track coherent structures in the ow. The PIV equipment is also used to evaluate the position of droplet groups by Mie scattering during the individual injection events. A high repetition rate (4.700 Hz) Laser Induced Fluorescence of hydroxyl radical (LIF-OH) system is applied to track the region with presence of OH in a vertical plane near the exit of the burner. Images of spontaneous chemical luminescence are acquired to analyze general aspects of the ames. The burner consists on a swirler and an automotive injector and is positioned in an open space with quiescent air. The analyzed injection frequencies are 100 Hz, 250Hz and 400 Hz, presenting characteristics ranging from a stable anchored ame to a lifted-like unstable ame. Liquid anhydrous ethanol is delivered to the injector with constant pressure and ow rate for all cases. Results show different spray densities produced by the change in the injection frequency. The case with lower injection frequency produced higher spray densities, in which the formation of large groups of droplets prevents the inuence of the recirculating airow on droplet dynamics. This case presented an anchored, elongated, narrow and stable ame. The case with higher injection frequency produced lower spray densities, in which droplet dynamics are more susceptible to the velocities of the airow. This inuence of the airow promotes spatial dispersion of droplets, been more prone to instabilities on the local combustion process. These local instabilities are associated to the inuence of periodic coherent structures of the ow passing through the reaction zone, which leads to the intermittent presence of OH and the absence of spontaneous chemical luminescence near the injection plane of the burner.

Le procédé de traitement du combustible du réacteur à sels fondus (réacteur de génération IV) est un procédé multi-étape dans lequell'extraction des actinides et des lanthanides utilise la technique d'extraction réductrice. Le développement d'un modèle analytique a montré que la mise en contact du sel combustible LiF-ThF4 avec une phase métallique constituée d'un mélange Bi-Li permet l'extraction sélective et quantitative des actinides dans un premier temps, puis l'extraction quantitative des lanthanides dans un second temps. La maitrise de ce procédé nécessite la connaissance des caractéristiques des phases salines impliquées dans le procédé. Les études des propriétés physico-chimiques des sels fluorures fondus ont permis de développer une technique de mesure de la fluoroacidité dans ces milieux via une mesure potentiométrique. Cette technique a permis d'établir un classement de différents mélanges de fluorures fondus en fonction de leur acidité relative. Par ailleurs, une méthode de détermination de la solvatation de solutés dans ces milieux a également été développée par électrochimie afin d'approfondir la connaissance du sel combustible (en particulier solvatation de ThF4 par les ions F-).L'extraction réductrice met également en jeu une phase métallique liquide. Une technique de préparation de cette phase a été développée par électro-réduction de lithium sur une électrode liquide de bismuth en milieu LiCl-LiF. Cette technique permet un bon contrôle de la fraction molaire de lithium introduite dans le bismuth, paramètre essentiel à l'efficacité de l'extraction.Enfin, afin d'optimiser le procédé général de traitement multi-étapes, des méthodes électrochimiques ont été proposées afin de régénérer les différentes phases liquides (salines et métalliques) mise en jeu lors de l'extraction.

