Gastrulation is an early phase in the embryonic development of vertebrates during which the three germ layers differentiate. The movement of the cells during this phase, is mediated by actin structures stimulated by integrins, extracellular matrix, and ligands. Migration of the cell is activated by Rho GTPases and focal interactions with cadherin-p120 catenin interaction stabilizing cell-to-cell adhesion. To be more specific, the protein, p120 catenin, is required for the extension of the dorsal axis and normal migration of the presomitic mesoderm. This lab studies the molecules that define the pathways of cell migration and tissue migration in zebrafish embryos. Specifically, we are studying how various mutations affecting the phosphorylation state of p120 catenin family genes were produced by undergraduate students and their respective effects on the dorsal axis extension were visually observed. In this experiment, zebrafish embryos were injected with mutated mRNA and analyzed using our in situ hybridization of two probes. Somites were stained using the myoD probe while the presomitic mesoderm was stained using the papC probe. Staining the mutated mRNA, allowed the team to witness phenotypic abnormalities of the dorsal axis much more clearly. Quantifications of the stained somites and presomitic mesoderm will allow a statistical analysis on various p120 catenin mutants. Overall, this experiment provides a deeper understanding of cell migration during developmental processes.

When a star undergoes the transition to the white dwarf stage of the stellar life cycle, an outburst of ionized gas is expelled in a planetary nebula. It is currently unknown if planets orbiting a white dwarf are able to survive this transition. However, between one fourth and one half of white dwarfs have elements in their atmospheres that are heavier than helium and will sink into the white dwarf quickly unless they are somehow being replenished. The recent detection of a disintegrating rocky planet orbiting a polluted white dwarf suggests that the source of metal pollution may be debris from these bodies. As the Sun will become a white dwarf in the distant future, our team began the search for an intact planet, which could provide a model for examining the future of our own planet and solar system. Using observations from the ARCSAT 0.5 m telescope at Apache Point Observatory, we examined fluctuations in brightness of a small sample of white dwarfs, over a period of several nights. We visually inspected the white dwarf light curves for strong dips in brightness that could indicate the transit of a planet, and promising candidates were further analyzed via Lomb-Scargle Periodograms. Most of the white dwarfs displayed periodicities in flux, but none were indicative of the presence of a planetary body. Included in our data was an eclipsing binary white dwarf system with a known periodicity that our function could not detect correctly. We have improved our search by using the Box-Least Squares method to detect weak transits instead of periodograms, but have yet to detect an intact planet orbiting a white dwarf.

With concern about climate change increasing, assessing how plant reproductive success is impacted by increases in temperature is crucial in order to fully understand how climate change influences ecosystem structure and dynamics. This study measures how population variation and the climate of different elevations affect the capsule size and seed mass of the high-elevation plant mountain monkeyflower, Mimulus tilingii, at Mt. Rainier National Park. If climate is the primary limiting factor determining the range of M. tilingii, plants within the species’ climate range will produce the most abundant capsules and seeds whereas plants grown outside the species’ distributional range will produce smaller amounts. Sample seedlings were planted at two elevation sites: 1)  M. tilingii’s range center and 2) below M. tilingii’s range to mimic the increase in temperature due to climate change. Populations were categorized as either within-population (for crosses within the same elevation site) or between-population (for crosses between the two elevation sites) with equal amounts at both sites. This study quantifies M. tilingii’s reproductive success both in the context of the site as well as the population type, allowing for comparisons on these two levels. Results show that parent plants from the lower site produced more flowers and grew faster, indicating that plants in the lower elevation site might be more successful. These unexpected results suggest that M. tilingii may be better adapted to a warmer environment and that climate change might even be beneficial for this reason. Determining the exact impact of temperature on reproductive success is crucial not only for measuring the species’ fitness but also for understanding how these new adaptations will play an even bigger role in competition for resources among different plant species and how those new interactions will alter the ecosystem as a whole as the climate warms.

Today's contemporary debate over the broken immigration system in the United States has placed politicians and policy makers at every level of government in a legal gridlock. It is estimated that 11 million undocumented immigrants reside in the U.S., and that at least a fifth of these are young people who have lived in this country since childhood. Of these 2.1 million youth nationwide, about 65,000 graduate from U.S. high schools every year and an even smaller percentage of these students go on to pursue a college education. President Obama's enactment of Deferred Action for Childhood Arrivals (DACA) in 2012 and its expansion in 2014 provided undocumented youth who meet certain criteria with deporation relief and temporary work permits, and with that a potential new outlook on their future prospects. Although there is a growing body of literature on undocumented youth's self-perceptions in American society and the challenges they face in their transition into adulthood, there is little academic work that focuses on undocumented students who have entered college environments. For this research project, I intend to capture the experiences of undocumented college students as they navigate through the university landscape. This process will include an ethnographic survey and semi-structured interviews with 18-30 undocumented university students at the University of Washington. My sampling strategy will involve contacting pro-immigrant groups, organizations, and clubs on campus to establish a pool of potential subjects in addition to snowball sampling. The results of these in-depth interviews and surveys will provide a unique insight into the lives of students and will contribute an additional narrative that will expand the body of knowledge on the subject at hand. The impact of DACA will be explored as well as its implications on undocumented students' educational trajectories and future aspirations. 

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 am assessing whether macroclimate influences the climate sensitivity of three prominent conifer species: Pacific silver fir (Abies amabilis), mountain hemlock (Tsuga mertensiana), and Alaska yellow cedar (Callitropsis nootkatensis) on Mt. Rainier. I am analyzing tree core data from three high elevation sites. Each site is located in a distinctly different climatic area on the mountain. I am comparing standardized tree growth to climate variables (mean growing-season temperature, total growing-season precipitation, snow duration, etc.) using linear mixed effect models. This will determine how influential climate variables differ by location and by species. 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. Range expansion of one species could lead to the crowding or elimination of another. For example, conifer range expansion on Mt. Rainier could negatively affect the alpine wildflower meadows, a major attraction for tourists. Widespread range contractions could also decrease forest density and 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.

Mass incarceration has disproportionally impacted communities of color over the past 41 years. Indeed, policies that are currently operating as a result of the “War on Drugs” have created large racial disparities in drug convictions. For an example, despite research suggesting that Blacks and Whites use marijuana at similar rates, Blacks have been convicted at a higher rate. Many proponents of criminal justice reform argue that just as certain policies have led to the creation of racial disparities in drug conviction, the creation of new policies and laws might also be able to reduce or eliminate these disparities. For this research project, I use longitudinal data of all Washington State convictions between 2000 and 2015 and ordinary least squares regression analysis to examine the association between the 2012 decriminalization of marijuana in Washington State, I-502, and in drug conviction rates by race. I also include a series of controls including: year of conviction, sex, county and prior convictions to test the robustness of this association. In particular, I am interested in understanding if racial disparities in drug convictions decreased post the implementation of marijuana legalization in Washington state. My results suggest that marijuana legalization leads to decreased disparities in drug conviction rates for Blacks. The overall lower conviction rates for all types of drugs may have been produced by the lessening of police contact with Blacks in Washington State for suspicion of marijuana possession.

