The circadian system provides living organisms a means to adapt their internal physiology to constantly changing environmental conditions that exists on our rotating planet, Earth. Clocks in peripheral tissues are referred to as peripheral which may participate in tissue-specific functions. The first step to investigating the circadian regulation in the peripheral tissues of avians was to examine for the presence of avian orthologs of core components of the molecular clock using Quantitative real time (qRTPCR) assays. We investigated the avian spleen for daily and circadian control of core clock genes and regulation of the inflammatory response by the spleen clock. The core clock genes, bmal1, bmal2, per2, per3 and clock displayed both daily and circadian rhythms. Proinflammatory cytokines TNFα, IL-1β, IL-6 and IL-18 exhibited daily and circadian rhythmic oscillations. A differential expression of proinflammatory cytokine induction was observed in the spleen undergoing lipopolysaccharide (LPS)-induced acute inflammation. Exogenous melatonin administration during inflammation seems to enhance some and repress a few inflammatory cytokines, implying that melatonin is pleiotropic molecule. To compare and contrast the role of peripheral clocks in regulating energy balance and reproduction in layer vs. broiler chicken, the visceral adipose tissue (VAT), ovary and hypothalamus were examined for the presence of core clock genes were investigated in these two lines of poultry birds. Quantitative RT-PCR was employed to examine daily control of core clock genes in these three peripheral tissues over a 24hr period. The layer hens exhibit rhythmic oscillations in the mRNA abundance of the core clock genes in the VAT, ovary and the hypothalamus. The hypothalamus and VAT of the broiler hens exhibit rhythmic mRNA abundance of the core clock genes. However, the clock genes in the ovary of the broiler pullets exhibit marked reduction in their amplitude and rhythms over a 24hr period. The broiler hens are prone to poor energy balance, obesity and reproductive capacity. In summary, these data provide evidence for a functional link between the circadian clock and the ovary by determining clock gene regulation under conditions of disrupted or eliminated reproductive function vs. normal reproductive output.

Os ciclos claro-escuro (CE) são considerados importantes pistas para o ajuste de relógios biológicos. Alças de retroalimentação positiva e negativa de transcrição e tradução de genes de relógio são a base molecular subjacente tanto a relógios centrais como periféricos. A opsina não visual, melanopsina (Opn4), expressa na retina de mamíferos, é considerada o fotopigmento circadiano pois é responsável pelo ajuste do relógio biológico endógeno. Este fotopigmento também está presente nos melanóforos de Xenopus laevis, onde ele foi descrito pela primeira vez, mas seu papel nestas células ainda não está completamente esclarecido. Espécies de vertebrados não mamíferos expressam duas ou mais melanopsinas e, no caso de X. laevis, há dois genes, Opn4m and Opn4x. Melanóforos de X. laevis respondem à luz com dispersão dos grânulos de melanina, a resposta máxima sendo atingida no comprimento de onda correspondente àquele de excitação máxima da melanopsina. Entre vários hormônios, endotelinas também dispersam os melanossomos em melanóforos de Xenopus através de via similar àquela evocada pela luz. Tendo esses fatos em mente, decidimos investigar se a luz e a endotelina modulam a expressão de genes de relógio em melanóforos de Xenopus, usando PCR quantitativo para avaliar os níveis relativos de RNAm de Per1, Per2, Clock e Bmal1. Ciclos CE promoveram alterações temporais na expressão de Per1, Per2 e Bmal1. Pulsos de 10 min de luz azul aumentaram a expressão de Per1 e Per2, diminuíram a expressão de Opn4x, mas não tiveram efeito sobre Opn4m. Ainda mais, diferentes localizações foram mostradas para cada melanopsina: imunorreatividade para OPN4x foi vista principalmente na membrana celular, enquanto OPN4m foi imuno-localizada no núcleo. Estes resultados em conjunto apontam para funções diferenciais das duas melanopsinas neste modelo. A translocação de grânulos de melanina foi maior quando um pulso de luz azul foi aplicado na presença de endotelina ET-3. E os níveis de RNAm de Clock exibiram variação temporal em melanóforos submetidos a