No description available.

<p>Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCF<super>Cdc4</super> ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that in the budding yeast,<italic> Saccharomyces cerevisiae</italic>, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APC<super>Cdh1</super>). I have determined, however, that the Kip1 and Cin8 proteins are actually present at wild-type levels in yeast in the absence of Clb/Cdc28 kinase activity. Here, I show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28, and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's <italic>in vivo</italic> localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, I have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCF<super>Cdc4</super> target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, I propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors. </p><p>In addition to the positive regulation of kinesin-5 function in spindle assembly, I have also found evidence that suggests CDK phosphorylation of kinesin-5 motors at different sites negatively regulates kinesin-5 activity to prevent premature spindle pole separation. I have also begun to characterize a novel putative role for the kinesins-5 in mitochondrial genome inheritance in <italic>S. cerevisiae</italic> that may also be regulated by CDK phosphorylation. </p><p>In the course of my dissertation research, I encountered problems with several established molecular biology tools used by yeast researchers that I have tried to address. I have constructed a set of 42 plasmid shuttle vectors based on the widely used pRS series for use in <italic>S. cerevisiae</italic> that can be propagated in the bacterium Escherichia coli. This set of pRSII plasmids includes new shuttle vectors that can be used with histidine and adenine auxotrophic laboratory yeast strains carrying mutations in the genes <italic>HIS2</italic> and <italic>ADE1</italic>, respectively. My new pRSII plasmids also include updated versions of commonly used pRS plasmids from which common restriction sites that occur within their yeast-selectable biosynthetic marker genes have been removed in order to increase the availability of unique restriction sites within their polylinker regions. Hence, my pRSII plasmids are a complete set of integrating, centromere and 2&#61549; episomal plasmids with the biosynthetic marker genes <italic>ADE2</italic>, <italic>HIS3</italic>, <italic>TRP1</italic>, <italic>LEU2</italic>, <italic>URA3</italic>, <italic>HIS2</italic> and <italic>ADE1</italic> and a standardized selection of at least 16 unique restriction sites in their polylinkers. Additionally, I have expanded the range of drug selection options that can be used for PCR-mediated homologous replacement using pRS plasmid templates by replacing the G418-resistance kanMX4 cassette of pRS400 with MX4 cassettes encoding resistance to phleomycin, hygromycin B, nourseothricin and bialaphos. Finally, in the process of generating the new plasmids, I have determined several errors in existing publicly available sequences for several commonly used yeast plasmids. Using updated plasmid sequences, I constructed pRS plasmid backbones with a unique restriction site for inserting new markers in order to facilitate future expansion of the pRS/pRSII series.</p> / Dissertation

La mitose assure la répartition égale du génome. La kinase mitotique Aurora B y joue un rôle majeur en contrôlant la fidélité de la ségrégation des chromosomes de par sa localisation aux centromères et aux microtubules, qui nécessite son ubiquitination par CUL3. Cependant, le mécanisme conduisant la forme ubiquitinée d’Aurora B sur ces structures mitotiques reste à déterminer. Dans ce contexte, j’ai pu identifier la protéine UBASH3B, qui contient un domaine de liaison à l’ubiquitine (UBD) comme un régulateur essentiel de la ségrégation chromosomique, agissant comme un récepteur de l’ubiquitine pour Aurora B. UBASH3B interagit directement avec Aurora B et cette interaction est dépendante de la modification d’Aurora B par l’ubiquitine ainsi que de CUL3. UBASH3B ne régule pas le niveau d’expression d’Aurora B. En revanche, UBASH3B se localise aux fuseaux mitotiques et est à la fois nécessaire et suffisant pour transférer Aurora B aux microtubules. De plus, la redistribution d’Aurora B des centromères vers les microtubules contrôle le déroulement et la fidélité de la ségrégation des chromosomes et donc le contenu correct du matériel génétique des cellules. Ainsi, mes résultats expliquent comment la modification par l’ubiquitine régule la localisation et la fonction d’Aurora B, reliant une voie de signalisation impliquant un récepteur à l’ubiquitine à la mitose. / Mitosis ensures equal segregation of the genome. The major mitotic kinase Aurora B controls fidelity of chromosome segregation by its localization to centromeres and microtubules, which requires CUL3-mediated ubiquitylation. However, it remains unknown how ubiquitylated Aurora B is targeted to mitotic structures. Here, I identify ubiquitin-binding domain (UBD) protein UBASH3B that critically regulates chromosome segregation, acting as ubiquitin receptor for Aurora B. UBASH3B directly binds Aurora B, and this interaction is dependent on CUL3 and on ubiquitin recognition. UBASH3B does not regulate protein levels of Aurora B. Instead, UBASH3B localizes to the mitotic spindle and is both required and sufficient to transfer Aurora B to microtubules. Moreover, redistribution of Aurora B from centromeres to microtubules controls timing and fidelity of chromosome segregation and thereby euploidy of cells. Thus, my findings explain how ubiquitin attachment regulates localization and function of Aurora B, linking receptor-mediated ubiquitin signaling to mitosis.

