Dissertations / Theses on the topic 'Circadianer Rhythmus'

Made available in DSpace on 2014-12-17T15:36:40Z (GMT). No. of bitstreams: 1 BrunoSBG_DISSERT.pdf: 3965357 bytes, checksum: 7e3aabdd040d50db3f4557799b032b1d (MD5) Previous issue date: 2013-04-03<br>Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico<br>Circadian rhythms are variations in physiological processes that help living beings to adapt to environmental cycles. These rhythms are generated and are synchronized to the dark light cycle through the suprachiasmatic nucleus. The integrity of circadian rhythmicity has great implication on human health. Currently it is known that disturbances in circadian rhythms are related to some problems of today such as obesity, propensity for certain types of cancer and mental disorders for example. The circadian rhythmicity can be studied through experiments with animal models and in humans directly. In this work we use computational models to gather experimental results from the literature and explain the results of our laboratory. Another focus of this study was to analyze data rhythms of activity and rest obtained experimentally. Here we made a review on the use of variables used to analyze these data and finally propose an update on how to calculate these variables. Our models were able to reproduce the main experimental results in the literature and provided explanations for the results of experiments performed in our laboratory. The new variables used to analyze the rhythm of activity and rest in humans were more efficient to describe the fragmentation and synchronization of this rhythm. Therefore, the work contributed improving existing tools for the study of circadian rhythms in mammals<br>Os ritmos circadianos s?o varia??es em processos fisiol?gicos que auxiliam os seres vivos na adapta??o aos ciclos ambientais. Esses ritmos s?o gerados e se sincronizam ao ciclo claro escuro por meio do n?cleo supraquiasm?tico. A integridade da ritmicidade circadiana tem grande implica??o na sa?de dos seres humanos. Atualmente sabe-se que dist?rbios nos ritmos circadianos est?o relacionados com alguns problemas da atualidade como a obesidade, propens?o a determinados tipos de c?ncer e transtornos mentais por exemplo. A ritmicidade circadiana pode ser estudada por meio de experimentos com modelos animais e diretamente nos seres humanos. Nesse trabalho utilizamos modelos computacionais para reunir resultados experimentais da literatura e explicar resultados de nosso laborat?rio. Outro foco desse trabalho foi na an?lise de dados de ritmos de atividade e repouso obtidos experimentalmente. Aqui fizemos uma revis?o sobre o uso de vari?veis utilizadas para analisar esses dados e por ?ltimo propomos uma atualiza??o na forma de calcular essas vari?veis. Os nossos modelos foram capazes de reproduzir os principais resultados experimentais da literatura e nos forneceram explica??es para resultados de experimentos realizados em nosso laborat?rio. As novas vari?veis utilizadas para analisar o ritmo de atividade e repouso em humanos se mostraram mais eficiente para descrever a fragmenta??o e sincroniza??o desse ritmo. Assim esse trabalho contribuiu aperfei?oando as ferramentas existentes para o estudo da ritmicidade circadiana nos mam?feros

Adipose tissue secretes many important biologically active proteins called adipokines. A subset of adipokines, called C1q tumor necrosis factor (TNF) related proteins (CTRPs), play a key role in metabolism, inflammation, and cell signaling. C1q TNF Related Protein 3 (CTRP3) increases hepatic fatty acid oxidation, decreases inflammation, and aids in cardiovascular recovery following a myocardial infarction. However, the mechanisms behind CTRP3’s protective effects on organ systems are unknown. This exploratory study aims to analyze the circulating oligomeric state of CTRP3 and the circadian regulation of CTRP3 to help understand the role of CTRP3 in preventing disease. METHODS: For analysis of the oligomeric state of CTRP3 non-fasting mouse serum was collected from high fat fed hyper-glycemic mice or low fat fed normoglycemic mice and was separated by size exclusion filtration. For analysis of the circadian regulation of CTRP3 serum samples were collected from mice at 4 different time points (2 dark cycle and 2 light cycle) throughout the day and circulating CTRP3 levels were analyzed by immunoblot analysis. RESULTS: In both high fat and low fat fed mice CTRP3 was found to circulate in both >300 kDa oligomers and >100kDa oligomers, with no detectable amount of CTRP3 less 100 kDa. Interestingly, although there was no difference in the total amount of CTRP3 between the high fat and low fat fed mice there was a higher abundance of CTRP3 >300 kDa in the high fat fed and a greater abundance of CTRP3 found 100-300 kDa. Additionally, we found that serum CTRP3 levels vary greatly throughout a 24-hour time-period within each mouse, but no consensus circadian pattern was observed. CONCLUSION: In vitro mammalian produced recombinant CTRP3 protein was found to exist as trimer, hexamer, and high molecule weight. This is the first study to indicate that CTRP3 circulates in different oligomeric states in vivo, and this is also the first study to observe a difference in the oligomeric state of CTRP3 related to metabolic state. Combined these findings indicate that oligomeric state of CTRP3 may be more metabolically relevant than total amount of circulating CTRP3. In addition, our finding of a high variability of CTRP3 within the same mouse at different times throughout the day indicates that is not regulated by circadian rhythms but is susceptible to variability due to some unknown regulatory factor. These findings have identified novel unknown aspects of CTRP3, which require further research to understand the role of CTRP3 in human health and disease.

Studies that show discrepancy between nocturnal and diurnal activity under laboratory and field conditions, respectively, have become increasingly common in rodents and suggest that the definition of temporal niche is far more plastic than originally suspected. Recently, it has been proposed that factors that challenge the animal\'s energy balance play an important role in temporal niche switches. Food availability and environmental temperatures could then be one of the fundamental differences between field and laboratory that could alter the temporal daily pattern of activity. In laboratory, animals are fed ad libitum, while in nature they need to expend energy for foraging. The \"circadian thermo-energetics hypothesis\" suggests that daytime activity could be a response to the high energetic costs of foraging, allowing the animal to save energy during the cooler night hours by resting and taking shelter in burrows where temperatures are higher than on the surface. In this thesis, we explored the interplay of plasticity in nocturnal/diurnal activity definition and energetic metabolism in two rodent species, tuco-tucos (Ctenomys aff. knighti) and laboratory mice (Mus musculus). Tuco-tucos are subterranean rodents which face peculiar energetic challenges in their habitat and were shown to be diurnal in the field and nocturnal in the laboratory. We characterized how their energy expenditure varies across day and night and described the peculiar finding of some factor inside the metabolic chamber being itself a trigger for the nocturnal to diurnal switch. Moreover, we estimated the amount of energy tuco-tucos would save by being diurnal in the field, by combining metabolic rate measurements at various ambient temperatures with records of environmental temperature in the tuco-tuco\'s natural habitat. We also described further investigations of circadian plasticity in both locomotor activity and body temperature of laboratory mice subjected to food restriction in semi-natural conditions. The findings of these three studies provided valuable evidence for the discussion of the role of environmental factors, particularly energetic challenges, in the plasticity of daily rhythms<br>Estudos que apontam discrepâncias entre atividade noturna e diurna, respectivamente, sob condições de laboratório e de campo, estão cada vez mais comuns em roedores e sugerem que a definição de nicho temporal é muito mais plástica do que se suspeitava inicialmente. Recentemente, foi proposto que fatores que desafiam o balanço energético do animal desempenham um papel importante em mudanças de nicho temporal. A disponibilidade de alimento e as temperaturas ambientais poderiam ser algumas das diferenças fundamentais entre campo e laboratório, os quais poderiam alterar o padrão temporal de atividade diária. No laboratório, os animais são alimentados ad libitum, enquanto na natureza eles precisam gastar energia para forrageamento. A \"hipótese circadiana termoenergética\" sugere que a atividade diurna pode ser uma resposta aos altos custos energéticos do forrageamento, permitindo que o animal economize energia durante as horas mais frias da noite, descansando e se abrigando em tocas onde as temperaturas são mais altas do que na superfície. Nesta tese, exploramos a interação entre a plasticidade da definição noturnalidade/diurnalidade e o metabolismo energético em duas espécies de roedores, o tuco-tucos (Ctenomys aff. knighti) e o camundongo de laboratório (Mus musculus). Tuco-tucos são roedores subterrâneos que enfrentam desafios energéticos peculiares em seu habitat e verificamos que são diurnos em campo e noturnos em laboratório. Nós caracterizamos a variação de seu gasto energético ao longo do dia e da noite e descrevemos o achado peculiar de que algum fator presente no interior da câmara metabólica pode ser um gatilho para a mudança de noturnalidade para diurnalidade. Além disso, estimamos a quantidade de energia que os tuco-tucos economizariam ao serem diurnos em campo, combinando medidas de taxa metabólica em várias temperaturas ambientes com registros dessa temperatura no habitat natural do tuco-tuco. Descrevemos também investigações adicionais sobre a plasticidade circadiana na atividade locomotora e na temperatura corporal de camundongos submetidos à restrição alimentar, em condições seminaturais. Os achados desses três estudos forneceram evidências valiosas para a discussão do papel dos fatores ambientais, particularmente os desafios energéticos, na plasticidade dos ritmos diários

