Circadian clocks are internal timekeepers that allow organisms to anticipate and exploit predictable daily changes in their environment, aiding survival. Clock-driven rhythms, such as asexual spore development (conidiation) in Neurospora crassa, show temperature compensated periodicity that persists in constant conditions and can be reset by environmental time cues. This ability of circadian clocks to maintain a constant period and phase of behaviour over a range of temperatures is important, and whilst much of the machinery making up the circadian clock is known, the mechanism that underpins temperature compensation is not well understood. Further, it is unknown how the clock can control conidiation in the face of changing temperatures. To investigate possible mechanisms underlying temperature compensation, I first explored how compensation may arise within the central clock machinery using a comprehensive dynamic model of the Neurospora crassa circadian clock. This clock incorporates key components of the clock, and I introduced known temperature-sensitive component changes based on experimental observations. This analysis indicated that temperature-dependent changes in the binding of CK-1a to the FRQ-FRH complex may be pivotal in the temperature compensation mechanism. Previous work has highlighted the importance of the blue-light photoreceptor VIVID (VVD), as VVD knockout strains show a temperature-dependent delay in the phase of peak conidiation. Next I explored this potential role using a theoretical output model. By incorporating regulation of this pathway by VVD, I found that VVD may contribute to phase control by increasing expression of genes or proteins that peak early on in the output pathway. RNA-Seq experiments were carried out to assess the contribution of VVD to the overall transcriptomic profile of Neurospora. The analysis highlighted several key genes through which VVD may regulate the conidiation pathway, including the clock-controlled genes eas and ccg-9, which both show temperature- and strain-dependent changes in expression patterns over the time course of conidiation. In conclusion, VVD may indeed have an important role in the temperature compensation of output pathways, though further work is needed to assess the specific
contributions of genes highlighted by my RNA-Seq analysis to the compensatory mechanism.

Many processes in mammalian body exhibit circadian rhythms. These rhythms are driven by an intricate system composed of the central pacemaker, suprachiasmatic nuclei (SCN) in the brain, which entrains the peripheral oscillators in various organs, such as pancreas, liver, colon and lungs. Circadian clocks are autonomously driven in each cell based on molecular circuits involving so called clock genes, such as BMAL, CLOCK, PER and CRY. Age- dependent impairment of physiological functions of mammalian body, such as behaviour and metabolic functions, has been well documented. However, it has not been fully elucidated whether the impairment is linked with worsening of the circadian clock function. The aim of our study was to find out whether i) aging affects basic properties of the circadian clock in SCN and peripheral organs, such as pancreas, colon, liver and lungs, ii) aging- induced changes in glucose homeostasis affect the properties of the circadian clock in the pancreas, and iii) the sensitivity of circadian clock in SCN and peripheral organs to disturbances in environmental lightning conditions is altered during aging. We used groups of adult (9 months) and aged (25 months) animals which were subjected to 3 different light regimes, namely to light/dark regime (LD 12:12), constant light (LL) and...

La séquence des événements moléculaires engendrés par des perturbations de signaux externes qui peuvent affecter les horloges circadiennes, et générer des pathologies restait peu connue. Durant ma thèse, j’ai démontré au niveau moléculaire, comment déplacer l’horaire de l'alimentation chez la souris de la phase active à la phase de repos, altère le métabolisme à la suite d’une hypoinsulinémie durant la phase active, ce qui provoque une activation de PPARα qui reprogramme le métabolisme et l'expression de RevErbα et qui de ce fait décale l’horloge de 12h dans les tissus périphériques. Notamment, l’absence de PPARα dans le noyau suprachiasmatique empêche le décalage de l’horloge centrale. Ainsi, les phases d’activité et de repos contrôlées par l’horloge centrale ne sont plus alignées avec l'expression des gènes contrôlée par les horloges périphériques. Ce non-alignement crée un syndrome métabolique similaire à celui observé chez des individus soumis à des horaires de travail décalés. / The sequence of molecular events through which alterations in externals cues may impinge on circadian clocks, and generate pathologies, was mostly unknown. During my thesis work, I have molecularly deciphered, how switching feeding in mice, from the “active” to the "rest" phase [Restricted Feeding (RF)] , alters the metabolism through hypoinsulinemia during the “active” phase, leading to increased PPARα activity, thereby reprograming both metabolism and RevErbα expression and leads to a 12h circadian clock-shift in peripheral tissues.Most notably, the lack of PPARα expression in the suprachiasmatic nuclei (SCN) prevents a shift of the central clock. Therefore, the “active” and “rest” phases controlled by the SCN clock and gene expression controlled by the peripheral circadian clocks are misaligned. Most interestingly, this misalignment generates a metabolic syndrome-like pathology, similar to that associated with shiftwork schedules.

