O relógio biológico é um mecanismo molecular autossustentado gerador de ritmos. Ele integra vias de percepção das condições ambientais com um oscilador central para gerar respostas fisiológicas rítmicas em escalas diária e sazonal. Nas plantas, o relógio biológico está associado a vias metabólicas e fisiológicas importantes, como fotossíntese. Na cana-de-açúcar, uma gramínea de grande interesse econômico, estudos realizados em condições circadianas mostraram que o relógio biológico tem uma influência superior àquela vista em outras plantas. Assim, este trabalho visa a compreender os mecanismos de funcionamento do oscilador central do relógio biológico da cana-de-açúcar crescida em campo. Para tanto, foram investigados o transcriptoma de diferentes órgãos da cana-de-açúcar; a expressão de isoformas alternativas e de múltiplos alelos dos genes do relógio biológico da cana; e o efeito do sombreamento mútuo das plantas em campo sobre o funcionamento do relógio biológico. Os resultados obtidos sugerem que o relógio biológico é funcional e sincronizado entre os diferentes órgãos da cana-de-açúcar analisados. Os transcritos regulados sinergicamente pelo relógio biológico e pelo ambiente flutuante pertencem a vias metabólicas, fisiológicas e de regulação gênica e epigenéticas todas essenciais à produtividade da cana-de-açúcar. O sombreamento mútuo observado em campo parece alterar a fase de expressão de genes do relógio biológico da cana-de-açúcar. Além disso, eventos de splicing alternativo foram observados nos genes do relógio biológico em condições de baixa temperatura e múltiplos alelos dos genes do relógio biológico são expressos e a regulação de sua expressão parece ser sazonal. / The circadian clock is a self-sustaining molecular mechanism that generates rhythms. It perceives the environmental conditions and connects this pathway with its central oscillator, generating daily and seasonal rhythms of physiological responses. In plants, the circadian clock is associated with major metabolic and physiological pathways. In sugarcane, an economically important grass, previous studies showed that the circadian clock has the largest influence on plants seen so far under circadian conditions. This work aims to understand how the central oscillator of the circadian clock works in field-grown sugarcane. Thus, the transcriptome from different sugarcane organs; the expression of alternative isoforms and multiple alleles of circadian clock genes; and the effect of mutual shading in the field on the circadian clock function were analyzed. The results suggest that there is a functional and synchronized circadian clock in the different sugarcane organs. The transcripts regulated synergistically by the circadian clock and the variable environment are related to metabolic, physiological, genetic or epigenetic pathways, all important to sugarcane productivity. Mutual shading observed in the field seems to change the phase of expression of the sugarcane circadian clock. Besides, alternative splicing events have been reported for circadian clock genes under low temperature conditions and multiple alleles of circadian clock genes are expressed and their expression is likely to be seasonally regulated.

O relógio biológico de plantas é uma rede regulatória de grande relevância para a adaptação dos organismos ao ambiente. Essa rede é composta por diversas vias de controle transcricional e pós-transcricional que se retroalimentam e geram ritmos biológicos. O controle exercido pelo relógio pode ser observado nos mais diversos aspectos da fisiologia e desenvolvimento de plantas. No presente projeto de pesquisa foi investigada a relação entre o relógio biológico e a sinalização por auxinas, uma classe de fitohormônios, em cana-de-açúcar. Foram utilizadas técnicas de expressão gênica, como RT-qPCR, para definição de um protocolo robusto de avaliação de respostas transcricionais a auxinas em plântulas de cana-de-açúcar geradas por organogênese direta. Após 1h da aplicação de 80 µM auxina sintética ácido 1-naftalenoacético, foi possível observar controle transcricional evidente exercido pela aplicação de auxina sobre alguns genes. Também foi observado variação na resposta obtida, dependendo do horário do ciclo circadiano em que o estímulo era oferecido. Esse fenômeno de controle temporal sobre a resposta a um estímulo é chamado gating, sendo de grande relevância para a atuação do relógio biológico de plantas. A partir dessas observações foram realizadas análises de expressão gênica em larga escala, usando oligoarranjos, para compreensão mais aprofundada da conexão entre o relógio biológico e a sinalização por auxinas em cana-de-açúcar. Entre os genes diferencialmente expressos após estímulo com auxina, foi verificado grande presença de genes relacionados a respostas contraestresse biótico. Além disso, as respostas observadas devem estar sobre o controle do relógio biológico de cana-de-açúcar. Diversos genes relacionados a combate a infecções, como quitinases e taumatinas, tiveram sua expressão alterada após aplicação de auxinas, sendo possível observar diferenças no padrão de expressão dos genes dependendo do horário em que auxina era aplicada. Dessa forma, o relógio biológico de cana-de-açúcar, a partir da sinalização por auxinas, deve exercer controle sobre as respostas a estresses bióticos nesse organismo. Os