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TRACKING AND MODULATING CIRCADIAN RHYTHMS IN CELL CULTURE MODELS

Abstract
Circadian rhythms are 24–hour activity cycles that exist for all organisms, from yeast and bacteria to mammals. Epidemiological data has shown that disruptions to circadian rhythms are associated with various diseases, including cancers. Circadian rhythms are regulated at the cellular level by a "molecular clock,” whose oscillations in protein transcription and translation control a range of downstream pathways. However, the connections between altered rhythms and diseases at the molecular level is unclear. Therefore, in this project, I tracked circadian rhythms in a high-resolution manner and thoroughly analyzed them to more completely reflect circadian behaviors and clock functioning at the molecular level. I applied these methods to study the circadian rhythms in different breast cancer subtypes, modulation following small molecule treatments, and alterations upon macrophage polarization or conditioned media treatments. To study whether there is a relationship between altered circadian rhythms and cellular phenotypes/oncogenic features, I tracked circadian rhythms in a high-resolution manner in commonly used model cell lines of breast cancer to determine whether different oscillations are correlated with cancer aggressiveness. By tracking circadian rhythms using luciferase reporters for Bmal1 and Per2 to generate high-resolution data, I found that oscillations for a less aggressive cancer type (MCF7) were rhythmic and had relatively normal periods of ~24 hours, while I observed no rhythms for highly aggressive cells (MDA-MB-231). I used similar tracking and analysis methods to determine the effects of nobiletin, a small molecule natural product targeting a circadian protein, in multiple cell lines with different circadian characteristics. I found that nobiletin can enhance the circadian rhythmicity of arrhythmic cells (MDA-MB-231), where it also reduces oncogenic effects. However, in cells with circadian rhythms (U2OS, MCF7), treatment resulted in no circadian enhancement or clear anti-oncogenic effects. Most small molecule modulators, including nobiletin, lack desired selectivity, and genetic approaches can result in undesirable effects. As an alternative strategy, I proposed the selection of nanobodies that can specifically bind to core circadian clock proteins Cryptochrome 1 and/or 2, and disrupt protein-protein interactions or be modified to yield protein degradation agents. Lastly, I studied the effects of phenotypic changes and cancerous environments on cellular circadian rhythms using a mouse macrophage model cell line (RAW 264.7). With high-resolution circadian data generated from luciferase reporters, I found that polarization of macrophages to pro- (M1) or anti-inflammatory (M2) subtypes differently alter their circadian rhythms and highly aggressive breast cancer conditioned media affects oscillations of the macrophages to a greater extent. Together, I have presented that circadian rhythms exist in the less aggressive breast cancer cell line MCF7 while they are absent in the highly aggressive breast cancer cell line MDA-MB-231, circadian rhythmicity enhancement and reduction of oncogenic features occur concurrently in MDA-MB-231cells, and macrophage polarization and breast cancer conditioned media affect macrophage circadian rhythms. I also provided a framework to generate target-specific nanobody-based circadian modulators.
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dissertation
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http://creativecommons.org/licenses/by/4.0/
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