Circadian clocks are responsible for daily rhythms in gastrointestinal function which are vital for normal digestive rhythms and health. The present study examines the roles of the circadian pacemaker, the suprachiasmatic nuclei (SCN), and the sympathetic nervous system in regulation of circadian gastrointestinal rhythms in Mus musculus. Surgical ablation of the SCN abolishes circadian locomotor, feeding, and stool output rhythms when animals are presented with food ad libitum, while restricted feeding reestablishes these rhythms temporarily. In intact mice, chemical sympathectomy with 6- hydroxydopamine has no effect on feeding and locomotor rhythmicity, but attenuates stool output rhythms. Again, restricted feeding reestablishes these rhythms. Ex vivo, intestinal tissue from mPer2LUC knockin mice expresses circadian rhythms of luciferase bioluminescence. 6-hydroxydopamine has little effect upon these rhythms, but timed administration of β−adrenergic agonist isoproterenol causes a phase-dependent phase shift in PERIOD2 expression rhythms. Collectively, the data suggest the SCN are required to maintain feeding, locomotor and stool output rhythms during ad libitum conditions, acting at least in part through daily activation of sympathetic activity. Even so, this input is not necessary for entrainment to timed feeding, which may be the province of oscillators within the intestines themselves or other components of the gastrointestinal system.

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.

Period 2 (Per2) is a core circadian factor responsible for its own negative regulation. It operates in the circadian clock, which affects multiple biological functions such as metabolic rate, hormone release, and core body temperature. The Per2 protein functions directly with factors in other biological functions such as tumor suppression, immune system, and metabolism. In many cases, the Per2 deficiency caused by disrupted expression is sufficient to create severe abnormalities in many of the mentioned functions. The sequence contains several domains and motifs in Per2 that are traditionally involved in protein interactions which suggests that Per2 serving a regulatory role by effecting downstream biological roles dependent on Per2 stability. In this work, we perform a two-hybrid screening assay using the C-terminal region of human Per2 and identified an extensive number of interactors. Utilizing a genetic ontology program, we assorted the list of clones into groups of proteins that are biologically relevant or operated in similar function. Through this program, we validated the two-hybrid screening by the clusters of biological function already attributed to hPer2 and identified new putative biological functions. We use the new putative interactors to gain further insight on the regulatory roles that hPer2 performs, in conjunction with operating as a core factor in circadian rhythmicity. We also show that Translationally Controlled Tumor Protein (TCTP) is capable of binding to hPer2 and is a novel interaction. When a sufficient amount of TCTP (1:1 molar stoichiometric ratio) is present in a system, a cleavage of hPer2 is observed in vitro. This cleavage occurs in reactions independent of ATP, ubiquitin, and the proteasome. The data points towards a method of cleavage similar to that of the archael lon-tk (Thermococcus kodakaraensis) that preferentially cleaved unstructured substrates in ATP-independent reactions. / Master of Science

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