Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 111-120). Also available on the Internet.

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 110-117). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Virginia, 1997. / Spine title: Control of anaphase onset checkpoint. Includes bibliographical references (172-192). Also available online through Digital Dissertations.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 197-228). Also available in electronic version.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 194-218). Also available in electronic version.

Aurora A is an important mitotic regulator that has been found to be up-regulated in a variety oftumours provoking a great deal of attention and the development of a number of small moleculeAurora kinase inhibitors. Most of these inhibitors though have predominantly targeted Aurora B,meaning that our understanding of the role of the kinase activity of Aurora A is comparatively lesswell developed.MLN8054 however, is a small molecule inhibitor that has been reported in vitro to have a highdegree of specificity towards Aurora A activity. In this thesis, I show in vivo that MLN8054 can beused to specifically inhibit Aurora A activity, and exploit this quality to probe the role of Aurora Aactivity in human cells. I was consequently able to show that Aurora A activity not only has a clearrole in spindle formation, where it is required for the determination of K-fibre length and in thedegree of centrosome separation, but also in the regulation of microtubule organisation. Despite thespindle deformities seen after inhibiting Aurora A activity, the majority of HeLa and DLD-1 cellswere still able to form bipolar spindles capable of attaching to kinetochores. These spindlestructures did not however, assert normal levels of force through the kinetochores, and cells wereconsequently unable to efficiently align their chromosomes, causing significant delays to mitoticprogression. Cells were still able to divide in the absence of Aurora A activity, although thedetection of segregation defects and aneuploid progeny indicates a role for Aurora A activity in thefaithful segregation of the genetic material. Importantly however, Aurora A activity was not foundto have a prominent role in the spindle assembly checkpoint.Increasing the potency of Aurora A inhibition by using a drug-resistant cell line confirmed theobservations made in HeLa and DLD-1 cells, emphasising that although Aurora A activity isrequired for spindle assembly, cells can still activate the spindle checkpoint and divide in itsabsence. I therefore propose that Aurora A activity is required for the formation of normal spindlestructures capable of efficiently aligning and evenly dividing chromosomes during cell division.These roles were attributed in part to the kinase activity of Aurora A in the regulation of TACC3and chTOG localisation on the spindle and centrosomes.Interestingly however, Aurora A activity did not appear to be required for spindle assembly in nontransformedcells, which were able to more efficiently align their chromosomes and dividefollowing Aurora A inhibition than the cancer cell lines. Furthermore, the non-transformed cellsaccumulated with 2N DNA after longer-term Aurora A inhibition, as opposed to the cancer celllines, which exhibited profound aneuploidy following the equivalent treatment. This finding isencouraging, as consistent with recently published reports, it indicates that Aurora A inhibitionmay be successfully used in order to specifically target cancer cells.

No description available.

