Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2024, College of Arts and Sciences

Fasting (F) offers many metabolic benefits but interestingly induces hepatic steatosis. Many fasting metabolic adaptations have largely been studied under experimental conditions where food is unexpectedly withheld. Such random fasting is in sharp contrast with Calorie restriction (CR), in which mice self-impose a periodic fasting cycle and spend most of their day without the food. Despite long fasting durations, CR mice are protected from hepatic steatosis via unclear mechanisms. Here, we utilized transcriptomic profiling to identify important players in the differential effect of CR and F on liver steatosis. Serum non-esterified fatty acids were elevated to similar concentrations by both CR and F. Both diets induced hepatic β-oxidation but the magnitude and kinetic were stronger in F than in CR. This revealed that liver energy utilization is a discriminating factor between both diets but did not explain differences in liver TAG accumulation. Our unbiased transcriptomic analysis identified some candidate genes that were induced by F but not CR, these genes code for liver fatty acid (FFA) transporters (Slc27a1 and Slc27a2), TAG synthesis (Gpat4), and lipid storage (Plin2 and Cidec). Correspondingly, liver FFAs increased in F (C16:0, C18:0, C18:1, C18:2), while only C18:2 was increased in CR. F increased liver TAG and lipid droplet accumulation while decreased profiles were observed in CR. We found that the tight control of liver lipid homeostasis in CR was achieved through entrained anticipation of the feeding/fasting cycle in CR. Specifically, when CR mice miss their anticipated daily meal or have an impaired circadian clock (Cry1,2 ablation), they began to accumulate liver TAG and induce metabolic profiles similar to F mice. Our study positions entrainment by the circadian clock in the mechanisms of CR.

Committee: Roman Kondratov (Advisor); Srinivasan Dasarathy (Committee Member); Crystal Weyman (Committee Member); Aaron Severson (Committee Member) Subjects: Molecular Biology

Doctor of Philosophy (PhD), Wright State University, 2024, Biomedical Sciences PhD

The circadian clock is a fundamental biological mechanism that regulates various physiological processes, including DNA repair, to synchronize with the day-night cycle. In human skin, exposure to ultraviolet (UV) light poses a significant challenge, inducing DNA damage that must be efficiently repaired to maintain genomic integrity and prevent carcinogenesis. This study delved into the complex interplay between the circadian clock, UV light exposure, DNA repair, and modulation of circadian transcriptional machinery in human skin. Initially, we examined the transcriptomic profile of the circadian clock in humans through in silico-based approaches and in vivo studies, revealing that core clock gene expression and downstream clock-controlled genes are influenced by factors such as age, sex, and UV exposure. Recognizing the potential to modify circadian clock output, we employed REV-ERB antagonist SR8278 and cryptochrome inhibitor KS15 to enhance clock function, thereby facilitating increased output of downstream processes like DNA repair. In our ex vivo model system, we found that SR8278 and KS15 were able to increase both the RNA and protein levels of ARNTL, XPA, and Wee1, as well as being able to decrease the amount of UV-induced apoptosis in some individuals. In keratinocytes in vitro, we found that treatment with SR8278 and KS15 led to significant increases in survival when exposed to UVB, but led to significant toxicity under UVA lights. Furthermore, we were able to modestly increase expression of ARNTL, XPA, Wee1 and p21 at the RNA and protein levels. However, we found that these increases did not translate to increased nucleotide excision repair capacity. Rather, we determined that the compounds are absorbing wavelengths of UV light and preventing damage formation initially. Further investigations are warranted to elucidate the precise mechanism of action of SR8278 and KS15 in modulating circadian clock function. Nevertheless, this study lays the groundwork for (open full item for complete abstract)

Committee: Michael G. Kemp Ph.D. (Advisor); Michael Leffak Ph.D. (Committee Member); Michael P. Markey Ph.D. (Committee Member); Yong-jie Xu M.D., Ph.D. (Committee Member); Jeffrey B. Travers M.D., Ph.D. (Committee Member) Subjects: Biomedical Research; Pharmacology

