Thesis (Ph. D.)--Virginia Commonwealth University, 2010. / Prepared for: Dept. of Human Genetics. Title from title-page of electronic thesis. Includes bibliographical references . Record unavailable until 5/13/2015.

Circadian rhythms have been implicated in severity and outcome of infection and disease. Commonly, LPS and bacterial infection have been used to identify the mechanisms behind the difference in immune responses depending on the time of day of the challenge. In this thesis, the colon dwelling nematode parasite Trichuris muris, which elicits a Th2 immune response in resistant mice, was used to identify if circadian rhythms influence infection outcome 3 weeks post infection. C57BL/6 mice infected with 200 eggs of T. muris at ZT0 (7am, lights on) expelled the parasite more efficiently than mice infected at ZT12 (7pm, lights off), which expelled with a delay of several days compared to ZT0 infected mice. Analysis of cell infiltration into the colon during the first days of infection suggested that there was no visible difference in the local immune response. There also were no differences in macrophage and dendritic cell numbers in colon tissue of naïve mice at ZT0 or ZT12. Further experiments examined immunomodulation of the immune response to T. muris by pushing the immune response towards a Th1, by low dose infection, or a Th2 response, by vaccination with excretory/secretory antigen. In both cases any circadian influence was overwritten. Mice infected at ZT0 or ZT12 with only 40 eggs of T. muris were equally susceptible to infection and mice infected at ZT3 10 days after vaccination at ZT0 or ZT12 were equally resistant to infection. Mice food restricted to mid-light phase and infected at ZT0 were not significantly delayed in their worm expulsion or polarised more towards a Th1 immune response compared to ZT0 infected mice fed during the dark phase. Therefore it is unlikely that feeding behaviour during the first days of infection is able to polarise towards a Th1 response and lead to delayed worm expulsion. Transgenic mice were used to dissect the mechanism underlying the delay in worm expulsion in ZT12 infected mice. mPer2::luc mice were used to confirm rhythmic Per2 expression in colon tissue and dendritic cells. Infection of mPer2::luc mice at ZT0 or ZT12 with T. muris showed similar worm expulsion, antibody and cytokine production when infected at ZT0 or ZT12. Bmal1floxLysMcre mice, which lack rhythmic clock gene expression in macrophages and granulocytes, produced a stronger Th2 antibody response in a primary infection at ZT3 than wild-type littermate controls. Newly generated mPer2::lucBmal1floxCD11ccre mice showed the no difference in worm burden and parasite specific antibody production between ZT0 and ZT12 infected mice. Only IL-10 and IL-6 levels were significantly lower in ZT12 infected mPer2::LucBmal1floxCD11ccre mice compared to ZT12 infected wild-type littermates. Confirmation of removal of exon 8 of the Bmal1 gene was not achieved; therefore it is not clear if circadian rhythm in dendritic cells has any impact on the immune response to T. muris or if the mPer2::LucBmal1floxCD11ccre mice and littermate controls both contain circadian rhythm in dendritic cells and therefore cannot be used to identify the role of the dendritic cell clock in the time of day differences in infection outcome. This thesis shows that time of day of the infection impacts on the outcome of infection with Trichuris muris.

No description available.

Master of Science / Department of Architectural Engineering / Fred Hasler and Russ Murdock / The correlation between natural and artificial lighting and the human circadian rhythm was researched to determine how changing artificial lighting design could improve the working environment in healthcare facilities. Research showed that human circadian rhythm is largely influenced by daylight and the accompanying light color (CCT) change in a day. Consequently, healthcare providers who are not exposed to daylight are isolated from this natural indicator. This can disjoint their circadian cycles from a normal rhythm and lead to physiological and psychological complications. Daylighting and standard artificial lighting design conditions were observed at Mercy Regional Medical Center (MRMC) in Manhattan, Kansas,. Then, healthcare providers at MRMC were anonymously surveyed about their perceived alertness throughout a typical working shift . The data was charted and plotted against a normal circadian rhythm to demonstrate whether a normal or disjointed cycle was experienced by healthcare providers. The comparison of this data to observed lighting conditions exhibited the necessary influence of daylight on achieving a normal circadian rhythm. This study reinforced the information available from many other sources connecting healthcare lighting and the circadian rhythm. Working on this premise, research suggested two lighting designs that would improve the working environment in healthcare facilities. The first of these would be the inclusion of circadian rooms. Special rooms in a healthcare facility would be available to staff in order to provide light therapy. Short wavelength blue light, experienced at optimal times throughout a shift, would act as stimulants (similar to daylight cues), adjusting employees' circadian cycles to normal when daylight exposure was unattainable. Alternately, a healthcare facility working on set, not-staggered, shifts could incorporate a variable lighting system. This system would rely on specialized lights to alter light color (CCT) throughout a shift to mimic daylight rhythms. Accordingly, staff would be exposed to daylighting cues from artificial sources and experience normal circadian rhythms.

