In order to adapt to changing external conditions, organisms developed the endogenous biological clock for predicting daily alterations. This so-called circadian system drives functions and processes in the whole body with an approximately 24h period. The central oscillator, located in hypothalamic suprachiasmatic nuclei (SCN), is synchronized by light and subsequently sends the information about the time of the day to the rest of the body. Even in the ontogenesis, the functional SCN clock is crucial for proper development as well as health later in life. Since the maturation of embryonic SCN is not completed before birth, maternal signals seem to play a fundamental role in setting and synchronizing the fetal clock. During my PhD studies, we focused on elucidating the nature of maternal signals and their diverse impact on fetal SCN of rat and mouse models. We have revealed that developing SCN is able to sense distinct signals related to various maternal behavioral regimes. Importantly, we have discovered eminent role of glucocorticoids in synchronizing the fetal SCN, along with their ability to accelerate SCN development. These observations point out the importance of regular daily routine and noxious effect of stress during pregnancy. Since the mother communicates with the fetus through placenta...

The circadian system has evolved in organisms as an adaptation to periodic changes in the environment. Its task is to ensure regular entrainment between the solar cycle and the internal period of the organism, and to generate signals that synchronize behavioral and physiological processes in the body with the solar cycle. The whole mechanism takes place at the cell level, where there are regular oscillations of the transcriptional translation loops of the clock genes occur within 24 hours, thus ensuring a regular rhythm of the organism. However, the circadian system may not generate the same length of period in humans and may differ in the degree of entrainment with the external cycle. Base on that there are developed so-called individual time preferences. These different preferences are referred to as chronotypes, which fall into five categories: extremely evening, moderate evening, intermediate, moderate morning, and extremely morning type. Clock gene polymorphisms are considered to be one of the possible causes of these differences. The association of selected clock gene polymorphisms with extreme chronotypes is the subject of this diploma thesis. We obtained a saliva sample for DNA isolation from volunteers with extreme chronotypes. Using molecular methods of PCR, restriction digest and...

No / Although the regulation of pigmentation is well characterized, it remains unclear whether cell-autonomous controls regulate the cyclic on–off switching of pigmentation in the hair follicle (HF). As human HFs and epidermal melanocytes express clock genes and proteins, and given that core clock genes (PER1, BMAL1) modulate human HF cycling, we investigated whether peripheral clock activity influences human HF pigmentation. We found that silencing BMAL1 or PER1 in human HFs increased HF melanin content. Furthermore, tyrosinase expression and activity, as well as TYRP1 and TYRP2 mRNA levels, gp100 protein expression, melanocyte dendricity, and the number gp100+ HF melanocytes, were all significantly increased in BMAL1 and/or PER1-silenced HFs. BMAL1 or PER1 silencing also increased epidermal melanin content, gp100 protein expression, and tyrosinase activity in human skin. These effects reflect direct modulation of melanocytes, as BMAL1 and/or PER1 silencing in isolated melanocytes increased tyrosinase activity and TYRP1/2 expression. Mechanistically, BMAL1 knockdown reduces PER1 transcription, and PER1 silencing induces phosphorylation of the master regulator of melanogenesis, microphthalmia-associated transcription factor, thus stimulating human melanogenesis and melanocyte activity in situ and in vitro. Therefore, the molecular clock operates as a cell-autonomous modulator of human pigmentation and may be targeted for future therapeutic strategies.