MS, University of Cincinnati, 2023, Arts and Sciences: Mathematical Sciences

The goal of this work is to perform a preliminary exploration of a potential model for the emergence of the circadian clock in mammalian cells. This will include an overview of the literature regarding the potential mechanisms of this emergence and existing models of the circadian clock. The full identification and validation of a true model for the emergence of the circadian clock is a lengthy process, far outside the scope allowable by the time frame this thesis was written in. It is for this reason that explorations are limited to only a few modifications to existing models, and only the preliminary steps at that. The novel model consists of a core transcriptional/translational feedback loop (TTFL) with a multi-step reaction of phosphorylation steps occurring within the cytoplasm, as well as Michaelis-Menten inspired degradation rates for proteins with enzyme-mediation degradation. The model also includes a stoichiometry-based transcription term, as described in a previous model by Kim and Forger. Parameter values are determined through an iterative search of the parameter space, then undergo stability analysis and Hopf bifurcation point identification. The parameters associated with the proposed mechanisms of emergence, posttranscriptional regulation of Clock mRNA and subcellular localization of PER, are further investigated to determine the suitability of the model. Further modifications to the model are proposed based on the analysis of the parameters of interest and existing literature.

Committee: Sookkyung Lim Ph.D. (Committee Chair); Benjamin Vaughan Ph.D. (Committee Member); Christian Hong Ph.D. (Committee Member) Subjects: Applied Mathematics

Master of Science (MS), Wright State University, 2023, Pharmacology and Toxicology

Cathepsin L is a lysosomal cysteine protease that is encoded by the CTSL gene in humans. Previous results have shown that CTSL expression and activity towards the DNA repair protein XPA are elevated in lysates of confluent/quiescent HaCaT keratinocytes. Because other nuclear proteins, including 53BP1 and histone H3, have also been reported to be CTSL substrates under conditions of cell differentiation, senescence, or quiescence, CTSL expression and activity (cleavage) of multiple nuclear proteins was examined in lysates from HaCaT cells and other cell lines at various days after plating and cell confluency. Our study found that adding CTSL inhibitors to a cell lysis buffer will block the artifactual cleavage of these nuclear proteins during cell lysis, which calls into question the interpretation of several previous publications. Furthermore, CTSL levels in human sera have been reported to display circadian rhythmicity, and we found that expression of CTSL in confluent cells can by controlled by clock modulating compounds. Specifically, we found that the REV-ERB antagonist SR8278 and the ROR agonist SR1078 cause a decrease in CTSL levels in a manner independent of the core clock protein BMAL1. These results provide greater insight into CTSL activity and regulation by the circadian clock.

Committee: Michael G. Kemp Ph.D. (Advisor); Ravi P. Sahu Ph.D. (Committee Member); Yong-Jie Xu M.D., Ph.D. (Committee Member) Subjects: Pharmacology; Toxicology

Master of Science (MS), Wright State University, 2021, Pharmacology and Toxicology

Cisplatin is a DNA damage-based chemotherapeutic drug widely used to treat various types of cancers; however, the treatment's toxicity restricts its efficiency. Studies have shown that the circadian rhythm controls the DNA damage response and affects the repair pathways of cisplatin-induced DNA damage. Circadian clock modulation, therefore, has been proposed to be a potential mechanism for enhancing cisplatin tolerability. Here we used clock-enhancing molecules to evaluate the effect of pharmacological clock modulation on cisplatin cytotoxicity. Using cultured human cell lines, cisplatin cytotoxicity was found to be attenuated following treatment with circadian-enhancing molecules KS15 and SR8278. Moreover, the protein and mRNA levels of cell cycle and apoptosis regulators, as well as clock-controlled genes, were modified in response to KS15 and SR8278. Those molecules were also able to enhance cisplatin-induced DNA adducts removal and induce G1-phase cell cycle arrest. Our findings suggest that the use of circadian clock modulators has promising implications for improving cancer care and treatment outcomes.

Committee: Michael G. Kemp Ph.D. (Advisor); Ravi P. Sahu Ph.D. (Committee Member); Yong-jie Xu M.D., Ph.D. (Committee Member) Subjects: Pharmacology; Toxicology

