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Hippocampus is one of the main components of the limbic system. It plays an important role in the process of memory formation and recent literature shows that, like in other peripheral organs and brain structures, there is a circadian clock present in the hippocampus. The aim of the thesis was to study the hippocampal circadian clock under various conditions. The first part of the thesis examines the influence of glucocorticoid hormones on the clock gene expression in the hippocampus. Glucocorticoids are mammalian steroid hormones secreted from the adrenal glands that affect many processes in the organism. The glucocorticoid secretion is under the control of the circadian system, causing their levels to exhibit a pronounced diurnal rhythm. The hippocampus provides feedback to the hypothalamo-pituitary-adrenal (HPA) axis and is thus involved in the regulation of glucocorticoid hormone secretion. Hippocampal neurons also express glucocorticoid hormone receptors (GR). This thesis explores the effect of the absence of glucocorticoid hormones on the clock gene expression in the hippocampus. The absence of glucocorticoids was due to adrenalectomy and its effect was further compared with the effect of endogenous glucocorticoid replacement by daily injections of the GR agonist dexamethasone, emulating...

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The main topic of this thesis are changes in Circadian rhythms caused by cerebellar disorders. Mice with Lurcher mutation, which have specifically degenerated Purkinje cells layer, were choosen as animal model. Our results show that mutation of the glutamate receptor GluRδ2, which causes gradual degeneration of Purkinje cells, leads to damage of Circadian system. Mice with this mutation have reduced capability to adapt to external conditions in different light modes. They are also showing increased variability in endogenous cycle. The mice are also unable to show anticipatory behavior in time-restricted feeding. Compared to control group, affected mice do not show significant rhythm in levels of protein of Bmal1 gene in suprachiasmatic nuclei, paraventricular nuclei nor in habenula. Phosphorylated kinases ERK1/2 and GSK3ß also had distorted rhythms in suprachiasmatic nuclei. Because Circadian oscillations in locomotor activity are partly preserved, Circadian system is likely not damaged on molecular level. Cerebellar mutation hampers synchronization between suprachiasmatic nuclei of neurons and can also affect processes in the ventromedial hypothalamus regulating food intake. Our findings are the first to suggest functional interactions between cerebellum and Circadian pacemaker in suprachiasmatic...

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...

The Dexras1 gene was identified after induction by glucocorticoid dexamethasone in pituitary tumor cells. Dexras1 has also been found in other brain regions and in the peripheral organs but its expression is rhythmic only in the suprachiasmatic nuclei of the hypothalamus (SCN), where the mammalian main circadian pacemaker is located. Dexras1 expression was also affected by stress, amphetamine or prenatal alcohol exposure. Its role in cells has not yet been explained. Dexras1 GTPase activity has been determined to be dependent on the NMDA receptor stimulation. Dexras1 acts as an activator of G protein signaling in cells. Its role has been detected in neuronal iron homeostasis or in the regulation of main circadian pacemaker sensitivity to photic and nonphotic synchronization cues during the day. The aim of our study was to describe the Dexras1 mRNA expression in the rat brain during ontogeny and during development after visual sensory deprivation by in situ hybridization. The earliest Dexras1 expression was detected on embryonic day 20, in the rat SCN and the ventral posteromedial thalamic nucleus. Postnatally, its expression also appeared in other sensory areas, motor thalamic areas, hypothalamic areas involved in the regulation of water homeostasis, or in limbic system. Our results further show...

Many actions we observe in nature show some kind of regularity, therefore we call them rhytms. Rhytms with a period of approximately 24 hours, so called circadian rhytms, can be distinguished in many physiological processes, with the sleep-wake cycle being one of the most prominent ones. Light is the main exogenous circadian synchronizator and thanks to the circadian rhytm influence on physiological function, it's also often spoken about an influence of light on the entire organism. Nowadays, when we spend most of the day indoors under artificial light, we often suffer from a lack of natural daylight and its synchronizing potential. This is even more prominent in elderly population living in nursing homes and in other social facilities. Lately, a new type of lighting, so called biodynamic, has been introduced. Biodynamic lighting can simulate changes in natural light conditions throughout the day and therefore partially compensate for the lack of natural daylight we suffer from, and also minimize risks of the night light. In this study we aimed to test changes in the circadian system of seniors living in the Retirement Home of TGM in Beroun using questionnaires and circadian markers. We have shown a positive effect of the installed biodynamic lighting on our participants' circadian markers and...

Tryptophanis anaminoacidwhichhasmanyfunctionsinthebody. Besidesitsparticipationin theproductionofproteins,itactsasasubstrateforthekynurenineandmethoxyindolemetabolic pathways. The kynurenine pathway ends with the production of nikotinamid e adenin dinukleotide ( NAD + ) ,whichisneededfortheproductionofcellularenergy. Thus,withincreased energy demand during immune system activation, the activity of the kynurenine pathway is increased. Dueto increasedactivity,itproduces more immunoactiveandneuroactivemetab olites suchaskynurenicacidandquinolinicacid.Thesemetabolitesareinvolvedinmanyprocessesin the body and affect the pathology of many diseases. Studies show that regulation of these metabolites could be a key innovation in the treatment of cance r, cardiovascular or neurodegenerative diseases. The methoxyindole pathway is another important tryptophan processingpathway.Itsbest - knownmetabolitesareserotonin,whichactsasaneurotransmitter, andmelatonin,ahormonewithimmunomodulatoryeffect sregulatedby thecircadianclock . This workdeals with thecircadian rhythmicityofenzymeexpressionofthesetwo metabolic pathways.Italsodescribestheeffectofsystemicadministrationoflipopolysaccharideendotoxin ontheexpressionofgenesoft heseenzymes.OurexperimentsusedWistarratsat30daysofage. The lipopolysaccharide was administered...

