Circadian Oscillatory System Research Articles

Pharmacokinetic Model-Based Control across the Blood-Brain Barrier for Circadian Entrainment.

The ability to shift circadian phase in vivo has the potential to offer substantial health benefits. However, the blood-brain barrier prevents the absorption of the majority of large and many small molecules, posing a challenge to neurological pharmaceutical development. Motivated by the presence of the circadian molecule KL001, which is capable of causing phase shifts in a circadian oscillator, we investigated the pharmacokinetics of different neurological pharmaceuticals on the dynamics of circadian phase. Specifically, we developed and validated five different transport models that describe drug concentration profiles of a circadian pharmaceutical at the brain level under oral administration and designed a nonlinear model predictive control (MPC)-based framework for phase resetting. Performance of the novel control algorithm based on the identified pharmacokinetic models was demonstrated through simulations of real-world misalignment scenarios due to jet lag. The time to achieve a complete phase reset for 11-h phase delay ranged between 48 and 72 h, while a 5-h phase advance was compensated in 30 to 60 h. This approach provides mechanistic insight into the underlying structure of the circadian oscillatory system and thus leads to a better understanding of the feasibility of therapeutic manipulations of the system.

Circadian clock proteins drive structural oscillations in colloidal fluids.

Cyanobacteria rely on a circadian oscillator system of proteins, KaiA, KaiB and KaiC, to regulate a variety of cellular processes. Powered by ATP phosphorylation, KaiA and KaiB proteins alternately bind to KaiC, resulting in the cyclical assembly and disassembly of KaiC-KaiB complexes. By biotinylating KaiB monomers, we repurpose the KaiC-KaiB complexes to function as crosslinkers with unique time-dependent properties. To demonstrate the efficacy of these ‘circadian crosslinkers’, we design suspensions of streptavidin-coated colloids that exhibit time-dependent structural changes driven by the oscillatory assembly and disassembly of KaiB-KaiC complexes. Further, using fluorescence microscopy, image analysis and biochemical assays, we show that the rate, efficacy and oscillatory signature of crosslinking can be tuned by altering the phosphorylation state of KaiC. Our findings demonstrate that a model circadian oscillator can drive time-varying restructuring of abiotic colloidal suspensions, providing new biochemical machinery for developing self-directed, programmable, and reconfigurable biomaterials. This work may one day enable engineers to incorporate biochemical circadian clocks into future exosuits, infrastructure, and deep sea and space exploration equipment.

Prevalence of Insomnia among Emergency Doctors and their Physiological Link with Serum Melatonin: Key to Better Sleep among Doctors of LUMHS

Background: As a consequence of the epidemic, health care workers have been subjected to unusual levels of stress, anxiety, and sleep issues. Chronic insomnia is usually linked to circadian rhythm abnormalities. Insomnia is a frequent indication of a lack of serum melatonin. Objective: To detect the prevalence of Insomnia among doctors at LUMHS and their physiological role with regard to serum melatonin levels. Methodology: This cross-sectional study was conducted involving 116 doctors, out of which male 48(41.3%) and female 68 (58.6%), working in all clinical wards of Liaquat University of Medical & Health Sciences, Jamshoro / Hyderabad from August 2021 to February 2022. Each patient's demographic information was collected. Serum Melatonin levels in the blood were measured using an ELISA KIT. Along with (Insomnia severity index) questioner was filled out by each participant. SPSS version 26 was used to analyze the collected data. Results: This study found that physicians' sleep and wake cycles are disrupted owing to their long, stressful working hours, out of 116 participants, 42.68 percent experienced insomnia. Regardless of age, gender, or BMI, Insomnia was shown to be co- related with a lower blood melatonin level (r value=-.585**, p=0.001). Conclusion: The findings of this investigation are in line with our hypothesis that reduced serum Melatonin levels in insomniacs lead to delayed signal transduction, causing circadian oscillator system anomalies. We have found that insomnia among doctors shows significant variations in relation to department, sex, designation, and working hours. Further addressing it requires molecular studies. Keywords: Insomnia, Prevalence, Serum Melatonin, sleep disturbance

