The structure of plastid DNA (ptDNA) provides insight concerning its replication, repair, maintenance, and stability. We are studying DNA molecules and their association with proteins in plastid nucleoids as maize seedlings develop from the newest cells in the basal meristem to the oldest in the leaf tip. When observing unbound ptDNA from light-grown seedlings, we have found that ptDNA copy number and molecular integrity decline from complex-branched forms (Type I) in the meristem, to a mixture of smaller complex and simple forms (Type II) in the stalk, and small simple forms (Type III) in the leaf blade. In contrast, preliminary examination of unbound stalk ptDNA showed that for the dark-grown seedlings ptDNA is maintained at high copy numbers, with large complex forms. Furthermore, DNA fibers connected to these complexes were longer in dark-grown than light-grown stalk. We will now continue to examine unbound ptDNA in the meristem and leaf blade from dark-grown seedlings. Once finished examining all samples of unbound dark-grown ptDNA, we will analyze nucleoid-associated ptDNA and deproteinized ptDNA of dark- and light-grown seedlings by gel electrophoresis and fluorescence microscopy. For the basal meristem of light- and dark-grown seedlings, in nucleoid-associated ptDNA, we expect similar high-integrity Type I ptDNA, since this is where replication is highest. In contrast, we expect major structural differences in the nucleoid-associated leaf blade ptDNA, with small Type III in light-grown and high-integrity complex Type I forms in dark-grown plants. We propose that replication and repair occurs exclusively on nucleoid-associated ptDNA and that unbound ptDNA is susceptible to nuclease degradation. Therefore, we expect complex forms for nucleoid-associated ptDNA and small, degraded molecules for the non-nucleoid fraction. On a larger scale, this research will be important for genetically modifying maize tissue, because we will know more about the structural integrity of the DNA we are targeting.

Giardiasis is a disease caused by the protozoan parasite Giardia lamblia which affects approximately 20,000 people per year in the United States alone. To this day, there are few anti-Giardiasis drugs available so new therapeutics to address toxicity and resistance issues are needed. In previous studies, we have examined seven protein kinases in Giardia’s genome and these kinases share an unusual structural feature in their active site, which results from an atypically small gatekeeper residue (amino acid residue). This feature confers sensitivity to a class of compounds called bumped kinase inhibitors (BKIs); BKIs demonstrated minimal toxicity and positive pharmacological properties. We repeated the search for small gatekeeper kinases using an updated multisequence alignment program, which identified four new small gatekeeper kinases. We developed a cloning strategy to 3HA tag each of these kinases. Then, we evaluated the expression and cellular localization of the proteins using Western blot and immunofluorescence (IF) assays. With the tagged kinase lines, we utilized translation blocking morpholinos to test the importance of each kinase using cell growth. This data will be used to prioritize protein purification for screening against a BKI library using thermal shift assays. These results may identify lead compounds toward novel treatment options for giardiasis.

Parkinson’s Disease (PD) is an age-related neurodegenerative disorder that leads to progressive decline in motor control. Tens of thousands of new cases are diagnosed each year. Many cases of PD are linked to chronic exposure to rotenone, a commonly used pesticide. As an inhibitor of complex I in the mitochondrial electron transport chain, rotenone causes deterioration of dopamine-producing neurons in the substantia nigra (part of the midbrain). Studies suggest that there is a genetic component to how likely one is to develop symptoms in response to rotenone exposure. To test this hypothesis, we used the Drosophila Genetic Reference Panel (DGRP), a set of 192 fully sequenced inbred lines of the fruit fly, Drosophila melanogaster. Exposure of Drosophila to rotenone has been shown to cause dopaminergic-specific degeneration, similar to the pathology observed in PD patients. When fed highly concentrated rotenone-infused food, we see significant variation in rates of survival among female flies from different lines of the DGRP, allowing us to capture natural variation in response to rotenone exposure. After most of the DGRP has been tested, we will carry out a genome-wide association study to identify specific genetic variants associated with rotenone resistance. In this way, we hope to uncover genetic factors that could point to specific mechanisms behind PD pathogenesis. With further related research, new genetic pathways could lead to new drug targets.

Traumatic brain injury (TBI) is an illness that affects people in all walks of life, from everyday citizens to soldiers in combat, and both young and old. Among all cases of TBI, there are some patients who recover quickly and some patients who take a long time to see any improvements, suggesting a range of what we call “TBI resistance”. To see if we could identify a genetic basis to this variation, we used the fruit fly, Drosophila melanogaster, as a model of TBI resistance. Specifically, we used a set of fly strains captured in Raleigh, North Carolina, and commonly known as the Drosophila Genome Reference Panel (DGRP). The DGRP is a sequenced set of inbred flies that researchers around the world experiment on, thus establishing a universal platform to identify genes associated with natural variation for a wide range of fly traits. For our study, we created a spring device to model TBI in flies. The device consists of vial of flies connected to a high resistant spring which propels it towards a hard pad causing damage. We compared the mortality rates between the different lines of flies. We hypothesized that we would see some lines display resistance and some lines display sensitivity. This turned out to be true, and we have already identified candidate genes associated with this variation. As a next step, to study TBI resistance on a cellular and molecular level, we will perform metabolomics with both resistant and sensitive lines to see if there is a significant difference between metabolite expression. The expression of certain metabolites in resistant flies, compared to sensitive flies, could hold the key to understanding how resistance is acquired against TBI and we will be one step closer towards understanding ways to protect people from the effects of TBI.

Research suggests that weathering by early cryptogams provided an overall negative feedback on atmospheric CO2 and an overall positive feedback on biologically available inorganic nutrients during the Ordovician. This process is considered to have contributed to global cooling and to the formation of Earth’s first soils. Although there is no known analogue to Earth’s first land plants, mosses possess the most shared spore and sporophyte features with the early cryptophyte fossil, Partitatheca, when compared to other bryophytes. They therefore present a plausible model organism for examining how the first land plants irreversibly altered Earth's geochemistry. However, before Earth's first land plants, lichens are known to have been colonizing the Earth’s surface since the Devonian. The role of lichens in this early Earth process is even more uncertain and in situ studies of lichen weathering have yet to be attempted. Here, I propose to extend the scope of a previous microcosm study of biologically induced weathering to investigate an additional lithic moss species and a lichen species, and to determine if an interaction exists between the groups. This study will enhance our understanding of the rate at which lichens and different lithic moss species weather bare rock and will provide insights into how mutualistic relationships could affect weathering in areas of primary succession.

Gene regulation lessens the cost of cellular operations by altering physiological processes in an as-needed manner depending on resource availability. Previously, it was shown that repeated ecologically-relevant shifts in combination with conditional gene regulation can lead to a population collapse phenomenon. In this prior work, wild-type and regulatory mutant strains of Desulfovibrio vulgaris Hildenborough (DvH) were grown in co-cultures together with wild-type Methanococcus maripaludis in an environment where they were continuously transitioned between sulfate respiration and syntrophy conditions. While the wild-type DvH co-cultures collapsed after few transitions in this environment, the regulatory mutant persisted and never collapsed. This work showed that conditional gene regulation can drive a microbial community to collapse when the environment fluctuates too rapidly. In addition, it provided us with an experimental framework for probing the impact of gene regulation in adaptation of microbial communities to rapidly fluctuating environments. In this work, we further combined the experimental framework of alternating transitions together with transposon mutant libraries to identify active gene regulatory networks. The random barcode transposon-site sequencing (Rb-TnSeq) DvH library was rapidly transitioned between high and low sulfate concentrations and samples were collected for barcode sequencing to determine transcription factors that were enriched over the course of transitions. We will present fitness data and a list of enriched regulatory genes to shed light on how the regulatory mutations impact the organism’s ability for adaptation to fluctuating environments.

Plants’ ability to sense ambient light conditions is critical for their fitness. Phytochromes (phy) are photoreceptors that translocate to the nucleus when activated by red light. One consequence of this translocation is rapid phosphorylation and turnover of basic helix-loop-helix (bHLH) transcription factors called Phytochrome-interacting factors (PIF). PhyB has been shown to recruit and inactivate PIF5 in seedlings and inhibit elongation of the stem (hypocotyl). Phytochrome mutants in Arabidopsis thaliana exhibit reduced CO₂ uptake, abnormal accumulation of sugars and metabolites during the day, and shade avoidance-like phenotypes. We aim to compare findings in A. thaliana with phenotypes of light signaling mutants in Brassica rapa, a related crop species. We have isolated phyB, and pif5 mutants and are growing them in ambient and high CO₂ conditions. Measurements of photosynthetic rate as well as levels of chlorophyll, sugars, total protein and starch will provide insights into the role of Phytochrome signaling on metabolic processes. Preliminary results show that phyB mutant seedlings exhibit longer hypocotyls in comparison to wild-type plants at ambient CO₂ but have a reduced growth response to elevated CO₂ compared to wild-type. Adult plants have a reduced photosynthetic rate compared to wild-type. In contrast, pif5 mutant seedlings have short stems and reduced root growth in comparison to wild type. Our results to date are consistent with previous studies in A. thaliana. Examining the role phytochromes play in resource allocation, specifically in response to Carbon availability will aid in identifying key phytochrome-mediated developmental pathways. This research has implications in engineering more efficient crops and in improving understanding of plant metabolism as a whole.

Predator-prey models provide invaluable insight into interspecies relationships and fluctuations in population sizes, but like all models, must rely on simplifying assumptions that reduce complex natural processes to functions with explanatory and predictive utility. Logistic equations for population growth predict future population size as a function of current population size and competition for limited resources; the related Lotka-Volterra equations use similar assumptions but with predator-prey interactions limiting population growth. In North America, the Canada Lynx-Snowshoe Hare cycle is an ideal demonstration of interconnected predator-prey population sizes. When vegetation is plentiful, hares have the resources to bear many litters of offspring and they provide a high level of nourishment to lynx. Lynx and hare populations increase in number until vegetation becomes scarce and predation by lynx reduces the size of the hare population. At this point, hares are too scarce to sustain lynx population growth and lynx numbers decrease while plants repopulate the region. Lotka-Volterra equations capture this cyclical relationship but assume genetic uniformity between individuals and no age structure in the population. Extensions of predator-prey models have incorporated age structure but not genetic variation. My model examines the dynamics of this system when both aging and genetic variation for fitness are incorporated into predator-prey dynamics. Using the statistical programming language R, I present a simulation of predator-prey dynamics with these two additional parameters—variation in relative fitness among individuals and age structure. Analysis of “the hare that never got caught” is an inquiry into the influence of age structure on fitness. More broadly speaking, this work attempts to model the effect of aging on the efficiency of natural selection and provides a basis for future research regarding the role of aging in evolutionary biology.

Alzheimer’s Disease (AD), the most common form of dementia, is the sixth leading cause of death in the United States. With a prevalence of over five million cases, the number of AD patients is expected to increase as a new diagnosis of AD is made every sixty-six seconds. Early symptoms include short-term memory loss and gradually progress to devastating deficits in cognition, temperament, and behavior. AD is a neurodegenerative disease linked to the accumulation and aggregation of two proteins, Aβ_1-42 and hyperphosphorylated Tau, which form the characteristic amyloid plaques and neurofibrillary tangles found in the brains of AD patients. Previous studies support the notion that genetic variation underlies the wide array of phenotypes observed among AD patients, and several genes have been discovered to cause early onset AD. In sporadic (late onset) AD patients, it remains unknown what effects natural variation may have on the expression and severity of the AD phenotype. To explore the influence of genes on AD, our study utilizes a Drosophila model of AD that allows us to express both the Aβ_1-42 and Tau peptides in the fly eye. Expression of AD-related proteins in the eye leads to degradation of the hundreds of ommatidia that make up the fly eye. To quantify the extent of ommatidial degradation, we use a computer program, ImageJ, which measures the size and circularity of each ommatidium. To introduce natural variation, the AD fly is crossed with flies from the Drosophila Genetic Reference Panel (DGRP), consisting of 192 fully sequenced isogenic lines. By comparing the Aβ_1-42 and Tau-induced degradation in each background, we can identify naturally occurring modifiers of the AD phenotype. We have already identified promising hits, which could eventually help in the development of novel targeted therapies.

Most organisms show a roughly 24-hour cycle in their physiological and behavioral processes, called circadian rhythms. Circadian rhythms are generated endogenously through the ~24-hour cyclic expression of genes known as clock genes. The expression of these genes oscillates in the master circadian clock of mammals – the suprachiasmatic nucleus (SCN) - but also in nearly every cell of the body. Typically, circadian clocks and the rhythms they sustain are ‘entrained’ by the 24-hour light-dark (LD) cycle. Our lab has found that fear can also act as an entraining factor; we observed that mice and rats subjected to random foot shocks during the dark phase of the LD cycle in their cages’ foraging area changed from their typically nocturnal foraging pattern to a diurnal one, avoiding the fear stimulus presented during the dark. The goal of my project is to understand the neural circuits and molecular processes that are involved in fear entrainment. I have analyzed the expression of clock genes in animals that are exposed to nighttime fear or in control animals exposed to daytime fear, using a technique called in situ hybridization, which labels mRNA levels. This allowed me to assess the circadian rhythm of expression of clock genes of interest (Per1 and Bmal1) in the SCN and the amygdala, the region of the mammalian brain that encodes fear stimuli. Our findings could not only help elucidate the circuits that are involved in fear entrainment but also help understand the mechanisms that link fear and anxiety disorders to problems with circadian rhythms and sleep.

Dozens of sculpin species inhabit the Salish Sea, and their heads range from short and squat to long and sleek to lumpy and oblong. Our study focused on jaw gearing, or the mechanical relationship between the movement of muscles and jaws, in five of these species. We measured 37 anatomic and 5 kinetic variables describing form and function in the jaws, and we phylogenetically corrected our data to track how jaws evolve. Anatomic gearing was measured in dissected specimens, and kinetic gearing in live feeding fish. Both lines of data revealed that evolutionary shifts to higher gear ratios correlated with shifts to shorter muscles. This coevolution of gearing and muscle length allows diverse jaw structures to maintain similar muscle strain magnitudes and preserve high feeding performance. This intraspecific, coevolutionary relationship may help explain how dozens of sculpin species are able to maintain distinct head morphologies and coexist in the Salish Sea.

Climate change is forecasted to have an impact in agricultural productivity. To ensure security of crop yields, we must develop a better understanding of flowering mechanisms in plants. Research on flowering mechanism has been performed mostly under simplified lab growing conditions. However, in the natural environment, plants experience dynamic changes in abiotic factors like temperature throughout the day. To better understand how flowering is regulated under natural conditions, we focused on the expression profiles of FLOWERING LOCUS T (FT), which correlates with the flowering time in plants. FT expression is known to peak in the evening under long day lab conditions (16 hours of light, 8 hours of darkness, 22^oC constant). However, under natural long day conditions (Summer Solstice in Seattle, 16 hours of light, 8 hours of darkness, average highest temperature 21^oC) we have observed an additional morning peak. We have been able to recreate the observed expression of FT in the lab by modulating temperature oscillation and light quality of long day lab conditions. In this project, we first asked if the observed FT pattern is widely conserved in different wild type accessions in Arabidopsis thaliana. We grew different accessions under re-created lab growth conditions and analyzed FT expression using quantitative PCR. So far we have seen similar FT expression patterns among some accessions, suggesting that morning peak of FT is conserved among these lines. Second, we studied which timing of the day temperature acts as a cue to affect morning FT expression. We grew plants under various temperature conditions and compared results to the observed FT expression in nature. The results from this project will contribute to the development of new modified lab growth conditions that represent natural conditions and give a better understanding of the mechanism that controls FT regulation in nature.

We know that climate sensitivity can vary within species, but the factors that influence which climatic variables influence population dynamics remain unclear. In this study, I assess whether macroclimate influences the climate sensitivity of four high elevation conifer species: Pacific silver fir (Abies amabilis), mountain hemlock (Tsuga mertensiana), Alaska yellow cedar (Callitropsis nootkatensis), and sub-alpine fir (Abies lasiocarpa). I also investigate if climate sensitivity of tree communities Mt. Rainier is changing over time. To address these questions, I analyzed tree core data from three high elevation sites at Mt. Rainier National Park. Each site is located in a distinctly different climatic zone on the mountain. I compared standardized tree growth to climate variables (mean growing-season temperature, total growing-season precipitation, annual snowpack, climate moisture deficit, etc.) using linear mixed effect models and “treeclim”, a tree ring analysis program in R. With these methods, I determine how influential climate variables differ by location, by species and over time. Gaining more insight into which climate variables have the greatest influence on growth in different areas will allow us to better predict how specific plant populations will react to ongoing climate change. Moreover, studying tree communities at high elevations is particularly important, since behavior at treeline will determine whether a species’ range expands, contracts or shifts, which in turn has implications for both that species and other species. For example, conifer range expansion on Mt. Rainier could positively increase carbon sequestration (by increasing woody cover in the park) while negatively encroaching on the alpine wildflower meadows, a major attraction for tourists. Widespread range contractions could potentially reduce the ability of those forests to sequester carbon from the atmosphere. Thus, with better predictions of tree shifts, we can make more informed management decisions for ecotourism and conservation.

Alternative pollinators such as the mason bee (Osmia spp.) are becoming increasingly important in agriculture as European honey bees (Apis mellifera L.) continue to decline. Osmia lignaria are especially important pollinators because they make contact with the anther and stigma during nearly every flower visit and are less likely to rob the plants of nectar without pollination. Previous studies indicate O. lignaria preferentially visit plants in the Rosaceae family, such as apple, cherry, pear and plum. The objectives of this study were (1) to analyze pollen loads in relation to proximity of natural areas surrounding the blueberry field and (2) to determine their pollen preference in a blueberry monoculture ecosystem. This study was conducted in a commercial blueberry field in southwestern Michigan. Pollen samples from flowering plants and O. lignaria nests were collected. Acetolysis was performed on all pollen samples, and pollen from O. lignaria nests was qualitatively and quantitatively analyzed. At this study site, O. lignaria collected pollen from white clover (Trifolium repens) and black cherry (Prunus serotine) most often. In spite of its availability, pollen collected from blueberries (Vaccinium corymbosum) accounted for significantly less of the total pollen. These findings are useful in determining the viability of Osmia species as crop pollinators. Based on the results, Osmia lignaria would not be a good option for blueberry pollination.

With increasing temperatures and altered precipitation patterns over the past several decades, montane environments are undergoing rapid anthropogenic changes. Climate controlled treelines are moving up in elevation worldwide, with dramatic consequences for alpine ecosystems, however little is known about the local drivers of variation that influence the rate of tree line change, like small-scale topography. We conducted a repeat photography study to assess the rate of tree encroachment into the subalpine meadows of Mt. Rainier National Park in various local landforms (coves, ridges, and slopes) as well as quantify the rate of treeline change over the 20th and early 21st century. We did this by aligning historical and repeat oblique photographs in a Geographic Information System and manually delineating tree encroachment within the subalpine zone. We also verified our results by comparing them to per-pixel classifications of aerial imagery collected in 1951 and 2013. Tree encroachment has occurred in all sites on Mt. Rainier National Park over the last century. Oblique photographs showed an approximately thirty percent increase in tree cover from the 1930s to 2016. Local topography did have a significant influence on the amount of tree encroachment. Small-scale topographic ridges were predicted to show the greatest amount of tree encroachment. Our results contradict this prediction, showing that sloped sites have the greatest increase in trees, followed by ridges, then topographic depressions (coves). Analyses of aerial photos are ongoing, but are expected to show  similar results. Per-pixel classifications or aerial images will help us more easily distinguish changes in the various environments at Mt. Rainier, as well as correlate them to environmental drivers like snow duration. This study helps fill an important gap in knowledge about how topography is related to tree line change as well as raises further questions about what management practices can be implemented.

Zinc is essential for the regulation of many biological processes in all known species. However aquatic species, especially invertebrates, are relatively sensitive to zinc toxicity compared to most terrestrial organisms. As oceans warm due to climate change, zinc previously trapped in ocean sediment will be released into the water column, exposing sensitive benthic and coastal species to elevated, potentially harmful zinc levels. Our study focuses on the impacts of elevated zinc on the calcium uptake of the pacific oyster. Previous research has shown that zinc can mimic other ions, disrupting calcium uptake in the gills. This is thought to be due to their similar size and charge, and could be especially harmful to shellfish and other organisms which use calcium to form their calcium carbonate (CaCO₃) shell. In our study, sixty-four oysters were randomly distributed into four tanks of varying elevated zinc concentrations. Following a 72 hour exposure period, the oysters were dissected and the gills were analyzed using Flame Atomic Absorbance Spectroscopy (FAAS) for calcium concentrations. The uptake of calcium through the gills of the oyster is expected to decrease as the concentration of zinc exposure increases. These results contribute to the growing body of knowledge concerning the interplay between zinc and calcium ions in aquatic organisms. Additional research is needed to characterize this effect on a wider variety of marine organisms, especially those in areas with significant run-off from urban or industrial localities.

Spontaneous electrical activity in the mouse neocortex has been extensively studied. Two main types of activity are observed: synchronous waves and asynchronous single cell activity. This synchronous activity has been believed to be unique to the developing brain. This research project investigated whether the phenomenon is as much a property of sleep as it is development. Synchronous waves exhibit remarkably similar dynamics to the up-down oscillations of slow wave sleep. Conversely, asynchronous single cell activity is very similar in form to the "persistent up" or wakeful brain state. Thus, the two types of spontaneous activity generally observed in the mouse neocortex may actually be a sign of overall brain state, with asynchronous single cell activity dominating during states of wakefulness and synchronous waves taking over during sleep states. Calcium imaging of slices of neonatal mouse cortex has revealed that application of curare, an acetylcholine antagonist, decreases asynchronous single cell activity and induces synchronous waves. As blocking acetylcholine puts the brain in a state associated with sleep, we hypothesize that the neurotransmitter may be involved in keeping the brain awake. Further experiments were conducted on the effect of curare on synchronous waves. According to our hypothesis, it should increase their frequency and/or propagation. The same experiments were performed with other acetylcholine antagonists, such as mecamylamine, to ensure the observed effect is not unique to curare as a drug. We also conducted experiments with acetylcholine agonists as well to see if they have the opposite effect. These synchronous waves are known to be involved in several developmental neural processes, and this research project may reveal they are a property of sleep, and could serve as precursors to adult slow-wave sleep.

Increasing atmospheric CO2 has lead to increased uptake of CO2 by oceans causing ocean acidification. Ocean acidification is the long-term reduction of pH that can have negative effects on marine organisms, especially young calcifying organisms. In Puget Sound, ocean acidification has already caused large-scale oyster larval mortality, negatively affecting a significant contributor to the Pacific Northwest economy. The native eelgrass in Puget Sound, Zostera marina, may be a local mitigation strategy for ocean acidification. Marine plants take up CO2 through photosynthesis to create biomass increasing the pH of the water. In this study, water samples were collected from several depths within the water column above eelgrass beds and surrounding mudflats in Padilla Bay. Water samples were analyzed for pH, total alkalinity, dissolved oxygen, dissolved inorganic carbon, and chlorophyll A. Eelgrass shoots and density were measured for leaf area index and the Padilla Bay National Estuary Research Reserve weather stations were used to calculate solar irradiance to estimate the photosynthetic activity of the eelgrass beds. Results suggest that eelgrass beds, in addition to providing critical habitat, may also help locally mitigate ocean acidification beyond the area of the eelgrass beds. Future research analyzing the input and output of carbon in eelgrass beds will be critical to developing large-scale carbon and pH models in marine environments.

Persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs), dichlorodiphenyl trichloreothane (DDTs), polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecanes (HBCDs) are toxic nonpolar molecules that can cause damage to the reproductive, developmental, behavioral, and endocrine systems. These pollutants accumulate within the fatty tissues of important species in Puget Sound, including orca whales, salmon, herring, and plankton. This study measured the concentrations of two banned POPs, PCBs and PBDEs, and one currently used POP, HBCD, in plankton communities in Possession Sound. POP concentration in the water was measured in situ using a Chemcatcher deployment device to passively sample water at five locations across Possession Sound. Gas chromatography-mass spectrometry (GC-MS) was used to identify and quantify the POPs collected on the Chemcatcher filters and in the plankton samples. This two-pronged approach to sample both the water and plankton communities helps better define the relationship between POP concentrations in the water and those in the plankton community. This relationship is critical as plankton serve as the primary entry point for POPs into marine food webs. Additional research is needed to help determine the pathways that these pollutants travel between organisms.

Composed of 5HT cells, the serotonergic system influences mood, social behavior, sleep, memory, libido, pain perception, motor activity, and respiration, among other functions. Understanding the regulation of proper serotonergic development then becomes vital in the development of pharmaceuticals and treatment of individuals suffering from the symptoms of a dysfunctional serotonergic circuit. Using mice as a model organism for the human brain, previous research work has shown that propagating waves of spontaneous intracellular calcium activity (SA) are necessary in the development of the serotonergic system within the hindbrain. Blocking SA with the drug ketanserin has been shown to reduce the number of of 5HT cells during embryonic development. To elucidate the second messenger pathway activated by the ketanserin-sensitive response, we carefully characterized the 5HT receptors responsible for SA. This research project focuses on characterizing whether 5HT2A, 5HT2C, and/or 5HT3 receptors regulate SA within the hindbrain. By using [Ca2+] imaging techniques to monitor SA in real time, drugs selective for specific 5HT receptors were applied to both acutely isolated hindbrains and also through 24hour cultures in order to pinpoint the receptor(s) regulating SA. Results currently show that antagonists of the 5HT2A receptor, M100907 and 4F 4PP, inhibit SA. Meanwhile, antagonists of the 5HT2C receptor, N-desmethylclozapine and SB242084, appear to have no effect. In addition, an antagonist of the 5HT3 receptor, zacopride hydrochloride, appears to have no effect as well. Therefore, results currently suggest the proper functioning of the 5HT2A receptor is important in the regulation of SA, thus playing a vital role in the proper development of the serotonergic system. In future experiments, we will determine the second messenger pathway underlying this regulation.

Contrary to common belief, all vertebrate brains are capable of adult neurogenesis, or the birth of new neurons in the adult brain. Few brains show as an extreme ability to generate and add new neurons as that of the songbird. Songbirds exhibit rapid changes in the rate of neurogenesis, survival of mature neurons, and as a result, total volume of brain regions that contribute to the development and production of song. For example, the songbird Gambel’s white-crowned sparrow (Zonotrichia leucophrys gambelli) is capable of adding more than 68,000 neurons to a region of the brain responsible for song production called HVC, all within four days of transition to breeding conditions. Along with this addition in new neurons, these sparrows also increase song production and song quality. To identify the dynamics between neurogenesis and the addition of new HVC neurons during rapid seasonal growth, we labeled dividing neural stem cells and their progeny, and quantified the incorporation of these progeny into HVC. Throughout the course of the experiment, we recorded individual bird’s song and analyzed the spectral features and stereotypy of whole songs. Ultimately, we aim to correlate improvements in song quality and rate with the cellular changes occurring HVC and the neural stem cell niche during seasonal rapid growth. Such correlations will provide a model for tracking the behavioral progress of the plastic neural circuit as well as for identifying and testing molecular and physiological mechanisms underlying neuronal birth, maturation, and survival.

Inflammation in the brain, called neuroinflammation, is typically considered a negative response following neuronal injury and damage. However, neuroinflammation can also play a beneficial role in the healthy adult brain. For example, one type of inflammatory cell, microglia, become polarized from the “inactive” surveying (or ramified) state following neuronal loss and promote the classic “negative” inflammatory response. Alternatively, in the healthy brain microglia elicit positive effects through neuronal and synapse pruning to maintain proper neuronal number and connections. To identify the dynamics of microglial responses between healthy and natural degenerative states, we employed the seasonal neuronal regression of avian motor pathways for song production in Gambel’s white crowned sparrow (Zonotrichia leucophrys gambelii). Neuronal number in the song control nucleus HVC (proper name) changes seasonally such that as male sparrows transition from nonbreeding to breeding conditions, HVC neuronal number increases by about 68,000 neurons. Useful for our study, an equal number of HVC neurons die through programed cell death as sparrows transition from breeding to nonbreeding conditions. Using this unique model of natural and rapid neurodegeneration, we quantified the number of ramified and activated microglia in HVC of birds maintained in breeding and nonbreeding conditions using immunohistochemistry. To identify microglial promotion of local neuroinflammation within HVC, we examined the number of ramified and activated microglia in HVC of birds treated with pro- and anti-inflammatory agents. Additionally, we identified and quantified a putative downstream effect of this local inflammation: reactive neurogenesis, or the birth of new neurons following neuronal loss. Our results indicate that microglia promote reactive neurogenesis through local inflammation, and suggest that discrete microglial activity could have therapeutic consequences for various neurological diseases.

Plants respond to abiotic factors in natural settings in order to regulate physical responses at the most appropriate timing. The timing of flowering affects seed and/or fruit production in many agriculturally relevant plants, so understanding its mechanism is essential for addressing agricultural needs as climate changes. To address this aim, we have used Arabidopsis thaliana as a model plant to understand the molecular mechanism of seasonal flowering. It has been shown that the expression of FLOWERING LOCUS T (FT), which encodes florigen, peaks in the evening under long-day lab conditions (16 hours light, 8 hours dark, 22°C) and promotes flowering in a day-length specific manner. However, our lab found that FT also peaks in the morning under outside long-day conditions over the summer solstice in Seattle (16 hours light, 8 hours dark, highest average temperature 21°C). This double-peak of FT can be re-created by changing the light quality and temperature settings of long-day lab conditions. In addition, we found that a mutant of PHYTOCHROME A (PHYA), which encodes a red/far-red light photoreceptor in plants, lacks FT expression in a morning-specific manner, suggesting that PHYA is involved in regulating morning expression of FT. In order to understand the molecular mechanism of PHYA-mediated FT regulation, we are in the process of identifying transcription factors and other molecules that work with PHYA to induce FT expression. We have successfully cloned various transcription factors that are reported to act in the flowering regulatory pathway and are in the process of testing protein-protein interaction against PHYA by yeast-two-hybrid methods. Identifying the components that act in the PHYA-FT pathway will allow us to further understand how light affects flowering regulation.

Zooplankton live throughout the water column and what they ingest gets passed to marine life in the higher trophic levels and can ultimately be consumed by humans. Microplastics, 5 mm in diameter or smaller, can contain and absorb high concentrations of toxins and have been consumed by zooplankton in laboratory studies. This research examines whether zooplankton are eating microplastics in Puget Sound. Zooplankton samples were collected from two locations on the east side of Whidbey Island, at an approximate depth of 180-200 meters, and preserved in formalin to prevent further ingestion or regurgitation of microplastics. Samples were then processed according to the National Oceanic and Atmospheric Administration’s methods for the analysis of microplastics in the marine environment. The zooplankton and other organic materials were dissolved and the remaining inorganic components were weighed and examined using light microscopy. The most abundant plankton collected were various species of amphipods, chaetognaths, copepods, and shrimp. Microplastic fibers were observed after completion of the chemical digestion. These results suggest that zooplankton are consuming microplastics in situ and provide evidence of the introduction of microplastics into the marine water column.

Spontaneous waves of electrical activity occur in many brain regions during early development and are critically important for many aspects of brain development. These waves are initiated primarily in the hippocampus and the piriform cortex and propagate throughout the neocortex during the first postnatal week. Most studies of spontaneous activity have utilized brain slice preparations and thus the properties of these waves in vivo are almost completely unknown. We are using calcium imaging in GCaMP transgenic mice to measure wave activity through the intact skull, which is transparent in neonatal animals. We have been able to measure cortical responses to somatosensory stimuli, and have detected widely propagating spontaneous waves similar to those seen in brain slices. We are now determining the developmental time course of spontaneous waves of activity and asking how they interact with responses to sensory stimuli. Preliminary results have shown a greater distance of wave propagation during sleep, implicating wave activity in the emergence of adult sleep-wake cycles. These experiments will determine, for the first time, the properties of spontaneous waves of activity in vivo, providing information about how sensory-driven and spontaneous activity cooperate to shape brain development

Plant phenology studies have shown that flowers are blooming earlier now than they did several decades ago. These shifts in bloom time are attributed to warming temperatures, earlier snowmelt, and other climatic factors related to temperature. One underutilized data source for phenology studies is herbarium specimens, which are plants that are collected, often during bloom, and preserved for the purpose of studying species traits and distributions. Since collection date and location is recorded, herbarium specimens can be used to study the relationship between climate change and long-term shifts in the timing of phenological stages such as flowering and fruiting. My goals for this study were to determine 1) What is the relationship between changes in climate over the past century and changes in bloom time? and 2) Do shifts in the timing of phenological stages in herbarium specimens match shifts reported by field studies? My study species is glacier lily (Erythronium grandiflorum), a subalpine plant native to the Pacific Northwest that is known from field studies to be sensitive to climate change because it blooms immediately after snowmelt. Specimen data from Pacific Northwest herbaria was accessed using the Consortium of Pacific Northwest Herbaria’s database. When not listed, phenological stages were classified using specimen images and geographic coordinates were determined using GEOLocate and descriptions of specimen collection locations. Historical climate information was gathered by entering specimen geographic coordinates and elevations into ClimateWNA, a spatial climate model. I hypothesized that warmer spring temperatures and less snowfall would result in earlier bloom times. I also expected that my results would support shifts in bloom times reported by field studies. My findings contribute to the collective understanding of the historical effects of climate change, which assists predictions and management strategies concerning the future effects of climate change on plant species and communities.

The patterning of skin appendages, such as hair, feathers, teeth, and scales, is crucial for a vertebrate’s ability to survive in their environment. For example, an increased density of eccrine sweat glands in humans has a large impact on our capability to maintain our body temperature. While some key developmental genes have been identified, little is known about the molecular mechanisms involved in skin appendage patterning. All skin appendages evolved from a single ancestor, and although they have different morphologies they share many developmental mechanisms. Zebrafish scales are simple skin appendages made of dentin-like extracellular matrix that form a partially overlapping pattern resembling roof tiles. While mouse and chick-models of skin appendage development require examination of fixed specimens, we can conduct conditional genetic manipulations while imaging the entire skin through scale patterning in live animals. Therefore, zebrafish scales are an ideal system to gain insight into mechanisms underlying skin appendage patterning. We manipulated conserved cell-cell signaling pathways and assayed scale patterning. The zebrafish genome harbors multiple Fgf receptors that are partially redundant, and removing one of them results in fewer, larger scales, while inhibition of all Fgf receptors completely abolishes scale development. This suggests that Fgf signaling, likely through multiple Fgf receptors, plays a role in scale initiation and patterning. Experimentally increasing Fgf signaling results in smaller and more numerous scales, likely due to delayed squamation, indicating that both Fgf and the timing of scale development ultimately determines density. This also implies that a specific level of Fgf signaling may be necessary for scales to form correctly. Inhibition of the Wnt signaling pathway also results in failed scale development. In summary, these results suggest that skin appendage density is dependent upon the timing of their development, and is controlled by a mechanism involving multiple signaling pathways.

The link between climate and range distributions is a key area of study for ecologists interested in predicting how species will respond to anthropogenic climate change. However, most studies only focus on abiotic factors (like climate), and ignore biotic determinants of species performance. In this study, I seek to understand how competition influences the success of a common conifer species in the Pacific Northwest. Specifically, I ask whether interspecific responses to competition lead to a predictable competitive hierarchy across different locations or whether competitive hierarchies vary. To address this question, tree cores from six focal conifer species, Abies amabilis, Tsuga heterophylla, Tsuga mertensiana, Thuja plicata, Pseudotsuga menziesii, and Callitropsis nootkatensis were collected from 18 stands of varying elevation and climatic conditions within Mt. Rainier National Park, with 20 individual trees cored per species and stand. Annual tree growth was used as a proxy for performance as growth is known to be sensitive to competition and results can be extrapolated to overall population performance. The competitive neighborhood of each cored tree was determined by identifying all individuals within a 10-meter radius by species, and quantifying the distance of competitors to focal individuals as well as their size. Growth rates for each individual tree within the stand were compared to the sum of the basal area of neighbors around that tree, using analysis of variance to determine the relationship between the two variables. These relationships were then compared across species to see which species were competitively superior, and then compared across stands to determine whether competitive hierarchies varied. The importance of this study is to identify which species are more sensitive to competition which would expand our knowledge on the biotic factors that may limit their ability to shift their range.

As our planet warms, it becomes increasingly important to study the impacts on biotic systems’ structure and function.  While there is consensus amongst the scientific community that changing climate will affect species’ ranges and distributions, there is a dearth of data on the intricacies of how such changes specifically alter the interactions among species. Notably, few studies simultaneously consider the effects of climate and competition on (current/existing) species performance, which is particularly important for generating a body of knowledge that relates climate change to shifts in species distributions.  This study seeks to investigate how competitive interactions vary across climate gradients for several conifer species common in the PNW and abundant in Mt. Rainier Park. Specifically, I am examining whether neighborhood effects on annual tree growth vary across elevational and climatic gradients. To accomplish this, tree cores were collected from Abies amabilis, Tsuga heterophylla, Tsuga mertensiana, Thuja plicata, Psuedosuga menziesii, and Callitropsis nootkatensis at 18 stands in Mt. Rainier Park, covering a large range of elevation (and thus climate). Additionally, the neighborhood of all cored trees, including the distance between cored trees and their neighbors, the species identity and diameter of neighbors was collected. Core samples were cross-dated by stand to ensure proper assignment of year of growth to each tree ring, and subsequently core and neighborhood data were analyzed using statistical models (multiple regressions) to extract relationship between competition, growth rates, elevational and climate variables. This is an ongoing project, whose growing body of data will be useful in predicting the way climate shifts may affect competition and thus growth of tree species in the PNW.

The Neotropics contain about 100,000 seed plant species, amounting to ca. 37% of the world’s species. The Olmstead lab is currently investigating Bignoniaceae, a plant family comprised of 82 genera and ca. 827 species distributed primarily in the Neotropics. There are 8 recognized clades in Bignoniaceae. One clade, Tecomeae, include 12 genera and approximately 55 species, with species distributed in North and South America, Southeast and Central Asia, and Australia. To create a phylogeny for Tecomeae, I gathered all available sequence data and DNA for species in the tribe from both the Olmstead lab and GenBank. Then, DNA from samples of additional species were extracted using the DNeasy Plant Mini Kits. Three different regions of DNA were sequenced, including two chloroplast regions (ndhF and rpl32-trnL) and one nuclear region (ITS). I amplified these regions via Polymerase Chain Reaction and sequenced the DNA using an ABI genetic analyzer in the UW Biology Department Center for Comparative Genomics. Sequences for all species were edited and aligned using Geneious. Phylogenetic analyses were performed in both Maximum Likelihood and Bayesian frameworks using RAxML and MrBayes. Creating a phylogeny for the Tecomeae is a crucial step toward deciphering the evolutionary history of Bignoniaceae. The overarching goal of our research is to not only create a phylogeny for Tecomeae, but for all 827 species within the Bignoniaceae family. Having a phylogenetic tree for Tecomeae allows us to identify when important divergences occurred, giving us an improved picture of how Bignoniaceae diversified and, ultimately, a better understanding of Neotropical diversification as a whole.

With concern about global warming rising, assessing how increases in temperature impact plant performance is crucial in understanding how plant communities may alter. Genetic diversity, local adaptation to climate, and gene flow may interact with the direct effects of climate to determine how plants fare in a warmer world (e.g. through range shifts and shifts in phenology), but these interactions are poorly understood. Studying local adaptation is crucial for predicting how local populations will fare with warming, as a high degree of local adaptation may cause populations at the range center to be negatively influenced by warming. Conversely, populations in newly climatically unsuitable habitats may be able to cope with warming by adaption. This study measures how the climate of different elevations affects population growth of the high-elevation plant mountain monkeyflower, Mimulus tilingii, at Mt. Rainier National Park. Utilizing data from the past two years, we will parameterize matrix population models to explore how climate influences fitness and population growth rates of different populations, thereby assessing how local adaptation contributes to population dynamics (and potentially, the impacts of climate change). Matrix models will also allow us to determine how local adaptation affects particular life stages, which climatic factors drive local adaptation, and how genetic diversity promotes local adaptation. Previous results show that plants growing in different environments (high vs. low elevation) did not significantly differ in reproductive success whereas different genotypes (crossed between 2 elevations vs. 1 elevation) did, suggesting genetic diversity is an important primary driver of fitness. We predict that vital rates will be greater for populations grown in their native range (high elevation) with genotypes matched to their environment (within 1 elevation). Through these analyses, our understanding of population response due to warming will help us make better ecological inferences of what to expect in the future.

A seed’s successful growth into a seedling depends on it finding a suitable site for germination. This may be limited by the parent’s ability to distribute seeds over a large area (dispersal), or by the seed’s ability to grow in given environmental conditions (microsite characteristics). This study attempts to answer the question: how is tree germination limited by dispersal and by the soil moisture of germination sites? A forest in a 25.6 hectare plot located in the Gifford Pinchot National Forest is the subject of this study. This forest is dominated by Tsuga heterophylla and Pseudotsuga menziesii, but also includes Thuja plicata, Abies amabilis, and Acer macrophyllum. Past surveys on trees, seeds, and seedlings within the plot have been conducted since 2014. Specifically, the species, size, and location of all trees are known. Forty paired seed traps and seedling quadrats throughout the plot provide data on seed and seedling densities by species, as well as soil moisture readings. The role of dispersal in germination success will be assessed by comparing the number of germinants within each seedling quadrat to the abundance of nearby parent trees. Soil moisture’s role will be determined by comparing germination rates (calculated by seedling densities divided by seed densities) to soil moisture in seedling quadrats. Results will be separated for each species. The species composition of these communities may prove to be determined largely by differences in dispersal and moisture requirements between species. For example: we expect to find a high dispersal capacity for Tsuga heterophylla, which would explain its dominance in the stand. We also predict that most species will fail to germinate in areas of low moisture. This might suggest serious implications for how these trees will respond to the warmer and drier conditions expected to accompany anthropogenic climate change in the Pacific Northwest.

As animals grow, scalar expansion of many types of neurons must be coordinated with animal growth to maintain receptive field coverage and proper connectivity, a process that is not well understood. Resulting from a genetic screen, the Parrish lab has generated and isolated a mutation that results in severe elongation of the ventral nerve cord. This mutation affects a predicted transcription factor of unknown function, senseless-2(sens-2). Although the origin of the defect is unknown, one possible explanation for the defect is that sens-2 regulates growth of the peripheral nerves. In the absence of sens-2 function, the peripheral nerves are unable to elongate properly and, as a result, larval growth leads to greater strain on the nerve cord, which leads to nerve cord elongation. An alternative explanation is that sens-2 is required in all the cells of the nerve cord (neurons and glia) for their growth. Thus, one major aim of this study is to produce antibodies that recognize the sens-2 protein. To accomplish this task, I will generate a plasmid vector for production of sens-2 protein in bacteria, perform a recombinant protein purification and submit the protein for antibody production. Upon receiving the antibody, I will perform a series of dissections accompanied by antibody staining and imaging in order to localize sens-2. A second major aim is to take an unbiased genetic approach to identify factors that interact with sens-2 to regulate nerve cord expansion. If we identify interactions with genes of known functions in growth control or patterning, it will allow us to formulate models to account for sens-2 function in nerve cord elongation. To accomplish this task, I will I will create a series of heterozygous sens-2/deficiency stocks and perform genetic screens to determine which genes in Drosophila interact with sens-2's molecular pathway. 

Seed masting occurs when seed production of a species is annually variable across large geographic regions, where years of extremely high seed production across individuals in a population is followed by one or more years of extremely low seed production. It occurs across large geographic regions and can occur within and amongst species. However, the drivers of masting and the implications of masting are often poorly known. Here, we use a long-term and spatially extensive data set of conifer seed production to A) test whether seed viability estimates are consistent with pollination efficiency (increased pollination success in high seed production years) driving masting; and B) explore the implications of masting for nitrogen dynamics. We found strong support for seed masting in six focal conifer species in Mt. Rainier national park from 7 years of data (2009-2015) collected from 18 locations. To further explore masting, we tested whether pollination efficiency is the driver of seed masting in Tsuga heterophylla, by testing whether seed viability (seed quality) is higher in mast years (using 4 years of data from 7 stands – which includes 2 mast years). Results indicate a higher proportion of filled seeds in mast years (i.e. higher viability), providing tentative support for pollination efficiency as the underlying cause of seed masting in these focal species. Germination tests of seeds in growth chambers will provide additional information. To determine the consequences of masting for nitrogen dynamics we measured the nitrogen content in seeds and leaf litter (across mast and non-mast years), and used these estimates to estimate total nitrogen content of leaf litter and seeds (i.e. nitrogen cycling). Understanding the mechanisms behind masting is important, as masting can control tree population demography, and influences ecosystem dynamics.

The Verbenaceae, commonly known as the verbena family, consists of mainly tropical trees, shrubs, lianas, and herbs. Verbenaceae’s diversity is highest in Latin America (where this family is primarily distributed) and Africa. Early classifications of this family relied on morphological traits. A molecular phylogenetic study was recently undertaken to assess and revise those classifications. Verbenaceae now include 35 genera and about 1000 species. In the current classification of Verbenaceae, there is one new tribe called Neospartoneae which consists of three small genera of Argentine species (Diostea, Neosparton, and Lampaya). According to the morphological traits, Diostea and Neosparton exhibit similar male reproductive characters and an ephedroid habit (many-branched shrubs with cylindrical, striate stems) while Diostea and Lampaya share fruit characteristics. Previous studies turned into the molecular data and tried to figure out the relationships between genera of this tribe by chloroplast DNA sequences (loci: ndhF, trnLF, ccsA, matK, rbcL, rpoC2, rps3). However, the evolutionary relationships remain are unresolved. In my study, I used nuclear DNA sequences (loci: ITS, ETS, PPR123, PPR42, PPR11, PPR70, PPR91) to provide a more robust molecular data and try to figure out the phylogenetic relationships in Neospartoneae. I extracted DNA from samples collected in Latin America. After doing PCR and purifying the PCR products, I conducted the sequencing reaction. After editing sequencing data manually, I aligned the nuclear DNA sequences to analyze data and produce the phylogenetic trees with maximum likelihood and Bayesian methods. The updated phylogeny improves our knowledge of evolutionary relationships among Diostea, Neosparton, and Lampaya.

FLAVIN-BINDING KELCH REPEAT F-BOX 1 (FKF1) is a blue light photoreceptor which induces photoperiodic flowering in Arabidopsis. Depending on blue light, FKF1, along with GIGANTEA, forms a complex to induce flowering by degrading a transcriptional repressor, CYCLING DOF FACTOR 1 (CDF1). FKF1 stays in light status for a long time, allowing plants to continue induce flowering. ZEITLUPE (ZTL), a close homolog of FKF1, is a circadian clock regulator with a faster photocycle LOV domain. A photocycle is the sequence of structural changes that takes place in molecules when exposed to light. ZTL has high affinity with GI in presence of light, but also degrades its targets quickly in the dark. The faster ZTL photocycle allows it to act as a sensor to the night periods in circadian oscillation phases. Therefore, we hypothesize that slower photocycles of FKF1 is necessary for FKF1 to keep light information and induce photoperiodic flowering. To test the effect of photocycle on the FKF1 LOV domain in photoperiodic flowering, we generated mutations on the FKF1 LOV domain based upon sequence information of the ZTL LOV domain, to speed up the photocycle kinetics. Quantitative real time PCR (qPCR) was used to select stable, transgenic lines expressing comparable levels of mutated FKF1. Then we analyzed samples for expressions of downstream genes CONSTANS, FLOWERING LOCUS T, along with flowering times to measure FKF1 functionality. As a control, wild type plants were used to provide regular flowering time data. We gathered samples to establish lines to test the effect of each mutation on FKF1 function. We expect to find that the mutations’ faster conversion to dark status decreases its affinity to GIGANTEA and CDF1 degradation, delaying the flowering time. This suggests the slow conversion of FKF1 LOV domain to dark status is necessary to induce photoperiodic flowering in Arabidopsis.

The plant hormone auxin controls nearly every aspect of plant growth and development. Proteins involved in the perception of auxin are closely studied in an effort to elucidate drivers of plant morphology. Potential applications of such research lie in engineering of plant architecture and ultimately maximizing crop yield. Auxin triggers binding between two proteins: auxin receptors (Auxin-signaling F-Boxes or AFBs) and transcriptional repressors (Aux/IAAs). This interaction leads to the ligation of ubiquitin to Aux/IAAs, triggering their degradation, and allowing transcription of auxin-induced genes. In the model plant Arabidopsis thaliana, the AFB family has six members (TIR1 and AFB1- AFB5). While the AFBs are highly similar in sequence to one another, individual AFBs may play specialized roles. In our preliminary studies, we have found that a reduction in AFB2 activity in the afb2-3 mutant produces a leaf phenotype not observed in TIR1 mutants. To confirm that AFB2 is responsible for this phenotype, we are engineering transgenic afb2 mutants that express a wild-type copy of AFB2. We expect that leaves of these transgenic plants will resemble those of wild-type. We are also analyzing the leaf phenotypes of other AFB mutants. To test whether small differences in AFB2 function can change leaf phenotype, we are currently transforming natural hypomorphic and hypermorphic AFB2 variants into afb2-3 mutants. By quantifying leaf morphology of these transgenic plants, we plan to map the relationship between AFB2 function and leaf phenotype.

Properly timed seasonal flowering in plants is necessary for reproductive viability in natural and agricultural settings. We use Arabidopsis thaliana as a model organism to study the molecular mechanisms that underlie the developmental transition to flowering, a process that is mediated by both internally propagated circadian cycles as well as information about day length (photoperiod) detected via photoreception. Transcriptional regulation of the FT (FLOWERING LOCUS T) gene is the primary mechanism of controlling the flowering response, and flowering occurs when requisite levels of FT proteins are transported from the leaves to apical stem cell tissue. FT expression is initiated by CO (CONSTANS), a transcription factor whose own expression is tightly regulated by molecular outputs of the endogenous circadian clock. One such output, CDF1 (CYCLING DOF FACTOR 1) is responsible for repression of CO and FT expression. We established that CDF1 forms a complex with the TPL (TOPLESS) protein, an interaction that is dependent on a conserved peptide sequence at the N-terminal of CDF1. We generated phloem companion tissue-specific mutants of TPL in a variety of flowering time genetic backgrounds and found that mutants of TPL consistently flowered earlier than nonmutants, similar to the phenotype witnessed in cdf mutants. tpl mutation had a similar effect on CO and FT expression across 24-hour timecourse to cdf-1,2,3,5 mutants in both long and short day conditions. We have shown that TPL protein associates with CO and FT promoters during the mornings of long days, and that this recruitment of TPL to the promoters is enhanced by the addition of excess CDF1 protein. We demonstrate that TPL protein is required for the repressive activity of CDF1 and are working to better understand the complex role it plays in regulating flowering time in Arabidopsis.

West Africa is a region characterized by high species diversity and endemism, making it an ideal region to study the mechanisms of speciation and the evolution of genetic and phenotypic differences. Species in these regions often vary in morphology, genetics, and other aspects of their population structure, however the specific drivers of these differences have not been clearly identified. This project uses landscape genetics to test for correlations between geographic distances, genetics, phenotypes, and ecology in rainbow lizards (Agama picticauda) in West Africa. Genetic data were collected from 90 specimens using the ddRADseq protocol, and the final data matrix included 2,614 single nucleotide polymorphisms (SNPs). To analyze the phenotypes, 10 morphological traits were measured from 45 specimens obtained from the UW Burke Museum, the LSU Museum of Natural Sciences, and the UCB Museum of Vertebrate Zoology. Phenotypic, geographic, and genetic data were used in the principal component analyses that generated plots showing the distribution of diversity. The preliminary results support four distinct groups with little overlap. Further research will be conducted to include environmental and landscape variables in the study. The results of this study will identify the factors that generate diversity in West Africa. Issues such as climate change and species degradation are becoming more prevalent, therefore an understanding of these factors is necessary for conservation efforts in ecologically diverse regions.

Knowing the age structure of a population improves demographic models, but birds lack many structures, such as teeth and otoliths, used to age other animals. Marking and following large numbers of individuals is required to know the age structure of a penguin population beyond the distinction between juvenile and adult plumage. In a large population of Magellanic penguins (Spheniscus magellanicus) at Punta Tombo, Argentina, we coded eye and foot color. The penguins ranged from less than one year to more than 28 years of age. We examined iris pattern and color, defining “distinct” eyes as reddish-colored irises with concentric red and pink rings and “indistinct” eyes as dark-brown irises. More than 75% of 2-3 year-old Magellanic penguins had distinct eyes, compared to 50-75% of 4-6 year-olds, 25-50% of 7-20 year olds, 10-25% of 21-24 year olds, and <10% of 25+ year olds (n=3262). Foot color progressed from mostly white to mottled to mostly black. We also examined foot color in a small known-age zoo population of Humboldt penguins (Spheniscus humboldti). In Humboldt penguins, feet became mostly black within a year of hatching, but continued to darken slowly with age (n=44, maximum age=25 years). In Magellanic penguins, feet took several years to become mostly black (n=190). Nestlings had mostly white feet with small specks of black (n=16). About 88% of adults 3-11 years old had mottled or mostly black feet, while the remaining 12% had white or fully black feet (n=90). 24 of 25 adults 23 years or older had black or almost black feet. Individual variation in both eyes and feet prevents aging an individual with a high degree of certainty, but a population may be split into broad adult age classes based on eye and foot color.

