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Discrete changes in the amplitude, frequency, and cellular localisation of calcium ion (Ca2+) transients encode information about numerous stimuli and function to mediate stimulus-specific responses. Cytoplasmic Ca2+ (Ca2+cyt) undergoes circadian oscillations in concentration that appear to be under the control of the same endogenous oscillator that regulates expression of genes in the photoperiodic-flowering pathway. It is currently not known whether these circadian [Ca2+cyt] oscillations are biochemical artefacts or are decoded and function to transduce clock dependent responses. Calmodulin (CaM) is a primary node in Ca2+ signalling in plants and as such is a promising target for investigating the role of Ca2+ in clock-controlled processes. In this study, Arabidopsis thaliana were treated with experimentally validated concentrations of pharmacological CaM inhibitors. Under inductive photoperiods (16 h light : 8 h dark), CaM inhibition was found to increase developmental flowering time, whilst under non-inductive photoperiods no such changes were evident. Inhibition of CaM led to changes in expression of the key clock gene TIMING OF CAB EXPRESSION 1 and flowering time genes, CONSTANS and FLOWERING LOCUS T and removed repression of flowering in darkness. These observations are consistent with CaM modulating the activity of the putative clock component GIGANTEA and the proteasomal targeting protein SUPPRESSOR OF PHYA-105. Due to the unwanted side effects often generated by chemical CaM inhibitors, a peptide inhibitor of CaM comprising a green fluorescent protein / calspermin fusion and labelled smGN was developed. Surface plasmon resonance analysis and affinity chromatography showed smGN to have extremely high selectivity for, and affinity to, CaM and to function as a powerful inhibitor of CaM in vitro. Further work on the methodology used to deploy smGN as a recombinant alternative to chemical CaM inhibitors in planta is also described.

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 circadian clock is an endogenous timekeeper that synchronises biological processes with daily external rhythms such as light and temperature cycles. It provides organisms with a competitive advantage by allowing anticipation of daily events. The circadian clock encompasses a network of transcription-translational feedback loops (TTFLs) that orchestrates rhythmic expression of a large part of the genome. This network is regulated at post-transcriptional and post-translational level. Post-translational regulation of clock proteins is essential to ensure stable rhythms and robust timekeeping. Unlike the genes in the TTFL network, modifiers of clock proteins at post-translational level are conserved across taxa. SUMO, a small ubiquitin-related post-translational modifier, regulates timekeeping in mammals through modification of the clock transcription factor BMAL. In this study, SUMO is shown to contribute to oscillator function in Arabidopsis plants. Methods have been developed to prove that mutant lines defective in SUMO machinery, including SUMO-ligase and -protease mutants, display long circadian rhythms. Additionally, sumoylation on the crucial plant clock transcription factor CCA1 is observed in vivo. A fraction of the protein is sumoylated across the expression window of CCA1, with the phase of peak sumoylation in advance of peak total CCA1. The effect of sumoylation of CCA1 was investigated with respect to localisation, stability and DNA binding affinity of the protein, as these are previously described possible effects of sumoylation. The subcellular location of CCA1-YFP fusions in protoplasts was not altered in mutant lines of the SUMO machinery. In vitro experiments show that sumoylation negatively affects the affinity of CCA1 to its cognate promotor element, suggesting that SUMO could act as a reversible attenuator of CCA1 activity. Furthermore, effects of SUMO machinery mutations appear to be differential across a range of physiologically relevant temperatures, implying that sumoylation could be involved in the response to or buffering against fluctuating ambient temperatures. There is an increasing amount of evidence to suggest that metabolic oscillations are not only driven by transcriptional outputs of the clock, but are to some extent self-sustained and can feed timing information back into the clock. Glutathione was investigated as a possible metabolic feedback signal. Expression of clock gene CCA1 was found to be abolished in a mutant of the rate-limiting enzyme for glutathione synthesis (pad2-1). Surprisingly however, the amount of glutathione was not found to oscillate. Combined, the results discussed in this thesis provide a substantial advance on our understanding of post-translational regulation and the integration of metabolic and environmental information into the plant circadian clock.

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