Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references (leaves 114-122).

Thesis (Ph. D.)--University of Chicago, Pritzker School of Medicine, Department of Molecular Genetics and Cell Biology, December 2000. / Includes bibliographical references (p. 81-88). Also available on the Internet.

Thesis (Ph. D.)--Miami University, Dept. of Chemistry and Biochemistry, 2005. / Title from second page of PDF document. Document formatted into pages; contains [3], vi, 124 p. : ill. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Also available online.

Drugs that alter microtubule dynamics are often used in chemotherapy regimes in combination with other agents in order to treat various cancers. Despite the success over many years, there remain problems in toxicity, resistance and predictability to the drugs. In order to overcome these problems, there is a need to gain an understanding of how these drugs kill cancer cells in cell culture. As microtubule function is particularly important for chromosome movement in mitosis, cells treated with these agents cause a mitotic arrest through activation of the spindle assembly checkpoint. Following induction of a mitotic arrest, cells can escape this arrest (mitotic slippage) or undergo mitotic death, determined in part by the response of the apoptotic network. Levels of an anti-apoptotic protein, Mcl-1, are often lost over time in mitosis. Using time-lapse analysis on a cell line unable to escape the mitotic arrest, this thesis shows that Mcl-1 protein contributes to cell death both in mitosis and the subsequent interphase in response to microtubule toxin, taxol. This suggests that inhibiting Mcl-1 may increase the efficacy of anti-mitotic agents. In addition, mitotic cell lines prone to mitotic slippage were found to have higher levels anti-apoptotic protein, Bcl-xL, in comparison to Mcl-1, indicating one way in which these cells can cope with loss of Mcl-1 during mitosis. Secondly, an evaluation of the contribution of the previously identified interaction between Mcl-1 and mitotic E3 ligase complex, the APC/C-Cdc20, to the rate of mitotic death and mitotic slippage was assessed. Inhibition of APC/C-Cdc20 activity or mutation of a Mcl-1 motif (RxxL) thought to engage with the APC/C-Cdc20 complex did not have a substantial effect on Mcl-1 degradation or mitotic death, thereby questioning the functional significance of this interaction. However, it appears that Mcl-1 protein levels can influence the rate of mitotic slippage and this influence was affected via Mcl-1’s RxxL motif within Mcl-1. This suggests that Mcl-1 protein may delay mitotic slippage via substrate competition for the APC/C-Cdc20 complex with Cyclin B1, whose degradation is required for mitotic exit. Further analysis of this effect showed that this interaction may not be a universal effect. This together with the specific functional effect on mitotic slippage rather than mitotic death, suggests that this is an indirect effect caused by network interference between the components of the death and slippage pathways.