dados obtidos são inéditos e podem contribuir para o aumento da produtividade de cana-de-açúcar assim como para o desenvolvimento de novas ferramentas biotecnológicas focadas nesse cultivar, o qual apresenta grande relevância econômica / The circadian clock is a regulatory network with great relevance to fitness of plants. This network creates biological rhythms, influencing plants metabolism and their interaction with the environment. The clock is composed of interlocking feedback transcriptional and post-transcriptional pathways. In the presente study, we investigated the interconnection between circadian clock and signaling through auxins, a group of phytohormones with great impact to plant biology. Using RT-qPCR, it was established a protocol to measure transcriptional responses after synthetic auxin 1-naphtalenacetic acid (NAA) treatment. The biological material used was leaves of sugarcane plantlets generated by direct organogenesis. After 1h treatment with 80 µM NAA, we observed obvious transcriptional responses in sugarcane plantlets. It was also possible to detect alterations of transcriptional responses according to the moment when the stimulus was offered. This temporal control is called gating and is of great relevance to plant circadian clocks. We then performed transcriptomic analysis, using oligoarrays, to get a deeper understanding of the results obtained. Indeed, it was verified that auxin stimulus is connected to biotic stress transcriptional responses and that these responses are clock-controlled. Transcripts coding for proteins like chitinases and thaumatins, which are related to biotic stress responses, were differentially expressed after auxin treatment. Also, the response of most genes was daytime-dependent. We conclude that sugarcane circadian clock, through auxin signaling, might exert control under biotic stressresponses in sugarcane. The data obtained are novelty and may contribute to increase sugarcane productivity and/or to development of new biotechnological tools dedicated to this cultivar.

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.

No description available.

O relógio biológico é um mecanismo molecular autossustentado gerador de ritmos. Ele integra vias de percepção das condições ambientais com um oscilador central para gerar respostas fisiológicas rítmicas em escalas diária e sazonal. Nas plantas, o relógio biológico está associado a vias metabólicas e fisiológicas importantes, como fotossíntese. Na cana-de-açúcar, uma gramínea de grande interesse econômico, estudos realizados em condições circadianas mostraram que o relógio biológico tem uma influência superior àquela vista em outras plantas. Assim, este trabalho visa a compreender os mecanismos de funcionamento do oscilador central do relógio biológico da cana-de-açúcar crescida em campo. Para tanto, foram investigados o transcriptoma de diferentes órgãos da cana-de-açúcar; a expressão de isoformas alternativas e de múltiplos alelos dos genes do relógio biológico da cana; e o efeito do sombreamento mútuo das plantas em campo sobre o funcionamento do relógio biológico. Os resultados obtidos sugerem que o relógio biológico é funcional e sincronizado entre os diferentes órgãos da cana-de-açúcar analisados. Os transcritos regulados sinergicamente pelo relógio biológico e pelo ambiente flutuante pertencem a vias metabólicas, fisiológicas e de regulação gênica e epigenéticas todas essenciais à produtividade da cana-de-açúcar. O sombreamento mútuo observado em campo parece alterar a fase de expressão de genes do relógio biológico da cana-de-açúcar. Além disso, eventos de splicing alternativo foram observados nos genes do relógio biológico em condições de baixa temperatura e múltiplos alelos dos genes do relógio biológico são expressos e a regulação de sua expressão parece ser sazonal. / The circadian clock is a self-sustaining molecular mechanism that generates rhythms. It perceives the environmental conditions and connects this pathway with its central oscillator, generating daily and seasonal rhythms of physiological responses. In plants, the circadian clock is associated with major metabolic and physiological pathways. In sugarcane, an economically important grass, previous studies showed that the circadian clock has the largest influence on plants seen so far under circadian conditions. This work aims to understand how the central oscillator of the circadian clock works in field-grown sugarcane. Thus, the transcriptome from different sugarcane organs; the expression of alternative isoforms and multiple alleles of circadian clock genes; and the effect of mutual shading in the field on the circadian clock function were analyzed. The results suggest that there is a functional and synchronized circadian clock in the different sugarcane organs. The transcripts regulated synergistically by the circadian clock and the variable environment are related to metabolic, physiological, genetic or epigenetic pathways, all important to sugarcane productivity. Mutual shading observed in the field seems to change the phase of expression of the sugarcane circadian clock. Besides, alternative splicing events have been reported for circadian clock genes under low temperature conditions and multiple alleles of circadian clock genes are expressed and their expression is likely to be seasonally regulated.