Multiple entrained oscillator model of food anticipatory circadian rhythms

For many animal species, knowing when to look for food may be as important as knowing where to look. Rats and other species use a feeding-responsive circadian timing mechanism to anticipate, behaviorally and physiologically, a predictable daily feeding opportunity. How this mechanism for anticipating a daily meal accommodates more than one predictable mealtime is unclear. Rats were trained to press a lever for food, and then limited to one or more daily meals at fixed or systematically varying times of day. The rats were able to anticipate up to 4 of 4 daily meals at fixed times of day and two ‘daily’ meals recurring at 24 h and 26 h intervals. When deprived of food, in constant dark, lever pressing recurred for multiple cycles at expected mealtimes, consistent with the periodicity of the prior feeding schedule. Anticipation did not require the suprachiasmatic nucleus circadian pacemaker. The anticipation rhythms could be simulated using a Kuramoto model in which clusters of coupled oscillators entrain to specific mealtimes based on initial phase and intrinsic circadian periodicity. A flexibly coupled system of food-entrainable circadian oscillators endows rats with adaptive plasticity in daily programming of foraging activity.

REFLEСTIONS ON IMPLEMENTING ELECTIVE COURSE IN MOLECULAR PATHOLOGY INTO THE EDUCATIONAL PROGRAM FOR MASTER’S DEGREE STUDENTS

Currently, the training of Master’s degree students in the specialty 222 “Medicine” does not provide the wide coverage of the molecular mechanisms of cellular disorders that would allow the students to get deeper understanding of modern approaches to the pathogenetic therapy of many diseases including type II diabetes mellitus, metabolic syndrome, coronary heart disease, etc. The curriculum of the mandatory component “Pathophysiology” implies the coverage of the above mentioned diseases and pathological conditions, however, because of the voluminous material the students need to assimilate, the course can not offer deeper insight into the molecular mechanisms of diseases and conditions. This article highlights the theoretical grounds for implementing the elective course “Molecular Pathology” into the training program for Master’s degree students in Medicine. The total workload of the elective course “Molecular Pathology” should not exceed 120 hours (4.0 ECTS credits). The study of the course can be divided into 2 modules of 60 hours each (2.0 ECTS credits): Module 1 “Pathophysiology of the cell” and Module 2 “Molecular mechanisms of the development of systemic pathologies”. Module 1 of the course “Molecular Pathology” is designed to promote the development of the following competencies including the ability to determine pathological changes in the cell during typical pathological processes; to assess the functional state of the antioxidant system of the body; to develop pathogenetically substantiated correction of oxidative damage to the body. Module 2 is elaborated to provide applicants with an opportunity to acquire the following competencies: analysis of the effect of cytokines on the development of typical pathologies in various organs and systems; make up a customized plan for pathogenetic therapy of patients with impaired insulin signalling; assessment of the functional state of the circadian oscillator system of the body. The implementation of the elective course of molecular pathology into the training program for Master’s medical students ensures the better understanding of pathological body processes, contributes to the high-quality education as well as increases the competitiveness of future specialists.

Detection of Uncoupled Circadian Rhythms in Individual Cells of Lemna minor using a Dual-Color Bioluminescence Monitoring System.

The plant circadian oscillation system is based on the circadian clock of individual cells. Circadian behavior of cells has been observed by monitoring the circadian reporter activity, such as bioluminescence of AtCCA1::LUC+. To deeply analyze different circadian behaviors in individual cells, we developed the dual-color bioluminescence monitoring system that automatically measured the luminescence of two luciferase reporters simultaneously at a single-cell level. We selected a yellow-green-emitting firefly luciferase (LUC+) and a red-emitting luciferase (PtRLUC) that is a mutant form of Brazilian click beetle ELUC. We used AtCCA1::LUC+ and CaMV35S::PtRLUC. CaMV35S::LUC+ was previously reported as a circadian reporter with a low-amplitude rhythm. These bioluminescent reporters were introduced into the cells of a duckweed, Lemna minor, by particle bombardment. Time series of the bioluminescence of individual cells in a frond were obtained using a dual-color bioluminescence monitoring system with a green-pass- and red-pass filter. Luminescence intensities from the LUC+ and PtRLUC of each cell were calculated from the filtered luminescence intensities. We succeeded in reconstructing the bioluminescence behaviors of AtCCA1::LUC+ and CaMV35S::PtRLUC in the same cells. Under prolonged constant light conditions, AtCCA1::LUC+ showed a robust circadian rhythm in individual cells in an asynchronous state in the frond, as previously reported. By contrast, CaMV35S::PtRLUC stochastically showed circadian rhythms in a synchronous state. These results strongly suggested the uncoupling of cellular behavior between these circadian reporters. This dual-color bioluminescence monitoring system is a powerful tool to analyze various stochastic phenomena accompanying large cell-to-cell variation in gene expression.