Asbesto é um nome geral dado a seis tipos de fibras minerais encontradas naturalmente na crosta terrestre. Estas fibras vêm sendo exploradas industrialmente desde 1970, porém diversos trabalhadores expostos às fibras apresentaram patologias no trato respiratório, como fibroses e carcinomas. Alguns tipos de fibra foram banidos do mercado, porém o tipo de asbesto crisotila ainda pode ser comercializado na maioria dos países. Estudos in vivo e in vitro tentam elucidar as alterações causadas pela exposição à asbesto nos tecidos e nas células que possam estar relacionadas ao aparecimento de doenças, e foi verificado que a exposição às fibras leva a quebras na dupla fita de DNA, estresse oxidativo, formação de células micronucleadas e células aneuploides. O presente estudo teve como objetivo verificar a presença de alterações causadas em células em cultura expostas à crisotila por 48 h e recuperadas em meio livre de fibras por 48 h, 4 dias e 8 dias, além de observar por microscopia em tempo real divisões aberrantes após a exposição as fibras por 24 e 48h. Foram verificadas alterações que permaneceram na cultura mesmo após 8 dias de recuperação, quando não foram mais observadas fibras na cultura, como formação de células aneuploides, diminuição de frequência de células em G0/G1, aumento de células em G2/M e aumento relativo de células em metáfase quanto à porcentagem de células em fases mais tardias da fase M do ciclo. Já aumento da frequência de células micronucleadas ocorreu apenas nos períodos quando foram observadas fibras na cultura. Para análise da formação de mitoses multipolares e destinos destas células foram construídos vetores para expressão de tubulinas fusionadas a proteínas fluorescentes RFP e GFP, padronizadas as condições de transfecção e de aquisição de imagens para que as células tratadas com crisotila fossem observadas por time-lapse. Alguns destinos de mitoses multipolares causadas pelo tratamento com crisotila foram observados, como morte em metáfase, divisão gerando duas ou três células filhas, fusão de células durante a telófase e retenção em metáfase. Os dados sugerem também a indução da amplificação centrossômica, que parece ocorrer inicialmente em células interfásicas, e também devido à fusão de células. / Asbestos is a general name given to six different fibrous silicate minerals found naturally in the earth\'s crust. These fibers are being exploited industrially since 1970, but several workers exposed to the fibers developed diseases in the respiratory tract, such as fibrosis and carcinomas. Some types of fiber were banished from the market, but the type of asbestos chrysotile can still be marketed in most countries. Studies in vivo and in vitro are trying to elucidate the asbestos effects in tissues and cells that could be related to the development of diseases, and these studies verified that asbestos exposure lead to DNA double strand breaks, oxidative stress, multinucleated and aneuploid cell formation. The present work aimed to verify the alterations in culture cells exposed to chrysotile for 48 h and recovered in fiber-free medium for 48 h, 4 days and 8 days, and also observe aberrant mitosis using time-lapse microscopy after 24 h and 48 h of chrysotile exposure. Some alterations were observed and remained in cell culture even after 8 days of recovery when chrysotile fibers were no longer observed - such as aneuploid cell formation, increased frequencies of G2/M cell, decreased frequencies of G1 cells, and increased frequencies of cells in early M phases as metaphase. The induction of micronuclei occurred only during the periods that fibers were observed in cell culture. For the analysis of multipolar mitosis formation and destinies of these cells after chrysotile treatment, DNA vectors for the expression of tubulins fused to fluorescent proteins (GFP and RFP) were constructed, and the conditions for cells transfection and image acquisition for time-lapse microscopy were established. The fate of some multipolar metaphases was observed: cell retention on metaphase, cell cycle progression generating two or three daughter cells, cell fusion during cytokinesis or during telophase after a multipolar anaphase, and cell death. The centrosome amplification was not observed during the M phase of cell cycle, and may occur in interphase, and also despite cell fusion.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18431号 / 生博第311号 / 新制||生||41(附属図書館) / 31289 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 豊島 文子, 教授 藤田 尚志, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

No description available.

During mitosis, replicated chromosomes are equally distributed among two daughter cells by means of a multi-component machine called the mitotic spindle. Spindle formation and function has been shown to involve numerous microtubule associated proteins and molecular motor proteins, including kinesins and dynein. One such kinesin, the plus-end directed, homotetrameric, Eg5, is involved in centrosome separation during spindle formation. In vitro, Eg5 crosslinks parallel and antiparallel microtubules, and localizes to spindle poles and microtubules in vivo. To further understand the function of Eg5 in mammalian cells, we determined its distribution and dynamics throughout mitosis using novel cloning techniques, fluorescence recovery after photobleaching, and total internal reflection fluorescence microscopy. Eg5-GFP was expressed from a mouse bacterial artificial chromosome to ensure the transgene’s expression was at or near endogenous levels. Our results confirm that Eg5 colocalizes with spindle, but not astral, microtubules and is enhanced at the spindle poles during prometaphase and metaphase. In early anaphase, Eg5 is localized near the poles transitioning to interzone microtubules with the exception of a 1 µm gap during late anaphase. Fluorescence recovery after photobleaching shows that Eg5 is rapidly turning over throughout mitosis with a recovery half time less than 10 s and extent of recovery greater than 85%. TIRF microscopy revealed a population of Eg5 that transiently binds to microtubules with a residency time of less than 6 seconds for all stages of mitosis. Eg5 remained stationary while bound to microtubules with no apparent directional motion. Treatment of cells expressing mEg5-GFP-LAP with the Eg5 inhibitor, STLC, caused Eg5 to no longer bind to microtubules and remain diffuse within the cell. TIRF microscopy also revealed Eg5-decorated tracks during interphase, which were abolished by treatment with STLC or Nocodazole suggesting that Eg5 is present on microtubules in interphase. Taken together, fluorescence recovery after photobleaching and TIRF microscopy reveal that Eg5 is highly dynamic in the mammalian spindle throughout mitosis.