京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第22046号 / 薬科博第112号 / 新制||薬科||12(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 土居 雅夫, 准教授 Fustin,Jean Michel, 教授 中山 和久 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

In order to adapt to changing external conditions, organisms developed the endogenous biological clock for predicting daily alterations. This so-called circadian system drives functions and processes in the whole body with an approximately 24h period. The central oscillator, located in hypothalamic suprachiasmatic nuclei (SCN), is synchronized by light and subsequently sends the information about the time of the day to the rest of the body. Even in the ontogenesis, the functional SCN clock is crucial for proper development as well as health later in life. Since the maturation of embryonic SCN is not completed before birth, maternal signals seem to play a fundamental role in setting and synchronizing the fetal clock. During my PhD studies, we focused on elucidating the nature of maternal signals and their diverse impact on fetal SCN of rat and mouse models. We have revealed that developing SCN is able to sense distinct signals related to various maternal behavioral regimes. Importantly, we have discovered eminent role of glucocorticoids in synchronizing the fetal SCN, along with their ability to accelerate SCN development. These observations point out the importance of regular daily routine and noxious effect of stress during pregnancy. Since the mother communicates with the fetus through placenta...

Despite recent interest, there still is relatively little known about the ecology and physiology of diel and circadian rhythms in spiders. However, previous work on spiders suggests that there is a striking amount of variation in circadian period both among, and within, species, when compared to model organisms. Whereas previous studies of behavioral rhythms in spiders focused on females, here we describe the diel and circadian patterns of locomotor activity in male Parasteatoda tepidariorum (C. L. Koch, 1841) (Theridiidae). We found that the males showed mostly nocturnal activity under a light:dark cycle, with activity peaking very early after lights off and steadily declining to near zero just prior to lights on. Under constant darkness most individuals showed significant circadian rhythmicity with a mean free-running period of about 21.2 h. Though not the shortest average free-running period described for spiders, being so out of resonance with the 24 h solar day strains conventional circadian rhythm theory. Our data also suggest that the phase angle of entrainment for locomotor activity is in the mid-to-late photophase, but that activity may be masked by light. Of particular note is that both the diel and circadian activity patterns reported here for male P. tepidariorum are similar to those reported elsewhere for females of the species. This study deepens our understanding of the nature and variation in circadian rhythm in spiders and builds a case for further developing spiders as a model system for research integrating the fields of chronobiology and ecology.

Circadian rhythms are endogenous cycles that control physiological and behavioral changes that can be affected by environmental factors which allow most eukaryotic organisms to synchronize their daily activities with the 24-hour day. Parasteatoda tepidariorum,the common house spider, demonstrates a short-period circadian clock averaging 21.6 hours when left in constant darkness, yet they are able to entrain to a 24-hour light cycle. We tested whether these spiders were able to use non-photic Zeitgebers to entrain to the 24-hour day. Periodic presentation of food and disturbance were not found to be effective cues for the spiders’ entrainment. A few individuals were clearly able to entrain to an 8 oC amplitude temperature cycle, while most did not.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第18924号 / 薬科博第38号 / 新制||薬||5(附属図書館) / 31875 / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 岡村 均, 教授 中山 和久, 教授 竹島 浩 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DGAM