Master of Science, The Ohio State University, 2021, Entomology

Diapause, a period of arrested development that allows mosquitoes to survive inhospitable conditions, is triggered by short daylengths in temperate mosquitoes. Different populations of mosquitoes initiate diapause in response to a specific photoperiod, or daylength, resulting in population-specific differences in annual cycles of abundance. The photoperiod that causes approximately 50% of a population to initiate diapause is known as the critical photoperiod (CPP). In the Northern Hemisphere, northern populations of mosquitoes experience lower temperatures earlier in the year and must be triggered into diapause by longer daylengths than southern populations. CPP is genetically based, but also adapts over time responding to the population's environment. Therefore, CPP has been shown to lengthen 1 hour with an increase of 5 °N latitude or an increase of 122 m altitude, following Bioclimatic Law. While the positive correlation between CPP and latitude/altitude has been established in a few mosquito species, including Aedes albopictus, Ae. triseriatus, Ae. sirennensis and Wyeomyia smithii, we do not know when most other species initiate their seasonal responses. As several of these species transmit important diseases, characterizing the CPP of arthropod vectors could improve existing control by ensuring that surveillance efforts align with the vector's seasonally active period. Additionally, better understanding when mosquitoes and other vectors initiate diapause can reduce the frequency of chemical applications, thereby ameliorating the negative impacts to nontarget insects. Females of Culex pipiens, the primary vector of West Nile virus, enter an adult reproductive diapause. The methods by which Cx. pipiens measures daylength are still unknown. However, clock genes which provide information on the time of day, may also be responsible for the regulation of diapause. Previous studies have shown that the mRNA of several circadian clock genes continue to oscillate throu (open full item for complete abstract)

Committee: Megan Meuti PhD (Advisor); Mary Gardiner PhD (Committee Member); Peter Piermarini PhD (Committee Member) Subjects: Entomology

Doctor of Philosophy, The Ohio State University, 2020, Neuroscience Graduate Studies Program

Cancer patients and survivors commonly report fatigue and cognitive impairments that can last months to years into remission. This cancer-associated cognitive impairment is commonly referred to as “chemobrain,” and these symptoms lead to decreased quality of life and poorer long-term survival outcomes. Inflammation has been posited as a key contributor to cancer-induced cognitive impairment, given that these symptoms persist following cessation of chemotherapy. Notably, increased fatigue and poorer cognition have been correlated with flattened cortisol slopes in cancer patients. As circulating cortisol, rest-activity cycles, and memory are all modulated by circadian, 24-hour rhythms, it stands to reason that cancer and cancer treatments may elicit chemobrain symptoms, in part, by disrupting the circadian clock. Here, I examine the extent to which paclitaxel chemotherapy, a microtubule-binding taxane agent, and non-metastatic mammary tumors disrupt circadian rhythmicity in a mouse model. First, in Chapter 2 I demonstrate that paclitaxel inhibits memory recall through a contextual fear conditioning task. This disrupted memory recall is accompanied by increased hippocampal and hypothalamic inflammation as measured by glial reactivity. Next, in Chapter 3 I demonstrate that individual CA1 pyramidal neurons exhibit 24-hour rhythms using a Per1-Venus clock gene reporter mouse. Other hippocampal cell types (astrocytes and GAD65/67-positive interneurons) exhibit clock gene expression and in vivo clock gene rhythms were observed at single-cell resolution in the posterior parietal cortex. In chapter 4, I demonstrate that paclitaxel chemotherapy ablates rhythms in circulating corticosterone, alters the free-running period of voluntary wheel running, and attenuates the amplitude of clock genes in multiple brain regions and the adrenal glands. Finally, in Chapter 5 I show that non-metastatic mammary tumors dysregulate time-of-day differences in hypothalamic clock gene expression (open full item for complete abstract)

Committee: Leah Pyter (Advisor); Karl Obrietan (Advisor); Fischer Andrew (Committee Member); Tedeschi Andrea (Committee Member) Subjects: Biology; Biomedical Research; Immunology; Neurobiology; Neurosciences

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2019, College of Sciences and Health Professions

Caloric restriction (CR) intervention has been demonstrated to improve health and extend lifespan. CR in mammals, imposes a short interval of feeding, called time restricted feeding (TR), which is followed by prolonged interval of fasting. TR or mealtime (MT), a form of periodic fasting, without reducing caloric intake, may contribute to improvement in metabolism. To dissect the contributions of reduced caloric intake and periodic fasting in health benefits mediated by CR, we measured physiological and metabolic parameters in mice subjected to CR and TR (without reduction in caloric intake). CR reduced blood glucose and insulin, and increased ketone levels across the day, significantly improved glucose and insulin sensitivity. TR did not affect blood glucose and glucose sensitivity, in contrast to CR, but reduced blood insulin and partially improved insulin sensitivity. Both the diets had little to no effect on phases of circadian clock genes, and CR significantly induced the expression of glucose metabolic genes, whereas TR did not, which correlates with modest effect of TR on glucose homeostasis. Therefore, we concluded that, TR is metabolically different from CR, and that periodic fasting contributed to some of the metabolic improvements on CR, independent from caloric intake. This may help provide a mechanistic explanation to differences in lifespan extension observed under CR and TR.

