No description available.

No description available.

The circadian clock drives ~24hr rhythms in a variety of processes, from gene expression through to behaviour, facilitating anticipation of daily changes in the external environment and temporal separation of internal processes. This pacemaker is a critical regulator of immune function and many inflammatory diseases show time-of-day variation in symptom severity. Disruption of the pacemaker by manipulation of the daily cycle of light and dark exposure (experimental 'jet lag') is known to exacerbate inflammatory responses to innate immune challenge, and recent evidence has highlighted immuno-modulatory roles for components of the molecular oscillator in peripheral tissues. The adrenal-derived glucocorticoid hormones are potent anti-inflammatory molecules and are capable of modulating circadian oscillations in peripheral tissues. This, along with their rhythmic secretion profile, makes them key candidates as mediators of circadian regulation of inflammatory signalling. Utilising adrenalectomy, timed glucocorticoid administration, hormone clamp and genetic targeting of the glucocorticoid receptor in mice, I present evidence for an interaction between glucocorticoid signalling and the circadian pacemaker in regulating the pulmonary inflammatory response to lipopolysaccharide (LPS) challenge. The neutrophilic response to aerosolised LPS exhibits a clear time-of-day effect in vivo, which is lost after disruption of endogenous glucocorticoid production via adrenalectomy. However, replacement of a rhythmic circulating glucocorticoid concentration with a flat daily average using a subcutaneous hormone clamp does not disrupt the inflammatory rhythm. Finally, a novel mouse strain was produced with disrupted expression of the glucocorticoid receptor (GR) in bronchial epithelial cells (Ccsp-GR-/-). These cells are critical regulators of circadian rhythmicity in the lung and drive rhythmic neutrophil influx in response to LPS stimulation through production of the chemokine CXCL5. Loss of GR in the bronchial epithelium was associated with a loss of rhythmic neutrophil influx after challenge, but anti-inflammatory sensitivity to the synthetic glucocorticoid dexamethasone remained. Collectively, these data show that appropriate temporal modulation of pulmonary inflammation requires functional glucocorticoid signalling, although the ligand itself does not need to oscillate. The retention of anti-inflammatory dexamethasone sensitivity suggests a role for cross-talk between the bronchial epithelium and additional cell populations, consistent with recent evidence for immuno-suppressive macrophage-epithelium communication in the lung. These are the first studies to dissect the mechanistic links between clocks, glucocorticoids and immunological responses in a target tissue.

Early light environment has been shown to alter locomotor activity behaviour in adult rats and mice when exposed to constant light (LL), constant darkness (DD) or 12:12 hour light-dark cycles (LD). In particular, exposure to LL during lactation results in an increased ability to cope with exposure to LL as adults, implying that the ability to interpret light information is altered depending on early light environment. Therefore, the aim of this project was to explore how early light environment affects the photic response of the circadian system. The retina forms the first component of the photic response of the circadian system. We wanted to know whether being raised in DD, LD or LL would alter retinal function or structure in adult CD1 (albino) and C57BL/6J (pigmented) mice. We found that in CD1 mice, being raised in LL caused significant retinal damage and a significant reduction in retinal function. In C57BL/6J mice, we saw no such changes, implying that any changes that we see in behaviour would be due to alterations further downstream, such as the suprachiasmatic nucleus (SCN), site of the master circadian clock in the mammalian brain. We next exposed C57BL/6J mice to LL and found that mice raised in DD had significantly longer taus than mice raised in LL, implying that mice raised in DD are more sensitive to light than mice raised in LL.Previous work has shown that early light environment alters neuropeptide and astrocyte expression in the SCN of C57BL/6J mice. Furthermore, early light environment produces opposite behavioural responses in CD1 and C57BL/6J mice when exposed to LD as adults. We therefore examined how neuropeptide and astrocyte expression would be affected by early light environment in CD1 mice and whether this would reflect the differential behavioural response. We found that neuropeptide and astrocyte expression in the SCN seemed to be affected by the level of retinal damage and/or the type and intensity of the light source used. This sensitivity to lighting environment makes CD1 mice unsuitable for further studies on the photic response of the circadian system. Pigmented mice were used for the remainder of this project. The photic response of the circadian system can be quantified using a phase response curve (PRC) which measures behavioural responses to light pulses administered at different times of day. We measured the effect of a light pulse on the delay and advance portion of the PRC and found no differences due to early light environment, implying that the phasic effect of light is not altered by early light environment. Light pulses administered during the subjective night result in the upregulation of SCN intracellular photic signalling pathways. After a light pulse given during the early subjective night, we found no differences in the upregulation of different components of the photic signalling pathway due to early light environment indicating early light environment does not seem to affect the initial photic signalling pathway in the SCN.Finally, recent advances in molecular biology allow for real-time monitoring of clock gene expression in vitro. Using mPer2::luc mice, we monitored in vitro PER2::LUC expression to determine the effects of early light environment on clock gene expression. In the SCN, we found that the amplitude of PER2::LUC expression was significantly reduced in mice raised in DD compared to mice raised in LD and LL. These results suggest that early light environment affects the coupling strength between SCN neurons and this may be the mechanism mediating the changes in behaviour we have measured. In peripheral tissue, we found altered PER2::LUC expression due to early light environment in the heart, lung and spleen, implying that early light environment not only alters behaviour but may also affect heart and lung function and the immune system.