Für medizinische Anwendungen sind Stammzellen aufgrund ihrer Eigenschaften (Selbsterneuerung, hohe Proliferationsrate und Differenzierungsmöglichkeit in verschiedene Zelltypen) beispielsweise in den Bereichen des regenerativen Gewebeersatzes und der Zelltherapie sehr interessant. In vivo umgibt die Stammzellen eine definierte Mikroumgebung, die sie unterstützt sich zu teilen, ihren undifferenzierten Status aufrecht zu erhalten und Tochterzellen für das Wachstum, die routinemäßige Erneuerung oder den Ersatz von Gewebe zu produzieren. Diese Mikroumgebungen werden als Stammzellnischen bezeichnet. Für die Kultivierung von Stammzellen in vitro muss die in vivo-Situation möglichst getreu nachgestaltet werden. Ziel der Forschung ist die Schaffung einer künstlichen Umgebung, die sowohl die funktionellen Eigenschaften einer Nische besitzt als auch frei von Risiken xenogener Pathogene oder Gewebeunverträglichkeiten für die Anwendung am humanen Organismus eingesetzt werden kann. Einen Ansatz dafür bietet beispielsweise die Kopplung von Faktoren, die für den Erhalt der Stammzelleigenschaften notwendig sind, an synthetische Oberflächen. Ausgehend vom Bedarf an Kultur- oder Modellsystemen für die Expansion von (embryonalen) Stammzellen sollte in dieser Arbeit analysiert werden, ob alternierende Maleinsäureanhydrid (MA)-Copolymere ein geeignetes Trägersystem für die biofunktionelle kovalente Immobilisierung spezifischer Zytokine sind und dadurch unter anderem als künstliche Stammzellnische Anwendung finden können. MA-Copolymere eignen sich aufgrund ihrer spontanen Reaktion mit Aminogruppen für die Immobilisierung von Proteinen. Das Zytokin LIF (Leukemia inhibitory factor) existiert in vivo auch in immobilisierter Form und ist in embryonalen Mausstammzellen (mESC) allein in der Lage, das Stammzellpotential dieser Zellen zu erhalten. Aus diesem Grund ist LIF für die Analyse der Aufgabenstellung geeignet. Nach der Charakterisierung LIF-modifizierter Oberflächen wurde die biologische Verfügbarkeit des kovalent immobilisierten Zytokins mit Hilfe von LIF-sensitiven Fibroblasten und mESC der Linie R1 überprüft. Anschließend wurde im Mausmodell in vivo der Erhalt der Pluripotenz der mESC durch immobilisiertes LIF analysiert. Dafür standen die Oberflächen Poly(ethylen-alt-maleinsäureanhydrid) (PEMA) und Poly(octadecen-alt-maleinsäureanhydrid) (POMA) jeweils ohne und mit Polyethylenglykol (PEG7)-Modifizierung zur Verfügung, an die LIF kovalent gekoppelt wurde. Zusätzlich wurde LIF physisorptiv an einer Kollagen-Fibronektin-Matrix über hydrolysiertem POMA immobilisiert. Mit Hilfe von radioaktiv markiertem LIF konnte gezeigt werden, dass die Gesamtbeladungsmenge mit Zytokin von den Eigenschaften der MA-modifizierten Träger abhing. Auf PEMA konnten mit steigenden Immobilisierungskonzentrationen höhere Belegungsdichten an der Oberfläche erreicht werden, die im analysierten Bereich eine lineare Abhängigkeit zeigten. Aufgrund der starken Quellung in wässrigen Lösungen war eine Einlagerung von LIF-Molekülen in die Polymerschicht möglich und führte bei hohen Immobilisierungskonzentrationen auch nach 3 Tagen Inkubation mit proteinhaltigem Medium noch zur Verdrängung nicht kovalent gebundener Zytokinmoleküle aus PEMA-Oberflächen. Obwohl ein Teil des LIF in die Polymerschicht eindrang, war der Großteil der Moleküle für einen spezifischen Antikörper zugänglich. Hydrophobe Oberflächen mit POMA konnten bei hohen Immobilisierungskonzentrationen weniger LIF binden und zeigten Sättigungsverhalten der Oberflächen bei einer Belegungsdichte von 178 ng/cm^2 LIF. Eine Freisetzung von LIF nach mehr als 3 Tagen konnte nicht beobachtet werden. Gleichzeitig war hier aufgrund der hydrophoben Polymerseitenketten die Antikörperzugänglichkeit deutlich reduziert. Wegen des geringen Quellungsverhaltens von POMA in wässrigen Lösungen konnte eine Einlagerung des immobilisierten Zytokins in die Polymerschicht aber ausgeschlossen werden. Die kovalente LIF-Immobilisierung über PEG7-Spacer führte im Vergleich zu den nicht PEG-modifizierten Oberflächen PEMA und