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Embryogenesis is one of the most delicate biological processes which requires precise control in various levels, including molecular distribution and gene expression, cellular orientation and specification, and tissue dynamics giving rise to proper morphology. The diverse animal morphology can be resulted from the difference during early embryonic cleavages. Spiral cleavage is a conserved embryonic patterning strategy used in the majority of the animal clade Spiralia. The specific cell positioning during cell division and quadrant-based clonal domain formation make the embryos with the blastomeres orientated in a spiral manner when viewing from the animal pole. Although spiral cleavage is conserved in many phyla, the detailed cellular, molecular and biophysical mechanisms for this left-right symmetry breaking event remain unclear. Here I studied the early development of the prototypic annelid spiral-cleaver Platynereis dumerilii, which performs two unequal embryonic cleavages followed by the first dextral spiral cleavages, and compared the mechanisms to other spiralians or to other cleavage types. First, I described the morphology of each cell cycle from the zygote until 64-cell stage by imaging the fluorescently labeled fixed embryos. Second, with mRNA injection, whole-embryo live-imaging with Selective Plane Illumination Microscopy (SPIM), and in silico cell tracking, I monitored these cleavages in 4-D, constructed the early cell lineages, and revealed the subtle asynchrony of the four quartets. Third, together with the spindle inclination angle measurement, I discovered the leading role of the D macromere during P. dumerilii spiral cleavage. I also confirmed that the dextral micromere orientation is neither affected by the eggshell nor the presence of all the neighbor macromeres, suggesting that this cellular property may be achieved by cell autonomous molecular mechanisms. In order to quantify the candidate cytoskeletal dynamics during spiral cleavage, I optimized the construction of the injected mRNAs and the injection protocol to achieve the highest translational level of the fluorescent protein within a given developmental time. Beside mRNA injection, I also established a protein expression and injection protocol for P. dumerilii protein injection in order to visualize the target gene as early as possible. Both techniques didn’t dramatically influence embryogenesis and allow for quantification of the protein dynamics. With these strategies, I discovered and measured the chiral counter rotational flow of cortical actomyosin in each spiral cleavage and revealed that it’s present in the first two spiral cleavages, especially of the macromeres. The biophysical force generated by actomyosin contributes in the cell deformation and spindle inclination, resulting in proper dextral micromere positioning, during the first spiral cleavage, confirmed by the chemical treatment to the P. dumerilii embryos. The asymmetric actomyosin distribution, nuclei migration, and the change of the cell axes during cytokinesis in the macromeres also suggests that the macromeres may play critical roles to lead spiral cleavage. This work is built on the knowledge of the spiral cleavage machinery and has extended it in multiple dimensions. The detailed phase-by-phase description of each cleavage increases the information of P. dumerilii embryogenesis. The established labeling and imaging techniques in this thesis are the important basis for investigation and comparisons of different spiralian development in the future. More broadly, the discovery of actomyosin dynamics shows conservation to the left-right symmetry breaking events of the animals which does not belong to Spiralia. These together bring insights to a global evolutionary speculation: a conserved mechanical force generation pathway, tuned by the upstream molecular signals, may be the key of the miscellaneous cleavage types, resulting in the astonishing variety of embryo patterning.

Genome regulation is an extremely complex phenomenon. There are various mechanisms in place to ensure smooth performance of the organism. Post-transcriptional regulation of gene expression is one such mechanism. Many proteins bind to mRNAs and regulate their translation. In this thesis, I have focused on the Cytoplasmic Polyadenylation Element Binding family of proteins (CPEB1-4); a group of sequence specific RNA binding proteins important for cell cycle progression, senescence, neuronal function and plasticity. CPEB protein binds mRNAs containing a short Cytoplasmic Polyadenylation Element (CPE) in 3’ untranslated Region (UTR) and regulates the polyadenylation of these mRNAs and thereby controls translation. In Chapter II, I have presented my work on the regulation of mitochondrial function by CPEB. CPEB knockout mice have brain specific defects in mitochondrial function owing to a reduction in Electron transport chain complex I component protein NDUFV2. CPEB controls the translation of this NDUFV2 mRNA and thus affects mitochondrial function. A consequence of this reduced bioenergetics is reduced growth and branching of neurons, again emphasizing the importance of this pathway. Chapter III focuses on the role of CPEB4 in neuronal survival and protection against apoptosis. CPEB4 shuttles between nucleus and cytoplasm and becomes nuclear in response to stimulation with ionotropic glutamate receptors, focal ischemia in vivo and when cultured neurons are deprived of oxygen and glucose; nuclear CPEB4 affords protection against apoptosis in ischemia model. The underlying cause for nuclear translocation is reduction in Endoplasmic Reticulum calcium levels. These studies give an insight into the function and dynamics of these two RNA binding proteins and provide a better understanding of cellular biology.

