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The Role of Dynamic Cdk1 Phosphorylation in Chromosome Segregation in Schizosaccharomyces pombe: A DissertationChoi, Sung Hugh 15 February 2010 (has links)
The proper transmission of genetic materials into progeny cells is crucial for maintenance of genetic integrity in eukaryotes and fundamental for reproduction of organisms. To achieve this goal, chromosomes must be attached to microtubules emanating from opposite poles in a bi-oriented manner at metaphase, and then should be separated equally through proper spindle elongation in anaphase. Failure to do so leads to aneuploidy, which is often associated with cancer. Despite the presence of a safety device called the spindle assembly checkpoint (SAC) to monitor chromosome bi-orientation, mammalian cells frequently possess merotelic kinetochore orientation, in which a single kinetochore binds microtubules emanating from both poles. Merotelically attached kinetochores escape from the surveillance mechanism of the SAC and when cells proceed to anaphase cause lagging chromosomes, which are a leading cause of aneuploidy in mammalian tissue cultured cells. The fission yeast monopolin complex functions in prevention of mal-orientation of kinetochores including merotelic attachments during mitosis. Despite the known importance of Cdk1 activity during mitosis, it has been unclear how oscillations in Cdk1 activity drive the dramatic changes in chromosome behavior and spindle dynamics that occur at the metaphase/anaphase transition. In two separate studies, we show how dynamic Cdk1 phosphorylation regulates chromosome segregation. First, we demonstrate that sequential phosphorylation and dephosphorylation of monopolin by Cdk1 and Cdc14 phosphatase respectively helps ensure the orderly execution of two discrete steps in mitosis, namely sister kinetochore bi-orientation at metaphase and spindle elongation in anaphase. Second, we show that elevated Cdk1 activity is crucial for correction of merotelic kinetochores produced in monopolin and heterochromatin mutants.
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Mitotic Response to DNA Damage in Early Drosophila Embroyos: a DissertationKwak, Seongae 30 April 2008 (has links)
DNA damage induces mitotic exit delays through a process that requires the spindle assembly checkpoint (SAC), which blocks the metaphase to anaphase transition in the presence of unaligned chromosomes. Using time-lapse confocal microscopy in syncytial Drosophila embryos, we show that DNA damage leads to arrest during prometaphase and anaphase. In addition, functional GFP fusions to the SAC components MAD2 and Mps1, and the SAC target Cdc20 relocalize to kinetochore through anaphase arrest, and a null mad2mutation blocks damage induced prometaphase and anaphase arrest. We also show that the DNA damage signaling kinase Chk2 is required for damage induced metaphase and anaphase arrest, and that a functional GFP-Chk2 fusion localizes to kinetochores and centrosomes through mitosis. In addition, in the absence of Chk2, we find that DNA damage sufficient to fragment centromere DNA does not delay mitotic exit. We conclude that DNA damage signaling through Chk2 triggers Mad2-dependent delays in mitotic progression, both before or after the metaphase-anaphase transition.
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Mechanisms of microtubule nucleation in metaphase spindles and how they set spindle sizeDecker, Franziska 25 September 2018 (has links)
Regulation of size and growth is a fundamental problem in biology and often closely related to functionality and fitness. A prominent example is the mitotic spindle, whose size needs to be perfectly tuned to ensure proper chromosome segregation during cell division. It is known that spindle size generally scales with cell volume, most likely as a result of limiting components. However, this relation breaks down in very large cells where spindles have a maximum size. How the size and microtubule mass are set and why spindles show an upper size limit in large cells is still not understood.
Spindles mainly consist of highly dynamic short microtubules that turn over very quickly in comparison to the lifetime of the entire structure. Thus, microtubules need to be constantly created throughout the spindle, a process called nucleation. Understanding the role of microtubule nucleation in setting the size of spindles is limited by the fact that little is known about the rate, distribution, and regulation of microtubule nucleation in these structures. This is partly due to the lack of methods to measure microtubule nucleation in spindles.
During this work, I developed an assay based on laser ablation to probe microtubule nucleation in monopolar spindles assembled in Xenopus laevis egg extract. Using this new method in combination with quantitative microscopy, I found that microtubule nucleation in these structures is spatially regulated. Furthermore, I observed that nucleation is stimulated by pre-existing microtubules leading to new microtubule growth in their physical proximity.
Combining my experimental results on nucleation with theory and further biochemical perturbations, I show that this autocatalytic nucleation mechanism is limited by the availability of active nucleators. In spindles, the amount of active nucleators decreases with distance from the chromosomes. Thus, this mechanism provides an upper limit to spindle size even when resources are not limiting.
