Tesi sul tema "Circadian rhythms"

Il comportamento è concepito come una relazione dipendente stimolo-risposta tra un input sensoriale e una risposta motoria. Nel passaggio da input a output, l’omeostasi interna è continuamente modellata per mantenere un equilibrio ottimale della spesa energetica. Lo scopo ultimo di mantenere l’omeostasi in relazione al mondo circostante viene raggiunto attraverso la produzione di comportamenti adattativi che permettono di incrementare la fitness alla luce della selezione naturale. L’ambiente circostante può essere sia prevedibile sia imprevedibile. La prima condizione ha portato all’evoluzione del ritmo circadiano che promuove la fase di attività durante il momento più favorevole della giornata, mentre la seconda si serve dell’asse dei glucocorticoidi per affrontare le sfide imprevedibili. Quindi, un dialogo tra il sistema circadiano e il sistema dei glucocorticoidi è mantenuto allo scopo di ottenere una regolazione ottimale dell’attività animale. Il mio obiettivo è quello di capire il dialogo tra i due sistemi monitorando il comportamento giornaliero e circadiano, e la sua controparte molecolare, in fase a differenti cicli di luce e cibo. La mia specie modello è lo zebrafish (Danio rerio), in particolare, ho utilizzato un mutante costruito con la tecnica CRISPR/Cas9 che manca della capacità di coordinare la via di trascrizione dei glucocorticoidi a causa della mancata funzionalità del loro recettore cognato tale che l’interazione ligando recettore non è mantenuta. Di conseguenza, i livelli circolanti di glucocorticoidi restano elevati, conferendo al mutante un fenotipo ansioso. Zebrafish gr-/- è stato costruito e gentilmente fornito dal laboratorio della Prof.ssa Luisa Dalla Valle, Università degli studi di Padova. L’analisi sistematica del comportamento in larve e adulti di gr-/- ha mostrato che l’attività locomotoria sincronizzata alla luce mantiene le sue proprietà oscillatorie endogene. Tuttavia, l’attività locomotoria giornaliera insorge con un ritardo di un giorno nei mutanti rispetto ai wild type. Questa insorgenza ritardata è associata a un rallentamento nello sviluppo del tessuto muscolare striato, la normale densità delle fibre muscolari viene ripristinata nei gr-/- al sesto giorno dopo la fertilizzazione. Inoltre, le larve gr-/- hanno mostrato differenze nei livelli di espressione e nelle relative acrofasi di elementi positivi (arntl1a and clock1a) e negativi (per1, per2a and cry1a) dell’orologio molecolare. Al di là degli elementi del cuore dell’orologio circadiano, un’analisi nel fegato di adullti gr-/- rivela un’abolizione dell’espressione di pck2, un gene implicato nella gluconeogenesi. In aggiunta, srebp1 ha un’acrofase anticipata nei mutanti. La sincronizzazione circadiana al cibo fallisce nei gr-/-, sia larve sia adulti producono profili anomali dell’attività locomotoria. L’analisi molecolare non associa la disfunzionalità comportamentale a quella genetica, infatti i geni orologio non mostrano alterate oscillazioni a eccezione di cry1a. Questi dati suggeriscono l’esistenza di un confine sfuocato tra il sistema circadiano e quello dei glucocorticoidi e una complessa organizzazione dei due ha prodotto un alterato output comportamentale negli zebrafish gr-/-. La causa prossima del disallineamento tra lo stimolo alimentare e la locomozione non è stata chiarita sebbene un passo avanti verso una maggiore comprensione del dialogo tra glucocorticoidi e orologio circadiano getta le basi per un’indagine più profonda.