Type 2 diabetes is a metabolic disorder characterized by increased glucose concentrations in the blood due to decreased insulin sensitivity. The worldwide incidence of diabetes has increased remarkably over the last two decades. Obesity, due to increased consumption of calorie dense diets, and sedentary life styles, is commonly cited as a primary cause. However, many epidemiological studies have established a relationship between insulin resistance and exposure to environmental chemicals such as persistent organic pollutants (POPs). The mechanisms by which POPs alter metabolism remain poorly understood, although their lipophilic nature suggests a role in adipose tissue function. The Tischkau lab has established a relationship between Aryl hydrocarbon Receptor (AhR) activation by different types of POPs and increased risk of insulin resistance. This dissertation, therefore, explored the effects of AhR activation by POPs on adipose tissue function. Adipose tissue regulates systemic glucose and lipid metabolism through production of hormones and cytokines that regulate appetite and energy homeostasis. It is well-known that impaired adipose function promotes systemic insulin resistance. The first specific aim examined the hypothesis that activation of AhR suppresses adipogenesis by lowering the rate of pre-adipocyte differentiation. Adipogenesis is a process by which mesenchymal stem cells (MSCs) and pre-adipocytes differentiate into mature adipocytes. Limitations in adipogenesis and accumulation of ectopic lipid have significant roles in decreasing insulin sensitivity. Thus, I hypothesized that POPs contribute to systemic insulin resistance by lowering the rate of MSCs and preadipocyte differentiation; the resulting large, poorly-functioning adipocytes increase serum lipids and promote lipid deposition in other tissues. MSCs derived from mouse bone marrow and pre-adipocytes were treated with different concentrations of AhR agonist, β-Naphthoflavone (BNF), and levels of transcripts associated with adipocyte differentiation were determined by using quantitative PCR. Oil red O staining and lipid content were observed to examine differentiation into mature adipocytes. Genes that promote adipogenesis, including peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (CEBPα), fatty acid binding protein 4 (FABP4), and adiponectin were downregulated in MSCs treated with BNF. Moreover, accumulation of triglycerides was decreased after BNF treatment. Recombinant lentivirus vector-mediated AhR knockdown blocked the effects of BNF on adipogenesis. Therefore, activation of AhR by exogenous ligands inhibits adipogenesis leading to impaired fat storage. Limitations in adipogenesis promotes accumulation of the excess lipid in non-fat tissue such as liver, muscle, and heart leading to decrease the insulin sensitivity and disrupt energy homeostasis. The second specific aim examined effects of AhR activation on circadian clock regulation in adipose tissue. A circadian clock essentially regulates systematic energy homeostasis; the central clock in the suprachiasmatic nucleus (SCN) works with the local clocks in peripheral tissues such as liver, muscle, and adipose tissue to regulate whole-body metabolism. The Tischkau lab has previously shown that AhR interacts with the core machinery of the circadian clock. Activation of AhR by environmental toxicants leads to a dampening of the rhythm expression of core clock genes or an alteration in the timing of their peak expression, which subsequently promotes metabolic disorders such as glucose insensitivity and hyperlipidemia. Given the importance of appropriately timed adipose tissue function to regulation of energy homeostasis, this study focused on mechanisms by which AhR may influence clock-controlled mature adipose tissue activity. Lipolysis is a clock-regulated process in adipose tissue that provides the necessary energy during periods of fasting and exercise. Thus, I hypothesized that AhR activation in adipose tissue would impair lipolysis by altering molecular circadian clock function. AhR activation was proposed to dampen adipose rhythms, leading to a decreased lipolysis rate during the absence of food, and subsequently, increased glucose concentrations in the blood. C57BL/6 mice were injected with vehicle or 50 mg/kg body weight of the AhR agonist, BNF, 48 hours after release into constant darkness. Mice were sacrificed, and epididymal adipose tissue was collected every 6 hours over a 24 hour period. Real-Time RT-qPCR was used to measure mRNA expression of genes responsible for lipolysis. To examine effects of AhR activation in vitro; mouse pre-adipocytes, 3T3-L1 cells, were differentiated into mature adipocytes for 12 days. Cells were then starved for 24 hours with DMEM media containing 1% FBS to induce lipolysis in the presence of 100, 200, 300 µM of BNF. RNA was then extracted and mRNA expression for genes responsible for circadian clock and lipolysis were determined by RT-qPCR. Alterations were observed in rhythms of core clock genes in wild type mice injected with BNF compared to wild type mice injected with vehicle. Rhythms of key enzymes controlling lipolysis including hormone sensitive lipase (HSL) and adipose triglycerides lipolysis (ATGL) was changed in wild type mice injected with BNF compared to wild type mice injected with vehicle. These effects were blocked in AhR deficient mice, suggesting that these effects were AhR dependent. Liver glycogen was decreased in mice injected with BNF compared to wild type mice injected with vehicle after 12 hour of food restriction but not in AhR null mice. Activation of AhR led to decreased expression of lipolysis genes in adipose tissue at CT6 (middle of the rest phase) as well as in 3T3-L1 cells. Recombinant lentivirus vector-mediated AhR knockdown blocked the effects of BNF on lipolysis in 3T3-L1 cell line. These data establish a link between environmental toxicants and impaired lipolysis, specifically by altering rhythms of clock genes in adipose tissue. In response to the decreased available energy from impaired lipolysis, the body increases glycogenolysis, thereby degrading more glycogen to provide the necessary energy. This process may lead to increased glucose level in the blood and development of type 2 diabetes. The data from this study suggest that activation of AhR by BNF increases the risk of insulin resistance and type 2 diabetes by impairing adipogenesis. Reduced adipogenesis likely decreases adipocyte capacity to capture triglycerides from the blood. These effects may disturb energy homeostasis and contribute to the development of metabolic syndrome. This study also establishes a link between environmental toxicants and impaired lipolysis, specifically by altering rhythms of clock genes in adipose tissue. In response to the decreased available energy from impaired lipolysis, the body increases glycogenolysis, thereby degrading more glycogen to provide the necessary energy. This process may lead to increased glucose level in the blood and development of type 2 diabetes. All together, these data suggest that environmental pollutants result in adipose tissue dysfunction by reducing adipogenesis and lipolysis. Therefore, activation of AHR by its exogenous ligands may increase the risk of insulin resistance and type 2 diabetes by impairing adipose tissue function. In particular, activation of AHR by exogenous ligands leads to impairment of free fatty acids storage during feeding and release during fasting to disturb energy homeostasis.