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Adaptive light sheet microscopy for the systematic analysis of mitotic spindle scaling in zebrafishBerndt, Frederic Carl 28 March 2019 (has links)
Multicellular life is formed by an orchestrated interplay of processes on different scales in space and time. Observing and quantitatively measuring these processes in an intact, living organism requires gentle and adaptive imaging.
One example of such a process is the scaling of the mitotic spindle during early development. The spindle segregates the chromosomes during cell division and the spindle length determines the positioning of the chromosomes in the successive daughter cells. Thus, adaptation of spindle size to cell size is crucial for proper functioning. Early development is an excellent phase to study spindle scaling since cells rapidly divide in the absence of growth. In this phase, the spindle can be studied in cells of the same organism changing its volume orders of magnitude.
During early zebrafish embryogenesis, the mitotic spindle only appears for three minutes out of the fifteen minutes cell cycle. Quantifying these short-lived events in a living embryo requires flexible and adaptive multi-resolution recordings, which are impossible with any state-of-the-art microscope. In this thesis, I present two new techniques to adaptively image biological samples based on light sheet fluorescence microscopy (LSFM).
First, I present a remote, contact-free positioning technique based on magnetic forces to orient the sample in the microscope. When imaging biological samples, there is often only one sample orientation that offers the best view on the region of interest. This preferred orientation typically changes over time as the specimen grows and develops. The contact-free positioning technique allows to always image specimens from the optimal viewing angle. I demonstrate the functionality of this method by 3D orientation of zebrafish embryos and zebrafish larvae.
Second, I present a new type of LSFM that autonomously adapts its detection scheme to the sample state. This microscope contains an adaptable magnification module to map the development of the millimeter-sized zebrafish embryo and measure single-molecule dynamics of individual spindles in a single experiment. To automatically adapt the detection scheme, I trained a Convolution Neural Network to detect the cell cycle state of individual cells from acquired fluorescence images. Using this new type of LSFM, I demonstrate autonomous measurements of the mitotic spindle scaling in freely developing zebrafish embryos. / Multizelluläres Leben wird durch ein orchestriertes Zusammenspiel von Prozessen auf verschiedenen Skalen in Raum und Zeit gebildet. Beobachtung und quantitative Messungen dieser Vorgänge in einem intakten, lebenden Organismus erfordern schonende und adaptive Bildgebung.
Ein Beispiel für einen solchen Prozess ist die Größenanpassung der mitotischen Spindel während der frühen Entwicklung. Die Spindel trennt die Chromosomen während der Zellteilung und die Spindellänge bestimmt die Positionierung der Chromosomen in den Tochterzellen. Daher ist die Anpassung der Spindelgröße an die Zellgröße entscheidend für die ordnungsgemäße Funktion. Die Phase der frühen Entwicklung eignet sich hervorragend zur Untersuchung der Spindel-Skalierung, da die Zellen sich schnell teilen ohne zu wachsen.
Während der frühen Zebrafischembryogenese erscheint die Spindel nur drei Minuten innerhalb des fünfzehnminütigen Zellzyklus. Die Quantifizierung dieser kurzlebigen Ereignisse in einem lebenden Embryo erfordert flexible und anpassungsfähige Aufnahmen mit variabler Auflösung, die mit keinem Mikroskop nach dem aktuellen Stand der Technik möglich sind. In dieser Arbeit präsentiere ich zwei neue Techniken zur adaptiven Abbildung biologischer Proben basierend auf der Lichtblatt-Fluoreszenzmikroskopie (LSFM).
Zuerst stelle ich eine berührungslose Positionierungstechnik vor, die auf Magnetkräften basiert, um die Probe im Mikroskop zu orientieren. Bei der Abbildung biologischer Proben gibt es oft nur eine Probenorientierung, welche die beste Sicht auf die Region von Interesse bietet. Diese Vorzugsorientierung ändert sich typischerweise mit der Zeit, wenn die Probe wächst und sich entwickelt. Die Positionierungstechnik ermöglicht es, Proben immer aus dem optimalen Betrachtungswinkel abzubilden.
Zweitens stelle ich einen neuen Typ von LSFM vor, der sein Detektionsschema autonom an den Probenzustand anpasst. Dieses Mikroskop enthält ein anpassbares Vergrößerungsmodul, um die Entwicklung des millimetergroßen Zebrafischembryos abzubilden und die Einzelmoleküldynamik einzelner Spindeln in einem einzigen Experiment zu messen. Um die Detektion automatisch anzupassen, trainierte ich ein Convolutional Neural Network, um den Zellzyklusstatus einzelner Zellen anhand der aufgenommenen Fluoreszenzbilder zu erkennen. Mit diesem neuen LSFM-Typ demonstriere ich autonome Messungen der Spindel-Skalierung in sich frei entwickelnden Zebrafischembryonen.
