Several studies have investigated the ecological factors that affect behavior in Micrathena gracilis, a diurnal orb-weaving spider that forages on flying insects during the day. However, none yet have considered how the temporal distributions of prey and predator occurrences shape their daily behavioral rhythms, especially web construction, which involves a heavy energetic investment well in advance of potential nutritional benefit. Recently, several orb-weaving spider species have been shown to exhibit a variety of abnormal rhythms, suggesting that circadian clock-controlled rhythms may play an unexpected role in behavioral evolution. Despite the appearance of significant insect abundance in the evenings, M. gracilis individuals stop foraging, take down their webs, and retreat before they can capitalize on this opportunity. Is the nutritional benefit of this forfeited prey significant compared to what they collect during the day, and if so, what potential cost might justify opting out of this potential gain? To investigate, sticky traps for prey collection and a camera array for recording predator activity were used at a local field site to survey what risks and rewards these spiders face throughout the 24-hour day. Spider activity and web captures in the field were also used to confirm behavioral patterns and capture success throughout the day. It was found that spiders begin foraging when prey becomes available but cease while prey is still abundant. These observations appear to support a theoretical model of behavioral decisions under predation risk. However, recorded predation events were rare, and predation was not confirmed outside of the foraging timeframe. These results support the notion that the circadian rhythm of Micrathena gracilis is shaped by factors other than prey availability, but the theoretical pressure from predation risk requires further investigation.

Circadian rhythm of parathyroid hormone (PTH) is well documented, but its physiological role is not fully understood. In healthy individuals, biochemical markers of bone remodeling follow a similar circadian rhythm to PTH with a nocturnal rise in bone resorption and formation. The loss of PTH diurnal variation was observed not only in primary hyperparathyroidism, but also in patients with postmenopausal osteoporosis. Continuously elevated concentrations of PTH lead to excessive stimulation of bone resorption, whereas intermittent PTH administration has a strong osteoanabolic effect in patients with osteoporosis. It has not been examined whether the skeletal sensitivity to PTH action depends also on the time of its application. The aim of our study was to verify the hypothesis that the application of teriparatide (TPTD, recombinant human PTH [1-34]) at different times of the day in the context of its diurnal variability affects the physiological circadian rhythm of bone remodeling and also the bone mineral density (BMD) after the long-term TPTD treatment. Fourteen women with postmenopausal osteoporosis treated with 20 micrograms of TPTD daily, applied subcutaneously either in the morning or evening, were included in the first study. The concentration of serum C-terminal telopeptide of type I collagen...

The diel locomotor activity patterns of wandering larvae in the flesh fly, Sarcophaga crassipalpis Macquart (Diptera: Sarcophagidae), were examined using a novel apparatus and shown to be primarily diurnal, but with a minority (37%) showing nocturnal activity. In response to the environmental stress of heat shock, a significantly larger proportion (72%) of the larvae became nocturnal. In comparison, adult circadian activity also was predominantly diurnal, but not correlated with the larval activity patterns. In addition, adult patterns showed age-related changes in entrainment and free running period. Finally, the phase of circadian-gated adult eclosion was shown to be entrained by a 3-day exposure to light-dark cycles delivered prior to pupariation, with the phase maintained throughout pupal-adult metamorphosis under constant dark conditions. These results demonstrate that environmental changes may have profound effects on the expression of 24-h activity patterns and circadian rhythms during different life stages throughout development.

Abstract. The circadian locomotor (walking) rhythms of forager honeybees (Apis mellifera ligustica L.) were entrained to eight different 24 h light‐dark cycles. The phases of activity onset, peak activity, and offset were correlated with the lights‐off transition, suggesting lights‐off as the primary zeitgeber for the rhythm. Further support for this hypothesis was provided by LD 1:23 experiments, in which entrainment occurred when the light pulse was situated at the end, but not at the beginning, of the subjective photophase. Steady‐state entrainment of the locomotor rhythm was achieved with square‐wave temperature cycles of 10oC amplitude under constant dark: most of the activity occurred within the early thermophase. Smaller amplitude temperature cycles yielded relative coordination of the rhythm. Interactions of temperature and light‐dark cycles resulted in entrainment patterns different from those elicited in response to either cycle alone or those formed by a simple combination of the two separate responses. Furthermore, temperature cycles having amplitudes insufficient for entrainment of the rhythm nevertheless modified the pattern of entrainment to light ‐ dark cycles, suggesting a synergism of light and temperature effects on the underlying circadian clock system.