Methodical approach to teaching students mechanisms of hemoblastosis development at the department of pathophysiology

This work is a fragment of the initiative research "The role of transcription factors, the circadian oscillator system and metabolic disorders in the formation and functioning of pathological systems" (№ 0119U103898).The article is devoted to the problem of methodical approach to teaching the topic "Hemoblastosis" at the Department of Pathophysiology. Module 2 "Pathophysiology of organs and systems" begins with the study of the submodule "Pathophysiology of the blood system" for all students majoring in 222 "Medicine", 228 "Pediatrics" and 221 "Dentistry". One of the difficult topics for students to understand is the topic of "Hemoblastosis".InUkraine, the system of higher medical education is undergoing active innovative changes. This process is characterized by the introduction of new technologies and techniques in the educational process. Innovative forms and methods of teaching are developed and implemented.The purpose of this work is to analyze the difficulties that students have when they studying the topic of "Hemoblastosis" and the development of methodological approaches to their elimination.According to the WHO: cancer is one of the leading causes of death in the world: in 2012, there were 8.2 million deaths from cancer. Hemoblastosis accounts for approximately 8% of all malignancies and all together they are among the 6 most common types of malignant tumors. Acute leukemias account for about 50-60% of all leukemias, with acute myeloblastic leukemia being slightly more common than acute lymphoblastic leukemia. Chronic leukemias account for about 40-50% of all leukemias, with chronic lymphocytic leukemia being slightly more common than chronic myelocytic leukemia. Hemoblastosis is a relatively rare disease; mortality from them is 1.7-3.1 per 100,000 population. Among therapeutic diseases, leukemia occurs in 1.5-2.6% of cases. Recently, the incidences of hemoblastosis, especially acute, have increased in all countries.

Noncoding RNAs: Bridging Regulation of Circadian Rhythms and Inflammation

The circadian oscillator system is known to affect various immune functions. These actions can be attributed to either direct effects of oscillator components, including those present in peripheral cellular clocks, or to indirect actions via strongl

The effects of desynchronosis on the gut microbiota composition and physiological parameters of rats

BackgroundAll living organisms experience physiological changes regulated by endogenous circadian rhythms. The main factor controlling the circadian clock is the duration of daylight. The aim of this research was to identify the impact of various lighting conditions on physiological parameters and gut microbiota composition in rats. 3 groups of outbred rats were subjected to normal light-dark cycles, darkness and constant lighting.ResultsAfter 1 and 3 months we studied urinary catecholamine levels in rats; indicators of lipid peroxidation and antioxidant activity in the blood; protein levels of BMAL1, CLOCK and THRA in the hypothalamus; composition and functional activity of the gut microbiota. Subjecting the rats to conditions promoting desynchronosis for 3 months caused disruptions in homeostasis.ConclusionsChanging the lighting conditions led to changes in almost all the physiological parameters that we studied. Catecholamines can be regarded as a synchronization super system of split-level circadian oscillators. We established a correlation between hypothalamic levels of Bmal1 and urinary catecholamine concentrations. The magnitude of changes in the GM taxonomic composition was different for LL/LD and DD/LD but the direction of these changes was similar. As for the predicted functional properties of the GM which characterize its metabolic activity, they didn’t change as dramatically as the taxonomic composition. All differences may be viewed as a compensatory reaction to new environmental conditions and the organism has adapted to those conditions.

Time zones of pancreatic islet metabolism.

Most living beings possess an intrinsic system of circadian oscillators, allowing anticipation of the Earth's rotation around its own axis. The mammalian circadian timing system orchestrates nearly all aspects of physiology and behaviour. Together with systemic signals originating from the central clock that resides in the hypothalamic suprachiasmatic nucleus, peripheral oscillators orchestrate tissue-specific fluctuations in gene transcription and translation, and posttranslational modifications, driving overt rhythms in physiology and behaviour. There is accumulating evidence of a reciprocal connection between the circadian oscillator and most aspects of physiology and metabolism, in particular as the circadian system plays a critical role in orchestrating body glucose homeostasis. Recent reports imply that circadian clocks operative in the endocrine pancreas regulate insulin secretion, and that islet clock perturbation in rodents leads to the development of overt type 2 diabetes. While whole islet clocks have been extensively studied during the last years, the heterogeneity of islet cell oscillators and the interplay between α- and β-cellular clocks for orchestrating glucagon and insulin secretion have only recently gained attention. Here, we review recent findings on the molecular makeup of the circadian clocks operative in pancreatic islet cells in rodents and in humans, and focus on the physiologically relevant synchronizers that are resetting these time-keepers. Moreover, the implication of islet clock functional outputs in the temporal coordination of metabolism in health and disease will be highlighted.