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.

Orientador: Michel Georges Albert Vincentz / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T14:21:25Z (GMT). No. of bitstreams: 1 Viana_AmericoJoseCarvalho_D.pdf: 5804951 bytes, checksum: a3c65fa34ad298641a9b177952050af3 (MD5) Previous issue date: 2014 / Resumo: O fator de transcrição do tipo basic leucine leucine zipper (bZIP) de Arabidopsis thaliana AtbZIP63 faz parte da via de resposta a carência energética coordenada pelas quinases KIN10/11, integradoras centrais dos sinais relacionados ao estado de privação de energia. O mutante de inserção de T-DNA atbzip63-2 apresenta uma redução do crescimento e desenvolvimento das folhas assim como um atraso do florescimento em comparação ao tipo selvagem (TS, acesso Ws) quando cultivado em fotoperíodo de dia curto (10 h/14 h). Condições de fotoperíodo de dia longo ou luz contínua promoveram uma reversão parcial ou completa, respectivamente, do fenótipo mutante para o tipo selvagem, levantando a possibilidade de que este fenótipo seja o resultado de uma carência energética. Plantas silenciadas para expressão de AtbZIP63 por RNAi apresentaram características similares a do mutante atbzip63-2 confirmando o envolvimento deste fator de transcrição no crescimento. O perfil de expressão gênica e os níveis de alguns metabólitos do mutante atbzip63-2 indicaram que AtbZIP63 participa do controle da degradação do amido, pois a expressão de alguns genes centrais na degradação deste carboidrato de reserva está desregulada neste mutante. Mostramos que as oscilações no nível do transcrito AtbZIP63 são reguladas pelo relógio circadiano e a fase da oscilação do AtbZIP63 é aparentemente influenciada pela disponibilidade de carboidratos na célula. Além de estar sob o controle do relógio, AtbZIP63 também atua como um ativador direto da expressão de PRR7, que codifica um dos componentes chave do oscilador central do relógio. Portanto, evidenciamos uma interação recíproca entre o relógio e AtbZIP63 que possivelmente está impactando o processo de degradação do amido à noite. Este conjunto de evidências revela novos aspectos do ajuste do relógio circadiano pelo status de açúcar na célula que estão de acordo com trabalhos recentes mostrando que os açúcares afetam diretamente o funcionamento do relógio. Nossa hipótese é que o AtbZIP63 está agindo como um mediador entre a disponibilidade de viii açúcar e o mecanismo oscilatório do relógio circadiano de A. thaliana. Adicionalmente, verificamos que o perfil de transcritos no final do dia no mutante atbzip63-2 é diferente do observado no final da noite, sugerindo a participação do AtbZIP63 na regulação de genes envolvidos em redes regulatórias distintas em função do período do dia. Dentre os genes desregulados no atbzip63-2 no final do dia, observamos um enriquecimento para genes relacionados com metabolismo secundário e síntese de trealose, o que sugere a participação do AtbZIP63 na regulação da síntese destes compostos durante o dia, e possivelmente reflete a ocorrência de stress no mutante / Abstract: he Arabidopsis thaliana basic leucine zipper domain (bZIP) AtbZIP63 transcription factor is part of the response pathway to energy shortage coordinated by kinases KIN10/11. The T-DNA insertion mutant atbzip63-2 shows a reduction in the growth and development of leaves, as well as a delay in flowering compared to wild type (WT; ecotype Ws), when grown in short-day conditions. Long day or continuous light conditions promoted a partial or complete reversion, respectively, of the mutant to wild-type phenotype, raising the possibility that this phenotype is the result of an energy shortage. Plants silenced for AtbZIP63 showed similar characteristics to the atbzip63-2 mutant, confirming the involvement of this transcription factor in the growth. The gene expression profile and the levels of some metabolites of the atbzip63-2 indicated that AtbZIP63 takes part in the control of starch degradation, regulating the expression of some key genes in starch degradation. Diurnal AtbZIP63 mRNA level fluctuation is regulated by the circadian clock, and the phase