POMA zu jeweils geringeren Belegungsdichten, ohne dabei den Charakter der Abhängigkeit von der Immobilisierungskonzentration zu verändern (linear für PEMA+PEG7, Sättigung für POMA+PEG7). Die schlechte Antikörperzugänglichkeit von immobilisiertem LIF auf POMA konnte durch die Einführung des PEG7-Spacers deutlich verbessert werden und erreichte einen Wert ähnlich dem der hydrophilen PEMA-Oberflächen. Kovalent immobilisiertes LIF zeigte auf den vier MA-Oberflächen homogene und definiert einstellbare Belegungsdichten auf den einzelnen Proben. Die physisorptive Immobilisierung von LIF an extrazelluläre Matrixkomponenten auf hydrolysiertem POMA führte zu inhomogenen und bereits bei geringen Immobilisierungskonzentrationen instabilen Belegungsdichten. Die Einstellung definierter Belegungsdichten und die homogene Verfügbarkeit des Zytokins sind für die spätere Anwendung bei der Kultivierung wichtig, da so allen Zellen die gleiche definierte Zytokindosis unabhängig von der Oberflächencharakteristik präsentiert wird und Populationsunterschiede vermieden werden. LIF-sensitive Mausfibroblasten der Linie NIH3T3 reagierten auf immobilisiertes LIF mit der Aktivierung des Signalwegproteins STAT3. Durch den direkten Vergleich von STAT3-Aktivierungsprofilen nach Stimulation mit gelöstem oder immobilisiertem LIF konnte gezeigt werden, dass durch beide Präsentationsformen innerhalb der ersten 15 Minuten nach Stimulationsbeginn eine starke Aktivierung von STAT3 erfolgt, die anschließend wieder abklingt. Die Profile beider Präsentationsformen unterschieden sich in ihren Intensitäten nur bei der starken STAT3-Aktivierung. Dabei ergaben sich bei gelöstem LIF aufgrund der größeren Kontaktfläche mit Zytokin (gesamte Zelloberfläche) etwas stärkere Aktivierungen. Durch die sehr ähnlichen Aktivierungsprofile konnte nachgewiesen werden, dass das Zytokin LIF für Zellen zugänglich an MA-Copolymere mit und ohne Spacer-Modifizierung immobilisiert werden kann. Dabei lag ein Teil der Moleküle in einer Konformation und Orientierung gebunden vor, die die Funktionalität des Zytokins erhalten konnten. Zwischen den Oberflächen mit kovalenter LIF-Immobilisierung konnten keine wesentlichen Unterschiede in der STAT3-Aktivierung festgestellt werden. LIF war an all diesen Oberflächen für die LIF-sensitiven NIH3T3 Mausfibroblasten biologisch verfügbar. LIF-abhängige embryonale Mausstammzellen (mESC) reagierten nach 72 Stunden LIF-Stimulation mit der Aktivierung von STAT3. Bei Belegungsdichten ab 8 ng/cm^2 kovalent immobilisiertem LIF auf POMA mit und ohne PEG7-Spacer konnten ähnliche Aktivierungen wie durch die Stimulation mit gelöstem LIF festgestellt werden. Dies bestätigte die biofunktionelle LIF-Immobilisierung. Zwischen den POMA-Oberflächen mit und ohne PEG7 war dabei kein deutlicher Unterschied erkennbar. Eine reduzierte Zugänglichkeit des Antikörpers auf POMA beeinflusste demnach die biologische Verfügbarkeit des Zytokins für die mESC nicht. Der Erhalt des Stammzellpotentials durch kovalent an POMA gebundenes LIF konnte in vitro durch die Präsenz von Oct4 im Zellkern der mESC nachgewiesen werden. Durch die instabile Immobilisierung bei physisorptiver Assoziation des Zytokins an Matrixkomponenten über hydrolysiertem POMA reduzierte sich der Erhalt des Stammzellpotentials auf diesen Oberflächen stark. Kovalent immobilisiertes LIF dagegen konnte auch während der Kultur über mehrere Passagen hinweg die Pluripotenz der murinen ESC erhalten. Nach der Fusion mit Blastozysten beteiligten sich diese kultivierten Zellen in vivo erfolgreich an der Bildung von Chimären. Dabei konnten keine Unterschiede der Chimärenhäufigkeit zwischen der Kultivierung der mESC mit gelöstem oder kovalent an POMA immobilisiertem LIF festgestellt werden. Kovalent an MA-Copolymere immobilisiertes LIF ist demnach in der Lage, gelöstes LIF vollständig zu ersetzen und über mehrere Passagen hinweg allein das Stammzellpotential von mESC zu erhalten. Die Experimente zeigten, dass sich MA-Copolymere für die funktionelle kovalente Immobilisierung von Signalmolekülen eignen. Dabei