This master´s thesis deals with the design of a cartridge spindle for milling machines. The aim of the work is to design a spindle with a power of 15 kW, which allows the automatic exchange of tools with ISO interface. In the beginning, the thesis deals with the description of the individual components related to the construction of machine tools spindle. A spindle design concept is chosen, for which a number of calculations were made concerning the structure itself. Based on the proposed design, a 3D model and drawing documentation were made.

The diploma thesis deals with the design of a built-in headstock of a turning center. The aim of the work is the design of a spindle for machines up to 10 kW. The spindle is driven by a two-speed gearbox and a belt drive. In the introduction, the work deals with the description of individual parts of the turning headstock. A design proposal is performed, accompanied by a number of technical calculations. The result of the work is a 3D model, drawing documentation and calculation report.

The thesis deals with the design of a headstock for a small lathe. The first part of the thesis deals with the description of individual structural nodes related to the construction of machine tools headstock. The second part includes an overview of similar machines available on the market. The final part deals with the calculation of necessary parameters, the verification of results using the finite element method and the construction of the proposed headstock. Based on the designed construction is created 3D model and drawing documentation.

The eukaryotic cell cycle is governed by molecular checkpoints that ensure genomic integrity and the faithful transmission of chromosomes to daughter cells. They inhibit the cycle until conditions prevail that guarantee accurate DNA duplication and chromosome segregation. Two major mechanisms are the ‘spindle assembly checkpoint’ and the ‘DNA damage checkpoint’. During pro-metaphase, the spindle checkpoint monitors the orientation process of chromatid pairs on the bipolar microtubule array nucleated by spindle pole bodies. In the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, six proteins are at the heart of spindle checkpoint function: Mad1, Mad2, Mad3, Bub1, Bub3 and Mph1/Mps1. The formation of spindle checkpoint complexes signals the presence of incorrect spindle microtubule attachments to kinetochores. These complexes cooperate to suppress the activity of the anaphase promoting complex (APC) and inhibit the onset of anaphase. By isolating these distinct complexes and analysing their composition by mass-spectrometry (MS) this work revealed several intriguing disparities between the two yeast species, and the way in which the Bub and Mad proteins cooperate to achieve inhibition. The ‘mitotic checkpoint complex’, which in S.cerevisiae consists of Mad2, Mad3, Bub3 and the APC activator Cdc20, was found to lack Bub3 in S.pombe. The S.pombe complex was shown to interact with the APC, but no stable interaction was found to be required in S.cerevisiae cells. And whereas Bub1 and Bub3 were found to form a complex with Mad1 in S.cerevisiae, in S.pombe they were shown to associate with Mad3 to form the ‘BUB+ spindle checkpoint complex’. In addition, MS analysis uncovered TAPAS: a novel S.pombe complex that was found to interact with the BUB+ complex and revealed to consist of Tfg3, Abo1 (gene product of SPAC31G5.19), Pob3 and Spt16. TAPAS mutant cells were shown to lose viability as a result of genotoxic stress, a phenotype that was surprisingly shared with bub1Δ and bub1kd ‘kinase dead’ mutants. Sensitivity of cells deficient in TAPAS or Bub1 did not appear to be due to the loss of DNA damage checkpoint or DNA replication checkpoint functions. Further examination revealed that Bub1 functions in the repair of DNA double strand breaks. Taken together, this work demonstrates that even though the molecular components of the spindle checkpoint pathway are conserved, their regulatory connections have to some extent diverged through molecular evolution. This process not only rewired, but entwined two molecular processes that together safeguard the genetic heritage of cells.