O relógio biológico de plantas é uma rede regulatória de grande relevância para a adaptação dos organismos ao ambiente. Essa rede é composta por diversas vias de controle transcricional e pós-transcricional que se retroalimentam e geram ritmos biológicos. O controle exercido pelo relógio pode ser observado nos mais diversos aspectos da fisiologia e desenvolvimento de plantas. No presente projeto de pesquisa foi investigada a relação entre o relógio biológico e a sinalização por auxinas, uma classe de fitohormônios, em cana-de-açúcar. Foram utilizadas técnicas de expressão gênica, como RT-qPCR, para definição de um protocolo robusto de avaliação de respostas transcricionais a auxinas em plântulas de cana-de-açúcar geradas por organogênese direta. Após 1h da aplicação de 80 µM auxina sintética ácido 1-naftalenoacético, foi possível observar controle transcricional evidente exercido pela aplicação de auxina sobre alguns genes. Também foi observado variação na resposta obtida, dependendo do horário do ciclo circadiano em que o estímulo era oferecido. Esse fenômeno de controle temporal sobre a resposta a um estímulo é chamado gating, sendo de grande relevância para a atuação do relógio biológico de plantas. A partir dessas observações foram realizadas análises de expressão gênica em larga escala, usando oligoarranjos, para compreensão mais aprofundada da conexão entre o relógio biológico e a sinalização por auxinas em cana-de-açúcar. Entre os genes diferencialmente expressos após estímulo com auxina, foi verificado grande presença de genes relacionados a respostas contraestresse biótico. Além disso, as respostas observadas devem estar sobre o controle do relógio biológico de cana-de-açúcar. Diversos genes relacionados a combate a infecções, como quitinases e taumatinas, tiveram sua expressão alterada após aplicação de auxinas, sendo possível observar diferenças no padrão de expressão dos genes dependendo do horário em que auxina era aplicada. Dessa forma, o relógio biológico de cana-de-açúcar, a partir da sinalização por auxinas, deve exercer controle sobre as respostas a estresses bióticos nesse organismo. Os dados obtidos são inéditos e podem contribuir para o aumento da produtividade de cana-de-açúcar assim como para o desenvolvimento de novas ferramentas biotecnológicas focadas nesse cultivar, o qual apresenta grande relevância econômica / The circadian clock is a regulatory network with great relevance to fitness of plants. This network creates biological rhythms, influencing plants metabolism and their interaction with the environment. The clock is composed of interlocking feedback transcriptional and post-transcriptional pathways. In the presente study, we investigated the interconnection between circadian clock and signaling through auxins, a group of phytohormones with great impact to plant biology. Using RT-qPCR, it was established a protocol to measure transcriptional responses after synthetic auxin 1-naphtalenacetic acid (NAA) treatment. The biological material used was leaves of sugarcane plantlets generated by direct organogenesis. After 1h treatment with 80 µM NAA, we observed obvious transcriptional responses in sugarcane plantlets. It was also possible to detect alterations of transcriptional responses according to the moment when the stimulus was offered. This temporal control is called gating and is of great relevance to plant circadian clocks. We then performed transcriptomic analysis, using oligoarrays, to get a deeper understanding of the results obtained. Indeed, it was verified that auxin stimulus is connected to biotic stress transcriptional responses and that these responses are clock-controlled. Transcripts coding for proteins like chitinases and thaumatins, which are related to biotic stress responses, were differentially expressed after auxin treatment. Also, the response of most genes was daytime-dependent. We conclude that sugarcane circadian clock, through auxin signaling, might exert control under biotic stressresponses in sugarcane. The data obtained are novelty and may contribute to increase sugarcane productivity and/or to development of new biotechnological tools dedicated to this cultivar.

