This dissertation includes three components. Component 1 provides an estima- tion procedure for circadian parameters in cyanobacteria. Component 2 explores the relationship between baseline and amplitude by model selection under the framework of smoothing spline. Component 3 investigates properties of hypothesis testing. The following three paragraphs briefly summarize these three components, respectively. Varying coefficient models are frequently used in statistical modeling. We pro- pose a semiparametric varying coefficient periodic model which is suitable to study periodic patterns. This model has ample applications in the study of the cyanobac- teria circadian clock. To achieve the desired flexibility, the model we consider may not be globally identifiable. We propose to perform local approximations by kernel based methods and focus on estimating one solution that is biologically meaningful. Asymptotic properties are developed. Simulations show that the gain by our proce- dure over the commonly used method is substantial. The methodology is illustrated by an application to a cyanobacteria dataset. Smoothing spline can be implemented, but a direct application with the penalty selected by the generalized cross-validation often leads to non-convergence outcomes. We propose an adjusted cross-validation instead, which resolves the difficulties. Biol- ogists believe that the amplitude function of the periodic component is proportional to the baseline function. To verify this belief, we propose a full model without any assumptions regarding such a relationship, and two reduced models with the ratio of baseline and amplitude to be a constant and a quadratic function of time, respectively. We use model selection techniques, Akaike information criterion (AIC) and Schwarz Bayesian information criterion (BIC), to determine the optimal model. Simulations show that AIC and BIC select the correct model with high probabilities. Application to cyanobacteria data shows that the full model is the best model. To investigate the same problem in component 2 by a formal hypothesis testing procedure, we develop kernel based methods. In order to construct the test statistic, we derive the global degree of freedom for the residual sum of squares. Simulations show that the proposed tests perform well. We apply the proposed procedures to the data and conclude that the baseline and amplitude functions share no linear or quadratic relationship.

Circadian rhythms of behavior have been well characterized in organisms including mammals, plants, insects, fungi, and photosynthetic bacteria. Cyanobacteria, such as the unicellular Synechococcus elongatus PCC 7942, display near 24-h circadian rhythms of gene expression. These rhythms persist in the absence of external cues, can be reset by the same stimuli to which they entrain, and are relatively insensitive to changes in ambient temperature within their physiological range. Key components have been identified as belonging to the central oscillator that comprises the timekeeping units, output pathways that relay temporal information to clock-controlled processes, and input pathways that synchronize the oscillator with local time. The emerging model of the cyanobacterial clock depicts the internal timekeeping elements KaiA, KaiB, and KaiC interacting with one another to form a large, multimeric complex that assembles and disassembles over the course of a day. Information is sent into and out of the oscillator via signal transduction pathways that include proteins involved in bacterial twocomponent systems. The research presented in this dissertation explores the regulatory mechanisms that exist at each level of the clock system. New components were identified that interact with an important protein in the input pathway; these new players are involved in clock-associated phenomena, such as resetting the internal oscillation to external stimuli and maintaining proper circadian periodicity, as well as the process of cell division. The model formerly associated with the temporal, transcriptional regulation of the kai genes was redefined to reflect the unique properties of the prokaryotic oscillator. The differential output of the clock was examined by studying the circadian regulation of the psbA gene family. Overall, these data provide insight into the complex molecular events that occur to create a circadian timing circuit in S. elongatus.

The genetic identification of molecular mechanisms responsible for circadian rhythm generation has advanced tremendously over the past 25 years. However the molecular identities of the avian clock remain largely unexplored. The present studies seek to determine candidate clock components in the avian species Gallus domesticus. Construction and examination of the transcriptional profiles of the pineal gland and retina using DNA microarray analysis provided a clear view into the avian clock mechanism. Investigation of the pineal and retina transcriptomes determined the mRNA profiles of several thousand genes over the course of one day in LD (daily) and one day in DD (circadian) conditions. Several avian orthologs of mammalian clock genes were identified and many exhibited oscillating patterns of mRNA abundance including several of the putative avian clock genes. Comparison of the pineal transcriptional profile to that of the retina revealed several intriguing candidate genes that may function as core clock components. Including the putative avian clock genes and several others implicated in phototransduction, metabolism, and immune response. A more detailed examination of several candidate photoisomerase/photopigment genes identified from our transcriptional profiling was conducted. These include peropsin (rrh), RGR-opsin (rgr), melanopsin (opn4) and cryptochrome 2 (cry2) genes. This analysis revealed several interesting patterns of mRNA distribution and regulation for these genes in the chick. First, the mRNA of all 4 genes is located within the Inner Nuclear Layer (INL) and Retinal Ganglion cell Layers (RGL) of the ocular retina, where circadian photoreception is present. Second, opn4 and cry2 mRNA is expressed in the photoreceptor layer of the chick retina where melatonin biosynthesis occurs. Lastly, the mRNA for all 4 candidate photopigment genes is regulated on a circadian basis in the pineal gland. As a whole these data yield significant insight into the mechanisms of the avian circadian system and present several candidate genes that may function to integrate photic information, and/or regulate circadian rhythm generation in birds.