Behavior is conceived as a stimulus-response dependent relationship between a sensory input and a motor output. While moving from an input to an output, internal homeostasis is continuously shaped to maintain an optimal energies expenditure balance. The ultimate purpose of enabling animals to adjust their homeostasis with the surrounding world is by producing adaptive behaviors in order to increase their fitness in light of natural selection. The environment can be either predictable or unpredictable. The former condition led to the evolution of the circadian rhythm to promote an active behavior at the time you mostly benefit from, while the latter take advantage of glucocorticoids axis to face sudden challenges. Thus, a crosstalk between the circadian and the glucocorticoid systems allows a fine tuning of animal’s activity. My goal is to understand the circadian-glucocorticoids dialogue by monitoring the locomotor daily/circadian behavior and its molecular oscillation counterpart under differentially phased light and feeding cycle. My model species is the zebrafish, particularly, I utilized a CRISPR/Cas9 mutant lacking the capability to coordinate glucocorticoids transcription because it lacks functional receptors which permit a correct ligand-receptor interaction. As a result, level of circulating glucocorticoids stays raised conferring an anxiety-related phenotype to the mutant. Zebrafish gr-/- has been built and kindly provided by Dr. Luisa Dalla Valle, University of Padua. Systematic behavioral analysis in gr-/- larvae and adults showed that the light entrainable locomotor activity is synchronized to the zeitgeber and maintain its oscillatory properties in absence of any cue. The onset of daily locomotor activity occurred one day later in mutants with respects to the wild type. This delay is linked to the slower striated muscle development in the gr-/- which recover regular fiber density at 6 days post fertilization. Furthermore, gr-/- larvae showed differences in the expression levels or in the peak phase of positive (arntl1a and clock1a) and negative (per1, per2a and cry1a) elements of the molecular clock. Outside the core clock network, an analysis on gr-/- adult livers reported an abolished daily expression of pck2, a gene involved in gluconeogenesis. In addition, srebp1 expression level has an anticipated acrophase in gr-/-. Feeding entrainment fails to occur in the mutants. Larvae and adults produced abnormal profiles of circadian locomotor activity. Further molecular investigation revealed this behavioral disruption wasn’t associated with a breakdown of molecular rhythms in the core clock genes. Nevertheless, the molecular phenotypes observed during feeding entrainment underlined a cry1a lack of rhythmicity. These data suggest the existence of a blurred boundary between the circadian-glucocorticoids crosstalk. A complex organization of the two produces an altered behavioral output in a food entrained schedule in gr-/- zebrafish. The proximate cause of input and output misalignment underlying food entrained locomotion has not been provided, but a step towards a more exhaustive comprehension about the circadian-glucocorticoids interaction paves the way for an in-depth investigation.

