Die intakte Signalübertragung im animalischen Nervensystem erfordert eine an richtiger Stelle ausgebildete funktionsfähige Synapse zwischen zwei Nervenzellen bzw. zwischen Nerv und Muskel. In der vorliegenden Arbeit wurde eine Mutante von Drosophila melanogaster untersucht, bei der es zu Veränderungen der Verteilung eines wichtigen Organisationsproteins der synaptischen aktiven Zone kommt. Ein wichtiges Ergebnis der Untersuchungen ist die Beobachtung, dass es in der Mutante zu einer ektopen Ausbildung von Elementen aktiver Zonen in Axonen kommt. In den Arbeitsgruppen von E. Buchner und S. Sigrist ist bereits das Protein Bruchpilot (BRP) charakterisiert worden, das Bestandteil der präsynaptischen Ribbons, bei Drosophila als T-bars bezeichnet, ist. Bei der Suche nach Interaktionspartnern von BRP, ist eine Serin-Arginin-Protein spezifische Kinase SRPK79D entdeckt worden, die offenbar an der Regulation des Aufbaus der Tbars beteiligt ist (Nieratschker et al., 2009). Es gibt vier verschiedene Isoformen der Kinase. Werden nur zwei Isoformen der Kinase (SRPK79D-RB und -RE) exprimiert bzw. das Gen der Kinase komplett ausgeschaltet, findet man Ansammlungen von BRP als immunreaktive Aggregate in der Immunfluoreszenz- Färbung von larvalen Motoneuron-Axonen (Nieratschker, 2008). Es ist unser übergeordnetes Ziel, die Funktion und den molekularen Signalweg der Kinase SRPK79D zu entschlüsseln. Ein Ziel der vorliegenden Arbeit war es, PB-Protein in Reinform für eine Affinitätsreinigung eines PB-Antikörpers zu gewinnen, um in nachfolgenden Untersuchungen die Lokalisation dieser Kinase-Isoform zu untersuchen. Die Proteinreinigung war erfolgreich, aber es gelang nicht, eine für eine Affinitätsreinigung ausreichende Menge des Proteins zu isolieren. Ein weiterer Versuch, Lokalisationsuntersuchungen zur Expression der Kinase in Drosophila- Embryonen durchzuführen, war ebenfalls nicht erfolgreich. Obwohl die Herstellung einer für die SRPK79D mRNA spezifischen RNA Sonde für die in-Situ-Hybridisierung gelang, war die Sensitivität dieser Sonde nicht hoch genug, um die Lokalisation vornehmen zu können. Eindeutige und aufschlussreiche Ergebnisse dagegen ergab die Untersuchung der Ultrastruktur der BRP-Ansammlungen in den larvalen Motornerven. Als deren Korrelat fanden sich elektronenmikroskopisch charakteristische Ansammlungen elektronendichter intraaxonaler Strukturen, deren Form Ähnlichkeiten zu T-bars aufwies und die von Vesikeln umgeben waren. Die elektronendichten Strukturen zeigten zahlreiche Formvariationen, die wie Ansammlungen von T-bars nebeneinander bzw. „miteinander verklebte“ T-bars oder wie zerstörte T-bars aussahen. In einer nachfolgenden Studie wurde durch eine immun-elektronenmikroskopische Untersuchung gezeigt, dass diese Strukturen in der Tat BRP enthalten (Nieratschker et al., 2009). Ergebnis der Untersuchungen der vorliegenden Arbeit war der Nachweis, dass prinzipiell ähnliche Aggregate auch im Wildtyp gelegentlich gefunden werden, dass sie aber in Mutanten signifikant häufiger vorkommen und auch einen signifikant höheren Durchmesser aufweisen. Doppelimmunreaktionen mit Antikörpern, die den C- bzw. N-terminalen Bereich von BRP erkennen, belegten darüber hinaus, dass in den Aggregaten das vollständige BRP-Protein vorliegt. Angeregt durch die Ultrastrukturbefunde von mit den elektronendichten Strukturen in den Aggregaten assoziierten Vesikeln wurde in weiteren Doppelimmunreaktionen untersucht, ob ein typisches Protein synaptischer Vesikel neuromuskulärer Synapsen in Drosophila, der vesikuläre Glutamattransporter (DVGlut), in den BRP-Ansammlungen nachweisbar ist. Während Kolokalisation von BRP und DVGlut in aktiven Zonen präsynaptischer Boutons nachgewiesen werden konnte, war der Vesikelmarker in BRP-Aggregaten nicht kolokalisiert. Die Ergebnisse belegen, dass die Kinase SRPK79D für die Vermeidung einer ektopen Bildung von BRP-enthaltenden, elektronenmikroskopisch