How do the brain and the immune system interact, and what are the consequences of this interaction on the physiology of an organism during infection? The main focus of my thesis is neuroimmune interaction, as studied in the following: (1) circadian regulation of immune system function, specifically phagocytosis by immune cells during bacterial infection; (2) the impact of circadian-regulated metabolism and feeding behavior on immunity and host tolerance of bacterial infection; and (3) immune system function in the context of Fragile X syndrome, a neurological disease known to cause circadian dysregulation. To investigate the interactions between these complex physiologies, I use the well-characterized and genetically tractable Drosophila melanogaster animal model. Each topic is briefly introduced in Chapter 1. Chapter 2 focuses on the body of work identifying the circadian regulation of the immune system, particularly phagocytosis, by immune cells during bacterial infection. Chapter 3 highlights findings regarding how diet and host metabolic state impact survival after infection. Chapter 4 illustrates phagocytic immune cell defects both systemically and in the brain in the Drosophila model of Fragile X syndrome. Lastly the conclusions discuss how these three works have built on our fund of knowledge of neuroimmune interactions and the future implications for these results.

Immunity, behavior, and circadian regulation are important ways that animals maintain homeostasis. Defects in these physiologies often lead to disease or even death, yet many questions remain about how these physiologies are related. I explored the interactions between innate immunity, behavior, and circadian regulation by using Drosophila melanogaster, a convenient, genetically tractable model organism with both functionally and molecularly conserved innate immune and circadian clock systems. In the first chapter, I show that feeding, a circadian-regulated behavior, increases immunity to a sepsis-like infection. In the second, I present evidence suggesting that aging-related changes in immunity may be linked to circadian defects. Finally, I use a novel automated method to demonstrate that reduced grooming is a conserved sickness behavior in Drosophila. The feeding project ultimately showed that mutating TORC2 components could increase the host’s ability to kill and clear a bacterial infection, as well as survive the pathogenic effects of infection. Therefore we have identified a possible drug target to create host-based therapies for sepsis patients. We also have established Drosophila as a model system for studying a conserved sickness behavior: reduced grooming. This experimental paradigm will allow researchers to isolate mutants that do not show reduced grooming, and investigate whether this sickness behavior is adaptive or not.

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Circadian rhythms refer to the physiological and biological processes that fluctuate over a 24-hour period. These rhythms are found in most living things such as animals, plants and fungi. In mammals, circadian rhythms are mainly generated and regulated by the suprachiasmatic nucleus (SCN).The period (Per1) gene in the SCN plays a key role in directing circadian rhythms. Per1 expression increases during the day and decreases at night. The neurons which express the Per1 gene show different behaviours to non-Per1 neurons during the day-night cycle. Per1 neurons are in a state of repetitive firing in the morning while in the late morning they display a bursting behaviour. In the afternoon, Per1 neurons divide into two groups: GA and GB, where GA is in a repetitive firing state and GB is silent. At night, these neurons are generally in a quiescent state but late at night they generate spikes. In this study, a standard Hodgkin-Huxley type model was used to study the behaviours of Per1 neurons over the day-night cycle using bifurcation analysis. In this model, the potassium and calcium currents carry the circadian rhythms which are modelled by their conductance. The currents had a significant impact on Per1 neuron behaviours. Furthermore, by changing some of the model parameters, different bistability mechanisms were examined. The study was extended to explore the effect of noise from other neurons on Per1 neuron behaviours. It was shown that noise plays a crucial role in inducing some of the Per1 neuron behaviours and that some Per1 neuron behaviours are fully induced by this noise, e.g. the late morning and late night behaviours. In contrast, the noise was found not to have any significant effect on other Per1 neuron behaviours other than the two behaviours that are observed in the afternoon.