L'orientation du fuseau mitotique joue un rôle essentiel dans le choix du destin cellulaire et dans l'homéostasie des tissus. Dans certains contextes, l'orientation du fuseau est contrôlée par le complexe moléculaire LGN, dont la localisation sous-corticale détermine le site de recrutement du moteur dyneine, lequel exerce des forces sur les microtubules astraux pour orienter le fuseau. Chez les vertébrés la régulation moléculaire de ce processus est cependant peu caractérisée. Nous avons décidé de chercher de nouveaux régulateurs de l'orientation du fuseau chez les vertébrés. Avec cet objectif, j'ai développé un modèle d'orientation du fuseau spécifiquement contrôlé par le complexe LGN. Avec ce modèle, j'ai réalisé un crible RNAi en évaluant 110 candidats incluant des moteurs moléculaires pour leur fonction dans l'orientation du fuseau. Notamment, ce crible a révélé que les régulateurs de la dyneine sont inégalement requis pour orienter le fuseau. De plus, entre les sous-unités de la dynactine, j'ai trouvé que la protéine du capping de l'actine, CAPZ-B, est un régulateur majeur de l'orientation du fuseau. La caractérisation de la fonction de CAPZ-B in vitro a révélé que CAPZ-B contrôle l'orientation du fuseau en régulant les complexes dyneine et dynactine ainsi que la dynamique des microtubules du fuseau, indépendamment de son rôle comme modulateur de l'actine. Finalement, nous avons démontré que CAPZ-B régule l'orientation planaire du fuseau in vivo dans le neuroépithelium. Je pense que mes travaux vont contribuer à la compréhension de la fonction de la dyneine dans l'orientation du fuseau chez les vertébrés, ouvrant la voie pour de nouvelles recherches dans le domaine. / Mitotic spindle orientation is involved in cell fate decisions, tissue homeostasis and morphogenesis. In many contexts, spindle orientation is controlled by the LGN molecular complex, whose subcortical localization determines the site of recruitment of the dynein motor which exerts forces on astral microtubules orienting the spindle. In vertebrates, there is missing information about the molecules regulating the formation of the complex and those working downstream of it. This prompted us to screen for new regulators of vertebrate spindle orientation. For this, I developed a novel model of spindle orientation specifically controlled by the LGN complex. Using this model, I performed a live siRNA screen testing 110 candidates including molecular motors for their function in spindle orientation. Remarkably, this screen revealed that specific dynein regulators contribute differentially to spindle orientation. Moreover, I found that an uncharacterized member of the dynactin complex, the actin capping protein CAPZ-B, is a strong regulator of spindle orientation. Analyses of CAPZ-B function in cultured cells showed that CAPZ-B regulates spindle orientation independently of its classical role in modulating actin dynamics. Instead, CAPZ-B controls spindle orientation by modulating the localization/activity of the dynein/dynactin complexes and the dynamics of spindle microtubules. Finally, we demonstrated that CAPZ-B regulates planar spindle orientation in vivo in the chick embryonic neuroepithelium. I expect that my work will contribute to the understanding of dynein function during vertebrate spindle orientation and will open the path for new investigations in the field.