An organism’s circadian clock exists as a self-regulating oscillator that can synchronize with its surroundings. This manifests as physiological and behavioral output which can anticipate changes in environment. These rhythms may even persist internally and are known to oscillate at a period (tau) of around 24 hours even in the absence of external cues. In the laboratory, such rhythmic output is known as a free-running period (FRP). Given that circadian rhythms are distributed in a number of taxa as well as their tendency to oscillate along with the solar day, it has been suggested that they result from natural selection. The argument that tone’s clock is adaptive is based on how it is advantageous: the clock instills temporal order among physiological processes as well as enabling one to anticipate external cues. Losing that order in one’s clock has also been associated with a number of metabolic and neurological pathologies. Along with adaptive significance, it has been surmised that an internal clock which synchronizes with one’s surrounding environment is beneficial to an individual. An organism whose free-running period closest matches the rhythmic output of its external environment will exhibit a higher relative fitness as compared to those whose periods deviate from 24 hours. This forms the basis of the ‘circadian resonance hypothesis’. Circadian resonance has been examined in a number of species, from Cyanobacteria to mammals. Collectively, experimental results have supported the rationale that an individual does best when its internal clock resonates with the 24 hour day. The bowl and doily spider, Frontinella communis, not only has its own endogenous rhythm (free-running period), it exhibits an average free-running period of 28.26 hours, deviating from a usual period of ~24 hours. Keeping in mind the circadian resonance hypothesis, an internal clock with such an extreme deviation from the 24 hour day should prove detrimental to one’s overall health. Despite this, Frontinella communis not only has a long clock; among the species, their clocks also appear to be highly variable, FRPs ranging from ~24 to ~33 hours. This study monitored locomotor activity of Frontinella communis to examine whether its free-running period, on average, remained the same throughout its active season (May-September). It was found that average free-running period in F. communis varied significantly over a five-month period. Average FRP appears to peak in June followed by a steady, linear decline as the season continues. A variety of organisms have been shown to exhibit seasonal responses that allow them to cope with environmental change. It is not known whether the change in Frontinella’s FRP is such an advantage or is merely coincidental. Any free running period detected under the alpha level of 0.05 was not ruled significant. Along with the rise and fall of average FRPs, the presence of FRP deemed significant was found to decline as the season ended- 42% of individuals (n= 19) reported as arrhythmic. While age has been found to correlate with circadian desynchrony in other taxa (rats, humans), an association in Frontinella remains to be tested.

Thesis (Ph. D.)--University of California, San Diego, 2009.<br>Title from first page of PDF file (viewed July 23, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 127-136).

Fatores ambientais podem modificar o desenvolvimento dos ritmos biológicos em neonatos, como já demonstrado em ratos. Neste contexto insere-se o estudo de recém-nascidos pré-termo mantidos em unidades de cuidado neonatal. Descrevemos neste trabalho a evolução da ritmicidade no ciclo vigília/sono, atividade/repouso, temperatura do punho e alimentação na fase neonatal. Paralelamente, caracterizamos o desenvolvimento dos ritmos biológicos em ratos mantidos sob luz constante durante a lactação e a atuação da melatonina e do exercício físico nessa evolução. Em um estudo longitudinal utilizando actímetros e termistores com memória, identificamos precocemente ritmo circadiano na temperatura do punho, enquanto na atividade motora há predomínio de ritmos ultradianos, bem como no ciclo vigília/sono e no comportamento alimentar, padrão este que se modifica logo após a alta hospitalar. Em ratos sob o paradigma de luz constante, oferecemos melatonina e uma roda durante a lactação e após o desmame, encontrando modificações na emergência do ritmo circadiano em ambos os grupos.<br>Environmental factors can change the development of biological rhythms in neonates, as has already been demonstrated in rats. In this context is the study of preterm newborns maintained in neonatal care units. We describe in the present work the evolution of rhythmicity in sleep/wake cycle, activity/rest, wrist temperature and feeding behavior along the neonatal phase. Simultaneously, we characterize the development of biological rhythms in rats maintained under constant light during lactation and the action of melatonin and physical exercise in this evolution. Through a longitudinal study using actimeters and thermistors with memory we identified precociously a circadian rhythm in wrist temperature, while in motor activity we found a dominant ultradian rhythm, as well as, in sleep/wake cycle and feeding behavior, with changes in this pattern just after hospital discharge. In rats reared under constant light, we offered melatonin and a wheel during lactation and after weaning, finding differences in the emergency of the circadian rhythm for both groups.

Circadian clocks are vital to the proper functioning of organisms’ internal processes and behavioral outputs and typically have endogenous periods that approximate (within 1-2 hours) the 24-hour solar day. Clocks that deviate significantly from about 24 hours are often associated with metabolic syndromes or other disease states. For instance, organisms with near-24-hour clocks have higher survivorship under 24-h light:dark (LD) cycles than with 22- or 26-hour cycles. Likewise, mutant organisms with 22-hour clocks survive better under 22-h cycles but fare poorly under 24- and 26-h cycles. In other words, organisms suffer if their circadian clocks do not “resonate” with environmental cycles. Organisms fail to synchronize (entrain) their activity with non-resonant LD cycles and this failure typically leads to a number of physiological disruptions. Interestingly, several spider species have endogenous circadian periods that deviate by several hours from the period of the Earth’s solar day. The object of the present study is to investigate whether the phenomenon of circadian resonance also pertains to these atypical spider circadian rhythms. We investigated three spider species, two of which have internal periods (τ) significantly different from 24 hours. Approximately 50 individuals of each species of spider (Frontinella communis: τ=29.05±0.62 hours; Metazygia wittfeldae: τ=22.74±0.24h; and Cyclosa turbinata: τ=18.54±0.28h) were placed into chambers with periods of 19 (9.5:9.5h L:D), 24 (12:12h L:D), or 29 hours (14.5:14.5h L:D). If resonance is pertinent for spiders, we would expect survivorship to decrease in non-resonant LD cycles. Instead, no spider species exhibited decreased longevity in non-resonant L:D cycles. These findings contradict all previous research into circadian resonance and suggest that spiders do not suffer the costs of extreme desynchronization. In a second experiment, 10-11 spiders from each species were placed into infrared activity monitors to determine if their locomotor activity could entrain to (synchronize with) the three different LD cycles. Individuals from all three spider species entrained to all LD period lengths, again in contrast with prior research in other species. These results indicate that spider circadian clocks have highly unusual limits of entrainment and suggest a remarkable level of plasticity in their release from the selective pressure to maintain an internal period of approximately 24 hours.

Daily rhythms of physiology and behaviour in mammals are orchestrated by a hierarchical network of cellular oscillators. The master pacemaker that defines local and systemic timing across the brain and body are the suprachiasmatic nuclei of the hypothalamus (SCN). Disruption to the timing of sleep and daily behavioural activity can manifest in a range of pathologies including neuropsychiatric disorders. Bipolar disorder (BPD) is once such neurological condition that exhibits profound associations with altered circadian rhythm generation and whose toolkit of pharmacological interventions impact upon circadian rhythm generation. Currently it is unclear exactly how changes to rhythmic physiology contribute to the aetiology and pathology of BPD. In recent years, rodent models possessing lesions within genes that make up the basic cellular oscillator are widely reported to exhibit concomitant changes in affective behaviours, namely mania-like phenotypes. Recently a mouse model possessing a mutation within the neuron-specific Na+/K+-ATPase (NKA) alpha3 subunit, known as Myshkin, was described as a model of the manic phase of BPD. The NKA alpha3 is not reported as a critical element of the circadian oscillator and we used this opportunity to characterise the behavioural and physiological circadian system of these animals. Under wheel-running paradigms Myk/+ animals exhibited a broad array of behavioural deficits including lengthened, low amplitude and labile free-running rhythms, altered phase re-setting and elevated metabolic activity. Physiological characterisation of the SCN revealed deficits in amplitude of electrical output and changes to post-synaptic signalling although the ex vivo molecular pacemaking of the SCN remained intact. Myshkin animals therefore represent a novel model in which changes to central output arise independently of changes to basic molecular pacemaking. Despite this seemingly distinct mechanism Myshkin animals share many mood and circadian phenotypes with other clock gene models of affective behaviours highlighting that changes to pacemaking output of the SCN may be a critical factor across animal models exhibiting circadian and mood deficits. In addition, the impact of the mood stabiliser lithium, commonly prescribed in BPD, on cellular pathways within the SCN was investigated. Lithium consistently lengthens the period of cellular and behavioural rhythms in mammals although the mechanism of this action is yet undefined. Glycogen synthase kinase 3β (GSK3β) and inositol monophosphatase (IMPase) are the major biochemical targets of lithium at therapeutic concentrations. GSK3β is known to shorten rhythms and this study targeted IMPase and inositol phosphate turnover in the period lengthening effects of lithium. We reveal that although inhibition of IMPase dampens SCN molecular rhythms, the period of oscillations remains unchanged and therefore lithium acts upon distinct cellular pathways within the SCN to exert effects on period.