The Spindle Assembly Checkpoint (SAC) delays the onset of anaphase in response to unattached kinetochores. The mechanism by which the SAC works is by inhibiting the activity of the Anaphase-promoting complex/cyclosome (APC/C), a large E3 ubiquitin ligase that targets several anaphase inhibitors for proteasome-mediated degradation, including securin and cyclin B. When the SAC is satisfied, the APC/C becomes active and this allows progression through the cell cycle. Work from the last decade identified the mitotic checkpoint complex (MCC) as the main transducer of the SAC. The MCC is composed of BubR1, Bub3, Mad2 and Cdc20 and it is a very potent inhibitor of the APC/C. When the SAC is active, the MCC binds the APC/C and it inhibits its activity. Once the SAC is satisfied, the MCC becomes disassembled, which allows APC/C activation and mitotic progression. However, the mechanisms that dictate MCC assembly and how it inhibits the APC/C remain to be understood. Here, I used a combination of cell biology and in vitro biochemistry to investigate the mechanism by which the MCC component BubR1 participates in the SAC. My data shows that through its interaction with Bub3, BubR1 localises to kinetochores and this event greatly facilitates its assembly onto the MCC and its SAC function. On the other hand, MCC formation and APC/C binding were only dependent on BubR1's N-terminus, therefore questioning the existence of a second Cdc20 binding site. Within this region, TPR domains and an N-terminal motif known as the KEN box (KEN1) mediates these interactions. By contrast, BubR1's second KEN box (KEN2) does not participate in MCC assembly or APC/C binding. However, both in cells and in vitro, the KEN2 box is required for APC/C inhibition. Indeed, I show that this second KEN box promotes SAC function by blocking the interaction of the APC/C with its substrates. Thus, both KEN boxes in BubR1 participate differentially in the SAC, the first to promote MCC assembly and the second one to block substrate recruitment to the APC/C.In addition, I investigated the mechanisms that mediate MCC inactivation, following SAC silencing. I observed that p31comet and APC/C activity cooperate to promote MCC turnover. The implication of these observations in our understanding of the SAC is discussed.

Accurate distribution of genetic material is critical for the formation of functional cells and their proliferation. During cell division, sister chromatids separate from each other and segregate to opposite poles. To ensure accurate chromosome segregation all sister chromatids should be attached to microtubules from opposite spindle poles, known as bi-orientation. Cohesin is a protein complex that holds sister chromatids together from the time of its replication in S phase until anaphase onset, and it is required for proper chromosome segregation both in mitosis and in meiosis. It is distributed intermittently along the full length of chromosomes with significant enrichment in the region surrounding the centromere, known as the pericentromere. This chromosome domain was shown to be crucial for chromosome bi-orientation. In my PhD I studied how the establishment of tension between sister chromatids in the process of bi-orientation affects the distribution of different pericentromeric proteins on budding yeast chromosomes. It was known that levels of cohesin at the pericentromere are decreased in response to the establishment of tension. I demonstrate that other proteins, such as subunits of condensin, members of the Chromosome Passenger Complex (CPC) and others, exhibit similar dynamics, and suggest a model to explain this phenomenon. Out of all studied proteins, Shugoshin (Sgo1) was the only one that was completely removed from the pericentromere in response to spindle tension establishment. There is evidence that Sgo1 plays a role in sensing spindle tension and halting the cell cycle until this has been achieved but how it does so is not known. Therefore, removal of Shugoshin from the pericentromere might be a signal for the cell that bi-orientation occurred. I then found that spindle tension itself is not sufficient for Sgo1 re-localization from the pericentromere, and there are other factors that affect it. I showed that deletion of RTS1, a highly conserved regulatory subunit of Protein Phosphatase 2A (PP2A), results in substantial enrichment of Shugoshin at the pericentromere in the situation when spindle tension is absent. In addition, Bub1 kinase, a protein that is required for Sgo1 localization, was found to be removed from the centromere in response to spindle tension as well as Sgo1. The role of Bub1 the in localization of Shugoshin is to phosphorylate histone H2A, which then becomes a mark for Sgo1 loading. Therefore, we assume that Sgo1 dynamics and, potentially, its role in sensing bi-orientation, are regulated through the array of phosphorylation and de-phosphorylation events at the pericentromeric area. Finally, I have also found that budding yeast Sgo1 undergoes the posttranslational modification as sumoylation. I showed that sumoylation of Shugoshin is not required for its removal from the pericentromere during biorientation. However, it might be important for the regulation of Sgo1 degradation and its role in the metaphase to anaphase transition in mitosis.