Committee: Roman Kondratov (Advisor); Crystal Weyman (Committee Member); Barsanjit Mazumder (Committee Member); Girish Shukla (Committee Member); Olga Stenina-Adognravi (Committee Member); Anton Komar (Other); Yana Sandlers (Other) Subjects: Biology

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2019, Biological Sciences

Circadian rhythms control cancer cell behavior in tumors and in vitro. Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are two key events of metastasis that lead to cancer progression and aggressiveness. Studies suggest that genes controlling circadian rhythms also regulate EMT, which generates mesenchymal cells (M-cells) with cancer stem cell (CSC) properties through dedifferentiation. MET enables post-EMT cells to differentiate back to epithelial cells at metastatic sites and initiate secondary tumors. If a circadian clock can be identified in M-cells, then it could be manipulated pharmacologically to more effectively target the cells with novel anticancer agents specific to CSCs or cause their differentiation into more easily treated and less aggressive cells before metastasis occurs. We tested for any role of circadian clocks in EMT and MET events by using cancer cell lines from two different tissues. We compared C6 rat glioma cells that have a well-established circadian clock with MCF-7 human breast tumor cells that are considered lacking a functional clock. EMT was induced in cell cultures by exchanging standard serum-containing medium (SM) with stem cell medium (SCM), a non-serum medium containing specific growth factors promoting CSC survival. Single-cell behavior and morphological states were quantified microscopically through time-lapse imaging. Expression of EMT markers ZEB1 and TWIST, mesenchymal markers vimentin and PDGFRA, and stem cell markers OCT4, nestin, MSI1 and CD133 were validated by immunocytochemistry. Both C6 and MCF-7 cultures showed circadian oscillations in the population size of post-EMT M-cells. MET was then induced by returning the cultures to SM from SCM. MET events observed in glioma CSCs clustered significantly at a particular phase of the circadian cycle. The cellular microenvironment also influenced migration properties of C6 cells with SM promoting faster closure in a standard wound healing assa (open full item for complete abstract)

Committee: Michael Geusz PhD (Advisor); Lynn Darby PhD (Other); Paul Morris PhD (Committee Member); Vipaporn Phuntumart PhD (Committee Member); Amit Tiwari PhD (Committee Member) Subjects: Biology; Biomedical Research; Cellular Biology; Health Sciences; Medicine; Molecular Biology; Neurosciences; Oncology; Pharmacology

Master of Science in Biology, Cleveland State University, 2017, College of Sciences and Health Professions

The rhythms in the expression of circadian clock genes are affected by calorie restriction (CR), a dietary paradigm known to increase lifespan. In our current study, we show that circadian rhythms are influenced by sex and the effects of CR are different between males and females. In particular, we found a group of clock genes which showed a sex-dependent difference in expression, as well as in response to CR (Rev-Erb α, Ror γ and both Cryptochromes: Cry1 and Cry2 genes). Two clock genes showed no difference in expression but their response to CR showed sexual dimorphism (Ror α and Rev-Erb β). Finally, we found some of the clock genes to be expressed in a sex-independent manner (Bmal1, Per1, Per2 and Per3). The response to CR for these genes did not show sexual dimorphism as well. Several genes were also previously reported to be regulated by CR. These genes showed a sex-dependent difference in expression as well as the sexual dimorphism in the response to CR in mouse liver (Fmo3, Mup4, Serpina12, Cyp4a12b and Cyp4a14a). IGF signaling plays an important role in aging and CR effects. Igf-1 expression is regulated by CR and by the circadian clock, we found that rhythms in Igf-1 expression have sexual dimorphism.We also investigated the differences in expression levels in young versus old mice to see if the effect of short-term CR differs from the effect of long-term CR. In all of the 4 analyzed genes, the expression does not change with age. The effect of CR on the expression of 2 out of 4 genes (Bmal1 and Per2) was lost upon long-term exposure to CR. For the other 2 genes (Per1 and Per3) the effect of CR persisted over time. Thus we emphasize that sex and age are important factors for consideration when administering CR.