O ciclo circadiano é um sistema adaptativo e vantajoso que permite a antecipação dos organismos e sincronização de suas atividades fisiológicas frente às variações ambientais que ocorrem ao longo do dia. Seu funcionamento molecular acontece pela geração dos ritmos circadianos, os quais surgem a partir da expressão cíclica dos genes do relógio em um feedback autoregulatório. Em insetos, os ritmos circadianos apresentam função importante em coordenar o timing do desenvolvimento e o comportamento. Nos últimos anos, estudos desvendaram que o relógio molecular de insetos sociais apresenta um funcionamento mais similar ao relógio de mamíferos do que com outros insetos como Drosophila melanogaster. Em especial, abelhas sociais têm sido ótimos modelos para investigar como os ritmos circadianos são modulados de acordo com as interações sociais entre os indivíduos, a plasticidade comportamental e a divisão social do trabalho entre as operárias. Operárias jovens (nutrizes) cuidam da cria no interior da colônia e geralmente apresentam uma atividade arrítimica ao longo de 24 horas, enquanto as abelhas mais velhas (forrageiras) são rítmicas e desenvolvem atividades complexas no ambiente externo. Nesse trabalho, nós caracterizamos os perfis de expressão dos genes do relógio period (per), cryptochrome mammalian-like (cry-m), clock (clk), cycle (cyc), timeout 2 (tim2), par domain protein 1 (pdp1), vrille (vri) e clockwork orange (cwo) durante todo o desenvolvimento de abelhas operárias de Apis mellifera. Verificamos que os genes do relógio são expressos antes mesmo da formação do sistema nervoso central no embrião e que seus transcritos podem ser herdados maternalmente. No desenvolvimento de larvas e pupas, revelamos que estes genes são diferencialmente expressos entre as fases investigadas e, com exceção dos genes cwo e tim2, todos respondem ao tratamento com o Hormônio Juvenil (HJ) na fase de pupa de olho branco. A resposta positiva dos genes clk, cyc e pdp1 frente ao tratamento hormonal pode estar relacionada com o envolvimento destes nas vias que respondem à sinalização do HJ, interagindo com os genes Kruppel (Kr-h1) e Methoprene-tolerant (MET). No desenvolvimento adulto, vimos que os genes per e cry-m são potenciais marcadores da plasticidade comportamental e divisão social do trabalho. Em um experimento usando single-cohort colony, estes genes apresentam níveis transcricionais que não oscilam em cabeças de operárias jovens (3 e 7 dias) ao longo de 24 horas, comparado aos níveis de expressão que oscilam de forma robusta em abelhas mais velhas (15 e 25 dias). Ainda, reconstruímos redes de interação proteína-proteína e miRNA-mRNA onde foram identificadas potenciais moléculas que atuam modulando os genes do relógio em nível póstranscricional e traducional. Dentre elas, validamos as interações entre os miRNA-34 e seus sítios de ligação que estão presentes nas 3`UTRS dos genes cyc e cwo, através do ensaio por luciferase, revelando que este miRNA é um regulador negativo da expressão desses genes. Pela primeira vez, realizamos uma análise ampla dos genes do relógio em um inseto social, além de identificar novas moléculas que podem atuar modulando os ritmos circadianos. Nosso trabalho demonstra a importâcia das abelhas sociais como modelos ideais para desvendar os mecanismos moleculares que regem os ritmos circadianos não só em abelhas, como também em outros organismos, inclusive mamíferos. / The circadian clock is an advantageous adaptive system that enables organisms to anticipate and syncronize their biological activities during the daily environmental changes. The circadian clock acts through the ontogeny of circadian rhythms, which are generated by the cyclic expression of the clock genes in an autregulatory feedback loop. In insects, the circadian rhythms have important roles in the coordination of the developmental timing and behavior, interacting with the endocrine system. In the last years, researchers revealed that the molecular clock of social insects is more similar to mammals than to insects. In particular, the social honeybee is an excellent model to investigate how the circadian rhythms are modulated accordingly to the social context, behavioral plasticity, and taskrelated activities. While young bees (nurses) work arrhythmically around the clock inside the colony in brood-care activities, old bees (foragers) need to be strongly rhythmic to develop complex tasks. In this work, we characterized the expression patterns of the clock genes period (per), cryptochrome mammalian-like (cry-m), clock (clk), cycle (cyc), timeout 2 (tim2), par domain protein 1 (pdp1), vrille (vri) e clockwork orange (cwo) in the entire development of Apis mellifera. Our results revealed that the clock genes are expressed before the formation of the central nervous system in embryos and that their transcripts might be inherited maternally. The clock genes are diferentially modulated during the larval and pupal development and, except for tim2 and cwo, all of them respond to the treatment with Juvenile Hormone (JH) in white-eyed pupae. The positive response to JH by clk, cyc and pdp1 might be related to the involvement of these genes on the pathways of the JH signaling, interacting with Kruppel (Kr-h1) and Methoprene-tolerant (MET) genes. In the adult development, the clock genes per and cry-m are potential molecular markers of the behavioral plasticity