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Temperature controls the entrainment of Drosophila melanogaster locomotor activity to different seasonal conditions through the regulation of splicing of the intron within the 3' UTR of period (per) mRNA. This analysis of per 3' splicing and behaviour in different genetic backgrounds revealed that at low temperatures per splicing is regulated primarily by the light input received via the visual system. At higher temperatures, regulation is more stringent, requiring the visual system input during the day and the circadian clock at night. This is surprisingly as cryptochome is assumed to be a circadian photoreceptor, yet regulates splicing primarily as a clock component. Conversely, the visual component, no-receptor-potential-A, appears to have an additional circadian role, probably in thermosensitivity, as a mutation generated levels of 3' splicing that could not be explained by its effect on the visual pathway.;This work also resulted in the identification of a unique circadian phenotype. per01; cryb mutants display an evening activity peak anticipating 'lights off' in light: dark cycles, which should be clock dependent, yet are arrhythmic under constant conditions, signifying the absence of a clock. This anticipation is not seen in tim01; cryb mutants, so is probably the result of the light dependent degradation of TIMELESS (TIM). Therefore, as per01; cryb flies have residual clock function even in the absence of PER, there must be a PER-independent role for TIM.;Finally, the per gene determines species-specific locomotor behaviour. This is not down to differential per splicing as an examination of D. pseudoobscura and D. virilis failed to discover regulated per splicing in either species. Instead, species-specific differences in behaviour seem to be determined by altered patterns of per mRNA expression. Thus, light and temperature sensitive per splicing has evolved in D. melanogaster, allowing it to 'fine tune' behaviour to the wide variety of habitats it occupies.

Endogenous circadian clocks generate rhythms of physiology and behaviour that are synchronised to the environment, principally through the light-dark cycle. In mammals, the circadian clock is dependent on interlocked feedback loops that involve several clock elements such as cryptochromes (Cryl and Cry2). Post-translational modifications control intracellular trafficking, functionality and degradation of CRY proteins which are keys to the functioning of the clock. CRY protein levels are dependent upon their timely degradation by F-box proteins. This has recently been shown in the afterhours (Afh) mutant carrying a mutation in the F -box gene, Fbxl3. Afh has been shown to lengthen circadian period by stabilising levels of CRY proteins across the circadian cycle. To understand the specific roles of each of the two CRY proteins in circadian regulation, we generated compound mouse mutants to investigate the behavioural and molecular consequences of stabilising either CRY1 or CRY2 protein levels in mice lacking the alternative form of Cry. The circadian wheel-running activity assessed in light:dark and constant environmental conditions for both Cryrl-;FbxI3Afh/Afh and Cry[I-;FbxI3Afh/Afh (stabilising CRY2 and CRY1 protein levels respectively); clearly show a gradual increase in period length in constant darkness as the dosage of Fbxl3Afh is increased. This would suggest that stabilisation of either CRY protein can lengthen the clock, presumably as a result of a prolonged phase of transcriptional repression by either protein. This effect seen in the compound mutants was confirmed at the gene and protein levels.

This thesis focuses on the design, construction and stable integration in mammalian cells of a natural microRNA-based genetic oscillator. This will help both in better understanding the rules underlying the periodic expression of genes observed in major biological processes, such as the circadian clock and cell-cycle, as well as, in generating new tools to probe and investigate the function of a gene in a cell, by allowing not only its over-expression or knock-down, but also its cyclic expression. The circuit involves a positive feedback loop, consisting of a transcription factor (TF) activating itself, and a negative feedback loop, using a natural micro RNA controlled by the TF, which induces degradation of the TF itself. The circuit was built in a modular way, and implemented it in two lentiviral vectors able to infect both dividing and non-dividing cells, hence suitable for many different applications. Since obtaining stable oscillations is non-trivial, a modified version of the oscillator was engineered, including an intermediate step between the TF and the microRNA, to increase the delay in the negative feedback loop. The oscillatory behavior was tested via in vivo time-lapse fluorescence microscopy in both versions of the oscillator, since both the TF(s) and the microRNA are expressed together with fluorescent reporters.

