Mitogen activated protein kinase (MAPK) pathways are conserved biochemical signal transduction pathways in eukaryotic organisms. These signaling pathways demonstrate great versatility in their ability to detect various environmental stimuli and direct an appropriate cellular response. The circadian clock is a timekeeping mechanism that temporally coordinates diverse biological functions in an organism with the environment. Thus, it is not surprising that MAPK pathways have been utilized by the circadian clock to regulate many essential functions. Due to the conserved nature of circadian clocks and MAPK signaling pathways in eukaryotes, it is possible to develop hypotheses in simple model organisms, such as the fungus Neurospora, that are relevant to more complex organisms. The OS-2 MAPK pathway in the filamentous fungus Neurospora is rhythmically activated by the circadian clock. In order to generate this rhythmic signal, the circadian oscillator directly regulates the rhythmic transcription of the os-4 MAPKKK and histidine phosphotransferase hpt-1, which are upstream regulators of the OS-2 MAPK. Also, the circadian rhythm in MAPK activation produces a more robust stress response during the time of the day that stress is most likely to be encountered. Based on these data, a model for the clock regulation of MAPK activation is presented, and a biological significance is assigned to the rhythms in this pathway. Informed by these findings in Neurospora, the related p38 MAPK pathway was studied in mammalian cell lines that represent functionally distinct tissues in regards to clock function. A rhythm in p38 MAPK activation was observed in cells derived from the suprachiasmatic nucleus and fibroblasts of a mouse, the master pacemaker and a peripheral tissue, respectively. In cells that lacked a functional circadian oscillator, the rhythm in p38 activation was absent, and overall levels of p38 protein were lower. These data demonstrate a circadian clock-dependent oscillation in p38 activity. These studies provide a basis to understand how the circadian clock generates endogenous rhythms in MAPK signal transduction pathways. Also, the characterization of clock-regulated stress response pathways provides an understanding of the adaptive advantage of the circadian clock.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Despite teleosts being the largest and most diverse group of vertebrates, fish models currently used to study photoperiodic effects on fish physiology have been limited to a few species, most of which are temperate seasonal breeders. The overall aim of this work was to expand our knowledge on circadian biology and environmental physiological effects in Nile tilapia (Oreochromis niloticus niloticus), a continuous breeding species of tropical-subtropical origin. The circadian light axis of Nile tilapia is described with regards to melatonin production. Circadian melatonin profiles of fish under 12L:12D photoperiods were observed to be low at day and high at night, suggesting melatonin to be an entraining signal as observed in all other vertebrates. When constant light (LL) was used, such day and night fluctuations where abolished. However when fish where exposed to constant darkness (DD) a strong robust endogenous melatonin rhythm was found, suggesting the presence of circadian oscillators in this species. Importantly, this endogenous rhythm was observed to be maintained for at least three weeks under darkness and proved to be circadian in nature. Moreover, although the melatonin system was able to produce day and night melatonin rhythms when exposed to a different (6L:6D) photocycle, the oscillator appeared to not be entrainable to such a short photo cycle when exposed to DD, as melatonin levels remained high. When comparing the circadian organization of different teleost species including Nile tilapia, preliminary studies showed at least three divergent circadian light organizations in teleosts. Nile tilapia was characterised by a pineal gland far less sensitive than in other fish species as demonstrated through in vitro studies. Furthermore, pineal melatonin production was clearly dependent on the light perceived by the eyes as ophthalmectomy resulted in basal plasma melatonin levels during the dark period. These findings are the first to be reported in a teleost and could be comparable to the circadian light organization of higher vertebrates such as mammals. The onset of puberty of Nile tilapia was studied with regards to the newly discovered Kiss1/GPR54 system. Such a system has recently been discovered in mammals and found to be the primary switch of the brain-pituitary-gonadal (BPG) axis. The results of this study not only suggest a link between the Kiss1/GPR54 system and the onset of III puberty in this tropical batch spawning teleost, that would be a highly conserved feature across vertebrates, but also that the transcriptional mechanisms regulating GPR54 expression could be directly or indirectly influenced by light. Finally, a study was conducted on the effects of different intensities of continuous light (LL) on the growth and sexual development of Nile tilapia up to first maturation. The results showed a significant growth response of fish in all LL treatments compared to control fish. Importantly, this confirmed that LL enhances growth in this species and suggests that it is the light regime more than the intensity which is having an effect. This work thus provides important basic knowledge of the light entrainment pathway and circadian melatonin rhythms in Nile tilapia. Of special importance is the discovery of a strong endogenous melatonin oscillator and a novel circadian organization in fish which would seem to be homologous to that observed in higher vertebrates. Moreover, this work provides evidence that the newly discovered Kiss1/GPR54 system has a similar role in fish as has been found in mammals and that such a system could be directly or indirectly regulated by light. If so, Nile tilapia and other fish species could become important models in the chronobiology and reproduction fields. Finally, this work not only increases our basic and applied knowledge of this species, but also broadens our understanding of the circadian light axis in teleosts and its mediatory effects on reproduction.