Brain Inflammaging: Roles of Melatonin, Circadian Clocks and Sirtuins

Inflammaging denotes the contribution of low-grade inflammation to aging and is of particular importance in the brain as it is relevant to development and progression of neurodegeneration and mental disorders resulting thereof. Several processes are involved, such as changes by immunosenescence, release of proinflammatory cytokines by DNA-damaged cells that have developed the senescence-associated secretory phenotype, microglia activation and astrogliosis because of neuronal overexcitation, brain insulin resistance, and increased levels of amyloid-β peptides and oligomers. Melatonin and sirtuin1, which are both part of the circadian oscillator system share neuroprotective and anti-inflammatory properties. In the course of aging, the functioning of the circadian system deteriorates and levels of melatonin and sirtuin1 progressively decline. Protective effects of melatonin and sirtuin1 are outlined and emphasis is given to possibilities of upregulating sirtuin1 by melatonin and circadian amplitude-enhancing actions of sirtuin1.

Glucose Homeostasis: Regulation by Peripheral Circadian Clocks in Rodents and Humans.

Most organisms, including humans, have developed an intrinsic system of circadian oscillators, allowing the anticipation of events related to the rotation of Earth around its own axis. The mammalian circadian timing system orchestrates nearly all aspects of physiology and behavior. Together with systemic signals, emanating from the central clock that resides in the hypothalamus, peripheral oscillators orchestrate tissue-specific fluctuations in gene expression, protein synthesis, and posttranslational modifications, driving overt rhythms in physiology and behavior. There is increasing evidence on the essential roles of the peripheral oscillators, operative in metabolically active organs in the regulation of body glucose homeostasis. Here, we review some recent findings on the molecular and cellular makeup of the circadian timing system and its implications in the temporal coordination of metabolism in health and disease.

Neural activity in the suprachiasmatic circadian clock of nocturnal mice anticipating a daytime meal

Circadian rhythms in mammals are regulated by a system of circadian oscillators that includes a light-entrainable pacemaker in the suprachiasmatic nucleus (SCN) and food-entrainable oscillators (FEOs) elsewhere in the brain and body. In nocturnal rodents, the SCN promotes sleep in the day and wake at night, while FEOs promote an active state in anticipation of a predictable daily meal. For nocturnal animals to anticipate a daytime meal, wake-promoting signals from FEOs must compete with sleep-promoting signals from the SCN pacemaker. One hypothesis is that FEOs impose a daily rhythm of inhibition on SCN output that is timed to permit the expression of activity prior to a daytime meal. This hypothesis predicts that SCN activity should decrease prior to the onset of anticipatory activity and remain suppressed through the scheduled mealtime. To assess the hypothesis, neural activity in the SCN of mice anticipating a 4–5-h daily meal in the light period was measured using FOS immunohistochemistry and in vivo multiple unit electrophysiology. SCN FOS, quantified by optical density, was significantly reduced at the expected mealtime in food-anticipating mice with access to a running disk, compared to ad libitum-fed and acutely fasted controls. Group differences were not significant when FOS was quantified by other methods, or in mice without running disks. SCN electrical activity was markedly decreased during locomotion in some mice but increased in others. Changes in either direction were concurrent with locomotion, were not specific to food anticipation, and were not sustained during longer pauses. Reduced FOS indicates a net suppression of SCN activity that may depend on the intensity or duration of locomotion. The timing of changes in SCN activity relative to locomotion suggests that any effect of FEOs on SCN output is mediated indirectly, by feedback from neural or systemic correlates of locomotion.

The clock protein PERIOD is expressed in goblet cells of the larval midgut in the silkworm, Bombyx mori

Insects have both a circadian oscillatory system in peripheral tissues and a master clock in the central nervous system. However, the distribution of oscillator cells in the digestive system that is indispensable for nutrient intake has not been described in detail. This study immunohistochemically investigates cellular localization of the clock protein PERIOD in the larval midgut of the silkworm Bombyx mori (Lepidoptera: Bombycidae). PERIOD-like immunoreactivity was not uniformly distributed and was located exclusively in goblet cells of the midgut epithelium. The primary role of goblet cells is the active transport of potassium from hemolymph into the gut lumen. However, these cells might function as a peripheral clock that regulates the daily rhythms of physiological activities in the midgut of silkworm larvae.