konnten keine starken Unterschiede bei der Reaktion der Zellen auf die Oberflächen PEMA oder POMA festgestellt werden. Auch die Einführung eines zusätzlichen Spacers war für die Signaltransduktion nach Stimulation mit kovalent immobilisiertem LIF nicht notwendig. Für künftige Arbeiten zur kovalenten Immobilisierung von LIF an MA-Copolymeren ist deshalb aus Stabilitäts- und Effizienzgründen die Oberfläche POMA zu bevorzugen. Diese Favorisierung kann jedoch aufgrund der unterschiedlichen Tertiärstruktur anderer Proteine und ihrer verschiedenen Steifigkeiten sowie bei der Verwendung anderer Zelltypen nicht automatisch für ein anderes Modellsystem übernommen werden. Die Verwendung hydrophiler Oberflächen oder die Kopplung über Spacer sollte demnach in Abhängigkeit vom zu immobilisierenden Protein und den auszusiedelnden Zellen geprüft werden. Die vorgestellte Kopplungsmethode umgeht die Modifikation des Proteins sowie Behandlungen zur Vernetzung des Zytokins. Die Immobilisierungsreaktion ist bei Raumtemperatur und Umgebungsdruck sowie unter sterilen Bedingungen durchführbar. Immobilisierte Zytokine werden homogen kovalent an der Oberfläche gebunden und sind dort für die Zellen zugänglich. Außerdem ermöglicht die Einstellung definierter Belegungsdichten die gezielte Applikation von Zytokindosen. MA-Copolymere sind somit nicht nur für die Kultivierung von Stammzellen unter Erhaltung des Stammzellstatus einsetzbar, sondern eignen sich auch für Differenzierungsstudien. Teilergebnisse dieser Arbeit wurden publiziert unter K. Alberti, R.E. Davey, K. Onishi, S. George, K. Salchert, F.P. Seib, M. Bornhäuser, T. Pompe, A. Nagy, C.Werner, and P.W. Zandstra. Functional immobilization of signaling proteins enables control of stem cell fate. Nat Methods, 5(7):645–650, Jul 2008 und T. Pompe, K. Salchert, K. Alberti, P.W. Zandstra, and C. Werner. Immobilization of growth factors on solid supports for the modulation of stem cell fate. Nat Protocols, 5(6):1042–1050, Jun 2010. / In vitro cultivation of (embryonic) stem cells requires a defined environment. Together different properties as cytokine supplement, extracellular matrix composition or topographic design can mimic this stem cell niche in an artificial system. For mouse embryonic stem cells the cytokine leukemia inhibitory factor (LIF) is able to keep those cells in undifferentiated state and to enhance self renewal without the supplement of other factors. In vivo LIF exists in both diffusible and extracellular matrix immobilized form. This work investigates whether LIF can be immobilized covalently to alternating maleic anhydride (MA)-copolymers in a functional manner. When bioavailable, covalently immobilized LIF should be able to interact with specific cytokine receptor subunits and provide information to keep murine embryonic stem cells in a pluripotent state. In aqueous solution with neutral pH (such as phosphate buffered saline, PBS) and ambient temperature and pressure MA-copolymers react spontaneously with aminogroups and therefore represent a useful support for covalent protein immobilization. Depending on the choice of co-monomer, properties of copolymer vary: ethylene results in hydrophilic poly-(ethylene-alt-maleic anhydride) (PEMA), octadecene in more hydrophobic poly-(octadecene-alt-maleic anhydride) (POMA). LIF can be covalently immobilized onto the MA-copolymers as shown by radiolabeling experiments. The amount of cytokine coupled to PEMA increased linear whereas on POMA saturation could be observed for higher concentrations. A subsequent coupling of a polyethylene glycol spacer (PEG7) further modified the properties and led to more hydrophilic surfaces. The amount of LIF per area decreased in comparison to MA-copolymers without the spacer but the graph characteristics remained unaltered (linear for PEMA+PEG7, saturation for POMA+PEG7). During the first three days in buffer solution supplemented with bovine serum albumin, unbound LIF was displaced and the amount of immobilized cytokine remained stable. This Stability after preincubation allowed to