Many studies have investigated the ecological factors that affect behavior in Micrathena gracilis, a diurnal orb-weaving spider that forages exclusively 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, other spider species have been found to express significant circadian plasticity, suggesting that circadian clock-controlled rhythms may play a larger role in niche partitioning than once thought. 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 in a lab environment and web captures in the field were also used to confirm behavioral patterns and nutrient uptake throughout the day. It was found that significant prey biomass is given up shortly after the time that spiders typically retreat, suggesting that the spiders truly forfeit this prey and do not simply retreat due to a gradual decrease in overall prey availability. Spiders reliably cease foraging in the early evening and show agitation throughout the night when not comfortably hidden, suggesting that significant extension of foraging behavior may be harshly punished. However, recorded predation events from the camera array were much rarer than anticipated, and no predation was confirmed in the evening. These results support the notion that these spiders’ circadian rhythms are shaped by factors other than prey availability, but more work is necessary to identify these factors

The natural daily cycles of light and dark have played a fundamental role in shaping the development of an adaptive intrinsic clock mechanism which allows organisms to coordinate the function of multiple organs by setting the correct circadian timing of cellular processes ensuring proper homeostasis. In mammalian skin, homeostasis is maintained by epidermal stem cells (epSCs). EpSCs localize to specialized niches where they undergo cycles of quiescence and proliferation. Several pathways are known to play essential roles in epSC function; however, how are these pathways spatiotemporally coordinated, and why not all stem cells within the niche behave in the same manner, is still poorly understood. We have analyzed the role of the molecular circadian clock in fine-­‐tuning the behavior of epidermal stem cells. Using a fluorescent circadian reporter mouse model, we demonstrate that the dormant epidermal stem cell compartment contains two co-­‐existing populations of stem cells in different clock states. Global comparative transcriptome analysis indicated that each clock population corresponds to a distinct predisposition state of response towards stem cell activating and dormancy cues. We provide evidence that the core circadian transcription factors BMAL1 and CLOCK bind to regulatory elements in the promoters of several of these stem cell homeostatic genes, thus being directly responsible for creating these two stem cell clock states. Unbalancing this clock driven equilibrium of epSCs in vivo resulted in progressive changes in the response of stem cells to activating or dormancy cues, which led to a progressive premature tissue aging, and a significant reduction in the development of cutaneous squamous cell carcinomas. Thus, our results indicate that the molecular clock machinery fine-­‐tunes the spatiotemporal behavior of epidermal stem cells within their niche, and that perturbation of this mechanism affects tissue homeostasis and the predisposition to neoplastic transformation.<br>Los ciclos naturales de luz y oscuridad han sido determinantes en el desarrollo de un reloj molecular intrínseco que permite coordinar la función de múltiples órganos para mantener la homeostasis global del organismo. La homeostasis del compartimento queratinocítico de la piel depende de una población de células troncales adultas epidermales (epSCs). Las epSCs están localizadas en nichos específicos y especializados desde dónde responden a las necesidades de repoblación celular del tejido mediante la alternancia de fases de quiescencia y proliferación. Varias rutas de señalización regulan el comportamiento de las epSCs; sin embargo, aún no entendemos bien porqué no todas las epSCs se comportan de la misma manera dentro de un mismo nicho troncal, y cómo están coordinadas a nivel espacio-­‐temporal. Hemos analizado el impacto del ritmo circadiano sobre las función de las epSCs. Mediante un ratón reportero fluorescente del ritmo circadiano hemos demostrado que el nicho troncal quiescente contiene dos poblaciones de epSCs en diferentes fases de su reloj molecular. El análisis comparativo global del transcriptoma de ambas poblaciones indicó que las dos poblaciones corresponden a dos estados opuestos de predisposición a responder a estímulos de activación y quiescencia. Mostramos resultados que demuestran que los factores de transcripción circadianos Bmal1 y Clock regulan directamente la expresión de genes que regulan el comportamiento de las epSCs. La arritmia in vivo en las epSCs resultó en una pérdida progresiva de la homeostasis tisular, un envejecimiento prematuro y una reducción significativa en el desarrollo de tumores escamosos de piel. Por lo tanto, nuestros resultados indican que la maquinaria del reloj molecular permite a las epSCs a anticiparse y coordinar su respuesta a estímulos locales del nicho, lo que constituye un mecanismo esencial para su correcta función en el tejido

Circadian regulation synchronises the sleep-wake cycle to the light-dark cycle. Light cues reach the hypothalamus, the site of the circadian clock, via the retino-hypothalamic tract. In turn, the hypothalamus projects to the pineal, regulating melatonin synthesis, which is high at night. The rhythms of plasma melatonin, its urinary metabolite 6-sulphatoxymelatonin (aMT6s) and plasma cortisol are reliable markers of ihe phase of the clock. Sleep-wake abnormalities are common in patients with cirrhosis and have traditionally been attributed to hepatic encephalopathy. Melatonin rhythm abnormalities have also been observed in these patients, and generally ascribed to impaired hepatic melatonin metabolism; their impact on sleep-wake behaviour remains unknown. The purpose of this thesis was to clarify the relationship between sleep-wake and neuropsychiatric disturbance in patients with cirrhosis and to determine the contribution of the circadian regulatory mechanisms. • Almost 70% of patients with cirrhosis showed significant sleep-wake disturbance. However, no relationship was observed between sleep-wake indices and the presence/degree of hepatic encephalopathy. • Patients with cirrhosis showed. evidence_ of impaired hepatic melatonin metabolism, namely reduced nocturnal melatonin clearance, delayed aMT6s peaks and significant correlations between circadian markers and indices of hepatic failure. • Patients with cirrhosis also showed evidence of central circadian disruption, with parallel delays in the onset of plasma melatonin/plasma cortisol rhythms and attenuated melatonin sensitivity to lignt. • Circadian rhythm delays were associated with delayed sleep habits, but not with impaired sleep quality. A subgroup of patients also exhibited a degree of desynchronisation between sleep and circadian timing. The association between circadian and sleep timing abnormalities observed in patients with cirrhosis is reminiscent of ‘delayed sleep phase syndrome', and probably susceptible to treatment. However, circadian deregulation does not offer a comprehensive explanation for the sleep quality impairment exhibited by these patients and alternative pathophysiological pathways should be explored.

Introduction: The first four postnatal months, for a newborn infant, is a period of rapid adaptation and change. Infants undergo a series of integrated physiological changes that culminate in mature physiological diurnal rhythms by which they establish equilibrium with the new environment, all of which are under the genetic control of the biological clock. This is a longitudinal study of 35 infants in which the age related changes in physiology, are assessed during night time sleep and related to circadian genes, melatonin and cortisol. Aim: The aim of the study is to monitor the physiological development of normal full-term human infants concomitantly assessing the expression of circadian genes. Method: Full term healthy infants were selected. Infants were recruited into the study from 6 weeks until 18 weeks of age. Fortnightly home visits were conducted in which the overnight deep body temperature of infants was monitored. On each night of study, actigraphy was used to study infant and maternal sleep. Longitudinal measurements of melatonin and cortisol secretion by paired day-night urine collection and peripheral gene expression using buccal swabs were taken by mothers. Results: There is evidence of a sequential and ordered development of circadian rhythms in human infant physiology. There was a temporal relationship demonstrated in the maturation of the infant circadian rhythms. Core body temperature demonstrated a robust rhythm, characterised by an abrupt change, the timing of which varied from infant to infant. Night time melatonin secretion increased with age. Cortisol played a key role. Infant sleep improved with physiological maturation. A number of complex relationships between aspects of the physiology and circadian gene expression were elucidated. Conclusion: There is a demonstrable integration of genetic and physiological changes, during the immediate postnatal period when infants are most vulnerable to illnesses and particularly to sudden and unexpected death.

