The soybean cyst nematode (SCN), Heterodera glycines, a plant parasitic pest, causes severe yield losses of soybean (Glycine max). Although a number of studies have identified various genes that function in defense, including a role for the vesicular transport machinery acting against H. glycines in infected roots, a regulatory mechanism occurring behind the transcriptional engagement of the vesicular transport system and delivery of the transported cargo proteins is not fully understood. The main goal of the current study is to determine the functional effect of genetically engineering the circadian clock gene, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) in G. max to examine a role on H. glycines parasitism. The outcome of the study has determined the functional effect of main clock component CCA1-1 along with other oscillator genes such as TIMING OF CAB 1 (TOC1-1), GIGANTEA (GI-1) and CONSTANS (CO-4) to enhance resistance against H. glycines parasitism. Further, the reduced level of the expression of Gm-CCA1-1 in infected roots, in comparison to uninfected roots, has demonstrated that clock components might have arrested and altered its expression during the nematode infection process. The study has also investigated the role of XYLOGLUCAN ENDO-TRANSGLYCOSYLASE /HYDROLASE (XTH), Gm-XTH43, during the resistance process soybean has to H. glycines. The results have demonstrated higher xyloglucan (XyG) amounts to be synthesized in the Gm-XTH43 overexpressing (OE) lines. In contrast, there is less XyG in the Gm-XTH43 RNA interference (RNAi) lines that have a negatively regulated XTH gene. These observations have led to elucidating the role in the potential cell wall rearrangement and the underlying metabolic processes required for the generation of the proper XyG architecture required for defense occurring outside of the plant cell. Furthermore, the observed result of lower level of weight average molecular weight (WAMW) of XyG in Gm-XTH43-OE and higher MW of XyG in Gm-XTH43-RNAi than respective control roots have demonstrated a key role in, presumably, changing the cell wall by the remodeling of the XyG chain as it relates to the cell wall architecture.

The circadian system in mammals is arranged as a hierarchical network of oscillators, with the master pacemaker of circadian rhythms located in the suprachiasmatic nuclei (SCN) of the hypothalamus and peripheral oscillators in most other organ and tissue systems of the body. The molecular machinery responsible for generating circadian rhythms is composed of interlocked transcriptional-translational feedback loops with the gene Brain Muscle Arnt-like 1 (Bmal1) functioning as a core positive regulator. Using the mouse, Mus musculus as a model system, we studied the post-transcriptional mechanisms regulating Bmal1 expression in the SCN pacemaker and in peripheral oscillators. Target prediction algorithms were used to identify microRNAs (miRNAs) predicted to target Bmal1. We profiled the temporal expression of miR-142-3p in the mouse SCN in vivo and in an immortalized SCN cell line and observed robust circadian rhythms in its expression in the SCN. Following luciferase-reporter and site-directed mutagenesis analyses, we identified miR-142-3p as a bona-fide post-transcriptional repressor of Bmal1. The temporal expression of potential Bmal1-targeting miRNAs was also examined in the circulation in mouse serum. In mice housed in a light-dark cycle, diurnal oscillations were observed in serum levels of miR-152 and miR-494, but not miR-142-3p expression. Luciferase reporter studies indicated that miR-494, both independently and synergistically with miR-142-3p, repressed the Bmal1 3′ UTR. Overexpression of these miRNAs disrupted ensemble circadian rhythms of PER2::LUCIFERASE activity in cultured fibroblasts. Overexpression of the miRNAs also increased their extracellular levels and their intracellular accumulation in recipient cultures exposed to conditioned medium. Furthermore, inhibition of exocytosis and endocytosis affected ensemble circadian rhythms in cultured fibroblasts. The results thus implicate miR-142-3p and miR-494 in the regulation of Bmal1 expression in the SCN and peripheral oscillators and suggest that miRNAs may function as both, intracellular and extracellular (cis- and trans- acting) signals, modulating the core clock mechanism in the SCN and in fine-tuning the synchronization of circadian rhythmicity between cell-autonomous oscillators in the periphery.