immobilize required amounts of LIF per area. Although hydrophilic surfaces with PEMA showed swelling behavior resulting in increased layer thickness after incubation in PBS, accessibility to LIF for an antibody was not impaired. The amounts per area detected by radiolabeling method and using the antibody were similar and indicated that LIF was not covered by copolymers. For cell culture addition of diffusible as well as immobilized growth factors or cytokines requires dosage control. Frequently it is necessary to provide homogeneous distribution of the factor of interest. In the present study analysis by fluorescence microscopy confirmed homogeneity for surfaces with covalently immobilized LIF (iLIF) but not for LIF physisorbed to extracellular matrix components collagen type I and fibronectin. LIF transduces signals via the JAK/STAT pathway. Preliminary experiments with LIF-sensitive fibroblasts showed similar activation of STAT3 after stimulation with immobilized or diffusible LIF. The results of STAT3 activation revealed an activation profile with high intensities within the first 15 minutes for both immobilized and diffusible LIF followed by decrease. STAT3 activation profiles were similar on different surfaces and independent of LIF presentation mode. These results revealed that fibroblasts could recognize covalently immobilized LIF onto MA-copolymers and were able to activate STAT3. In the absence of LIF mESC start to differentiate within 24 to 36 hours and loose their pluripotency. To confirm the functional immobilization of LIF mouse embryonic stem cells (mESC) were cultivated on iLIF-modified POMA or POMA+PEG7 surfaces for 72 hours and stained for activated STAT3. Results showed a dose-dependent activation increasing with the iLIF amount per area. Higher amounts (8 and 75 ng/cm^2) of iLIF activated STAT3 similar to 10 ng/ml diffusible LIF. Introduction of PEG7 spacer did not further increased STAT3 activation. Both, the amount of ESC marker Oct4 and the percentage of Oct4-positive cells increased with higher amounts of iLIF and showed similar results as obtained with 10 ng/ml diffusible LIF. Murine ESC cultivated on LIF physisorbed to matrix components expressed similar amounts of transcription factor Oct4 compared to unstimulated cells. STAT3 activation and Oct4 expression in the absence of diffusible cytokine indicated a functional covalent immobilization of LIF. To confirm the pluripotency, mESC were stimulated for 6 to 8 subcultures only with iLIF, cell aggregates were fused with mouse embryos and implanted in pseudopregnant surrogate mothers. Three weeks after birth the contribution of mESC aggregates to chimera was evaluated. ESC stimulated with iLIF only contributed to chimera formation with around the same frequency as mESC cultivated with 10 ng/ml diffusible LIF. Thus, iLIF maintained pluripotency of mESC during in vitro expansion and could replace diffusible LIF. As shown by the experiments, MA-copolymers provide a support to covalently immobilize cell signaling molecules in a functional manner. This method of coupling does not need any protein modification or cross-linking treatment after protein incubation. Reaction can be carried out under sterile conditions at ambient temperature and pressure. The immobilized ligand is distributed equally on the supporting copolymer and the adjustment of required ligand amounts is possible. These properties characterize MA-copolymers as a suitable support to immobilize cell signaling molecules not only for keeping the stem cell fate but also for differentiation studies. Parts of this work were published: K. Alberti, R.E. Davey, K. Onishi, S. George, K. Salchert, F.P. Seib, M. Bornhäuser, T. Pompe, A. Nagy, C.Werner, and P.W. Zandstra. Functional immobilization of signaling proteins enables control of stem cell fate. Nat Methods, 5(7):645–650, Jul 2008. T. Pompe, K. Salchert, K. Alberti, P.W. Zandstra, and C. Werner. Immobilization of growth factors on solid supports for the modulation of stem cell fate. Nat Protocols, 5(6):1042–1050, Jun 2010.