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

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.

Orientador: Milva Maria Figueiredo De Martino
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: Esta pesquisa teve como objetivo verificar a variabilidade circadiana das temperaturas timpânica, oral e axilar; correlacionar as medidas da temperatura timpânica considerando o ângulo de posicionamento e comparar as medidas entre si, em pacientes adultos hospitalizados. Participaram, 15 pacientes do sexo masculino sem sinais de processos infecciosos, com idade entre 22 a 75 anos com diversos diagnósticos clínico e cirúrgico, internados nas enfermarias de Cardiologia, Gastroclínica e Enfermaria Geral de Adultos (EGA). Foram medidas as temperaturas ao longo do período de vigília, iniciando às 6 horas da manhã e a última às 22 horas, com um total de nove medidas. Verificou-se também a temperatura ambiente nas enfermarias durante o período das 5h30, às 14 horas e às 20 horas. Os resultados mostraram que houve diferença significativa entre as médias dos termômetros; as médias dos horários medidos; às médias entre as temperaturas dos termômetros no período noturno e entre as médias nos períodos matutino e vespertino (p-value=0,0001). Não houve diferença significativa entre os horários medidos no período noturno (p-value=0,8) e entre as médias das temperaturas nos períodos matutino e vespertino (p-value=0,4), quando utilizada a técnica paramétrica de análise de variância e o teste de Tukey para comparações múltiplas. O nível de significância adotado foi ? = 0,05. O termômetro timpânico registrou a variabilidade circadiana dos pacientes e seus valores de temperatura foram maiores em relação aos outros locais de medida
Resumo: Esta pesquisa teve como objetivo verificar a variabilidade circadiana das temperaturas timpânica, oral e axilar; correlacionar as medidas da temperatura timpânica considerando o ângulo de posicionamento e comparar as medidas entre si, em pacientes adultos hospitalizados. Participaram, 15 pacientes do sexo masculino sem sinais de processos infecciosos, com idade entre 22 a 75 anos com diversos diagnósticos clínico e cirúrgico, internados nas enfermarias de Cardiologia, Gastroclínica e Enfermaria Geral de Adultos (EGA). Foram medidas as temperaturas ao longo do período de vigília, iniciando às 6 horas da manhã e a última às 22 horas, com um total de nove medidas. Verificou-se também a temperatura ambiente nas enfermarias durante o período das 5h30, às 14 horas e às 20 horas. Os resultados mostraram que houve diferença significativa entre as médias dos termômetros; as médias dos horários medidos; às médias entre as temperaturas dos termômetros no período noturno e entre as médias nos períodos matutino e vespertino (p-value=0,0001). Não houve diferença significativa entre os horários medidos no período noturno (p-value=0,8) e entre as médias das temperaturas nos períodos matutino e vespertino (p-value=0,4), quando utilizada a técnica paramétrica de análise de variância e o teste de Tukey para comparações múltiplas. O nível de significância adotado foi ? = 0,05. O termômetro timpânico registrou a variabilidade circadiana dos pacientes e seus valores de temperatura foram maiores em relação aos outros locais de medida
Abstract: The aim of this research was to verify the daily variation of the tympanic, oral and axillary temperatures, and correlate measurements of the Tympanic temperature considering the positioning angle and to compare the set of measurements in adult volunteer patients during treatment in the Clinics Hospital of Universidade Estadual de Campinas, São Paulo. The results refer to fifteen male in patients, 22 to 75 years old with no signal of infectious processes, having different clinical and cirurgic diagnostics in the Cardiology, Gastroclinics, and Adult General Nursery. The temperatures were measured nine times between 6 am and 10 pm. The ambient nurserys temperature was also monitored, at 5:30 am, 2 pm, and 8 pm. The results show that there was a significant difference between: the mean measured temperatures in different positions; the mean values of the different scheduled times; the mean values of the morning and afternoon periods (p-value=0,0001). When using the parametric technique of analysis of variance and the Tukey¿s test of multiple comparation, there was no significant difference between the measured values (p-value=0,8). The significance level adopted was ? = 0,05. The tympanic thermometer has registered the daily variation of the patients¿ temperature and its values were bigger than the measured by the other places of measurement
Abstract: The aim of this research was to verify the daily variation of the tympanic, oral and axillary temperatures, and correlate measurements of the Tympanic temperature considering the positioning angle and to compare the set of measurements in adult volunteer patients during treatment in the Clinics Hospital of Universidade Estadual de Campinas, São Paulo. The results refer to fifteen male in patients, 22 to 75 years old with no signal of infectious processes, having different clinical and cirurgic diagnostics in the Cardiology, Gastroclinics, and Adult General Nursery. The temperatures were measured nine times between 6 am and 10 pm. The ambient nurserys temperature was also monitored, at 5:30 am, 2 pm, and 8 pm. The results show that there was a significant difference between: the mean measured temperatures in different positions; the mean values of the different scheduled times; the mean values of the morning and afternoon periods (p-value=0,0001). When using the parametric technique of analysis of variance and the Tukey¿s test of multiple comparation, there was no significant difference between the measured values (p-value=0,8). The significance level adopted was ? = 0,05. The tympanic thermometer has registered the daily variation of the patients¿ temperature and its values were bigger than the measured by the other places of measurement
Mestrado
Enfermagem e Trabalho
Mestre em Enfermagem

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

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.