Errors in chromosome segregation during the first division of female meiosis are very common in humans and result in aneuploidy leading to reproduction problems. Chromosome segregation depends on the formation and function of the meiotic spindle as well as the structure of chromosomes, which need to condense to be able to orient and segregate properly. It is important to understand the mechanisms underlying the female meiotic spindle function and chromosome condensation to gain insight into female fertility problems. The female meiotic spindle assembles without centrosomes, so the mechanisms ensuring microtubule nucleation, spindle assembly and establishment of bipolarity act differently from those of mitosis or male meiosis. I identified a set of genes that are required for microtubule nucleation, spindle maintenance and centromere orientation in Drosophila female meiosis. This was accomplished by mapping previously uncharacterized Drosophila mutants and depleting already known genes by RNAi. I discovered that several proteins have a different role in female meiosis as compared to mitosis, which provides insight into the major differences between these systems. Little is known about the molecular mechanisms of chromosome condensation. The roles of only a few factors, such as condensin complexes, have been studied previously, and the evidence suggests that there are more molecular players required for chromosome condensation. To discover molecular mechanisms critical to this process, I depleted various chromosomal proteins by RNAi and screened for abnormalities of metaphase chromosome morphology in Drosophila oocytes by immunostaining and live imaging. I found that the conserved kinase NHK-1 plays a role in chromosome condensation in female meiosis. BAF is a critical NHK-1 substrate in this process and its phosphorylation is required for detachment of the chromosomes from the nuclear envelope to allow proper condensation. Also, I discovered that the nucleosome remodelling complex NuRD is crucial for chromosome condensation, especially for the chromosome arms. As a result of my PhD project I identified multiple factors required for meiotic spindle function. I also discovered two novel pathways of chromosome condensation that require the NuRD complex and NHK-1 activity.

The accurate segregation of DNA during cell division is essential for the viability of future cellular generations. Genetic material is packaged in the form of chromosomes during cell division, and chromosomes are segregated equally into two daughter cells. Chromosome mis-distribution leads to genetic disorders (e.g. Down's syndrome), aneuploidy and cancer. The spindle checkpoint ensures proper chromosome segregation by monitoring kinetochore-microtubule interactions. Upon checkpoint activation, unattached kinetochores recruit checkpoint proteins that combine to form a diffusible inhibitor (the Mitotic Checkpoint Complex-MCC). The MCC delays anaphase, thus giving cells time to fix attachment errors. Although the major checkpoint proteins were identified several years ago, we have only just begun to understand how they assemble at unattached kinetochores to generate the checkpoint signal. Yeast genetics and proteomics have revealed that kinetochores are highly complex molecular machines with almost 50 kinetochore components and ~10 components of the spindle checkpoint machinery. Such complexity makes the separation of error correction, kinetochore bi-orientation and microtubule attachment functions very challenging. To circumvent this complexity, a synthetic version of the spindle checkpoint (SynCheck), based on tetO array was engineered at an ectopic location on a chromosome arm away from kinetochores in S. pombe. This work describes that combined targeting, initially of KNL1Spc7 with Mps1Mph1 and later of Bub1 (but not Mad1) with Mps1Mph1 fragments, was able to activate the spindle checkpoint and generate a robust arrest. The system is based on, soluble complexes, which were formed between KNL1Spc7 or Bub1 with Mps1Mph1. The synthetic checkpoint or 'Syncheck' is independent of localisation of the checkpoint components to the kinetochores, to spindle pole bodies (SPBs) and to nuclear pores. By using the synthetic tethering system a Mad1-Bub1 complex was identified for the first time in S.pombe. Bub1- Mad1 complex formation is crucial for checkpoint activation. Bub1-Mad1 gets phosphorylated itself and is thought to act as an assembly platform for MCC production and thereby generation of the "wait anaphase" signal. The ectopic tetO array is an important tool, not only for generating MCC formation and activating the spindle checkpoint, but also for providing a nice system for analysing in vivo protein-protein interactions. The ectopic array is capable of not only recruiting checkpoint components, but also recruiting them in a physiological manner (similar to the unattached kinetochores). For this reason it was decided to adopt this system to examine the role of the conserved Bub1TPR domain in the recruitment of other spindle checkpoint proteins. This work represents two novel functions for the S. pombe Bub1TPR domain. For the first time in S. pombe, both in vivo tethering and in vitro experiments with purified, recombinant proteins showed that the Bub1 has the ability to homodimerise and to form a complex with Mad3BubR1 through its TPR domain. These results revealed that complex formation of Bub1 with Mad3BubR1 is important for checkpoint signalling and that the highly conserved TPR domains in BubR1Mad3 and Bub1 have key roles to play in their interactions.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available online.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.