Protein kinases play essential roles in orchestrating almost every step during mitosis. Aberrant kinase activity often leads to errors in the cell cycle progression which consequently becomes the underlying cause for developmental defects or abnormal cell proliferation leading to cancer. Tank Binding Kinase 1 (TBK1) is overexpressed in certain cancer types and is activated on the centrosomes during mitosis. Loss of TBK1 impairs cell division resulting in growth defects and the accumulation of multinucleated cells. Therefore, proper activation and localization of TBK1 are essential for mitotic progression. Yet, the upstream regulation of TBK1 and the function of activated TBK1 on the centrosomes is unknown. Also, the cause and consequences of overexpression of TBK1 in cancers remain to be explored. Activation of TBK1 depends on its binding to an adaptor protein which induces a conformational change leading to trans autophoshorylation on serine 172 of its kinase domain. We identified that an established innate immune response protein, NAK Associated Protein1 (NAP1/AZI2), is the adaptor required for binding and activating TBK1 during mitosis. Loss of either NAP1 or TBK1 results in the accumulation of binucleated and multinucleated cells, possibly due to several mitotic and cytokinetic defects seen in these knockout (KO) cells. We establish NAP1 as a cell cycle regulated protein which colocalizes with activated TBK1 on the centrosomes during mitosis. Furthermore, by performing an unbiased quantitative phosphoproteomics analysis during mitosis, the substrates discovered reveal that TBK1 also regulates other known cell cycle regulating kinases such as Aurora A and Aurora B. TBK1 is also an established autophagy protein and since the autophagy machinery is often impaired or remodeled to facilitate rapid cell division, we evaluated the underlying connection between TBK1 activation and autophagy. The data shows that cells lacking the essential autophagy proteins FIP200 or ATG9A exhibit overactivation and mislocalization of TBK1. By using both genetic and pharmacological inhibition of autophagy processes, we found that impaired autophagy leads to a significantly higher number of micronuclei – a hallmark for tumorigenesis that correlates with defects in mitosis and cytokinesis. Taken together our work has uncovered a novel function for the NAP1-TBK1 complex during mitosis and establishes that overactivation and mislocalization of TBK1 is a direct consequence of impaired autophagy which causes micronuclei formation. / Doctor of Philosophy / Defective cell division is the underlying cause for many human health maladies such as birth defects and cancer. Investigation into the proteins that are abnormally expressed in cancer can help us identify their physiological roles in regulating the cell cycle. Tank Binding Kinase 1 (TBK1) is often overexpressed in several types of cancer such as glioblastomas, breast, and lung cancers. It has also been extensively studied in the process of removing damaged cytosolic components from cells called autophagy. During cancer progression, cells often hijack the autophagy machinery to their advantage for abnormal cell proliferation. However, we do not completely understand the role of TBK1 in cancer pathogenesis or during normal cell division. Each cell duplicates its genomic contents and divides its organelles and cytosolic components during cell division. Centrosomes organize microtubules to attach to the duplicated genomic material to equally segregate the DNA between two daughter cells. Previous studies have shown that TBK1 is active on the centrosomes during mitosis, and the loss of TBK1 leads to reduced cell proliferation. However, the function of TBK1 and what regulates its activation on the centrosomes are unknown. Using a combination of genetic, biochemical, and molecular biology techniques, we found that an immune response protein Nak Associated Protein 1 (NAP1/AZI2) binds to TBK1 and activates it on the centrosomes during cell division. Furthermore, our study demonstrates that the loss of either NAP1 or TBK1 exhibits a multitude of different types of defects in the process of cell division. We further identified TBK1 substrates in a phosphoproteomic screen indicating that TBK1 regulates the activity of other major cell division kinases. We show that defects in autophagy machinery result in the mislocalization and overactivation of TBK1 resulting in defects during chromosome segregation, and in the formation of micronuclei. Together our study shows that an established immune response protein NAP1 regulates the function of TBK1 during cell division and there exists a connection between TBK1 activity and disrupted autophagy.

No description available.

Entry into mitosis in eukaryotes depends on the activation of the Cyclin-dependent kinase 1 (Cdk1), which phosphorylates many mitotic protein substrates. Activation of Cdk1 requires formation of a complex with Cyclin B (CycB), which gradually rises in concentration during interphase. However, in most organisms Cdk1 activation is not gradual but switch-like, because phosphorylation of the Cdk1-CycB complex by the Wee1 kinase normally keeps Cdk1-CycB inactive during interphase. Mitotic entry is induced when rapid dephosphorylation of Cdk1-CycB by the Cdc25 phosphatase causes abrupt activation of Cdk1-CycB. Cdk1-CycB in turn phosphorylates both Wee1 and Cdc25 leading to Cdc25 activation and Wee1 inhibition. This regulation creates both a positive and a double-negative feedback loop in the system, which are thought to generate a sharp, bistable switch that controls mitotic entry. Bistability is known to require positive feedback and ultrasensitivity, however, how ultrasensitivity arises in the mitotic switch is subject to extensive research efforts both experimentally and theoretically. In this thesis I explore several possible sources of ultrasensitivity in the mitotic switch through mathematical modelling. Based on theoretical considerations and experimental evidence, I show that the existence of multiple positive feedback loops, multisite phosphorylation, and Cdk1-CycB-dependent regulation of Cdk1-counteracting phosphatase activity can all contribute to ultrasensitivity and bistability in the mitotic switch. I analyse models of the mitotic switch including these bistability-generating mechanisms, to simulate and explain experimental data and make testable predictions. I argue that it is unlikely that a single mechanism is responsible for ultrasensitivity in this system, and that bistability requires a combination of different sources, including the ones studied here and others such as enzyme saturation and sequestration effects. I also highlight the importance of network architecture and coherent regulation of opposing reactions in generating efficient biochemical switches. Finally, I draw on recent experimental evidence and ideas derived from this analysis to propose a revised network of the mitotic switch.