Landscape, Flux, Correlation, Resonance, Coherence, Stability, and Key Network Wirings of Stochastic Circadian Oscillation

Circadian rhythms with a period of ∼24 h, are natural timing machines. They are broadly distributed in living organisms, such as Neurospora, Drosophila, and mammals. The underlying natures of the rhythmic behavior have been explored by experimental and theoretical approaches. However, the global and physical natures of the oscillation under fluctuations are still not very clear. We developed a landscape and flux framework to explore the global stability and robustness of a circadian oscillation system. The potential landscape of the network is uncovered and has a global Mexican-hat shape. The height of the Mexican-hat provides a quantitative measure to evaluate the robustness and coherence of the oscillation. We found that in nonequilibrium dynamic systems, not only the potential landscape but also the probability flux are important to the dynamics of the system under intrinsic noise. Landscape attracts the systems down to the oscillation ring while flux drives the coherent oscillation on the ring. We also investigated the phase coherence and the entropy production rate of the system at different fluctuations and found that dissipations are less and the coherence is higher for larger number of molecules. We also found that the power spectrum of autocorrelation functions show resonance peak at the frequency of coherent oscillations. The peak is less prominent for smaller number of molecules and less barrier height and therefore can be used as another measure of stability of oscillations. As a consequence of nonzero probability flux, we show that the three-point correlations from the time traces show irreversibility, providing a possible way to explore the flux from the observations. Furthermore, we explored the escape time from the oscillation ring to outside at different molecular number. We found that when barrier height is higher, escape time is longer and phase coherence of oscillation is higher. Finally, we performed the global sensitivity analysis of the underlying parameters to find the key network wirings responsible for the stability of the oscillation system.

Quantitative Analysis of Phase Wave of Gene Expression in the Mammalian Central Circadian Clock Network

BackgroundThe suprachiasmatic nucleus (SCN), the master circadian clock, is a heterogeneous oscillator network, yet displays a robust synchronization dynamics. Recent single-cell bioluminescent imaging revealed temporal gradients in circadian clock gene expression in the SCN ex vivo. However, due to technical difficulty in biological approaches to elucidate the entire network structure of the SCN, characteristics of the gradient, which we refer to as phase wave, remain unknown.Methodology/Principal FindingsWe implemented new approaches, i.e., quantitative analysis and model simulation to characterize the phase waves in Per2::Luciferase clock reporter gene expression of the rat SCN slice. Our quantitative study demonstrated not only a high degree of synchronization between the neurons and regular occurrence of the phase wave propagation, but also a significant amount of phase fluctuations contained in the wave. In addition, our simulations based on local coupling model suggest that the intercellular coupling strength estimated by the model simulations is significantly higher than the critical value for generating the phase waves. Model simulations also suggest that heterogeneity of the SCN neurons is one of the main factors causing the phase wave fluctuations. Furthermore, robustness of the SCN network against dynamical noise and variation of the natural frequencies inherent in these neurons was quantitatively assessed.Conclusions/SignificanceTo our knowledge, this is the first quantitative evaluation of the phase wave and further characterization of the SCN neuronal network features generating the wave i.e., intercellular synchrony, phase fluctuation, strong local coupling, heterogeneous periodicity and robustness. Our present study provides an approach, which will lead to a comprehensive understanding of mechanistic and/or biological significance of the phase wave in the central circadian oscillatory system.

Entrainment of circadian clocks in mammals by arousal and food

Circadian rhythms in mammals are regulated by a system of endogenous circadian oscillators (clock cells) in the brain and in most peripheral organs and tissues. One group of clock cells in the hypothalamic SCN (suprachiasmatic nuclei) functions as a pacemaker for co-ordinating the timing of oscillators elsewhere in the brain and body. This master clock can be reset and entrained by daily LD (light-dark) cycles and thereby also serves to interface internal with external time, ensuring an appropriate alignment of behavioural and physiological rhythms with the solar day. Two features of the mammalian circadian system provide flexibility in circadian programming to exploit temporal regularities of social stimuli or food availability. One feature is the sensitivity of the SCN pacemaker to behavioural arousal stimulated during the usual sleep period, which can reset its phase and modulate its response to LD stimuli. Neural pathways from the brainstem and thalamus mediate these effects by releasing neurochemicals that inhibit retinal inputs to the SCN clock or that alter clock-gene expression in SCN clock cells. A second feature is the sensitivity of circadian oscillators outside of the SCN to stimuli associated with food intake, which enables animals to uncouple rhythms of behaviour and physiology from LD cycles and align these with predictable daily mealtimes. The location of oscillators necessary for food-entrained behavioural rhythms is not yet certain. Persistence of these rhythms in mice with clock-gene mutations that disable the SCN pacemaker suggests diversity in the molecular basis of light- and food-entrainable clocks.