oscillation is influenced by the availability of carbohydrates. In addition, to be controlled by the circadian clock, AtbZIP63 directly regulates the expression of PRR7 which encodes one of the key regulators of the core clock. We have therefore identified a reciprocal interaction between the clock and AtbZIP63 which is probably affecting the starch degradation process. This set of evidence reveals new aspects of the entrainment of the circadian clock by sugars, and is consistent with recent studies showing that sugars directly regulate the circadian clock. Our hypothesis is that AtbZIP63 is acting as a mediator between the energy status (availability of sugar) and the oscillatory mechanism of the A. thaliana circadian clock. Additionally, we found that the profile of transcripts at the end of the day in atbzip63-2 mutant is different from that observed in the end of the night, suggesting the involvement of AtbZIP63 in the regulation of genes involved in distinct regulatory networks according to the period of day. Among the genes deregulated in atbzip63-2 at the end of the x day, an enrichment for genes related to secondary metabolism and trehalose biosynthesis was observed. Suggesting the involvement of AtbZIP63 in regulating the synthesis of these compounds during the day, and probably reflects the occurrence of stress in the mutant / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

No description available.

Les rythmes circadiens contrôlent de nombreux aspects de la physiologie chez les mammifères. Parmi ces processus physiologiques, les horloges circadiennes contrôlent entre autres la réponse immunitaire innée et adaptative. Depuis des décennies, de nombreuses études ont commencé à couvrir ce sujet. Cependant, le contrôle circadien de la réponse adaptative reste peu étudié. Dans le cadre de ce projet de recherche de doctorat, nous avons exploré le rôle des rythmes circadiens dans la réponse des lymphocytes T CD8 à la présentation antigénique par des cellules dendritiques. Des travaux du laboratoire publiés par Erin E. Fortier et al. ont mis en évidence une différence jour/nuit dans l’expansion des lymphocytes T CD8 dont le récepteur T (TCR) est spécifique au complexe KbOVA exprimé par les cellules dendritiques ainsi que dans le nombre de lymphocytes T CD8 CD44hi IFN+ spécifiques pour l’antigène de l’ovalbumine (OVA)1. En effet, la réponse des lymphocytes T CD8 est plus importante après une vaccination faite en milieu de jour (zeitgeber time (ZT) 6) par rapport à une vaccination faite en milieu de nuit (ZT18). Cependant, ces travaux de recherche n’ont pas démontré le rôle des horloges circadiennes dans le rythme de réponse des lymphocytes T CD8 à la présentation antigénique. Mes travaux de recherche de doctorat ont dans un premier temps confirmé l’implication des horloges circadiennes dans le rythme de réponse des lymphocytes T CD8 à la présentation antigénique. Nous avons ensuite démontré la contribution des horloges circadiennes des cellules dendritiques ainsi que le rôle essentiel des horloges circadiennes des lymphocytes T CD8 dans ce rythme de réponse. De plus, nous avons montré que ce rythme avait un impact sur la capacité des lymphocytes T CD8 à contrôler une infection bactérienne (Listeria monocytogenes). En effet, les variations jour/nuit de la charge bactérienne dans la rate et le foie des souris de type sauvage étaient abolies dans les souris déficientes pour le gène des horloges circadiennes Bmal1 dans les lymphocytes T CD8. Dans un second temps, nous avons mis en évidence suite à l’analyse du transcriptome des lymphocytes T CD8 de souris naïves collectés toutes les 4 heures sur 48 heures que ces cellules sont plus enclines à être activées le jour et à l’opposé plus enclines à être inhibées la nuit. Ces résultats corrèlent avec le rythme de réponse des lymphocytes T CD8 à la vaccination. Dans un dernier temps, nous avons confirmé que le rythme de réponse des lymphocytes T CD8 à la présentation antigénique agissait de manière précoce dans l’activation de ces cellules. Pour cela nous avons irradié des souris de type sauvage et nous les avons ensuite