Committee: Roman Kondratov PhD (Committee Chair); Crystal Weyman PhD (Committee Member); Aaron Severson PhD (Committee Member) Subjects: Biology; Molecular Biology

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2016, College of Sciences and Health Professions

Calorie Restriction (CR) is a powerful paradigm known to delay aging and thus increase longevity in several organisms, from yeast to non-human primates. Many molecular pathways have been proposed to mediate the beneficial effects of CR, however, the mechanism is still unknown. Circadian clock which is an internal time keeping system is regulated by feeding. Thus our aim was to study the effect of CR on the circadian clock. Here we show that CR significantly affects the expression of circadian clock genes in mice at the mRNA and protein levels, suggesting that CR reprograms the clocks at the transcriptional and post-transcriptional level. CR also affected the circadian output through up- or down-regulation of the expression of several clock-controlled transcriptional factors and the longevity candidate genes. CR-dependent effects on some clock gene expression were impaired in the liver of mice deficient for BMAL1, suggesting importance of this transcriptional factor for the transcriptional reprogramming of the clock, however, BMAL1-independent mechanisms exist too. We have shown that Bmal1 deficient mice develop premature aging phenotype and have a shortened lifespan. We decided to apply 30%CR to these mice and found that CR did not increase the lifespan of these Bmal1 mutants, further suggesting that BMAL1 is necessary for full benefits of CR. We also analyzed the plasma levels of IGF-1 and insulin, which were found to be impaired in Bmal1 deficient mice on 30%CR. We propose that CR recruits biological clocks as a natural mechanism of metabolic optimization and synchronization of the several downstream pathways under limited nutrient conditions.

Committee: Roman Kondratov PhD (Advisor); Barsanjit Mazumder PhD (Committee Member); Aaron Severson PhD (Committee Member); William Baldwin M.D., PhD (Committee Member) Subjects: Biology; Molecular Biology

PHD, Kent State University, 2015, College of Arts and Sciences / Department of Biological Sciences

The suprachiasmatic nucleus(SCN) of the hypothalamus acts as an endogenous circadian clock. Individual neurons of the SCN are capable of acting as independent circadian oscillators, but the SCN exhibits a specific structural organization, with subregions that can be defined by a variety of criteria, including neuropeptide expression and cellular responses to stimuli. individual subsets of cells can show different rhythms of gene expression. Individual genes have been identified that are differentially expressed in three specific SCN subregions. 1) The ventral SCN, characterized by expression of vasoactive intestinal polypeptide (VIP), 2) the central SCN, characterized by expression of gastrin-releasing peptide (GRP), and 3) the dorsomedial SCN, characterized by expression of arginine vasopressin (AVP).In this study we hypothesized that these 3 regions were not only defined by their neuropeptide expression, but other circadian and non-circadian gene expression, thus revealing previously unknown details of the organization of the neuronal components of the SCN.. We have identified several transcripts, some not previously identified in the SCN, that are circadian across all three regions. Our current data suggests that these three SCN subregions are characterized primarily by differences in neuropeptide expression and not by any other large-scale differences in gene expression. The robustness of rhythmic expression across SCN subregions may be an indicator of genes that have critical core functions in rhythmicity or rhythmic output, and may serve to identify good target genes for investigation in rhythmic control in the SCN. In addition, we examined the potential for estrogenic regulation of circadian clock function. There is strong evidence that estrogen influences circadian clock function, at both the behavioral and molecular levels. Several previous studies have shown that estrogen affects the expression of clock genes including Per1, Per2 and Cry2. However (open full item for complete abstract)

Committee: Eric Mintz (Advisor) Subjects: Bioinformatics; Biology; Neurosciences

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2015, Biological Sciences

Understanding how the circadian clock controls delivery and activity of chemotherapies could optimize cancer treatments. Most anti-cancer drugs are targeted to the nucleus and their movement through the nuclear envelope could be controlled by the circadian clock. Circadian regulation of cell toxicity was tested by measuring the ability of the phytochemical curcumin to kill C6 glioma cells at different phases of the circadian cycle. Mitotic and cell death events were counted every 5 minutes for 5 days. An initial low curcumin treatment (5 µM) caused an elevated cell death rate in C6 cells at rhythmic intervals with a period of 24.4 hours. The rhythm was disrupted at 10 µM. These results revealed a sensitive phase of the circadian cycle that could be exploited in therapies based on curcumin or its analogs. Curcumin's persistence in cells was imaged for at least 24 hours using autofluorescence. The observed higher stability of the curcumin congeners demethoxycurcumin and bisdemethoxycurcumin suggested that they too may produce sustained cancer cell toxicity. Circadian regulation of anti-cancer agents may also occur in the nuclear transport mechanism through nuclear pore complexes (NPCs). The transport mechanism could be regulated by cell calcium ion stores. A single-molecule imaging technique provided the kinetic parameters as well as spatial locations of transported molecules at altered calcium ion store concentrations. This three-dimensional, high-speed, super-resolution imaging indicated that transport was altered. It is possible that previously described circadian calcium oscillations regulate NPC activity. Quantitative RT-PCR was used to test whether with the circadian clock controls expression of the Crm1 gene. CRM1 protein is a nuclear exporter that may generate circadian rhythms in cargo concentrations in the cytoplasm. The phase of the circadian rhythm detected suggested that Crm1 could be controlled by the circadian clock protein BMAL1. To test whethe (open full item for complete abstract)