La division cellulaire asymétrique est un mécanisme fondamental qui assure la diversité cellulaire, le renouvellement des cellules souches et le maintien de l’identité cellulaire. Elle dépend du bon positionnement du fuseau mitotique car il dicte le plan de division des cellules. La première division des embryons de C. elegans, est asymétrique et génère deux cellules fille de taille et devenir différents. Elle consiste en deux étapes : la centration des pronoyaux en prophase puis le déplacement postérieur du fuseau mitotique en anaphase. Lors de l'anaphase le fuseau subit des oscillations transverses plus marquées au pôle postérieur qu’au pôle antérieur. Ces mouvements sont contrôlés par des forces de traction agissant sur les microtubules astraux. Les générateurs de force ont été moléculairement identifiés et sont évolutivement très conservés. Un complexe composé de protéines Gα, liées à GPR (protéine à domaine GoLoco, homologue de LGN/Pins), à LIN-5 (protéine à domaine super-enroulé, homologue de NuMA/Mud) et à la dynéine serait ancré au cortex et activé en début de mitose pour tirer le fuseau. En analysant la première division d’une espèce proche de C. elegans : C. briggsae, on observe des variations de trajectoire du fuseau. Les embryons de C. briggsae présentent un décalage antérieur des noyaux en prophase et les oscillations du fuseau sont réduites en anaphase. La combinaison de perturbations physiques et l'analyse de mutants dans ces espèces, ont montré que ces différences s’expliquent par des changements dans la régulation du complexe ternaire. Mais, nous avons découvert que dans les deux espèces 1) un switch positionnel conservé contrôle le démarrage des oscillations du fuseau, 2) la localisation postérieure de GPR détermine ce switch positionnel, et 3) l'amplitude maximum des oscillations est déterminée en partie par le temps passé dans la phase oscillatoire. Nous avons utilisés ces variants pour corréler les phénotypes, la localisation de GPR et la divergence de séquence entre espèces afin d’identifier les éléments de régulation de cette protéine. Nous avons alors échangé les protéines et construits des protéines chimères entre les deux espèces. Enfin, par optogénétique, nous avons essayé de contrôler la localisation temporelle de GPR et analyser les conséquences sur les mouvements des noyaux et du fuseau. En étudiant la microévolution d'un processus sous-cellulaire, nous avons identifié de nouveaux mécanismes qui contribuent à la compréhension du positionnement du fuseau. / Asymmetric cell division is a fundamental mechanism essential in all organisms to assure cell diversity, stem cell renewal and cellular identity maintenance. It is relying on proper mitotic spindle positioning because it dictates the cell division plan. In C. elegans one-cell embryos, the first division is asymmetric and gives rise to two daughter cells of unequal size and fate. It occurs in two steps: pronuclei centration during prophase and spindle posterior displacement during anaphase. During anaphase, the mitotic spindle undergoes transverse oscillations that are more pronounced for the posterior than the anterior pole. These movements are controlled by pulling forces acting on astral microtubules. The force generators are identified and are evolutionary conserved. A complex made of Gα proteins, linked to GPR (a GoLoco containing protein, the LGN/Pins homologues), LIN-5 (a coiled-coil protein, the NuMA/Mud homologues) and dynein is thought to be anchored at the cortex and activated at the onset of mitosis to pull on the spindle. We identified variations in spindle trajectories by analyzing the outwardly similar one-cell stage embryo of a close relative of C. elegans, C. briggsae. Compared to C. elegans, C. briggsae embryos exhibit an anterior shifting of nuclei in prophase and reduced anaphase spindle oscillations. By combining physical perturbations and mutant analysis in both species, we show that differences can be explained by inter-species changes in the regulation of the cortical Gα/GPR/LIN-5 complex. However, we uncover that in both species 1) a conserved positional switch controls the onset of spindle oscillations, 2) GPR posterior localization may set this positional switch, and 3) the maximum amplitude of spindle oscillations is determined in part by the time spent in the oscillating phase. Interestingly, GPR is poorly conserved at the amino acid level between these species. We use these variants to correlate phenotypes, GPR localization and sequence divergence to identify GPR regulatory elements. To this end, we performed protein replacement between species, as well as analysis of protein chimeras. Finally we tried to use optogenetics in order to control GPR localisation temporally and analyze the consequences on pronuclei and spindle movements during the first division. By investigating microevolution of a subcellular process, we identified new mechanisms that are instrumental to decipher spindle positioning.