Circadian clocks are biological signalling networks which have a period of ~24 hours under constant environmental conditions. They have been identified in a wide range of organisms, from cyanobacteria to mammals and through the temporal co-ordination of biological processes are believed to increase individual fitness. The mechanisms which generate these self-sustained rhythms, the pathways of entrainment and the target outputs of the clock are all areas of great interest to circadian biologists. The plant circadian clock is believed to comprise of interlocking feedback loops of transcription and translation. The morning MYB-transcription factors CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) bind to the promoter of TIMING OF CAB2 1 (TOC1) and repress its expression, as well as their own. As levels of CCA1 and LHY fall, TOC1 is expressed and activates the expression of its repressors. This is a simplified version of the known clock components and the current model contains this core loop as well as an interlocked morning and evening loop, which also incorporates some post-translational modification (Chapter 1). Understanding the plant circadian network and its entrainment are the topics of this thesis. The study has focused on two plant species, the land plant Arabidopsis thaliana and the picoeukaryotic marine algae Ostreococcus tauri. In both of these species light-mediated entrainment of the clock has been investigated (Chapter 8), as well as the core circadian mechanism. In A. thaliana the role of a circadian associated gene, EARLY FLOWERING 3 has been a particular focus for investigation, through both experimentation and mathematical models (Chapters 4 and 5). In O. tauri the responses to light signals have been tested, as have the circadian responses to pharmacological manipulation (Chapters 6, 7 and 8). The work presented identifies a role for ELF3 in the repression of circadian genes and also links it with the regulation of protein stability. Likewise, in O. tauri the regulation of protein stability is identified to be a key mechanism for sustaining circadian rhythms. As well as investigating the clock in plants, certain photoreceptors have been characterised in S. cerevisiae with the aim of linking them to a synthetic oscillator. Together the work presented in this thesis provides evidence for the circadian community to aid with the understanding of circadian rhythms in plants, and possibly other organisms.

This thesis focuses on the circadian clock genes and their involvement in the photoperiodic time measurement in the linden bug, Pyrrhocoris apterus. Application of the molecular biology methods enabled us to propose the architecture of circadian clockwork. We also investigated the role of several previously undescribed players in the circadian clock. Furthermore, by using molecular biology methods we focused on the involvement of core circadian clock genes in the photoperiodism.

The circadian system controls 24-hour cycles of behavior and physiology, such as rest-activity and feeding rhythms. The human circadian system synchronizes with, or entrains to, the light/dark cycle (sunrise/sunset) to promote activity and food consumption during the day and rest at night. However, strict work schedules and nighttime light exposure impair proper entrainment of the circadian system, resulting in chronic circadian misalignment. Numerous studies have shown that chronic circadian misalignment results in poor health. Therefore, therapeutic interventions that could shift circadian rhythms and alleviate circadian misalignment could broadly impact public health. Although light is the most salient time cue for the circadian system, several laboratory studies have shown that exercise can also entrain the internal circadian rhythm. However, these studies were performed in controlled laboratory conditions with physically-active participants. The purpose of this study was to determine whether timed exercise can phase advance (shift earlier) the internal circadian rhythm in sedentary subjects in free-living conditions. Fifty-two young, sedentary adults (16 male, 24.3±0.76 yrs) participated in the study. As a marker of the phase of the internal circadian rhythm, we measured salivary melatonin levels (dim light melatonin onset: DLMO) before and after 5 days of timed exercise. Participants were randomized to perform either morning (10h after DLMO) or evening (20h after DLMO) supervised exercise training for 5 consecutive days. We found that morning exercisers had a significantly greater phase advance than evening exercisers. Importantly, the morning exercisers had a 0.6h phase advance, which could theoretically better align their internal circadian rhythms with the light-dark cycle and with early-morning social obligations. In addition, we also found that baseline DLMO, a proxy for chronotype, influenced the effect of timed exercise. We found that for later chronotypes, both morning and evening exercise advanced the internal circadian rhythm. In contrast, earlier chronotypes had phase advances when they exercised in the morning, but phase delays when they exercised in the evening. Thus, late chronotypes, who experience the most severe circadian misalignment, may benefit from exercise in the morning or evening, but evening exercise may exacerbate circadian misalignment in early chronotypes. Together these results suggest that personalized exercise timing prescriptions based on chronotype could alleviate circadian misalignment in young adults.