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available online through Digital Dissertations.

Introdução: Os seres vivos sincronizam suas atividades com os ciclos ambientais influenciados por fatores externos como as condições de luz, horários de alimentação, interações sociais e por fatores endógenos como os genéticos, níveis hormonais e apetite, entre outros. Sensores percebem a variação temporal informando o estado de iluminação ambiental através da rede neural ou sistema endócrino mediado pelos relógios circadianos. Os genes do relógio pertencem a quatro famílias: CLOCK, BMAL1, Cryptochromes e Period onde a formação, expressão e supressão da transcrição destes e de seus heterodímeros resultam na ritmicidade de 24h. O relógio circadiano modula o metabolismo de energia através do controle da atividade de diversas enzimas, dos sistemas de transporte e de receptores nucleares envolvidos no metabolismo dos nutrientes. Alterações nos horários de alimentação podem modificar a relação entre o relógio central e or relógios periféricos podendo causar mudanças no metabolismo e afetar o sistema circadiano. Em algumas desordens alimentares como a Síndrome do Comer Noturno (SCN), reconhecida como um atraso de fase da alimentação, o ritmo de alimentação está alterado. Assim sendo, a SCN pode ser um fator que influencia na obesidade, modificando padrões do sono, padrão alimentar, apetite e regulação neuroendócrina. Objetivos: Estudar os aspectos cronobiológicos na SCN. Métodos: Características, como aspectos emocionais, cronotipo e qualidade do sono foram avaliadas por meio de questionários. Variáveis de ritmicidade da atividade e exposição à luz foram avaliadas por actigrafia e a expressão dos genes do relógio CLOCK, BMAL1, Cry 1 e Per 2 foi medida em leucócitos. Resultados: 28 pacientes (14 com SCN - 10 mulheres e 14 controles - 9 mulheres) participaram deste estudo. A média de idade foi de 40,71 ± 12,37 anos e IMC foi de 26,8 ± 5,7kg/m². Não foi encontrada nenhuma evidência cronobiológica nas análises realizadas relacionada à SCN. Conclusão: Nossos resultados não corroboram a hipótese de que o the time system pode ser ligado à fisiopatologia da SCN, pois a associação de sintomas que definem a SCN como uma síndrome. Os tempos e a qualidade da alimentação devem ser mais profundamente estudados para esclarecer a relação entre a alimentação e o fato de que se os seres humanos alocam a sua principal alimentação no turno da noite pode trazer consequências ao metabolismo e refletir mudanças no comportamento e contribuir no controle da obesidade. Este é o primeiro estudo em humanos para relacionar os genes do relógio e a SCN. / Introduction: Living beings synchronize their activities with environmental cycles influenced by external factors such as light conditions, feeding schedules, social interactions and endogenous factors such as genetics, hormone levels and appetite, among others. Sensors perceive the temporal variation informing the state of ambient lighting through the neural network or endocrine mediated by circadian clocks. The clock genes belong to four families: CLOCK, BMAL1, Cryptochromes and Period where the formation, expression and suppression of these transcriptions and their heterodimers result in 24h rhythmicity. The circadian clock modulates energy metabolism by controlling the activity of several enzymes, transport systems and nuclear receptors involved in the metabolism of nutrients. Changes in feeding schedules may modify the relationship between the central clock and peripheral clocks and cause changes in metabolism and affect the circadian system. Some eating disorders, such as Night Eating Syndrome (NES) recognized as a phase delay in the feed, the feed rthythmicity coul be changed. Thus, NES may be a factor that influences in obesity, changing sleep and eating patterns, appetite and neuroendocrine regulation. Objectives: Cross-sectional study to examine the chronobiological aspects of NES. Methods: Features such as emotional aspects, chronotype and sleep quality were assessed by questionnaires. Variables rhythmicity of activity and light exposure were assessed by actigraphy and expression of clock genes CLOCK, BMAL1, Cry 1 and Per 2 was measured in leukocytes. Results: 28 patients (14 with SCN - 10 women and 14 controls - 9 women) participated in this