This thesis is concerned with textile production in Middle and Late Bronze Age on the Greek continent and western Anatolia. First part explains the description of methodology of approach to textile manifacture in the studied area, followed by description and properties of threads used to weave the cloths, as well as technologies used in their making. Terms related to textile are mentioned on clay tablets with linear writing B, hence the thesis engages in their interpretation and comparison between particular centres. Second part analyses tools used in textile manifacture, namely whorls and loom weights based on their shape. Then follows topographic overview of the most prominent localities of textile manifacture in which textile-making tools were found. Localities not included in the overview are mentioned in the table. The overview describes locality, number and types of whorls and loom weights and what materials are they made of. Whenever possible, the most important specification is mentioned - the weight - based on which the type of weaved textile is described. The aim of the thesis is to create overview of localities with tools used in textile making as well as their analysis and comparison. Collected data are converted into tables, graphs and maps. In the last part called discussion,...

La kinase Aurora A est une protéine essentielle au cycle cellulaire et plus particulièrement lors de la mitose. En effet, Aurora A est nécessaire à l'entrée en mitose et joue un rôle dans la maturation des centrosomes. Elle participe à l'assemblage du fuseau mitotique et est nécessaire à la réussite de la cytodiérèse. Elle est également nécessaire à l'égale répartition des mitochondries dans les cellules filles et joue un rôle dans l'épissage alternatif des ARNm de facteurs apoptotiques. Au-delà de ses fonctions mitotiques, plusieurs études récentes indiquent qu'Aurora A présente des fonctions supplémentaires dans les cellules en interphase. Elle est notamment essentielle au désassemblage du cil primaire et joue un rôle dans la dynamique des microtubules et la migration cellulaire. Enfin, une dérégulation de son expression, de sa stabilité et/ou de son activité perturbe le déroulement du cycle cellulaire ce qui conduit à la transformation des cellules et favorise l'apparition de cancers. Ses fonctions normales ainsi que ses fonctions lors de la carcinogenèse sont conduites à travers les nombreux partenaires protéiques qui entrent en interaction avec elle. Ils modulent son activité, sa localisation et sa stabilité. En retour Aurora A phosphoryle un bon nombre d'entre eux régulant ainsi leur activité, localisation et stabilité. Cependant, l'analyse des interactions déjà connues d'Aurora A ne permet pas d'expliquer tous les phénotypes observés lors de sa dérégulation. Afin de mieux comprendre les fonctions d'Aurora A, les mécanismes qui la régulent et mettre en évidence ses multiples rôles au sein de la cellule, j'ai construit puis analysé un interactome d'Aurora A généré à partir d'une méthode de purification d'affinité couplée à la spectrométrie de masse en tandem. J'ai identifié 477 partenaires potentiels dont 180 présentant une forte probabilité d'être des partenaires directs de la kinase. L'analyse bioinformatique approfondie de cet interactome a permis de révéler les partenaires associés à des mécanismes liés à la mitochondrie et l'épissage des ARN messagers mettant en évidence une implication potentielle d'Aurora A dans ces mécanismes. Pour valider cet interactome, j'ai choisi d'étudier plus précisément deux partenaires identifiés dans cette étude : les protéines WDR62 et CEP97. J'ai montré que ces deux partenaires co-localisent avec Aurora A et sont phosphorylés par la kinase. Ainsi, ce travail de thèse a permis de mettre en évidence un nombre important de nouveaux partenaires d'Aurora A associés à de nouvelles fonctions. L'étude de ces nouvelles fonctions liées aux mitochondries et à l'épissage des ARN, constitue deux nouveaux projets actuellement menés par des collaborateurs au sein de notre institut. / The kinase Aurora A is an essential mitotic cell cycle protein. Aurora A is necessary for mitotic entry and for the maturation