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Simulationsgestützte Entwicklung eines modellbasierten Reglers zur Vorspannkraftregelung in einer adaptronischen HauptspindelIvanov, Georg 24 May 2023 (has links)
Ausgangspunkt der hier vorgestellten Entwicklungen ist eine am ICM – Institut Chemnitzer Maschinen- und Anlagenbau e.V. entwickelte adaptronische Werkzeugmaschinen-Hauptspindel zur variablen Einstellung der Lagervorspannkräfte. Diese neuartige Werkzeugmaschinenspindel bietet Anwendungspotentiale in den Bereichen: verbessertes Hochdrehverhalten, erhöhte Flexibilität des Bearbeitungsspektrums, Lebensdauererhöhung der Wälzlager durch verbessertes Bohr-Roll-Verhältnis sowie einer optimierten Spindeldynamik durch gezielte Verschiebung der Eigenfrequenzen. Ziel war die Entwicklung einer möglichst dynamischen und genauen Ansteuerung des in der Hauptspindel enthaltenen hydraulischen Vorspannelementes. Dieser Beitrag fasst das Vorgehen und die bisherigen Ergebnisse zusammen. Auf die Eigenentwicklung eines hydraulischen Aktuators sollte verzichtet werden, sodass die Ansteuerung über ein direktgesteuertes Regelventil erfolgt. Mit einer einfachen Druckregelung mit PID-Regler konnten nur unzureichende Ergebnisse hinsichtlich Dynamik und Regelkreisstabilität erzielt werden. Hinzu kommt die geringe Genauigkeit der Krafteinstellung bei Anwendung einer einfachen Druckregelung. Zur Verbesserung der Ansteuerungsdynamik und -genauigkeit des Vorspannelementes sollten erweiterte Regelungsstrukturen zur Anwendung kommen. Im Projekt wurde hierzu ein modellbasierter Regler entwickelt und in einer dem Experiment vorgelagerten Untersuchung an einem Systemsimulationsmodell getestet und optimiert. Ausgangspunkt für die simulationsgestützte Reglerauslegung war die Entwicklung eines Regelstreckenmodells in SimulationX. Die Modellentwicklung umfasste den Vergleich zweier grundlegender Modellierungsansätze, eines bidirektionalen Modells mit einem linearen Signalflussmodell hinsichtlich Modellgenauigkeit und Rechenperformance. Zur Untersuchung der Reglerfunktionalität sowie zur Optimierung der Reglerparameter wurde die in SimulationX vorhandene COM-Schnittstelle genutzt und eine vereinfachte Optimierungsfunktion in Matlab umgesetzt. Für die experimentelle Validierung des Ansteuerungssystems wurde die entwickelte modellbasierte Reglervorsteuerung in der Programmierumgebung LabView umgesetzt. Erste Tests erfolgten zunächst an der stehenden Hauptspindel. Diese zeigten ein sehr dynamisches und stabiles Regelungsverhalten, sodass neben einfachen Sprungvorhaben auch Trajektorienvorgaben mit hoher Dynamik geregelt werden können. Es konnte eine deutliche Verbesserung gegenüber der zu Beginn vorhandenen einfachen Druckregelung erzielt werden. Umfangreichere experimentelle Untersuchungen an der drehenden Spindel für unterschiedliche Regelungs- und Bearbeitungsszenarien sollen in Zukunft durchgeführt werden. Dabei stehen vor allem die Verkürzung der Hochdrehzeiten für Bearbeitungsprozesse mit geringer Lagervorspannung sowie die anwendungsspezifische Optimierung der Spindeldynamik durch Eigenfrequenzverschiebungen im Vordergrund. / The starting point of the developments presented here is an adaptronic machine tool main spindle developed at the ICM - Institute Chemnitz Machine and Plant Construction e.V. for variable adjustment of the bearing preload forces. This new type of machine tool spindle offers application potential in the areas of: improved high-speed behavior, increased flexibility of the machining spectrum, increased service life of the roller bearings through improved drilling-rolling ratio and optimized spindle dynamics through targeted shifting of the natural frequencies. The aim was to develop the most dynamic and precise control possible for the hydraulic pretensioning element contained in the main spindle. This article summarizes the procedure and the results so far. The in-house development of a hydraulic actuator should be avoided, so that the control takes place via a directly controlled control valve. With a simple pressure control with a PID controller, only insufficient results could be achieved in terms of dynamics and control loop stability. Added to this is the low accuracy of the force setting when using a simple pressure control. Extended control structures should be used to improve the control dynamics and accuracy of the preload element. For this purpose, a model-based controller was developed in the project and tested and optimized on a system simulation model in an investigation prior to the experiment. The starting point for the simulation-based controller design was the development of a controlled system model in SimulationX. The model development included the comparison of two basic modeling approaches, a bidirectional model with a linear signal flow model in terms of model accuracy and computational performance. The COM interface available in SimulationX was used to examine the controller functionality and to optimize the controller parameters, and a simplified optimization function was implemented in Matlab. For the experimental validation of the control system, the developed model-based controller pre-control was implemented in the LabView programming environment. The first tests were initially carried out on the stationary main spindle. These showed a very dynamic and stable control behavior, so that in addition to simple jump projects, trajectory specifications can also be controlled with high dynamics. A clear improvement could be achieved compared to the simple pressure control that was available at the beginning. More extensive experimental investigations on the rotating spindle for different control and processing scenarios are to be carried out in the future. The main focus here is on reducing the ramp-up times for machining processes with low bearing preload and the application-specific optimization of the spindle dynamics through natural frequency shifts.