Thesis (Ph.D.)--Tufts University, 2000.
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;

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.
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 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.

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 A. thaliana 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 A. thaliana, 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.

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.
Master of Science

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.

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.

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.

Thesis (MSc)--Stellenbosch University, 2014.
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.
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.
National Research Foundation

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.

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.

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.

Un extracte de proantocianidinas extretes de la llavor del raïm (GSPE) ha estat relacionat amb un ampli rang de efectes beneficiosos per la prevenció i tractament de les alteracions metabòliques hepàtiques induïdes per l’obesitat, p.e., la resistència a la insulina o l’esteatosi. A més, el GPSE és capaç de modular els ritmes circadians del fetge, els quals es troben desajustats a l’obesitat, suggerint que el GSPE pot regular parcialment el metabolisme de lípids i glucosa modulant els ritmes circadians hepàtics. La metabolòmica per RMN pot ser una tècnica adequada per l’estudi de la interacció entre l’efecte de les proantocianidines i els ritmes circadians del metabolisme hepàtic. En aquest sentit, aquesta tesi te com a objectius avaluar si el GSPE té diferents efectes sobre el metabolisme hepàtic depenent del temps d’administració en animals que pateixen la síndrome metabòlica. Addicionalment, es va estudiar la disrupció dels ritmes circadians del metabolisme hepàtic, causats per l’obesitat, a ambdós gèneres per a eludir si aquesta disrupció és depenent del sexe. Les femelles van mostrar una major resistència i flexibilitat quan eren alimentades amb una dieta obesogénica. L’administració crònica de GSPE va presentar efectes diferents en rates mascles obeses depenent del temps d’administració. Moltes dels efectes beneficiosos es van trobar en els animals que van ser tractats al principi de la fase lluminosa. A més, també es va observar un possible efecte antioxidant i una millora en la sensibilitat hepàtica a la insulina en aquest animals. Els resultats d’aquesta tesi ens mostren la importància del temps d’administració del GPSE. A més, aquesta tesi ens ensenya que les rates femelles obeses tenen una millor flexibilitat dels ritmes circadians hepàtics.
El extracto de proantocianidinas procedente de la semilla de uva (GSPE) se ha asociado a un amplio rango de efectos beneficiosos para la prevención o tratamiento de las alteraciones metabólicas hepáticas causadas por la obesidad, p.ej. resistencia a la insulina o esteatosis. Además, el GPSE es capaz de modular los ritmos circadianos hepáticos, los cuales son alterados por la obesidad. Por lo tanto, esto sugiere que el GPSE puede regular parcialmente el metabolismo lipídico y glucídico a través de la modulación de los ritmos circadianos. La metabolòmica basada en RMN es una técnica adecuada para el estudio de las interacciones entre los efectos de las proantocianidinas i los ritmos circadianos del metabolismo hepático. El objetivo de esta tesis es evaluar si los efectos del GSPE son diferentes dependiendo del momento del día de administración en animales que padecen síndrome metabólico. También se han estudiado las alteraciones en los ritmos circadianos del metabolismo hepático provocadas por una dieta obesogénica para determinar si, dicha alteración, es diferente en función el género. Las ratas hembra obesas mostraron una mayor resistencia y flexibilidad en los ritmos circadianos del metabolismo hepático. La administración crónica de GSPE presentó diferentes efectos en ratas macho obesas en función del momento de su administración. La mayoría de los efectos beneficiosos fueron hallados cuando el GSPE fue dado al inicio de la fase lumínica. Solo en dichos animales se observó un posible efecto antioxidante y una mejora en la sensibilidad a la insulina en el hígado. Los resultados de esta tesis eluciden la importancia del momento de administración del GSPE. Además, esta tesis demuestra una mayor flexibilidad en los ritmos circadianos en ratas hembra obesas.
A grape seed proanthocyanidin extract (GSPE) has been associated with a widely range of beneficial effects for the prevention and treatment of hepatic metabolic disturbances induced by obesity, such as insulin resistance or steatosis. Moreover, GSPE is capable to modulate the clock system in the liver, which is also disrupted in an obesity status, thus suggesting that GSPE can partially regulate lipid and glucose metabolism by modulating the hepatic circadian rhythms. NMR-based metabolomics strategy is an adequate approach to study the interaction between the proanthocyanidin effects and the circadian rhythmicity of the hepatic metabolism. In this regard, this thesis aims to evaluate whether a grape seed proanthocyanidin extract (GSPE) has different effects on the hepatic metabolism depending on the administration time, in a metabolic syndrome situation. The circadian rhythm disruption of the hepatic metabolism, caused by obesity, was studied in both genders in order to elucidate whether this disruption is gender-dependent. Female animals showed to be more resistance and flexible against an obesogenic diet. The chronic administration of GSPE presented different effects in obese male rats depending on its administration time. A large amount of its beneficial effects were found when GSPE was given at the beginning of the light phase. Possible antioxidant effects and an improvement in hepatic insulin sensitivity were only observed in those animals. The results of this thesis elucidate the importance of the administration time of GSPE. Additionally, this thesis shows the better circadian rhythm flexibility of obese female rats.

