京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第18923号 / 薬科博第37号 / 新制||薬||5(附属図書館) / 31874 / 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 / DFAM

Circadian rhythm is a ~24-h mechanism that keeps our physiology and behavior in synchrony with environmental changes. PERIOD2 (PER2) is a core component of the circadian clock and a candidate tumor suppressor as its knockout expression results in a cancer-prone animal. p53 is an effector in the DNA damage response and regulates downstream effectors by trans-activation. Recent studies in our lab show that PER2 can bind to p53, and regulates the trans-activation function. This project studied the subcellular distribution of PER2 in response to DNA damage, and explored the role of p53 in the regulation of PER2 subcellular distribution. We found that PER2 accumulates in the nucleus in response to DNA damage, and such accumulation is independent of p53. In addition, we analyzed Single Nucleotide Polymorphisms (SNP) of PER2 in the 1000 Genome project to gain insight onto how missense mutations in PER2 lay at the interface of p53:PER2 binding. In a separate project, we also performed bioinformatics analysis on the iron related genes to discuss the circadian regulation of iron genes in the liver. These findings shed light on the regulation of PER2 under genotoxic stress, genetic variations of Per2 in normal human population, and expression of circadian genes under iron controlled diets. / Master of Science / Circadian rhythm is a ~24-h mechanism that keeps the body in synchrony with the environment. Period2 (Per2) is a gene at the core of circadian rhythm in mammals. In this work, we found that PER2 accumulates in the nucleus of cells in response to DNA damage. In addition, we analyzed the genetic variation of PER2 in general human population to gain insight onto how mutations in PER2 affect the risk of cancer that’s associated to circadian disruption. In a separate project, we performed bioinformatics analysis on the genes related to iron metabolism, and showed pattern of circadian regulation of iron genes in the liver. These findings shed light on how circadian rhythm responds to genotoxic stress, and summarized genetic variations of Per2 in normal human population, and the expression of circadian genes under iron-controlled diets.

The present thesis is based on four studies focusing on alcohol consumption among Swedish adolescents, and therewith related psychosocial and genetic factors. One main objective was to study the reasons for drinking alcohol among different population - representative samples of adolescents in order to identify motives for drinking. Relationships between these drinking motives, alcohol consumption, and alcohol - related problems were also investigated. Three motives emerged from this study: social - enhancement, coping and dominance. The association with alcohol consumption and alcohol - related problems was positive for social - enhancement and coping motives, but negative for the dominance motive. A significant heritability of alcohol use disorders has been demonstrated by family, adoption and twin studies. Environmental influences have also been acknowledged to play an important role in the development of alcohol use disorders. Moreover, the interaction between genetic and environmental factors is likely to influence the risk - resilience for alcohol use disorders. In view of this knowledge, plausible candidate polymorphisms were considered in gene - environment interaction models. An effect of the genetic polymorphisms was only present when a G x E model was considered. A genetic variant of the clock gene Period2, in an interaction with sleep problems, was studied in relation to alcohol consumption among adolescents. High alcohol consumption was associated with the AA genotype of the PER2 SNP10870 polymorphism, in an interaction with several and frequent sleep problems, among adolescent boys. A genetic variant in the opioid µ receptor 1 gene, in an interaction with alcohol consumption, was studied in relation to depressive symptoms. Depressive symptoms were predicted by the G allele of the OPRM1 A118G polymorphism, in an interaction with high alcohol consumption, among adolescent girls. Additionally, the PER2 SNP10870 and the OPRM1 A118G polymorphisms were studied in a sample of severely alcoholic females. Furthermore, alcohol consumption was assessed by using different instruments, such as biomarkers and surveys. Comparisons were carried out to identify the most suitable method to assess alcohol consumption among adolescents. Questionnaire and interview seemed more suitable tools than biomarkers in this regard.The results eventually support the importance of psychosocial and genetic influences, and their interaction effect on alcohol consumption among adolescents.