Contemporary understanding of the brain indicates that a reciprocal relationship exists between mind and body. Biological functioning adjusts to the consequences of our behavior and our behavior is influenced by our biology. This is the case with the stress responsivity system. The stress hormone cortisol follows a biologically-predetermined daily cycle of secretion (controlled by circadian rhythm) that correlates with expected activity throughout the day, however this cycle can accommodate to different environmental changes that can occur. It has been noticed that individuals who report stress problems also report sleep problems. I hypothesized that sleep quality can predict maladjustments in cortisol’s rhythm. All participants provided saliva samples and had to take the Pittsburgh Sleep Quality Index (PSQI). Salivivay cortisol was measured via was enzyme-immuno-assayed for cortisol. I analyzed the data for three independent studies: (1)12 samples were taken for basal and lab days in 65 individuals. People who scored worse in total PSQI showed decreased stress reactivity (γ 21=-.02, t(63)=-2.27, p=0.026) and faster recovery (γ31=-0.102, t(608)=-2.044, p=0.041). (2)6-8 samples per day across 5 days in 120 maltreated or control adolescents. I used a 3-level hierarchical linear model to examine rhythms within each day and within each individual. The cortisol rhythm was flattened on days when adolescents had poor sleep latency (β;=.013, p=.025 for time-since-waking, β=-.0008, p=.039 for quadratic time-since-waking). (3) 10 samples were taken in 44 skydivers for jumping and basal days. Those who scored worse in sleep latency had slower reactivity (γ31=-0.16, t(284)=-3.701, p<.001) and slower recovery (γ31=0.22, t(284)=3.311, p<0.001). Stress and sleep problems are related to cognitive and physiological issues; finding an appropriate connection between them can be elemental in preventing problems.

Reabsorbing ~7% of the sodium load, the distal convoluted tubule plays key roles in blood pressure (BP) homeostasis. Here, Na-Cl co-transport (NCC) is the major route for apical Na entry making thiazide diuretics (NCC inhibitors) a mainstay hypertension treatment. Predictive adaptations of sodium excretory rhythms are supported by an intrinsic renal clock, which regulates transporter activity according to physiological need. Peripheral clocks can be influenced by glucocorticoids, which also have a circadian rhythm. We therefore hypothesized that NCC’s diurnal rhythm is regulated by glucocorticoids. C57BL6 mice were kept on a 12h light cycle with subjective day starting at 7am. Urine was collected in 12h periods and kidneys harvested at 1am (night) and 1pm (day). Slc12a3 (NCC encoding gene) mRNA and NCC protein abundance were similar between day and night but NCC phosphorylation at threonine 53 was significantly higher at night compared to day. Plasma and urinary corticosterone levels were elevated at night. Glucocorticoid inducible leucine zipper (GILZ) and serum and glucocorticoid inducible kinase (SGK1) transcripts also increased at night. Chronic corticosterone infusion flattened the plasma corticosterone rhythm within an intermediate physiological range. The diurnal rhythm of pT53 NCC was dampened in these mice but not in vehicle-treated mice. Blood pressure was monitored in the mice by radiotelemetry. After 2 weeks of baseline measurements mice received chronic corticosterone or vehicle for 3weeks, during the last 10 days of which they received ~80 mg/kg hydrochlorothiazide in their drinking water. At night systolic BP (SBP) was unaffected by any treatment whereas during the day SBP significantly increased following corticosterone but was unaffected by vehicle. Cosinor analysis of SBP in corticosterone and vehicle treated mice showed a marked reduction in rhythmicity, increased MESOR and reduced amplitude. In animals receiving corticosterone HCTZ partially rescued daytime SBP. This manoeuvre also improved SBP rhythmicity, reduced MESOR and increased amplitude. These data indicate that NCC phosphorylation has a diurnal rhythm that is in part regulated by glucocorticoids. They also show that alteration of the glucocorticoid rhythm affects the blood pressure rhythm in part through its effect on NCC phosphorylation. These findings may be clinically relevant in the pathogenesis of hypertension in conditions associated with elevated glucocorticoid levels such as metabolic syndrome and chronic stress.