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.

Investigation into the impact of ocular disease on sleep and mood has shown that in humans eyes have an important role, and that absence of eyes or interference with light reaching the retina can have deleterious effects. Light is the main zeitgeber 'time-giver' used by most species for the regulation of circadian rhythms and is detected by rods, cones and photosensitive retinal ganglion cells (pRGCs) in mammals. The aims of this research project were to investigate this from three different perspectives. Three prospective studies were undertaken. The first, studied the impact of ocular disease on the sleep/wake cycle in diabetic retinopathy (DR) and in bilateral anophthalmia. There was no significant difference found between the severity of DR and global sleep scores, however the acquired anophthalmic groups have significantly raised global sleep scores compared to controls and the congenital anophthalmic group. Both anophthalmic groups had varying sleep/wake cycles on their actograms depending on the lifestyle (independent of the urinary melatonin). All the anophthalmic participants showed a non 24 hour sleep-wake rhythm disorder after melatonin profiling. The second study investigates the evidence for the presence of extraocular circadian photoreceptors (EOCP) in participants with anophthalmia and sighted controls. Changes in brain activity using a functional MRI scan was assessed, when a bright white light is shining in different locations. This study did not reveal any evidence of EOCP.Finally, structural brain MRI differences in anophthalmic groups were investigated. While similar changes in structural reorganisation occur in all anophthalmic groups in the occipital cortex, the acquired anophthalmic groups show an inverse relation with the time since becoming anophthalmic and the volume of optic radiation and optic nerve volume. The acquired anophthalmic group did not show increase in hippocampal volume (memory areas) or in the precuneus (spatial navigation) contrast to the congenital anophthalmic groups.