La Protéine Prion (PrP) est une glycoprotéine ubiquitaire attachée au feuillet externe de la membrane plasmique par une ancre glycosylphosphatidylinositole (GPI). Cette dernière est l’agent infectieux responsable de la maladie Creutzfeld-Jacob ou « maladie de la vache folle ». Cette protéine existe sous sa forme cellulaire mais également sous sa forme infectieuse, nommée PrPSc (Scrapie). Alors que la fonction de PrPSc est établie au cours de la pathogenèse, la fonction de la protéine cellulaire est beaucoup plus énigmatique notamment chez les mammifères. Il est clairement admis que la forme infectieuse découle d’un changement de conformation de la forme cellulaire. Ainsi afin de mieux appréhender le rôle de la protéine prion dans les cellules saines mais également lors de la pathogenèse il apparaît essentiel d’étudier le trafic de cette protéine. La protéine prion est exprimée dans les cellules neuronales qui sont comme les cellules épithéliales des cellules polarisées. J’ai au cours de ma thèse étudié le trafic de la protéine prion dans les cellules polarisées MDCK. MDCK est la lignée épithéliale sur laquelle nous avons la plus grande connaissance. Dans mon travail j’ai utilisé des cellules MDCK polarisées classiquement en culture bidimensionnelle (2D) mais également en culture tridimensionnelle (3D) où les cellules forment des kystes, structures hautement polarisées, physiologiquement proches de l’épithélium in vivo. Il apparaît que dans les cellules MDCK polarisées sur filtre (en 2D) la localisation de la PrP est controversée. Nous avons trouvé que, contrairement à la majorité des protéines à ancre GPI, la PrP suit la voie de transcytose. La PrP qui se retrouve à la membrane basolatérale est transcytosée vers la membrane apicale. De plus la PrP envoyée à la surface apicale est clivée (clivage alpha) générant deux fragments distincts : le fragment C1, pourvu de l’ancre GPI qui reste associé à la surface apicale et le fragment soluble N1 qui est sécrété dans le milieu de culture des cellules MDCK cultivées en 2D ou dans le lumen des cellules MDCK cultivées en 3D. Mon travail permet de mieux comprendre les études réalisées auparavant mais surtout révèle l’existence d’un mécanisme de transcytose de la protéine prion dans les cellules épithéliales. Cette information est essentielle et nous permet de supposer que ce mécanisme pourrait être également utilisé par les cellules neuronales. / The Prion Protein (PrP) is a ubiquitously expressed glycosylated membrane protein attached to the external leaflet of the plasma membrane via a glycosylphosphatidylinositol anchor (GPI). While the misfolded PrPSc scrapie isoform is the infectious agent of “prion diseases” the cellular isoform (PrPC) is an enigmatic protein with unclear function. Prion protein has received considerable attention due to its central role in the development of Transmissible Spongiform Encephalopathies (TSEs) known as “prion diseases”, in animals and humans. Understanding the trafficking, the processing and degradation of PrP is of fundamental importance in order to unravel the mechanism of PrPSc mediated pathogenesis, its spreading and cytotoxicity. The available data regarding PrP trafficking are contradictory. To investigate PrP trafficking and sorting we used polarized MDCK cells (two-dimensional and tree-dimensional cultures) where the intracellular traffic of GPI-anchored proteins (GPI-APs) is well characterized. GPI-APs that are sorted in the Trans Golgi Network follow a direct route from the Golgi apparatus to the apical plasma membrane. The exception to direct apical sorting of native GPI-APs in MDCK cells is represented by the Prion Protein. Of interest, PrP localization in polarized MDCK cells is highly controversial and its mechanism of trafficking is not clear. We found that full-length PrP and its cleavage fragments are segregated in different domains of the plasma membrane in polarized cells in both 2D and 3D cultures and that the C1/PrP full-length ratio increases upon MDCK polarization. We revealed that differently from other GPI-APs, PrP undergoes basolateral-to-apical transcytosis in fully polarized MDCK cells and is α-cleaved during its transport to the apical surface. This study not only reconciles and explains the different findings in the previous literature but also provides a better picture of PrP trafficking and processing, which has been shown to have major implications for its role in prion disease.