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Melatonin is rhythmically synthesized and released by the pineal gland and, in some species, retina during the night and regulates many physiological and behavioral processes in birds and mammals. Chick diencephalic astrocytes express two melatonin receptor subtypes in vitro, and melatonin plays a role in regulating metabolic activity. We examined the role of glial cells in circadian function and asked if melatonin modulated glial functions within the retina and the brain. Calcium waves were potentiated by physiological concentrations of melatonin. Melatonin increased resting calcium levels and reduced gap junctional coupling among astrocytes at these same concentrations. Both mouse and chick diencephalic and telencephalic astrocytes express melatonin receptor protein. Nanomolar melatonin modulated astrocytic calcium waves of the mouse and chick diencephalon but not waves of the telencephalon. Mammalian intercellular calcium waves spread farther than avian calcium waves, and the nature of the spread of the waves differed between telencephalic and diencephalic mammalian astrocytes. These differences in propagation were abolished by melatonin. Using northern analysis, we identified period2, period3, cryptochrome1, cryptochrome2, clock, melanopsin and peropsin within chick diencephalic astrocytes. The clock genes cry1 and, per2 were expressed rhythmically in a LD cycle, but metabolic activity was not rhythmic. When cells were placed in constant darkness and rhythmically administrated melatonin, a robust rhythm in glucose uptake was induced without a coordinated clock gene rhythm, suggesting rhythmic clock gene expression and metabolic activity are separable processes. Melatonin affected visual function as assessed by electroretinogram. Circadian rhythms of a- and b-wave implicit times and amplitudes were observed. Melatonin (1 mg/kg and 100 ng/kg) decreased a- and b-wave amplitudes greater during the night than during the day and it increased a- and b-wave implicit times while 1 ng/kg melatonin had little to no effect over the saline controls. These data indicate that melatonin modulates glial intercellular communication, affects metabolic activity in astrocytes, and may play a role in regulating a day and night functional shift in the retina, at least partially through Müller glial cells. Thus, melatonin can regulate glia function and thereby, affect outputs of the vertebrate biological clock.

Circadian rhythms in biological processes occur in a wide range of organisms and are generated by endogenous oscillators. In Neurospora crassa, the FRQ-oscillator (comprised of FRQ, WC-1 and WC-2) is essential for rhythms in asexual sporulation and gene expression. How this oscillator signals to the cell to control rhythmicity is unknown. Furthermore, under certain growth conditions, rhythms are observed in FRQ-null strains, indicating the presence of one or more FRQ-less oscillators (FLOs). Interestingly, while circadian rhythms are observed in the related Aspergillus spp., they lack the frq gene, leading to the hypothesis that a FLO is responsible for rhythms in Aspergillus. Thus, Aspergillus provides a useful organism to investigate the components of the FLO. To investigate how an oscillator controls circadian output, we characterized the role of N. crassa NRC-2. The nrc-2 gene is under control of the clock and encodes a putative serine-threonine protein kinase. In a NRC-2-null strain cultured in low glucose conditions, FRQ-oscillator-dependent outputs are arrhythmic, but are rhythmic in high glucose. Our data suggests a model whereby NRC-2 relays metabolic information to the FRQ-oscillator to control rhythmic output. To understand the role of FLO(s) in the N. crassa circadian system, we examined regulation of the ccg-16 gene. We show that ccg-16 transcript rhythmicity is FRQ-independent, but WC-1-dependent. Furthermore, in contrast to current models for the FRQ-oscillator, we observed that rhythms in WC-1 protein accumulation persist in the absence of FRQ. These data support a new model involving two oscillators that are coupled through the WC-1 protein and that regulate different outputs. One approach to identify components of the FLO involved characterizing circadian rhythms in Aspergillus spp, which lacks FRQ. We find that A. flavus and A. nidulans, display circadian rhythms in sporulation and gene expression, respectively. Together, these findings provide a foundation for the identification of FLO components in both Aspergillus and N. crassa, that will ultimately lead to an understanding of how a multi-oscillator system can generate and coordinate circadian rhythmicity.

