Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Microtubules are tubular polymers that are involved in a variety of cellular processes such as cell movement, mitosis and intracellular transport. The dynamic behavior of microtubules makes this possible because all of these processes require quick responses. Embryonic stem (ES) cells were first isolated from mouse embryos and they have two unique characteristics; they can be kept undifferentiated for many passages with a stable karyotype and they can be differentiated into any type of cells under appropriate conditions. The pluripotency of ES cells, their ease of manipulation in culture, and their ability to contribute to the mouse germ-line provides us a model of differentiation both in vitro and in vivo. In my thesis I focused on the cell division and neuronal differentiation of ES cells and developed two methods to understand the effects of microtubule dynamics in spindle assembly and chromosome segregation and to reveal the roles of different Microtubule Associated Proteins (MAPs) in the neuronal morphology formation. In the first part, we developed a live-cell imaging method for ES cells to visualize, track and analyze the single cell behavior in a cell population over a time period. So far many techniques have been adapted and combined for imaging of cell lines, mainly for the cancer or immortalized ones. However, because ES cells are very prone to apoptosis, tend to form spheres and hard to stably label, it is quite tricky to image them in culture conditions. In our system, we combined the BAC-based gene expression with wide-field deconvolution microscopy for ES cells that are plated onto the laminin-511 coated surface and kept in CO2 independent culture conditions. This combined technique does not interfere with the growth of cells and keeps them healthy up to 24 hours on the microscope stage. In the second part, we analyzed the effects of MAPs chTOG, EB1, Kif18A and MCAK in the overall spindle morphology and mitotic progression in mES cells. For this purpose, we utilized our stable TUBB-GFP and H2A-GFP cell lines along with our live-cell imaging set-up to reveal the effects of the above-mentioned proteins and the interplay among each other. By using RNAi method we either single or co-depleted the genes by siRNAs and measured the spindle length and width in RNAi conditions. We further analyzed the mitotic progression in H2A-GFP cell line in terms of the metaphase timing and the percentage of chromosome segregation errors. Our results showed that, EB1 depletion did not cause any significant changes in the overall spindle morphology or in the metaphase timing. However, the co-depletion of EB1 with chTOG partially rescued the sichTOG specific mini-spindle phenotype. siKif18A produced longer spindles without any change in the spindle width. Surprisingly, the co-depletion of antagonistic chTOG and Kif18A proteins had additive effects on the spindle dynamics and on mitotic progression in a way that spindle assembly was severely disrupted by the absence of these two proteins and as a result of this, both metaphase timing and chromosome missegregation levels increased significantly. These results overall indicate that MAPs have important roles in the regulation of dynamic instability and these proteins have an interplay among each other to be able to control the morphology of the spindle as well as the correct segregation of chromosomes into daughter cells. In the last part, I will introduce you a new ES cell based differentiation and morphology model, which brings the advantages of high resolution imaging capacity, control over development and easy genetic manipulation and culturing. We have generated Tet-induced shRNA cell lines against chTOG, Kif18A and MCAK, which are also stably expressing TUBB-GFP. These labeled cells were mixed with unlabeled wild-type mES cells before differentiation at 1:1000 ratio and then they were differentiated into mouse cortical cells and spinal motor neurons. Our results showed that, all of the three genes could be successfully knocked-down by shRNA after 48 hours of Tet induction. After mixing the labeled and unlabeled cells, single neurons could be imaged at high resolution and their skeletons could be generated afterwards. The RNAi studies in shchTOG cell line showed that, the knock-down of this gene in early differentiation interferes with the neuronal differentiation.

Cette thèse contribue au domaine de la morphologie mathématique et illustre comment la statistique multivariée et les techniques d'apprentissage numérique peuvent être exploitées pour concevoir un ordre dans l'espace des vecteurs et pour inclure les résultats d'opérateurs morphologiques au processus d'analyse d'images multivariées. En particulier, nous utilisons l'apprentissage supervisé, les projections aléatoires, les représentations tensorielles et les transformations conditionnelles pour concevoir de nouveaux types d'ordres multivariés et de nouveaux filtres morphologiques pour les images multi/hyperspectrales. Nos contributions clés incluent les points suivants :* Exploration et analyse d'ordre supervisé, basé sur les méthodes à noyaux.* Proposition d'un ordre nonsupervisé, basé sur la fonction de profondeur statistique calculée par projections aléatoires. Nous commençons par explorer les propriétés nécessaires à une image pour assurer que l'ordre ainsi que les opérateurs morphologiques associés, puissent être interprétés de manière similaire au cas d'images en niveaux de gris. Cela nous amènera à la notion de décomposition en arrière plan. De plus, les propriétés d'invariance sont analysées et la convergence théorique est démontrée.* Analyse de l'ordre supervisé dans les problèmes de correspondance morphologique de patrons, qui correspond à l'extension de l'opérateur tout-ou-rien aux images multivariées grâce à l'utilisation de l'ordre supervisé.* Discussion sur différentes stratégies pour la décomposition morphologique d'images. Notamment, la décomposition morphologique additive est introduite comme alternative pour l'analyse d'images de télédétection, en particulier pour les tâches de réduction de dimension et de classification supervisée d'images hyperspectrales de télédétection.* Proposition d'un cadre unifié basé sur des opérateurs morphologiques, pour l'amélioration de contraste et pour le filtrage du bruit poivre-et-sel.* Introduction d'un nouveau cadre de modèles Booléens multivariés en utilisant une formulation en treillis complets. Cette contribution théorique est utile pour la caractérisation et la simulation de textures multivariées.