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第23142号 / 薬科博第141号 / 新制||薬科||15(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, 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 / DFAM

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / A saudável interação entre o indivíduo e o meio depende do alinhamento entre a dinâmica fisiológica do primeiro e os periódicos movimentos da natureza. A interação entre tais ritmos por sua vez constitui-se em base e derivação do processo de evolução. O comprometimento de tal alinhamento representa um risco para a sobrevivência das espécies. Neste contexto, os organismos alinham seus ritmos fisiológicos a diferentes ciclos externos. Desta forma, ciclos endógenos são coordenados por relógios biológicos que determinam em nosso organismo, específicos ritmos em fase com a natureza, tais como ritmos circadianos (RC), cujo período aproxima-se de 24 horas. O peso corporal, a ingestão de alimentos e o consumo de energia são processos caracterizados pelo RC e a obesidade está associada a uma dessincronização deste processo. A modulação do RC é resultado da expressão dos clock gens CLOCK e BMAL1 que formam um heterodímero responsável pela transcrição gênica de Per1, Per2, Per3, Cry1 e Cry2. As proteínas codificadas por estes genes, uma vez sintetizadas, formam dímeros (PER-CRY) no citoplasma que, a partir de determinada concentração, retornam ao núcleo, bloqueando a ação do heterodímero CLOCK/BMAL1 na transcrição dos próprios genes, formando assim uma alça de retroalimentação negativa de transcrição e tradução. Estes genes asseguram a periodicidade e são significativamente expressos no núcleo supraquiasmático (SCN) do hipotálamo. Para estudar esse processo em camundongos normais e hiperalimentados, saciados e em estado de fome, foi utilizado um método de registro do comportamento alimentar baseado no som produzido pela alimentação dos animais, e a correlação destes estados metabólicos com a expressão de CLOCK, BMAL1, Per1, Per2, Per3, bem como das proteínas Cry1 e Cry2 no SCN, por análise de imagens obtidas em microscopia confocal. Camundongos suíços controle em estado de fome (CF) e saciados (CS) foram comparados com animais hiperalimentados com fome (HF) e saciados (HS). Nenhum grupo demonstrou diferença nos conteúdos CLOCK e BMAL1, indicando capacidade potencial para modular os ritmos biológicos. No entanto, as proteínas Per1, Per2, Per3 e Cry1 apresentaram menor expressão no grupo CS, mostrando uma diferença significativa quando comparados com o grupo CF (P<0,05), diferença esta não encontrada na comparação entre os grupos HF e HS. A quantidade de proteína Cry2 não foi diferente na mesma comparação. Os resultados do estudo indicaram que as alterações dos ritmos endógenos e exógenos, refletido pelo comportamento hiperfágico observado em camundongos hiperalimentados, pode ser devido a um defeito no mecanismo de feedback negativo associado ao dímero Cry-Per, que não bloqueia a transcrição de Per1 Per2, Per3 e Cry1 pelo heterodímero CLOCK-BMAL1. / The healthy interaction between the subject and the environment depends on the alignment between the physiological dynamics of the first one and the periodical movements of nature. The interaction between these rhythms in turn is based on the derivation and evolution process. The involvement of such an alignment is a risk to the survival of species. In this context the bodies line up their physiological rhythms to different external cycles. Thus, endogenous cycles are coordinated by biological clocks which determine in our organism specific rhythms in phase with the nature, such as Circadian Rhythms (CR) whose period is close to 24 hours. The body weight, the food intake and the energy consumption are processes characterized by the CR and the obesity is associated with a different timing of this process. The CR modulation is a result of the formulation of clock-gens CLOCK and BMAL1 who form an heterodimer responsible for the gene transcription of Per1, Per2, Per3, Cry1 e Cry2. The proteins encoded by these genes, once synthesized, form dimers (PER-CRY) in the cytoplasm that, depending on a given concentration, return to the core blocking the action of the CLOCK/BMAL1 heterodimer in the transcription of its own genes, thus forming a negative feedback loop of transcription and translation. These genes secure the periodicity and are significantly expressed in the hypothalamus suprachiasmatic