Le travail présenté ici s'insère dans le cadre des études amont financées par le CNES et SNECMA qui visent à mieux appréhender, et simuler les écoulements cavitants dans les inducteurs à l'amont des turbopompes spatiales. Compte tenu de la complexité des mécanismes mis en jeu dans ce type de géométrie, il a été décidé de réaliser une étude dans une géométrie académique de type marche descendante cavitante comportant les différents mécanismes physiques inhérents aux écoulements de backflow (écoulements instationnaires décollés) présents dans les inducteurs. Cette géométrie simplifiée a permis la mise en place d'une instrumentation de pointe telle que la densitométrie par absorption de rayons X résolue en temps ou encore la PIV-LIF 2D, 3C haute cadence pour la mesure du champ de vitesse liquide. Ces techniques étant couplées à la mesure du champ de pression pariétale, il a donc été possible d'estimer les interactions mutuelles entre la turbulence et la cavitation dans ce type d'écoulement.Les mesures couplées aux champ de pression ont également permis d'utiliser des techniques de traitement POD-LSE pour estimer des termes de corrélations densité-vitesse nécessaires à la validation des modèles de type transport de taux de vide. De manière générale, la cavitation engendre des modifications notables d'origine dilatatoire sur l'agitation turbulente et modifie la dynamique tourbillonnaire. Ces résultats permettent de tirer des conclusions sur les stratégies de modélisation. En effet en régime cavitant l'augmentation de l'énergie cinétique turbulente étant décorrélée du cisaillement moyen, les modèles basés sur une viscosité turbulente semblent inadaptés . D'autre part à fort taux de vide les résultats semblent être en contradiction totale avec les modèles barotropes. / The present study is inserted in the framework of previous research which aim to get a better understanding of turbulent and cavitating phenomena which occur in the spatial turbopump. Actually, the specific topology of the detached flow which takes place upstream the inducer is today miss-predicted by the standard numerical models.Taking into account the huge complexity of such a flow it has been decided to experimentally investigate the backward-facing step flow which is recognized as a benchmark for numerical simulations and cover a large area of similitude with the backflow upstream the inducers.For this purpose, the mean and turbulent quantities of the liquid phase in the presence of the vapor phase as well as spatio-temporal correlations have been investigated using stereoscopic time resolved PIV, the void ratio has been determined using X-Ray attenuation techniques and measurements have been made for different cavitation levels. The main originality of the present work is based on instantaneous measurements of the wall pressure signals correlated with high speed visualizations, PIV and X-Ray measurements. Specific signal processing as Proper Orthogonal Decomposition and Linear Stochastic Estimation has been performed in order to estimate the velocity-density correlations useful to evaluate the void ratio transport. In this experimental work the physical mechanisms of the vapour phase effects on the large vortex structures, shear layer instability, reattachment wall and reverse flow have been investigated to determine what physical assumptions can be applied in the usual cavitation and turbulence models.It has been found for the cavitating cases that the growth of turbulent kinetic energy is not correlated to mean and turbulent shear stresses. This experimental observation is in contradiction with the Boussinesq hypothesis used for the linear eddy viscosity models.Moreover concerning the phase-change modelization for high void fraction, the barotropic models seem to disagree with our experiments.

Em nosso trabalho anterior mostramos que dentre as citocinas analisadas, os níveis do mRNA de LIF nas células estromais pediátricas, de SMD e de SMD-LMA foram maiores quando comparados às células estromais de crianças saudáveis. No presente estudo, observamos um aumento tempo dependente nos níveis da proteína LIF após adição de SFB em todas as células analisadas (células estromais de crianças saudáveis, de SMD e de SMD-LMA). O envolvimento de p38, ERK e JNK na expressão LIF nestas células foi determinado pelo uso de inibidores dos membros das proteínas quinase ativadas por mitógenos: ERK (PD98059), p38 (SB302580) e JNK (SP600125) os quais inibiram a produção de LIF nas células estromais de crianças saudáveis, após estas serem estimuladas por SFB. No entanto, os níveis da expressão de LIF-induzido por soro nas células estromais de SMD e de SMD-LMA tratadas com SB302580 (p38) foram significativamente diminuídos, em comparação com a inibição observada no tratamento com PD98059 