atypischen aktiven Zonen ähnelnden Strukturen in larvalen Motoneuronaxonen notwendig ist. Die in diesen Aggregaten regelmäßig zu beobachtenden Vesikel ähneln morphologisch synaptischen Vesikeln, besitzen aber keine dafür typischen Vesikelmarker. / Intact signal transmission in an animal’s nervous system requires a properly localized and functional synapse between two neurons or between neuron and muscle. This dissertation is part of the investigation of a Drosophila melanogaster mutant which displays alterations in the distribution of a synaptic active zone protein. An important result of the present study is the documentation of an ectopic formation of active zone structural elements in this mutant. Analyses carried out in the laboratories of E. Buchner and S. Sigrist contributed to the characterization of the protein Bruchpilot (BRP), a constituent of the T-bar, the characteristic presynaptic ribbon in Drosophila. Searching for interaction partners of BRP, a serine-arginine-protein specific kinase was identified that apparently regulates T-bar assembly (Nieratschker et al., 2009). There are four kinase isoforms. Knocking out two of these isoforms (SRPK79D-RB and -RE) results in accumulations of BRP-immunoreactive aggregates in the larval ventral nerves (Nieratschker, 2008). Further studies were designed to identify the function and molecular signalling pathways of the kinase SRPK79D. One objective of the present experiments was to produce purified PB-protein in order to enable affinity-purification of an antibody against this isoform of the kinase for subsequent specific immunohistochemical localization analyses. Although production of the antigen was successful, the amount of protein produced was too low to allow efficient affinity purification. An attempt to show the expression pattern of the kinase in Drosophila embryos with in-situ hybridization resulted in production of a SRPK79D specific RNAprobe, however, the probe sensitivity was not high enough to yield conclusive results for mRNA localization. Ultrastructural analyses of the BRP-ir aggregates in the larval ventral nerves, on the other hand, yielded definite and conclusive results. These aggregates corresponded to extensive intraaxonal electron-dense, ribbon-like structures surrounded by vesicles. These electron-dense structures were differently shaped and resembled accumulations of regularly shaped, clotted or dysmorphic T-bars, which in subsequent immuno-electronmicroscopic analyses carried out by another investigator were proven to contain BRP (Nieratschker et al., 2009). An important result of the present study was the observation that similar intraaxonal aggregates were occasionally also present in wild type nerves, however, the aggregates found in the mutants were significantly more frequent and of significantly larger size than those observed in wild-type larvae. Moreover, double-immunostaining using BRP-antibodies recognizing specifically the C- and the N-terminal part of the protein, respectively, provided evidence that the complete BRP protein is localized in the aggregates. Since electron microscopy had showed that numerous vesicles were associated with the electron dense aggregates, we tested whether the vesicular glutamate transporter (DVGlut), a marker protein for synaptic vesicles of motoneurons in Drosophila, could be localized in BRP-ir aggregates. While colocalization of BRP and DVGlut was observed at the presynaptic active zones, no colocalization of the synaptic vesicle marker was observed in the BRP-ir aggregates in the larval nerves. In conclusion, the results provide evidence that the kinase SRPK79D is required for the prevention of ectopic formation of BRP-containing ribbon-like structures in larval ventral nerves. These structures include vesicles resembling synaptic vesicles, which however do not display immunoreactivity for a typical synaptic vesicle marker protein.