To characterise the circadian transcriptome, the expression profiles of transcripts in whole leaves of mature, soil grown <i>A. thaliana </i>plants were analysed. Circadian-regulated transcripts constituted approximately 9.5% of the transcripts detected, and were found to encode proteins involved both a well-described circadian processes and in pathways that have not been previously identified as having circadian regulation. Transcripts encoding proteins involved in core metabolic processes and stress response pathways were particularly highly representative in the dataset, which suggested a potential basis for the fitness benefits associated with the possession of a functional biological clock. To determine the correlation between rhythms in transcript abundance in whole leaves and in a single cell type, the stomatal guard cell was selected as a model system. However, assessment of two published methods of guard cell isolation, epidermal fragmentation and guard cell protoplasting, revealed that neither method was suitable for analysis of circadian rhythms in transcript abundance. Consequently, single cell analysis was not pursued. Nevertheless, bio-informatic analysis of the whole leaf circadian transcriptome and published microarray data was employed in order to characterise components of the intra-cellular circadian signalling pathway. This analysis revealed a relationship between the circadian oscillator and the regulator of [Ca²⁺]_cyt release, cyclic adenosine disphosphate ribose (cADPR). Evidence is presented suggesting that circadian [Ca²⁺]_cyt oscillations form a component of the oscillator that maintains the periodicity of circadian rhythms in transcript abundance. Collectively the data presented provide an overview of the biological clock in <i>A. thaliana</i>, and form a resource for further analysis of the structure of the clock and its role in integrating diverse cellular and physiological processes into a coherent biological programme.

Circadian clocks are responsible for daily rhythms in gastrointestinal function which are vital for normal digestive rhythms and health. The present study examines the roles of the circadian pacemaker, the suprachiasmatic nuclei (SCN), and the sympathetic nervous system in regulation of circadian gastrointestinal rhythms in Mus musculus. Surgical ablation of the SCN abolishes circadian locomotor, feeding, and stool output rhythms when animals are presented with food ad libitum, while restricted feeding reestablishes these rhythms temporarily. In intact mice, chemical sympathectomy with 6- hydroxydopamine has no effect on feeding and locomotor rhythmicity, but attenuates stool output rhythms. Again, restricted feeding reestablishes these rhythms. Ex vivo, intestinal tissue from mPer2LUC knockin mice expresses circadian rhythms of luciferase bioluminescence. 6-hydroxydopamine has little effect upon these rhythms, but timed administration of β−adrenergic agonist isoproterenol causes a phase-dependent phase shift in PERIOD2 expression rhythms. Collectively, the data suggest the SCN are required to maintain feeding, locomotor and stool output rhythms during ad libitum conditions, acting at least in part through daily activation of sympathetic activity. Even so, this input is not necessary for entrainment to timed feeding, which may be the province of oscillators within the intestines themselves or other components of the gastrointestinal system.

The circadian clock drives ~24hr rhythms in a variety of processes, from gene expression through to behaviour, facilitating anticipation of daily changes in the external environment and temporal separation of internal processes. This pacemaker is a critical regulator of immune function and many inflammatory diseases show time-of-day variation in symptom severity. Disruption of the pacemaker by manipulation of the daily cycle of light and dark exposure (experimental 'jet lag') is known to exacerbate inflammatory responses to innate immune challenge, and recent evidence has highlighted immuno-modulatory roles for components of the molecular oscillator in peripheral tissues. The adrenal-derived glucocorticoid hormones are potent anti-inflammatory molecules and are capable of modulating circadian oscillations in peripheral tissues. This, along with their rhythmic secretion profile, makes them key candidates as mediators of circadian regulation of inflammatory signalling. Utilising adrenalectomy, timed glucocorticoid administration, hormone clamp and genetic targeting of the glucocorticoid receptor in mice, I present evidence for an interaction between glucocorticoid signalling and the circadian pacemaker in regulating the pulmonary inflammatory response to lipopolysaccharide (LPS) challenge. The neutrophilic response to aerosolised LPS exhibits a clear time-of-day effect in vivo, which is lost after disruption of endogenous glucocorticoid production via adrenalectomy. However, replacement of a rhythmic circulating glucocorticoid concentration with a flat daily average using a subcutaneous hormone clamp does not disrupt the inflammatory rhythm. Finally, a novel mouse strain was produced with disrupted expression of the glucocorticoid receptor (GR) in bronchial epithelial cells (Ccsp-GR-/-). These cells are critical regulators of circadian rhythmicity in the lung and drive rhythmic neutrophil influx in response to LPS stimulation through production of the chemokine CXCL5. Loss of GR in the bronchial epithelium was associated with a loss of rhythmic neutrophil influx after challenge, but anti-inflammatory sensitivity to the synthetic glucocorticoid dexamethasone remained. Collectively, these data show that appropriate temporal modulation of pulmonary inflammation requires functional glucocorticoid signalling, although the ligand itself does not need to oscillate. The retention of anti-inflammatory dexamethasone sensitivity suggests a role for cross-talk between the bronchial epithelium and additional cell populations, consistent with recent evidence for immuno-suppressive macrophage-epithelium communication in the lung. These are the first studies to dissect the mechanistic links between clocks, glucocorticoids and immunological responses in a target tissue.

The aim of this investigation was to explore the nature and the severity of circadian abnormalities and attentional deficit in type 1 narcolepsy. In three studies, narcolepsy patients were compared with patients suffering from other central disorders of hypersomnolence and healthy controls on attentional functions and circadian rhythms. Study 1 evaluated the sensibility of actigraphic monitoring in distinguishing the features of daytime and nighttime sleep between adult patients with type 1 Narcolepsy, Idiopathic Hypersomnia and healthy controls. Actigraphy provides a reliable assessment of sleep quality and daytime napping behavior able to distinguish central disorders of hypersomnolence and identify Narcolepsy Type 1 patients. Study 2 describes the features of circadian activity rhythm of narcolepsy type 1 children with recent disease onset. Type 1 narcolepsy children and healthy children were monitored for seven days during the school week, circadian activity rhythms were analyzed through functional linear modeling. Children with type 1 narcolepsy present an altered rest-activity rhythm characterized by enhanced motor activity throughout the night and blunted activity in the first afternoon. The observation of a discrete circadian profile provides new insight on the nature of diurnal variations and suggested that the quantitative assessment of motor activity is a promising behavioral biomarker of Type 1 narcolepsy. The aim of Study 3 was to explore the nature and the severity of attentional Deficits of Narcoleptic patients. This study examined whether narcoleptic patients would exhibit impairments in alerting, orienting, and executive control of attention relative to healthy controls. Narcoleptic patients present a deficit in alerting network, while orienting and executive control networks resulted preserved. Moreover the alerting network efficiency significantly correlate with levels of subjective sleepiness. Results indicates that in narcolepsy the unstable tonic component of alerting process make necessary monitoring and compensation strategies.

Circadian rhythms are periodic physiological cycles that recur about every 24 hours, by means of which organisms integrate their physiology and behavior to the daily cycle of light and temperature imposed by the rotation of the earth. Circadian derives from the Latin word circa "about" and dies "day". Circadian rhythms have three noteworthy properties. They are endogenous, that is, they persist in the absence of external cues (in an environment of constant light intensity, temperature, etc.). Secondly, they are temperature compensated, that is, the nearly 24 hour period of the endogenous oscillator is remarkably independent of ambient temperature. Finally, they are phase shifted by light. The circadian rhythm can be either advanced or delayed by applying a pulse of light in constant darkness. Consequently, the circadian rhythm will synchronize to a periodic light-dark cycle, provided the period of the driving stimulus is not too far from the period of the endogenous rhythm. A window on the molecular mechanism of 24-hour rhythms was opened by the identification of circadian rhythm mutants and their cognate genes in Drosophila, Neurospora, and now in other organisms. Since Konopka and Benzer first discovered the period mutant in Drosophila in 1971 (Konopka and Benzer, 1971), there have been remarkable developments. Currently, the consensus opinion of molecular geneticists is that the 24-hour period arises from a negative feedback loop controlling the transcription of clock genes. However, a better understanding of this mechanism requires an approach that integrates both mathematical and molecular biology. From the recent discoveries in molecular biology and through a mathematical approach, we propose that the mechanism of circadian rhythm is based upon the combination of both negative and positive feedback.<br>Master of Science

Circadian Rhythms (CR) are driven by a biological clock called as suprachiasmaticnucleus (SCN), located in a brain region called the hypothalamus. These rhythms are very much necessary in maintaining the sleep and wake cycle at appropriate times in a day. As a starting step towards non-invasive investigation of CR, aim is to study changes in the physiological processes of two Regions of Interest (ROI), the hypothalamus and the visual cortex. This was studied using a functional Magnetic Resonance Imaging (fMRI) technique to investigate for any changes or differences in the Blood Oxygen Level Dependent (BOLD)signals extracted from the ROI during a visual stimulation. We acquired and processed fMRI data to extract BOLD signals from ROI and the extracted signals are again further used to study the correlation with the experimental ON-OFF design paradigm. The extracted BOLD signals varied a lot between the two specified brain regions within the same subject and between three types of fMRI data. These variations were found in terms of number of activated voxels and also Signal to Noise ratio(SNR) level present in the signals. The number of activated voxels and SNR werehigh in visual cortex whereas low number of activated voxels and low SNR were found in hypothalamus. The correlation between BOLD responses from primaryvisual cortex were shown as positive with the experimental stimulation whereas BOLD responses extracted from hypothalamus have shown a negative correlation in time with the experimental stimulation. As a start up of the project, these BOLD responses can provide references for a future use in research studies, especially to further study about change in phase of the BOLD signal extracted exactly from the SCN. These phase responses can then be used to study physiological processing in subjects affected by sleep disorders.