The majority of our physiological and metabolic processes are coordinated by an internal clock, which has evolved as an adaptive response to the daily light-dark cycles. Thus, several physiological and behavioral activities display an oscillatory rhythmic period of 24 hours. This highly conserved molecular mechanism is achieved through a specific program of gene expression, characterized by a complex interaction between clock-core proteins, chromatin remodelers and epigenetic events associated with the oscillatory nature of circadian transcriptional activity in the genome. Clock disruption leads to a wide spectrum of severe health problems including chronic metabolic disorders, muscle waste and cardiopathies. Previous studies revealed that each cell and organ possess an intrinsic clock and that coordination between central versus peripheral clocks is key for health. Furthermore, it has been found that under nutritional challenge such as High Fat Diet (HFD), the circadian transcriptome and metabolome are rapidly remodeled in the mouse model. Surprisingly, metabolome and gene expression analysis on various tissues revealed that skeletal muscle is the most affected under HFD. Mechanisms that regulate circadian cycle and stress induced rapid adaptation and in particular metabolic stress at the chromatin level are largely unknown. In this study, we investigated the role of Polycomb proteins group (PcG) mediate cell memory system by maintaining transcriptional gene silencing, in particular the PRC2-Ezh1. We hypothesized that Ezh1 could play an important role in circadian clock regulation in post-mitotic skeletal muscle, and this pathway has never been explored in this context. We explored the circadian role of PRC2-Ezh1 in the mouse skeletal muscle. Intriguingly, we found that the oscillatory profile of a novel isoform of Ezh1 (Ezh1beta), localized specifically in the cytoplasm and controlling stress induced nuclear PRC2 activity, was completely disrupted under HFD. More interestingly, the circadian pattern of core clock components was impaired in Ezh1 depleted cells. Our data unveils an interesting physiological role of the PcG memory system, from cytoplasm to chromatin, which could indicate a new link between the chromatin remodeler Polycomb proteins and the endogenous clock in adaptation mechanism in skeletal muscle.

No description available.