reconstituées avec une moelle osseuse contenant 1% de précurseurs de cellules OT-I (lymphocytes T CD8 spécifiques au complexe KbOVA, exprimant la chaîne du TCR V5. Après vaccination de ces souris en milieu de jour subjectif (circadian time (CT) 6) ou en milieu de nuit subjective (CT18), nous avons observé un rythme de la réponse des lymphocytes CD8 pour différents marqueurs impliqués dans la réponse précoce des lymphocytes T CD8, telles que CD69, CD5, IRF4, et la phosphorylation de S6 (marqueur de l’activité de mTOR) et de AKT. L’ensemble des recherches de mon doctorat ont permis de mettre en évidence un tout nouveau mécanisme impliquant les horloges circadiennes dans la réponse des lymphocytes T CD8 en réponse à une vaccination. Une meilleure compréhension du fonctionnement des horloges circadiennes dans la réponse immunitaire permettra de mettre en place des nouveaux traitements personnalisés en fonction du type d’infection et du type de maladie, délivré à un certain moment de la journée dans le but d’améliorer l’efficacité tout en réduisant les effets secondaires. / Circadian rhythms control various aspects of the physiology in mammals. Among these processes, circadian clocks control the innate and the adaptive immune responses. Since few decades, numerous studies started to uncover the role of the circadian system in the immune response. However, the circadian control of the adaptive immune response remains poorly studied. My PhD work focused on the circadian control of the CD8 T cell response to vaccination by dendritic cells. Erin E. Fortier et al. published in The Journal of Immunology that wild type mice vaccinated with antigen presenting cells loaded with the OVA peptide present a day/night variation of the CD8 T cell response after a vaccination done during the middle of the day (zeitgeber time (ZT) 6) compared to a vaccination done during the middle of the night (ZT18)1. Indeed, the proportion of CD8 KbOVA+ cells and the proportion of CD8 CD44hi IFN+ T cells were higher during the middle of the day than the middle of the night. However, this work showed a diurnal but not a circadian rhythm, that remained to be confirmed. The first part of my PhD research confirmed a role of the circadian system in the rhythm of the CD8 T cell response to vaccination. We showed a contribution of the dendritic cell clock as well as an essential role of the CD8 T cell clock. Moreover, this rhythm impacts the ability to control an infectious challenge as shown by a circadian variation in bacterial load (Listeria monocytogenes) in wild type but not in mice lacking clock in mature CD8 T cells. The second part of my research focused on the analysis of the transcriptome of CD8 T cells from naive mice collected every 4 hours over 48 hours. We showed that CD8 T cells are more prone to be activated during the day and at the opposite are more prone to be inhibited during the night. These results are correlated with the rhythm of the CD8 T cell response to vaccination. Finally, during the third part of my PhD, we confirmed that the rhythm of the CD8 T cell response to antigen presentation was acting at the early stage of the CD8 T cell activation. We used wild type mice reconstituted with bone marrow cells containing 1% of OT-I precursor cells (CD8 T cells restricted for the KbOVA complex, expressing the receptor chain of the TCR for the OVA peptide, V5) and vaccinate these mice during the middle of the subjective day (CT6) or during the middle of the subjective night (CT18). At the early stage of the CD8 T cell response to vaccination, we showed a higher expression of several activation markers after a vaccination done during the middle of the day than during the middle of the night, such as CD5, CD69, IRF4 and the phosphorylation of S6 (marker of the mTOR activity) and AKT. Altogether, my PhD work highlights a new mechanism involving the circadian system in the control of the immune response. A better understanding of how circadian clocks act on the immune response will allow implementing new treatment strategies in order to increase their efficacy as well as to decrease side effects.