Thesis (Ph.D.)--Tufts University, 2000.<br>Adviser: David Harder. Submitted to the Dept. of Psychology. Includes bibliographical references (leaves 90-116). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Orientador: Milva Maria Figueiredo De Martino<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas<br>Made available in DSpace on 2018-08-04T03:28:42Z (GMT). No. of bitstreams: 1 Silva_ClaudiaAparecidaRosada_M.pdf: 5933281 bytes, checksum: abd9b797717c0af73f50c3227041de8e (MD5) Previous issue date: 2005<br>Resumo: Este estudo foi conduzido entre 53 enfermeiros, trabalhadores em turnos, com faixa etária de 32 a 40 anos, de um hospital universitário de Campinas - São Paulo. O objetivo foi estudar as características cronobiológicas do ciclo vigília-sono, o estado de sono atual e os estados emocionais presentes. Os questionários utilizados foram: Levantamento de dados da população, Avaliação do ciclo vigília-sono, Caracterização do padrão de sono e Lista de Estados Emocionais Presentes. Os resultados mostraram que o turno matutino é formado por enfermeiros em sua maioria casados, com filhos e que não realizam outra ocupação, o turno vespertino é formado por enfermeiros mais jovens, com menor tempo de formado e de serviço no turno, o turno noturno é formado por enfermeiros com maiores idades e que realizam outra ocupação. As análises dos padrões de sono dos enfermeiros revelaram que os enfermeiros do matutino acordaram mais cedo, porém não anteciparam o horário de dormir, os enfermeiros do turno vespertino apresentaram padrão normal do ciclo vigília-sono, com relação ao sono noturno os enfermeiros do turno noturno apresentaram padrão normal do ciclo vigília-sono, com relação ao sono diurno este é mais curto que o sono noturno e fracionado em até três episódios. Em relação à percepção do estado de sono atual os três turnos classificaram o sono como regular. Os resultados da Lista de Estados Emocionais Presentes, independente do turno de trabalho, mostraram um perfil emocional estável dos enfermeiros, quase ausência de alguns estados emocionais e algumas locuções representaram efeitos específicos do turno com diferenças significativas entre o início e o final do turno<br>Abstract: This study was conducted among 53 nurses, shift workers, aging between 32 and 40 years old, of a University Hospital in Campinas - São Paulo. The objective was study the chronobiological characteristics of the sleep wake cycle, the current sleeping pattern and present mood states. The questionnaires used were: Survey of population data, Evaluation of sleep-wake cycle, Characterization of sleep standard and Present Mood States List. The results demonstrated that the morning shift is most made up of marriage and with children nurses and that do not carry through another occupation, the afternoon shift is made up of younger nurses, more recently graduated and with less time working at this shift. The night shift is made up of older nurses and that they carry through another occupation. The analysis about the nurses standard sleep showed that the nurses of the morning had waked up more early, however had not anticipated the schedule to sleep, the nurses of the afternoon shift had presented normal standard of the sleep-wake cycle, with relation to night sleep the nurses of the night shift one had presented normal standard of the sleep-wake cycle, with relation to daily sleep this is shorter than night and fragmented in up to three episodes. In relation to the perception of the current sleeping pattern the three shifts had classified sleep as to regulate. The results of the Present Mood States List, independent of the shift work, showed a stable emotional profile of the nurses, almost absence of some emotional states and some locutions had represented specifics effects of the shift, with significant differences between the beginning and end of the shift<br>Mestrado<br>Enfermagem e Trabalho<br>Mestre em Enfermagem

Objetivo: Avaliar a influencia da iluminação artificial nos ritmos biológicos e metabolismo por meio de dois experimentos em ratos Wistar. O primeiro avalia mudanças de fotoperíodo que mimetizem a sazonalidade; o segundo, a qualidade da iluminação artificial (espectro de cor) no ciclo claro/escuro (LD). Métodos: Experimento1 - Três grupos de animais: Controle (CL; n=6, ciclo LD de 12/12); grupo que inicia com fotoperíodo longo (LP/SP; n=7; LD 16.5:7.5); grupo que inicia com fotoperíodo curto (SP/LP; n=7; LD 7.5:16.5). Os grupos experimentais passaram por 18 dias no fotoperíodo inicial, 17 dias de redução ou aumento gradual do fotoperíodo, 18 dias no fotoperíodo inverso ao que iniciou. Experimento 2 - 36 animais foram mantidos 108 dias em ciclo LD 16:8h, divididos em 2 grupos: Standard Light (SL; n=18), mantidos sob iluminação com espectro de cor padrão (LED, 4000K); e Circadian Light (CL; n=18) com alterações de espectro de cor ao longo do dia (LED, 2700-6500K). Em ambos os estudos, níveis de atividade e temperatura, além de melatonina e corticosterona sérica, foram mensurados. No Experimento 2, além das pesagens semanais, após eutanásia a gordura visceral foi medida. Os parâmetros circadianos foram obtidos por meio da análise de séries temporais. Na análise estatística foram usados os testes paramétricos ou não paramétricos, de acordo com a normalidade dos dados. Resultados: A quantidade de atividade noturna, além dos níveis de corticosterona foram menores no grupo SP/LP (p<0.05). Portanto, os animais demonstraram pior adaptação dos ritmos à transição do fotoperíodo de dia curto para longo (SP/LP). A qualidade de iluminação também influenciou o comportamento animal. O grupo CL apresentou melhores parâmetros rítmicos que o grupo SL, por exemplo, menor intracyclevariability, maior amplitude e quantidade de atividade (p<0.05). Apesar de o peso corporal ter sido similar, o grupo SL apresentou maior quantidade de gordura visceral (p<0.05). Parâmetros rítmicos de atividade correlacionaram com a concentração de melatonina somente no grupo CL, enquanto que parâmetros rítmicos correlacionaram com a concentração de corticosterona principalmente no grupo SL. Conclusões: Nosso estudo reforça a relevância da iluminação como um fator importante na regulação do comportamento e metabolismo, sugerindo que o a iluminação artificial comumente utilizada, sem variação de espectro de cor, é um forte fator facilitador do processo de cronodisrupção e aumento de gordura visceral. Ainda, o sistema de iluminação utilizado frequentemente nos alojamentos experimentais podem ser subótimas para simular o ambiente natural. Apoio: FIPE/HCPA, CNPq, CAPES e Luxion Iluminação.<br>Objective:To evaluate the influence of artificial illumination on biological rhythms and metabolism by two experiments whit Wistar rats. The first one evaluated changes in photoperiod that mimetics seasonality; the second one, the quality of artificial light (color spectrum) on light/dark cycle (LD). Methods: Experiment 1 – Three animal groups: Control (CL; n=6, LD cycle 12/12); group that started with long photoperiod (LP/SP; n = 7; LD 16.5:7.5); group that started with short photoperiod (SP/LP; n=7; LD 7.5:16.5). Experimental groups passed for 18 day in a start photoperiod, 17 days of gradual increase or decrease of photoperiod, 18 days on inverse photoperiod to what start.Experiment 2 – 36 animals were kept for 108 days in a LD cycle of 16:8h, divided in 2 groups: Standard Light (SL; n=18), kept under illumination with standard color spectrum (LED, 4000K); and Circadian Light (CL; n=18) with changes of color spectrum during the day (LED, 2700-6500K). In both studies, activity and temperature levels, as well as serum melatonin and corticosterone, were measured. On Experiment 2, in addition to weekly weighing, after euthanasia the visceral fat was measures. The circadian parameters were obtained by temporal series analyses. In statistical analyses were used parametric or non-parametric tests, according the normality of data. Results:Amount of activity on dark, besides corticosterone levels were lower on SL/LP group (p<0.05). So, animals showed low rhythms adaptation to photoperiod transitions from short to long light (SL/LP). The quality of illumination also influenced in animal behavior. The CL group presented better rhythmic parameters than SL group, for example, low intracycle variability, high amplitude and quantity of activity (p<0.05). Although body weight was similar, SL group presented higher amount of visceral fat (p<0.05). Rhythmic parameters of activity correlated with the melatonin concentration just in CL group, whereas rhythmic parameters correlated whit corticosterone concentration principally in SL group. Conclusions: Our study reinforces the relevance of illumination as an important factor on metabolic and behavioral regulation, suggesting that artificial illumination commonly used, without color spectrum variation, is a strong facilitating factor on the process of chronodisruption and increase of visceral fat. Thus, the illumination system frequently used in experimental accommodation could be suboptimal for to simulate the natural environment. Support:FIPE/HCPA, CNPq, CAPES andLuxionIluminação.