The suprachiasmatic nucleus receives dense serotonergic projections from the raphe nuclei and this input has been implicated in the modulation of circadian rhythms. This input appears to have many functions including the transmission of non-photic information during the day and the modulation of photic information at night. However, it has emerged that this input may also be involved in the transmission of light information with activation of 5-HT2C receptors at night having a photo-mimetic effect. The studies described in this thesis aim to clarify the role of 5-HT2C receptors in the control of circadian rhythms in the rat model and compare their actions to light. The acute effects of 5-HT2C receptor agonist administration on clock gene expression were investigated in the rat SCN. Systemic administration of the 5-HT2A/2C agonist DOI to rats during early night induced c-fos, Per1 and Per2 expression in a manner similar to light. This response was time of day dependent with maximal induction occurring in the early night, and no response during the day. The role of 5-HT2C receptors in this response was confirmed with the use of the selective 5-HT2C receptor agonist RO-60 0175. The effect of 5-HT2C receptor activation on the phase of expression of various circadian rhythms including temperature, melatonin and clock gene expression in the SCN and periphery was examined. Both DOI administration and light exposure at night phase delayed rhythms of melatonin and temperature. Similarly, the selective 5-HT2C receptor agonist RO-60 0175 phase delayed rhythms of 6-sulphatoxymelatonin, a response which was antagonised by the 5-HT2C receptor antagonist SB-242084. The expression of functional and clock genes within the pineal was also phase delayed following both light and 5-HT2C receptor agonist administration. However, the phase of expression of clock genes within the SCN or liver did not shift in response to either a single nocturnal light pulse or agonist administration. To investigate the site of action of 5-HT2C receptor agonists, rat SCN explants were maintained in culture allowing exposure of agonists to denervated tissue. The acute effect of DOI administration at various circadian times on c-fos and Per1 expression was assessed. 5-HT2C receptor activation significantly increased Per1 expression when administered during early subjective night, but had no effect during either subjective day or late subjective night, similar to that observed in vivo. Finally, the suitability of immortalised rat SCN cells for investigation of the intracellular actions of 5-HT2C receptors in the circadian system was assessed. Using RT-PCR the expression of various serotonin receptors in the SCN2.2 cell line was compared with that observed in punches of adult rat SCN. The mRNA for 5-HT1B and 5-HT2A receptor was expressed in both the SCN2.2 cell line and the adult rat SCN. However, 5-HT2C receptor mRNA along with 5-HT3 receptor, 5-HT5A receptor and 5-HT7 receptor mRNA was expressed in the adult rat SCN tissue but not the SCN2.2 cells. These significant differences in serotonin receptor expression limit the usefulness of this cell line for further investigation. Together these experiments further implicate 5-HT2C receptors in the control of circadian rhythms. The role of these receptors appears limited to early night, with activation showing photo-mimetic responses. Furthermore, the location of action appears to be post-synaptic within the SCN, altering the core clock genes, which in turn phase delay various circadian rhythms. / Thesis(Ph.D.)-- School of Paediatrics and Reproductive Health, 2008