New Vistas on Oxidative Damage and Aging

Age-associated rises in oxidative damage are assumed to be a central phenomenon of aging. Their attenuation is an aim for both healthy aging and life extension. This review intends to critically discuss the potential of anti-oxidant actions, but even more to direct the attention to the modes of radical avoidance and to regulatory networks involved. Mitochondria seem to play a decisive role in radical formation and cellular decline. Avoidance and repair of disruptions in the electron transport chain reduce electron leakage and, thus, oxidative damage. Several low molecular weight compounds, such as melatonin, its metabolite N1-acetyl-5-methoxykynuramine, resveratrol, α-lipoic acid, and various mitochondrially targeted nitrones are capable of supporting mitochondrial electron flux. Some of them have been successfully used for extending the lifespan of experimental animals. Importantly, chemopreventive effects of these substances against cancer development should not be confused with a slowing of the aging process. We also focus on connections between these compounds and mitochondrial biogenesis, including the roles of sirtuins and signaling via peroxisome proliferator-activated receptor-γ coactivator-1α, the participation of the circadian oscillator system in radical avoidance, as well as the potentially beneficial or detrimental effects of NO, as either a regulator or a source of mitochondrial dysfunction. Especially in the central nervous system, anti-excitatory actions by melatonin, kynurenic acid and theanine are discussed, which seem to prevent calcium overload that results in mitochondrial dysfunction. New findings on direct binding of melatonin to the amphipathic ramp of Complex I may indicate an additional regulatory role in the avoidance of electron leakage.

Neuroprotection by radical avoidance: search for suitable agents.

Neurodegeneration is frequently associated with damage by free radicals. However, increases in reactive oxygen and nitrogen species, which may ultimately lead to neuronal cell death, do not necessarily reflect its primary cause, but can be a consequence of otherwise induced cellular dysfunction. Detrimental processes which promote free radical formation are initiated, e.g., by disturbances in calcium homeostasis, mitochondrial malfunction, and an age-related decline in the circadian oscillator system. Free radicals generated at high rates under pathophysiological conditions are insufficiently detoxified by scavengers. Interventions at the primary causes of dysfunction, which avoid secondary rises in radical formation, may be more efficient. The aim of such approaches should be to prevent calcium overload, to reduce mitochondrial electron dissipation, to support electron transport capacity, and to avoid circadian perturbations. L-Theanine and several amphiphilic nitrones are capable of counteracting excitotoxicity and/or mitochondrial radical formation. Resveratrol seems to promote mitochondrial biogenesis. Mitochondrial effects of leptin include attenuation of electron leakage. Melatonin combines all the requirements mentioned, additionally regulates anti- and pro-oxidant enzymes and is, with few exceptions, very well tolerated. In this review, the perspectives, problems and limits of drugs are compared which may be suitable for reducing the formation of free radicals.

The effects of temperature change on the circadian clock of Neurospora.

The phase resetting of the circadian oscillatory system by pulses of increased temperature (zeitgebers) and the temperature compensation of its period length during longer exposures are major features of the system, but are not well understood in molecular terms. In Neurospora crassa, the effects of pulses of increased temperature on the circadian rhythm of conidiation were determined and possible inputs to the oscillatory system tested, including changes in cyclic 3',5'-adenosine monophosphate (cAMP), inositol 1,4,5-trisphosphate and H+ concentrations, as well as changes of phosphorylation, synthesis and degradation of proteins. Following the kinetics of these parameters during exposure to increased temperature showed transient changes. Experimental manipulation of cAMP, Ca2+ and H+ levels, and of the synthesis and, possibly, degradation of proteins, resulted in phase shifts of the oscillatory system. It is assumed that the temperature signal affects the oscillator(s) by multiple pathways and shifts the whole state of the oscillatory system. Second messenger levels, protein synthesis and protein degradation show adaptation to longer exposures to elevated temperature which may be involved in the temperature compensation of the period length. The temperature compensation is also proposed to involve a shift in the state of all or most oscillator variables.