and division of labor in a single-cohort colony, once they did not exhibit transcriptional oscillations in heads of young bees (3 and 7 days-old) during 24h, compared to the robust transcriptional oscillation in old bees (15 and 25 days-old). Additionally, we reconstructed protein-protein and miRNA-mRNA interaction networks and identified putative molecules involved in the post-transcriptional and translational regulation of the clock genes. Among those molecules, we validated interactions between the miR-34 and its binding sites in the 3`UTR of cyc and cwo by luciferase assay, showing that this miRNA is a negative regulator of both clock genes. We showed for the first time a broad analysis of the circadian clock elements in a social insect, and also identified news molecules with potential to act as modulators of the circadian rhythms. This work expands the knowledge about the biological roles of the circadian clock in honeybees. Our work also contributes to highlight the importance of honeybees as an ideal model to uncover the molecular mechanisms that govern the circadian rhythms, not only in bees, but in other organisms, including mammals.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Melatonin is hormone regulated by the light/dark cycle. It is considered to be a signaling pathway of the circadian clock located in the hypothalamic suprachiasmatic nucleus. Melatonin also regulates functions involving the energetic and behavioral metabolism of mammals. However, the melatonin synthesis can suffer influence from other hormones, like insulin and other glucocorticoids, in pathological conditions and stress. Studies have shown that glucocorticoids seem to modulate clock genes in peripheral tissues leading to metabolic diseases. The objective of the present study was to evaluate the functionality of the pineal gland and clock genes modulation in rats treated chronically with dexamethasone. Male Wistar rats (200-250g) were divided in to two groups: Control (CON) and Dexamethasone-treated (DEX). Animals from DEX group had 2mg/kg body weigh of dexamethasone administered intraperitoneally for 10 consecutive days and control animals received equal volume of saline solution also intraperitoneally. The circadian profile of blood glucose was evaluated and presented increased points of hyperglycemia during the night (p<0,05). The animals were sacrificed at each time point and had their pineal gland and plasma collected. The obtained data demonstrate a significant hyperinsulinemia in DEX animals (p<0,05). The findings show a reduction in the melationin content (p<0,05) associated with a decrease in the enzymatic activity of AANAT (p<0,05). Also, in these points, DEX animals presented a reduction in the glucose transporter (Glut1) in the pineal gland and insulin receptor (Insr) (p<0,05). The levels of mRNA of the clock genes Bmal1, Per1, Per2, Cry1, Cry2 and Rev-erbα in the isolated pineal glands demonstrated an increase in the gene expression (p<0,05). These results suggest that chronic treatment with dexamethasone is capable of modulating melatonin synthesis, as well as the clock genes. This evidence also suggests the possible presence of a responsive element to the functional glucocorticoid in the promoter region of these genes in the pineal gland. / A melatonina é um hormônio regulado pelo ciclo claro/escuro. Sendo considerado um sinal de saída do relógio circadiano localizado no núcleo supraquiasmático do hipotálamo. A melatonina regula funções envolvendo o metabolismo energético e comportamental em mamíferos. A síntese de melatonina pode ser influenciada por outros hormônios, a exemplo da insulina e dos glicocorticoides em condições patológicas e estresse. Estudos têm demonstrado que os glicocorticoides parecem modular os genes relógio em tecido periférico ocasionando doenças metabólicas. O presente estudo teve como objetivo avaliar a funcionalidade da glândula pineal e a modulação dos genes relógio em ratos tratados cronicamente com dexametasona. Ratos Wistar (200-250g) foram divididos em 2 grupos: Controle (CON) e Tratados com dexametasona (DEX). Os animais DEX receberam 2mg/kg de peso de dexametasona intraperitonial durante 10 dias consecutivos e os animais CON receberam o volume correspondente de solução salina intraperitonialmente. Os animais de ambos os grupos tiveram o perfil de glicose avaliado e apresentaram pontos de hiperglicemia acentuada durante a noite (p<0,05). Os animais de ambos os grupos foram sacrificados nos ZT17 ao ZT22 e tiveram a glândula pineal e o plasma coletados. Os dados obtidos mostram um quadro de hiperinsulinemia nos animais DEX (p<0,05). Os animais do grupo DEX apresentaram uma redução no conteúdo de melatonina total (p<0,05) associada à diminuição da atividade enzimática da AANAT (p<0,05). Nesses mesmos pontos os animais DEX apresentaram uma redução do transportador de glicose (Glut1) na glândula pineal e receptor de insulina (Insr) (p<0,05). Os níveis de RNAm dos genes do relógio Bmal1, Per1, Per2, Cry1, Cry2 e Rev-erbα em pineais isoladas apresentaram aumento da expressão gênica (p<0,05). Estes resultados sugerem que o tratamento crônico com dexametasona é capaz de modular a síntese de melatonina, bem com também os genes relógio, estas evidências sugerem a possível presença de um elemento responsivo ao glicocorticoide funcional na região promotora desses genes na glândula pineal.