La miniaturisation des dispositifs élémentaires de la technologie CMOS impose le remplacement de l'oxyde de silicium pour l'élaboration de l'oxyde de grille. Par l'identification des conditions de formation du silicate de baryum au contact direct du substrat de silicium, cette étude a révélé un candidat potentiel. En premier lieu, la réaction entre Ba et SiO2 aboutissant à la formation d'un silicate de baryum a été mise en évidence in-situ par XPS et SR-PES. Dans un second temps, des films de silicate de baryum ont été élaborés par co-déposition de baryum et d'oxygène à une température de 580 °C. Des traitements thermiques sous vide ont montré que le silicate de baryum est stable jusqu'à 900 °C. Des analyses ex-situ par SIMS et MET ont révélé une interface abrupte avec le substrat. Enfin, un dispositif dédié à la réalisation de croissances par MOCVD a été développé. Il a permis de montrer la possibilité de former un silicate de baryum. La réaction est favorisée lorsque le dépôt se déroule à température élevée, sous une pression partielle d'oxygène.

<p>Viruses modulate biosynthetic machineries of the host cell for a rapid and efficient virus replication. One important way of modulating protein activity in eukaryotic cells is by reversible phosphorylation. In this thesis we have studied adenovirus and vaccinia virus, two DNA viruses with different replication stategies. Adenovirus replicates and assembles new virions in the nucleus, requiring the host cell transcription and splicing machinieries, whereas vaccinia virus replicates in the cytoplasm, only requiring the cellular translation machinery for its replication. </p><p>Adenovirus uses alternative RNA splicing to produce its proteins. We have shown that adenovirus takes over the cellular splicing machinery by modulating the activity of the essential cellular SR family of splicing factors. Vaccinia virus, that does not use RNA splicing, was shown to completely inactivate SR proteins as splicing regulatory factors. SR proteins are highly phosphorylated, a modification which is important for their activity as regulators of cellular pre-mRNA splicing. We have found that reversible phosphorylation of SR proteins is one mechanism to regulate alternative RNA splicing. We have demonstrated that adenovirus and vaccinia virus induce SR protein dephosphorylation, which inhibit their activity as splicing repressor and splicing activator proteins. We further showed that the adenovirus E4-ORF4 protein, which binds to the cellular protein phosphatase 2A, induced dephosphorylation of a specific SR protein, ASF/SF2, and that this mechanism was important for regulation of adenovirus alternative RNA splicing.</p><p>Inhibition of cellular pre-mRNA splicing results in a block in nuclear- to cytoplasmic transport of cellular mRNAs, ensuring free access of viral mRNAs to the translation machinery. We propose that SR protein dephosphorylation may be a general viral mechanism by which mammalian viruses take control over host cell gene expression.</p>

<p>Pre-mRNA splicing and alternative pre-mRNA splicing are key regulatory steps controlling geneexpression in higher eukaryotes. The work in this thesis was focused on a characterization of thesignificance of exonic and intronic splicing enhancer elements for pre-mRNA splicing.</p><p>Previous studies have shown that removal of introns with weak and regulated splice sitesrequire a splicing enhancer for activity. Here we extended these studies by demonstrating thattwo "strong" constitutively active introns, the adenovirus 52,55K and the Drosophila Ftzintrons, are absolutely dependent on a downstream splicing enhancer for activity <i>in vitro</i>.</p><p>Two types splicing enhancers were shown to perform redundant functions as activators ofSplicing. Thus, SR protein binding to an exonic splicing enhancer element or U1 snRNP bindingto a downstream 5'splice site independently stimulated upstream intron removal. The datafurther showed that a 5'splice site was more effective and more versatile in activating splicing.Collectively the data suggest that a U1 enhancer is the prototypical enhancer element activatingsplicing of constitutively active introns.</p><p>Adenovirus IIIa pre-mRNA splicing is enhanced more than 200-fold in infected extracts. Themajor enhancer element responsible for this activation was shown to consist of the IIIa branchsite/polypyrimidne tract region. It functions as a Janus element and blocks splicing in extractsfrom uninfected cells while functioning as a splicing enhancer in the context of infected extracts.</p><p>Phosphorylated SR proteins are essential for pre-mRNA splicing. Large amount recombinantSR proteins are needed in splicing studies. A novel expression system was developed to expressphosphorylated, soluble and functionally active ASF/SF2 in <i>E. Coli</i>.</p>

Pre-mRNA splicing and alternative pre-mRNA splicing are key regulatory steps controlling geneexpression in higher eukaryotes. The work in this thesis was focused on a characterization of thesignificance of exonic and intronic splicing enhancer elements for pre-mRNA splicing. Previous studies have shown that removal of introns with weak and regulated splice sitesrequire a splicing enhancer for activity. Here we extended these studies by demonstrating thattwo "strong" constitutively active introns, the adenovirus 52,55K and the Drosophila Ftzintrons, are absolutely dependent on a downstream splicing enhancer for activity in vitro. Two types splicing enhancers were shown to perform redundant functions as activators ofSplicing. Thus, SR protein binding to an exonic splicing enhancer element or U1 snRNP bindingto a downstream 5'splice site independently stimulated upstream intron removal. The datafurther showed that a 5'splice site was more effective and more versatile in activating splicing.Collectively the data suggest that a U1 enhancer is the prototypical enhancer element activatingsplicing of constitutively active introns. Adenovirus IIIa pre-mRNA splicing is enhanced more than 200-fold in infected extracts. Themajor enhancer element responsible for this activation was shown to consist of the IIIa branchsite/polypyrimidne tract region. It functions as a Janus element and blocks splicing in extractsfrom uninfected cells while functioning as a splicing enhancer in the context of infected extracts. Phosphorylated SR proteins are essential for pre-mRNA splicing. Large amount recombinantSR proteins are needed in splicing studies. A novel expression system was developed to expressphosphorylated, soluble and functionally active ASF/SF2 in E. Coli.