study. The age average was 40.71 ± 12.37y and BMI was 26.8 ± 5.7kg/m². We found no evidence in chronobiological analyzes related to SCN. Conclusion: Our results do not support the hypothesis that the time system can be conected to the pathophysiology of NES, because the association of symptoms that define the NES as a syndrome mainly by the characteristic temporal power that is possibly involved with the time system. The timing and quality of food should be further studied to clarify the relationship between food and the fact that humans allocate their main food in the night shift may have consequences for metabolism and reflect changes in behavior and contribute to the control of obesity. This is the first study in humans to relate the clock genes and NES.

Introdução: Os seres vivos sincronizam suas atividades com os ciclos ambientais influenciados por fatores externos como as condições de luz, horários de alimentação, interações sociais e por fatores endógenos como os genéticos, níveis hormonais e apetite, entre outros. Sensores percebem a variação temporal informando o estado de iluminação ambiental através da rede neural ou sistema endócrino mediado pelos relógios circadianos. Os genes do relógio pertencem a quatro famílias: CLOCK, BMAL1, Cryptochromes e Period onde a formação, expressão e supressão da transcrição destes e de seus heterodímeros resultam na ritmicidade de 24h. O relógio circadiano modula o metabolismo de energia através do controle da atividade de diversas enzimas, dos sistemas de transporte e de receptores nucleares envolvidos no metabolismo dos nutrientes. Alterações nos horários de alimentação podem modificar a relação entre o relógio central e or relógios periféricos podendo causar mudanças no metabolismo e afetar o sistema circadiano. Em algumas desordens alimentares como a Síndrome do Comer Noturno (SCN), reconhecida como um atraso de fase da alimentação, o ritmo de alimentação está alterado. Assim sendo, a SCN pode ser um fator que influencia na obesidade, modificando padrões do sono, padrão alimentar, apetite e regulação neuroendócrina. Objetivos: Estudar os aspectos cronobiológicos na SCN. Métodos: Características, como aspectos emocionais, cronotipo e qualidade do sono foram avaliadas por meio de questionários. Variáveis de ritmicidade da atividade e exposição à luz foram avaliadas por actigrafia e a expressão dos genes do relógio CLOCK, BMAL1, Cry 1 e Per 2 foi medida em leucócitos. Resultados: 28 pacientes (14 com SCN - 10 mulheres e 14 controles - 9 mulheres) participaram deste estudo. A média de idade foi de 40,71 ± 12,37 anos e IMC foi de 26,8 ± 5,7kg/m². Não foi encontrada nenhuma evidência cronobiológica nas análises realizadas relacionada à SCN. Conclusão: Nossos resultados não corroboram a hipótese de que o the time system pode ser ligado à fisiopatologia da SCN, pois a associação de sintomas que definem a SCN como uma síndrome. Os tempos e a qualidade da alimentação devem ser mais profundamente estudados para esclarecer a relação entre a alimentação e o fato de que se os seres humanos alocam a sua principal alimentação no turno da noite pode trazer consequências ao metabolismo e refletir mudanças no comportamento e contribuir no controle da obesidade. Este é o primeiro estudo em humanos para relacionar os genes do relógio e a SCN. / Introduction: Living beings synchronize their activities with environmental cycles influenced by external factors such as light conditions, feeding schedules, social interactions and endogenous factors such as genetics, hormone levels and appetite, among others. Sensors perceive the temporal variation informing the state of ambient lighting through the neural network or endocrine mediated by circadian clocks. The clock genes belong to four families: CLOCK, BMAL1, Cryptochromes and Period where the formation, expression and suppression of these transcriptions and their heterodimers result in 24h rhythmicity. The circadian clock modulates energy metabolism by controlling the activity of several enzymes, transport systems and nuclear receptors involved in the metabolism of nutrients. Changes in feeding schedules may modify the relationship between the central clock and peripheral clocks and cause changes in metabolism and affect the circadian system. Some eating disorders, such as Night Eating Syndrome (NES) recognized as a phase delay in the feed, the feed rthythmicity coul be changed. Thus, NES may be a factor that influences in obesity, changing sleep and eating patterns, appetite and neuroendocrine regulation. Objectives: Cross-sectional study to examine the chronobiological aspects of NES. Methods: Features such as emotional aspects, chronotype and sleep quality were assessed by questionnaires. Variables rhythmicity of activity and light exposure were assessed by actigraphy and expression of clock genes CLOCK, BMAL1, Cry 1 and Per 2 was measured in leukocytes. Results: 28 patients (14 with SCN - 10 women and 14 controls - 9 women) participated in this study. The age average was 40.71 ± 12.37y and BMI was 26.8 ± 5.7kg/m². We found no evidence in chronobiological analyzes related to SCN. Conclusion: Our results do not support the hypothesis that the time system can be conected to the pathophysiology of NES, because the association of symptoms that define the NES as a syndrome mainly by the characteristic temporal power that is possibly involved with the time system. The timing and quality of food should be further studied to clarify the relationship between food and the fact that humans allocate their main food in the night shift may have consequences for metabolism and reflect changes in behavior and contribute to the control of obesity. This is the first study in humans to relate the clock genes and NES.

Introdução: Os seres vivos sincronizam suas atividades com os ciclos ambientais influenciados por fatores externos como as condições de luz, horários de alimentação, interações sociais e por fatores endógenos como os genéticos, níveis hormonais e apetite, entre outros. Sensores percebem a variação temporal informando o estado de iluminação ambiental através da rede neural ou sistema endócrino mediado pelos relógios circadianos. Os genes do relógio pertencem a quatro famílias: CLOCK, BMAL1, Cryptochromes e Period onde a formação, expressão e supressão da transcrição destes e de seus heterodímeros resultam na ritmicidade de 24h. O relógio circadiano modula o metabolismo de energia através do controle da atividade de diversas enzimas, dos sistemas de transporte e de receptores nucleares envolvidos no metabolismo dos nutrientes. Alterações nos horários de alimentação podem modificar a relação entre o relógio central e or relógios periféricos podendo causar mudanças no metabolismo e afetar o sistema circadiano. Em algumas desordens alimentares como a Síndrome do Comer Noturno (SCN), reconhecida como um atraso de fase da alimentação, o ritmo de alimentação está alterado. Assim sendo, a SCN pode ser um fator que influencia na obesidade, modificando padrões do sono, padrão alimentar, apetite e regulação neuroendócrina. Objetivos: Estudar os aspectos cronobiológicos na SCN. Métodos: Características, como aspectos emocionais, cronotipo e qualidade do sono foram avaliadas por meio de questionários. Variáveis de ritmicidade da atividade e exposição à luz foram avaliadas por actigrafia e a expressão dos genes do relógio CLOCK, BMAL1, Cry 1 e Per 2 foi medida em leucócitos. Resultados: 28 pacientes (14 com SCN - 10 mulheres e 14 controles - 9 mulheres) participaram deste estudo. A média de idade foi de 40,71 ± 12,37 anos e IMC foi de 26,8 ± 5,7kg/m². Não foi encontrada nenhuma evidência cronobiológica nas análises realizadas relacionada à SCN. Conclusão: Nossos resultados não corroboram a hipótese de que o the time system pode ser ligado à fisiopatologia da SCN, pois a associação de sintomas que definem a SCN como uma síndrome. Os tempos e a qualidade da alimentação devem ser mais profundamente estudados para esclarecer a relação entre a alimentação e o fato de que se os seres humanos alocam a sua principal alimentação no turno da noite pode trazer consequências ao metabolismo e refletir mudanças no comportamento e contribuir no controle da obesidade. Este é o primeiro estudo em humanos para relacionar os genes do relógio e a SCN. / Introduction: Living beings synchronize their activities with environmental cycles influenced by external factors such as light conditions, feeding schedules, social interactions and endogenous factors such as genetics, hormone levels and appetite, among others. Sensors perceive the temporal variation informing the state of ambient lighting