Sleep and circadian rhythm disruption (SCRD) is frequently found to co-occur in psychotic disorders. This can include a range of phenotypes such as insomnia, circadian delays, deficits in sleep spindles, and sleep-dependent cognitive impairments. However, increasing evidence suggests that psychosis occurs across a continuum of severity within the general population, yet few studies have investigated sleep and circadian rhythms prior to clinical diagnosis. Furthermore, SCRD and psychosis are posited to share underlying neuropathologies and although increasing evidence implicates shared genetic influence, little is known of the shared environmental risk. This thesis investigates sleep and circadian rhythms at multiple levels, from their occurrence in the general population, their disruption in high risk individuals, and a focus on sleep spindle oscillations in the brain. Firstly, the relationship between subjective measures of sleep and circadian rhythms, risk factors for psychosis, and psychotic-like experiences (PLEs) were studied in a large population sample. All three were highly related, with a subset of risk factors showing a strong association to both PLEs and SCRD. Secondly, sleep and circadian rhythms were assessed in individuals at high risk for developing psychosis based upon having a high load of risk factors and sub-clinical psychotic symptoms. High risk individuals showed subjective SCRD but this was not reflected in objective measures assessed by actigraphy and polysomnography. A subset of high risk individuals further showed substantially later melatonin rhythms compared to a low risk group. Thirdly, high and low risk individuals were assessed for measures of declarative and procedural sleep dependent memory consolidation. High risk individuals showed no evidence for sleep dependent cognitive impairment but did show a potential sensitivity in performance to the amount of sleep, not seen in low risk individuals. Finally, recordings in the somatosensory cortex (SCx) of mice were used as a model to explore the spatio-temporal dynamics and functional significance of sleep spindles. Distinct highly localised spindle events were discovered in the anterior SCx, with their complete absence just a fraction of a millimetre away, and their occurrence of which coincided with unique responses to global vigilance states. Together, this work pioneers research into sleep and circadian phenotypes associated with both sub-clinical psychotic symptoms and risk factors for disease and furthers our knowledge of particular sleep processes which could collectively help us to understand why SCRD and psychosis develop.