Viruses modulate biosynthetic machineries of the host cell for a rapid and efficient virus replication. One important way of modulating protein activity in eukaryotic cells is by reversible phosphorylation. In this thesis we have studied adenovirus and vaccinia virus, two DNA viruses with different replication stategies. Adenovirus replicates and assembles new virions in the nucleus, requiring the host cell transcription and splicing machinieries, whereas vaccinia virus replicates in the cytoplasm, only requiring the cellular translation machinery for its replication. Adenovirus uses alternative RNA splicing to produce its proteins. We have shown that adenovirus takes over the cellular splicing machinery by modulating the activity of the essential cellular SR family of splicing factors. Vaccinia virus, that does not use RNA splicing, was shown to completely inactivate SR proteins as splicing regulatory factors. SR proteins are highly phosphorylated, a modification which is important for their activity as regulators of cellular pre-mRNA splicing. We have found that reversible phosphorylation of SR proteins is one mechanism to regulate alternative RNA splicing. We have demonstrated that adenovirus and vaccinia virus induce SR protein dephosphorylation, which inhibit their activity as splicing repressor and splicing activator proteins. We further showed that the adenovirus E4-ORF4 protein, which binds to the cellular protein phosphatase 2A, induced dephosphorylation of a specific SR protein, ASF/SF2, and that this mechanism was important for regulation of adenovirus alternative RNA splicing. Inhibition of cellular pre-mRNA splicing results in a block in nuclear- to cytoplasmic transport of cellular mRNAs, ensuring free access of viral mRNAs to the translation machinery. We propose that SR protein dephosphorylation may be a general viral mechanism by which mammalian viruses take control over host cell gene expression.

La caractérisation du micro-endommagement et la compréhension de son rôle dans le métabolisme ou dans la fragilisation osseuse restent des challenges, tout particulièrement en raison de l'absence de techniques de mesures bien adaptées à son étude. Il apparaît donc nécessaire de développer de nouvelles techniques non-invasives pour détecter et suivre l'accumulation de micro-endommagement osseux, en particulier celui qui se manifeste par la présence de microfissures. L'objectif de la thèse est d'évaluer la sensibilité de la spectroscopie ultrasonore non-linéaire (NRUS) à l'accumulation du micro-endommagement osseux. La sensibilité et la reproductibilité de la technique NRUS ont tout d'abord été optimisées. Puis, deux groupes d'échantillons d'os cortical ont été prélevés sur des diaphyses fémorales humaines. Les spécimens du premier groupe ont été endommagés progressivement par fatigue en flexion 4-points. Le second groupe a subi un test de ténacité pour initier et propager de manière contrôlée une fissure unique. Nos résultats montrent que la non-linéarité mesurée des échantillons fatigués et fissurés augmente de manière significative après les étapes de fatigue ou le test de ténacité. De plus, nous observons une corrélation significative entre la variation relative du paramètre non-linéaire et l'augmentation de la densité de petites fissures (évaluée par microtomographie par rayonnement synchrotron). Enfin, le niveau de non-linéarité des spécimens fissurés est significativement corrélé à la longueur totale de la fissure. Ces résultats suggèrent que la technique NRUS optimisée est sensible à l'accumulation du micro-endommagement osseux.

The assemblage of ca. 6–4 Ma volcanic rocks exposed at the Sagehen Research station in the Truckee-Tahoe region of the northern Sierra Nevada, United States, is interpreted to be, within the Ancestral Cascades volcanic arc, a Lassen-type stratovolcano complex. Sagehen is of particular importance because it is one of the few Tertiary arc volcanic centres in California which has not been heavily glaciated during the Pleistocene. The volcanic rocks are variably porphyritic or aphanitic, including abundant plagioclase with clinopyroxene and amphibole. The rocks range from basalt to basaltic-andesite to andesite in composition. Basalts are olivineand clinopyroxene-bearing with minor phenocrysts of plagioclase. The basaltic-andesites are primarily pyroxene bearing while the andesites contain pyroxene-, plagioclase- and hornblende porphyritic phases. Sagehen arc lavas are calc-alkaline and enriched in the large ion lithophile elements and depleted in High Field Strength Elements. The basalts are depleted in Zr and Hf while the andesites are enriched with Zr and Hf relative to the middle rare earth elements. Compared to previously studied Ancestral Cascade arc samples, Sagehen region basalts have lower 143Nd/144Nd isotopic values that do not correspond to proposed mantle-lithosphere mixing lines, while the andesite samples appear to represent the interplay of these two components on a 87Sr/86Sr vs. 143Nd/144Nd. The trace element data and isotopic plots suggest that the melts that produced the basalts are from subduction modified mantle wedge peridotites that ponded near the base of the lithosphere similar to the generation of other subduction related calc-alkaline lavas along convergent continental margins. The andesitic samples appear to be the result of further modification through crustal assimilation as seen in the higher isotopic Sr contents in the andesites and Ce/Smpmn vs. Tb/Ybpmn plots. Finally, the proposed map units from Sylvester & Raines (2007) were found to contain various geochemical facies based on the samples collected indicating that some map units may have to be redefined or sub-divided.