Selon l'organisation mondiale de la santé, le diabète de type II (DT2), caractérisé par un défaut de contrôle de la glycémie, est une des causes principales de décès dans le monde. Le GLP-1, sécrété par l'intestin après un repas, contribue au contrôle de la glycémie en stimulant la sécrétion d'insuline par le pancréas et en inhibant la vidange gastrique et la prise alimentaire. Ces actions sont principalement médiées par le nerf vague selon un axe intestin-cerveau-organes périphériques, bien que l'hormone puisse aussi agir de manière endocrine directement sur ses organes cibles via son récepteur (GLP-1r). Des stratégies thérapeutiques basées sur le GLP-1 sont donc utilisées pour traiter les patients diabétiques, mais les réponses sont hétérogènes voire inefficaces pour le contrôle glycémique. Les mécanismes moléculaires responsables sont inconnus mais pourraient être en lien avec la modification du microbiote intestinal, élément déterminant dans le développement des maladies métaboliques. Nous avons d'abord montré que des souris rendues diabétiques (régimes riches en graisse) perdent leur sensibilité aux actions hypoglycémiantes du GLP-1 et présentent une neuropathie entérique, une baisse de l'expression du GLP-1r intestinal et vagal et une altération de l'axe intestin-cerveau. De plus, dans les neurones entériques en culture primaire issus de ces souris diabétiques, la production de NO induite par le GLP-1, est diminuée. Tous ces effets sont retrouvés chez des souris axéniques ou traitées aux antibiotiques sous régime normal démontrant l'implication du microbiote. À l'inverse, des souris sous régime gras traitées aux antibiotiques ont une amélioration de l'action du GLP-1. Cette action hormonale intestinale pourrait aussi dépendre du cycle nycthéméral pour lequel nous avons observé une oscillation de la sécrétion d'insuline, de l'expression du GLP-1r et des bactéries intestinales. De plus, les souris contrôles répondent moins bien à l'hormone au cours du jour que de la nuit et les souris diabétiques, axéniques et antibiotiques - modèles résistants au GLP-1 - ont des variations très marquées et communes de l'expression des "clock genes". L'ensemble de ces résultats montre qu'au cours diabète, l'action du GLP-1 est diminuée. Cette diminution peut s'expliquer par une baisse de l'expression neuronale du GLP-1r et une diminution de la voie de signalisation dépendant du NO capable de réguler la sécrétion d'insuline induite par le GLP-1. Le microbiote et/ou la régulation circadienne semblent déterminants dans la sensibilité au GLP-1. / According to the World Health Organisation, Type II Diabetes, characterized by an alteration of glycemic control, causes numerous death around the world. After a meal, gut secretes Glucagon-Like Peptide one (GLP-1) which regulates glycemia by stimulation of insulin secretion and inhibition of gastric emptying and food intake. Although GLP-1 acts as an endocrine hormone on its target organs through the GLP1 receptor, its action is mainly mediated by nervous pathway involving vagus nerve and gut-brain-periphery axis. Thus, GLP-1 based therapies are used to control glycaemia in type 2 diabetic patients, but, efficiency of the treatment is heterogeneous defining a state of GLP-1 unresponsiveness. Molecular mechanisms involved in this unresponsiveness are not known but could be linked to the changes in gut microbiota (dysbiosis), key element in the development of metabolic diseases. We first found that diabetic mice (high fat diet) are unresponsive to hypoglycemic action of GLP-1 and present enteric neuropathy, impaired gut-brain axis and reduction of GLP-1r and neuronal NO synthase expression in the ileum. In addition, GLP-1-induced nitric oxide production in primary neuron culture is decreased. These effects were also found in germ-free or antibiotic-treated mice under normal chow diet, indicating the involvement of gut microbiota. By contrast, high fat diet mice treated with antibiotics show an improvement of GLP-1 action. This gut incretin action could also depend on the circadian cycle for which we observed a wavering of insulin secretion, GLP-1r expression and gut microbiota. Moreover, the GLP-1 response of control mice is better in the day than in the night and the different mice model resistant to GLP-1 (HFD, axenic or antibiotics) present the same marked variations in the expression of major clock genes. Overall our results show that in type 2 diabetes GLP-1 action is lowered and can be explained by decreased neuronal expression of GLP-1r as well as the NO-dependent signaling pathway regulating insulin secretion induced by GLP-1. Microbiota or the circadian clock seems essential in this GLP-1 sensitivity.