Committee: Michael Geusz (Advisor); George Bullerjahn (Committee Member); Scott Rogers (Committee Member); Vipaporn Phuntumart (Committee Member); Lewis Fulcher (Other) Subjects: Biology

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2015, Biological Sciences

Adult neurogenesis creates new neurons and glial cells from stem cells in the human brain throughout life, and it is best understood in the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ). Recent studies have described possible interactions between the molecular mechanism of circadian clocks and the signaling pathways that regulate stem cell differentiation. Circadian rhythms have been identified in the olfactory bulb and the hippocampus, but the role of any endogenous circadian oscillator cells in neurogenesis and their importance in learning and memory remains unclear. Circadian rhythms have not been examined well in neural stem cells and progenitor cells that produce new neurons and glial cells during adult neurogenesis. To evaluate circadian timing abilities of cells undergoing neural differentiation, neurospheres were prepared from mPer1::luc mouse SVZ and DG. Circadian bioluminescence rhythms were recorded in neurospheres maintained in culture medium that induces neurogenesis but not in one that maintains the stem cell state. Cell types were also characterized by confocal immunofluorescence microscopy at early and late developmental stages in vitro. Evidence was provided that neural stem progenitor cells (NSPCs) derived from the SVZ and DG of adult mice are self-sufficient clock cells capable of producing a circadian rhythm without input from known circadian pacemakers of the organism. Extremely rare percentages of mature neuronal cells were observed during ontogeny of rhythms. The bulk of the neurosphere cells were undifferentiated, indicating that they are the circadian clock cells producing timing signals. This conclusion was supported by immunocytochemistry for mPER1 protein that was localized to the inner, more stem cell-like neurosphere core. To further test whether circadian clocks in NSPCs are necessary for growth, differentiation and cell survival, neurospheres were cultured from Bmal1-/- and Cry1-/-,2-/- knockout mice. Neur (open full item for complete abstract)

Committee: Michael, E. Geusz Dr. (Advisor); Roudabeh, J. Jamasbi Dr. (Committee Member); Scott, O. Rogers Dr. (Committee Member); Vipa Phuntumart Dr. (Committee Member); Robert Midden Dr. (Other) Subjects: Biology; Molecular Biology; Neurosciences

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2014, College of Sciences and Health Professions

Understanding mechanisms of aging is important for the treatment and prevention of age-associated pathologies. However, these mechanisms are not well understood. Recently we have demonstrated that the circadian clock (an internal time keeping system) regulates longevity in mammals, but the molecular mechanisms are not known. The aim of our current study is to investigate a possible interconnection between the circadian clock and mTORC1 (mammalian target of Rapamycin) signaling pathway. mTORC1 pathway is a nutrient response pathway involved in many cellular processes; many recent studies indicate a role of mTORC1 pathway in aging. Here we demonstrate that circadian system regulates mTORC1 signaling in vivo. Analysis of liver, heart and spleen from WT mice reveals circadian rhythms in phosphorylation of known mTOR targets such as S6K1 and 4EBPs. These rhythms are disrupted in the tissues of BMAL1 KO mice providing potential mechanistic explanation of reduced longevity of these mice. Further analysis of expression of mTORC1 complex components and upstream regulators demonstrated that some of them have circadian rhythms at protein and mRNA levels. Taken together, these results suggest that circadian clock controls aging by regulating mTOR signaling pathway through BMAL1 dependent control of gene expression.