e SP600125 (p <0001, teste ANOVA). Em adição analisamos as formas fosforiladas de p38 e ERK, após 48hs na ausência ou na presença de soro por diferentes tempos. Níveis de atividade de ERK e do p38 foram inicialmente elevados na ausência de soro. A atividade de p38 foi sustentada após tratamento com SFB, entretanto, ERK apresentou uma variação de atividade durante o tratamento. Sugerimos que a sinalização das MAPKs (p38, ERK e JNK), em resposta a fatores de crescimento presentes no soro, parece desempenhar um papel importante na expressão da LIF em células estromais de crianças saudáveis, mas a sinalização do p38 parece ser funcionalmente mais importante nas mielodisplasias ou naquelas associadas à LMA / Our previous report showed that among the cytokines analysed, LIF mRNA levels in stromal cells from pediatric MDS and MDS-AML were higher as compared to those found in healthy stromal cells. In the present study, we have observed an increased protein LIF levels in a time dependent manner after FCS stimulation in all stromal cells analysed (MDS, MDS-AML and healthy children) and the involvement of p38, ERK and JNK pathways in the LIF expression in these cells was determined. In stromal cells from two healthy children, LIF production was equally inhibited in a dose dependent manner after FCS stimulation by mitogen-activated protein kinase (MAPKs) members inhibitors: ERK (PD98059), p38 (SB302580) and JNK (SP600125). However, in MDS and MDS-AML stromal cells, the levels of LIF-induced by serum, were significantly decreased by SB302580, as compared with the inhibition observed by treatment with PD98059 and SP600125 (p <0,001, ANOVA test). In addition we have analysed the presence of p38 and ERK phosphorylated forms in stromal cells, after 48hs of serum starvation or in the presence of FCS for different times. Activated ERK and p38MAPK levels were initially elevated in the absence of serum. p38MAPK activation was sustained after treatment with FCS, whereas ERK presented a variation of the activated forms during treatment. We suggest that the signalling of the MAPKs (p38, ERK and JNK) in response to growth factors present in the serum, seems to play an important role in the LIF expression by stromal cells of healthy children, but p38 MAPK signalling appears to be functionally more important in MDS and MDS-AML

<p>This thesis is focused on studies of substituted Hg-based superconducting copper oxides ((Hg_1-x<i>M</i>_x)Ba₂Ca_n-1Cu_nO2_n+2+δ). These compounds are promising objects of investigation, not only from a fundamental point of view but also because of their high values of superconducting transition temperature (<i>T</i>_c) and irreversibility field (<i>H</i>_irr).</p><p>The first part of the thesis is devoted to optimization of the synthesis procedure for Hg-based cuprates. The influence of different parameters (<i>T</i>,<i> t</i>, <i>p</i>(Hg),<i> p</i>(O₂)) on the synthesis of these compounds in sealed silica tubes was studied. Optimal conditions yielded samples containing up to 95% of HgBa₂Ca₂Cu₃O_8+δ (Hg-1223). The formation of solid solutions with the formula (Hg_1-xCu_x)Ba₂Ca₂Cu₃O_8+δ (where x <= 0.5) was also established. Another technique was developed, using LiF as a flux, for synthesis of samples containing up to 90% of the HgBa₂CaCu₂O_6+δ (Hg-1212) phase.</p><p>The second part concerns synthesis and studies of oxyfluorides using Hg-1212 and Hg-1223 as starting materials together with XeF₂ as a fluorinating agent. It was found that oxyfluorides of both phases have a parabolic dependence of T_c vs. <i>a</i> parameter as well as enhanced <i>T</i>_c values (Δ<i>T</i> ≈ 3-4 K) in comparison with optimally doped non-fluorinated analogues. The crystal structure of Hg-1223 oxyfluoride was studied by X-ray powder and neutron diffraction methods. It is suggested that chemical modification of the crystal structure leads to a decrease in Cu-O distance without noticeable change in Cu-O-Cu angle (in the (CuO₂) layers), which may be the significant factors influencing this <i>T</i>_c increase. Hg-1223 oxyfluoride was also studied under high pressure for first time. It was found that this compound has a record-high <i>T</i>_c value (≈ 166 K) at <i>P</i> ≈ 23 GPa.</p><p>The last part describes the investigation of substituted Hg-based superconductors in the series (Hg_0.9M_0.1)Ba₂CuO_4+δ {(Hg,<i>M</i>)-1201}, where <i>M</i> = Tl, Pb, W, Mo, Nb and V. A comprehensive study of these compounds by various methods (X-ray powder diffraction, EDX, IR-, EXAFS- and XANES -spectroscopy) indicated that the change of charge carrier doping level is a crucial factor determining the irreversibility line. (Hg_0.9Mo_0.1)Ba₂CuO_4+δ showed the most improved irreversibility line position among the (Hg,<i>M</i>)-1201 compounds studied in this series.</p>