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La protéine Staufen1 contrôle la localisation des ARN spécifiques sur le fuseau mitotique dans les cellules de cancer colorectal humain HCT116Hassine, Sami 04 1900 (has links)
La protéine de liaison à l’ARN double-brin Staufen1 (STAU1) est exprimée dans les cellules de mammifères de manière ubiquitaire. STAU1 est impliqué dans la régulation post-transcriptionnelle de l’expression génique grâce à sa capacité de lier les ARN et moduler leur épissage, leur transport et localisation, leur traduction ainsi que leur dégradation. Des études récentes de notre laboratoire indiquent que l’expression de STAU1 est régulée durant le cycle cellulaire, ayant une abondance maximale au début de la mitose. En prométaphase, STAU1 est lié à des ARNm codant pour des facteurs impliqués dans la régulation de la prolifération, la croissance et la différenciation cellulaires. De plus, des analyses protéomiques menées sur des cellules humaines ont permis d’identifier STAU1 comme un composant de l’appareil mitotique. Cependant, l’importance de cette association n’a pas été investiguée. Par ailleurs, il a été montré qu’une défaillance dans l’expression ou les fonctions de STAU1 pourrait contribuer au développement et l’accélération de plusieurs maladies débilitantes, dont le cancer. Dans cette thèse, nous avons montré la localisation de STAU155 sur le fuseau mitotique dans les cellules de cancer colorectal HCT116 et les cellules non transformées hTERT-RPE1. Nous avons également caractérisé le déterminant moléculaire impliqué dans l’interaction entre STAU155 et les microtubules mitotiques, soit la séquence située dans les 88 premiers acides aminés N-terminaux de RBD2, un domaine qui n’est pas requis pour l’activité de liaison à l’ARN de STAU1. Nous avons ainsi montré que la fraction de STAU1 enrichie sur le fuseau colocalise avec des ribosomes dans des sites actifs de traduction. De plus, notre analyse transcriptomique du fuseau mitotique montre que 1054 transcrits (ARNm, pré-ARNr, lncRNA et snoRNA) sont enrichis sur l’appareil mitotique. De façon intéressante, le knockout de STAU1 entraine la délocalisation des pré-ARNr et de 154 ARNm codants pour des protéines impliquées dans l’organisation du cytosquelette d'actine et la croissance
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cellulaire. Bien que STAU1 n’est pas essentiel pour la survie et la prolifération des cellules cancéreuses HCT116, nos résultats mettent clairement en évidence l’implication de STAU1 dans la régulation des ARN spécifiques en mitose et suggèrent un nouveau rôle de cette protéine dans la progression mitotique et la cytokinèse par la régulation de la maintenance des pré-ARNr, la ribogenèse et/ou la reconstitution de l’enveloppe nucléaire. / Staufen1 (STAU1) is a double-stranded RNA-binding protein that is ubiquitously expressed in mammals and known for its involvement in the post-transcriptional regulation of gene expression such as splicing, transport and localization, translation, and decay. It has been demonstrated that STAU1 protein expression level is modulated through the cell cycle with peak abundance by the onset of the mitotic phase after which it is degraded. Genome-wide analysis revealed that in prometaphase, STAU1 bound with mRNAs code for factors implicated in cell differentiation, cell growth as well as for cell proliferation. Interestingly, previous large-scale proteomic studies identified STAU1 as a component of the human mitotic spindle apparatus. Altering STAU1 expression patterns or functions may lead to several debilitating human diseases including cancer. In this thesis, we further elucidated the localization of STAU1 at the mitotic spindle of the colorectal cancer HCT116 and the non-transformed hTERT-RPE1 cells. Next, we characterized the molecular determinant required for STAU1/spindle association within the first 88 N-terminal amino acids, a domain that is not required for the RNA binding activity. RNA-Seq analysis of purified mitotic spindles reveals that 1054 mRNAs as well as the precursor ribosomal RNA, lncRNAs and snoRNAs are enriched on spindles compared to cell extracts. Spindle-associated STAU1 partly co-localizes with ribosomes and active sites of translation. Interestingly, the knockout of STAU1 delocalizes pre-rRNA and 154 mRNAs coding for proteins involved in actin cytoskeleton organization and cell growth. Our results highlighting a role for STAU1 in mRNA trafficking to the spindle. These data demonstrate that STAU1 controls the localization of sub-populations of RNA during cell division and suggests a novel role of STAU1 protein in mitotic progression and cytokinesis by regulating pre-rRNA maintenance, ribogenesis and/or nucleoli reassembly.