The fruit fly Drosophila exhibits robust daily behavioral rhythms, which are driven by a network of circadian pacemaker neurons in the fly brain. The Narrow Abdomen (NA) sodium leak channel functions rhythmically in pacemaker neurons, downstream of the molecular circadian clock, to depolarize resting membrane potential and promote neuronal excitability. Loss of NA function (NA-LOF) strongly disrupts behavioral rhythms, and these behavioral phenotypes are consistent with decreased circadian neuronal activity. Yet despite some recent advances, the mechanisms of NA channel function and regulation in the circadian system are still not well understood. To further elucidate the role of the NA channel in the circadian neuronal network, we generated mutated versions of the NA transgene and assessed the effects of transgene expression in Drosophila circadian pacemaker neurons. Expression of a putative gain of function na transgene (na-GOF) in pacemaker neurons generates unique behavioral phenotypes, suggesting novel effects on neuronal excitability or/and the molecular circadian clock. Next, we investigated how NA-LOF and NA-GOF mutations affect circadian neuron activity through optical recording of fluorescent voltage and calcium sensors expressed in these neurons. Using the fluorescent voltage sensor ArcLight, we find that both NA-LOF and NA-GOF manipulations suppress spontaneous membrane activity in clock neurons in the Drosophila brain. This finding was surprising because the behavioral effects of NA-LOF and NA-GOF are quite distinct. However, the information provided from these spontaneous assays may be a combination of neuronal input and output, and in some cases information is combined from multiple cells. To further characterize the neurophysiological effects of NA channel manipulation, we next paired optical recording with pharmacology in brain explants. Here we find that both wild-type and NA-LOF DN1p clock neurons are strongly depolarized by the acetylcholine receptor agonist nicotine, while NA-GOF neurons show little response. This suggests that NA-GOF expression already depolarizes the membrane potential of these neurons. We also assessed intracellular calcium levels in the DN1p clock cells after applying the inhibitory neurotransmitter glutamate at either morning (peak) or evening (trough) timepoints. We find that wild-type DN1p neurons show a strong decrease in calcium at the peak timepoint and a much smaller decrease at the trough. In contrast, NA-GOF DN1p neurons show decreases at both timepoints, indicating that they have elevated calcium levels (and elevated activity) at the trough time. Through immunostaining, we find that NA-GOF expression alters the core clock protein PERIOD levels in sLNv and LNd neurons during early day. Taken together, this study shows that overexpression of NA-GOF ion channel in Drosophila pacemaker neurons induce unique behavioral phenotypes, likely by depolarizing membrane potential and increasing neuronal activity. We propose that these changes in neuronal activity may feedback to alter the oscillation of molecular clocks. While these transgenic studies have been informative, we have also established gene-editing methods in order to distinguish the effects of gene mutation from effects of overexpression. We have used the CRISPR-Cas9 system to target the endogenous na locus. In the initial step, we replaced na exons 1-13 with a fluorescent marker flanked by attP integration sites. Through subsequent integrase-mediated recombination, we hope to generate a series of na mutations of interest, including gain-of-function mutations, for future studies.

Chemoreception is a primitive sense universally employed by organisms for finding and selecting food, rejecting toxic chemicals, detecting mates and offspring, choosing sites for egg-laying, recognizing territories and avoiding predators. Chemosensory responses are frequently modulated based on the internal environment of the organism. An organism’s internal environment undergoes regular changes in anticipation and in response to daily changes in its external environment, e.g., light-dark cycle. A resettable timekeeping mechanism called the circadian clock internally drives these cyclical changes with a ~24 hour period. Using electrophysiological, behavioral and molecular analyses, I tested where and how these two conserved processes, viz., the circadian timekeeping mechanism and the chemosensory pathway, intersect each other at organismal and cellular levels. The presence of autonomous peripheral oscillators in the chemosensory organs of Drosophila, prompted us to test whether chemosensory responses are under control of the circadian clock. I found that local oscillators in afferent (primary) chemosensory neurons drive rhythms in physiological and behavioral responses to attractive and aversive chemical signals. During the middle of the night, high level of G proteincoupled receptor kinase 2 (GPRK2), a clock controlled signaling molecule present in chemosensory neurons, suppresses tastant-evoked responses and promotes olfactory responses. G-protein mediated signaling was shown to be involved in generating optimal response to odorants. Multifunctional chemosensory clocks exert control on feeding and metabolism. I propose that temporal plasticity in innate behaviors should offer adaptive advantages to flies.

No description available.

MAGEL2 is one of the five genes inactivated in Prader-Willi Syndrome (PWS), a genetic disorder, manifesting with symptoms of developmental delay and morbid obesity. Magel2 is highly expressed in the suprachiasmatic nucleus, which is the location of the central clock or circadian pacemaker. Magel2 knockout mice exhibit defects in circadian rhythm. I hypothesized that Magel2 plays a role in one of the inter-connecting feedback loops that control circadian rhythm in suprachiasmatic neurons. I determined that Magel2 acts as a repressive protein in the cycles feedback loop using a luciferase assay. Magel2 exerts this effect by restricting the movement of Bmal1 into the nucleus. Magel2 levels are then reduced by increasing Per2, associated with increase movement into the nucleus, as determined by experiments examining subcellular localization and effects on protein levels. Loss of Magel2 in PWS may contribute to sleep abnormalities in this disorder, specifically the cycling between different sleep stages.