nucleus. In order to study this process in regular, hyper-fed, hungry and satiated mice, we used a registration method of feeding behavior based on the sound produced by animal feeding and the relation between the metabolic states with the expression CLOCK, BMAL1, Per1, Per2, Per3, as well as the Cry1 and Cry2 proteins in the SCN, by analysis of images obtained in confocal microscopy. Control Swiss mice in state of hunger/ satiated were compared to hyper-fed animals in the same conditions. None of them showed difference in the CLOCK and BMAL1 contents, showing a potential capacity to modulate the biological rhythms. However, the Per1, Per2, Per3 and Cry1 proteins showed a minor expression in the CS group and a significant difference when compared to the CF group (P<0,05). This difference cant be found in the HF and HS groups. The results of the studies indicated that the endogenous and exogenous changes, reflected by the hyperphagic behavior observed in hyper-fed mice, may be due to a defect in the mechanism of negative feedback associated to the Cry-Per dimer, which has abolished the blocking mechanism of Per1 Per2, Per3 and Cry1 by the CLOCK-BMAL1 heterodimer.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / A saudável interação entre o indivíduo e o meio depende do alinhamento entre a dinâmica fisiológica do primeiro e os periódicos movimentos da natureza. A interação entre tais ritmos por sua vez constitui-se em base e derivação do processo de evolução. O comprometimento de tal alinhamento representa um risco para a sobrevivência das espécies. Neste contexto, os organismos alinham seus ritmos fisiológicos a diferentes ciclos externos. Desta forma, ciclos endógenos são coordenados por relógios biológicos que determinam em nosso organismo, específicos ritmos em fase com a natureza, tais como ritmos circadianos (RC), cujo período aproxima-se de 24 horas. O peso corporal, a ingestão de alimentos e o consumo de energia são processos caracterizados pelo RC e a obesidade está associada a uma dessincronização deste processo. A modulação do RC é resultado da expressão dos clock gens CLOCK e BMAL1 que formam um heterodímero responsável pela transcrição gênica de Per1, Per2, Per3, Cry1 e Cry2. As proteínas codificadas por estes genes, uma vez sintetizadas, formam dímeros (PER-CRY) no citoplasma que, a partir de determinada concentração, retornam ao núcleo, bloqueando a ação do heterodímero CLOCK/BMAL1 na transcrição dos próprios genes, formando assim uma alça de retroalimentação negativa de transcrição e tradução. Estes genes asseguram a periodicidade e são significativamente expressos no núcleo supraquiasmático (SCN) do hipotálamo. Para estudar esse processo em camundongos normais e hiperalimentados, saciados e em estado de fome, foi utilizado um método de registro do comportamento alimentar baseado no som produzido pela alimentação dos animais, e a correlação destes estados metabólicos com a expressão de CLOCK, BMAL1, Per1, Per2, Per3, bem como das proteínas Cry1 e Cry2 no SCN, por análise de imagens obtidas em microscopia confocal. Camundongos suíços controle em estado de fome (CF) e saciados (CS) foram comparados com animais hiperalimentados com fome (HF) e saciados (HS). Nenhum grupo demonstrou diferença nos conteúdos CLOCK e BMAL1, indicando capacidade potencial para modular os ritmos biológicos. No entanto, as proteínas Per1, Per2, Per3 e Cry1 apresentaram menor expressão no grupo CS, mostrando uma diferença significativa quando comparados com o grupo CF (P<0,05), diferença esta não encontrada na comparação entre os grupos HF e HS. A quantidade de proteína Cry2 não foi diferente na mesma comparação. Os resultados do estudo indicaram que as alterações dos ritmos endógenos e exógenos, refletido pelo comportamento hiperfágico observado em camundongos hiperalimentados, pode ser devido a um defeito no mecanismo de feedback negativo associado ao dímero Cry-Per, que não bloqueia a transcrição de Per1 Per2, Per3 e Cry1 pelo heterodímero CLOCK-BMAL1. / The healthy interaction between the subject and the environment depends on the alignment between the physiological dynamics of the first one and the periodical movements of nature. The interaction between these rhythms in turn is based on the derivation and evolution process. The involvement of such an alignment is a risk to the survival of species. In this context the bodies line up their physiological rhythms to different