Thesis (MSc)--Stellenbosch University, 2014.<br>ENGLISH ABSTRACT: Introduction: Mammalian circadian rhythms form an integral physiological system allowing for the synchronisation of all metabolic processes to daily light/dark cycles, thereby optimising their efficacy. Circadian disruptions have been implicated in the onset and progression of different types of cancers, including those arising in the breast. Several links between the circadian protein Per2 and DNA damage responses exist. Aberrant Per2 expression results in potent downstream effects to both cell cycle and apoptotic targets, suggestive of a tumour suppressive role for Per2. Due to the severe dose limiting side effects associated with current chemotherapeutic strategies, including the use of doxorubicin, a need for more effective adjuvant therapies to increase cancer cell susceptibility has arisen. We therefore hypothesize, that the manipulation of the circadian Per2 protein in conjunction with doxorubicin may provide a more effective chemotherapeutic strategy for the treatment of breast cancer. The aims of this project were thus to: (i) Characterize the role of Per2 in normal breast epithelial cells as well as in ER+ and ER- breast cancer cells; (ii) to determine the role of Per2 in doxorubicin-induced cell death, (iii) to determine the role of Per2 in autophagy and finally (iv) to assess whether the pharmacological inhibition of Per2 with metformin, can sensitize chemo-resistant MDA-MB-231 breast cancer cells to doxorubicin-induced cell death. Methods: An in vitro model of breast cancer was employed using the normal MCF-12A breast epithelial, estrogen receptor positive (ER+) MCF-7 and estrogen receptor negative (ER-) MDA-MB-231 breast adenocarcinoma cell lines. Circadian rhythmicity of Per2 protein expression was determined using western blotting, and Per2 cellular localization was assessed using fluorescent confocal microscopy. Per2 was then silenced by means of an endoribonuclease-prepared siRNA, and silencing efficiency was determined with the use of western blotting. The roles of Per2 in doxorubicin-induced cell death and autophagy were assessed by treating MDA-MB-231 breast cancer cells under the following conditions (1) Control, (2) 2.5 μM doxorubicin or 10 nM bafilomycin A1 (3) 30 nM esiPer2 and (4) 30 nM esiPer2 in combination with 2.5 μM doxorubicin or 10 nM bafilomycin A1. Following treatments cell viability was assessed using the MTT assay, western blotting for markers of apoptosis including p-MDM2 (Ser166), p-p53 (Ser15), cleaved caspase-3 and –PARP as well as markers of autophagy (AMPKα, mTOR and LC3). Furthermore, cell cycle analysis, G2/M transition and cell death (Hoechst 33342 and propidium iodide staining) were assessed by means of flow cytometry. The pharmacological inhibition of Per2 was achieved by treating MDA-MB-231 cells with 40 mM metformin as well as in combination with 2.5 μM doxorubicin. MTT cell viability assays, cell cycle analysis (flow cytometry) and western blotting for apoptosis (Per2, p-AMPKα (Thr172), p53, caspase-3 and PARP) were assessed. Results and discussion: A circadian pattern of Per2 protein expression was observed in the normal MCF-12A and MDA-MB-231 cancer cells with protein levels peaking at ±700% and ±500% of baseline was observed. However, no rhythmic expression was observed in the MCF-7 cancer cells. Immunostaining for Per2 showed localization OF Per2 in the cytoplasm as well as in the nucleus of both the MCF-12A and MDA-MB-231 cells. Concentration curves showed a significant reduction in cell viability following 2.5 μM doxorubicin treatment for 24 hours. Per2 protein expression was significantly reduced with both esiPer2 and metformin treatment. Silencing of Per2 in combination with doxorubicin treatment resulted in cell cycle arrest with a significant increase in apoptosis, indicating that Per2 silencing effectively sensitized the MDA-MB-231 cancer cells to the anti-carcinogenic properties of doxorubicin. Modulation of Per2 protein expression was effectively achieved with the use metformin although this decrease occurred independently of AMPKα phosphorylation. A significant increase in apoptosis was observed following treatment with metformin in combination with doxorubicin treatment. However, no changes in cell cycle regulation were observed. Per2 appears to be involved in the regulation of autophagy as a significant increase in autophagy flux was observed when Per2 was silenced. Additionally, this increase in autophagic flux resulted in a significant increase in MDA-MB-231 cancer cell death which was enhanced further when autophagy was inhibited with bafilomycin A1 subsequent to Per2 silencing. Conclusions: Per2 protein expression was shown to display a 24 hour circadian rhythm in the MCF-12A cells, and to a lesser extent in the MDA-MB-231 cells. However, the MCF-7 cells failed to show rhythmic changes in Per2 protein expression. Per2 was shown to be located predominantly in the cytoplasm, with nuclear localization observed when cytoplasmic fluorescent intensity was lower. Per2 silencing effectively sensitized the chemo-resistant MDA-MB-231 breast cancer cells to both doxorubicin-induced cell death and autophagic inhibition.<br>AFRIKKANSE OPSOMMING: Inleiding: Sirkadiese ritmes vorm ‘n integrale fisiologiese sisteem wat die sinkronisasie van alle metaboliese prosesse asook lig/donker siklusse se effektiwiteit optimaliseer. Onderbreking van hierdie sirkadiese ritmes word geïmpliseer in die ontstaan en bevordering van verskillende kankertipes, insluitend borskanker. Verskeie raakpunte bestaan tussen die sirkadiese proteïen, Per2, en die DNA skade-respons. Abnormale Per2 uitdrukking veroorsaak afstroom effekte op beide die selsiklus en apoptotiese teikens wat moontlik aanduidend van ‘n tumor-onderdrukkende rol vir Per2 kan wees. Daar bestaan ‘n groot nood vir meer effektiewe adjuvante terapieë om kankersel vatbaarheid vir chemoterapie te verhoog as gevolg van dosis-beperkende newe-effekte wat geassosieer word met huidige chemoterapeutiese strategieë, insluitende dié van doxorubicin. Ons hipotese is dus dat die manipulering van die sirkadiese Per2 proteïen tesame met doxorubicin ‘n meer effektiewe chemoterapeutiese strategie vir die behandeling van borskanker sal wees. Die doelwitte van hierdie projek was dus om: (i) Die rol van Per2 in normale borsepiteelselle sowel as in ER+ en ER- borsepiteel kankerselle te karakteriseer; (ii) die rol van Per2 in doxorubicin-geïnduseerde seldood te bepaal; (iii) te bepaal of Per2 ‘n rol in autofagie speel en laastens (iv) te bepaal of die farmakologiese inhibisie van Per2 met metformin chemo-weerstandbiedende MDA-MB-231 kankerselle kan sensitiseer vir doxorubicin-geïnduseerde seldood. Metodes: ‘n In vitro model vir borskanker is gebruik wat normale MCF-12A borsepiteelselle, estrogeen reseptor positiewe (ER+) MCF-7 en estrogeen reseptor negatiewe (ER-) MDA-MB-231 bors adenokarsenoomselle insluit. Sirkadiese ritmisiteit van Per2 proteïen uitdrukking is deur middel van die westelike kladtegniek bepaal en die sellulêre ligging van Per2 deur middel van fluoresensie mikroskopie. siPer2 is voorberei deur middel van endoribonuklease-siRNA en die effektiwiteit daarvan is deur middel van westelike kladtegniek getoon. Die rol van Per2 in doxorubicin-geinduseerde seldood en autofagie is bepaal deur MDA-MB-231 borskankerselle onder die volgende omstandighede te toets: (1) Kontrole, (2) 2.5 μM doxorubicin of 10 nM bafilomycin A1 (3) 30 nM esiPer2 en (4) 30 nM esiPer2 in kombinasie met 2.5 μM doxorubicin of 10 nM bafilomycin A1. Na die behandeling, is sellewensvatbaarheid bepaal deur gebruik te maak van ‘n MTT toets; westelike kladtegniek is gebruik om vir merkers van apoptose soos p-MDM2 (Ser166), p-p53 (Ser15), gekliefde caspase-3 en -PARP asook vir merkers van autofagie (AMPKα, mTOR en LC3) te toets. Die selsiklus, G2/M oorgang en seldood (Hoechst 33342 en propidium iodide kleuring) is deur middel van vloeisitometrie bepaal. Per2 is ook farmakologies geïnhibeer deur MDA-MB-231 selle met 40 mM metformin asook in kombinasie met 2.5 μM doxorubicin te behandel. Daarna is sellewensvatbaarheid (MTT) sowel as die selsiklus (vloeisitometrie) en apoptose (westelike kladtegniek vir Per2, p-AMPKα (Thr172), p53, caspase-3 and PARP) gemeet. Resultate en bespreking: ‘n Sirkadiese patroon vir Per2 proteïen uitdrukking is in die normale MCF-12A selle asook in die MDA-MB-231 kankerselle waargeneem met proteïenvlakke wat hul piek by ±700% and ±500% onderskeidelik in vergelyking met basislyn bereik het. Geen ritmiese patroon van Per2 proteïen uitdrukking is egter in die MCF-7 kankerselle waargeneem nie. Immunokleuring om Per2 ligging te bepaal het getoon dat Per2 in the sitoplasma sowel as in die nukleus in beide MCF-12A en MDA-MB-231 selle voorgekom het. Konsentrasie kurwes het aangetoon dat daar ‘n insiggewende vermindering in sellewensvatbaarheid voorgekom het na die behandeling van die selle met 2.5 μM doxorubicin vir 24 uur. Per2 proteïen uitdrukking is insiggewend verlaag met beide esiPer2 en metformin behandeling van die selle. esiPer2 aleen of in kombinasie met doxorubicin behandeling het selsiklus staking tot gevolg gehad asook ‘n beduidende toename in apoptose veroorsaak wat dus aangedui het dat esiPer2 effektief was om MDA-MB-231 kankerselle te sensitiseer vir die anti-karsinogeniese doxorubicin behandeling. Modulering van Per2 proteïen uitdrukking was effektief met metformin behandeling, alhoewel die afname onafhanklik van AMPKα fosforilasie plaasgevind het. ‘n Insiggewende toename in apoptose is waargeneem na metformin behandeling in kombinasie met doxorubicin. Geen veranderinge in die selsiklus is egter onder hierdie omstandighede waargeneem nie. Per2 blyk betrokke te wees in die regulering van autofagie aangesien ‘n insiggewende verhoging in autofagie omsetting waargeneem is na esiPer2 behandeling. Die toename in autofagie omsetting is geassosieer met ‘n insiggewende toename in seldood in MDA-MB-231 kankerselle wat verder verhoog is wanneer autofagie met bafilomycin A1 (autofagie inhibitor) in kombinasie met esiPer2 behandel is. Gevolgtrekkings: Per2 proteïen uitdrukking het ‘n 24 uur sirkadiese ritme in die MCF-12A normale selle, en tot ‘n mindere mate in die MDA-MB-231 selle getoon. Die MCF-7 selle het egter geen ritmiese patroon van Per2 proteïen uitdrukking getoon nie. Per2 kom hoofsaaklik in die sitoplasma voor en het slegs in die nukleus voorgekom wanneer die sitoplasmiese fluoresensie intensiteit laer was. esiPer2 was dus effektief om die chemo-weerstandbiedende MDA-MB-231 borskankerselle te sensitiseer vir doxorubicin-geïnduseerde seldood.<br>National Research Foundation