Ectopic calcification can cause pain and limit mobility. Studies suggest that circadian genes may play a role in the calcification process. Core circadian genes Clock, Npas2, and Bmal1 are transcription factors that form CLOCK:BMAL1 or NPAS2:BMAL1 transactivator complexes that drive the rhythmic expression of circadian oscillator genes and output genes. Circadian oscillator genes Period1-3 and Cryptochrome1-2 encode proteins that form transcription repressor complexes that feedback to inhibit CLOCK/NPAS2:BMAL1 activity, thus completing the feedback loop that is the basis of the molecular circadian clockwork. Arrhythmic Bmal1-/- mice exhibit site-specific, age-dependent arthropathy. While studying the circadian phenotype of Clock-/-;Npas2m/m double mutant mice, we discovered that these double mutant mice develop site-specific arthropathy similar to the arthropathy described in Bmal1-/- mice. Based on the circadian clockwork mechanism, we hypothesized that CLOCK/NPAS2:BMAL1 transactivator complexes drive the expression of a gene (or genes) that prevents age-dependent arthropathy. To investigate Clock-/-;Npas2m/m double mutant mouse arthropathy, we evaluated mutant mice using X-ray, micro-computed tomography, and histology, and found that Clock-/-;Npas2m/m double mutant mice exhibit age-dependent, site-specific arthropathy that phenocopies that of Bmal1-/- mice. The costosternal junction and calcaneal tendon are most prominently affected, in that calcification of those tissues is detectable as early as 4-5 weeks and 11-12 weeks, respectively. The arthropathic lesions in these tissues consist of calcium phosphate vii deposits, and in Bmal1-/- costosternal junction calcifications, the deposits contain calcium pyrophosphate dihydrate crystals. Mechanical stress, disregulation of centrally-regulated circadian rhythms, and systemic serum mineral imbalances likely do not contribute to this pathology. In vitro micromass cultures generated from Clock-/-;Npas2m/m double mutant mouse embryonic fibroblasts do not exhibit irregular chondrocyte differentiation compared to wild-type cultures, suggesting that chondrocyte cell-autonomous mechanisms are insufficient to induce this arthropathy. Analysis of Clock-/-;Npas2m/m double mutant intersternebral tissue RNA did not reveal significant changes in chondrocyte or calcification-related gene expression. Histological stains showed an absence of osteoblasts and osteoclasts around costosternal junction calcifications, suggesting that these cell types are not contributing to this pathology. Instead, chondrocytes are localized to the costosternal junction but there were no significant changes in the distribution of chondrocyte markers in this tissue, as evaluated by immunohistochemistry. These findings suggest that Clock or Npas2, and Bmal1, regulate ectopic calcification through a combination of systemic and local factors, and that the cells affected by Clock and Npas2, or Bmal1, disruption are a subset of the cells distributed in specific tissues that develop age-dependent arthropathy. The significance of these findings is that “circadian genes” play a role in the regulation of ectopic calcification in a non-oscillator capacity. Understanding this new mechanism by which ectopic calcification is controlled could lead to novel approaches for the treatment of some human calcification diseases.