Legionella pneumophila est un pathogène opportuniste avec un cycle de vie intracellulaire obligatoire, ils arrivent à se répliquer dans leurs cellules hôtes comme des protozoaires, en exploitant les protéines et les voies de signalisation de l’organisme infecté pour échapper à sa réponse immunitaire. L. pneumophila est décrite comme un organisme sans oscillations circadiennes, pourtant elle possède des gènes homologues aux gènes circadiennes kaiBC de cyanobactéries. En appliquant un système d’infections in vitro et in vivo ainsi qu’une approche de génomique comparative et fonctionnelle sur l’ organisme modèle Legionella pneumophila mon projet a pour objectif de caractériser le rôle des gènes kaiBC de L. pneumophila. Les protéines KaiABC de cyanobactéries encodent un oscillateur circadien permettant la coordination et l’optimisation temporelle de divers processus biologiques ainsi que l’adaptation aux fluctuations quotidiennes (comme la production d’oxygène via la photosynthèse pendant le jour et la fixation d’azote dans l’obscurité). Notre étude montre que kaiC, kaiB, avec le lpp1114 (codant pour une protéine à plusieurs domaines), l’ensemble constitue une unité transcriptionnelle sous la commande du facteur sigma RpoS. Les souches mutantes de l'opéron kai affichent une haute sensibilité sous conditions de stress par le paraquat ou le sel en comparaison avec la souche sauvage. En effect, nos données provenant d’une expérience utilisant des systèmes de double hybride suggèrent que les proteines KaiC et KaiB de L. pneumophila n’interagissent pas comme ceux de cyanobactéries. Cependant, une version étendue de L. pneumophila KaiB contenant des résidus de C-terminal de T. elongatus est capable d’interagir avec KaiC. Nous démontrons aussi que la structure cristalline de KaiB de L. pneumophila révèle un pliage pareil a ceux de thiorédoxine (protéine d'oxydoréduction) mais manque les résidus de l'extrémité C-terminale important pour l'interaction avec KaiC. En revanche, L. pneumophila KaiC conserve l'activité d'autophosphorylation, mais KaiB ne declénche pas la phosphorylation de KaiC comme chez les cyanobacteries. L'analyse phylogénétique des protéines de Kai indique qu'elles ont été transférés à L. pneumophila et ont évolué de Synechosystis KaiC2B2 et pas du copie circadienne KaiB1C1. Il semble que les protéines Kai de L. pneumophila améliorent son adaptation à des conditions stressantes et aux changements environnementaux. / Legionella pneumophila is an opportunistic pathogen with an intracellular life cycle that uses aquatic protozoa as replication niche and protection from harsh environments. Although L. pneumophila is not known to have a circadian clock, it encodes homologues of the KaiBC proteins of Cyanobacteria that regulate circadian gene expression. By using a wide range of in vitro, in vivo and in silico approaches I characterized the KaiB and KaiC proteins of L. pneumophila The proteins KaiABC of cyanobacteria coordinate a circadian oscillator that regulates many physiological functions in the cells according to the day and the night time induce by the rotation of the Earth (e.g. they do photosynthesis during the day and nitrogen fixation during the night). We show that L. pneumophila kaiB, kaiC and the downstream gene lpp1114, are transcribed as a unit under the control of the stress sigma factor RpoS. Mutant analyses revealed that the kai operon-encoded proteins increase fitness of L. pneumophila in competitive environments, and confer higher resistance to oxidative and sodium stress. Indeed, KaiC and KaiB of L. pneumophila do not interact as evidenced by yeast and bacterial two- hybrid analyses. Fusion of the C-terminal residues of cyanobacterial KaiB to Legionella KaiB restores their interaction. The crystal structure of L. pneumophila KaiB suggests that it is an oxidoreductase-like protein with a typical thioredoxin fold. In contrast, KaiC of L. pneumophila conserved autophosphorylation activity, but KaiB does not trigger the dephosphorylation of KaiC like in Cyanobacteria. The phylogenetic analysis indicates that L. pneumophila KaiBC resemble Synechosystis KaiC2B2 and not the circadian KaiB1C1 copy. Thus, the L. pneumophila Kai proteins do not encode a circadian clock, but enhance stress resistance and adaption to changes in the environments.