Melatonin is rhythmically synthesized and released by the pineal gland and, in some species, retina during the night and regulates many physiological and behavioral processes in birds and mammals. Chick diencephalic astrocytes express two melatonin receptor subtypes in vitro, and melatonin plays a role in regulating metabolic activity. We examined the role of glial cells in circadian function and asked if melatonin modulated glial functions within the retina and the brain. Calcium waves were potentiated by physiological concentrations of melatonin. Melatonin increased resting calcium levels and reduced gap junctional coupling among astrocytes at these same concentrations. Both mouse and chick diencephalic and telencephalic astrocytes express melatonin receptor protein. Nanomolar melatonin modulated astrocytic calcium waves of the mouse and chick diencephalon but not waves of the telencephalon. Mammalian intercellular calcium waves spread farther than avian calcium waves, and the nature of the spread of the waves differed between telencephalic and diencephalic mammalian astrocytes. These differences in propagation were abolished by melatonin. Using northern analysis, we identified period2, period3, cryptochrome1, cryptochrome2, clock, melanopsin and peropsin within chick diencephalic astrocytes. The clock genes cry1 and, per2 were expressed rhythmically in a LD cycle, but metabolic activity was not rhythmic. When cells were placed in constant darkness and rhythmically administrated melatonin, a robust rhythm in glucose uptake was induced without a coordinated clock gene rhythm, suggesting rhythmic clock gene expression and metabolic activity are separable processes. Melatonin affected visual function as assessed by electroretinogram. Circadian rhythms of a- and b-wave implicit times and amplitudes were observed. Melatonin (1 mg/kg and 100 ng/kg) decreased a- and b-wave amplitudes greater during the night than during the day and it increased a- and b-wave implicit times while 1 ng/kg melatonin had little to no effect over the saline controls. These data indicate that melatonin modulates glial intercellular communication, affects metabolic activity in astrocytes, and may play a role in regulating a day and night functional shift in the retina, at least partially through Müller glial cells. Thus, melatonin can regulate glia function and thereby, affect outputs of the vertebrate biological clock.

Circadian rhythms in biological processes occur in a wide range of organisms and are generated by endogenous oscillators. In Neurospora crassa, the FRQ-oscillator (comprised of FRQ, WC-1 and WC-2) is essential for rhythms in asexual sporulation and gene expression. How this oscillator signals to the cell to control rhythmicity is unknown. Furthermore, under certain growth conditions, rhythms are observed in FRQ-null strains, indicating the presence of one or more FRQ-less oscillators (FLOs). Interestingly, while circadian rhythms are observed in the related Aspergillus spp., they lack the frq gene, leading to the hypothesis that a FLO is responsible for rhythms in Aspergillus. Thus, Aspergillus provides a useful organism to investigate the components of the FLO. To investigate how an oscillator controls circadian output, we characterized the role of N. crassa NRC-2. The nrc-2 gene is under control of the clock and encodes a putative serine-threonine protein kinase. In a NRC-2-null strain cultured in low glucose conditions, FRQ-oscillator-dependent outputs are arrhythmic, but are rhythmic in high glucose. Our data suggests a model whereby NRC-2 relays metabolic information to the FRQ-oscillator to control rhythmic output. To understand the role of FLO(s) in the N. crassa circadian system, we examined regulation of the ccg-16 gene. We show that ccg-16 transcript rhythmicity is FRQ-independent, but WC-1-dependent. Furthermore, in contrast to current models for the FRQ-oscillator, we observed that rhythms in WC-1 protein accumulation persist in the absence of FRQ. These data support a new model involving two oscillators that are coupled through the WC-1 protein and that regulate different outputs. One approach to identify components of the FLO involved characterizing circadian rhythms in Aspergillus spp, which lacks FRQ. We find that A. flavus and A. nidulans, display circadian rhythms in sporulation and gene expression, respectively. Together, these findings provide a foundation for the identification of FLO components in both Aspergillus and N. crassa, that will ultimately lead to an understanding of how a multi-oscillator system can generate and coordinate circadian rhythmicity.

Thesis (M.A.) - Simon Fraser University, 2006. / Theses (Dept. of Psychology) / Simon Fraser University. Also issued in digital format and available on the World Wide Web.

Thesis (Ph.D.)--York University, 2008. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 165-180). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR39028

Thesis (M.S. in Human Systems Integration)--Naval Postgraduate School, December 2009. / Thesis Advisor(s): Miller, Nita Lewis. Second Reader: Schiffman, David L. "December 2009." Description based on title screen as viewed on January 26, 2010. Author(s) subject terms: Navy Standard Workweek, fatigue, sleep, circadian rhythm. Includes bibliographical references (p. 95-97). Also available in print.

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Alcoholism is a devastating inheritable disease that causes a large fiscal and societal impact worldwide. The fruit fly, Drosophila melanogaster, has proven to be a useful model system in identifying genetic factors underlying ethanol-associated behaviors. Many genes have been identified in the fly that are involved in the acquisition of tolerance, or the reduced response of an effect of a drug caused by previous exposure. In this thesis, I investigated whether circadian genes are involved in tolerance to ethanol in the fly. Circadian genes had previously been implicated in cocaine sensitization in flies, as well as some ethanol responses in mammals. I developed a novel assay using a bootstrapping paradigm to analyze tolerance to ethanol in the fly that allows for the investigation of multiple components of tolerance. I then used this assay to test whether circadian genes were necessary for the acquisition of tolerance in the fly. Interestingly, only some circadian genes affect tolerance to ethanol. These results argues that circadian genes are involved in tolerance to ethanol, but because some mutants are arrhythmic but still acquire tolerance, that they are acting in a role outside of the circadian system. While in the course of this work, I intended to investigate if these mutations affected ethanol preference in a two-choice assay. Before employing this assay, however, we wished to determine if flies prefer ethanol for its pharmacological effect or for its value as a food. Weperformed experiments in which flies had a choice between food supplemented with ethanol and food supplemented with an isocaloric carbohydrate. When presented with the isocaloric alternative, flies no longer demonstrated preference for ethanol. Flies will even stop showing preference for ethanol when switched to a balanced assay after preference has already been attained. We conclude that the flies prefer ethanol not because of its effect as a drug, but as a food source. / text