CE após tratamento com ET-3 10-9M, enquanto a expressão de Per1 não foi afetada pelo tratamento hormonal. Em adição, ensaios farmacológicos indicaram que as respostas de Per1 e Per2 à luz azul são evocadas através da ativação da via de fosfoinositídeos, com crosstalks com GMPc/proteina quinase G (PKG) para ativar os genes de relógio. Estes dados sugerem a participação de melanopsina na foto-ativação de genes de relógio, e apontam para uma participação menor de endotelina como sincronizador desta linhagem celular. Nossos resultados constituem uma importante contribuição ao campo emergente dos relógios periféricos os quais, em espécies de não mamíferos têm sido mais extensivamente estudados em Drosophila melanogaster e Danio rerio. Dentro deste contexto, nós mostramos que os melanóforos de Xenopus laevis representam um modelo ideal para a compreensão da modulação de ritmos circadianos por luz e hormônios / Light-dark cycles (LD) are considered important cues to entrain biological clocks. Positive and negative feedback loops of clock gene transcription and translation are the molecular basis underlying the mechanism of both central and peripheral clocks. The non-visual opsin, melanopsin (Opn4), expressed in the mammalian retina, is considered a circadian photopigment because it is responsible of entraining the endogenous biological clock. This photopigment is also present in the melanophores of Xenopus laevis, where it was first described, but its role in these cells is not fully understood. Non-mammalian vertebrate species express two or more melanopsins, and in X. laevis there are two melanopsin genes, Opn4m and Opn4x. X. laevis melanophores respond to light with melanin granule dispersion, the maximal response being achieved at the wavelength of melanopsin maximal excitation. Among various hormones, endothelins also disperse melanosomes in Xenopus melanophores through a similar pathway as light does. Therefore, we decided to investigate whether light and endothelin modulate clock gene expression in Xenopus melanophores, using quantitative PCR to evaluate the relative mRNA levels of Per1, Per2, Clock and Bmal1. LD cycles elicited temporal changes in the expression of Per1, Per2 and Bmal1. A 10 min pulse of blue light increased the expression of Per1 and Per2, decreased Opn4x expression, but had no effect on Opn4m. In addition, a different localization was shown for each melanopsin: immunoreactivity for OPN4x was mainly seen in the cell membrane, whereas OPN4m was immunolocalized in the nucleus. These results taken together point to a differential role for each melanopsin in this model. Melanosome translocation was greater when a blue light pulse was applied in the presence of endothelin ET-3. And mRNA levels of Clock exhibited temporal variation in melanophores under LD cycles after 10-9 M ET-3 treatment, whereas Per1 expression was not affected by the hormone treatment. In addition, pharmacological assays indicated that Per1 and Per2 responses to blue light are evoked through the activation of the phosphoinositide pathway, which crosstalks with cGMP/protein kinase G (PKG) to activate the clock genes. These data suggest the participation of melanopsin in the photo-activation of clock genes and point to a minor role of endothelin as synchronizer for this cell line. Our results add an important contribution to the emerging field of peripheral clocks, which in non-mammalian species have been mostly studied in Drosophila melanogaster and Danio rerio. Within this context, we show that Xenopus laevis melanophores represent an ideal model to understanding circadian rhythms modulation by light and hormone

Os ciclos claro-escuro (CE) são considerados importantes pistas para o ajuste de relógios biológicos. Alças de retroalimentação positiva e negativa de transcrição e tradução de genes de relógio são a base molecular subjacente tanto a relógios centrais como periféricos. A opsina não visual, melanopsina (Opn4), expressa na retina de mamíferos, é considerada o fotopigmento circadiano pois é responsável pelo ajuste do relógio biológico endógeno. Este fotopigmento também está presente nos melanóforos de Xenopus laevis, onde ele foi descrito pela primeira vez, mas seu papel nestas células ainda não está completamente esclarecido. Espécies de vertebrados não mamíferos expressam duas ou mais melanopsinas e, no caso