Energy homeostasis and metabolic processes have been shown to be under 24-hour control. Hormones secreted from adipocytes and about 20% of the genes in white adipose tissue exhibit daily variations. Changes in metabolism are able to influence the clock in adipose tissue and disruption of the clock mechanism has been shown to lead to metabolic consequences. The aims of this PhD are firstly to investigate links between peripheral clocks and metabolism by using in vivo human and in vitro experimental models and secondly to investigate a cell model, which might reflect adipocyte biology better than the currently available cell lines. The influence of overweight and type 2 diabetes mellitus on plasma hormones was investigated in men over a 24-hour time course under highly controlled conditions. /' Significantly higher nocturnal plasma melatonin concentrations were observed in obese compared to lean or diabetic subjects. All participants showed clear diurnal variation in plasma leptin, but there was surprisingly no significant difference between the groups, irrespective of whether the data were plotted relative to external clock time or endogenous melatonin phase. Rhythmicity and phase of the expression of core clock genes and clock controlled genes, in a novel adipocyte model derived from IMMORTO™ mouse pre-adipocyte cells, support the presence of a molecular oscillator in those cells. Rhythms were phase advanced by about 5 hours in adipocytes, compared to the pre-adipocytes. Preliminary results revealed that lipolysis in those cells may be gated by the adipocyte clock. Our results suggest that the timing of the leptin secretion into the blood is not influenced by the metabolic state under well controlled conditions whilst the concentration of plasma melatonin is increased in overweight participants. The in vitro results show for the first time a molecular clock in primary-like murine adipose cells. The IMW AT cells appear to be a better model because of the robust rhythmicity of the clock genes. Moreover, the preliminary lipolysis data suggests circadian gating for lipolysis. The different phasing of gene expression in pre-adipocytes and adipocytes suggests possible differences in the response to entraining stimuli and that the molecular oscillator may be influenced by the metabolic or differentiation state of the cell.

Chez les mammifères dont l’Homme, les rythmes circadiens physiologiques et comportementaux sont régulés par l’horloge centrale, localisée dans les noyaux suprachiasmatiques de l’hypothalamus. Possédant une période endogène proche mais pas exactement de 24 heures, cette horloge est constamment synchronisée à la période terrestre par le cycle lumière-obscurité perçu au niveau de l’oeil. Cette synchronisation entraîne l’expression de rythmes appropriés (hormonaux, veille-sommeil, température corporelle, etc.). Les hypothèses de ma thèse sont : 1- une exposition chronique à un spectre lumineux appauvri en longueurs d’ondes courtes, causée par l’opacification du cristallin chez le sujet âgé ou par l’exposition chronique à des lumières artificielles blanches, est à l’origine d’une altération de la réponse du système circadien à la lumière ; 2- une exposition chronique à un spectre lumineux enrichi en longueurs d’ondes courtes chez le sujet jeune, améliore la synchronisation du système circadien, la vigilance, les performances cognitives et la qualité du sommeil. L’objectif de ma thèse est d‘évaluer ces hypothèses selon deux approches : 1. Une approche physiologique : chez le sujet âgé sain, le brunissement physiologique du cristallin oculaire conduit à une filtration des longueurs d’ondes courtes du spectre lumineux. Cette approche inclus la mise au point et la validation d’un système de mesure de transmittance du cristallin in vivo. Ce système est nécessaire pour quantifier la qualité spectrale de la lumière atteignant la rétine. 2. Une approche artificielle : chez des sujets jeunes exposés de manière chronique (63 jours) à des lumières ambiantes blanches ou enrichies en longueurs d’ondes courtes / Physiological and behavioral circadian rhythms in mammals and humans are under the control of a central clock located in the suprachiasmatic nuclei of the hypothalamus. This endogenous clock has a period close to but not exactly 24 hours and therefore needs to be constantly entrained to the 24-h period of the earth, by the light-dark cycle. Light is perceived through the eyes and implicates all the retina’s photoreceptors (rods, cones, melanopsin ganglion cells (ipRGCs)). A properly entrained circadian system leads to an appropriate rhythmic expression of many physiological functions (hormonal secretion, sleep/wake cycles, core body temperature …). My project’s hypotheses are: 1- a chronic exposure to blue deprived light, as occurring in the aged due to lens filtration or under standard indoor lighting, leads to a decreased nonvisual sensitivity to light.; 2- exposure to blue enriched white light in the young subjects enhances non-visual responses to light such as, entrainment of the circadian system, vigilance, mood, sleep quality and cognitive performance. The aim of my thesis is to evaluate these hypotheses using two approaches : 1. A physiological approach: In the aged subject, in whom the ocular crystalline lens specifically filters short wavelength lights, known to be crucial for circadian entrainment. This approach includes the development and clinical validation of a scotopic heterochromatic flicker photometry technique to assess lens transmittance in vivo. This technique is essential to evaluate individual light spectra reaching the retina. 2. An artificial approach: In young subjects chronically exposed (63 days in the Concordia base, Antarctica) solely to standard white or blue enriched white light