No description available.

La rotation de la Terre oblige les organismes vivants à s’adapter aux modifications cycliques de l’environnement, et tout particulièrement aux changements de lumière et de température. Des unicellulaires à l’Homme, la plupart des espèces ont développé des horloges circadiennes, qui leur permettent d’anticiper les transitions jour-nuit. La lumière constitue le signal majeur pour la synchronisation de l’horloge. En cycles jour-nuit, les drosophiles présentent un profil d’activité locomotrice bimodal, avec un premier pic autour de l’aube et le deuxième au crépuscule. Chez cet insecte, la perception de la lumière est assurée à la fois par un système complexe, constitué des yeux composés, des ocelles et de l’eyelet d’Hofbauer-Buchner. Ces organes contiennent des photorécepteurs (PRs) exprimant six protéines photosensibles différentes, les rhodopsines (Rh1 à Rh6). Une septième rhodopsine (Rh7) a été décrite dans quelques neurones de l’horloge cérébrale. La lumière est également perçue directement dans la plupart des neurones d’horloge grâce à une protéine photosensible, le cryptochrome (Cry). Les différentes études du rôle de la lumière sur l’entraînement de l’horloge ont essentiellement porté sur la voie cry-dépendante, en utilisant de courts flashs lumineux pour recaler l’horloge cérébrale. Notre étude s’est intéressée à l’entraînement de l’horloge via les rhodopsines. Quels types de photorécepteur sont impliqués ? Après l’activation de la cascade de phototransduction et la libération de l’histamine par les photorécepteurs, quels neurones, exprimant les récepteurs à l’histamine Ort et Hiscl1, participent à l’entraînement de l’horloge circadienne ? Une première partie présente l’étude de l’implication des 6 rhodopsines dans l’entraînement circadien. Tout d’abord, nous avons mis en évidence la fonction de photorécepteurs spécifiques (exprimant Rh1 ou Rh6) dans la voie NorpA-dépendante (Saint-Charles et al. J Comp Neurol 2016). Nous avons ensuite généré des lignées de drosophiles n’exprimant aucune ou qu’une seule rhodopsine. Sans rhodopsine ni Cry les mouches sont incapables de se synchroniser sur les cycles jour-nuit, quelle que soit l’intensité lumineuse. En lumière faible, l’input pour l’entraînement vient principalement des photorécepteurs exprimant Rh1 et Rh6. En forte lumière, chacune des 6 rhodopsines des différents photorécepteurs est capable d’entrainer l’horloge, Rh1, Rh5 et Rh6 étant les plus efficaces ( Alejevski et al., in prep). Une deuxième partie présente la caractérisation des voies neuronales connectant directement ou indirectement les PRs à l’horloge cérébrale. L’horloge circadienne de mouches mutantes, à la fois pour le cryptochrome et les 2 récepteurs à l’histamine, est « aveugle » alors que les mutantes pour Cry mais possédant l’un ou l’autre récepteur à l’histamine sont capables de se synchroniser sur les cycles de lumière. La ré-expression chez les mutants de Ort ou Hiscl1 dans les neurones d’horloge ne restaure pas l’entraînement, suggérant ainsi l’absence de connexions directes entre les PRs histaminergiques et les neurones d’horloge. Nos expériences de sauvetage comportemental mettent en évidence des connexions fonctionnelles entre certains interneurones Ort des lobes optiques et les neurones d’horloge. En revanche et de façon inattendue, nous n’observons d’entraînement circadien que lorsque nous ré-exprimons Hiscl1 dans les seuls PRs Rh6. Nos résultats révèlent que les photorécepteurs interviennent dans l’entraînement à la fois comme photorécepteurs et comme interneurones, cibles d’input histaminergique, rappelant ainsi le double rôle des cellules ganglionnaires de la rétine exprimant la mélanopsine chez les mammifères (Alejevski et al. Nat