Alors que les citoyens français se montrent plutôt enclins à la communauté européenne, et que la France poursuit son intégration et sa coopération régionales, les candidats aux élections présidentielles parlent peu de l’Europe comme d’un véritable enjeu électoral. Cette thèse étudie la place de l’Europe comme enjeu, thème et sujet électoral dans les différentes structurations du débat médiatico-politiques. Au cœur des programmes, des allocutions et des débats des candidats de diverses familles politiques, elle démontre que l’Europe est un sujet glissant, souvent relégué à la dernière place des préoccupations électorales. Un sujet qui dérange le candidat et le citoyen car son traitement politique présuppose une définition stricte du rôle de la France dans l’Europe et de la finalité de l’Europe elle-même. Un sujet qui est aussi au cœur de clivages politiques flottants. A la croisée des notions de souveraineté, d’indépendance, de grandeur voire d’identité, les analyses de la place de l’Europe dans les élections présidentielles démontrent un espace de confrontation politique quasi vide, non investi par les candidats et les électorats. Cette étude démontre que cette immersion au cœur de la problématique de la place de l’intérêt national rend compte d’un processus de désidéologisation du thème européen latent et, au regard de l’écologie électorale aux référendums européens et aux élections présidentielles suivantes, d’un repli populiste en puissance dont le traitement politique du sujet européen n’est peut-être pas le dernier responsable. Quelle France souverainiste ou européiste s’exprime dans les élections présidentielles françaises de la Ve République, pour quelle Europe ? / French citizen since the 1960s have seemed to be generally in favour of the idea of a European community, and later, the European Union. However, French politicians who have run for presidential elections did not feel the need to include Europe as an electoral topic in their campaigns. If in the 1990s, Europe did become a programmatic data, it was still not considered as a topic that could create political cleavages. This thesis studies the place of Europe as an electoral issue and as a programmatic data inside the political debates. Through the analysis of the electoral programmes, the campaign speeches, and the debates, this essay demonstrates that Europe was a delicate topic and a political space of confrontation that candidates have invested very little. Europe disturbed the candidates as well as the citizen because its political treatment presupposed a strict definition of France’s role in Europe and the purpose of Europe itself. At the crossroads of the notions of sovereignty, independence, grandeur and identity, this thesis analyzes the place of Europe in the national presidential elections. Our immersion in the heart of the issues of national interests reveals a process of desideologisation of the European theme. It also shows, in regard to the electoral ecology, that there was, during the following European referendums and presidential elections, a potential populist tendency which the political treatment of the European issue was maybe not the only culprit. Which of soverainist France or europeanist France expressed itself in the French presidential elections from the Ve republic, for which Europe?