Chemical signaling, in the form of pheromones, is an important mechanism of sexual selection in reptiles, as it provides a means of communicating mate receptivity and quality during the mating season. Previous research on the chemical profile of Striped Plateau Lizard (Sceloporus virgatus) skin lipids found that lower levels of oleic and stearic acid in female skin lipids correlate with larger egg clutch sizes, suggesting that low levels of the fatty acids might be an attractive mating cue for males. The purpose of this research was therefore focused around two aims: 1) Determine if male lizards respond to manipulation of oleic and stearic acid ratios, as well as between presence and absence of the fatty acids when presented as an isolated cue; 2) Investigate how altering the levels of the two fatty acids in the skin lipids of female lizards affects male courting behavior.  To address these aims we recorded male chemosensory behaviors (nose taps, tongue flicks, and air tasting), as well as escape behaviors (scurrying and jumping) during 40 minute behavioral trials. Although there was no significant difference in male chemosensory behavior between low and high lipid ratio treatments when the cues were isolated, results from the manipulated female trials show that male lizards demonstrated significantly more escape behaviors when presented with cues from a fatty acid spiked female than from an un-manipulated female. This suggests that male lizards were less interested in fatty acid spiked females—possibly due to the unattractive nature of a high lipid ratio cue.

Climate change is threatening global food security. There is a pressing need for better crops that can prevent severe losses. We hope to apply our current understanding of plant developmental mechanisms that are involved in responses to harsh external stimuli to engineer more resistant crop varieties. The plant hormone auxin plays an important role in organogenesis, the process of new organ formation. Auxin controls organogenesis by accumulating in specific tissues at specific developmental times. These accumulations are due to the polar flow of auxin through the plant. These flows are in turn reinforced by auxin through a feedback mechanism called canalization. PIN1 is a an auxin efflux transporter that is required for polar auxin transport. Loss of PIN1 results in several developmental defects. These phenotypes and other analyses have led to mathematical models of organogenesis. We used these models to predict plant phenotype in response to increased canalization. We have engineered transgenic plants with a library of synthetic promoters with varying auxin sensitivity driving expression of PIN1. We are characterizing the shoot architectures of these plants, specifically the number of branches and the pattern of lateral organ initiation. We are also studying how the increased canalization affects the distribution of auxin in our plant lines using fluorescent reporters. We hope that our results will demonstrate how plant synthetic biology and predictive models can be used to rationally design plant developmental phenotypes.

Crop production will have to double by 2050 to meet projected global food demands. Insect herbivory currently accounts for nearly 15% of global crop losses; thus, plants better able to repel insect attack should have a significant impact on food security. One successful strategy for engineering plants is the constitutive expression of insecticidal toxins such as Bacillus thuringiensis endotoxin (BT). However, the constant production of these toxins decreases crop yield and increases the development of BT-resistant pests. When plants are damaged by insects, they produce the phytohormone jasmonate. We propose a new plant engineering strategy that links the expression of BT to jasmonate signaling. To create this link we have built a synthetic transcription factor called a Jasmonate Degradable CRISPR Transcription Factor (JDCTF). This transcription factor is easily retargeted to any gene of interest by changing the dcas9-associated guide RNA. The JDCTF responds to jasmonate via a jasmonate sensitive degradation domain. Using the plant model Arabidopsis thaliana, we’ve generated transgenic plant lines expressing a JDCTF that targets a Venus-Luciferase reporter. We have shown the system works using a bioluminescence time course assay on seedlings treated with the jasmonate analog coronatine. After induction with coronatine, we observed an increase in bioluminescence in the treated seedlings and no increase in biolumescence for the control. Currently we are characterizing the dynamic response to both exogenous jasmonate, as well as simulated and real insect herbivory on mature leaves. Future work will focus on regulating BT expression with the JDCTF system. Our system has the potential to increase yields, increase consumer safety, and slow down the development of insecticide resistance in pests.

Mutations have a large range of effects on an organism’s observable characteristics, from neutral (no effect) to deleterious (lower an organism’s fitness) to beneficial (increase an organism’s fitness). I engineered three mutations into the virus phi-6 and evaluated their effects on viral thermotolerance and growth. Because these mutations were found in a population that had experienced high temperature heat shocks, I hypothesized that the mutant viruses would have a higher thermotolerance than the ancestral virus. To test the thermotolerance of these mutations, I heat shocked bacteria-free lysates of mutant virus and ancestral virus at various temperatures and then calculated the percent survival of the lysate at each temperature. The mutants were also evaluated for their fitness through growth competitions with a common competitor. I found that only one mutation conferred a higher thermotolerance than the ancestor. However, fitness competitions showed that all mutations and combinations of mutations did increase viral growth. This suggested that additional selective pressures may have been present in the evolution experiment. Specifically, the viruses had been grown at low temperatures between heat shocks, which may have resulted in the evolution of higher growth rates, even at the cost of thermotolerance. My research shows that all selective pressures must be taken into consideration when studying evolutionary trajectories. We cannot accurately predict how organisms will evolve in response to one selective pressure such as increased environmental heat without taking in to account the other selective pressures they experience.

Cell-to-cell interactions are necessary for proper cell function. In the peripheral nervous system, interactions between sensory neurons and the epidermis influence neuronal growth, survival and function. However, mediators of these neuron-epidermis interactions are largely unknown. Drosophila melanogaster provides a tractable genetic system to study these cell-cell interactions. In Drosophila, dendrites of larval sensory neurons are sandwiched between the epithelial cell and the extracellular matrix. Occasionally the epithelial cells form invaginations at the location of dendrites, and eventually the dendrites become embedded inside the epithelial cell. In a screen for markers that label plasma membrane-associated proteins that localize to sites of dendrite embedding, we identified components of gap junctions as candidate regulators of dendrite-epidermis interactions. Gap junctions connect two cells via their cytoplasms and allow signals to be passed from one cell to another. Innexins are a family of gap junction proteins in Drosophilia, one of which is encoded by the gene innexin3 (inx3). Our preliminary data showed that, when overexpressed, GFP-tagged Inx3 protein can localize to sites of epithelial dendrite ensheathment, thus we hypothesize that inx3 contributes to formation/stabilization of membrane invaginations that enclose dendrites. However, no tool exists to visualize endogenous Inx3 distribution, nor is there an Inx3 antibody for immunostaining. Using CRISPR/CAS9 genome editing we plan to generate a Drosophila line with endogenous Inx3 tagged with GFP (Inx3-GFP). Using this line, we will determine whether Inx3 indeed localizes to sites of epithelial dendrite ensheathment. If we observe Inx3 enrichment at sites of dendrite ensheathment, we will perform expansion microscopy, a technique that our lab has recently adapted for use in Drosophila, to determine whether Inx3 labels discrete domains in the epithelial membrane invaginations. Finally, using our inx3-GFP fly line, we will conduct genetic mosaic analysis to determine whether inx3 is necessary for epithelial enclosure of dendrites.

Plasmids are small circular pieces of DNA that replicate separately from the chromosome. Plasmids often contain antibiotic resistance genes that allow their host bacteria to grow in environments containing that antibiotic. They can be transmitted both vertically during cell division, and horizontally via conjugation to new bacterial hosts, and thus spread antibiotic resistance through populations. Plasmids are often costly to their bacterial hosts when not in the presence of antibiotics, but the coevolution of a bacteria and plasmid can reduce these costs through compensatory mutations. Typically a reduction or an amelioration of plasmid cost is seen during plasmid/host coevolution, but not a fitness benefit. However, preliminary data has shown evidence of a fitness advantage to containing a plasmid for the bacterial host Pseudomonas sp. nov. H2 after it was evolved with the plasmid RP4. In this project, I determined whether the fitness benefit seen in the aforementioned plasmid/host pair also occurs in three other bacterial species with different plasmid types. For each of these pairs the plasmids initially conferred a cost to the host. We hypothesized that compensatory mutations (on either the host chromosome or the plasmid) would result in the reduction, amelioration or reversal of the initial high cost of maintaining a plasmid for all three pairs. To test this, we evolved three plasmid/host pairs in antibiotic-containing media for several hundred generations, selecting for antibiotic resistance genes on the plasmids. We ran competitions between an evolved host/evolved plasmid pair and an evolved host with the plasmid removed, to determine whether there remained a cost to containing a plasmid, or whether that cost has been reduced, ameliorated, or reversed. Our experiments will help explain why plasmid-conferred antibiotic resistance persists in bacterial populations.

Ocean warming, due to climate change, is currently altering marine ecosystems. In the Salish Sea, temperature anomalies of up to 4°C per year have been reported. Sea urchins hold an important ecological role as community engineers with the ability to alter the ecosystem in which they are found. The green sea urchin (Strongylocentrotus droebachiensis) relies on phytoplankton as a food source for development in the field, but has been shown to consume kelp detritus in the laboratory. Studies have separately shown the effects of different diets and temperatures on larval growth but have not studied the stresses of increasing ocean temperature with changing food sources and abundance. Here we studied the combined effects of food concentration, food type, and temperature on sea urchin larval development. Contrary to prior studies, our results show that at warmer temperatures, larvae can develop under lower food concentrations, but in agreement with past studies we confirm that larvae develop best under a high concentration phytoplankton diet. Interestingly, we saw increased mortality for larvae grown under warm temperatures and fed a high concentration kelp detritus diet, potentially caused by the introduction of microbes that thrive at warmer temperatures. Warming ocean conditions, causing nutrient composition shifts, could be harmful for sea urchin larvae.

NASA confronts a forward contamination problem with the launch of spacecraft and probes into extraterrestrial environments with potential for detection of life. Microbial control is required in order to avoid cross contamination that would compromise data collected. Currently approved decontamination methods are costly and involve high heat and chemical treatments which risk damaging thermally sensitive spacecraft materials and components. Alternative methods, including atmospheric pressure plasma jets (APPJs), show promise as effective technologies for microbial control. Researchers at Edmonds Community College partnered with Eagle Harbor Technologies (EHT) of Seattle, WA to test the capabilities of an APPJ developed by EHT, performing characterization of killing efficacy and demonstrating proof of principle for large-area spacecraft sterilization. Research demonstrated killing capabilities of APPJ when applied to various conditions including different surface materials, meshes, tapered and elevated holes. The plume of the APPJ traveled through tapered and elevated holes yielding greater than 99% killing of endospores. Application of APPJ as a “brush” demonstrated greater than a 6 log reduction of endospores over 56.75 cm²/10 minute exposure. Findings suggest a brush composed of multiple jets could be effective for spacecraft sterilization over large surface areas of differing materials with capability to penetrate unusual shapes and surfaces. Anticipated improvements in killing efficacy resulting from better characterization of increased voltage, frequency, humidity, and the inclusion of low O₂ concentrations should lead to the development of an APPJ brush effective at spacecraft sterilization.