L'intestin assure l'absorption des lipides alimentaires et doit faire face aux variations d'apport en lipides, entre les périodes inter-prandiales et post-prandiales. J'ai donc étudié dans les cellules Caco-2/TC7, la réponse des entérocytes à différents modes d'apports en lipides. Par une approche transcriptomique, j'ai montré que les lipides modulent l'expression de nombreux gènes entérocytaires. Les profils géniques obtenus diffèrent entre l'apport luminal ou sérique en lipides. La comparaison des apports apicaux de micelles inter- ou post-prandiales (MPP) a montré que les MPP modulent spécifiquement l'expression de 46 gènes majoritairement impliqués dans la transduction de signaux, le métabolisme lipidique, et l'architecture cellulaire. Ces résultats, s'ajoutant aux effets spécifiques des MPP obtenus dans l'équipe, suggéraient une détection entérocytaire des lipides alimentaires apportés par ces MPP. J'ai alors montré que les MPP se lient à une protéine dont le poids moléculaire correspond à celui du récepteur SR-BI, induisant sa clusterisation à la membrane apicale et son adressage vers les rafts. Seules les MPP induisent, le trafic de l'apoB, de la membrane apicale vers les compartiments sécrétoires et l'activation de kinases. Ces effets sont abolis quand SR-BI est bloqué par un de ses ligands ou invalidé par ARN interférence. Ces travaux montrent pour la première fois que les entérocytes sont capables de détecter spécifiquement les lipides alimentaires sous forme micellaire. Cette détection implique SR-BI et induit des voies de signalisation aboutissant à la mise en place de paramètres morphologiques et fonctionnels nécessaires au transfert des lipides alimentaires.

The perovskite structure has an incredible versatility that results in myriad compounds with varied and eccentric behaviors. Perovskite oxides have been extensively studied and used for over 60 years. In order to expand on our already thorough knowledge of these compounds, it is necessary to use modern and creative experimental techniques. High-pressure synthesis and high oxygen-gas pressure annealing techniques are used to synthesize oxygen stoichiometric RNiO₃ (R = lanthanide). The particularly rich phase diagram of this compound allows for the study of the crossover from localized to itinerant electronic behavior and from an enhanced Pauli to a Curie-Weiss law paramagnetism. Single crystals of RFeO₃ are grown in order to analyze the spin canting in these antiferromagnetic samples. The size of the rare earth-cation is used to tune the magnitude of octahedral-tilt distortions. This tuning allows distinguishing between the two possible drivers for spin canting and weak ferromagnetism in these compounds, the octahedral-tilt-dependent single-ion anisotropy and the octahedral-tilt-independent Dzyaloshinskii-Moriya interaction. Although it is a fluoride compound, KCuF₃ has been used as an analogue to transition-metal oxide perovskites such as LaMnO₃ because of the similarity of their orbital ordering. Through the use of high-temperature neutron diffraction, it is shown that the orbital ordering and Jahn-Teller distortion in this compound are not lifted at the predicted temperature. Another mechanism for orbital ordering is identified. La₂[subscript-x] Sr [subscript x] CuO₄ has long been of interest as the progenitor system of the highTc superconductors. Despite having an exceedingly well-studied phase diagram in the over-doped region of its superconducting dome, little is known about this system in the region x > 0.3 because of the difficulty of synthesizing fully oxygen-stoichiometric samples. With high-oxygen-gas-pressure annealing and high-pressure synthesis, the completion of the phase diagram up to x = 1.0 is attempted. Finally, like many iridates, post-Perovskite CaIrO₃ exhibits a very strong spinorbit coupling of its 5d electrons. Because its magnetism is very weak, traditional methods to measure the magnitude of its orbital moment and spin-orbit coupling, such as neutron powder diffraction, are not viable. In order to address this issue, direct measurement of the orbital moments was conducted by using x-ray absorption spectroscopy and x-ray magnetic circular dichroism techniques. / text