and separation of centrosomes. It participates in mitotic spindle assembly and chromosome biorientation, and it is essential for the completion of cytokinesis. Furthermore, Aurora A activity is necessary for the equal distribution of mitochondria to daughter cells and, through its role in the alternative splicing of mRNA of apoptotic factors, it provides a link between cell cycle control and apoptosis. Beyond its mitotic functions, several recent studies suggest that Aurora A is also important during interphase. Notably, it influences microtubule dynamics, promotes cell migration and polarity control and is essential for primary cilia disassembly. Reflecting the fact that Aurora A is found to be up-regulated in many cancers, deregulation of Aurora A activity can result in an aberrant cell cycle, ultimately leading to malignant transformation of cells. The crucial regulation of Aurora A’s numerous functions is achieved through its interaction with several protein partners, which modulate its activity, localisation and stability. Aurora A in turn phosporylates a number of them, thus regulating their activity, localisation and stability. However, the known interactions of Aurora A cannot explain all the phenotypes that have been described of its deregulation.To better understand the functions of Aurora A, the regulation mechanisms governing it, and to expose its multiple roles in the cell, I have built and analysed an Aurora A interactome using tandem affinity purification coupled with mass spectrometry. This resulted in the identification of 477 potential interacting partners, of which, 180 were determined to have a high probability of interacting directly with the kinase.In-depth bioinformatic analysis of this interactome has revealed the associated partners to be related to mitochondria and mRNA splicing, highlighting the potential involvement of Aurora A in these mechanisms. To validate the interactome, two of the proteins identified in this study, WDR62 and CEP97, were examined in detail. Here I show that these two proteins colocalise with Aurora A, and are phosphorylated by the kinase.WDR62 is implicated in microcephaly and is deregulated in certain cancers. I have shown that Aurora A phosphorylates WDR62 during mitosis, and that this phosphorylation is necessary for its localisation to the centrosomes. CEP97 is a poorly charactarised protein of the primary cilium, abnormalities of which are associated with ciliopathies. I have shown that Aurora A phosphorylates CEP97 in vitro, and that the inhibition of Aurora A activity in vivo perturbs the localisation of CEP97 to cilia and centrosomes.This study has identified a number of new Aurora A-interacting proteins, implicating the kinase with novel functions. These functions, related to mitochondria and mRNA splicing have opened up a new area for further investigation.

Autosomal recessive primary microcephaly (MCPH) is defined by congenital microcephaly and associated mental retardation with head circumference of the affected individual at least 3 standard deviations below age- and sex-means. It is a disorder of abnormal fetal brain growth which is a consequence of impaired neurogenesis. It is genetically heterogeneous with seven known loci and genes for all the seven loci have been identified: MCPH-1-MCPH1, MCPH2-WDR62, MCPH3-CDK5RAP2, MCPH4-CEP152, MCPH5-ASPM, MCPH6-CENPJ, and MCPH7-STIL. All the seven MCPH proteins localize at the centrosome. Apart from MCPH, many other proteins associated with the phenotype microcephaly have been localized to the centrosome or linked to it functionally. For example, Microcephalic osteodysplastic primordial dwarfism type II protein PCNT and Seckel syndrome protein ATR are also centrosomal proteins. All of the above findings show the importance of centrosomal proteins as the key players in neurogenesis and brain development. However, the exact mechanism as to how the loss-of-function of these proteins leads to microcephaly remains to be elucidated. The study of MCPH genes can also provide insights into the basics of neurogenesis that lead to a normal brain size. The most common cause of MCPH is mutations in the ASPM (abnormal