Committee: ROMAN KONDRATOV (Advisor); CRYSTAL WEYMAN (Committee Member); ALEXANDRU ALMASAN (Committee Member); VALENTINE BOERNER (Committee Member) Subjects: Biology; Cellular Biology; Molecular Biology

MS, Kent State University, 2013, College of Arts and Sciences / Department of Biological Sciences

TOPACIO, TRACEY KAREN, M.S., DECEMBER, 2013 BIOLOGICAL SCIENCES CIRCADIAN DISRUPTION, DIET, AND EXERCISE (88 pp.) Director of Thesis: Eric Mintz Circadian rhythms studies in rodents are conducted using a wide range of housing conditions, with animals housed in cages of different sizes that may or may not have a running wheel for assessing locomotor activity. I examined whether housing conditions influence the core clock gene mechanism that drives circadian rhythms. C57BL/6J mice were maintained under 12:12 LD in either small cages, large cages, or large cages with a running wheel. Mice were euthanized at four, equally spaced time points throughout the 24-hours. The brain, liver, lung, and heart tissue were collected and analyzed by real-time PCR to assess the expression of Per1, Per2, Clock, and Bmal1 at each time point. Gene expression varied with a daily rhythm as expected in each tissue. Significant differences in Clock and Bmal1 expression between housing conditions were observed in the liver, lungs, and the suprachiasmatic nucleus (SCN), but not the heart. Differences were observed in the middle and end of the night and were mostly a result of differences in cage size rather than by the presence or absence of a running wheel. These data suggest that the size of the cage appreciably alters Clock and Bmal1 expression in the liver, lungs, and SCN primarily during the active time points, and that care should be taken when comparing the results of studies using different housing conditions. Circadian disruption can have a variety of negative physiological consequences including metabolic impairment. I asked whether weight gain on a high fat diet (HFD) would be increased under simulated chronic jet lag conditions. Male C57BL/6J mice were housed in cages with running wheels and given either normal or HFD for 7 weeks. After the first week, animals were kept in 12:12 LD, or the lights were either advanced (PA) or delayed (PD) by 6 hours (open full item for complete abstract)

Committee: Eric Mintz (Advisor); Colleen Novak (Committee Member); Wilson Chung (Committee Member) Subjects: Biology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2010, Neuroscience Graduate Studies Program

Circadian (~24 hr) rhythmicity is a prevalent feature of virtually all the living organisms on the earth. This intrinsic property influences every aspect of life activities. In mammals, the master internal clock is localized in the suprachiasmatic nucleus (SCN) of the hypothalamus. Rhythmic clock gene expression constitutes the molecular basis of clock oscillation. As an important feature of the clock, it can be reset by signaling events that impinge on clock gene expression. There has been significant interest in unraveling the intracellular signal transduction pathways that couple extracellular signals to clock gene expression. Along these lines, our lab has identified a pivotal role of the p42/44 mitogen-activated protein kinase (MAPK) signaling pathway in the clock resetting process. As a continuation of this line of research, my thesis project is focused on the role of the mammalian target of rapamycin (mTOR) pathway in the circadian clock timing and entrainment. I found that brief light exposure during the subjective night, but not during the subjective day, triggered rapid phosphorylation (a marker of catalytic activity) of the mTOR translation effectors p70 S6K, ribosomal S6 protein (S6) and 4E-BP1 and MAPK cascade is an essential intermediate that couples light to mTOR. I also found that mTOR inhibition led to a significant attenuation of the phase-delaying effect of early-night light. Conversely, disruption of mTOR during the late night augmented the phase-advancing effect of light. To assess the role of mTOR signaling within the context of molecular entrainment, the effects of rapamycin on light-induced expression of PERIOD1 and PERIOD2 were examined. At both the early- and late-night time points, abrogation of mTOR signaling led to a significant attenuation of light-evoked PERIOD protein expression. These results also reveal that light-induced mTOR activation leads to the translation of mRNAs with a 5'-terminal oligopyrimidine tract such as eukaryotic el (open full item for complete abstract)

Committee: Karl Obrietan PhD (Advisor); Randy Nelson PhD (Committee Member); John Oberdick PhD (Committee Member); Glenn Lin PhD (Committee Member) Subjects: Neurosciences