In cells, phosphorylation of p120 catenin causes it to dissociate from cadherin transmembrane proteins, which leads to the activation of downstream pathways and cell motility. We have mutated amino acids in p120 catenin in order to locate the functions of specific sites of phosphorylation in zebrafish embryos. The phosphorylated tyrosine amino acid, Y335, in p120 catenin is a target of the phosphatase "PTP-PEST” and controls epithelial cell motility and Rho GTPase activity in colon carcinoma cells. We designed a knock-out of normal activity by mutating the tyrosine to a phenylalanine, which cannot to be phosphorylated. We also mutated Y335 in p120 catenin to a glutamic acid (E), which mimics the negatively charged phosphate. These mutants were transformed into bacterial cells and the isolated DNAs were transcribed into mRNA. We have also injected zebrafish eggs with the mRNAs to analyze the effect of these mutants on development. We have also mutated several other serine and threonine phosphorylation sites on p120 catenin including, S252, T310, and S312 to determine if the mutation to an alanine or to a glutamine will result in altered downstream activity. We have injected mRNAs with the S252A and S252E mutants into zebrafish eggs and determined that the S252E mutation rescues a knockdown of the endogenous p120 catenin, while the S252A mutation does not. The results suggest that phosphorylation of S252E is required for the migration of presomitic cells to the dorsal axis. In fibroblast cells, phosphorylation of p120 at T310 promotes adhesion junction disassociation, allowing cells to mobilize in a “treadmill” fashion. We used p120 catenin mRNAs with the mutations T310A and T310E to measure their impact on cell motility in zebrafish. These individual mutations may give insight on the downstream effects p120 catenin in cell motility, including morphological bone abnormalities and metastasizing cancer cells.

Tree lines play a vital role in plant community composition in montane ecosystems. Over the past several decades, increasing annual temperatures have led to shifts in tree lines in montane environments. However, little is known about how local topography may have an effect on tree line advance. My research incorporates historical and repeat, ground based and aerial photography to assess the change of alpine tree line in a range of topographical conditions in Mount Rainier National Park over the past century. My study quantifies changes in tree cover and tree-line elevation in ridge and cove environments of the park. To track treeline change, historical and recent photographs were aligned, and areas of the photograph were broken up into polygons based on their small-scale topography. Random points were overlaid in these polygons to assess the rate of change of tree line shift. By looking at the change in tree cover in each random point relative to the polygon type, we are able to determine the amount of tree thickening that has occurred. We predicted that tree lines on sun exposed ridges will shift more quickly than those in cove environments. Snow is prevalent for longer periods of time in concave valleys limiting the amount by which the trees can shift up. Our results thus far indicate that small-scale ridge regions have the highest rate of change for tree line shift followed by cove. Subalpine meadows often occur in cove topography. Tree thickening in these regions over time may impact the wildflower populations and decrease species richness in subalpine meadows. In all, our research adds to the growing body of evidence that tree lines are moving up in elevation as well as provides the National Park Service with information about tree line influences on the plant communities of the park.

We are investigating adverse event (AE) reporting in clinical trials on cystic fibrosis (CF) to address whether specific approaches to coding AEs prior to un-blinding of study data is informative. While attribution standards are available in the Common Toxicity Criteria; there are no formal guidelines on how this determination should be made. The process of reporting attributions increases both time and cost of the trial although it is uncertain whether this process is accurate or valuable. This is a retrospective data repeated measures data analysis with pooled clinical trial data from four randomized controlled trials involving patients with CF. We will employ descriptive statistics to describe the overall population and demographics from individual studies who experienced at least one adverse event and summarize the adverse event rates over all and per study by individual rates. We will also employ repeated multiple variable regression techniques to describe associations of demographic and clinical characteristics with the attribution of AE’s. We hypothesize that physicians are not very good at attributing these events when the patient is on the placebo. We currently have available data on 11,974 adverse events in 785 subjects in the four randomized controlled trials. This study could have implications to how clinical trials are conducted in the United States. A future goal would be to clarify the value of attribution of adverse events in clinical trials.

Masting refers to annually variable seed production; whereby plant species release large quantities of seeds one year, followed by one or more years of significantly lower seed production. Seed masting behavior is thought to be evolutionarily favorable, a technique for survival. Two main theories exist to explain mast seeding, predator satiation and increased pollination. The purpose of this research study was to determine A) if a group of six conifer species within Mt. Rainier National Park exhibit mast seeding behavior (i.e. synchronized seed production over large areas, annual variation in seed production exceeds annual climate variation); B) Whether seed viability is greater in 'boom' years than 'bust' years (which would support wind pollination efficiency as an evolutionary driver of masting); and C) whether these patterns vary by species and/or elevation. We examined six species of conifers: Abies amabilis, Callitropsis nootkatensis, pseudotsuga menziesii, Thuja plicata, Tsuga heterophylla, and Tsuga mertensiana. Data was collected from 108 seed traps located in 18 stands at multiple elevation ranges within Mt. Rainier National Park. Seeds were sorted based on tree species, and classified as filled (viable) vs. unfilled (not viable). We then calculated mean total seed production for each stand and used correlations to assess how correlated annual seed production is across 18 stands. Next, we compared annual variation in climate within each stand, to assess whether species showed higher variation in seed production in comparison to climatic variation. Finally, we compared results for our six focal species to assess species specific similarities and differences. Understanding the mechanisms behind masting is important, as masting can control tree population demography, and influences ecosystem dynamics. Moreover, our study may provide greater understanding of the implications of climate change on masting dynamics for forests at Mt. Rainier National Park.

Sea ice plays a vital role in the Arctic ecosystem and is a good indicator of climate change, so it is important to understand the physical processes that control sea ice evolution in order to get a better sense of the detrimental effects of climate change on the environment. Thus, accurate measurements of the features of ice – such as ice concentration – are needed to provide a clearer picture of how and when the ice evolves. This project aims to document and quantify ice variability from airborne imagery during the summer melting season in the Beaufort Sea. Images were taken from US Coast Guard Arctic Domain Awareness (ADA) flights in June, July, August, and October of 2014, with data collection to be continued in coming summers, including this summer, the summer of 2015. Algorithms were developed to automate the identification and quantification of features of the sea ice and open water such as ice concentration, floe size, melt pond coverage, and melt pond size. This data will be charted over time and space (latitude) to identify and analyze patterns in the evolution of Arctic sea ice. Results from this study will be useful for verifying satellite data, evaluating models, and initializing predication schemes. This information will be helpful for quantifying and analyzing the effects of climate change on the Arctic ecosystem.

Cell adhesion and motility are important cell processes that are necessary for embryo morphogenesis. Prevous studies have identified p120-catenin as a key player in coordinating cell movements by either stabilizing E-cadherin complexes to strengthen adhesion or activating cytoplasmic CDC42 and Rac1 GTPases to initiate motility. Most of these studies were done in-vitro using cells grown in a 2-dimensional plane. Comparatively, few studies have attempted to describe p120-catenin's function in a 3-D space. In an effort to investigate p120-catenin's function in embryos, we are co-immunoprecipitating p120-catenin from developing zebrafish embryos. We are specifically investigating how the tyrosine 228 (Y228) residue of p120-catenin mediates binding to E-cadherin. The hypothesis is that phosphorylation of Y228 causes p120-catenin to bind to E-cadherin. To test this, we are injecting zebrafish embryos with p120-catenin containing either an Y228F of Y228E mutation. If our hypothesis is correct, the Y228F mutation would prevent phosphorylation of the 228 residue and decrease affinity between E-cadherin. The Y228E mutation mimics the negative charge of phosphorylation and would instead cause p120-catenin to bind to E-cadherin. 

Lost, confused, lonely, and out of place: these are sentiments felt by many transfer students as they try to acclimate to the University of Washington. Adjusting to a new institution can be a difficult process to undergo alone. For transfer students, the inability to acclimate or the delay in acclimation can result in higher levels of stress, poor academic performance, psychological instability, and unbalanced social well-being. In response to these issues, my senior project investigates how current University of Washington transfer students can help incoming transfer students acclimate to the university. Drawing on the Transfer Students United organization (the first transfer student club at the UW), I conducted surveys and focus group interviews with current UW transfer students in order to better understand their experiences acclimating to the UW and identify their specific needs as incoming transfer students. Based on the results of this research, I then proposed several social and psychological focus areas for the Transfer Students United organization to incorporate as they create resources and services for transfer students.
 

“Human Libraries” are interactive, community-based events in which ordinary people gather to share information and discuss ideas face-to-face with members of the community they wouldn't typically interact with. Created in Denmark in 2000, they have since spread around the globe, reaching the U.S. in 2008. This project examines several Human Libraries that have taken place across the U.S. and tries to determine which elements are most responsible for the events’ success. Findings for this research are based on interviews with several Human Library organizers, participant observations at a Human Library event in Vancouver, B.C., and an analysis of the commonalities shared between multiple Human Library events throughout the United States. The results of this research will help Human Libraries reach their full potential by providing organizers with a strengthened set of best practices and successful case studies on which to model their own events.

One of the central tenants of modern urban planning is that communities and citizens should be involved in the planning process, in part so that planners can focus on problems and solutions that people care most about. However, one group that is often missing from these conversations is children, even though children often have specific needs, experiences, and ways of interacting with the built environment that are quite different from adults. To help fill this gap in planning knowledge, this project uses an age-appropriate method to uncover children’s perceptions of and experiences with the city. Specifically, this project worked with students from two local elementary schools to produce drawings of their streets and neighborhoods as they see them now, and envision them changing for the better. These drawings were then analyzed for common patterns, elements, and themes, and used to develop recommendations of child-centric items that planners can further focus on. Additionally, the maps were collected in a bound book that was provided to the children to reinforce the value of their own experiences and perceptions of the built environment, and to demonstrate to planners that children’s views of the city also matter.

In the United States today, biking is a gendered activity. Nationwide, far fewer women bike commute or ride their bikes in urban areas than men. This gap holds true even in bicycle-oriented cities such as Seattle, where the majority of people who ride their bikes in the city identify as male. This research investigates the cause of this gap and identifies steps that cities and communities can take to encourage more women to cycle. The research includes a review of the literature on bicycle commuting, with a focus on known barriers to and attitudes about bicycle commuting; in depth interviews with leaders in Seattle’s bike community; and an analysis of other American communities known for strong bike infrastructure. Findings from this research are then used to inform several policy, practice, and design recommendations aimed at closing the gender gap by increasing the number of women who regularly commute or bicycle in urban areas.
 

In preparation for the 2021 opening of a new light rail station, the University District community in Seattle has begun to address safety and cleanliness concerns in the alleyways around the station and in the neighborhood as a whole. This project contributes to that effort by identifying areas of particular concern and recommending steps that the community and city can take to address them. Specifically, working with the U District Partnership, a local neighborhood non-profit, I have focused on cleanliness and safety in the alleyways. I then completed a more specific analysis of two alleyways in Seattle identified as being particularly clean and safe. The findings of this analysis were combined with best practices derived from literature on neighborhood safety and used to develop several design and policy solutions for the neighborhood alleys. Finally, these solutions were applied to a single alleyway closest to the future light rail station, providing both a guide for the development of this space as well as a case study for other alley improvements in the University District.

As a result of rapid urbanization, Lake City, a neighborhood in north Seattle, has experienced a significant shift in its demographics, local socio-political dynamics, cultural values, economic conditions, and spatial boundaries. As an effort to better represent and improve the Lake City area, residents created the Lake City Neighborhood Alliance (LCNA), a network of 28 neighborhood organizations, including multiple neighborhood associations, a parent teacher association, a food bank, and a safe-street advocacy program, among others. Such alliances are often championed by neighborhood planning advocates as an effective way to increase citizen participation, enhance community representation, and improve neighborhood conditions. However, very few studies have looked closely at the challenges and internal dynamics of such alliances. This research fills that gap by understanding the benefits and difficulties that come with a parent organization, such as the LCNA, working to improve a community through collaboration between member organizations. Through a series of in depth interviews with LCNA members and participant observations, this project uses the LCNA as a case study to explore why organizations and their representatives continue to be a part of such coalitions, and how the structure of a neighborhood alliance can influence its ability to effectively address local problems and strengthen the community overall.

For many immigrants to the United States, the process of acculturation, adapting to the ideals, values, and behaviors of a new culture and balancing them with those of a culture of origin, can be mentally and emotionally challenging. One potential response to these problems is art therapy. In the west, art therapy has been used as a successful intervention for a range of mental, emotional, and social problems, however there is little research on how making and sharing art may specifically affect immigrants. This research aims to examine the effectiveness of art making and sharing as a coping mechanism for acculturation. After reviewing the literature on the impacts of immigration and the benefits of art making, I interviewed seven immigrant artists from different countries, focusing on the role that art plays in their personal acculturation process. The results of my interviews provide a deeper insight into the effectiveness of using art as an individual and community coping mechanism and suggest several lessons that recreational or community-based art programs could use to ease the difficulty of acculturation for other immigrants.

Developmental phenotypic plasticity can be a vital means to buffer the sudden and persistent effects of anthropogenic climate change, through facilitation of species persistence and possible facilitation of species range shifts. Integrating our understanding of plastic physiological growth mechanisms with functional traits may prove useful for interpretation of the ecological impacts of climate change. We investigated the influence of water-stress and light limitation on leaf mass per area (LMA), leaf anatomy, and leaf gas exchange of juvenile Populus tremuloides trees. Leaves that developed while water-stressed had significantly elevated LMA, due to increased leaf density from significantly higher spongy mesophyll surface area per leaf area (A_smes/A) and reduced stomatal conductance (g_s) but only marginally reduced photosynthesis (Pmax). In shade, leaves had significantly reduced LMA, due to reduced thickness, density, and combined A_mes/A, but maintained the same A_smes/A, and reduced palisade mesophyll surface area per leaf area (A_pmes/A), which was coupled with a reduction of g_s and P_max. Furthermore, water-stressed leaves had elevated intrinsic water use efficiency (WUE_i: P_max/g_s) while the shaded leaves, despite reduced g_s, maintained similar WUE_i. Our results suggest that, under water-stress and full sunlight, greater A_smes/A conferred maintenance of photosynthetic rate possibly through reduction of mesophyll resistance (r_m), at the cost of reducing expansive leaf area (and increasing density) as leaves were more dense but were 20 % the area of control leaves. In shade, suppressing A_pmes/A while maintaining A_smes/A may be a plastic strategy to increase laminar light capture, as leaves had reduced density and thickness but were still 50 % the area of control leaves . Together, in P. tremuloides saplings, these stresses induced developmental plasticity in LMA with differing plastic underlying anatomy and function; we discuss future implications specifically in the context of developmental plasticity, growth trade-offs, and the ecological impacts of climate change.

The scope of this project is to invite an expert to make a speech focused on water mismanagement and global climate change and to conduct pre-speech and post-speech surveys to explore the effects of the speech on knowledge and attitude change regarding these two issues. One common assumption among those combating climate change is that providing the public with expert knowledge on the subject will change their opinions and behaviors. This project tests whether that assumption is correct by measuring the impact of a single speech on the knowledge and attitudes of college-aged students. For the application of my research, I planned and organized the event inviting 300 students to listen to a 1-hour speech delivered live by a Senior member of the United Nations on climate change and global water issues, and conduct pre-speech and post-speech surveys that were administered to determine what effect, if any, the speech had on the students’ knowledge and attitudes about the topics. 197 pre-surveys were completed while 130 attendees were present at the event and 60 attendees completed the post-survey. The post-survey results are in the process of cross comparison with the initial pre-survey. I hypothesized that the results speech measured by the pre-surveys and post-surveys will show significant positive change in both knowledge and attitude change. The implications of my research results will be used to suggest broader implications for scientific and policy communication to a lay audience in order to continuously educate and successfully convey knowledge and information while advocating for positive change in attitude to further advance our efforts in solving the global threat of climate change and water management.

The ability to recapitulate the dynamic presentation of signals in a stem cell’s microenvironment remains a major hurdle in tissue engineering. Controlling cell growth and differentiation in 4 dimensions (i.e., time and 3D space) would allow for heterogeneous synthetic tissues to be produced that match the complexity of their native counterparts. Combining strategies in both light-programmable hydrogels and recombinant protein engineering, we control the cellular microenvironment using proteins site-specifically modified with a bioorthogonal handle and able to covalently bind to a photocaged reactive group in the hydrogel. Previously we’ve demonstrated the ability to photopattern gels with fluorescent proteins that excite at different wavelengths, and have shown that multiple proteins can be patterned independently within the same material with 4D control. These techniques have many potential applications, including improving joint replacement. Current joint replacement therapies often involve use of metals, plastics, and ceramics, and commonly require future revision surgeries. We propose that through our techniques, we can generate a patterned bone/cartilage interface to improve joint replacement, creating a longer-term option for joint replacement. Towards this, we have generated two photopatternable recombinant growth factors, BMP-2 (bone morphogenic protein 2) and TGF-β (transforming growth factor β), known to direct human mesenchymal stem cell (hMSC) osteogenesis and chondrogenesis. I will encapsulate hMSCs in a hydrogel and direct cell differentiation and growth in 4D using a combination of photopatterned BMP-2 and TGF-β proteins. This research will have significant impact in tissue engineering, as it will enable recreation of complex physiological structures, grown outside the body with the patient’s own cells, that can be used for personalized medicine. Our approach is unique in that it allows for unprecedented control over microscale tissue structures, ultimately matching the complexity of native tissue.

Current chemotherapies exploit the larger pore size of tumor vasculature to reach cancer cells. However, this rudimentary form of targeting offers low selectivity and carries the risk of severe systemic toxicity. In addition, chemotherapy distribution to tumors is limited by the large distance between blood vessels in tumors, increased interstitial fluid pressure, and physiological resistance by the extracellular matrix (ECM). With the goal of addressing these limitations, researchers have developed drug-bearing polymers of various sizes and architectures that selectively target tumor cells. The Pun lab previously produced a novel drug delivery polymer, the sunflower polymer, that can be utilized for anticancer drug-delivery. The sunflower polymer is a macrocyclic brush polymer that can be functionalized with a folic acid targeting ligand and an anticancer drug, doxorubicin, that is released in the acidic tumor microenvironment. In order to maximize the potential of anti-cancer drug-delivery polymers such as this, the impact of polymer size and architecture on tumor penetration must be better understood. This is because the efficacy of any anticancer drug, regardless of its mechanism of action, is limited by its ability to navigate the tumor microenvironment and reach all viable cells in a tumor. This project seeks to discover how polymer size and architecture impacts tumor penetration by using a 3D in vitro perfusion chip model of the tumor microenvironment. The model consists of microfluidic channels seeded with KB carcinoma cells and ECM, and has been optimized using model polymers of polyethylene glycol (PEG) and dextran. Tumor penetration is studied by perfusing fluorescently labelled polymers of various morphologies through the seeded microfluidic channels. The information obtained from this project could potentially be used to optimize the size and architecture of drug-delivery polymers for tumor penetration. In turn, these optimizations could present a significant contribution to the development of anticancer drug-delivery polymers.

Due to dwindling non-renewable energy sources, people have started to harvest clean and sustainable energy. Utilized to capture solar energy, organic photovoltaics (OPVs) are developed due to their ability to be printed on a flexible substrate, ease of production, and lightweight. Current researches are focused on improving the device efficiency and durability. Therefore, OPVs may compete with their inorganic counterparts, which are currently more popular although limited by the expensive material and manufacturing process. Phenyl-C61-butyric acid methyl ester (PC61BM) and poly(3-hexylthiophene-2,5-diyl) (P3HT) nanowires are widely-used acceptor and donor polymers, respectively, in an OPV’s active layer. A desirable morphology of the active layer is a bulk heterojunction structure. One of the factors that affect the morphology is the processing of these polymers. The purpose of this project is to investigate how the PC61BM addition affects the P3HT nanowire formation. The experiment involves nanowire development in three cases: (1) pure P3HT, (2) P3HT with PC61BM added after the formation, and (3) P3HT with PC61BM added before the formation. Samples from the three cases were characterized using UV-Visible spectroscopy and X-ray Diffractometer to determine the crystallinity and morphology of the polymer chains. The morphology of polymers is discussed as a function absorbance ratio, which indicates chain planarity, and crystallite size. The results were then compared with those from previously published papers. Due to considerable differences observed, the regioregularity of the P3HT precursor was tested using Nuclear Magnetic Resonance spectroscopy. The result was a lower regioregularity than that of P3HT which is typically used for photovoltaic applications. Therefore, a different batch of P3HT was used to investigate the morphological differences between the three cases. Assessment of the polymers’ morphology were correlated with OPV device performances in published literatures. A determination of the PC61BM-addition effect may assist future fabrication of OPV with superior performance.

Stille coupling, Suzuki coupling, and Kumada coupling are some of the most commonly used reactions in the synthesis of polymers for organic solar cells and organic electrical devices. They are highly effective in creating new carbon-carbon (C-C) bonds. However, when synthesizing a conjugated polymer for organic solar cell applications through these conventional reactions, the process can be challenging and lead to many synthetic steps. In addition, some of the materials used are expensive and extremely toxic to both human health and the environment. In this project, I work individually to synthesize a new benzobisoxazole-based compound which can potentially serves as the monomer for conjugated polymer of organic polymer. The goal of the project is to create a compound in which the inert carbon-hydrogen (C-H) bonds are strongly weakened and easily broken. Potentially, organic solar cells production via this new compound can be more efficient, and as a result, reduces the cost and makes it more environmentally friendly. Recently, the lab’s collaborators at Emory University synthesized a benzobisthiazole-based monomer with two solubilizing alkyl thiophene groups. However, the monomer has limited solubility in organic solvents as well as C-H activation ability. To improve the monomer, I attached a further 2-ethyl hexyl chain to the 2-position on each thiophene solubilizing group and also replaced thiazole with the more electron deficient oxazole unit. Right now, I have successfully synthesized the desired compound and been in the process of activating it using a Gold-based catalyst. If C-H activation is possible for this compound, oligomers of at least three monomers are expected to form.

Therapsids (mammal-like reptiles) document the transition to modern mammals and are an important study tool in examining how the past informs the present. Understanding diseases today requires understanding the deep evolutionary history in our fossil ancestors. We present an instance of dental pathology in a gorganopsid therapsid (~260 million years old). Using fossil histological techniques, we examine a gorganopsian canine and preliminary results reveal abnormal mixture of dental tissue next to the functional tooth. These individual ectopically expressed teeth have preserved tissue layers such as enamel, dentine and a pulp cavity. Based off of the histological characters of our specimen as compared to modern clinical studies we hypothesized two possible diagnoses: odontoma and enamel pearls. Enamel pearls show dento-enamel pearl nodules protruding from the enamel of a functional tooth. Odontoma presents as individual teeth with their own histological packaging (i.e. dentine and enamel). These results suggest that our specimen most likely had odontoma. In modern cases, this results in excess dentinal tissue growth and un-alignment of teeth. The cause of this odontoma in modern clinical practices is a result of either trauma or rapid tooth replacement. The canine tooth of this gorgonopsid was prone to trauma since it served to pierce prey and was subjected to intense forces. Alternatively, gorgonopsids replaced their teeth continuously. This continous replacement could have gone awry resulting in this disease. This study presents the oldest possible case of cancer in the fossil record and elucidates the evolution of such pathologies. This study has relevance in dental clinical studies because understanding how diseases operate in humans requires an understanding of how they evolved in our deep evolutionary history. 

Chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) is the gold standard for assaying the binding sites of chromatin-binding proteins and their modifications, such as histone tail modifications. One major limititation for this method is that it requires large amounts of starting material, therefore limiting applications in those cases where only a limited number of cells can be recovered, e.g. rare cell populations obtained directly from patients. In the last few years, great efforts have been made to develop new ChIP-seq protocols to overcome this limitation. In my lab, they have developed a method called carrier ChIP-seq (cChIP-seq). The method is based on the use of a DNA-free carrier to enhance recovery of DNA after IP. Using cChIP-seq they have been able to perform ChIP-seq on 10,000 cells for several histone tail modifications. I tested a series of modifications on the cChIP-seq protocol. The goal of my project was to develop a protocol that can be performed in one day. Currently the cChIP-seq protocol takes 3 days to be complete. This is mainly due to two overnight incubations. The first one is the immunoprecipitation, so I performed multiple trials to identity the minimum time sufficient for immunoprecitating the chromatin. The second overnight incubation is to reverse crosslink immuneprecipitated chromatin. I tested a version of the cChIP-seq protocol for native chromatin, meaning the DNA is not crosslinked. By optimizing and combining these two modifications I was able to shorten the cChIP-seq to a single day. Through my project I contributed to a larger project with the goal of scaling down the number of cells used for ChIP-seq.

Proteins are the executors of life’s most essential processes, and the ability to express and purify proteins of interest is critical to studying their structure and function. Genetic fusions to small ubiquitin-like modifier (SUMO) tags generally increase the solubility and stability of target proteins during recombinant expression. Following purification, the fusion protein can be cleaved by the highly specific SUMO protease, leaving the target protein intact and unmodified. This specificity attests to the use of the SUMO protease as a powerful biotechnical tool. The current SUMO system relies on the Saccharomyces cerevisiae protease Ulp1. However, this protein has considerable limitations in that it is temperature sensitive and requires detergent to maintain stability, which can interfere with downstream applications. Furthermore, Ulp1 instability often leads to incomplete digestion of SUMOylated constructs and can nucleate aggregation of target proteins. We sought to improve the current system by two orthogonal approaches. First, we mined genomic information from thermophilic organisms for homologous proteins. We have identified and experimentally characterized a novel SUMO-protease system from, Chaetomium thermophilium, a eukaryote that can grow in compost up to 60°C. Second, we used computational protein design with Rosetta to reengineer S. cerevisiae Ulp1 to enhance the stability and solubility of this protein. Using a novel fluorescence resonance energy transfer (FRET) based reporter assay that we have developed to monitor protein cleavage, we can characterize the SUMO systems and evaluate their efficacy. This assay allows for assessment and optimization of protease digestion in varying temperature, pH, and salinity conditions. These SUMO protease systems that we have uncovered and redesigned allow for the ability to circumvent many of the issues associated with the current Ulp1 system by offering an alternative, enhanced tool for the purification and expression of recombinant protein in laboratory research settings.

The mitochondrial unfolded protein response (UPR^mt) is a key cellular mechanism for maintaining mitochondrial function in the face of oxidative and proteotoxic stress. This pathway upregulates mitochondrial chaperones and proteases that aid in folding and degradation of aberrant proteins. Induction of the UPR^mt was observed to correlate with increased longevity in the nematode worm Caenorhabditis elegans, a model whose genetic system is widely used to study the molecular mechanisms of aging. Through a genome-wide RNAi screen for regulators of the UPR^mt, we found that deficiency in the transaldolase (tald-1) resulted in activation of the UPR^mt. TALD-1 is responsible for the production of NADPH in the oxidative phase of the pentose phosphate pathway (PPP). In subsequent studies, we have found that knockdown of transaldolase reduces in vivo mitochondrial respiration, changes the overall metabolism of lipids, and affects the activity of transcription factors that regulate a starvation response and expression of metabolic genes. Among the genes transcriptionally altered by transaldolase knockdown is the flavin-containing monooxygenase-2 (fmo-2) gene, which has been previously found to be regulated by starvation. Like starvation, tald-1(RNAi) induced nuclear localization of HLH-30, a transcription factor responsible for the expression of autophagy genes. HLH-30 is also required for fmo-2 up-regulation. Epistasis lifespan analyses using tald-1 (RNAi) and deletion mutant strains of C. elegans, quantitative gene expression studies, and fluorescence reporter assays were used to determine the mechanistic relationship between tald-1, hlh-30, and fmo-2 as regulators of longevity in C. elegans.