external cycles. Thus, endogenous cycles are coordinated by biological clocks which determine in our organism specific rhythms in phase with the nature, such as Circadian Rhythms (CR) whose period is close to 24 hours. The body weight, the food intake and the energy consumption are processes characterized by the CR and the obesity is associated with a different timing of this process. The CR modulation is a result of the formulation of clock-gens CLOCK and BMAL1 who form an heterodimer responsible for the gene transcription of Per1, Per2, Per3, Cry1 e Cry2. The proteins encoded by these genes, once synthesized, form dimers (PER-CRY) in the cytoplasm that, depending on a given concentration, return to the core blocking the action of the CLOCK/BMAL1 heterodimer in the transcription of its own genes, thus forming a negative feedback loop of transcription and translation. These genes secure the periodicity and are significantly expressed in the hypothalamus suprachiasmatic nucleus. In order to study this process in regular, hyper-fed, hungry and satiated mice, we used a registration method of feeding behavior based on the sound produced by animal feeding and the relation between the metabolic states with the expression CLOCK, BMAL1, Per1, Per2, Per3, as well as the Cry1 and Cry2 proteins in the SCN, by analysis of images obtained in confocal microscopy. Control Swiss mice in state of hunger/ satiated were compared to hyper-fed animals in the same conditions. None of them showed difference in the CLOCK and BMAL1 contents, showing a potential capacity to modulate the biological rhythms. However, the Per1, Per2, Per3 and Cry1 proteins showed a minor expression in the CS group and a significant difference when compared to the CF group (P<0,05). This difference cant be found in the HF and HS groups. The results of the studies indicated that the endogenous and exogenous changes, reflected by the hyperphagic behavior observed in hyper-fed mice, may be due to a defect in the mechanism of negative feedback associated to the Cry-Per dimer, which has abolished the blocking mechanism of Per1 Per2, Per3 and Cry1 by the CLOCK-BMAL1 heterodimer.

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus has long been considered a master circadian pacemaker that drives rhythms in physiology and behavior in mammals. The recent discovery of self-sustained and cell-autonomous circadian oscillators in peripheral tissues has challenged this position. This dissertation tested the general hypothesis that the SCN has properties that distinguish it from other oscillators, thereby positioning it atop a circadian hierarchy. The general approach was to compare the consequences of altering the molecular circadian clock on tissue-autonomous rhythmicity in mice. In the first experiments, the role of the SCN as a master clock was tested by manipulating the expression of a circadian gene in the brain. Specifically, the expression of the short period tau mutation of casein kinase-1-epsilon (CK1ε) was controlled in an anatomically- and a temporally-specific manner via a tetracycline transactivator regulatory system. This inducible expression of CK1εtau affected the period of activity rhythms when expressed in the SCN, but did not affect the tissue-autonomous rhythmic properties in the peripheral tissues. Second, real-time bioluminescence imaging of tissues from PER2::LUCIFERASE mice revealed that period and phase of different circadian oscillators were tissue specific. Various circadian gene mutations (Cry1-/-, Cry2-/-, Cry1-/-;Cry2-/-, Clock∆19/∆19) produced little difference in rhythmic properties between the SCN and peripheral oscillators, although Cry1-/- SCN had more robust and persistent rhythms compared with the periphery. Third, the loss of Bmal1, which produces behavioral arrhythmicity, eliminated rhythms in the peripheral tissues, but not in the SCN. Bmal1-/- SCN rhythms were highly variable in period and amplitude, fitting a stochastic, but not a deterministic model of rhythm generation. Unlike mutations in other circadian genes, rhythmicity was completely abolished in single SCN neurons in Bmal1-/- mice, indicating that rhythms in Bmal1-/- SCN tissue are a property of the tissue organization rather than an averaging of single-cell autonomous rhythms. The SCN, therefore, has a unique anatomical organization that contributes to long-term stability and temporal organization of the circadian hierarchy.

