The development of new medications is critical for treating disease and extending life. Approximately 40-60% of drugs target a specific type of membrane protein termed G-Protein Coupled Receptors (GPCRs), which permit cells to communicate with each other (by binding neurotransmitters) and regulate body physiology. The Hague lab studies the molecular mechanism of drugs targeting these receptors. Specifically, they examine a small, yet important structural portion of the receptor - the PDZ binding motif - which acts a protein-protein interaction site. Interestingly, of the ~800 different GPCRs in the body, 30 have this PDZ binding motif, including those activated by the neurotransmitter 5-hydroxytryptamine (5-HT), which is commonly known as serotonin. Serotonin regulates many key CNS processes including mood, sleep, and appetite. Current 5-HT medications that are on the market are used to treat depression, generalized anxiety disorder, and social phobia. Two common antidepressants that affect serotonin GPCRs are Selective Serotonin Reuptake Inhibitors, or SSRIs, and Monoamine Oxidase Inhibitors, or MAOIs. My goal was to examine the necessity of the PDZ binding motif for 5-HT serotonin receptor function in human cells. To do this, HEK293 cells expressing the wild-type 5HT receptor (WT) or mutated 5-HT receptor missing the PDZ binding motif (ΔPDZ) were subjected to diagnostic assays including: microscopy, protein biochemical analysis, proteomics, and cellular signaling. In summary, the data suggest the PDZ-binding motif is not essential for 5-HT serotonin receptor function in HEK293 cells, although further experiments are necessary to solidify this conclusion.

Because a protein’s three-dimensional structure defines its function, improved methods for resolving structure are an important objective in molecular biology. For example, the structures of many pharmaceutically relevant proteins are difficult to characterize with current experimental approaches. Computational techniques that predict structures from amino acid sequences obviate problematic physical manipulation of proteins, but are unreliable. Computational prediction improves, however, when supplemented with limited structural data. We propose generating data that describes spatial constraints with deep mutational scanning, a method we developed to measure the functional consequences of hundreds of thousands of variants simultaneously. I will use large-scale mutagenesis to create single- and double-mutant variants of two essential yeast proteins, cdc42 and guk1. These constructs will be transformed into Tet-Off yeast, in which the endogenous cdc42 or guk1 promoter is replaced with a repressible tet promoter. I will then employ high-throughput DNA sequencing to track variant frequencies before and after competitive growth in doxycycline. Stable, functioning variants should rescue growth and increase in frequency while deleterious variants will decrease in frequency. From these frequencies, I will derive functional scores. We hypothesize that functional scores given by two single mutations will predict the functional score of those mutations combined in a double mutant; double mutants with unexpectedly high or low scores would suggest interaction between the mutated positions. Interacting pairs associate amino acids in space, revealing spatial constraints that may enhance computational approaches for determining otherwise intractable three-dimensional protein structures.

The North Creek Wetlands restoration on the University of Washington Bothell campus harbors a seasonally variable roost of American Crows (Corvus brachyrhynchos), of between 2,000 and 16,000 crows. I investigated the seasonal changes of crows’ movements within and around the North Creek Wetlands roost by making observations of their evening arrival times, numbers, and directions of origin. I investigated seasonal fluctuations in area of the roost by mapping the perimeter on foot; the nocturnal activities of crows in this roost by placing trail cameras; and the distances traveled daily by crows commuting to this roost by making field observations of their flight vectors at varying distances and directions from the roost. The nightly times of arrival of crows coming to their nearby pre-roost (R² = 0.955), their move from pre-roost to roost (R² = 0.992), and the time that >99% of crows are in the roost (R² = 0.9942) are strongly correlated with photoperiod. The roost area changes both seasonally and annually, averaging approximately 45,330m² in Winter of 2013, 127,880m² in Fall 2013, and 65,960m² in Winter of 2014. Population density also varies seasonally. Field cameras show little nocturnal movement of crows after arrival in the tree tops, and never show birds moving to the understory, shrub layer, or forest floor. While flight paths to and from the roost are highly variable in direction, observations suggest that some crows are commuting to this roost nightly from at least 30 kilometers, and possibly greater than 40 kilometers away. This study may help us to understand why the crows have chosen this, or other, wetland roost sites, and may be useful in crow management decisions and the planning of future restoration sites.