The circadian system is a mechanism designed to generate circadian time and to synchronize it with the solar cycle. Its function is to adjust to behavioral and physiological function with the 24-hour period. The adjustment is performed using a so-called zeitgeber or synchronizer. The main circadian clock is in the suprachiasmatic nuclei (SCN) in the hypothalamus. Prolonged exposure of the organism to constant light conditions results in desynchronization of the circadian clock, which can lead to many pathologies. The impact of light at night on the organism has been studied for a long time, but the question of the impact of constant light on the development of the circadian system of the organism has been less studied. My thesis deals with this issue. Using RT-qPCR I investigated how the rhytm changes in the expression of selected clock genes in selected parts of the rat's brain, which has been kept in constant light sice birth. I also tested the impact of exposure to constant light on the early development of rhytm in locomotor activity later in the rat's life. Keywords: circadian system, photic entrainment, desynchronization under constant light, development, rat

L’horloge circadienne permet de régler avec précision les anticipations physiologiques et comportementales face à un environnement perpétuellement oscillant entre jour et nuit. Cette capacité endogène n’est utile que si les processus biologiques restent synchronisés sur le temps solaire. La lumière représente le stimulus le plus efficace pour informer l’horloge des cycles environnementaux.
Chez la drosophile (Drosophila melanogaster) la synchronisation des rythmes veille/sommeil par la lumière est assurée par la molécule photosensible CRYPTOCHROME et par le système visuel. Alors que le cryptochrome agit dans les neurones d'horloges, le système visuel renseigne ces derniers par des voies qui restent à découvrir. La drosophile possède trois organes photorécepteurs, l'oeil composé, les ocelles et l'eyelet de Hofbauer-Buchner, qui expriment chacun une ou plusieurs rhodopsines. La cascade de phototransduction activée par la lumière dépend de la phospholipase C-ß NORPA et conduit à une libération d’histamine.
Dans notre étude, nous avons tenté de caractériser la contribution de chaque rhodopsine dans l’entraînement circadien, mais également de déterminer leur contribution norpA-dépendante en condition de faible lumière.
L’analyse de mutants a montré que les 6 rhodopsines du système visuel constituaient les seules molécules photosensibles capables d’informer l’horloge et que la RH2 et la RH5 seules étaient capables d’entraîner l’horloge en fonction des conditions expérimentales. Nous avons également pu mettre en évidence le fait que les RH1, RH3, RH4 et RH6 utilisaient une voie NORPA-dépendante pour informer l’horloge, alors que la RH2 ne semblait pas le faire. Des doutes subsistent quant à l’existence d’une voie NORPA- dépendante de la RH5 pour informer l’horloge. Nous avons également caractérisé la contribution des récepteurs à l’histamine ORT et HISCL1 dans les processus circadiens: en l'absence de cryptochrome, chacun des deux récepteurs suffit à synchroniser l'horloge et la perte des deux rend les mouches circadiennement aveugles De plus, nous avons constaté que la connexion des photorécepteurs à l’horloge ne se faisait pas directement mais par l’intermédiaire de voies glutamatergiques ou cholinergiques. L’ensemble de ce travail a permis de faire une 1er ébauche des circuits nécessaires à la transmission de l’information lumineuse à l’horloge cérébrale et d’identifier les opsines ainsi que les interneurones impliqués. / The circadian clock allowed physiologic and behavioural anticipation against the day/night oscillation. Light is the most powerful clue for living organism. In the fly Drosophila melanogaster, the rest-activity is synchronized by light and pass through the cryptochrome and the visual system. CRYPTOCHROME act directly in the clock neurons to inform the clock but little is known about the visual system. Drosophila posses tree structures: the ocelli, the compound eye and the eyelet of Hofbauer-Buchner, each structure expressed one or multiple rhodopsins. The phototransduction cascade is activated by light and depend one a phospholipase C-ß NORPA, this lead to histamine realised. Study of mutants show that the 6 rhodopsines represent the only photo-sensible molecule for the clock and the RH2 and the RH5 alone could entrain the clock. We have also find that the RH1, RH3, RH4 and RH6 use a NORPA-dependant way to inform the clock whereas the RH2 does not. Some doubt is still present regarding the RH5 NORPA-dependant way. We have determined that the two-histamines receptor ORT and HISCL1 are involved in the circadian process. Besides, we have shown that there is no direct connexion between the clock and the photoreceptors but the information is relay on a glutamatergique and a cholinegique pathway. This thesis draws the circuit by which the light informed the clock and identified the opsines and the interneurons involved.