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus has long been considered a master circadian pacemaker that drives rhythms in physiology and behavior in mammals. The recent discovery of self-sustained and cell-autonomous circadian oscillators in peripheral tissues has challenged this position. This dissertation tested the general hypothesis that the SCN has properties that distinguish it from other oscillators, thereby positioning it atop a circadian hierarchy. The general approach was to compare the consequences of altering the molecular circadian clock on tissue-autonomous rhythmicity in mice. In the first experiments, the role of the SCN as a master clock was tested by manipulating the expression of a circadian gene in the brain. Specifically, the expression of the short period tau mutation of casein kinase-1-epsilon (CK1ε) was controlled in an anatomically- and a temporally-specific manner via a tetracycline transactivator regulatory system. This inducible expression of CK1εtau affected the period of activity rhythms when expressed in the SCN, but did not affect the tissue-autonomous rhythmic properties in the peripheral tissues. Second, real-time bioluminescence imaging of tissues from PER2::LUCIFERASE mice revealed that period and phase of different circadian oscillators were tissue specific. Various circadian gene mutations (Cry1-/-, Cry2-/-, Cry1-/-;Cry2-/-, Clock∆19/∆19) produced little difference in rhythmic properties between the SCN and peripheral oscillators, although Cry1-/- SCN had more robust and persistent rhythms compared with the periphery. Third, the loss of Bmal1, which produces behavioral arrhythmicity, eliminated rhythms in the peripheral tissues, but not in the SCN. Bmal1-/- SCN rhythms were highly variable in period and amplitude, fitting a stochastic, but not a deterministic model of rhythm generation. Unlike mutations in other circadian genes, rhythmicity was completely abolished in single SCN neurons in Bmal1-/- mice, indicating that rhythms in Bmal1-/- SCN tissue are a property of the tissue organization rather than an averaging of single-cell autonomous rhythms. The SCN, therefore, has a unique anatomical organization that contributes to long-term stability and temporal organization of the circadian hierarchy.

Tesis por compendio de publicaciones / La gran preocupación que existe actualmente alrededor del peso corporal, está colaborando a la proliferación de innumerables dietas de adelgazamiento entre las personas “no satisfechas con su peso”. En este sentido hemos estudiado la obesidad desde un punto de vista cronobiológico, para poder dilucidar la importancia que tiene la alteración circadiana de ciertos ritmos biológicos en la ganancia de peso o la no pérdida del mismo. El acúmulo de grasa corporal y el grado de lipogénesis en el tejido adiposo podría ser diferente a distintas horas del día, ingiriendo las mismas calorías. A su vez uno de los aspectos más interesantes, es poder conseguir una caracterización cronobiológica de cada individuo, establecer mejoras en las terapias de comportamiento alimentario, introducir nuevos índices que permitan detectar pacientes de riesgo y poder establecer pautas alimentarias y hábitos de vida individualizados que ayuden a estos pacientes a alcanzar la meta de peso propuesta. / The great controversy now there about body weight, is collaborating with the proliferation of countless diets among people "not satisfied with their weight." In this sense, we have studied obesity from a chronobiological viewpoint, to elucidate the importance of the alteration of circadian biological rhythms in weight gain or no loss. The accumulation of body fat and the degree of lipogenesis in adipose tissue may be different n accordance with the time of day, even eating the same calories. In turn one of the most interesting aspect, is to be able to characterize the individual chronobiology of each patient, establish improved therapies feeding behavior, introducing new indices to detect patients at risk and to establish dietary patterns and lifestyle habits that help these patients to achieve the proposed weight goal.

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