Ecologically, spiders are both predators and prey. Therefore, they must balance being aggressive enough to forage successfully, but not so aggressive that they become overly exposed to predation. Some species of spiders actively forage during clearly defined periods of the day, leading to the hypothesis that they should be less aggressive (or more defensive) during periods when they are not foraging, predicting that antipredator behaviour should be more pronounced during inactive foraging times. We tested the antipredator 'huddle response' in a nocturnal foraging orb-weaver, Larinioides cornutus, and found that, as predicted, the spiders huddled longer in the day than at night. We then conducted tests to determine whether the cycling of the response was regulated by an internal clock (circadian), and we found that huddle duration continued to cycle under constant dark (with periodicity significantly less than 24. h) as well as under constant light (periodicity nonsignificantly longer than 24. h). This work adds a novel behaviour to the list of behaviours under circadian control and also to the surprisingly short list of studies demonstrating circadian rhythm in spiders.

Honey bee (Apis mellifera) foragers can remember both the location and time of day food is collected and, even in the absence of a reward, reconnoiter the food source at the appropriate time on subsequent days. This spatiotemporal memory (time-memory) is linked to the circadian clock and enables foragers to synchronize their behavior with floral nectar secretion rhythms, thus eliminating the need to rediscover productive food sources each day. Here, we asked whether the establishment of one time-memory influences the formation of another time-memory at the same time of day. In other words, can two time-place memories with the same ‘time-stamp’ coexist? We simultaneously trained two groups of foragers from a single hive to two separate feeders at the same restricted time of day. After 5 days of training, one feeder was shut off. The second feeder continued being productive 4 more days. Our results showed that (1) foragers with high experience levels at the first source were significantly more likely than low-experience foragers to maintain fidelity to their original source and resist recruitment to the alternative source, (2) nearly one-third of foragers demonstrated multiple, overlapping time-memories by visiting both feeders at the correct time and (3) significantly more high-experience than low-experience foragers exhibited this multitasking behavior. The ability to maintain and act upon two different, yet contemporaneous, time-memories gives the forager bee a previously unknown level of versatility in attending to multiple food sources. These findings have major implications for understanding the formation and management of circadian spatiotemporal memories.

Circadian rhythms are self-sustained 24-hour period oscillations present in most organisms, from bacteria to human. They can be synchronized to external cues, thus allowing organisms to anticipate environmental variations and optimize their performance in nature. In Drosophila, the molecular pacemaker consists of two interlocked transcriptional feedback loops. CLOCK/CYCLE (CLK/CYC) sits in the center and drives rhythmic transcription of period (per), timeless (tim), vrille (vri) and PAR domain protein 1 (Pdp1). PER and TIM negatively feedback on CLK/CYC transcriptional activity, forming one loop, while VRI and PDP1 form the other by regulating Clk transcription negatively and positively, respectively. Posttranscriptional and posttranslational regulations also contribute to circadian rhythms. Although much has been learned about these feedback loops, we are still far from understanding how stable 24-hour period rhythms are generated. My thesis work was to determine by which molecular mechanisms kayak-α (kay-α) and Ataxin-2 (Atx2) regulate Drosophila circadian behavior. Both genes are required for the precision of circadian rhythms since knocking down either gene in circadian pacemaker neurons results in long period phenotype. The work on kay-α constitutes the first half of my thesis. We found that the transcription factor KAY-α can bind to VRI and inhibit VRI’s repression on the Clk promoter. Interestingly, KAY-α can also repress CLK’s transcriptional activity on its target genes (e.g., per and tim). Therefore, KAY-α is proposed to bring precision and stability to the molecular pacemaker by regulating both transcriptional loops. The second half of my thesis focuses on ATX2, an RNA binding protein whose mammalian homolog has been implicated in neurodegenerative diseases. We found that ATX2 is required for PER accumulation in circadian pacemaker neurons. It forms a complex with TWENTY-FOUR (TYF)—a crucial activator of PER translation—and promotes TYF’s interaction with Poly(A)-binding protein. This work reveals the role of ATX2 in the control of circadian rhythms as an activator of PER translation, in contrast to its well-established role as a repressor of translation. It also further demonstrates the importance of translational regulation on circadian rhythms. Finally, it may help understanding how ATX2 causes neuronal degeneration in human diseases.