As the primary producers of energy (ATP) in a cell, the mitochondria are key players in controlling cell aging and death. Interestingly, mild oxidative stress, derived from mitochondrial dysfunction or exogenous agents, evokes adaptive stress response pathways that can increase healthspan and lifespan in the nematode worm Caenorhabditis elegans. One of the pathways that responds to mitochondrial stress is the mitochondrial unfolded protein response (UPR^mt). The UPR^mt responds to stress from unfolded proteins or oxidative damage by upregulating expression of chaperones that stabilize and assist folding of unfolded proteins as well as proteases that can degrade proteins within the mitochondria. To search for genes that regulate the UPR^mt, we performed a genome-wide RNAi screen and found that knockdown of the tald-1 gene activates the UPR^mt. The tald-1 gene encodes transaldolase, an enzyme in the pentose phosphate pathway (PPP). One role of the PPP is to generate NADPH, which is required for cellular redox homeostasis. Although transaldolase deficiency has been linked to mitochondrial dysfunction in humans and mice, how the PPP controls mitochondrial function is not well understood. The focus of our study was to determine the relationship between PPP inhibition, mitochondrial function, and longevity by investigating the role of transaldolase in redox homeostasis and regulation of redox sensitive signaling pathways. Here we report that transaldolase deficient worms induced a starvation-like metabolic response that requires JNK MAPK signaling, a pathway that is directly activated by oxidative stress, to increase lifespan. Since the UPR^mt and PPP are correlated with several age-related diseases such as cancer and neurodegenerative disease, understanding the underlying genetics and molecular mechanisms of these pathways and their role in aging is likely to be relevant for human health.
 

In mammals, fundamental differences evolved between the sex chromosomes of males (XY) and females (XX), resulting in specific regulatory mechanisms, which restore a balanced expression of X-linked genes between the sexes. To do this, one of the female’s X chromosomes is silenced (i.e. X inactivation). However, some genes escape this inactivation and remain bi-allelically expressed in females, resulting in higher expression levels potentially contributing to sex differences. The Car5b gene, which encodes a mitochondria carbonic anhydrase important for heart function, escapes X inactivation in both mouse and human. The CCCTC-binding factor (CTCF) often binds near the boundaries between escape and silenced genes. Car5b is inactivated in mouse brain, but it escapes silencing in embryonic kidney (Patski) cells derived from a F1 hybrid female mouse. Selective CTCF binding at a site near Car5b in Patski cells but not in brain suggests the possibility that a chromatin loop that includes the Car5b promoter may facilitate escape in the cell line. To test this looping escape model, we are deleting the CTCF binding site on the inactive X (BL6 allele) and will evaluate its effects on escape. To establish an allelic deletion at the Car5b locus, a CRISPR/cas9 approach (DNA modifier) is being used with two BL6-specific small guide RNAs spanning the CTCF binding site upstream of the Car5b promoter. We’ve completed single-cell cloning for Patski cells transfected with CRISPR constructs. These cell clones are currently being verified for correct deletion by PCR and Sanger sequencing. Results have shown a positive deletion in the cell cloning colony. Allelic expression of Car5b will then be examined in the clones to investigate whether deletion of the CTCF binding site affects its escape (chromatin architecture). If results are conclusive, such an approach will be utilized to examine other escape gene regions.

Hydrostatic pressure is important to many vascular processes, including endothelial activation (inflammation), vasodilation, vasoconstriction and angiogenesis (branching of new blood vessels from existing ones). The problem of pressure regulation in in vitro studies is usually solved by syringe pump based systems. Harvard Apparatus provides a catalogue of “Constant Pressure Syringe Pumps,” the cheapest of which costs $4500. This is prohibitively expensive for many applications. Thus, there is a need to create an affordable device that can reliably regulate hydrostatic pressure. I present a syringe pump based system that maintains liquid pressure within a flow chamber at a fraction of the cost of commercially available devices. My pump was designed from open-source and off the shelf components, including a pressure transducer and Arduino Microcontroller, at a total cost of ~$350. I have tested the pump’s ability to maintain constant liquid pressure across a high-impedance microfluidic system and have shown that it maintains pressure at a user-defined set point between 0 and 70 cmH2O of gauge pressure. This device illustrates the power of open-source software and hardware, 3D-printing and rapid prototyping to allow labs to design customized hardware to meet specific research needs at a reasonable price. Once completed, I will use it to study the pressure dependence of angiogenesis and vasculogenesis (formation of new blood vessels by endothelial cells embedded in interstitium). We will vary hydrostatic pressure to optimize and document blood vessel network formation. This is relevant to studying embryonic development and tumor vascularization and to engineering grafts to repair the damage from myocardial infarction (heart attack). Other possible applications include eliminating the confounding variable of pressure variation in Doppler echocardiography or in studies of endothelial cell response to variation in microvessel geometries.

Imidacloprid (IMI) is being sought as an alternative to the carbamate pesticide, carbaryl to control burrowing shrimp (ghost shrimp, Neotropea californiensis) in Willapa Bay and Grays Harbor. The shrimp destabilize sediments resulting in poor survival and low yields of the commercially harvested Pacific oyster. Previous laboratory tests indicate ghost shrimp are overtly affected (immobilized) when exposed to IMI at concentrations up to 1,500 ppb in artificial seawater (SW), but not killed and subsequently recover. Our objective was to determine the concentrations of IMI in SW that result in mortality of the shrimp and compare these levels to those known to affect other marine invertebrates, particularly Crustacea. Additionally, we quantified time to recovery in shrimp exposed to the concentrations tested. We exposed adult non-gravid females to 0, 10, 100, 1000, 10000, and 1000000 ppb (1,000 ppm) of the active ingredient (a.i.) (Nuprid® 2F) in SW for 48 h (24-h static renewal; 5 individual shrimp per concentration) and quantified overt behavior, survival and water quality. All shrimp at concentrations > 1 ppb were overtly affected, but none died. In the recovery test, 6-8 individual shrimp were exposed to 0, 10, 100, 1000, and 10000 ppb a.i. for 24 h (static) followed by 120 h in clean SW (static renewal). One shrimp exposed to 100 ppb died and time to recovery in survivors was concentration dependent. Results suggest ghost shrimp are very sensitive to IMI in terms of immobilization, but concentrations necessary to kill the shrimp exceed those for other marine invertebrates. The neurophysiology of Crustacea may not match the mode of action of IMI because most neural receptors in these species use glutamate as the neurotransmitter and not acetylcholine. Our results, coupled with the less than desired efficacy in the field, suggest IMI may not be an effective alternative to carbaryl.
 

Making the surrounding of a yellow target brighter will make the target appear darker and change its hue to brown. These changes are called darkness induction and brightness induction, respectively. Brown induction is interesting because the shift from yellow to a different dark hue, brown, is not found for the other basic colors—red, green, and blue. To assess whether the same neural and perceptual processes mediate brown and darkness induction, we explore the possible parallels with a checkerboard variation of White’s Illusion, which produces unexpected and theoretically controversial darkness induction effects. Surround stimuli consisted of a 20x10 black and white checkerboard grid. In a portion of the checkerboard grid, either the white or black squares were replaced by a 4x4 set of target squares, leaving the target squares surrounded by either only black or only white squares, respectively. For the darkness induction task, gray target squares were adjusted in luminance (physical light level) so that targets on white matched targets on black in perceived darkness. For the brown induction task, yellow/brown target squares were adjusted in luminance so that each target was right at the boundary between pure brown and yellowish brown, the “brown boundary.” The magnitude of the White’s Illusion effect was expressed as the difference in luminance between the two darkness-matched targets (darkness induction) or the two brown-adjusted targets (brown induction). Preliminary results show the White’s Illusion effect for brown induction to generally be in the same direction, but much weaker in magnitude, compared to darkness induction. This suggests that brown induction cannot simply be concluded as being darkness induction. These results help us to begin to understand the differences in the underlying perceptual and neural processes that mediate the two key properties of human vision, darkness induction and brown induction.