spindle-like, microcephaly-associated protein) gene. The main aim of this study was to gain insight into the function of ASPM using the yeast two-hybrid technique. The main findings of the study are listed below. To find novel interacting proteins for SPM, a GAL4 based yeast two-hybrid system was used. The 3,477 amino acid long ASPM was divided into eight different baits and each bait was individually used for screening a human fetal brain cDNA library cloned in the pACT2 vector. To generate baits, the different regions were amplified from human fetal brain cDNA and cloned in-frame with the GAL4-DNA binding domain in the pGBKT7 vector. Screening with a C-terminus ASPM bait (pGBKT7-CTR) identified Angelman syndrome protein ubiquitin protein ligase E3A (UBE3A) as an ASPM interactor. A region of UBE3A from amino acids 639-875 was found to interact with ASPM. The identification of UBE3A as an ASPM interacting partner was interesting as more than 80% of Angleman syndrome patients are reported to have microcephaly. Screening with the baits pGBKT7-1.4 kb ASPM and pGBKT7-2.1 kb ASPM harboring parts of IQ domain identified calmodulin as an ASPM interating partner. The full length calmodulin was found to interact with the IQ domain of ASPM. The interactions identified in the yeast two-hybrid assay were confirmed in vivo by co-immunoprecipitation studies. For this, a rabbit polyclonal anti-ASPM antibody was raised against the N-terminal region of ASPM (from amino acids 544-1059). The specificity of the antibody was tested by Western blot analysis and immunofluorescence microscopy. ASPM antibody recognized the 410 KDa fulllength ASPM protein in lysates from human fetal tissues and different cell lines. Immunofluorescence analysis in HEK293 cells with the antibody revealed centrosomal staining of ASPM throughout mitosis and midbody staining in cytokinesis, as reported previously. Using antibodies against ASPM and UBE3A and human fetal kidney lysate, ASPM and UBE3A interaction was confirmed in vivo by co-immunoprecipitation. The interaction between ASPM and calmodulin was confirmed similarly. The relevance of the interaction between ASPM and UBE3A was pursued further Like ASPM, UBE3A localized to the centrosome throughout mitotic progression. ASPM levels were found to be unaffected upon overexpression of UBE3A in HEK293 cells, indicating that ASPM is not degraded by a UBE3A-dependent proteasomal pathway or the degradation may be spatial-temporal control. Further, immunofluorescence analysis of UBE3A overexpressing HEK293 cells revealed that UBE3A does not affect either the ASPM localization or its protein level at the centrosome. Synchronization of HEK293 cells in different cell cycle phases revealed that UBE3A is a cell cycle dependent protein and its level peaks in mitosis To explore the functional role of UBE3A’s increased level in mitosis, UBE3A was depleted in HEK293 cells with a shRNA construct and stable clones were generated. HEK293- UBE3A shRNA knockdown cells were examined for normal mitotic progession and spindle defects. There was a 3.81- to 5.52-fold increase in the frequency of anaphase/telophase cells with missegregated chromosomes in UBE3A knockdown clones as compared to scrambled clones. Hence, we identified a definitive role of UBE3A in chromosome segregation. Defective chromosome segregation has been reported in many studies associated with microcephaly-related proteins. Interestingly, chromosome malfunctioning has also been reported in Drosophilia asp mutants (ASPM orthologue) and Celegans aspm-1 knockdown cells. Therefore, the loss of both ASPM and UBE3A leading to chromosome segregation defects reveals the existence of a molecular pathway common to both ASPM and UBE3A As a consequence of chromosome missegregation, UBE3A knockdown cells were found to undergo abnormal cytokinesis and apoptosis. The percentage of apoptotic cells in UBE3A knockdown clones was 1.25- to 3.04-fold higher as compared to scrambled clones. Interestingly, an extensive apoptosis has been found in the neural folds of MCPH7 gene STIL null mice embryos. Thus, the present study links Angleman syndrome protein UBE3A to ASPM, centrosome and mitosis for the first time.