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Protein 14-3-3 (YWHA) isoforms and their roles in regulating mouse oocyte maturationDe, Santanu 02 July 2014 (has links)
No description available.
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The Arabidopsis nucleoporin NUA is involved in mRNA export and functionally interacts with spindle assembly checkpoint proteinsMuthuswamy, Sivaramakrishnan January 2009 (has links)
No description available.
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Konstrukce frézovací hlavy pro CNC soustruh s nastavitelným sklonem / Design of CNC lathe milling head with adjustable tiltHorák, Vítězslav January 2020 (has links)
This thesis deals with design of CNC lathe milling head with B axis. Thesis includes research in CNC turning machines, their tool systems and overview of manufacturers of milling B axis. It also includes calculation of machining forces, design of spindle and design of B axis including necessary calculations. Part of this thesis is also 3D model and assembly drawing, 2 manufacturing drawings and list of items.
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Spår av textilproduktion : En analys av sländtrissor och vävtyngder från yngre järnålderslokaler i Birkas omland.Björklund Andersson, Simone January 2015 (has links)
Denna kandidatuppsats behandlar sländtrissor och vävtyngder från vikingatida lokaler samt yngre järnålderslokaler som angränsar till den vikingatida handelsplatsen Birka. Syftet är att studera sländtrissor och vävtyngder från vikingatida fyndlokaler i Birkas omland; vilket åsyftar geografiska områden i Södermanlands län (Södermanland), Stockholms län och Uppsala län (Uppland). Sländtrissor påverkar de textila råmaterialen och de spunna trådarna, vidare påverkar vävtyngderna varpen och de vävda tygerna. Vilka trådar och vilka tyger som har producerats inom vikingatida lokaler i angränsning till Birka diskuteras utifrån sländtrissornas och vävtyngdernas vikt, form och storlek (trots att fragmentariska artifakter förekommer). Studien omfattar spår av textilproduktion inom vikingatida lokaler i Birkas omland, samt en undersökning gällande om det har förekommit inhemska importer av textilier från fastlandet till Birka under vikingatiden. Resultatet påvisar om att gårdsbaserad textilproduktion har förekommit i Birkas omland och ett fåtalet gårdar i omlandet tolkas ha haft möjlighet att exportera textilier till Birka. / This bachelor thesis is regarding spindle whorls and loom weights from late Iron Age-sites and Viking Age-sites in adjoining relation to the Viking Age town Birka. The basis of this thesis concerns in which amount spindle whorls and loom weights occur at Viking Age-sites in Birka's hinterland; it refers to geographical areas in Södermanland County and Uppland County. Spindle whorls influences the textile raw materials and the spun threads, furthermore loom weights influences the warp and the woven cloths. Which threads and which cloth that were produced at the Viking Age-sites in adjoining relation to Birka will be discussed by regarding the weight, form and size of the spindle whorls and loom weights (though fragmental artifacts occur). The thesis will concern trails of textile production from Birka's hinterland and furthermore an investigation about domestic imports of textiles from the mainland to Birka could have oocurred during the Viking Age. The Results indicates that farmbased textileproduction has occured in Birka's hinterland and furthermore numerous of the farms in the hinterland can be discussed as farms with the possibility to export textiles to Birka.
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