Though researchers have been studying marijuana for decades, little attention has been given to the role marijuana may play in an individual’s self-concept. Previous research has shown that drinking identity (the extent to which an individual views alcohol as part of his/her identity), as measured by the Alcohol Self-Concept Scale (ASCS), predicts alcohol consumption and alcohol-related problems. The current study sought to explore whether a similar pattern of results would be found for marijuana. Drawing from the ASCS, the newly developed Marijuana Self-Concept Scale (MSCS) seeks to evaluate how strongly individuals identify with marijuana. Stronger marijuana identity was predicted to be associated with more use and use-related problems. Men and non-college individuals were predicted to identify with marijuana more strongly than women and college students. Individuals of legal age (>21) were also hypothesized to be more likely to identify with marijuana. To test these hypotheses, participants (N=380, 54.8% female, ages 18-24) from the Seattle area completed a lab-based survey, which was a part of a larger study assessing the young adults’ developmental transitions and how those transitions relate to substance use. The MSCS and measures of marijuana use and problems were included in the survey. As expected, people who identified with marijuana tended to use more frequently and report more related problems. Men were more likely to identify with marijuana than women. However, we found no significant differences between non-college individuals and college students. In addition, being of legal age to use marijuana did not reveal a significance in identity. Findings suggest the MSCS may be a useful screening tool in future research and/or clinical application. By examining the relationship between marijuana identity and its subsequent use, we may also have the potential to develop more effective therapies and interventions for those who are at risk.

The switch from vegetative to reproductive development in flowering plants is tightly regulated by a complex gene network, yet little is known about how this network evolved. The Arabidopsis thaliana flower meristem identity gene LEAFY (LFY) encodes a transcription factor that activates floral organ identity genes to initiate flower development; loss of function mutants develop leaf-like structures in place of floral organs. Genes homologous to LFY have been identified in representatives of more basal land plant lineages, including gymnosperms, ferns, and mosses. Moss LFY functions in early sporophyte development; knock-outs result in the zygote being arrested at the single-cell stage with no further development. To investigate the evolution of LFY, we have generated transgenic lines in the emerging model fern Ceratopteris richardii that use RNAi to silence CrLFY expression. We have validated lines with reduced LFY expression by RT-PCR showing a delay in sporophyte development and distinct leaf development. Our working hypothesis is that fern LFY may be crucial for early sporophyte development. To find evidence for this, we are currently analyzing gametophyte and early sporophyte development in detail using microscopy to determine the timing and location of the developmental defect (i.e. in gametangia, during fertilization, or in the early cell divisions of the zygote). Our research seeks to bridge the evolutionary gap between LFY function in moss and flowering plants by looking at its function in ferns (sister taxa to all seed plants) and to ultimately investigate how it became specified for flower development.

Sea otters (Enhydra lutris spp.) play a significant role as a keystone species in shaping nearshore marine ecosystems of the northern Pacific coast through their primary diet of bottom-dwelling invertebrates, like sea urchins. Northern sea otters (Enhydra lutris kenyoni) were hunted to extinction from Washington State in 1911 due the fur trade of the 18th and 19th centuries, and remained locally extinct until their reintroduction to Washington State from Alaska in 1969 and 1970. We studied the foraging ecology of this species since this reintroduction, specifically how the diversity of prey species consumed by sea otters has changed from the 1990s to the present day. The prey switching hypothesis suggests that predators preferentially consume the most abundant prey species, then as a given prey item becomes rare, the predator will add a prey item of the next highest rank to their diet, increasing their diet diversity. Based off of this hypothesis, we expected sea otters in the 1990s to consume a lower diversity of prey species compared to recent years. Using sea otter diet data we collected from 2014-2016 as well as data collected by the U.S. Geological Survey from 2010-2011, we compared prey species diversity from 1990s data to present day data with the Shannon-Wiener Diversity Index. Preliminary analyses suggest that sea otter diet in the 1990s consisted primarily of clams, while it consisted primarily of snails in the 2010s.

There are many mutualistic interactions that occur naturally in the world. Some of these mutualistic relationships evolve to be obligate, where the presence of one species is dependent on the other for its survival. We study the evolution of an obligate relationship with two model organisms, a sulfate reducer, Desulfovibrio vulgaris, and a methanogen, Methanococcus maripaludis. 22 populations of these organisms have evolved in an environment that has forced their cooperation. When we sequenced these populations, we discovered that at least 13 of the 22 populations of D. vulgaris have lost the ability to reduce sulfate To confirm if these mutations are beneficial or not, we measured the fitness of mutants with knockouts in the sulfate-reducing gene pathway relative to the wild-type strain with either an ancestral or evolved M. maripaludis partner. The final population size of each D. vulgaris mutant will be measured using FREQ-Seq; a technique that quantifies frequencies of various mutations in an amplicon. In previous iterations of this experiment, results presented a discrepancy between replicates (I and II) due to a failed cell lysis technique. We found that only a small fraction of the cells were lysed. With a better cell lysis protocol, we expect to see an increase in D. vulgaris mutant frequencies when paired with either an ancestor or evolved M. maripaludis compared to the wild-type. The data collected from these experiments will help us have a better understanding whether losing the ability to reduce sulfate is a beneficial mutation for D. vulgaris or not.
 

Cryopreservation is a technique that uses very low temperatures in order to store and preserve biological material for an extended period of time without deterioration. With the use of cryoprotectants, a substance that prevents the damage of freezing, cells are protected from the normally lethal effects of freezing. In order to understand the effects of cryopreservation in marine microorganisms, we are developing this technique with Phaeodactylum tricornutum, a marine pennate diatom with a sturdy body type. Diatoms are important drivers for biogeochemical cycles and the versatility of Phaeodactylum tricornutum in different environments allow it to be used as a model organism in studies about global phytoplankton primary production. By using a cryoprotectant such as DMSO, and a two-step freezing protocol, we allow the cells to experience freezing at a gradual rate giving it as stress-free of an environment as possible. Once frozen, the Phaeodactylum will be preserved until the thawing process begins, using a protocol that allows the rate of thawing to be gradual as well. This technique could allow for successful preservation and could be applied to many aspects of biology, in our case a laboratory model for ecological work.