de X. laevis, há dois genes, Opn4m and Opn4x. Melanóforos de X. laevis respondem à luz com dispersão dos grânulos de melanina, a resposta máxima sendo atingida no comprimento de onda correspondente àquele de excitação máxima da melanopsina. Entre vários hormônios, endotelinas também dispersam os melanossomos em melanóforos de Xenopus através de via similar àquela evocada pela luz. Tendo esses fatos em mente, decidimos investigar se a luz e a endotelina modulam a expressão de genes de relógio em melanóforos de Xenopus, usando PCR quantitativo para avaliar os níveis relativos de RNAm de Per1, Per2, Clock e Bmal1. Ciclos CE promoveram alterações temporais na expressão de Per1, Per2 e Bmal1. Pulsos de 10 min de luz azul aumentaram a expressão de Per1 e Per2, diminuíram a expressão de Opn4x, mas não tiveram efeito sobre Opn4m. Ainda mais, diferentes localizações foram mostradas para cada melanopsina: imunorreatividade para OPN4x foi vista principalmente na membrana celular, enquanto OPN4m foi imuno-localizada no núcleo. Estes resultados em conjunto apontam para funções diferenciais das duas melanopsinas neste modelo. A translocação de grânulos de melanina foi maior quando um pulso de luz azul foi aplicado na presença de endotelina ET-3. E os níveis de RNAm de Clock exibiram variação temporal em melanóforos submetidos a CE após tratamento com ET-3 10-9M, enquanto a expressão de Per1 não foi afetada pelo tratamento hormonal. Em adição, ensaios farmacológicos indicaram que as respostas de Per1 e Per2 à luz azul são evocadas através da ativação da via de fosfoinositídeos, com crosstalks com GMPc/proteina quinase G (PKG) para ativar os genes de relógio. Estes dados sugerem a participação de melanopsina na foto-ativação de genes de relógio, e apontam para uma participação menor de endotelina como sincronizador desta linhagem celular. Nossos resultados constituem uma importante contribuição ao campo emergente dos relógios periféricos os quais, em espécies de não mamíferos têm sido mais extensivamente estudados em Drosophila melanogaster e Danio rerio. Dentro deste contexto, nós mostramos que os melanóforos de Xenopus laevis representam um modelo ideal para a compreensão da modulação de ritmos circadianos por luz e hormônios / Light-dark cycles (LD) are considered important cues to entrain biological clocks. Positive and negative feedback loops of clock gene transcription and translation are the molecular basis underlying the mechanism of both central and peripheral clocks. The non-visual opsin, melanopsin (Opn4), expressed in the mammalian retina, is considered a circadian photopigment because it is responsible of entraining the endogenous biological clock. This photopigment is also present in the melanophores of Xenopus laevis, where it was first described, but its role in these cells is not fully understood. Non-mammalian vertebrate species express two or more melanopsins, and in X. laevis there are two melanopsin genes, Opn4m and Opn4x. X. laevis melanophores respond to light with melanin granule dispersion, the maximal response being achieved at the wavelength of melanopsin maximal excitation. Among various hormones, endothelins also disperse melanosomes in Xenopus melanophores through a similar pathway as light does. Therefore, we decided to investigate whether light and endothelin modulate clock gene expression in Xenopus melanophores, using quantitative PCR to evaluate the relative mRNA levels of Per1, Per2, Clock and Bmal1. LD cycles elicited temporal changes in the expression of Per1, Per2 and Bmal1. A 10 min pulse of blue light increased the expression of Per1 and Per2, decreased Opn4x expression, but had no effect on Opn4m. In addition, a different localization was shown for each melanopsin: immunoreactivity for OPN4x was mainly seen in the cell membrane, whereas OPN4m was immunolocalized in the nucleus. These results taken together point to a differential role for each melanopsin in this model. Melanosome translocation was greater when a blue light pulse was applied in the presence of endothelin ET-3. And mRNA levels of Clock exhibited temporal variation in melanophores under LD cycles after 10-9 M ET-3 treatment, whereas Per1 expression was not affected by the hormone treatment. In addition, pharmacological assays indicated that Per1 and Per2 responses to blue light are evoked through the activation of the phosphoinositide pathway, which crosstalks with cGMP/protein kinase G (PKG) to activate the clock genes. These data suggest the participation of melanopsin in the photo-activation of clock genes and point to a minor role of endothelin as synchronizer for this cell line. Our results add an important contribution to the emerging field of peripheral clocks, which in non-mammalian species have been mostly studied in Drosophila melanogaster and Danio rerio. Within this context, we show that Xenopus laevis melanophores represent an ideal model to understanding circadian rhythms modulation by light and hormone