Doctor of Philosophy, The Ohio State University, 2008, Entomology

A non-diapausing (nd) strain of the flesh fly, Sarcophaga bullata, loses the normal daily adult eclosion rhythm as well as the normal photoperiodic diapause response. The inheritance mode of diapause was investigated by crossing this mutant strain with two other rhythmic diapausing strains having different diapause capacities. The crossing results consistently indicated that diapause incidence is inherited in a simple Mendelian pattern, thus a single gene or a small gene cluster controls the seasonal response of diapause in the nd strain. The essential circadian clock genes, period, timeless, cycle and cryptochrome were originally targeted as candidates of the single malfunctioning gene in the nd strain. As the initial step to study the genetic differences among strains, the full length cDNA sequences of the cryptochrome and the cycle genes in S. bullata were obtained. The cDNA of the cryptochrome gene is 1629 base pairs long, which encodes a protein of 542 amino acids. The cycle gene cDNA is 1517 base pairs long, and its product consists of 410 amino acids. Some functional domains are highly conserved. However, no significant differences at the cDNA level were discovered in the four circadian clock genes between the nd strain and the wild-type flies, suggesting that none of these clock genes is the major cause of circadian rhythm loss in the nd strain. Meanwhile, various mutations on the period gene were discovered using another strain of S. bullata. Among these mutations, perd interferes with light detection when determining time of dawn eclosion, while perm2 shifts the daily eclosion time window significantly earlier. These mutations help detailed investigation of functional motifs on the period gene. More interestingly, deletion and insertion mutations at the C-terminal region, as well as their correlations with diapause capacities, were discovered. This region on the period gene is involved in photoperiodism in S. bullata, suggesting that circadian and photoper (open full item for complete abstract)

Committee: David Denlinger (Advisor); Thomas Wilson (Committee Member); Glen Needham (Committee Member) Subjects: Entomology

Doctor of Philosophy, The Ohio State University, 2006, Molecular, Cellular, and Developmental Biology

The circadian oscillator of Arabidopsis thaliana builds upon interlocked transcriptional feedback loops onto which a diverse array of period-affecting factors are incorporated to culminate into a 24-hour periodicity. A subset of the period-affecting factors function in light input pathways which are specifically connected to the clock, thereby modulating circadian period in a light-dependent manner. A novel F-box protein, ZEITLUPE (ZTL) which controls circadian period in a fluence-rate dependent way, falls into this category. The primary focus of this dissertation is to characterize the light-regulated proteolytic pathway mediated by ZTL. At first we identified the SCFZTL complex in planta and established that the formation of SCFZTL complex is important for proper regulation of circadian clock. SCFZTL complex targets an essential oscillator component, TOC1 for its proteasome-dependent degradation. TOC1/PRR1 is the founding member of a small gene family of Pseudoresponse Regulators (PRRs) which consists of additional four genes (PRR3,PRR5,PRR7 and PRR9). The transcriptional regulations among PRR members partially define the interlocked transcriptional feedback loops which are at the core of central oscillator. In this study we characterized the expression pattern of all PRR members and identified two post-translational regulatory events which are shared by four PRR members (PRR1/3/5/7): phosphorylation and proteasome-dependent protein turn over. In addition, we identified PRR5 as a novel interactor of ZTL. In order to further characterize biochemical properties of ZTL and more broadly, to better understand the ZTL-mediated light signal transduction pathway which is specifically coupled to circadian clock, we took both forward and reverse genetic approaches. First, we dissected the roles of three distinctive domains of ZTL in the regulation of circadian period and photomorphogenesis. We found that the C-terminal F/KELCH domain could act together to alter circadian pe (open full item for complete abstract)

Committee: David Somers (Advisor); Greg Armstrong (Other); David Bisaro (Other); David Mackey (Other) Subjects: Biology, Plant Physiology

BS, Kent State University, 2012, College of Arts and Sciences / Department of Chemistry and Biochemistry

Circadian rhythm is one of the most fundamental regulators of physiology and behavior. The transcription-translation feed-back loop of clock genes control metabolic activity by regulating the transcription of key proteins and enzymes. The transcription proteins BMAL1 and CLOCK are two of the proteins that are involved in translation-transcription feed-back loop. They dimerize and bind to promoter regions and positively regulate the expression of other clock genes. The expressed clock proteins in turn can negatively regulate the action of CLOCK and BMAL1 and/or regulate the expression of other metabolic proteins. Because BMAL1 is an integral part of the circadian rhythm and its roles in whole-body metabolism, the modification of its expression can disrupt muscle metabolism. The overexpression of BMAL1 in skeletal muscles may have a broader impact on whole-body metabolic regulation.

Committee: Colleen Novak PhD (Advisor); Paul Sampson PhD (Committee Member); Eric Mintz PhD (Committee Member); Roger Gregory PhD (Committee Member) Subjects: Biology