Orientador: João Manuel Marques Cordeiro / Abstract: The energetical and environmental problematic related to the cement production is a very sensitive issue. As every other field, the construction industry must go through drastic change in the design of concrete and cement-based materials. The understanding of the physical and chemical properties of the Portland cement (PC) clinker is important to improve its design. Tricalcium silicate C3S, or alite, is the main phase of PC clinker and has been largely studied since it is the first responsible for the strength development of the cement paste. On the other hand, the development of computational methods at the molecular scale has made possible the modelling of structural, dynamical and energetic properties, sometimes hardly measurable by experimental means. Such methods are relatively new in the field of cement chemistry, but have been increasingly employed over the last 15 years. In this project, density functional theory (DFT), classical molecular dynamics (MD), and ab initio molecular dynamics (AIMD) are employed towards a better understanding of mechanical, thermal, and superficial properties of monoclinic C3S, as well as C3S/water interface features. The present thesis consists of five chapters. The first chapter presents a review of the literature on the chemistry of cement, and more particularly on the hydration process modeling. The various phases which compose the Portland cement clinker are introduced, then the the different polymorphs of C3S are described, in particu... (Complete abstract click electronic access below) / Resumo: A produção de cimento envolve questões energéticas e ambientais muito relevantes. Em função disso, a indústria da construção deve sofrer mudanças radicais na concepção de concreto de materiais cimentícios. A compreensão das propriedades físicas e químicas do clínquer de cimento Portland (PC) é importante para melhorar seu design. O silicato tricálcico (C3S), ou alita, é a fase principal do clínquer de PC e tem sido amplamente estudado uma vez que é o principal responsável pelo desenvolvimento da resistência da pasta de cimento. Por outro lado, o desenvolvimento de métodos de cálculo na escala molecular possibilitou a modelagem de propriedades estruturais, dinâmicas e energéticas, às vezes difícil de medir por meios experimentais. Esses métodos são relativamente novos no campo da química do cimento, mas tem sido cada vez mais empregados nos últimos 15 anos anos. Neste trabalho, a teoria de funcional da densidade (DFT), a dinâmica molecular clássica (MD) e a dinâmica molecular ab initio (AIMD) são utilizadas para permitir uma melhor compreensão das características mecânicas, térmicas e de superfície do C3S monoclinico, bem como propriedades da interface C3S/água. Esta tese consiste em cinco capítulos: O primeiro capítulo apresenta uma revisão da literatura sobre a química dos cimento e, mais particularmente, sobre o processo de hidratação e sua modelagem. Introduzimos as diferentes fases que compõem o clínquer de cimento Portland, em seguida, a estrutura e os diferentes polimor... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Model organism studies have been fundamental in understanding evolutionary and developmental biology. However, non-model organisms present opportunities to study unique characteristics and as comparisons to model organisms, leading us toward broader and more relevant perspectives on diversity. The Euphilomedes genus of ostracods is an example of a non-model group with potential for evolutionary and developmental studies. Ostracoda is an ancient, basally branching lineage of Crustaceans with a complete and prodigious fossil record. Despite the group’s promise for evolutionary studies, much remains unknown about the basic biology of this clade. There are a limited number of embryogenesis studies in Ostracoda; here, I study development in Euphilomedes. In Chapter 1, I study the main events in Euphilomedes’ embryology, focusing on cleavage and cell migration. I describe the general embryology of Euphilomedes, and devise a visual staging scheme for their development. Using fluorescent nuclear staining and microscopy, I visualize nuclei in cleavage throughout development of nuclear divisions and migrations during development. The meroblastic cleavage observed in Euphilomedes resembles that of another Myodocopid ostracod, Vargula hilgendorfii. Finally, immunostaining for acetylated-alpha tubulin and phalloidin staining are used to visualize the general anatomy of the embryonic brain. This provides new protocols for visualizing the nervous system, enabling more detailed nervous system studies in the future. In Chapter 2, I explore differential gene expression patterns in the developing eyes of juvenile Euphilomedes. Euphilomedes have sexually dimorphic eye types – males have lateral compound eyes, while females instead have eye rudiments. Previous studies in E. carcharodonta show that genes in the retinal determination and phototransduction gene networks have differential expression in males and females during eye development. In this thesis, we attempt to compare these patterns to expression in a sister species, E. morini.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18324号 / 農博第2049号 / 新制||農||1021(附属図書館) / 学位論文||H26||N4831(農学部図書室) / 31182 / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 髙野 俊幸, 教授 西尾 嘉之, 教授 梅澤 俊明 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19239号 / 工博第4074号 / 新制||工||1628(附属図書館) / 32238 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 村上 正浩, 教授 吉田 潤一, 教授 松田 建児 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21120号 / 工博第4484号 / 新制||工||1697(附属図書館) / 京都大学大学院工学研究科物質エネルギー化学専攻 / (主査)教授 大江 浩一, 教授 辻 康之, 教授 中尾 佳亮 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM