Like most organisms, Drosophila melanogaster can synchronize its physiological and behavioural processes by possessing internal circadian clock that regulates. Naturally fluctuating timing cues, like light and temperature (also known as Zeitgebers), synchronize these endogenous and self‐sustained clocks with external time. In Drosophila, synchronization of the circadian clock by light has been studied in detail, but much less is known about the molecular mechanisms underlying temperature entrainment. Previous data from our lab shows that Nocte, a Chordotonal organ (Ch organ) located protein, is required for normal temperature entrainment in Drosophila. However, neither the function of Nocte in temperature entrainment nor the molecular underlying mechanisms are clear. To address these issues, a proteomics strategy of combing co‐immunoprecipitation and MS/MS sequencing was applied to isolate potential interactors of Nocte. IR25a was one of the most promising candidates, which was later confirmed by behavioural tests using RNA interference: Reducing IR25a expression in Chorgan resulted in abnormal behaviour during temperature cycles, similar to what had been described for Nocte mutant. To further confirm the interaction between Nocte and IR25a, I showed that IR25a physically interacts with Nocte in vivo. Moreover, using an IR25a‐gal4 line, I was able to show that IR25a is expressed in subsets of chordotonal organs (Ch organ) including Johnston's Organs (JO), where Nocte is also highly expressed. These results, along with the behavioural data mentioned above are consistent with the proteomics results and suggest that Nocte and IR25a physically and functionally interact. IR25a mutants were employed to further investigate the function of IR25a in temperature entrainment. First of all, I found that both central and peripheral clocks in wild type flies can be synchronized to temperature cycles with only two degree differences (12h: 12h, 27 °C: 25 °C). In contrast, synchronization of locomotor activity rhythms in the IR25a null mutants to the same temperature cycles and other TC's with 2°C amplitude was eliminated. Under the same conditions, the oscillations of the core clock proteins TIMLESS (TIM) and PERIOD (PER) that normally occur in fly heads were completely abolished inIR25a null mutants, suggesting that IR25a is required for temperature entrainment of peripheral clocks. In the central brain pacemaker neurons, the oscillations of TIM in dorsal and lateral neurons were also affected by the IR25a mutants. On the contrary, IR25a is not required for light entrainment and temperature compensation, suggesting that IR25a is specifically involved in temperature synchronization. Moreover, temperature entrainment of the IR25a null mutants can be partially restored by applying larger temperature intervals (29°C: 25°C) indicating that IR25amay function as amplitude detector independent of absolute temperature values. Finally, neuronal activity in IR25a+ neurons is crucial for the synchronization of circadian clocks to low amplitude temperature cycles. Re‐constitution of functional olfactory receptors required the assembly of IR25a with IR76a and IR76b. Interestingly, IR76a and IR76b are neither required for temperature entrainment at the behavioural level nor expressed in the Ch organs. To check if other potential IRs interacting with IR25a exist, I screened the expression pattern of most divergent IRs using IR‐gal4/UAS‐GFP flies. IR56a was isolated as a potential partner of IR25a because it is also expressed in the femur chordotonal organs. To investigate the function of IR56a in temperature entrainment, I generated a null mutant of IR56a. Surprisingly, this gene is not required for synchronizing clocks to a temperature cycle (27°C: 25°C) at the behavioural level. However, the behavioural and molecular phenotypes of IR56a mutant under different temperature cycles need to be further characterized.

INTRODUÇÃO: A associação entre saúde e fatores como parâmetros de sono, autoeficácia percebida, e rotinas de trabalho é bem estabelecida, e relacionada a importantes desfechos clínicos. Contudo, não constam na literatura estudos que analisem associações entre todos esses fatores, tampouco a inter-relação destes entre si. OBJETIVO: Investigar a inter-relação de bem-estar psicológico com parâmetros de sono, níveis de autoeficácia percebida e rotinas de trabalho. MÉTODOS: Trata-se de um estudo transversal, realizado no Vale do Taquari-RS, Brasil, com 987 indivíduos (66,9% mulheres; idade média=43,9 anos). A atividade de trabalho predominante foi ligada à agricultura (46%), e a maior parte dos indivíduos exercia atividades de trabalho sete dias por semana (69,1%). Foram utilizadas as escalas World Health Organization five-item well-being index (WHO-5), Munich Chronotype Questionnaire (MCTQ), a Escala de Autoeficácia Percebida (General Self-Efficacy Scale – GSE), além de questionários de dados demográficos e de rotinas de trabalho. As análises estatísticas foram realizadas por meio de regressão linear hierárquica, e testes de modelos de mediação e de moderação. RESULTADOS: A análise de moderação demonstrou interação do horário de término do trabalho sobre a relação entre o horário de início do sono e o bem-estar psicológico (R=0,147; F=23,77; P<0,001). O modelo de regressão hierárquica final, incluindo a análise de moderação, demonstrou associação de bem-estar psicológico com sexo (B=-28,554; P=0,004), horário de início do sono (B=-10,132; P=0,011), autoeficácia (B=0,174; P<0,001) e com a variável de interação entre horário de início do sono e horário de término do trabalho (B=-3,460; P=0,030). A análise de mediação não mostrou efeitos indiretos estatisticamente significativos. CONCLUSÃO: O modelo final mostrou que, quando controlada para o efeito de moderação, a relação entre rotinas de trabalho e bem-estar psicológico é dependente da interação com parâmetros de sono individuais. Nossos achados chamam a atenção para a importância da autoeficácia percebida e da interação entre ritmos individuais e ritmos de trabalho sobre desfechos relacionados ao bem-estar psicológico. / BACKGROUND: The importance of sleep-wake patterns, self-efficacy and work related parameters, in relation to health outcomes, is well established. To the best of our knowledge, there are no studies analyzing the inter-relationship between these factors. OBJECTIVES: Investigate the inter-relationship between psychological well-being and sleep-wake patterns, general self-efficacy and work routine parameters. METHODS: This cross-sectional study was performed in a rural area of Brazil. A sample of 987 individuals (66.9% women; mean age = 43.9 years) was analyzed. Most participants were farmers (46%), and most worked 7 days a week (69.1%). The World Health Organization Five-item Well-being Index (WHO-5) was used to assess our outcome, and the Munich Chronotype Questionnaire (MCTQ), the General Self-Efficacy Scale (GSE), and a demographic and work routine questionnaire were used to assess the variables of interest. To better understand the inter-relationship between variables and outcome, mediation and moderation models were tested. RESULTS: The moderation model showed an effect of work end time on the relationship between sleep onset time and psychological well-being (R²=0.147; F=23.77; P<0.001). The final regression model showed an association of psychological well-being with sex (B=-28.554; P=0.004), sleep onset time (B=-10.132; P=0.011), self-efficacy (B=0.174; P<0.001), and with the interaction variable between sleep onset time and work end time (B=-3.460; P=0.030). The mediation model showed no statistically significant effects. CONCLUSIONS: Our final model showed that, when controlled for the moderation effect, the relationship between worse psychological well-being and later work end times is significant only when there is interaction to sleep onset times. These findings draw the attention to the importance of the perceived self-efficacy alone and the interaction between sleep-wake and work routine rhythms in relation to psychological well-being.

INTRODUÇÃO: Pacientes com fibromialgia (FM) apresentam distúrbios no sistema de resposta ao estresse, o qual é composto pelo eixo hipotalâmico-pituitário-adrenal (HPA) e pelo sistema nervoso autônomo. Recentemente, tem havido muito interesse na possível função do sistema nervoso autônomo na patogênese da FM. O objetivo deste estudo foi avaliar a interação entre os sistemas simpático e parassimpático, em mulheres com FM e mulheres saudáveis, utilizando a análise da variabilidade da freqüência cardíaca (VFC). MÉTODOS: Foram estudadas 20 mulheres com FM com idades entre 35 e 55 anos, e 20 controles saudáveis pareados pela idade, gênero e índice de massa corporal. A VFC foi analisada sobre gravações eletrocardiográficas, obtidas através da monitorização eletrocardiográfica ambulatorial (Holter) de 24 horas, e avaliada pelos índices da VFC no domínio do tempo (SDNN, SDANN, SDNNi, RMSSD e pNN50) e no domínio da freqüência (LF, HF, WF e LF/HF). A VFC foi analisada durante o período de 24 horas e também durante o período noturno, entre 01:00 e 04:00 h AM, consideradas como horas de sono. O equilíbrio simpato-vagal foi analisado através da razão LF/HF, sendo as faixas de freqüências da LF (0,04-0,15 Hz) considerada como predominantemente simpática, e da HF (0,15-0,50 Hz) considerada como predominantemente parassimpática. RESULTADOS: Não houve diferença de idade entre pacientes com FM e o grupo controle (44,40 ± 5,01 e 44,65 ± 5,32 anos, respectivamente; p=0,879). Os índices que refletem o sistema nervoso parassimpático, mostraram um comportamento similar entre pacientes com FM, mas revelaram atividade significativamente diminuída quando comparado ao grupo controle, ambos durante o período noturno e durante o período de 24 horas (p<0,05). Não houveram diferenças entre os índices que refletem o sistema simpático entre os grupo FM e controle (p>0,05), assim como não mostraram hiperatividade simpática. Contudo, a razão LF/HF foi significativamente maior em pacientes com FM, quando comparado ao grupo controle, ambos durante o período de sono (p=0,015) como durante o período de 24 horas (p=0,025), sugerindo predominância simpática em indivíduos com FM. CONCLUSÃO: Nossos resultados sugerem que pacientes com FM apresentam predominância da atividade simpática, associado ao tônus parassimpático diminuído. Sob condições basais não foi detectada hiperatividade simpática, uma vez que a atividade simpática não mostrou alterações significantes. / INTRODUCTION: Patients with fibromyalgia (FM) exhibit disturbances of the stress-response system, which is composed by hypothalamic-pituitary-adrenal axis (HPA) and autonomic nervous system. Recently, much interest has been expressed in the possible role of the autonomic nervous system in the pathogenesis of FM. The aim of this study was to assess the interation between sympathetic and parasympathetic systems, in FM and health women, using heart hate variability (HRV) analysis. METHODS: It was studied 20 women with FM aged between 35 and 55 years-old, and 20 healthy controls matched for age, sex and body mass index. HRV was assessed over electrocardiographic recordings, obtained by 24-hours ambulatory electrocardiography monitoring (Holter), and evaluated by time domain indexes (SDNN, SDANN, SDNNi, RMSSD e pNN50) and frequency domain indexes (LF, HF, WF e LF/HF). HRV was analyzed over the 24-hours period and also over the night period, between 01:00 and 04:00 AM, considered as sleep hours. Sympathovagal balance was analysed by LF/HF ratio, with LF band (0.04-0.15 Hz) considered as sympathetic predominance, and HF band (0.15-0.50 Hz) considered as parasympathetic predominance. RESULTS: There was no age difference between FM patients and control group (44.40 ± 5.01 and 44.65 ± 5.32 years, respectively; p=0.879). The indexes that reflect parasympathetic nervous system, showed a similar behavior among FM patients, but revealed a significantly decreased activity when compared to control group, both during the nocturnal period as well during the 24h period (p<0.05). There was no difference between the indexes that reflect sympathetic system in FM patients and controls (p>0.05), as did not show sympathetic hyperactivity. However, the ratio LH/HF was significantly higher in FM patients, when compared to control group, both during the sleep period (p=0.015) as well as over the 24h period (p=0.025), suggesting a sympathetic predominance in FM subjects. CONCLUSION: Our data suggest that FM patients present a predominance of sympathetic activity, associated with a reduced parasympathetic tonus. Under basal conditions sympathetic hyperactivity was not detected, since sympathetic activity did not show significant alterations.

INTRODUÇÃO: Pacientes com fibromialgia (FM) apresentam distúrbios no sistema de resposta ao estresse, o qual é composto pelo eixo hipotalâmico-pituitário-adrenal (HPA) e pelo sistema nervoso autônomo. Recentemente, tem havido muito interesse na possível função do sistema nervoso autônomo na patogênese da FM. O objetivo deste estudo foi avaliar a interação entre os sistemas simpático e parassimpático, em mulheres com FM e mulheres saudáveis, utilizando a análise da variabilidade da freqüência cardíaca (VFC). MÉTODOS: Foram estudadas 20 mulheres com FM com idades entre 35 e 55 anos, e 20 controles saudáveis pareados pela idade, gênero e índice de massa corporal. A VFC foi analisada sobre gravações eletrocardiográficas, obtidas através da monitorização eletrocardiográfica ambulatorial (Holter) de 24 horas, e avaliada pelos índices da VFC no domínio do tempo (SDNN, SDANN, SDNNi, RMSSD e pNN50) e no domínio da freqüência (LF, HF, WF e LF/HF). A VFC foi analisada durante o período de 24 horas e também durante o período noturno, entre 01:00 e 04:00 h AM, consideradas como horas de sono. O equilíbrio simpato-vagal foi analisado através da razão LF/HF, sendo as faixas de freqüências da LF (0,04-0,15 Hz) considerada como predominantemente simpática, e da HF (0,15-0,50 Hz) considerada como predominantemente parassimpática. RESULTADOS: Não houve diferença de idade entre pacientes com FM e o grupo controle (44,40 ± 5,01 e 44,65 ± 5,32 anos, respectivamente; p=0,879). Os índices que refletem o sistema nervoso parassimpático, mostraram um comportamento similar entre pacientes com FM, mas revelaram atividade significativamente diminuída quando comparado ao grupo controle, ambos durante o período noturno e durante o período de 24 horas (p<0,05). Não houveram diferenças entre os índices que refletem o sistema simpático entre os grupo FM e controle (p>0,05), assim como não mostraram hiperatividade simpática. Contudo, a razão LF/HF foi significativamente maior em pacientes com FM, quando comparado ao grupo controle, ambos durante o período de sono (p=0,015) como durante o período de 24 horas (p=0,025), sugerindo predominância simpática em indivíduos com FM. CONCLUSÃO: Nossos resultados sugerem que pacientes com FM apresentam predominância da atividade simpática, associado ao tônus parassimpático diminuído. Sob condições basais não foi detectada hiperatividade simpática, uma vez que a atividade simpática não mostrou alterações significantes. / INTRODUCTION: Patients with fibromyalgia (FM) exhibit disturbances of the stress-response system, which is composed by hypothalamic-pituitary-adrenal axis (HPA) and autonomic nervous system. Recently, much interest has been expressed in the possible role of the autonomic nervous system in the pathogenesis of FM. The aim of this study was to assess the interation between sympathetic and parasympathetic systems, in FM and health women, using heart hate variability (HRV) analysis. METHODS: It was studied 20 women with FM aged between 35 and 55 years-old, and 20 healthy controls matched for age, sex and body mass index. HRV was assessed over electrocardiographic recordings, obtained by 24-hours ambulatory electrocardiography monitoring (Holter), and evaluated by time domain indexes (SDNN, SDANN, SDNNi, RMSSD e pNN50) and frequency domain indexes (LF, HF, WF e LF/HF). HRV was analyzed over the 24-hours period and also over the night period, between 01:00 and 04:00 AM, considered as sleep hours. Sympathovagal balance was analysed by LF/HF ratio, with LF band (0.04-0.15 Hz) considered as sympathetic predominance, and HF band (0.15-0.50 Hz) considered as parasympathetic predominance. RESULTS: There was no age difference between FM patients and control group (44.40 ± 5.01 and 44.65 ± 5.32 years, respectively; p=0.879). The indexes that reflect parasympathetic nervous system, showed a similar behavior among FM patients, but revealed a significantly decreased activity when compared to control group, both during the nocturnal period as well during the 24h period (p<0.05). There was no difference between the indexes that reflect sympathetic system in FM patients and controls (p>0.05), as did not show sympathetic hyperactivity. However, the ratio LH/HF was significantly higher in FM patients, when compared to control group, both during the sleep period (p=0.015) as well as over the 24h period (p=0.025), suggesting a sympathetic predominance in FM subjects. CONCLUSION: Our data suggest that FM patients present a predominance of sympathetic activity, associated with a reduced parasympathetic tonus. Under basal conditions sympathetic hyperactivity was not detected, since sympathetic activity did not show significant alterations.

Family and twin studies have shown a heritable component to seasonal affective disorder (SAD). While a few studies have examined individual genetic variants in SAD, many methodological issues exist in the current literature. First, most studies combined major depression (MDD) and bipolar (BD) cases in the genetic analysis of SAD. This makes it difficult to differentiate the effect from MDD and BD. Second, most studies adopted a candidate gene approach and used fairly small sample sizes. This does not allow for testing across a wide variety of genes, and it yields less robust P-values. Third, healthy controls have been used, but not case comparisons, which makes it difficult to differentiate the effects of seasonality from that of the primary illness (MDD and BD). To overcome these issues, seasonal MDD and BD cases were separated into two different studies in this thesis; sample sizes for both studies are the largest in the current SAD molecular genetics literature; GWAS was used to test for potential risk loci in a hypothesis-free fashion; case comparisons were incorporated to exclude potential genetic contributions related generally to the primary diseases themselves (MDD and BD). For MDD, we performed a GWAS with 562 seasonal MDD cases and 1,225 comparison cases with non-seasonal MDD. Subjects were drawn from two iterations of the Genetics of Recurrent Early Onset Depression (GenRED) study. Seasonal cases were those whose depressive episodes typically started in fall or winter. A mega-analysis of the two GWAS datasets was done using SNPTEST. We found that two single nucleotide polymorphisms (SNPs), rs149882931 and rs77073398, on chromosome 16p12.1 were associated with seasonal depression, at a genome-wide significant level (OR= 1.66, P= 3.59 x 10-8 and OR=1.62, 4.76 x 10-8, respectively). Since SAD is more prevalent in females, a female-specific analysis was carried out. The two variants were more significant in this analysis: P=2.18x10-9 (OR=1.89) and P=2.79x10-9 (OR=1.82), respectively, and a significant sex-by-SNP interaction was observed. These SNPs are located in a conserved intergenic region between the genes HS3ST4 and C16orf82. The protein product of HS3ST4 modifies the side chains of heparan sulfate proteoglycans. We therefore tested the hypothesis that the heparan sulfate biosynthesis pathway would be enriched in nominally significant SNPs using the SNP ratio test, and found evidence for such enrichment (P=0.008, SNP ratio test, P=0.027, SKAT). For BD, the GWAS analysis of 818 seasonal BD cases and 1,515 healthy controls showed that BD-S is most strongly associated with two SNPs within the ZBTB20 genes. BD subjects were drawn from NIMH Bipolar Genetics Study (BIGS), and seasonal cases were defined as those with depressive episodes starting in fall or winter. An association study was carried out with SNPTEST, and we found two single nucleotide polymorphisms (SNPs) in the intronic region of ZBTB20 gene to be associated with BD-S (rs7646282, OR=2.34, P= 7.23 x 10-8 and rs139459337, OR=2.37, 8.05 x 10-8). A similar case-only study was carried out with 818 BD-S cases and 1239 cases without seasonal depressive symptoms (non-BDS), though no SNP was found to be significantly associated in this analysis. rs7646282 is the strongest SNP in cis-association with ZBTB20 gene expression, and ZBTB20 has been shown to affect the neural development of the hippocampus, a brain region implicated in the pathophysiology of BD. Finally, we sought to determine whether there is a role for circadian rhythm genes in BD susceptibility. In this study, we used a discovery set of 189 exome-sequenced BD patients and 105 healthy controls to look for circadian genes associated with BD. We found the DRD2 gene to be the circadian gene most strongly associated with BD. Among the rare damaging variants in the DRD2 gene, the S311C variant was the predominant SNP. To test whether this variant segregates in family members with BD, we genotyped the family members of probands from the discovery sample. This data was used for a linkage and family-based association study. Even though the linkage analysis was only very weakly positive, the family-based association study showed significant segregation of the variant in family members with BD (P< 0.05). To follow up on this finding, we further genotyped 2,185 unrelated BD cases and 1,982 healthy controls. We found no support for the S311C variant in this replication dataset. Sub-phenotype study of psychotic features and mood-incongruence also did not show significant association. Meta-analysis with 2,994 BD cases and 3,661 controls, however, revealed no association between the S311C variant and BD.