Die Leistungsfähigkeit vieler kognitiver Funktionen zeigt tageszeitliche Schwankungen, welche auf dem Zusammenspiel der im 2-Prozess-Modell der Schlafregulation beschriebenen Prozes- se basieren: dem homöostatischen Schlafdruck (Prozess S) sowie dem circadianen Schrittma- cher (Prozess C). Darüberhinaus existieren verschiedene Chronotypen, welche oftmals einen synchrony-effect, also eine bessere Leistung zu für sie optimalen Tageszeiten im Vergleich zu nicht-optimalen Tageszeiten, zeigen. In der vorliegenden Arbeit wurde untersucht, in- wieweit die Leistungsfähigkeit der mittels des Attention Network Task (ANT) gemessenen Aufmerksamkeitsfunktionen tonische und phasische Alertness, Orientierung und exekutive Aufmerksamkeit aufgrund homöostatischer und circadianer Faktoren bei extremen Chrono- typen tageszeitabhängige und/oder chronotypabhängige Variationen unter synchronisierten Bedingungen (also einem normalen Tag-Nacht-Rhythmus) mit selbstgewählten Schlafzeiten zeigen. Zu diesem Zweck wurden je 20 ausgeprägte Morgen- bzw. Abendtypen zu fünf ver- schiedenen Uhrzeiten (9:00, 12:00, 15:00, 18:00 und 21:00 Uhr) in randomisierter Reihenfolge getestet. Phasische Alertness, Orientierung sowie die exekutive Aufmerksamkeit zeigten sich bei beiden Chronotypen im Tagesverlauf stabil. Die in einer ähnlichen Studie gefundenen Schwankungen der phasischen Alertness der Morgen-/Neutraltypen sowie der exekutiven Auf- merksamkeit bei beiden Chronotypen konnten nicht repliziert werden, was wahrscheinlich auf eine unterschiedliche Einteilung in Chronotypgruppen sowie ein unterschiedliches Studiende- sign zurückzuführen ist. Möglicherweise kann dies aber auch darauf hinweisen, dass es sich bei der Chronotyp-Dimension auf behavioraler Ebene nicht um ein Kontinuum handelt. Wäh- rend die tonische Alertness bei den Morgentypen gleich blieb, zeigte sich bei den Abendtypen ein synchrony-effect, d.h. sie zeigten eine Verbesserung der Leistung im Tagesverlauf, wobei nicht auszuschließen ist, dass neben der nicht-optimalen circadianen Phase der Abendtypen am Morgen auch sleep inertia sowie partielle Schlafdeprivation zu diesem Verlauf beigetragen haben können. Darüberhinaus zeigten die Morgentypen unabhängig von der Tageszeit eine generell schlechtere Orientierungsfunktion als die Abendtypen, was die Hypothese einer ver- schiedenartigen hemisphärischen Dominanz bei den verschiedenen Chronotypen unterstützt. Die Ergebnisse der vorliegenden Studie unterstützen die These, dass verschiedene kognitive Prozesse selektiv durch homöostatische und circadiane Prozesse moduliert werden, da selbst so ähnliche Funktionen wie die tonische und die phasische Alertness unterschiedliche Ver- läufe zeigen und in einer früheren Studie Schwankungen der phasischen Alertness und der exekutiven Aufmerksamkeit bei moderaten Morgen-/Neutraltypen bzw. moderaten Abendty- pen beschrieben wurden. Ob die unterschiedlichen Ergebnisse der vorliegenden und früherer Studien tatsächliche Unterschiede zwischen verschiedenen Chronotypen reflektieren oder Un- terschieden des jeweiligen Studiendesigns geschuldet sind, ist in weiterführenden Studien zu untersuchen.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the master mammalian pacemaker, co-ordinating the multitude of cell-autonomous circadian oscillators across the body to ensure internal synchrony, as well as maintaining an adaptive phase relationship with the light-dark cycle via projections from the retina. Intercellular communication between SCN clock neurons synchronises their oscillations, resulting in coherent output signals to the periphery. Vasoactive intestinal peptide (VIP), a neuropeptide expressed in the retinorecipient ventrolateral region of the SCN, is vital to this circuit-level co-ordination by signalling to its cognate VPAC2 receptor. In addition, VIP is important for the integration of light input into the SCN oscillation. The aims of the work presented in this thesis were to determine the roles of the VIP and VPAC2 cells in controlling circadian rhythmicity, and to elucidate the mechanisms of VIP signalling that underpin these roles. The first two experimental chapters utilise intersectional genetics and viral transduction to address separable roles for the VIP and VPAC2 cell populations. By diphtheria toxin-mediated cell ablation, or by adjusting cell-autonomous periodicity or rhythmicity specifically in these cell populations, I have identified that the VPAC2 cells are important for period setting and rhythmicity of both the