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The circadian system is an important coordinator of physiological functions of a mammalian organism. It comprises of a central oscillator represented by cells in the suprachiasmatic nuclei of hypothalamus (SCN) and peripheral oscillators in most if not all cells of peripheral tissues. The peripheral oscillators, similarly to the central ones, generate circadian oscillations at the level of so called clock genes and their protein products. In peripheral tissues, oscillations in expression of the individual clock genes are autonomous, however, they need to be synchronized to ensure their robust rhythmic expression. The peripheral clocks are synchronized mainly by rhythmical signals from the SCN, including signals regulating food intake. Disturbances in the clock gene expressions, as well as impaired synchronization signals, can result in various pathophysiological states. Spontaneously hypertensive rat (SHR) strain is a convenient animal model to study potential connection between the disturbed circadian system and progressive development of hypertension and metabolical diseases in mammals. Various studies have shown differences in the rhythmical expression of clock genes between SHR strain and normotensive Wistar/Wistar-Kyoto strain. The aim of this thesis is to provide insight into the early...

A melatonina (MEL), hormônio sintetizado pela glândula pineal apenas durante a noite, é um marcador circadiano interno. A sua produção depende de uma via neural temporizada pelo núcleo supraquiasmático (NSQ), que culmina na liberação rítmica noturna de noradrenalina na pineal, através de fibras simpáticas vindas do gânglio cervical superior (GCS). Sabendo que a pineal é essencialmente rítmica, foram estudados o relógio molecular da glândula pineal de ratos após GCSx e seus efeitos no relógio molecular do fígado e do NSQ. A GCSx alterou o relógio molecular da pineal, além de reduzir e tornar arrítmica a via de síntese de MEL. O fígado, um oscilador periférico, manteve a robustez de seu relógio, enquanto que o NSQ, oscilador central, teve sua expressão alterada, tanto no grupo GCSx, quanto após a remoção da pineal (PINX). O NSQ coordena os ritmos de atividade motora e temperatura, os quais exibiram alterações após a GCSx, sendo tais alterações ainda mais intensas após a PINX, com a redução da temperatura e aumento da atividade ao longo do tempo. Em conjunto, esses dados demonstram a complexidade do sistema circadiano e a importância da inervação simpática da pineal para, direta ou indiretamente, mantê-la inalterada. / Melatonin (MEL), a hormone synthesized by the pineal gland on the dark phase, is an internal circadian marker. Its production relies on a neural pathway timed by suprachiasmatic nuclei (SCN), which ends with the rhythmic release of norepinephrine by sympathetic fibers from the superior cervical ganglia (SCG). Since the pineal gland is essentially a rhythmic structure, the aim of this study were evaluate the molecular clock of pineal gland after the superior cervical ganglia ablation (SCGx) and its effects on liver and SCN molecular clocks. The SCGs modified the molecular clock of pineal gland and, in addition, the MEL synthesis pathway was decreased and became arrhythmic. Besides that, the molecular clock of liver maintained its robustness, while the SCN\'s, the master clock, was changed in SGCx and after pineal removal. The SCN coordinates the rhythms of motor activity and temperature. Those are changed after SCGx, although the removal of pineal gland had changed them more, decreasing the temperature and increasing motor activity over the time. Together, these data show the complexity of the circadian system and the relevance of pineal sympathetic innervation to, direct or indireclty, maintain it unaltered.