Temporal control, inherent in all biological processes, relies on intrinsic systems to govern periodical behaviors and physiological responses. The circadian clock, a vital timekeeper, enables organisms to anticipate and adjust to daily environmental changes. In mammals, the circadian clock is organized hierarchically, with a central master clock in the hypothalamic suprachiasmatic nucleus regulating peripheral clocks distributed across the body. To maintain coherent circadian rhythms at the tissue level, peripheral oscillators exchange intercellular coupling factors by paracrine signaling pathways to synchronize. At the individual cell scale, the circadian clock interacts with another periodical biological process: the cell cycle. However, the mechanisms governing this interplay remain poorly elucidated. Here, we explore the influence of extracellular circadian synchronization on the intracellular coordination between the circadian clock and the cell cycle. To do so, we combined a mathematical model and long-term live-imaging recordings at the single-cell and population level of a human cell line. We show that the global circadian and cell cycle coordination within individual cells is disrupted when the extracellular circadian synchronization is lost, obstructing collective tissue growth. Populations with coherent circadian rhythms display rhythmic growth oscillations, uncovering a novel global regulator of tissue dynamics. Knocking down core circadian elements abolished these effects, revealing the fundamental role of circadian clock control as a timing mechanism. These findings advance our understanding of how biological systems maintain equilibrium and regulate proliferation in normal and pathological conditions. The circadian clock plays a crucial role in orchestrating cell proliferation, impacting tumor initiation, growth, and treatment responses. Recent research has reported significant changes in drug response for different administration hours throughout the day, highlighting the benefits of aligning treatment strategies to the inherent circadian rhythm. However, chronotherapy is still omitted in clinical practice, primarily due to a lack of understanding of the underlying mechanisms driving time-dependent drug responses. Currrently, no standardized protocols exist for identifying these temporal factors. Therefore, we developed a combined mathematical and experimental approach to identify the factors influencing time-dependent drug sensitivity in human cells. Our results show how circadian and drug properties independently shape time-of-day drug responses, offering novel insights into the time-dependent treatment outcome. This framework holds potential for developing personalized treatment schedules aligned with the internal circadian clock, optimizing cancer therapeutical strategies. On the other hand, tissue growth masks heterogeneous proliferation patterns at the single-cell level, potentially jeopardizing the treatment outcome, which cannot be exclusively attributed to circadian clock regulation. Clustering the cells upon their overall number of divisions, the proliferative patterns remain strikingly constant across different tissues, a phenomenon reported by several recent studies. This consistency implies the existence of a common underlying mechanism that is currently unknown. Proliferation control relies on a set of checkpoint mechanisms that accurately and quickly detect DNA damage. The onset of cellular stress triggers the activation of the p53 protein, orchestrating the expression of hundreds of genes responsible for cell cycle regulation or apoptosis, among other functions. Here, we present evidence that changes in cellular stress levels contribute to the gradual proliferation variability. Specifically, different DNA damage levels are encoded quantitively into signal parameters of p53 and p21 proteins in a gradual manner, shaping proliferation activity proportionally. These results propose a novel function of the p53-p21 signaling network in deciphering and decoding the magnitude of DNA damage to adjust and control proliferation. / This study examines how extracellular circadian synchronization affects the coordination between the circadian clock and the cell cycle. By combining mathematical modeling and long-term live imaging of human cells, we show that loss of synchronization disrupts global circadian and cell cycle coordination in individual cells, hindering tissue growth. When circadian rhythms are coherent, rhythmic growth oscillations occur, indicating a global tissue dynamics regulator. Knocking down key circadian elements abolished these effects, emphasizing the circadian clock's timing role. These findings enhance our understanding of biological balance and proliferation regulation in both normal and pathological states. The circadian clock is also vital in cell proliferation, influencing tumor growth and treatment responses. Drug responses vary depending on the time of day, highlighting the importance of aligning treatments with circadian rhythms. However, chronotherapy is not widely used in clinical practice due to insufficient understanding of the underlying mechanisms. Our approach identifies factors affecting time-dependent drug sensitivity, offering insights into personalized treatment schedules. Tissue growth masks single-cell proliferation patterns, essential for effective treatment. By clustering cells based on division numbers, we found consistent proliferation patterns across tissues, suggesting an unknown underlying mechanism. The p53-p21 signaling network regulates proliferation by quantifying DNA damage and adjusting cell cycle responses. This study reveals how p53-p21 signaling decodes DNA damage levels to control proliferation. / Diese Studie erforscht, wie extrazelluläre zirkadiane Synchronisation die Abstimmung zwischen der zirkadianen Uhr und dem Zellzyklus beeinflusst. Durch mathematische Modellierung und langfristige Live-Bildgebung an menschlichen Zellen zeigt sie, dass der Verlust der Synchronisation die zirkadiane und Zellzykluskoordination stört und Gewebewachstum hemmt. Bei kohärenten zirkadianen Rhythmen entstehen rhythmische Wachstumsoszillationen, die eine globale Steuerung der Gewebedynamik andeuten. Das Ausschalten zirkadianer Elemente beseitigt diese Effekte und verdeutlicht die zeitliche Rolle der zirkadianen Uhr. Diese Ergebnisse liefern Einblicke in die biologische Balance und Regulierung der Proliferation in normalen und pathologischen Zuständen. Die zirkadiane Uhr beeinflusst die Zellproliferation, das Tumorwachstum und die Wirkung von Behandlungen. Arzneimittelreaktionen variieren tageszeitabhängig, was die Relevanz der Chronotherapie unterstreicht – der Anpassung von Therapien an zirkadiane Rhythmen. Allerdings wird Chronotherapie selten klinisch genutzt, da die zugrundeliegenden Mechanismen nicht ausreichend verstanden sind. Diese Studie identifiziert Faktoren, die die zeitabhängige Arzneimittelempfindlichkeit beeinflussen, und bietet Perspektiven für personalisierte Therapien. Gewebewachstum verdeckt Proliferationsmuster einzelner Zellen, die für Behandlungen entscheidend sind. Durch Clusteranalysen der Zellteilungen zeigten sich konsistente Muster in Geweben, was auf unbekannte Mechanismen hindeutet. Das p53-p21-Signalnetzwerk reguliert die Proliferation, indem es DNA-Schäden bewertet und Zellzyklusreaktionen anpasst. Die Studie zeigt, wie dieses Netzwerk DNA-Schäden interpretiert, um Zellwachstum zu steuern.