SCN ex vivo and mouse behaviour in vivo, while the VIP cells play a vital role in behavioural rhythmicity and phase coherence across the SCN. The next two chapters use application of VIP to SCN slices to address mechanisms of phase-resetting through pharmacological manipulation and microarray analysis. I find that VIP has long lasting effects on all major circadian parameters of the SCN slice oscillation at both the cellular and circuit levels, and that it achieves this through a diversity of molecular pathways, in particular through cAMP/Ca2+ response elements within gene promoters. The final chapter focuses primarily on DUSP4, a negative regulator of the MAP kinase pathway that I have demonstrated to be upregulated by VIP. Here I demonstrate that DUSP4 affects the steady-state period of SCN slices, as well as influences phase shifting characteristics of both slices and mice. To conclude, the work presented here furthers our knowledge of neuropeptidergic communication in mammalian pacemaking. I have undertaken extensive characterisation of the molecular mechanisms through which the VIP neuropeptide influences SCN oscillators, and I have determined differential roles for the VIP and VPAC2 neurons in circadian timekeeping.

Subterranean rodents are interesting model group for studying of the circadian timing system due to their cryptic lifestyle and challenging living conditions. The filed data indicate that the locomotor activity of the silvery mole-rat (Heliophobius argenteocinereus) is influenced by temperature cycle, but the confirmation in laboratory study was to date missing.

xii, 109 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / There are two fundamental environmental rhythms that organisms in nature encounter: (1) the daily rhythm of light and dark that is due to the rotation of the earth about its axis and (2) the yearly seasonal rhythm due to the angle of the earth's rotation relative to the plane of its orbit around the sun. All eukaryotes have an endogenous circadian (daily) clock that allows for the timing of biological events within the context of the daily light:dark cycle. A wide diversity of plants and animals in temperate regions use photoperiodic (daylength) cues to time life history events, such as reproduction and diapause (insect dormancy) within the context of the yearly seasonal cycles. This dissertation focuses on the relationship between the circadian clock, photoperiodic time measurement and diapause. Chapter I serves as an introduction to biological timing and briefly summarizes the chapters that follow Chapter II outlines why Drosophila melanogaster , the workhorse of modern insect genetics, is not an appropriate system for the study of photoperiodism. Chapter III defines the Nanda-Hamner response, the circadian phenotype used in this dissertation, and proposes that the NH response is due to a rhythmic level of circadian disorganization in response to environmental cycle length. Chapters IV and V deal primarily with the long-held proposition that the circadian clock forms the causal basis of photoperiodic time measurement. I show that variation in the circadian clock does not covary with photoperiodic phenotypes among natural populations of Wyeomyia smithii , and thus these two processes are evolutionarily independent. Chapter VI describes the first forward genetic screen for candidate genes involved in photoperiodism and diapause termination in any animal. Chapter VII is a discussion of the complexity involved in studies of the genetics of photoperiodism and diapause and how historical inertia of scientific hypothesis acts to confound, rather than clarify, the relationship between genotypes and phenotypes. Chapter VIII is a concluding discussion of the implications of the work presented. This dissertation includes both previously published and co-authored material. / Committee in charge: William Cresko, Chairperson, Biology; William Bradshaw, Advisor, Biology; Patrick Phillips, Member, Biology; Eric Johnson, Member, Biology; Stephen Frost, Outside Member, Anthropology