Commun, in revision). / The rotation of the earth forces living organisms to adapt to its cyclic environment, in particular light and temperature changes. From unicellular organisms to humans, almost all species have evolved circadian clocks, which allow them to anticipate day-night transitions and use light as the most powerful synchronizing cue. In light-dark cycles, D. melanogaster flies display a bimodal locomotor activity with peaks around dawn and dusk. To perceive light, Drosophila has evolved a complex visual system, composed of compound eyes, ocelli and Hofbauer-Buchner eyelet. These organs contain photoreceptors (PRs) expressing six different light receptors named rhodopsins (Rh1 to Rh6). In addition, one rhodopsin (Rh7) is found in some of the clock neurons in the brain. Most of the clock cells also express another type of light receptor, Cryptochrome (Cry). Most studies about clock entrainment by light have focused on the Cry-dependent light input, which allows short light pulses to reset the brain clock. The present thesis focuses on the entrainment of the brain clock through rhodopsins. In photoreceptors, rhodopsins capture photons and activate a transduction cascade, where a key player is the phospholipase C (PLC) encoded by norpA. Mutants deficient for Cry and NorpA do not synchronize at low light intensity but still entrain with high light, indicating that an unknown NorpA-independent pathway is also used by the clock. Light induces a depolarization of the PRs, which release histamine as a neurotransmitter, but their role in circadian entrainment is unknown. Which type of rhodopsine-expressing photoreceptors are implicated? After the phototransduction cascade activation and the release of histamine from the photoreceptors, which downstream neurons expressing the histamine-gated chloride channels Ort and Hiscl1 (whose function has been studied in the visual behavior) are involved in the circadian entrainment? The first part of the thesis was to study the function of the 6 PR rhodopsins in circadian entrainment. I first contributed to studying the function of the specific photoreceptors in the NorpA-dependent pathway (Saint-Charles et al. J Comp Neurol 2016). Then, we generated genotypes having either none or only one of the six PR rhodopsins. Mutants with no Cry and none of the 6 PR rhodopsins could not synchronize with light-dark (LD) cycles (low light or high light). In low light, Rh1 and Rh6 were the main light input for entrainment. In high-light, each one of the 6 PR rhodopsins can provide entrainment, with Rh1, Rh5 and Rh6 being the most efficient (Alejevski et al., in prep).The second part of the work was to identify the neuronal pathways that connect the PRs to the brain circadian clock. Flies deficient for Cry and the two histamine receptors are circadianly blind, whereas Cry mutants having either Ort or Hiscl1 are able to entrain. Thus, each one of the two receptors supports circadian entrainment. Rescuing Ort or Hiscl1 in the clock cells could not restore entrainment, indicating that there is no direct histaminergic connection between PRs and clock neurons. Our rescue experiments revealed several pathways in otic lobes that rely on Ort-expressing interneurons to entrain the clock. In contrast and unexpectedly, we observed that the expression of Hiscl1 in PRs but not in interneurons was involved in circadian entrainment. In fact, only Hiscl1 expression in Rh6 PRs mediates entrainment. Our work thus reveals Rh6-expressing PRs as both photoreceptors and histamine-receiving interneurons in the rhodopsin-dependent entrainment pathway, which recalls the role of melanopsin-expressing retinal ganglion cells in the mammalian retina (Alejevski et al. Nat Commun, in revision).