This thesis explores a design project concerned with the relationship between the person and nature in the context of achieving a state of symbiosis between the two – a state which can be reached through highlighting the relationship between the person and the rhythmic characteristics of natural light. The project originated from a concern with modern society’s constant separation from the natural environment and the resulting sense of placelessness often experienced in the spaces created. In response, a desire arose to investigate the effect that natural light has on the person and contribute to the design of naturally enriched spaces where light is used as the link between the person and the natural environment. We are constantly influenced by the prevailing conditions of light. Our biorhythms are in tune with the natural changes from day to night, the duration and intensity of sunlight and the spectral composition of light. However, in an attempt to create the optimum formula for comfort, efficiency, and productivity, we are using advanced lighting technology to create uniform interior spaces detached from the everchanging exterior environment. The outcomes of the study are to inform the practice of design and architecture and to use the findings in a beneficial manner towards the design of educative spaces. In response to the need for a secondary school in Fond des Blancs, Haiti, I want to focus on the chosen site and program, and investigate the rhythms of light and their effects as they are related to the specific location and the purpose of education. Moreover, I want to use the results to create a set of design guidelines for the specific location and function of the buildings to find out how biorhythmic design can be used for the creation of an educative environment where natural light is channeled, maximized and utilized for the goals of the learning process?

Most members of the PAS (PER-ARNT-SIM) protein family are transcription factors, mediating development and adaptive responses to the environment, such as circadian rhythms and toxin responses. Because the PAS domain mediates protein-protein interactions and functional cross-talk between distinct biological processes, we hypothesized that PAS genes in the circadian clockworks, namely Per1 and Per2, may be involved in development and toxin responses, which are modulated by other PAS members. To explore the possible role of clock genes in development, we examined mammary epithelial cells in vitro and the mouse mammary gland in vivo for evidences of changes in clock gene expression during different stages of development and differentiation. Our results showed that Per1 and Bmal1 expression were up-regulated in differentiated HC-11 cells, whereas Per2 mRNA levels were higher in undifferentiated cells. A similar differentiation-dependent profile of clock gene expression was observed in mouse mammary glands; Per1 and Bmal1 mRNA levels were elevated in late pregnant and lactating mammary tissues, whereas Per2 expression was higher in proliferating virgin and early pregnant glands. These data suggest that circadian clock genes may play a role in mouse mammary gland development. To examine clock gene function in toxin responses, we evaluated whether disruption or inhibition of Per1 and/or Per2 alters toxin-induced activity of the AhR signaling pathway in the mouse mammary gland and liver. We assessed the activation of the AhR signaling pathway in response to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a prototypical AhR agonist, by analyzing the mRNA abundance of its two target genes, cytochrome P450, subfamily I, polypeptide 1 (Cyp1A1) and Cyp1B1. Our results showed that the targeted disruption of Per1, but not Per2, significantly increases the TCDD-induced p450 expression in the mammary gland and liver in vivo. Similar changes in TCDD-mediated p450 expression were observed in vitro using mammary primary cultures of mammary cells derived from from Per1ldc, Per2ldc and Per1ldc/Per2ldc mutant mice and Hepa1c1c7 cells subjected to siRNA-mediated inhibition of Per1 or Per2. These discoveries suggest that the clock gene Per1 may modulate toxin responses perhaps by functioning as a negative regulator for TCDD-mediated activation of the AhR signaling pathway.