When isolated subpopulations adapt to the particular features of the predators, prey, or cooperative partners that are specific to their local environment, populations may become specialized to their local partner and less able to interact with other populations. To what extent might this hypothesis explain the diversification of microbial species, which cooperate or compete with each other in complex communities? We tested whether specialization was a common outcome of evolution between the bacterium Desulfovibrio vulgaris and the archaeon Methanococcus maripaludis after they evolved together in a beneficial interaction for 1000 generations. To test if D. vulgaris populations tended to become specialized for the M. maripaludis populations they evolved with (and vice versa), we revived 6 independently-evolved communities, separating the D. vulgaris and M. maripaludis populations from one another, and then paired each population back up with its sympatric partner (evolved in the same community) or with 5 allopatrically-evolved partners (evolved in different communities). We used microscopy to quantify the density of D. vulgaris and M. maripaludis to test whether the absolute fitness of either species was higher in sympatric versus allopatric pairings. We also competed allopatrically-evolved and sympatrically-evolved D. vulgaris against one another. We conclude that variation across cocultures in population yield of D. vulgaris or M. maripaludis is not necessarily correlated with whether they evolved in sympatry or allopatry, suggesting that the first 1000 generations of adaptation to a new mutualism may not be characterized by specialization. Our results show that some sympatric pairings were more efficient than allopatric pairings;however no distinct trend was identified. Broader implications of our research include roles in  byproduct inhibition which is an important factor in global carbon cycling within diverse anaerobic environments, including bio-digestion for fuels, wastewater treatment, and experimental evolution. 

Do species evolve in diverse communities, similar to the simple community of our research? We are looking at the evolution that we've seen in a species of sulfate reducers found in our simple laboratory environment and comparing this evolution to that of a large, more complex anaerobic digester of the wastewater treatment plant. In our 'simple community', a methanogen, Methanoccocus maripaludis, and a sulfate reducer, Desulfovibrio vulgaris, were paired. When two species are forced to live together over time we expect to see evolution take place. It was found that after 1000 generations D. vulgaris loses its ability to reduce sulfate in this paired relationship. Using this laboratory pairing as a model, we have looked at the evolution of sulfate reducers in  these two different anaerobic digesters from the University of Washington and Brightwater Municipal Waste Water Treatment Center. Both digesters have similar conditions; they are both anaerobic with low levels of sulfate present. To test our hypothesis we have currently been extracting DNA from weekly samples of both digesters. The dsrAB gene is first amplified, as it is a conserved region throughout sulfate reducers and their relatives. We are now doing Illumina sequencing in order to better determine the diversity of sulfate reducers present. Using this information, we will be able to design primers for the sat apsA and apsB genes. This will show us if any mutations are present among the population of sulfate reducers in both digesters. This will be means for the comparison between real world and experimental conditions. Future work will look for mutations in the sat, apsA and apsB genes. By identifying these loss-of-function mutations in both the digesters, we will be able to see the impact of evolution on sulfate reducers in complex communities.

An ion trap uses electromagnetic fields to trap charged particles in vacuum. We use an ion trap in this way to implement qubits (quantum bits) for quantum operations. Barium (Ba) and ytterbium (Yb) are promising candidates because their photoionization and cooling laser, optics and fiber optics are commercially available and in the visible spectrum. The micro-fabricated surface ion trap confines long chains of ions which are potentially scalable for running real and useful quantum algorithms and building a quantum computer. The current trap can confine Ba-Yb/Ba-Ba ion chains in a harmonic oscillator. As the number of ions increases, the ion chain will form a zigzag pattern and make uniquely addressing an individual ion for quantum gates with a laser beam impossible. But, quartic anharmonic oscillator can produce equal ion-ion spacing. My project can minimize quantum operation errors for quantum gates operation. I find the solutions for the DC electrode voltages numerically, using and improving the existing Boundary Element Method to generate a quartic anharmonic trap. Then, starting from the initial harmonic trap, I use the solution to morph the real trap potential into the quartic one. The changes of ion-ion positions are compared and verified using an EMCCD camera. Then, I determine the resonant frequencies of the ion chains in both the original (harmonic) and the modified (anharmonic) traps. The resonant frequency stays the same for the harmonic trap, but decreases in the quartic anharmonic trap as more ions are added to the chain. In conclusion, the quartic anharmonic trap has constant ion-ion spacing and overcome the zigzag issue which increases our ability to control qubits for quantum operations.

As a consequence of unhealthy lifestyles, the prevalence of hypertensive heart disease is rising both in the United States and globally. Hypertension often results from the chronic activation of adrenergic receptors found in the walls of blood vessels. These receptors are normally activated in times of extreme stress by the sympathetic nervous system. However, chronic activation leads to elevated blood pressure which puts extra stress on the heart and can lead to complications that kill millions of people annually. Drugs such as beta-blockers treat this disease by blocking the endogenous ligand, epinephrine, from binding to the receptor, which relaxes the blood vessels and lowers blood pressure. However, this class of drugs ignores another important receptor found in vessel walls, the alpha-1D adrenergic receptor (ADRA1D). ADRA1D is known to contribute to hypertension and benign prostatic hypertrophy in humans, but its mechanism of action is poorly studied. The Hague Lab conducts pharmacological studies on ADRA1D to elucidate how this receptor confers disease. My project focuses on understanding how the receptor localizes to the surface of the cell where it can be activated. Recently, our lab conducted proteomic screenings to identify intracellular binding partners. The results showed that ADRA1D binds to two unique proteins, a signaling protein, alpha-syntrophin (SNTA1), and a cellular polarization protein, Scrbble (SCRIB). This interaction was later attributed to a conserved binding region known as a PDZ domain that interacts with the C-terminus of ADRA1D. SNTA1 has one of these domains while SCRIB has four. My project attempts to quantify the strength of these interactions to give a better understanding of ADRA1D’s intracellular landscape. By better understanding how this receptor locates to the surface and how it signals, drugs can be created to disrupt these processes and better treat patients with hypertensive heart disease.

In 2015, the American Heart Association (AHA) stated that sudden cardiac arrest is still a primary cause of death in the United States. During cardiac arrest, lack of intervention to maintain the perfusion of tissues with oxygenated blood leads to irreparable damage to the brain and vital organs within minutes. In 2010, the A-B-C (airway, breathing, circulation) of CPR changed to C-A-B (circulation, airway, breathing) due to strong evidence that supports the idea that high-quality CPR chest compressions help sustain life until the underlying causes of cardiac arrest are addressed. The purpose of this study was to use technology to measure and review chest compression data collected during cardiac resuscitations, and evaluate specific compression parameters, in relation to AHA standards, for overall quality improvement. The CODE-STAT™ technology used in this study measured changes in thoracic pressure during a cardiac resuscitation, and then generated a report stating rate of chest compressions per minute, and total time spent on chest compressions. The data was then given to the hospital resuscitation teams so that they could visualize and discuss the quality of their performance in comparison to AHA and hospital standards. Thus far, results of the study have shown that in-hospital resuscitation teams have compression rates that are too fast and there are too many pauses, or times without chest compressions. A compression rate above 120 beats per minute can decrease sufficient blood return to the heart from the rest of the body, making subsequent compressions less effective at dispersing blood. Too many pauses in chest compressions can negatively impact the pressure gradients in the thoracic cavity that are required to maintain adequate cerebral and coronary blood flow. Hospitals must continue to implement technologies that measure CPR performance so that cardiac arrest patients receive the best possible outcomes for survival.