The circadian oscillator of the cyanobacterium Synechococcus elongatus is composed of only three proteins, KaiA, KaiB, and KaiC, which together with ATP can generate a self-sustained ~24 hour oscillation of KaiC phosphorylation for several days. KaiA induces KaiC to autophosphorylate whereas KaiB blocks the stimulation of KaiC by KaiA, which allows KaiC to autodephosphorylate. We propose and support a model in which the C-terminal loops of KaiC, the “A-loops”, are the master switch that determines overall KaiC activity. When the A-loops are in their buried state, KaiC is an autophosphatase. When the A-loops are exposed, however, KaiC is an autokinase. The data suggest that KaiA stabilizes the exposed state of the A-loops through direct binding. We also show evidence that if KaiA cannot stabilize the exposed state KaiC remains hypophosphorylated. We propose that KaiB inactivates KaiA by preventing it from stabilizing the exposed state of the A-loops. Thus, KaiA and KaiB likely act by shifting the dynamic equilibrium of the A-loops between exposed and buried states, which shifts the balance of autokinase and autophosphatase activities of KaiC. A-loop exposure likely moves the ATP closer to the sites of phosphorylation and we show evidence in support of how this movement may be accomplished. Density functional theory calculations of isolated Watson–Crick A:U and A:T base pairs predict that adenine 13C2 trans-hydrogen bond deuterium isotope shifts due to isotopic substitution at the pyrimidine H3, 2hΔ13C2, are sensitive to the hydrogen-bond distance between the N1 of adenine and the N3 of uracil or thymine, which supports the notion that 2hΔ13C2 is sensitive to hydrogen-bond strength. Calculated 2hΔ13C2 values at a given N1–N3 distance are the same for isolated A:U and A:T base pairs. Replacing uridine residues in RNA with 5-methyl uridine and substituting deoxythymidines in DNA with deoxyuridines do not statistically shift empirical 2hΔ13C2 values. Thus, we show experimentally and computationally that the C7 methyl group of thymine has no measurable affect on 2hΔ13C2 values. Furthermore, 2hΔ13C2 values of modified and unmodified RNA are more negative than those of modified and unmodified DNA, which supports our hypothesis that RNA hydrogen bonds are stronger than those of DNA. It is also shown here that 2hΔ13C2 is context dependent and that this dependence is similar for RNA and DNA.

Circadian clocks are present in most eukaryotes and some prokaryotes and control rhythms in behavior, physiology and gene expression. One well-characterized circadian clock is that of Neurospora crassa. In addition to the well-described N. crassa FRQ/WCC oscillator, several lines of evidence have implied the presence of other oscillators which may have important functions in the N. crassa circadian clock system. However, the molecular details are only known for the core FRQ/WCC oscillator. The light mutant oscillator (LMO) was identified by two mutations (LM-1 and LM-2) and shown to control developmental rhythms in constant light (LL), conditions in which the FRQ/WCC oscillator is not functional. The objective of this project was to determine whether the developmental rhythms driven by the LMO are circadian, whether the components of the LMO communicate with components of the FRQ/WCC oscillator, and to begin to define the molecular nature of the LMO. First, the conditions for growth of the LM-1 mutant strain that reveals the best circadian rhythm of development in LL were found. Second, the LMO was determined to display the three properties required of a circadian oscillator. Third, the LMO was shown to function independently of the FRQ/WCC oscillator to control developmental rhythms in LL. However, evidence suggests that the FRQ/WCC oscillator and the LMO communicate with each other. Finally, using Cleaved Amplified Polymorphic Sequence (CAPS) markers, the LM-1 mutation was genetically mapped to the right arm of linkage group I within a 1069 kb region. Together, these results provide a start towards understanding of the complexity of oscillators that form a circadian clock in organisms.

Diverse forms of life have evolved 24-hour or circadian timekeeping systems serving to coordinate internal biological events with the daily solar cycle. The generation of circadian rhythms by this timekeeping system ensures that internal processes occur at the appropriate time of day or night in relation to the environmental cycle and to other functionally-affiliated events. For mammals, endogenous oscillations in gene expression are a prevalent feature of oscillatory cells residing in the suprachiasmatic nucleus (SCN) and non-SCN tissues. To determine whether immortalized cells derived from the rat SCN (SCN2.2) retain the intrinsic rhythm-generating properties of the SCN, oscillatory behavior of the SCN2.2 transcriptome was analyzed and compared to that found in the rat SCN in vivo. In SCN2.2 cells, 116 unique genes and 46 ESTs or genes of unknown function exhibited circadian fluctuations for 2 cycles. Many (35%) of these rhythmicallyregulated genes in SCN2.2 cells also exhibited circadian profiles of mRNA expression in the rat SCN in vivo. To screen for output signals that may distinguish oscillatory cells in the mammalian SCN from peripheral-type oscillators, the rhythmic behavior of the transcriptome in forskolin-stimulated NIH/3T3 fibroblasts was analyzed and compared relative to SCN2.2 cells in vitro and the rat SCN in vivo. Similar to the circadian profiling of the SCN2.2 and rat SCN transcriptomes, NIH/3T3 fibroblasts exhibited rhythmic fluctuations in the expression of the core clock genes and 323 (2.6%) functionally diverse transcripts. Overlap in rhythmically expressed transcripts among these different oscillator models was limited to the clock genes and four genes that function in metabolism or transcription. Coupled with evidence for the rhythmic regulation of the inducible isoform of nitric oxide synthase (Nos) in SCN2.2 cells and the rat SCN but not in fibroblasts, studies examining the effects of antisense oligonucleotide-mediated inhibition of Nos2 suggest that the gaseous neurotransmitter nitric oxide may play a key role in SCN pacemaker function. Thus, our comparative analysis of circadian gene expression in SCN and non-SCN cells has important implications in the selective analysis of circadian signals involved in the coupling of SCN oscillators and regulation of rhythmicity in downstream cells.