Mikrotubuli sind filamentöse intrazelluläre Polymere, die als grundlegende Bestandteile subzellulärer Strukturen in Eukaryoten dienen. Diese Studie verwendet einen vergleichenden Ansatz, um zu untersuchen, wie sich die intrinsischen dynamischen und biochemischen Eigenschaften von Tubulin zwischen verschiedenen Spezies unterscheiden, und zeigt ihre Konsequenzen in zwei verschiedenen physiologischen Kontexten: 1) Bestimmung der Spindelgröße bei Fröschen der Gattung Xenopus und 2) Spezifität von Mikrotubuli-Inhibitoren für Plasmodium falciparum-Mikrotubuli über denen ihres menschlichen Wirts. In den Eiern der Froschgattung Xenopus wird die Länge der meiotischen Spindel biochemisch festgelegt und erreicht unabhängig von räumlichen Einschränkungen eine Obergrenze. Messungen der Dynamik von Xenopus-Mikrotubuli zeigen, dass X. laevis-Mikrotubuli sowohl schneller wachsen als auch länger leben als die von X. tropicalis. Darüber hinaus spielt die Quantifizierung der Länge und Massenverteilung der Xenopus-Mikrotubuli zusammen mit den Reaktionen der Eiextrakt-Spindelanordnung eine Rolle für die intrinsische Dynamik der Mikrotubuli bei der Modulation der Spindellänge. Mikrotubuli sind auch Wirkstofftargets bei Pilz- und parasitären Helmintheninfektionen und haben in den letzten Jahrzehnten die Aufmerksamkeit als potenzielles Wirkstoffziel beim Malariaparasiten Plasmodium falciparum auf sich gezogen. Um die Dynamik und Medikamentspezifität von Mikrotubuli von P. falciparum zu charakterisieren, haben wir Tubulin direkt von den Parasiten gereinigt. Zum ersten Mal wurden hier dynamische P. falciparum-Mikrotubuli in vitro rekonstituiert und eine parasitenspezifische Unterdrückung der Dynamik von Mikrotubuli durch Oryzalin und Amiprofos-Methyl direkt nachgewiesen. Diese Studie legt einen experimentellen Rahmen fest, um direkt auf parasitenspezifische Hemmung von Mikrotubuli zu testen, die bisher unter Verwendung bestehender in-vitro-Ansätze nicht beobachtet wurden. / Microtubules are filamentous intracellular polymers that are fundamental components of subcellular structures including the spindle, the cytoskeleton, and flagella in eukaryotes. This study uses a comparative approach to investigate how the intrinsic dynamic and biochemical characteristics of tubulin vary between species and demonstrates their consequences in two different physiological contexts: 1) Spindle size control in Xenopus frogs, and 2) The specificity of microtubule inhibitors for Plasmodium falciparum microtubules over those of their human host. In Xenopus frog eggs, the length of the spindle is biochemically controlled and reaches an upper limit independent of spatial constraints. In this study, in vitro measurements of Xenopus microtubule dynamics show that X. laevis microtubules are both faster-growing and longer-lived X. tropicalis, independent of the influence of microtubule-associated proteins. Furthermore, quantification of Xenopus microtubule length and mass distributions, combined with egg extract spindle assembly reactions, establishes a role for intrinsic microtubule dynamics in modulating spindle length. Microtubules are also established drug targets in fungal and parasitic helminth infections and have in the past decades drawn attention as a potential drug target in the malaria parasite Plasmodium falciparum. In order to characterize P. falciparum microtubule dynamics, structure, and drug specificity, we have used an affinity chromatography-based approach to purify tubulin directly from blood-stage parasites. For the first time, dynamic P. falciparum microtubules have been reconstituted in vitro and parasite-specific suppression of microtubule dynamics by oryzalin and amiprofos methyl has been directly demonstrated. This study establishes an experimental framework to directly test for parasite-specific microtubule inhibition, microtubule structure, and interactions with MAPs that previously have not observed using existing in vitro approaches.

This master’s thesis deals with design of a headstock for a machine tool. Theoretic part of this thesis provides basic description of a horizontal machine tool. Further it is dedicated to description of a headstock of machines with extensionable work spindle. In a design part firstly optimal variant is chosen by using multicriterial analysis. Necessary calculations were done and chosen variant was developed. Design is realised by 3D CAD model and drawing documentantion.