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Circadian clocks enable organisms to anticipate predictable environmental changes over 24-hour day-night cycles on Earth to promote health and fitness. Since the discovery of the first clock gene in Drosophila melanogaster, Drosophila has been a model organism revealing the molecular underpinnings of animal circadian clocks. The first two chapters of this thesis will investigate the regulation of molecular clocks by posttranslational mechanisms. In particular, we will investigate the functions of core clock protein phosphorylation in Drosophila. Despite the importance of rhythmic gene expression programs produced by the molecular clock in generating daily biological rhythms, it has now been established that phosphorylation of core clock proteins that make up the molecular clock represent central and conserved timing mechanisms across organisms. Chapter One will investigate mechanisms by which phosphorylation regulates the major circadian transcriptional activator CLOCK (CLK) of rhythmic genes in D. melanogaster. CLK phosphorylation states exhibit daily rhythms and regulate its abundance, subcellular localization, and transcriptional activity. We characterized the role of casein kinase 1 alpha (CK1a) as a novel CLK kinase. We identified CK1a-dependent CLK phosphorylation sites using mass spectrometry proteomics. We next characterized the function of phosphorylation at Serine 13 (S13) residue. Our results showed that CLK(S13) phosphorylation reduces the binding activity of CLK to circadian promoters, therefore reducing CLK transcriptional activity. We also showed that CK1a-CLK interaction is dependent on PERIOD (PER), the circadian transcriptional repressor that is known to inhibit CLK function. We revealed a mechanism by which repressor-dependent phosphorylation of an activator inhibits its transcriptional activity and thus closing the transcriptional-translational feedback loop (TTFL) of the molecular clock. In Chapter Two, we focused on a comprehensive investigation of phosphorylation on TIMELESS (TIM), the heterodimeric partner of PER. We first identified phosphorylation sites on PER-bound TIM using mass spectrometry. We found that abolishing phosphorylation at some of these residues caused altered circadian behavioral rhythms. Phosphorylation at Serine 1404 (S1404) residue promotes TIM nuclear accumulation by reducing its interaction with exportin 1 (XPO1), a nuclear export machinery. Reduced nuclear localization of TIM in S1404A nonphosphorylatable fly mutants not only resulted in lower level of nuclear PER and TIM, but also showed dampened daily rhythms in CLK phosphorylation. This is likely caused by reduced recruitment of CLK kinase by PER-TIM complex. Taking Chapter One and Two together, we provide mechanistic insights into CLK and TIM phosphorylation and how these posttranslational modifications are indispensable for the maintenance of 24-hour molecular oscillation to regulate circadian rhythms. Chapter Three will review our current understanding of the function of Drosophila TIM (dTIM) and mammalian TIM (mTIM). dTIM is a cardinal clock protein, whereas the role of mTIM in regulating circadian rhythms are under debate since it was first identified over two decades ago. We will summarize the circadian and non-circadian roles of the two TIM paralogs and discuss the potential mechanisms by which mTIM regulates circadian rhythms via its non-circadian roles. We will conclude Chapter Three by summarizing recent findings about potential functional parallel between mTIM and dTIM. Overall, the research included in this thesis will provide mechanistic insights into the regulation of animal circadian clocks and contribute to future development of therapeutics for clock-related human diseases including cancer.

The Architect's first drawn line marks a significant moment where alteration to the site is conceived and intervention with nature is beset. Equilibrium of the natural order; vegetative, habitat, hydrology, and geology are all in a vulnerable state. Rarely do these develop into harmonious balances. More often they are imposed instances. The Industrial Revolution forever changed the relationship between humans and nature, tilting the weight of power towards man. While humans capacity for innovation and destruction have grown enormously, our dependence on the natural cycles and resources of the planet remain and grow more voracious. Yet simultaneously, modern progress has facilitated the physical and psychological detachment of that interdependence. The fundamental elements of our existence are veiled through the efficiency of urbanization and its derivatives of specialization, mass-production, and globalization. This project is an examination of the interrelationship between humans and nature through the lens of civic architecture within a naturalistic setting. The fundamental thesis of this project is that there is a primal biological thread that connects human beings to the natural order, whether on a visceral or conscious level. This project explores the belief that humans intrinsically yearn to reinforce that bond - awakening primordial instincts developed over millions of years of evolutionary survival that have been suppressed by the artifice of modern life. Through a process of retreat and contemplation, this project offers the opportunity of individuals to evaluate and rebalance their own scales with nature and find their own accord and harmony.

The northern house mosquito, Culex pipiens L., is a vector of several diseases including West Nile virus, St. Louis encephalitis, and filariasis. Adult females of this species enter an overwintering dormancy known as diapause. Like most temperate insects, diapause in Cx. pipiens is programmed by the short daylengths of late summer and early fall although the molecular mechanism by which mosquitoes and other insects measure day length is unknown. Like other insects, Cx. pipiens, have circadian clocks which are composed of several genes whose mRNA transcripts and proteins oscillate throughout the day and, in doing so, regulate daily behaviors. In this thesis, I explore the possibility that circadian clock genes, which provide Cx. pipiens with information on the time of day, are also involved in measuring day length and hence initiating the adult overwintering diapause in this species.The clock genes in Cx. pipiens are highly similar to the circadian clock genes in the other mosquitoes and more distantly related insects. This suggests that the circadian clock in Cx. pipiens is similar in structure to other, non-drosophilid insects. To determine whether clock genes are differentially expressed during diapause, their expression was measured over a 24-hr period in nondiapausing mosquitoes as well as in females of Cx. pipiens throughout the diapause program and after diapause termination. I found that clock genes continue to cycle robustly throughout the diapause program, although early in diapause the expression of several core circadian clock genes show an altered expression profile with peak mRNA levels occurring later in the night than anticipated. This shift in clock gene expression may explain the shift in flight and feeding activity that has been previously observed in diapausing females of Cx. pipiens early in diapause. Using RNA interference (RNAi), the expression of several circadian clock genes was suppressed and the ability of females of Cx. pipiens to enter diapause was assessed. RNAi against negative circadian regulators, the genes period, timeless and cryptochrome2, caused females that were reared under short day, diapause-inducing conditions to avert diapause. In contrast, RNAi against the circadian-associated gene, pigment dispersing factor, caused females that were reared under long day, diapause-averting conditions to enter a diapause-like state. Together, these results suggest that clock genes control diapause initiation and perhaps diapause maintenance in Cx. pipiens.Additionally, the location of the circadian clock in the brains of Cx. pipiens was investigated by using immunohistochemistry with a primary antibody for the clock protein TIMELESS (TIM) derived from Drosophila melanogaster. TIM positive cells were found in the region between the optic lobes and the central brain, the subesophageal ganglion, and the pars intercerebralis (PI). Previous studies have demonstrated that the PI regulates the adult diapauses of other insects. Therefore TIM staining in the PI is the first step in establishing a physical connection between the circadian clock and the regions of the mosquito brains that control diapause in Cx. pipiens. Taken together, these experiments implicate circadian clock genes in the initiation and regulation of overwintering diapause in this important disease vector.