The Earth’s oblique rotation results in changes in light and temperature across the day and time of year. Living organisms evolved rhythmic behaviours to anticipate these changes and execute appropriate responses at particular times. The current paradigm for the biological clocks in several branches of life is an underlying biochemical oscillator mainly composed by a network of repressive transcription factors. The slow decay in their activity is fundamental for generating anticipatory dynamics. Interestingly, these dynamics can be well appreciated when the biological system is left under constant environmental conditions, where oscillation of several physiological readouts persists with a period close to 24 hours, hence the term “circadian clocks”, circa=around dian=day. In plants the model species Arabidopsis thaliana has served as an invaluable tool for analysing the genetics, biochemical, developmental, and physiological effects of the oscillator. Many of these experimental results have been integrated in mechanistic and mathematical theories for the circadian oscillator. These models predict the timing of gene expression and protein presence in several genetic backgrounds and photoperiodic conditions. The aim of this work is the introduction of a correct mass scale for both the RNA transcript and protein variables of the clock models. The new mass scale is first introduced using published RNA data in absolute units, from qRT-PCR. This required reinterpreting several assumptions of an established clock model (P2011), resulting in an updated version named U2017. I evaluate the performance of the U2017 model in using data in absolute mass units, for the first time for this clock system. Introducing absolute units for the protein variables takes place by generating hypothetical protein data from the existing qRT-PCR data and comparing a data-driven model with western blot data from the literature. I explore the consequences of these predicted protein numbers for the model’s dynamics. The process required a meta-analysis of plant parameter values and genomic information, to interpret the biological relevance of the updated protein parameters. The predicted protein amounts justify, for example, the revised treatment of the Evening Complex in the U2017 model, compared to P2011. The difficulties of introducing absolute units for the protein components are discussed and components for experimental quantification are proposed. Validating the protein predictions required a new methodology for absolute quantification. The methodology is based on translational fusions with a luciferase reporter than has been little used in plants, NanoLUC. Firstly, the characterisation of NanoLUC as a new circadian reporter was explored using the clock gene BOA. The results show that this new system is a robust, sensitive and automatable approach for addressing quantitative biology questions. I selected five clock proteins CCA1, LHY, PRR7, TOC1 and LUX for absolute quantification using the new NanoLUC methodology. Functionality of translation fusions with NanoLUC was assessed by complementation experiments. The closest complementing line for each gene was selected to generate protein time series data. Absolute protein quantities were determined by generation of calibration curves using a recombinant NanoLUC standard. The developed methodology allows absolute quantification comparable to the calibrated qRT-PCR data. These experimental results test the predicted protein amounts and represent a technical resource to understand protein dynamics of Arabidopsis’ circadian oscillator quantitatively. The new experimental, meta-analysis and modelling results in absolute units allows future researchers to incorporate further, quantitative biochemical data.

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.

Shiftwork, particularly that involving night work and/or transmeridian flight, forces a mismatch between the sleep/wake cycle and the endogenous circadian timing system. Specifically, shiftworkers are often required to sleep at a phase in the circadian cycle when they would usually be active, and to work at a phase in the circadian cycle when they would usually be asleep. The current thesis describes a series of five studies designed to examine the disruption and adaptation associated with shiftwork, with an emphasis on night work and, to a lesser extent, transmeridian flight. The first study (Chapter 3), conducted in the field, was designed to examine the effects of break duration and time of break onset on the amount of sleep that shiftworkers obtain between consecutive work periods, and to consider the role that pineal production of melatonin may play in this process, through its regulation of sleep. Not surprisingly, total sleep time (TST) increased with break duration for breaks that began at similar times of day. Importantly though, TST was greater for breaks that occurred during the night-time than for breaks that occurred during the daytime. These results indicated that the minimum-length break requirements contained in prescriptive duty hours regulations might not necessarily protect shiftworkers from being exposed to unacceptable levels of fatigue. In addition, there was a temporal relationship between the circadian rhythms of sleep duration, sleep quality, and 6-sulphatoxymelatonin excretion, such that sleep was longer and of better quality when melatonin production was relatively high. This data did not prove a causal link, but it did provide further indication that melatonin may be involved in the regulation of sleep. The aim of the second study (Chapter 4) was to examine the effects of time of day, shift duration, and prior sleep length on self-assessed alertness and neurobehavioural performance of shiftworkers in a real work setting. Cosinor regression models fitted to the data indicated that time of day had a significant effect on alertness and performance, with both reaching nadirs in the early morning. Indeed, the cosinor regression lines of best fit explained more than 90% of the within-subjects variability in both the alertness and performance measures. In addition, alertness declined as shift duration increased and rose as prior sleep length increased, and there was a decline in performance across work periods that was greater for extended shifts. However, the results indicated that time of day was the most important determinant of subjective alertness and neurobehavioural performance. Consequently, the fatigue associated with night work can never be eliminated, only minimised through the application of risk management strategies. The aim of the third study (Chapter 5) was to quantify the effects of fatigue on performance in a simulated work environment, i.e. a rail simulator, and to compare them with the effects of alcohol intoxication. Reaction time (RT) performance on a visual psychomotor vigilance task (PVT) was also assessed. Rather than cause a general decline in performance as was hypothesised, fatigue impaired some safety and efficiency measures (i.e. number and duration of extreme speed violations increased, average speed reduced, brake use increased), but not others (i.e. fuel use, inter-train forces, and minor and moderate speed violations were unaffected). The reduction in safety and consequent increase in risk due to fatigue reached levels equivalent to those associated with moderate levels of alcohol intoxication (i.e. -05?-10%). The results indicated that fatigue caused participants to disengage from operating the simulator such that safety was traded off, not necessarily deliberately, against some aspects of efficiency. RT performance on the PVT was also significantly impaired by fatigue, similar to the magnitude of impairment associated with moderate levels of alcohol intoxication (i.e. -05?-10%). However, the PVT results could not predict the complex relationship between simulator safety and efficiency measures. This indicated that the effects of fatigue on performance in the workplace cannot necessarily be derived on the basis of simple performance measures such as RT. The fourth study (Chapter 6), conducted in the laboratory, was designed to assess adaptation to a simulated night work schedule using salivary dim light melatonin onset (DLMO) as the circadian phase marker. Participants worked seven consecutive simulated 8-hour night shifts (i.e. 23:00?07:00h). This resulted in a mean total phase delay in DLMO of 5.5 hours, equivalent to an average delay of 0.8 hours per day. In addition, baseline DLMO was significantly correlated with mean wake time over the previous seven days. These results indicated that partial circadian adaptation occurred in response to the simulated night work schedule, and that baseline DLMO was reliably predicted by the mean wake up time for the preceding week. The radioimmunoassay used proved to be a sensitive measure of melatonin concentration in saliva for the determination of DLMO, and thus provides an alternative phase marker to core body temperature. The last study (Chapter 7) was designed to examine the adaptation of a RAAF aircrew to several small time zone transitions using salivary melatonin onset as the marker of circadian phase. In addition, the effects of the aircrew?s work schedule on their sleep/wake patterns and subjective alertness were assessed. During the first six days of a routine surveillance patrol (SURPAT), the aircrew travelled eastward and melatonin onset occurred progressively earlier (i.e. phase advanced). During the second six days of the SURPAT, the aircrew travelled westward but melatonin onset did not significantly shift. Night-time sleep duration was shorter prior to work days than prior to rest days, and subjective alertness was not significantly affected by either the duration of night-time sleep prior to work, or the duration of flight. The melatonin onset results indicated that participants? body clocks adapted well to several small time zone transitions when initially travelling eastward, but did not adapt to a similar pattern of time zone transitions when subsequently travelling westward. This was contrary to expectations based on studies of single acute time zone transitions, which indicate that adaptation to westward flight is more rapid than adaptation to eastward flight. Taken together, the results of these five studies confirm that shiftwork provides a considerable source of disruption to shiftworkers? sleep/wake patterns. Whilst this disruption to shiftworkers? sleep may impair subjective alertness, the greatest influence on alertness and performance is exerted by time of day. Furthermore, the combined effects of sleep disruption and time of day may result in a level of performance impairment in a simulated work environment similar to that associated with moderate levels of alcohol intoxication. Finally, night work and transmeridian flight provide a source of circadian disruption, the adaptation to which can be assessed in both laboratory and field settings by examining changes in the timing of nocturnal melatonin onset. / thesis (PhD)--University of South Australia, 2001.