(測定・評価)

reinhardtii, a unicellular green alga, is a model organism to study the circadian clock. Cryptochromes are the blue light photoreceptors that entrain the clock in some organisms. The CPH1 protein of C. reinhardtii resembles the cryptochromes of the plant model Arabidopsis, but whether CPH1 entrains the circadian clock in C. reinhardtii is not yet known. Recent reports have suggested the existence of one more cryptochrome in C. reinhardtii, which resembles the cryptochromes of animals. However, the amino acid sequence of this protein shows even higher sequence similarity with the 6-4 DNA photolyase of Arabidopsis. DNA photolyases are involved in the repair of UV light-induced DNA damage using the energy of blue light. In order to determine, if the “animal cryptochrome” gene of C. reinhardtii actually encodes a 6-4 DNA photolyase rather than a photoreceptor, an experimental design was developed to test whether the protein product is able to rescue an E. coli mutant defective in its DNA photolyase gene. The design is as follows: In a first step, the coding region of the “animal cryptochrome” cDNA is cloned. In a second step, the cDNA is inserted in-frame into an E. coli expression vector. In a third step, the construct is transformed into an E. coli photolyase mutant, its expression induced, and the strain tested for better survival after UV light exposure. To accomplish the first step, the cloning of “animal cryptochrome” cDNA, total RNA was successfully extracted from C. reinhardtii 4 hrs into the light phase of a 12 h light/12 h dark cycle and reverse transcribed into cDNA using oligo(dT) primers. After initially unsuccessful attempts at amplifying animal cryptochrome from cDNA or genomic template with a variety of primers and conditions, a short fragment with the expected size of 186 bp was amplifiable with both templates. However, even this fragment was not reliably obtained in every PCR assay. Because of this difficulty, real-time PCR was finally performed in the presence of DMSO (Dimethylsulfoxide) and Betaine. These two adjuvants were reported to improve amplifications particularly for GC-rich templates. C. reinhardtii DNA is especially GC-rich with an average of 64% Gs and Cs. The improved conditions allowed the reliable amplification of the 186 bp fragment from genomic template. It also enabled the amplification of a larger fragment of 528 bp from the same template. The results suggest that a combination of 5% DMSO and 1M Betaine is optimal for the amplification of C. reinhardtii DNA and thus can serve as the basis for successful amplification of the entire 1788 bp coding region of the animal cryptochrome cDNA.

Most members of the PAS (PER-ARNT-SIM) protein family are transcription factors, mediating development and adaptive responses to the environment, such as circadian rhythms and toxin responses. Because the PAS domain mediates protein-protein interactions and functional cross-talk between distinct biological processes, we hypothesized that PAS genes in the circadian clockworks, namely Per1 and Per2, may be involved in development and toxin responses, which are modulated by other PAS members. To explore the possible role of clock genes in development, we examined mammary epithelial cells in vitro and the mouse mammary gland in vivo for evidences of changes in clock gene expression during different stages of development and differentiation. Our results showed that Per1 and Bmal1 expression were up-regulated in differentiated HC-11 cells, whereas Per2 mRNA levels were higher in undifferentiated cells. A similar differentiation-dependent profile of clock gene expression was observed in mouse mammary glands; Per1 and Bmal1 mRNA levels were elevated in late pregnant and lactating mammary tissues, whereas Per2 expression was higher in proliferating virgin and early pregnant glands. These data suggest that circadian clock genes may play a role in mouse mammary gland development. To examine clock gene function in toxin responses, we evaluated whether disruption or inhibition of Per1 and/or Per2 alters toxin-induced activity of the AhR signaling pathway in the mouse mammary gland and liver. We assessed the activation of the AhR signaling pathway in response to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a prototypical AhR agonist, by analyzing the mRNA abundance of its two target genes, cytochrome P450, subfamily I, polypeptide 1 (Cyp1A1) and Cyp1B1. Our results showed that the targeted disruption of Per1, but not Per2, significantly increases the TCDD-induced p450 expression in the mammary gland and liver in vivo. Similar changes in TCDD-mediated p450 expression were observed in vitro using mammary primary cultures of mammary cells derived from from Per1ldc, Per2ldc and Per1ldc/Per2ldc mutant mice and Hepa1c1c7 cells subjected to siRNA-mediated inhibition of Per1 or Per2. These discoveries suggest that the clock gene Per1 may modulate toxin responses perhaps by functioning as a negative regulator for TCDD-mediated activation of the AhR signaling pathway.