My research sought to study the effect of removing the egg jelly layer from the eggs of the sea urchin species Lytechinus pictus, on chemoattractant release. Sea urchins are broadcast spawners, meaning the males and females of the species release their sperm and eggs into the water to independently find each other and form zygotes. L. pictus eggs release a chemoattractant peptide called speract, which binds to receptors on sperm cells in order to guide their movements toward the eggs. Little is known about the mechanism of release or the site of production for these peptides. The purpose here was to further illuminate the egg jelly layer’s role in speract release. I removed the egg jelly layer in order to identify if the attractant is actively released from the egg, or simply stored in the egg jelly layer. To do this, I quantified the concentration of speract released from eggs that had their egg jelly removed and compared this to eggs with intact egg jelly as a control, both from the same female, over the course of half an hour. I used column chromatography to purify the peptides released from artificial sea water and high performance liquid chromatography (HPLC) to identify the specific peptides present. The results showed that lack of a jelly layer lowered the absolute concentration of speract but did not significantly change the rate of chemoattractant release. Understanding the workings of an important factor in fertility can give insight into urchin species’ reproduction, biodiversity and evolution.

NASA confronts a forward contamination problem with the launch of spacecraft and probes into extraterrestrial environments with potential for detection of life. Earth-origin microbes risk threatening planetary systems, compromising the data obtained, and require implementing microbial control for planetary protection from cross contamination. While generally effective, currently approved decontamination methods are costly, involve high heat and chemical treatments, and risk damaging thermally sensitive spacecraft materials and components. Alternative methods, including atmospheric pressure plasma jets (APPJs), show promise as effective technologies for microbial control. Researchers at Edmonds Community College partnered with Eagle Harbor Technologies (EHT) of Seattle, WA to test the capabilities of an APPJ developed by EHT, performing characterization of killing efficacy. Bacillus atrophaeus endospores were spread on tryptic soy agar plates to quantify killing efficacy of over 900 combinations of APPJ parameters including pulse width, frequency, voltage, distance, time, electrode placement, jet configuration, gas composition, and flow rate. Unprecedented independent control of APPJ pulse width, frequency, and voltage allowed researchers to identify combinations of these parameters resulting in greater than 4-log reduction of endospores. A decimal reduction time (D-value) was additionally determined at an average of 160 seconds on aluminum. High humidity and low concentrations of O₂ were identified as conditions that enhanced killing of endospores. Further characterization of higher voltage, frequency, and humidity, as well as variable pulse width and low O₂ should lead to improved APPJ killing efficacy with decreased exposure time and allow for development of an APPJ optimized for microbial reduction for large-area spacecraft.

Brassinosteroids and auxin are plant hormones that are crucial in plant growth development. Although it is known that brassinosteroids and auxin signaling interact, it is not known exactly how. We are focusing our attention on the question of how auxin and brassinosteroids interact during the formation of lateral roots. Lateral roots are roots that extend horizontally from the primary root and the developmental tradeoff between the primary and lateral root formation helps the plant optimize its water and nutrient uptake. Previous studies have demonstrated that auxin promotes specification of founder cells, the first step in making a new root. To test if brassinosteroids are also involved in this process, I am quantifying the density of founder cells in seedlings treated with brassinosteroids alone, auxin alone or both hormones together. In this study, I will be able to distinguish whether the hormones act additively or synergistically (a much greater effect than a simply addition of the effect of each hormone individually). Our study will deepen our understanding of root formation which will have agricultural significance as strong roots lead to effective nutrient and water intake and stability for the plant under varying weather conditions.

Understanding how genes and regulatory networks evolve to promote innovation is a key question in evolution and development. Flowers represent a classic example of evolutionary innovation, one that propelled angiosperms into one of the most notable adaptive radiations. In angiosperms, the flower meristem identity gene LEAFY (LFY) is expressed after a shoot meristem is induced by long days, allowing flowers to develop. In the absence of LFY function, additional shoots with leaves develop instead of flowers. Surprisingly, knockouts of LFY homologs of moss, a representative of an early plant lineage, result in an arrested zygote that does not get passed the one-cell stage. Since ferns lie midway in the evolutionary tree between mosses and angiosperms, and are the sister group to seed plants, a functional study in the emerging model fern Ceratopteris richardii will provide much needed insight as to how this important gene evolved. We predict that in angiosperms LFY may have been recruited from a broader role as a meristem gene in the shoot apical meristem (SAM) and in indeterminate compound leaves, common in ferns. Expression data backs up this prediction, with CrLFY being found in the embryo SAM and later in the growing areas of compound leaves. We have taken a loss-of-function approach to elucidate the role of LFY homologs in ferns via RNA interference (RNAi). Currently, we are investigating the embryo within knock-down transgenic lines to determine whether it develops normally or if it arrests at any stage, and we are following its development into the compound leaf stage. By providing insight into the evolution of a master regulator of flowering, this projects has potential applications towards agriculture, where most of the crops for human consumption consist of grains or fruits that derive from flowers.

Didelphodon vorax is a large-bodied, North American metatherian, closely related to today's marsupials. D. vorax is posited to have been durophagous (i.e. feeding on hard-shelled organisms or bone), and is considered one of the largest mammalian forms of the Late Cretaceous, living among the dinosaurs. Fossil finds for this taxon are rare and geographically constrained to the North American Western Interior, which often times impedes comparative intraspecific studies. Through quantitative and qualitative measures, including both linear measurements and comparative description, we compiled an extensive review of multiple specimens. We analyzed each specimen, comprised of various mandibular elements, in the context of ontogeny (i.e. the development of an organism from early stage to maturation) utilizing a principal components analysis. We seek to investigate the morphological modifications which characterize and differentiate juvenile and adult forms of D. vorax. Our results shed light on the relationship between ontogeny and morphology in this taxon. 

Both content knowledge and problem-solving strategies are important components for academic success in biology. All problem-solving strategies begin with the ability to understand the problem being presented. In the context of biology, this means identifying the core concept being asked in complex questions. Identifying keywords, words that are important or significant, can help students identify these core concepts. The purpose of this study was to explore how well students can identify keywords and if this ability is correlated to exam performance. Students in an Introductory Biology course took weekly practice exams (PE), after each PE they either graded their own or a peer’s PE. In addition, students were asked to “identify the keywords in the question that should have caused you to provide the complete and correct answer given in the key”. This experimental design also allowed us to explore how grading an anonymous PE affects quality of students’ keyword selections. Students’ keyword selections were compared to a keyword rubric created by the research team. Data was coded by two researchers to 90% inter-rater reliability. On average, students only correctly identified 39% of keywords each week. Multiple regression models were used to explore correlations between correct identification of keywords and performance on both PEs and course exams. Interestingly, students who self-graded were more consistently engaged with the keyword activity as compared to peer-graders. In both cases, percent keywords correct could predict overall exam performance. These results indicate that if instructors want to help students improve their problem-solving skills of complex biological problems, helping students recognize keywords may be one solution. Furthermore, identifying which students have trouble simplifying complex exam problems and helping these students improve their ability to identify keywords may be beneficial in addressing learning disparities within large introductory classes.

Climate change has lowered the reproductive success of the Magellanic penguin (Spheniscus magellanicus) population at Punta Tombo, Argentina contributing to the population’s 37% decline since 1987. While increased rainfall and storm intensity directly affect reproductive success through chick mortality, the indirect effects of weather can also impact fitness. We predicted that temperature would alter penguin behavior in two ways: 1) fewer penguins would leave the colony on hot days since traveling in heat requires more energy, and 2) penguins leave earlier on hot days to escape the highest temperatures of the day. Automatic scales that record weight, time, and direction were placed where penguins frequently exit and enter the colony. The scales also recorded the Radio Frequency Identification (RFID) number of the penguins for 5,000 crossings, allowing us to calculate their trip duration. Days were considered hot if the maximum temperature was greater than two standard deviations above mean maximum temperature (>28℃, n=11 days) and cold days were less than two standard deviations below (<20℃, n=11 days). Temperature did not affect when penguins exited or entered the colony, however, more penguins exited and entered the colony on hot days than on cold. A greater number of penguins took short trips (<= 12 hours) on hot days than on cold days, suggesting they only go to the beach to cool down, rather than to forage. Increasing temperatures could lead to parents leaving their chicks alone at the nest for longer durations of time without foraging which could increase chick mortality.

We are studying the regulation of p120 catenin in zebrafish embryonic development. P120 catenin plays an essential role in the pathway for cell migration and normal embryonic development. Our research on cell migration and the extension of the dorsal axis can be applied to all vertebrates. These functions are also relevant to many clinical issues such as Spina Bifida in humans, wound healing, and cancer metastasis. The particular protein we're studying (p120 catenin) facilitates the activation of Rac1 GTPase when it is bound to both Vav2 and Rac1 GTPase. Vav2 is a guanine nucleotide exchange factor (GEF) that activates Rac1 by exchanging GTP for GDP. Rac1 GTPase activates the actin polymerization and lamellipodia. There has been research suggesting that the dephosphorylation of certain tyrosine residues in p120 catenin is necessary for this binding and subsequent Rac activation. Rac activation promotes cell migration in zebrafish embryonic development. This research has prompted us to ask how phosphorylation at Y217 and co-phosphorylation at Y228-Y217 of p120 catenin affect gastrulation of zebrafish embryos. Based on previous research, we expect embryonic injection of mRNA containing Y217E and Y228E-Y217E mutations to result in abnormal gastrulation. Abnormal phenotypes exhibit delays with cell migration and yoke constriction. In contrast, mutations Y217F and Y228F-Y217F should result in a rescue phenotype similar to the wild type, since the phenyalannine residues are not negatively charged. In order to test this, we designed primers for each mutation, and inserted them using PCR. We used gel electrophoresis to confirm the effectiveness of the PCR by comparing the size of the PCR product with its expected length. We amplify the DNA in E. coli, followed by DNA purification, and sequencing. With confirmed mutations, the next step is linearization of the DNA followed by production of mRNA. We are now in the process of synthesizing mRNA for the multiple DNA mutations we have created. We will then inject them into zebrafish embryos with knocked down wild type p120 catenin and look for resulting phenotypes.