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

Thesis (M.S.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains xiii, 119 p. : ill. (some col.), col. maps. Includes abstract. Includes bibliographical references (p. 105-110).

Daily (circadian) rhythms exist at molecular, physiological, and behavioral levels and coordinate many life functions. This coordination is believed to contribute to an organism's fitness, however, such contributions have not been convincingly demonstrated in any animal. The most significant measure of fitness is the reproductive output of the individual and species. In this thesis I examine the consequences of loss of clock function on reproductive fitness in Drosophila melanogaster. I demonstrated that single mating among couples with mutated period (per���), timeless (tim���), cycle (cyc���), and Clock (Clk[superscript Jrk]) genes resulted in approximately 40% fewer progeny compared to wild-type flies. Male and female contribution to this phenotype was demonstrated by a decrease in reproductive capacity among per��� and tim��� flies mated with wild-type flies of the opposite sex. The important role of clock genes for reproductive fitness was confirmed by reversal of the low fertility phenotype in flies with rescued per or tim function. These results prompted an investigation to determine the relative contribution of each sex to the fertility phenotype. Males lacking a functional clock showed a significant decline in the quantity of sperm released from the testes to seminal vesicles (SV), suggesting that this peripheral oscillator is involved in sperm maturation. We found that clock genes are rhythmically expressed in these tissues and the cycling of per and tim expression continued in vitro, hence the testes and SV complex contained an autonomous circadian clock. In contrast to males, PER and TIM were constantly present in the cytoplasm of follicular cells in fly ovaries. Ovarian expression of per and tim is not disrupted by constant light and females lacking per and tim produced nearly 50% fewer mature oocytes then wild-type flies. These results suggest that per and tim are acting in a non-circadian pathway in the ovaries. Taken together, this data demonstrates that circadian clock genes significantly contribute to the fitness of Drosophila melanogaster by affecting the fecundity of both sexes. / Graduation date: 2003