Thesis (Ph. D.)--University of Wisconsin--Madison, 1937. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 34-38).

Thesis--Universiteit van Amsterdam. / "Literatuur-overzicht" : p. 80-3.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1951. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 53-57).

Antimitotics are used in cancer chemotherapy for the treatment of cancers such as breast, ovarian, lung and prostate. Despite the success of agents such as Taxol, problems have emerged such as side effects, resistance and the lack of ability to predict patient responsiveness. As a result, a class of second-generation inhibitors have been developed with the aim to overcome or improve these issues. Such inhibitors target proteins and kinases involved in the control of mitosis and the cell cycle. However, these have yet to be clinically successful and therefore, this highlights the requirement for an increased understanding of the mitotic process and how antimitotics truly elicit their action. Reasons for the lack of efficacy may be due to the absence of biomarkers to stratify patients into those likely to respond to treatment. It may also be possible that other targets are required. Our understanding of the action of antimitotics is therefore paramount to improving cancer chemotherapy. By exploiting mitosis and understanding what happens when mitosis goes wrong, this thesis aims to explore new and improved methods of targeting, but also proposes to improve our understanding of the consequences of aberrant mitoses through the use of small molecule inhibitors. In the first case the thesis investigated the targeting of the spindle checkpoint protein Bub1 with 2OH-BNPP1, where previously inhibitors against the kinase were not acknowledged. However the inhibitor used was not effective in cells and therefore further experimentation was not possible. Secondly, to explore the consequences of mitotic perturbation, an assay to explore aneuploidy was established. To do this a Cenp-E inhibitor GSK923295 was synthesised, which was subsequently used in assays with the Mps1 inhibitor AZ3146 to generate aneuploidy progeny. The Cenp-E inhibitor was then used as an antimitotic agent in the final chapter to explore the mechanism of action of mitotic blockers and drivers often used in cell biology and clinical settings. Evidence suggests that the intrinsic apoptotic pathway is activated upon exposure to these agents. With focus on this pathway, the importance of Bcl-xL on cell survival was considered, revealing particular importance in the post-mitotic response. Ultimately, this thesis should contribute to devising new and improved anti-cancer strategies.

In cells undergoing mitosis with unreplicated genomes (MUG), anaphase is successfully initiated despite the abundance of kinetochores that are attached to microtubules emanating from both spindle poles (merotely). In cultured cells, merotely is associated with lagging at the metaphase plate. Treatment with microtubule-perturbing drugs alters the frequency of lagging, but the effect of these drugs on MUG cells is unclear. In this study, low doses of a microtubule-stabilizing drug, taxol, or a microtubule-destabilizing drug, nocodazole, dramatically increased the frequency of lagging kinetochores in the midbody of MUG daughter cell pairs. Likewise, increasing the kinetochore number increased the frequency of lagging kinetochores. In this thesis, these data are used to propose a model of mitosis in which the bipolar attachments of MUG cells are reduced to monopolar attachments that are stabilized by their perpendicular orientation with respect to the kinetochore, allowing for spindle assembly checkpoint satisfaction without centromeric tension.

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Microtubules are essential for many dynamic processes occurring within eukaryotic cells including organelle and vesicular trafficking, motility of cilia and flagella, and mitosis. Microtubules operate in conjunction with the kinesin superfamily of microtubule-dependent motor proteins, which use the energy from ATP hydrolysis to "walk" along microtubule tracks, and in doing so generate force for the transport of cellular cargo and mitosis. The goal of this project was to define the microtubule interactions and regulation of two kinesin-like proteins (KLPs), the Homo sapiens mitotic kinesin-like protein-1 (HsMKLP-1) and the Arabidopsis thaliana kinesin-like calmodulin-binding protein (KCBP). Functional domains of HsMKLP-1 and KCBP were heterogeneously expressed in insect cells (HsMKLP-1) and/or E. coli (HsMKLP-1, KCBP) and used to examine the microtubule binding and ATPase activity of HsMKLP-1 and KCBP catalytic domains. Overall, the HsMKLP-1 catalytic domain was found to operate in a similar fashion to other KLPs with respect to microtubule binding and ATP hydrolysis, but HsMKLP-1 exhibited enhanced microtubule binding of the dimer and weaker affinity for ATP that functionally distinguishes it from other KLPs. HsMKLP-1 proteins were also used to generate HsMKLP-1 specific antibodies to be used as a tool for characterizing native HsMKLP-1. To define the role of nuclear localization in regulating the activity of HsMKLP-1 during interphase, sequences directing nuclear localization of HsMKLP-1 were identified. Mutation of the nuclear localization sequence 799PNGSRKRR806 to 799PNGSRTSR806 or removal of AA's 830-856 of HsMKLP-1, which contains the nuclear localization sequence 851PKRKKP856, were sufficient to abolish nuclear localization. In the absence of a functional nuclear localization sequence HsMKLP-1 localized to microtubule plus ends, suggesting that nuclear localization serves to limit the interaction of HsMKLP-1 with the interphase microtubule array. The KCBP catalytic domain, which contains a calmodulin-binding site, was used to determine the effect of Ca2+/calmodulin on the microtubule binding and ATPase activity of KCBP. Ca2+/calmodulin was found to inhibit the binding of KCBP to microtubules and reduced the motor's microtubule-stimulated ATPase activity, which suggests that Ca2+/calmodulin may modulate the activity of KCBP in vivo by regulating the motor's association with microtubules. / Ph. D.