As twilight falls on the University of Washington Bothell campus, the skies turn black with the arrival of 10-15,000 crows (Corvus brachyrhynchos) that use the restored North Creek Wetlands as their winter nocturnal roost.  The crows leave large amounts of nitrogenous waste under their roost, and the impact this has on the biodiversity of flora in the wetlands is unknown.  Using simple random sampling, we surveyed flora composition and collected soil samples at sites on- and off-roost across seasons (spring 2013, fall 2013, and winter 2014).  We analyzed soil from each site for moisture, pH, and levels of nitrates.  Across all seasons, significantly higher levels of nitrates and lower pH levels were found on the roost compared to non-roost locations; pH levels varied both with season and by location, but there was no significant interaction effect. Plant community composition was not significantly different on and off the roost, nor did it vary significantly across seasons.  Although the crows are fundamentally changing the chemical composition of the soil under their roost, they are not yet affecting plant biodiversity or abundance.  Birds, however, are vectors for invasive plant species, and these variables are predicted to change over the long term, which will have important management implications.  The North Creek Wetlands are an exemplar of ecological restoration, surrounded by rapidly-developing urban centers, so any future impacts the crows have on these wetlands could cause larger ripple effects over the remaining fragmented forests, streams, and wetlands of the Lake Washington watershed.

Testosterone plays a major role in aggressive and territorial behavior in many male vertebrates during the breeding season. Song sparrows, Melospiza melodia morphna, are not only aggressive in the breeding season but also in the non-breeding period, when their testes are regressed and plasma testosterone levels are low. Previous studies suggest that this territorial aggression is mediated in part by high levels of an androgen and estrogen precursor, dehydroepiandrosterone (DHEA), produced by the adrenals. In this study, we aim to determine whether DHEA exerts these effects via an androgen or estrogen receptor mediated mechanism, or both. Male song sparrows were captured, kept on a short day cycle (8L: 16D) typical of the non-breeding season, and implanted subcutaneously with either empty or DHEA-filled silastic implants for fourteen days. DHEA implanted birds had significantly higher circulating DHEA and testosterone levels and showed increased aggression in a laboratory-based aggression test. Brains were extracted and in situ hybridization was used to label androgen receptor (AR) and aromatase mRNA. Aromatase is the enzyme that catalyzes the transformation of androgens to estrogens. Using dark field microscopy, AR and aromatase mRNA expression in brain regions previously associated with aggressive and reproductive behaviors were quantified. In the ventromedial nucleus of hypothalamus (VMH), AR expression was significantly greater in DHEA birds compared to controls. However, in the preoptic area (POA), aromatase mRNA was significantly higher in DHEA birds over controls. In the medial preoptic area (mPOA), neither AR nor aromatase expression differed between DHEA and control birds. We conclude that DHEA may cause non-breeding aggression via both androgen and estrogen receptor mediated mechanisms. Deeper understanding of how exogenous DHEA affects the brain and behavior of song sparrows may provide useful insights for biomedical researchers interested in investigating the health benefits and side effects of DHEA supplements.

Graphite Oxide combined with selective laser scribing were used to create laser scribed graphene based electrochemical electrodes. Combining multiple electrodes with varying types of gelatinous and aqueous electrolyte a highly conductive, flexible, and robust capacitor was produced. The primary goal of this research was to discover an optimal combination between variables including graphite oxide density, substrate type, laser scribing patterns, and electrolyte porperties. A range of possible combinations were selected to be tested based on prior resaerch and their probability to produce an increase in energy density. To insure consistency, isolation of a single variable insured each capacitor was constructed with the same methods and regiment. In order to isolate a single variable and maintain uniformity within the test capacitor, a single sheet of laser scribed graphene electrodes were used to construct capacitors of a similar size and shape while simultaneousy adjusting a single parameter at any one time. Initial research was used to select starting values for variables, which were then individually adjusted, with a new capacitor constructed for each test run. Further research with seek to discover the feasibility of maintaining the combinations discovered through current work.