The day-to-day physiologies are largely influenced by circadian rhythms. Disruptions of such rhythms are associated with many diseases. Adjusting them to a healthy one can be promising to treat different circadian rhythms disruption associated diseases such as sleep disorders, cardiovascular disorders, metabolic disorders. The regulations underlying the circadian rhythms are much complicated and systematic which may involve thousands of genes. In mammals, these robust circadian rhythms are primarily intended by the concerted molecular interplays, knowingly, transcriptional-translational feedback loops (TTFLs). The collaborative interactions among a large number of genes intend to sustain the TTFLs. It facilitates to generate the primary transcriptional oscillations among the clock genes and genome-wide rhythmic oscillations. The collaborative transcriptional events act as dominant driving forces underpinning such rhythmic expressions. The mode of the transcriptional regulations depends on the concentration of the transcriptional factors (TFs) at the promoter region at a particular time point. The inclusive mechanisms of their regulations and the associated circadian rhythmic outputs across the physiology are not well defined yet. However, temporal recruitment of core-clock proteins, different transcriptional and translational regulators and chromatin modifications are imperative towards a comprehensive understanding of the spatio-temporal regulation of such complex rhythms. Despite many experimental signs of progress about the circadian transcriptional controls, there is still an interesting question remains unexplored that how do these few components belonging to the same molecular architecture are capable to govern such divergent gene expressions? Nevertheless, how they are being regulated and the landscape of their combinatorial regulatory controls have not gained any inclusive attention yet. Thus, a systematic understanding considering all-encompassing circadian TFs and their relational interplay could help us to decode the intricated transcriptional regulatory logics composed by different TFs. Such comprehensive understanding may lead to unleashing their potential to therapeutically modulate the circadian rhythms. Experiments alone are indeed quite challenging to achieve this. Decoding the inclusive transcriptional insights along with multifaceted molecular regulations remained out of reach with prevailing approaches. They are limited by the complexities of more integrative algorithms that accommodate different layers of molecular information quantitatively into a single framework. Studies indicated the knockout of the circadian TFs results in changing the rhythms. And, rescuing them helps to regain the circadian functionality substantially. However, knocking out all possible combinations of circadian TF-genes experimentally is merely very tedious, time-consuming and expensive. And, some essential genes cannot be knocked out. The magnitude of disruption of the circadian rhythmic fluctuations may also vary in disease conditions and even from individual to individual. These are serious concerns which were weakly understood. Due to the lack of advanced quantitative approach, these have remained a great challenge with traditional practices for reversing the disrupted circadian rhythms. Another level of challenge is not only aligning the rhythms but also, having a strong understanding of the directionality of the alignment varying in different clinical contexts is the most crucial. Consequently, a thorough quantitative understanding at the molecular level of the clock control mechanisms is essential. To address these ambiguities, a quantitative understanding of the circadian gene regulation and the molecular interplay among the key regulators are quite important. An alternative yet the operative approach is the reconstruction of transcriptional network with those genes having circadian fluctuations by computational simulations. It may capture a systematic snapshot of such gene regulation network at a dynamic scale. Inferring them is again a complicated task as the large numbers of variables are unknown in the systems. There is also a lack of tools to capture and integrate the dynamic view which is biologically relevant. Virtual knockout experiments leverage in inferring such dynamic transcriptional regulatory networks iteratively and effectively. The molecular machinery underpinning the circadian rhythms possess high-temporal resolutions. Thus, it is also quite challenging to construct the network of those genes under the influence of TTFLs at dynamic scale using existing methods. Most of the prevailing approaches are quite limited by the quantitative understanding of the transcriptional landscape thoroughly. Recently, one of our computational approaches, LogicTRN was proposed for modelling the transcriptional regulatory networks quantitatively. Deploying the high-resolution temporal gene expression data and the TF-DNA binding data, it calculates the TF-DNA binding occupancy, which is a quantitative estimation. It also predicts the all possible combinatorial TF-logics influencing those target genes' regulations. Here, we introduced an extended computational approach based on LogicTRN to decode the quantitative transcriptional regulatory landscapes of circadian genes. We introduced the reconstruction of quantitative transcriptional regulatory networks (qTRNs) for circadian gene regulations using LogicTRN framework. The qTRNs facilitated to discover a wide range of genes exhibiting circadian fluctuations. Their dynamic behaviours and the cis-regulatory logics in the networks were also estimated precisely. Based on quantitative knowledge from qTRNs, we have further developed a method for virtually knocking out the core clock component TFs to estimate the influence to perturb the circadian rhythmic fluctuations at a dynamic scale. Consecutively, the method of single/multiple genes virtual knockout was developed and used to screen the best TF/TFs combination that effectively modulates the circadian rhythmic output at a dynamic scale. They were also ordered by their influence to perturb the circadian fluctuations in the qTRNs. In future, it may indicate a way to target the molecular regulators to therapeutically modulate the circadian period lengths in a specific direction based on an individual's clinical conditions. Our results indicate the reconstruction of highly accurate quantitative regulatory networks for the transcriptional controls of the circadian gene regulation at a dynamic scale. We have also identified the best plausible transcription factors or their combinations those can effectively modulate the circadian rhythms. Of them, the CLOCK and CRY1 double knockout preserve the highest capacity to modulate the circadian rhythm dynamics. Besides, all possible TFs/TF-combinations were ordered in terms of their capacity to influence the qTRNs at dynamic scales. Finally, our quantitative framework offers a quick, robust, and physiologically relevant way to screen and identify the most effective TFs/TF-combinations to modulate circadian rhythms. This foundation may potentially enable us to engineer the molecular regulators underpinning the circadian rhythms. This potentially indicates a clue towards adjusting the circadian rhythmic phases in desired directions depending on clinical requirements

