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.

No description available.

No description available.

No description available.

No description available.

Proper cell division is a fundamental process for the development and sustainability of healthy living organisms. Defective cell division can have deleterious effects on tissue homeostasis and can represent the first step towards disease development. The overall goal of this work was to develop and validate a new, mitosis-based, single-cell toxicity approach. This contributes to the current need of toxicology research to replace animal testing with predictive in vitro models. Cell division-based assays would be better at predicting risk than other commonly used in vitro measurements, such as persistent cell cycle arrest or cell death. Finally, single-cell microscopic analysis provides far deeper insight into the underlying toxicity mechanism(s) than bulk cell population measurements. To meet our goal, we investigated the toxicity of silver nanoparticles (AgNPs) on immortalized human retinal pigmented epithelial (RPE-1) cells. AgNPs are a major nanomaterial employed in product manufacturing due to desirable antimicrobial properties, yet toxicity reports are still confounding. RPE-1 cells were cultured in the presence of low and high doses of polyvinylpyrrolidone (PVP)-coated AgNPs for a single 24-hour treatment (acute treatment), for six 24-hour treatments administered over a period of 3 weeks (moderate treatment), or for twelve 24-hour treatments administered over a period of 6 weeks (chronic treatment). Time-lapse, phase-contrast microscopy of acutely treated cells showed that 100% of cells engulfed AgNPs, which was further confirmed by electron microscopy. Moreover, we found that higher concentrations of AgNPs resulted in large numbers of acutely treated cells becoming arrested in mitosis, dying, or dividing abnormally. In contrast, untreated cells displayed normal mitotic behavior. High-resolution fluorescence microscopy performed in treated cell populations identified an increased percentage of abnormal nuclear morphologies compared to the untreated cells. Further live-cell analysis indicated that treated cells failed cytokinesis or slipped out of mitosis more often than untreated cells. Overall, our results indicate that AgNPs impair cell division, not only further confirming toxicity to human cells, but also revealing previously unreported toxicity mechanisms and highlighting the propagation of adverse phenotypes within the cell population after exposure. Furthermore, this work illustrates that cell division-based single-cell analysis could provide an alternative to animal experimentation in the future. / Master of Science / Multiple agencies, including the U.S. Environmental Protection Agency and the National Academy of Science, are urging for a radical paradigm shift from standard, whole-animal testing to alternative and novel technologies. To meet this urgent need, we aimed to develop a new, cell division-focused toxicity assay by investigating the mechanism of toxicity from silver nanoparticles (AgNPs) on human retinal pigment epithelial (RPE-1) cells. Cultured RPE-1 cells were treated with varying concentrations of AgNPs and live-cell microscopy was used to analyze the behavior of cells undergoing cell division over a 24 hour time period. Physical interaction between cells and particles was visually observed and 100% of treated cells appeared to engulf particles. We found that higher concentrations of AgNPs resulted in large numbers of cells stalling in mitosis and/or dying. In contrast, untreated cells displayed normal mitotic behavior. High-resolution fluorescence microscopy performed in chronically treated cell populations identified an increased percentage of binucleated cells. Further live-cell analysis indicated that one major cell division defect could explain the binucleated cell phenotype. Indeed, treated cells failed cytokinesis (cytoplasmic division following mitotic chromosome segregation) more often than control cells. Overall, our results indicate that AgNPs specifically impair cell division, not only further confirming toxicity to human cells, but also revealing specific, previously unreported toxicity mechanisms and highlighting the propagation of adverse phenotypes within the cell population after exposure. Furthermore, this work illustrates that cell division-based assays and ingle-cell analysis could greatly benefit chemical safety experimentation in the future.