The passage of No Child Left Behind has increased the level of accountability for all educators. There are many factors that affect student achievement. One factor that may be overlooked is the schedule configuration of schools. Addressing student needs through scheduling options may assist school systems and students in performing at the level they are being held accountable. The population for this study was students from a rural East Tennessee middle school with a population of approximately 700 students. The low socioeconomic students represent 68% of the school total enrollment while 18% of the students have an individual education plan (IEP). The gender of the school is nearly 50% male and female. Looking at 2 research questions, an independent t test was used to determine if there was a significant difference in reading-language arts and mathematics Tennessee Comprehensive Assessment Program (TCAP) scores after implementing a rotating schedule. Subgroups used in this study were: students with an Individual Education Plan (IEP), low socioeconomic students, male and female students. Results of this study were mixed. Students with an IEP showed an increase in both reading-language arts and mathematics. For all subgroups in reading, there was an increase in achievement although the results showed that there was not a significant relationship between the rotating schedule and student achievement. The only group to show gains in mathematics after implementation of the rotating was those students with an IEP. Each of the 3 remaining subgroups actually showed a loss and there was a significant relationship between the rotating schedule and student achievement.

Biological rhythms are thought to have evolved to enable organisms to organise their activities according to the Earth’s predictable cycles, but quantifying the fitness advantages of rhythms is challenging and data revealing their costs and benefits are scarce. More difficult still is explaining why parasites that exclusively live within the bodies of other organisms have biological rhythms. Rhythms exist in the development and traits of parasites, in host immune responses, and in disease susceptibility. This raises the possibility that timing matters for how hosts and parasites interact and, consequently, for the severity and transmission of diseases. Despite their obvious importance in other fields, circadian rhythms are a neglected aspect of ecology and evolutionary biology. The ambitions of this thesis are to integrate chronobiology, parasitology and evolutionary theory with mathematical models to obtain a greater understanding about how and suggest why malaria parasites have rhythms as well as the effect of infection on host rhythms. First, I identify how malaria parasites lose their developmental rhythms in culture, when they lack any potential time cues from the host. Next, I characterise parasite rhythms inside the mammalian host in terms of synchrony and timing and demonstrate there is genotype by environment interactions for characteristics of parasite rhythms. Then, I investigate the effect that parasite infection has on host rhythms and show there is variation between parasite genotypes in their effect on host locomotor activity and body temperature rhythms during infections. Finally, I explore which host rhythms may be driving parasite synchrony and timing and demonstrate the importance of peripheral host rhythms for the timing of malaria parasite developmental rhythms. The data presented here provides novel and important information on the role of rhythms during disease and opens up a new arena for studying host-parasite coevolution.

Critical care patients commonly experience sleep fragmentation, in which sleep quality is poor and distributed throughout the 24 hour cycle. This irregular sleep wake pattern is a form of circadian rhythm sleep disorder. The causes of sleep disturbances are multifactorial and contribute to patient morbidity. Conventional hypnotic treatment is often ineffective and, indeed, may cause delirium and reduced sleep quality. Administration of exogenous melatonin has been shown to re-enforce circadian rhythm disorders and improve sleep in other patient groups. An open evaluation of 5 mg oral melatonin was undertaken in a group of 12 critical care patients exhibiting sleep disturbances resistant to conventional hypnotics. Melatonin significantly increased observed sleep quantity by night 3, compared to baseline. An oral solution of melatonin was formulated to allow administration by enteral feeding tubes. It was shown to have a 1 year shelf life when refrigerated and protected from light. A randomised controlled trial was undertaken in 24 critical care patients weaning from mechanical ventilation. Melatonin 10 mg orally increased nocturnal bispectral index sleep quantity over nights 3 and 4 compared to placebo. Agreement of the other sleep measurement techniques with the bispectral index was poor. Actigraphy was not a useful measure of sleep in critical care patients and nurse observation overestimated sleep quantity. The clearance of melatonin appeared to be decreased in critical care patients compared to that in healthy subjects. Doses of 1-2 mg should be used in future critical care studies. 11 Acute administration of melatonin did not have a significant effect over placebo on rest-activity rhythms, which remained delayed, fragmented and reduced. Similar disturbances were present in plasma melatonin and cortisol rhythms, which were no longer phase locked. Melatonin therapy may prove beneficial in the treatment of sleep and circadian rhythms in critical care patients, and further larger studies should be pursued.