Ce travail avait pour objet d’étudier les propriétés d’horloge et de synchronisation de la peau, un modèle potentiel d’horloge périphérique. L’activité rythmique a été analysée par bioluminescence en temps réel, sur des explants de peau abdominale et des fibroblastes dermiques primaires, isolés à partir de rats transgéniques Per1-luciférase. Nous avons montré que des explants de peau présentent une activité rythmique soutenue en culture, indiquant une importante synchronisation interne dans le tissu. Cette synchronisation se manifeste au cours du développement post-natal à partir de 1 mois et augmente jusqu’à 6 mois, avant de décroître, laissant place à des rythmes altérés à l’âge de 2 ans. Nous avons aussi établi que les fibroblastes dermiques présentent la propriété de compensation thermique commune à toutes les horloges circadiennes, et qu’ils sont potentiellement synchronisables par la mélatonine puisque celle-ci augmente leur amplitude en culture. Nous avons aussi préparé un vecteur lentiviral exprimant le gène rapporteur luciférase sous le contrôle du promoteur du gène horloge Bmal1, un nouvel outil pour compléter l’étude des rythmes dans les cellules de la peau. / This work aimed to investigate the skin as a potential model of peripheral clock by characterizing its rhythmic and synchronization properties. Circadian activity was examined in abdominal skin explants and fibroblasts derived from Per1-Luciferase transgenic rats by real-time recording of bioluminescence. First, the skin clock was characterized from early postnatal to old age. Low amplitude oscillations appeared at 1 month only and their robustness increased until 6 months. In 1-2 year-old rats, skin circadian rhythms showed decreasing amplitude and abnormal cycles. Primary fibroblasts derived from the skin at the same ages demonstrated similar pattern of clock activity. Temperature compensation, an intrinsic clock feature, was shown the first time in skin and primary fibroblasts. Secondly, we demonstrated a phase-dependent effect of melatonin to increase the amplitude of oscillations in skin primary fibroblasts, indicating it displays a synchronising role in the circadiansystem. Finally, to facilitate our studies on the multioscillatory skin tissue, we constructed a lentivirus carrying a Bmal1-luciferase reporter, to measure clock genes activities in human skin cells.

AABBSSTTRRAACCTT The molecular circadian clock enables anticipation of environmental changes. In mammals, clocks are ubiquitously present in almost all tissues and they are comprised of transcriptional-translational feedback loops of the so-called clock genes. The central clock represents the intrinsic pacemaker which is located in suprachiasmatic nuclei (SCN) of hypothalamus and synchronizes peripheral clocks. Clockwork system in alimentary tract and its regulatory link to intestinal functions are poorly understood. Therefore the objective of the thesis was to characterize molecular clock in particular parts of the rat intestine and to elucidate its link to the intestinal transport, regulation of cell cycle and neoplastic transformation in colonic tissue. We used quantitative RT-PCR (qPCR) to determine circadian profiles of mRNA expression of clock genes in the epithelium of duodenum, jejunum, ileum, and colon of rat. Furthermore, we analysed the expression of genes coding sodium chloride transporters and channels as well as cell cycle regulators in colon. To focus more precisely on different structures of intestinal epithelia we used laser capture microdissection. In addition, we performed Ussing chamber measurements to determine the colonic electrogenic transport. To study the contribution of circadian...

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