We investigated how grading treatment, self-evaluation versus peer-evaluation, on weekly practice exams (PE) in an Introductory Biology course affects the advice students give to improve academic performance. We hypothesize that students who suggest more resources and study strategies perform better on course exams because they are aware of how improvements need to be made and what resources to use. Students in the class (approximately 550) take a PE on Thursday and then grade the PE responses on Friday. The grading groups were split up into two treatments: half of the class self-evaluated whereas the other half evaluated their peers’ responses. In addition, they were prompted to answer two questions: 1) Pick a single thing that you/this student should work on. 2) What resource should they consult before the next exam? We used this information to explore how study strategies and resources that students suggest relate to their test scores. To analyze the responses, we created specific codes corresponding to student answers. Results of the codes indicate that the students’ responses fall into four categories for question 1 and two sets of categories for question 2 (with three and two codes in each set). When asked to provide ways to improve PE performance, the four specific codes were being descriptive, increasing knowledge of content, interpreting the question, and time management. When providing resources, the two sets of categories were active versus passive resources and expert, peer, and self resources. Over the course of ten weeks, students who suggested more strategies and resources tended to perform better on exams. As well, both self-evaluators and women in the course suggested more strategies and resources than peer-evaluators and men respectively. Given this study, instructors can better understand what resources students are utilizing but also which study strategies tend to be more successful for students.

The Hille lab studies the movement of cells during embryogenesis using the model organism of zebrafish. Currently, we are focusing on how the phosphorylation of certain residues on the protein p120 catenin affects both cell movement and cell-cell adhesion during the early stages of embryonic development. Protein p120 catenin is imperative in the motility or adhesion of cell-cell junctions, and knocking it down results in wider somites and abnormal notochord protrusions as discussed in the Hsu et al paper published earlier by the lab. We are focused on how specific mutations in the genetic sequence of this protein affect cell migration and adhesion in the early development of zebrafish. Other members of the lab have mutated a phosphorylated tyrosine residue (Y228) within the DNA encoding for p120 to either phenylalanine and glutamic acid residues. We are then taking the mutated embryos and staining the somites and presumptive slow muscle cells using a myoD probe in order to visualize the resulting phenotype. This process involves a four-day protocol that is repeated weekly throughout the quarter in order to produce the best staining and results. After hybridization is complete, we then dissect the embryos under a dissecting scope and image them using a high-powered microscope. Using software, we analyze the images, measuring somite width as well as the number of somites in order to determine the developmental stage of the embryo. We then compare somite length and mesodermal formation between both mutated and uninjected, control embryos to gain a better understanding of which amino acid residues in the p120 protein are important in its function during embryogenesis.

Student-graded practice exams (PEs) have been used to facilitate learning in large classrooms. In a large introductory biology class, we investigated the impact of self vs peer-grading of PEs. We hypothesized that students who graded their own PEs would grade more accurately than peer graders because they are more motivated and reflective in their grading. Over time this more accurate grading would lead to increased performance on course exams. One question from each week’s PE was graded by a team of experts. We calculated PE grading accuracy by comparing the expert grade to the grade assigned by the student. We analyzed the correlation of PE grading accuracy with exam performance. For each metric we investigated the impact of treatment, incoming GPA, and student demographics (e.g., gender, socioeconomic status, first generation, race/ethnicity) using general linear models. There was no difference in average expert PE grade between the two treatments. Incoming GPA was the only significant predictor of expert PE grade with GPA positively correlated with expert PE grade. There was a significant interaction between incoming GPA and treatment on grading accuracy. Students with high GPAs who graded peers consistently underestimated their peers’ PE performance. Students with lower GPAs who graded peers either graded similarly to experts or slightly overestimated their peers’ PE performance. Students who graded their own PEs more accurately predicted their PE performance regardless of incoming GPA. There was an interaction between GPA and treatment on course exam performance, as high GPA self-graders earned more course exam points than high GPA peer-graders. Average GPA students earned the same amount of course exam points regardless of treatment. Given these findings and the logistical complications of peer-grading in large classes, instructors can use the more easily administered self-grading method of providing feedback on low-stakes practice exams.

During embryogenesis, phosphorylation of serine and threonine residues on p120 catenin causes disassociation from cadherin transmembrane proteins and activation of downstream pathways leading to cell motility. We have mutated a single amino acid on p120 catenin, T310, to determine its involvement in cell migration of the dorsal axis during zebrafish embryogenesis in vivo. T310 is a target of glycogen synthase kinase 3 (GSK3) and phosphorylation of this site facilitates disassociation from membrane-bound N-cadherin in astrocytes, decreasing cell-cell adhesion and activating the cell motility pathway. We created two mutations of p120 catenin: T310E that mimics the negatively charged phosphate and T310A containing a neutral residue that cannot be phosphorylated. Either mutated or wild type mRNA was co-injected with morpholino, which knocks down endogenous p120 catenin, to determine the injected protein’s ability to rescue the short-tailed knockdown phenotype. As expected, co-injection of wild type p120 catenin mRNA and morpholino rescued the damaged phenotype resulting in embryos with normal tail extension. Co-injecting either T310A or E with morpholino resulted in partial rescue, suggesting that phosphorylation at this site may be variable during development or that the effects cannot be measured by phenotypic quantification methods. We are currently studying the effects of phosphorylation at other sites, S268 and S269, which may give insight on the downstream effects p120 catenin in cell motility, including morphological bone abnormalities and metastasizing cancer cells.

Brain computer interfaces (BCIs) hold an amazing potential to increase our knowledge of the brain and to help improve the lives of countless people. However, almost all work currently being done with BCIs is performed in a strictly controlled laboratory setting. In order to create BCIs that can function robustly in the real world we must examine brain signals during naturalistic tasks like speech. Through our partnership with Harborview Neurology we are able to get week long continuous audio/visual/and electrocorigraph (ECoG) data from long term monitoring of epilepsy patients. A significant problem with this data set is that there is so much data that manual annotation of the subjects' speech would be incredibly time consuming. Our work focuses on using high throughput analysis techniques to automatically annotate our data set in order to efficiently study the brain. We present a pipeline to perform automatic speaker tracking of the implanted patients in our clinical recordings. Speaker tracking is the task of figuring out all the times in which a particular speaker spoke during a recording or across several recordings. We have developed an automatic pipeline that extracts all human speech from a recording and then uses a support vector machine (a machine learning algorithm) to differentiate the subject's speech from anyone else that might speak inside the hospital room. Accuracy for every step is above 80%. The methods developed in the study will allow us to automatically generate timestamps of subject speech across our entire data set. This will allow us to perform useful analysis on the ECoG signal in order to better understand the underlying neural basis of natural speech and develop more robust BCIs to improve the lives of countless people.

Understanding cold stress response pathways in plants is key to the development of new agricultural techniques, which help prevent crop loss or degeneration due to cold snaps or early frosts. Epigenetic modifications by histone acetyltransferases appear to regulate these pathways. HAC1 is a histone acetyltransferase identified in Arabidopsis thaliana that is involved in cold stress response, and binds with transcription factors to mediate gene expression. CsWRKY46, a transcription factor recently identified in cucumbers, was found to be deeply involved in cold stress response. We identified the WRKY71 gene as the Arabidopsis homolog most similar in sequence to CsWRKY46. The WRKY71 transcription factor has also been identified as part of the response pathway to general abiotic response. We hypothesized that HAC1 regulates cold response in A. thaliana by localizing with WRKY71 and acetylating histones of nearby cold response genes such as COR47. We predicted that in cold temperature, WRKY71 and COR47 transcripts would be up-regulated only if the HAC1 protein is present. We designed an experiment with 6 A. thaliana plants: 3 wild type and 3 Salk_080380 hac1 mutants. Wild type Colombia ecostrain and hac1 A. thaliana were subjected to a 4°C cold temperature treatment of 0, 2, or 6 hours under constant light cycle conditions. RNA was extracted from the leaf tissue of each plant and RT-PCR and gel electrophoresis were performed to measure gene expression of HAC1, WRKY71, COR47, and e1f4-III (control gene). Results from this pilot study of HAC1/WRKY71 interaction with RT-PCR assays were inconclusive due to nonspecific primers. Without RT-PCR results with which to analyze the relative expression levels of eIF4-III, COR47 and WRKY71 throughout cold stress, we can neither support nor refute our hypothesis. We propose repeating the experiment with larger, more specific primers and examining WRKY71-HAC1 protein interaction through a co-immunoprecipitation assay or similar test.

Extant mammals derive from three lineages: monotremes, marsupials, and placentals. The divergence of marsupials and placentals is estimated to have occurred 160 million years ago. Marsupials typically have very small, underdeveloped offspring born after a short gestation period, which are then nursed for long periods of time in a pouch. In contrast, placental mammals give birth to large, more developed young, which are nursed for a shorter period of time. Sexual dimorphism, the anatomical differences between males and females, has been demonstrated in the pelves of many species, both marsupial and placental, but no studies to date have compared the degree of sexual dimorphism in the pelves of these two groups. This study compares representative pelves from 30 species, 9 marsupial and 21 placental, across a wide range of taxa. One male and one female pelvis from each species was scanned using either a laser scanner or a microCT scanner to create a 3D surface scan. A set of landmark points were placed on each pelvis and the distances between points were calculated. The pelves were compared to calculate a value representing the amount of sexual dimorphism in each species. These values were then compared between placentals and marsupials using a t-test. Locomotion, adult female size, offspring size, number of offspring per litter, were included in the analysis as possible influencing factors. Placentals are expected to show a greater degree of sexual dimorphism due to the burden on the females for bearing larger, more developed offspring. More specialized modes of locomotion are expected to reduce the amount of sexual dimorphism. This study provides important insight into the evolutionary development of the two dominant mammalian lineages today. To our knowledge, no other study has examined sexual dimorphism of the pelvis in such a broad range of taxa.

Cell movement during development is crucial as the location of a cell determines what that cell will ultimately become. Cells require both adhesion and motility for movement. Adhesion is cells holding on to each other via proteins called cadherins. Motility is movement through projections called fillopodia and lamelliopodia. We are interested in the protein p120-catenin, which determines how cells move. We purpose that phosphorylation, or the addition of negative PO4- groups at specific amino acid residues changes p120 catenin’s function. Specifically we are interested in tyrosine (Y) residues 228 and 335 which likely effect p120 antagonistically, by mediating adhesion and motility, respectively. In order to mimic the process of phosphorylation we mutated each residue to either glutamate (E) or phenylalanine (F). Glutamate, which is charged, mimics phosphorylation, while phenylalanine cannot be phosphorylated, but is otherwise similar to tyrosine. To examine which pathway is activated, we quantified whether proteins associated with motility or adhesion were interacting with p120 catenin for each mutation. The primary technique we used to explore this is coimmunoprecipitation. Coimmunoprecipitation works by using antibodies to link p120 catenin to magnetic beads, as well as any proteins interacting with it. The beads can then be extracted and we can determine the amount of each protein interacting with p120 catenin through a process called western blot. So far we have verified that our mutations for Y228 and Y335 produce proteins, which guarantees that any later results are due to phosphorylation. Also preliminary coimmunoprecipitations suggest Y228 does mediate adhesion. Additionally injection of Y228E in embryos, results in embryos with shortened tails and poor yolk constriction, which are associated with excessive adhesion. Taken together these results strongly support our hypothesis for Y228 mediating adhesion. Based on these results, in the future we plan on examining Y335 and verifying our Y228 results.