BMAL1, une protéine centrale de l'horloge circadienne.L’inactivation de BMAL1 (BMAL1-KO) entraîne une perte complète de la rythmicité dans les horloges central et périphérique. Le travail de ma thèse se concentre sur le rôle du gène BMAL1 dans la développement et le traitement des cancers du sein et du côlon.1. Pharmacodynamique in vitro de l’Everolimus en fonction du temps d’administration malgré une horloge circadienne défectueuse ((Zhang et al., 2018) (Zhang, Levi and Chang, 2018)L’everolimus (EV) est un inhibiteur de la mTOR chez les mammifères et il est utilisé pour traiter le cancer du sein positif aux oestrogènes (ER+). Nous avons focalisé nos recherches sur la chronopharmacologie de l’Everolimus administré sur des cellules MCF-7 (ER+). Les MCF-7 présentent une oscillation circadienne de l’activité de mTOR sans mise en évidence d’une oscillation des gènes d’horloge. L’oscillation d’activité de mTOR induirait une oscillation de synthèse et/ou de phosphorylation de protéines importantes dans la progression de la phase G1, notamment la Cycline D1 et RB phosphorylée. Ces variations rythmiques des MCF-7 synchronisées expliquent la chrono-efficacité de l’Everolimus selon des temps différents d’administration.Ce travail a révélé que même dans un système de cellules cancéreuses dont l’horloge était perturbée, l'intégration d'autres rythmes cellulaires dans la chronothérapie pouvait augmenter l'efficacité du médicament. Ce principe peut être appliqué à des traitements du cancer pour optimiser la chronothérapie du cancer.2. Le Knockdown BMAL1 a déclenché différents destins de cellules du carcinome du côlon (CRC) en modifiant l'équilibre délicat entre les voies AKT / mTOR et P21 / P53 (Article soumis)Premièrement, nos résultats ont révélé que le knockdown BMAL1 par le shRNA (BMAL1-KD) avait déclenché une activation plus évidente de l’AKT / mTOR dans deux lignées cellulaires primaires (HCT116 et SW480) que une lignée métastatique de CRC, SW620. De plus, bien que les deux lignées cellulaires primaires de CRC aient présenté une augmentation significative de l'activité de l'AKT/mTOR, elles avaient des statuts différents de P53 (WT ou mutant). Dans ce contexte, les cellules SW480 BMAL1-KD avec P53 mutant présentaient une sénescence accrue, mais les cellules HCT116 BMAL1-KD avec P53 WT présentaient d’abord une apoptose transitoire, puis un taux de prolifération plus élevé.Ainsi, nos travaux ont révélé le rôle crucial de BMAL1 pour équilibrer un régulateur central du métabolisme AKT / mTOR et une voie de réponse au stress P53 / P21 dans des lignées cellulaires de CRC, ce qui met en évidence l’importance de BMAL1 dans le développement de CRC et la progression du vieillissement.3. BMAL1 renforce les propriétés épithéliales et diminue la chimiorésistance des cellules du CRC (article en préparation)La transition épithélo-mésenchymateuse (EMT) est un événement critique dans l'invasion et la métastase des carcinomes, y compris le CRC.Dans ce travail, nous avons étudié comment BMAL1 knockdown (Bmal1-KD) altère l’équilibre délicat entre les propriétés épithéliales et mésenchymateuse de trois lignées cellulaires de CRC (HCT116, SW480 et SW620).Après BMAL1-KD, la diminution de l’expression Twist, un facteur de transcription favorisé l’EMT et des marqueurs mésenchymateux (N-Cadhérine, Vimentine) étaient associées à une expression accrue des marqueurs épithéliaux (E-cadhérine, CK20 et EpCAM). De manière constante, l'augmentation de l'expression de l’E-cadhérine après BMAL1-KD était accompagnée d'une co-localisation membranaire accrue de la β-caténine avec l'E-cadhérine, ainsi que d'une diminution de la localisation nucléaire de la β-caténine, suggérant une diminution de l'activation de la voie Wnt. De plus, les cellules BMAL1-KD ont montré une diminution des capacités de migration et de la résistance aux médicaments.Au total, ces données soulignent l’importance de BMAL1 dans l’EMT des cellules de CRC. / BMAL1 is a core circadian clock