Le moment, la qualité et la quantité de sommeil dépendent de l'interaction fine entre l'horloge circadienne et la machinerie homéostatique (Borbely A. et al., 1982, Daan S. et al., 1984, Borbely et Achermann, 1999). Au cours des dernières années, l'utilisation de divers organismes modèles a fourni de nouvelles perspectives sur les mécanismes neuronaux et moléculaires de la régulation du sommeil (Miyazaki S. et al., 2017). Cependant, les bases moléculaires de l'homéostasie du sommeil et les circuits neuronaux sous-jacents à son interaction avec le réseau circadien n'ont pas été établis en détail.Dans ce travail de thèse, j'ai utilisé la mouche Drosophile melanogaster comme système modèle pour étudier la fonction d'un sous-ensemble de neurones d'horloge, les DN1ps, dans la mise en place du sommeil. Des études antérieures ont suggéré un rôle de ces neurones circadiens dans la régulation du sommeil (Kunst et al., 2014, Guo et al 2016, Lamaze et al., 2017, Guo et al., 2017). J’ai ainsi démontré que les cellules d'horloge DN1ps DH31 (+) CRY (+) sont impliquées dans la suppression du sommeil. Par ailleurs, j’ai mis en évidence un circuit en aval des DN1ps, qui comprend le groupe dopaminergique postérieur apparié latéral 1 (PPL1) et les neurones dorsaux en forme d’éventail (dFSB), un centre homéostatique récemment décrit pour la régulation du sommeil chez la drosophile (Donlea JM et al., 2011, Liu S. et al., 2012, Ueno et al., 2012, Donlea JM et al., 2014, Pimentel et al., 2016, Qian et al., 2017, Donlea JM. et al., 2018). Nos résultats indiquent que la suppression du sommeil nocturne nécessite la signalisation DH31-R2 dans une sous-population des neurones dopaminergiques PPL1, qui projette au dFSB. Fait intéressant, la perte de sommeil de jour et de nuit médiée par les DN1ps dépend de l'inhibition du dFSB. Néanmoins, nous suggérons que les neurones DN1ps CRY (-) favoriseraient le sommeil, en concordance avec d'autres travaux (Guo et al., 2016; Guo et al., 2017).Ces résultats fournissent de nouvelles données sur le lien entre l'horloge circadienne et l'homéostasie du sommeil, impliqué dans la régulation du comportement sommeil-éveil chez Drosophile melanogaster. / The timing, quality and quantity of sleep depend on the fine interaction between circadian clock and homeostatic machinery (Borbely A. et al., 1982; Daan S. et al., 1984; Borbely and Achermann, 1999). In the recent years, the employment of various model organisms has provided new insights into the neuronal and molecular mechanisms of sleep regulation (Miyazaki S. et al., 2017). However, the molecular basis of the sleep homeostat and the neuronal circuitry underlying its interaction with the circadian network haven’t been established in details.In this work, I use the fruit fly Drosophila melanogaster as a model system to investigate the sleep function of a subset of clock neurons, the DN1ps. Previous studies have already suggested a sleep-regulating role for these circadian neurons (Kunst et al. 2014, Guo et al. 2016; Lamaze et al., 2017; Guo et al. 2017). Here, we report the DH31-positive CRY-positive DN1ps as sleep suppressing clock cells. Furthermore, we identify a sleep-relevant circuit downstream of the DN1ps which includes the paired posterior lateral 1 (PPL1) dopaminergic cluster and the dorsal Fan-shaped body projecting (dFSB) neurons, a recently described homeostatic center for sleep regulation in Drosophila (Donlea JM. et al., 2011; Liu S. et al., 2012; Ueno et al., 2012; Donlea JM. et al., 2014; Pimentel et al., 2016; Qian et al., 2017; Donlea JM. et al., 2018). Our results indicate that the night-time sleep suppression requires DH31-R2 signaling in the PPL1-to-dFSB dopaminergic neurons. Interestingly, both day and night-time DN1ps-mediated sleep loss rely on the inhibition of the dFSB. Nevertheless, we suggest the CRY-negative DN1ps as sleep promoting clock neurons, in concordance with other works (Guo et al. 2016; Guo et al. 2017).These findings provide a novel link between circadian clock and sleep homeostat, in the regulation of sleep-wake behavior in Drosophila melanogaster.