Este estudo tem como objetivo introduzir ao leitor a influência da luz sobre os usuários residenciais, mostrando que o avanço das tecnologias ao longo da história e o acesso à iluminação artificial elétrica proporcionaram maior conforto às atividades relacionadas a sua moradia. Novas tecnologias, mais econômicas do ponto de vista energético, possibilitaram acesso a maiores quantidades de luz pelos usuários com diferentes tons de luz branca, permitindo uma maior escolha, porém podendo influenciar na produção do hormônio melatonina, o qual regula o relógio biológico, provocando distúrbios diversos. O trabalho de campo foi realizado em residências, dividido em duas linhas de raciocínio, sendo o primeiro à leitura em uma amostra de residências aleatórias e o segundo com base em uma residência utilizada como modelo. Entretanto, nesse último caso foram alteradas as tecnologias empregadas. Em ambos os casos foram feitas medições na sala de estar em frente à televisão. Esses estudos foram fundamentados no método de Circadian Stimulus (Rea M.S., 2005, 2011), baseado em diversos estudos laboratoriais, que demostraram a influência da luz sobre a produção de melatonina, onde foi empregado nos dois procedimentos adotados. Conclui-se que, apesar da estimativa dos níveis de supressão encontrados serem baixas, as tecnologias com temperaturas de cor mais elevadas, ou seja, luzes mais brancas, tem índices de inibição maiores e devem ser evitadas, assim como as quantidades de luz praticadas para os ambientes residenciais devem ser cuidadosamente dimensionadas. Esse trabalho não tem caráter conclusivo e sim introduz uma metodologia que ainda está em fase final de estudos, porém pode contribuir para apontar diretrizes até então pouco abordadas na prática profissional e terem o intuito de orientar os usuários finais.<br>This study aims to introduce the reader to the influence of light on residential users, showing that the advancement of technologies throughout history and access to electric artificial lighting have provided greater comfort to the activities related to their homes. New technologies, more economically energetic, allowed access to greater amounts of light by users with different white light tones, allowing a wide choice, but may influence the production of the hormone melatonin, which regulates the biological clock, causing many disturbances. Fieldwork was carried out in residences, divided into two lines of reasoning, the first to be read in a sample of random households and the second based on a residence used as a model. However, in the latter case, the technologies employed were altered. In both cases measurements were taken in the living room in front of the television. These studies were based on the Circadian Stimulus method (Rea M.S., 2005, 2011), based on several laboratory studies, which demonstrated the influence of light on the production of melatonin, where it was used in the two adopted procedures. It is concluded that, although estimates of suppression levels found are low, technologies with higher color temperatures, meaning whiter lights, have higher inhibition rates and should be avoided, as well as the amounts of light practiced for residential environments must be carefully dimensioned. This work does not have a conclusive character but rather introduces a methodology that is still in the final phase of studies, but it can contribute to point out guidelines that have not been approached in the professional practice and are intended to guide end users

Orientador: Everardo Magalhães Carneiro<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia<br>Made available in DSpace on 2018-08-24T14:27:08Z (GMT). No. of bitstreams: 1 Borck_PatriciaCristine_M.pdf: 1384245 bytes, checksum: 89fa526347ae455e0cfcf6e3bd414018 (MD5) Previous issue date: 2014<br>Resumo: Os processos fisiológicos como ciclo sono-vigília e o metabolismo estão sujeitos a oscilações circadianas e são regulados por um conjunto de genes conhecidos como genes do relógio, ou clock genes. Mutação nesses genes em camundongos reduz a secreção de insulina e a proliferação das células ? pancreáticas promovendo intolerância a glicose e hiperglicemia. Distúrbios nutricionais em fases iniciais da vida estão associados com o aparecimento do Diabetes Mellitus tipo 2 na vida adulta. Camundongos submetidos a restrição proteica intrauterina apresentam expressão alterada dos clock genes e maior suscetibilidade ao ganho de peso e intolerância a glicose. Neste trabalho tivemos como objetivo determinar a expressão diária dos clock genes em tecidos periféricos, hipotálamo e ilhotas pancreáticas de camundongos submetidos a restrição proteica. Avaliamos também o perfil oscilatório da secreção de insulina estimulada por glicose e pelo agonista colinérgico carbacol nesse modelo animal. Camundongos submetidos a restrição proteica (R) apresentaram características típicas de desnutrição como menor peso corpóreo, hipoinsulinemia, hipoproteinemia e maior tolerância a glicose e a insulina. Camundongos R apresentaram maior consumo alimentar, acompanhado de alterações no perfil oscilatório de genes hipotalâmicos Pomc (Pro-opiomelanocortina) e Npy (Neuropeptídeo Y). Nesse tecido, somente o gene do relógio Rev-erb? teve sua expressão influenciada pela restrição proteica. Camundongos R apresentaram, no fígado e músculo perda do perfil oscilatório para os genes Bmal1 e Clock. Ainda, no fígado e ilhotas pancreáticas a expressão de Rev-erb? foi alterada, com redução no conteúdo de mRNA. Em relação ao gene Per1, camundongos R exibiram adiantamento na expressão desse gene no tecido adiposo. No músculo e ilhotas houve perda da oscilação diária para esse gene. Camundongos R exibiram, no músculo e tecido adiposo, adiantamento na expressão do gene Per2. Ilhotas isoladas de camundongos controle (C) apresentaram padrão oscilatório de secreção de insulina sendo os maiores níveis atingido nos ZT 2 e 14 e redução no ZT 8. Contudo, camundongos R apresentaram redução na secreção de insulina estimulada por glicose, e perda do seu perfil oscilatório. O grupo R não apresentou alteração na liberação de insulina na presença do agonista e antagonista de Rev-erb?. Além disso, a expressão dos genes Sintaxina, Sinaptotagmina, e Insulina, estão reduzidos em camundongos R. O grupo R também apresentou perda oscilatória da secreção de insulina na presença de glicose associada ao Carbacol e redução na expressão do Receptor Muscarínico de Acetilcolina. Com os presentes resultados podemos concluir que camundongos submetidos a restrição proteica apresentaram características típicas de desnutrição com alteração na homeostase glicêmica e secreção de insulina. Ademais, camundongos R exibiram perda do perfil de secreção desse hormônio ao longo de 24 horas, o qual está relacionado com as alterações na expressão de Rev-erb?. Além disso, houve alteração no perfil de expressão dos genes clock, em especial Rev-erb?, Per1 e Per2 nos tecidos periféricos, fato que pode estar relacionado com as alterações na tolerância a glicose e insulina em camundongos R<br>Abstract: The physiological processes such as sleep-wake cycle and metabolism are subject to circadian fluctuations and are regulated by a group of genes known as clock genes or genes clock. Mutations in these genes in mice reduces insulin secretion and ?-pancreatic cell proliferation promoting impaired glucose tolerance and hyperglycemia. Nutritional disorders in the early stages of life are associated with the onset of type 2 diabetes in adulthood. Mice subjected to intrauterine protein restriction have altered expression of clock genes and increased susceptibility to weight gain and glucose intolerance. In this study we aimed to determine the daily expression of clock genes in peripheral tissues, hypothalamus and pancreatic islets of mice subjected to protein restriction. We also evaluated the oscillatory profile of the glucose stimulated insulin secretion and the cholinergic agonist carbachol in this animal model. Mice subjected to protein restriction (R) showed typical features of malnutrition as lower body weight , hypoinsulinemia , hypoproteinemia and increased glucose tolerance and insulin. R mice had higher food consumption, accompanied by changes in the oscillatory profile to Pomc and Npy gene in the hypothalamus. In this tissue, only the expression Rev- erb? gene was influenced by protein restriction. Mice R showed in the liver and muscle loss of the oscillatory profile to Clock and Bmal1 gene. Still, in liver and pancreatic islets the expression of Rev- erb? was changed, with reduction in mRNA content. Regarding the Per1 gene, R mice exhibited advance in the expression of this gene in adipose tissue. In muscle and islets there was loss of daily fluctuation for this gene. R mice exhibited, muscle and adipose tissue, in advance of Per2 gene expression. Islets isolated from control mice (C) showed oscillatory pattern of insulin secretion with the highest levels attained in the ZT 2 e 14 and reduction in the ZT 8. However, R mice had reduced glucose stimulated insulin secretion and loss of its oscillatory profile. R group showed no change in insulin release in the presence of Rev- erb? agonist and antagonist. Furthermore, the expression of Syntaxin, Synaptotagmin, and Insulin genes are reduced in R mice. R group also exhibited oscillatory loss of insulin secretion in the presence of glucose linked Carbachol and the reduction in the expression of Muscarinic Acetylcholine Receptor. With these results we can conclude that mice subjected to protein restriction showed typical features of malnutrition with alterations in glucose homeostasis and insulin secretion. Moreover, R mice exhibited loss of secretion of this hormone profile over 24 hours, which is associated with changes in the expression of Rev- erb?. In addition, there were changes in expression profile of clock genes, especially Rev-erb?, Per1 and Per2 in peripheral tissues, which may be related to changes in glucose tolerance and insulin in R mice<br>Mestrado<br>Fisiologia<br>Mestra em Biologia Funcional e Molecular