protein, forming a heterodimer with CLOCK to initiate the transcription of circadian and output genes. Among canonical clock genes, only BMAL1 knockout results in complete loss of rhythmicity in both the SCN and peripheral tissues. My thesis work focuses on exploring the important role of BMAL1 in human breast and colon cancer progression and treatment. My work is divided into three main parts:1. Dosing time dependent in vitro pharmacodynamics of Everolimus despite a defective circadian clock (Zhang et al., 2018)(Zhang, Levi and Chang, 2018) Everolimus (EV) is an inhibitor of mammalian target of Rapamycin (mTOR) and is used to treat estrogen positive (ER+) breast cancer. Here, we investigated whether EV efficacy varied according to administration timing by using the ER+ breast cancer cell line MCF-7 as a model system. Serum shock synchronization induced a circadian oscillation in mTOR activity in MCF-7 cells, which rhythmically regulated the synthesis or phosphorylation of key G1 progression proteins, such as Cyclin D1 and phosphorylated RB, ultimately resulting in different G0/G1 blockage efficiency according to different EV administration timing. Thus, the different delivery schedule of EV presented different efficacy in G0/G1 phase blockage in serum shocked MCF-7 cells.This investigation revealed that, even in a breast cancer cell system with disrupted circadian organization, modulating drug administration according to other protein rhythms could still increase drug efficacy. This principle may be applied to many other cancer systems and treatment types to optimize cancer chronotherapy.2. Knockdown BMAL1 triggered different colon carcinoma cells fates by altering the delicate equilibrium between AKT/mTOR and P21/P53 pathways (Article in preparation)We tried to evaluate in vitro how knockdown BMAL1 (BMAL1-KD) by shRNA influences human colorectal cancer cell (CRC) behavior.The results revealed that BMAL1-KD triggered different CRC cell fates based on distinct p53 status in different cell lines. First, after BMAL1 knockdown, two primary CRC cell lines (HCT116 and SW480) presented a more evident AKT/mTOR activation than the metastatic colon carcinoma cell line, SW620. Furthermore, although both primary CRC cell lines presented a significant increase of AKT/mTOR activity, they had different P53 status (WT or mutant) and activation pattern. Under these context, SW480 BMAL1-KD cells exhibited increased senescence but HCT116 BMAL1-KD cells showed firstly a transient apoptosis and then higher proliferation rate.Thus, our work uncovered the crucial role of BMAL1 to balance a central metabolism regulator AKT/mTOR and a stress response pathway P53/P21 in CRC cell lines, which highlighted the importance of BMAL1 in CRC development and aging progression.3. BMAL1 knockdown leans epithelial–mesenchymal balance toward epithelial properties and decreased the chemoresistance of colon carcinoma cell (Article in preparation)Epithelial-mesenchymal transition (EMT) is a critical early event in the invasion and metastasis of carcinoma, including colorectal cancer (CRC). In this work, we studied how BMAL1-KD alters the delicate equilibrium between epithelial and mesenchymal properties of three colon carcinoma cell lines (HCT116, SW480 and SW620).The results showed the molecular alterations after BMAL1-KD promote mesenchymal-to-epithelial transition-like changes mostly appeared in two primary CRC cell lines (HCT116 and SW480) compared to the metastatic cell line SW620. Subsequently, BMAL1-KD HCT116 and SW480 cells harbored a decreased migration, invasiveness and drug resistance capacities relative to their scramble counterpart cells. All these data suggested the importance of BMAL1 on EMT inducing in colon carcinoma cells.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13242号 / 論農博第2867号 / 新制||農||1069(附属図書館) / 学位論文||H31||N5166(農学部図書室) / (主査)教授 久米 新一, 教授 松井 徹, 教授 廣岡 博之 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

No description available.