Les granulats naturels alluvionnaires sont utilisés actuellement à hauteur de 70 à 80% en volume dans la composition d'un béton et représentent désormais une ressource non renouvelable dont l'accessibilité diminue. Les formulations actuelles de béton visent à augmenter la part de granulats de roche massive concassés. Ces matériaux, de par leur histoire de fabrication, n'ont pas du tout la même morphologie. La substitution des granulats roulés par des granulats concassés affecte considérablement la mise en œuvre du matériau. Dans un premier temps, nous étudions spécifiquement l'influence de la morphologie des granulats sur les propriétés d'empilement des grains. Nous montrons que, le facteur d'aspect des grains affecte fortement la compacité dense et lâche aléatoire. À l'inverse, pour des grains de facteur d'aspect identique, l'impact de la convexité sur la mesure de la compacité dense est faible. Nous étendons, dans un deuxième temps, notre étude au comportement rhéologique de nos systèmes composés d'émulsion inverse et de sables monodisperses. Pour cela, nous construisons des courbes d'écoulement à l'échelle locale grâce à un imageur à résonance magnétique. Nous montrons que cette méthode est, de nos jours, la plus précise pour des mesures de viscosités, car elle prend en compte la migration et la localisation du matériau durant l'écoulement. Nous montrons également que le facteur d'aspect contrôle tout d'un point de vue rhéologique dans le régime où il n'y a pas de réseau percolé de contacts entre grains

A fundamental problem in biology is the reconstruction of the relatedness of all (extant) species. Traditionally, systematists employ visually recognizable characters of organisms for classification and evolutionary analysis. Recent developments in molecular and computational biology, however, lead to a whole different perspective on how to address the problem of inferring relatedness. The discovery of molecules, carrying genetic information, and the comparison of their primary structure has, in a rather short period of time, revolutionized our understanding of the phylogenetic relationship of many organisms. These novel approaches, however, turned out to bear similar problems as previous techniques. Moreover, they created new ones. Hence, taxonomists came to realize that even with this new type of data not all problematic relationships could be unambiguously resolved. The search for complementary approaches has led to the utilization of rare genomic changes and other characters which are largely independent from the primary structure of the underlying sequence(s). These “higher order” characters are thought to be evolutionary conserved in certain lineages and largely unaffected by primary sequence data-based problems, allowing for a better resolution of the Tree of Life. The central aim of this thesis is the utilization of molecular characters of higher order in connection with their consistent and comparable extraction from a given data set. Two novel methods are presented that allow such an inference. This is complemented with the search for and analysis of known and novel molecular characteristics to study the relationships among Metazoa, both intra- as well as interspecific. The first method tackles a common problem in phylogenetic analyses: the inference of reliable data set. As part of this thesis a pipeline was created for the automated annotation of metazoan mitochondrial genomes. Data thus obtained constitutes a reliable and standardized starting point for all downstream analyses, e.g. genome rearrangement studies. The second method utilizes a subclass of gaps, namely those which define an approximate split of a given data set. The definition and inference of such split-inducing indels (splids) is based on two basic principles. First, indels at the same position, i.e. sharing the same end points in two sequences, are likely homologous. Second, independent single-residue insertions and deletions tend to occur more frequently than multi-residue indels. It is shown that trees based on splids recover most of the undisputed monophyletic groups while influence of the underlying alignment algorithm is relatively small. Mitochondrial markers are a valuable tool for the understanding of small and large scale population structure. The non-coding control region of mitochondrial DNA (mtDNA) often contains a higher amount of variability compared to genes encoding proteins and non-coding RNAs. A case study on a small scale population structure investigates the control region of the European Fire-bellied Toad in order to find highly variable parts which are of potential importance to develop informative genetic markers. A particular focus is placed on the investigation of the evolutionary dynamics of the repetitive region at an inter- and intraspecific level. This includes understanding mechanisms underlying its evolution, i.e. by exploring the impact of secondary structure on slipped strand mispairing during mtDNA replication. The 7SK RNA is a key player in the regulation of polymerase II (Pol-II) transcription, interacting with at least three known proteins: It mediates the inhibition of the Positive Transcription Elongation Factor b (P-TEFb) by the HEXIM1/2 proteins, thereby repressing transcript elongation by Pol-II. A highly specific interaction with LARP7 (La-Related Protein 7), on the other hand, regulates its stability. 7SK RNA is capped at its 5’ end by a highly specific methyltransferase MePCE (Methylphosphate Capping Enzyme). Employing sequence and structure similarity it is shown that the 7SK RNA as well as its protein binding partners have a much earlier evolutionary origin than previously expected. Furthermore, this study presents a good illustration of the pitfalls of using markers of higher order for phylogenetic inference.

Techn. Universiẗat, Diss., 2007--Darmstadt.

Zugl.: International Faith Theological Seminary (IFTS), Diss., 2009. / 979338697! ; Bd. 10.

Th. doct.--Morphol. math.--Paris--Ecole nationale supérieure des mines, 1990. / Bibliogr. p. 279-287. Résumé en anglais.

Stuttgart, Univ., Diss., 2004.