Doctor of Philosophy / Humans and all mammals have an internal timekeeping mechanism named the circadian clock that enables anticipation and response to the approximately 24-hour solar day and other environmental conditions. The circadian clock is self-sustained and coordinates rhythmic physiological functions such as the sleep/wake cycle, body temperature, hormone production, and metabolism, together forming the organism's "circadian rhythm." Chronic disruption of the circadian rhythm is known to be carcinogenic , but the molecular explanation for this phenomenon remains elusive. The purpose of my dissertation work was to investigate the role of mutations commonly associated with cancer as a potential molecular mechanism of circadian clock dysfunction. The PER2 gene produces the PER2 protein, which our laboratory has previously shown to interact with p53, a key "tumor suppressor" that responds to DNA damage. When not functional, these tumor suppressors can lead to uncontrolled cell division and eventually cancer. We focused on a mutation in p53 that changes p53's function and its interaction with PER2 in a manner that also prevents PER2 from functioning normally within the circadian clock. This dual dysregulation leads to the loss of rhythmic clock gene expression, and in turn, changes to cellular fitness, metabolism, and proliferation.

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Breast cancer is the second most common cancer in women worldwide and the second leading cause of all-cancer mortality. Breast cancer is a multifactorial disease with several elements that contribute to its development. Several lines of evidence suggest that disruptions in circadian rhythm may increase the risk of breast cancer, primarily through decreasing the body’s production of melatonin, a molecule that combats tumorigenesis through antioxidant, anti-angiogenic, and anti-aromatase properties. Sleep characteristics like insomnia, sleep duration, chronotype, and participating in night shift work have all been studied to determine their impact on breast cancer risk. While there is research both in support of and against each of the previously mentioned sleep characteristics’ role in breast cancer development, the majority of studies that have been carried out are supportive of some form of causal association between sleep disturbances and breast cancer incidence and progression. The variability in various studies suggests that sleep disturbance may need to be particularly severe or persist for many years in order to have tumorigenic effects. This connection between sleep and breast cancer suggests that maintaining healthy sleep habits may reduce the risk of breast cancer over time. Taking melatonin supplements, participating in cognitive behavioral therapy, exercising regularly, and limiting exposure to blue light have all been shown to be useful practices for improving sleep quality; thus, these techniques may be useful for reducing breast cancer risk in women who experience sleep disturbances.

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京都大学 / 新制・課程博士 / 博士(人間健康科学) / 甲第23824号 / 人健博第95号 / 新制||人健||7(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 十一 元三, 教授 林 悠, 教授 小林 恭 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第24558号 / 薬科博第175号 / 新制||薬科||19(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 土居 雅夫, 教授 竹島 浩, 教授 中山 和久 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Circadian rhythms are self-sustained, 24h, biological oscillatory processes that are present in organisms ranging from bacteria to human. Circadian rhythms, which can be synchronized by external cues, are important for organisms to adjust their behavior, physiological activity, and metabolic reactions to changes in environmental conditions. Another well-established oscillatory mechanism that shares common organizational and regulatory features with the circadian system, is the cell division cycle. Recent findings reveal that some essential regulators are common to both the cell cycle and the circadian clock. The first half of my thesis (Chapter 2-3) focuses on the function of Period 2 (Per2), a key regulatory component of the negative feedback arm of the clock and tumor suppressor protein, as a modulator of cell cycle response. We found that Per2 binds the C-terminus end of the tumor suppressor p53 thus forming a trimeric complex with p53's negative regulator Mdm2 and preventing Mdm2-mediated p53's ubiquitination and degradation. Thus, Per2 stabilizes p53 under unstressed conditions allowing for basal levels of the protein to exist and be available for a rapid response to take place in case of any stressed signals. Our experiments prove that Per2 plays an indispensible role in p53 signaling pathway. The second half of my thesis (Chapter 4-5) focuses on how Mdm2 and Per2 interplay regulate Per2 availability and its impact on circadian clock function. My research found that Mdm2 targets Per2 for ubiquitination as Mdm2 depletion stabilizes Per2 and, conversely, Mdm2 ectopic expression shorten Per2's half-life. Accordingly, association of Per2 to Mdm2 maps C-terminus of the p53 binding region in Mdm2 and thus, the RING domain remains accessible. Next, we tested the hypothesis that Mdm2-dependent ubiquitination of Per2 directly impacts circadian clock period length. Accordingly, addition of sempervirine nitrate (SN), a specific molecular inhibitor of Mdm2, to MEF cells abrogated Per2 ubiquitination leading to the accumulation of a stable pool of Per2. By recording the oscillatory behavior of the Per2:Luc reporter system in MEF cells treated with SN at different circadian times, we found that inhibition of Mdm2 E3 ligase activity promoted phase advance only when treatment took place during the degradation period. This is in agreement with our findings that radiation, but not light pulses, causes the same phase behavior. Considering the established role of both Mdm2 and p53 in the response of cells to genotoxic stress and Per2 in modulating the clock, the existence of the Mdm2-Per2-p53 complex opens the possibility of various stimuli triggering regulatory mechanisms converging in a critical node. Overall, our work provides a holistic view of how signals are integrated at multiple levels to ensure that environmental signals are sense and responses triggered timely. / Ph. D.