through the neural network or endocrine mediated by circadian clocks. The clock genes belong to four families: CLOCK, BMAL1, Cryptochromes and Period where the formation, expression and suppression of these transcriptions and their heterodimers result in 24h rhythmicity. The circadian clock modulates energy metabolism by controlling the activity of several enzymes, transport systems and nuclear receptors involved in the metabolism of nutrients. Changes in feeding schedules may modify the relationship between the central clock and peripheral clocks and cause changes in metabolism and affect the circadian system. Some eating disorders, such as Night Eating Syndrome (NES) recognized as a phase delay in the feed, the feed rthythmicity coul be changed. Thus, NES may be a factor that influences in obesity, changing sleep and eating patterns, appetite and neuroendocrine regulation. Objectives: Cross-sectional study to examine the chronobiological aspects of NES. Methods: Features such as emotional aspects, chronotype and sleep quality were assessed by questionnaires. Variables rhythmicity of activity and light exposure were assessed by actigraphy and expression of clock genes CLOCK, BMAL1, Cry 1 and Per 2 was measured in leukocytes. Results: 28 patients (14 with SCN - 10 women and 14 controls - 9 women) participated in this study. The age average was 40.71 ± 12.37y and BMI was 26.8 ± 5.7kg/m². We found no evidence in chronobiological analyzes related to SCN. Conclusion: Our results do not support the hypothesis that the time system can be conected to the pathophysiology of NES, because the association of symptoms that define the NES as a syndrome mainly by the characteristic temporal power that is possibly involved with the time system. The timing and quality of food should be further studied to clarify the relationship between food and the fact that humans allocate their main food in the night shift may have consequences for metabolism and reflect changes in behavior and contribute to the control of obesity. This is the first study in humans to relate the clock genes and NES.

Every fall, Northeastern America monarch butterflies (Danaus plexippus) undergo an extraordinary migration to their overwintering site in Central Mexico. During their long migration, monarch migrants use sun compass to navigate. To maintain a southward flying direction, monarch migrants compensate for the continuously changing position of the sun by providing timing information to the compass using their circadian clock. Animal circadian clocks depend primarily on a negative transcriptional feedback loop to track time. I started my work to re-construct the monarch butterfly circadian clock negative feedback loop in cell culture, focusing on homologs of Drosophila clock genes. It turned out that in addition to a Drosophila-like cryptochrome (cry1) gene, a second mammalian-like cry2 gene exists in monarch butterflies and many other insects, except in Drosophila. The two CRYs showed distinct functions in our initial assays in cultured Drosophila Schneider 2 (S2) cells. CRY2 functions as a potent transcriptional repressor, while CRY1 is light sensitive but shows no obvious transcriptional activity. The existence of two cry genes in insects changed the Drosophila-centric view of insect circadian clock. During the course of my study, our lab obtained a monarch cell line called DpN1 cells. These cells possess a light-driven clock and contributed tremendously to the research on monarch circadian clock. Using this cell line, I provided strong evidence supporting monarch CRY2’s role as a major circadian clock repressor and identified a protein-protein protective interaction cascade underlying the CRY1-mediated resetting of the molecular oscillator in DpN1 cells. I continued my work trying to understand how insect CRY2 inhibits transcription. I provided evidence suggesting the involvement of monarch PER in promoting CRY2 nuclear entry in both S2 cells and DpN1 cells. Finally, I mapped CRY2’s transcriptional inhibitory activity onto its N-terminal domain. Collectively, my research helped to change our view of insect clocks from a Drosophila-centric standpoint to a much more diverse picture. My studies also advanced the understanding of monarch circadian clock mechanism, and provides a foundation for further studies.