Drug-induced liver injury (DILI) is a serious adverse drug reaction (ADR) that is frequently encountered during drug development, representing a major cause of drug attrition. Furthermore, DILI is also a serious concern in the clinic, accounting for approximately half of all acute liver failure cases. Paracetamol overdose (acetaminophen; APAP) accounts for the majority of DILI-associated cases of ALF encountered in patients. The identification and development of novel biomarkers of DILI that are sensitive, specific, and rise early during hepatotoxicity are urgently required in the clinic and in the laboratory. Two liver-specific microRNAs (miRNAs) have recently been described that serve as sensitive and early markers of APAP-induced acute liver injury (APAP-induced ALI) in a mouse model. Together with the superior liver-enrichment of some liver-enriched miRNAs, these potential markers need to be assessed in patients for the clinical promise. Moreover, further work is warranted to test these potential markers in alternative pre-clinical models with other compounds to gain a better understanding regarding sensitivity of release, mechanism of release and circulatory kinetics. Furthermore, in separate work discussed in this thesis, the mammalian biological clock has been found to exert a powerful influence on the physiology of mammalian systems. This regulation hinges on the complex interplay between the clock genes and their products that oscillate over a twenty-four hour period and promote a diurnal variation in numerous output pathways. Emerging evidence suggests that the efficacy and toxicity of many drugs follow a diurnal rhythm and that this may be at least partly attributable to the clock-mediated regulation of drug targets and pathways of drug metabolism. APAP and FS represent two compounds that elicit hepatotoxicity in the mouse through two distinct mechanisms. Both APAP and FS are known to exhibit circadian variation in their toxicology and/or pharmacology. However, little is known about the molecular mechanisms that govern these differences in circadian variation. Two liver-enriched miRNAs (miR-122 and miR-192) were tested alongside serum ALT activity, the gold-standard marker of ALI, for sensitivity and time of release in a mouse model of APAP-induced ALI. At 2 hours after APAP administration, miR-122 (ΔΔCt 75.0, P=0.02) was significantly higher compared to controls (ΔΔCt 4.1) while ALT levels were in the normal range (21 U/L) indicating earlier release of miR-122. In a sensitivity study, miR-122 was not more sensitive than ALT at a 300 mg/kg dose of APAP compared to controls (mean values 300 mg/kg vs 0 mg/kg: ALT = 491 U/L vs 38.1 U/L; miR-122 = ΔΔCt 572.9 vs ΔΔCt 209.4). In patients, serum miR-122 and miR-192 were substantially higher in APAP-induced ALI patients, compared to healthy controls (median ΔΔCt miR-122: 1,265 versus 12.1, P < 0.0001; miR-192: 6.9 versus 0.44 P < 0.0001). A heart-enriched miR-1 showed no difference between APAP-ALI patients and controls, whereas miR-218 (brain-enriched) was slightly higher in the APAP-ALI cohort (ΔΔCt 0.17 versus ΔΔCt 0.07 P = 0.01). In a cohort comprised of patients who presented early (median time of presentation since APAP overdose = 8 hours), miR-122 was significantly raised in patients who develop ALI (> 3 x ULN serum ALT activity) compared to those that did not (median ΔΔCt 3.48 vs ΔΔCt 0.16, P<0.0001). In contrast, presentation ALT levels were not different between patients who developed ALI compared to those that did not (median ALT = 21 U/L vs 19 U/L). Moreover, miR-122 was significantly raised in patients who develop coagulopathy (INR > 1.3) compared to those that did not (ΔΔCt vs 3.48 vs ΔΔCt 0.17, P=0.0004). In contrast, presentation ALT levels were not different between patients who developed ALI iv compared to those that did not (median ALT = 21 U/L vs 19 U/L). In chronotoxicity studies, both APAP exhibited greater toxicity in the evening (mean ALT = 12785, 66% survival) compared to morning (mean ALT = 380, 100% survival) whereas FS showed greater toxicity after morning administration (mean ALT=561, 100% survival) compared to evening administration (mean ALT = 69.2, 100% survival). Circadian variation in APAP-induced ALI was associated with 38% lower (P=0.003) GSH levels and 20% higher (P=0.024) Cyp2e1 levels at 21:00h compared to 09:00. This work confirms that miR-122 is released earlier than ALT in a young mouse model of APAP-induced ALI. Furthermore, it is shown for the first time that circulating liver enriched miRNAs are higher in patients following an APAP overdose. Also, plasma miR-122 is raised at emergency room presentation when serum ALT activity is in the normal range. Further clinical development of blood-based miR-122 is warranted, this work suggests that miR-122 analysis at the point of hospital admission can predict risk of subsequent liver injury in patients. Finally, APAP and FS exhibit circadian variation in their toxicity in a mouse model associated with circadian variation with genes involved in drug metabolism and drug detoxification. Profiling of the hepatic proteome over the circadian phase is now warranted.