Circadian rhythms in mammals are self-sustained repeating activities driven by the circadian gene expression in cells, which is regulated at both transcriptional and posttranscriptional stages. In this work, we first used mathematical modeling to investigate the transcriptional regulation of circadian gene expression, with a focus on the mechanisms of robust genetic oscillations in the mammalian circadian core clock. Secondly, we built a coarse-grained model to study the post-transcriptional regulation of the rhythmicities of poly(A) tail length observed in hundreds of mRNAs in mouse liver. Lastly, we examined the application of Sobol indices, which is a global sensitivity analysis method, to mathematical models of biological oscillation systems, and proposed two methods tailored for the calculation of circular Sobol indices. In the first project, we modified the core negative feedback loop in a mathematical model of the mammalian genetic oscillator so that the unrealistic tight binding between the repressor PER and the activator BMAL1 is relaxed for robust oscillations. By studying the modified extended models, we found that the auxiliary positive feedback loop, rather than the auxiliary negative feedback loop, makes the oscillations more robust, yet they are similar when accounting for circadian rhythms (~24h period). In the second project, we investigated the regulation of rhythmicities in poly(A) tail length by four coupled rhythmic processes, which are transcription, deadenylation, polyadenylation, and degradation. We found that rhythmic deadenylation is the strongest contributor to the rhythmicity in poly(A) tail length and the rhythmicity in the abundance of the mRNA subpopulation with long poly(A) tails. In line with this finding, the model further showed that the experimentally observed distinct peak phases in the expression of deadenylases, regardless of other rhythmic controls, can robustly cluster the rhythmic mRNAs by their peak phases in poly(A) tail length and abundance of the long-tailed subpopulation. In the last project, we reviewed the theoretical basis of Sobol indices and identified potential problems when it is applied to mathematical models of biological oscillation systems. Based on circular statistics, we proposed two methods for the calculation of circular Sobol indices and compared their performance with the original Sobol indices in several models. We found that though the relative rankings of the contribution from parameters are the same across three methods, circular Sobol indices can better quantitatively distinguish the contribution of individual parameters. Through this work, we showed that mathematical modeling combined with sensitivity analysis can help us understand the mechanisms underlying the circadian gene expression in mammalian cells. Also, testable predictions are made for future experiments and new ideas are provided that can enable potential chronopharmacology research. / Doctor of Philosophy / Circadian rhythms are repeating biological activities with ~24h period observed in most living organisms. Disruption of circadian rhythms in humans has been found to be promote cancer, metabolic diseases, cognitive degeneration etc. In this work, we first used mathematical modeling to study the mechanisms of robust oscillations in the mammalian circadian core clock, which is a molecular regulatory network that drives circadian gene expression at transcriptional stage. Secondly, we built a coarse-grained model to investigate the post-transcriptional regulation of the rhythmicities in poly(A) tail length, which are observed in hundreds of mRNAs in mouse liver. Lastly, we examined the application of Sobol indices, which is a global sensitivity analysis method, to mathematical models of biological oscillation systems, and proposed two methods tailored for the calculation of circular Sobol indices. In the first project, we modified a previous mathematical model of the mammalian genetic oscillator so that it sustains robust oscillation with more realistic parameter values. Our analysis of the model further showed that the auxiliary positive feedback loop, rather than the auxiliary negative feedback loop, makes the oscillations more robust. In the second project, we found that rhythmic deadenylation, among the coupled transcription, polyadenylation, and degradation processes, mostly controls the rhythmicity of poly(A) tail length and mRNA subpopulation with long poly(A) tails. Lastly, we reviewed the theoretical basis of Sobol indices and found potential problems when it is applied to mathematical models of biological oscillation systems. Based on circular statistics, we proposed two circular Sobol indices, which can better distinguish the contribution of individual parameters to model outputs than the original Sobol indices. Altogether, we used mathematical modeling and sensitivity analysis to investigate the regulation of circadian gene expression in mammalian cells, providing testable predictions and new ideas for future experiments and chronopharmacology research.