Living organisms are composed of cells; thus, cells are called the basic building blocks of life. In order for an organism to grow, cells undergo mitosis. During mitosis, chromosomal DNA is replicated and segregated into two daughter cells. Errors in segregation can lead to the development of some forms of cancer. Because of this, equal chromosome segregation is a critical mitotic process. Microtubules are fibril-like cytoskeletal structures that are responsible for chromosome separation. They originate from two centrosomes. The growth and extension of microtubules push the two centrosomes apart, creating two poles in a mitotic cell. Kinetochores bind to these microtubules and align at the center of the cell. Disassembly of these microtubules is coupled to the equal segregation of sister chromatids. In Saccharomyces cerevisiae, correct kinetochore-microtubule attachment is mediated by the Ndc80 and Dam1 complexes. The heterotetrameric and rod-like Ndc80 complex is the main microtubule-binding component of the kinetochore. The heterodecameric Dam1 complex forms oligomeric rings around microtubules and enhances the microtubule-binding capacity of the Ndc80 complex. In vitro, the Ndc80 complex bridges two Dam1 complex rings, yielding a specific 33 nm inter-ring distance. To investigate the importance of this distance, we constructed various mutant Ndc80 complexes to further increase the Dam1 complex inter-ring distance. Specifically, each Ndc80 complex mutant contained an 8, 10, or 12 heptad repeat insertion that elongated the length of the coiled-coil domain of the Ndc80 complex by 8, 10, and 12 nm, respectively. We tested if these mutant Ndc80 complexes support cell division by transforming a yeast strain that does not contain wild-type Ndc80 gene and selecting for growth on SD-ura low adenine plates. These produced non-sectoring red colonies; therefore, the constructs did not support cell division. This suggests the specific 33 nm Dam1 complex inter-ring distance is important for cell viability.

Adrenergic receptors, a family of G-protein coupled receptors, are a common target by pharmaceutical drugs for cardiovascular disease. With cardiovascular disease being the leading cause of death in the United States, adrenergic receptor targeting drugs are increasing in use. Phenylephrine has long been known to be an agonist of the specific α-1 adrenergic receptor. Past research shows that cancer cells do not contain α-1 adrenergic receptors. Although, using dynamic mass redistribution (DMR), when we introduced U251 human glioma cells to increasing concentrations of phenylephrine, we found evidence of a conformational change in the U251 cells. Other adrenergic receptor agonists (norepinephrine, isoproterenol, and clonidine) exhibited a similar response. This suggests that U251 cells may have α-1 adrenergic receptors. We hypothesize that U251 glioma cells exhibit a structural change in the presence of phenylephrine because they contain α-adrenergic receptors that recognize the ligand and trigger a cellular response. The effects of phenylephrine on U251 cancer cell viability and cell proliferation is still unknown, allowing for a possible future in generating therapies to combat cancer.

Approximately 40% of over-the-counter drugs target G protein-coupled receptors (GPCRs), which are seven-transmembrane proteins that become activated when drugs, hormones, or neurotransmitters bind to the receptor. One class of GPCRs, known as adrenergic receptors (ARs), are highly targeted by the endogenous catecholamines, epinephrine and norepinephrine. ARs are comprised of three unique subtypes: α1ARs, α2ARs, and βARs. Focusing on the α1ARs, the different types are α1A, α1B, and α1D. In cancer biology, most research has focused on βARs since many people take medications that specifically target βARs. In my research, I focus on α1BARs due to a recent publication of The Hague Lab, which recently discovered α1BARs in colorectal cancer cells. Reading this publication led me to search for α1BARs in various cancer cell lines by using the Label Free Dynamic Mass Redistribution (EPIC-DMR) method. After adding specific drugs, the EPIC-DMR tracks any conformational change of individual cells to indicate the cellular response to the drugs. Using this method, I discovered α1ARs are present in HCT116 cells, a colorectal cancer cell line. In the future, I intend to determine the α1AR subtype by performing radio-ligand binding, using Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR), and looking at agonist and antagonist responses. This discovery could potentially play an important role in therapeutic drug development for many cancer patients.

Anterior cervical discectomy and fusion (ACDF) is a well-established surgical technique used for the treatment of various disorders of the cervical spine. The two most common ACDF procedure methods are: standalone systems and traditional plating techniques. Dysphagia, the inability to properly swallow, is an extensively described complication; it has been proposed that mechanical irritation, additional dissection, or displacement of the esophagus by plate placement may contribute to a greater incidence of postoperative dysphagia. The aim of this retrospective cohort study is to assess differences in pain, clinical outcomes, and dysphagia, including complication and readmission rates, following ACDF using either a standalone cage system or a cage with anterior plating (traditional plating). Between 2014 and 2015 we identified 377 consecutive patients meeting study criteria (standalone, n= 211; plate and cage, n= 166). Pre-operatively scores were collected for patient specific characteristics, surgical characteristics, and Numeric Pain Rating Scale (NRS). In addition, complication and readmission rates, the Dysphagia Disability Index (DDI), and NRS scores were collected at one year and two years post-operatively. There was significantly greater improvement in neck pain scores for the plate and cage group after one and two years post-surgery ( 2.3 and 3.1 versus 1.6 and 1.5, respectively; p <0.01). This could presumably result from biomechanical advantages when compared to stand-alone cage systems. There was no statistical difference between DDI scores either postoperatively or at two years post-surgery. These results indicate that mechanical irritation may not differ between procedure types. Further research is needed in order to determine factors that may lead to a higher rate of nonunion for patients undergoing standalone cage placement. 

The Westphalian state system—i.e., the global division of power in to roughly 195 sovereign nation-states—is proving inadequate in the face of rapid globalization. As our world becomes increasingly integrated, the political tools of the past, which uphold division and absolute autonomy, cannot offer a viable vision for the future. This is particularly salient in the case of global climate change: our commitment to statehood and international competition is preventing us from seeing the ways in which we are deeply interconnected, effectively imperiling current and future generations. Indeed, how should we respond to the 200-250 million climate refugees that will be displaced in the next 50-70 years due to climate change? What duties of justice are they owed? In this project, I assess the social contract tradition in political theory which offers the main philosophical grounds for the contemporary state system. I argue that, for a number of different reasons, social contract theory fails to offer a desirable framework for the new global political order necessitated by globalization. A theoretical framework that recognizes the ways in which humans are fundamentally in nature, and thus widens the scope of justice, offers a more promising alternative for regulating global cooperation. Indeed, if we, the human race, see ourselves as a unified group playing a small part in the larger play that is the global ecosystem, then our vision will clear and we will begin to see the appropriate duties of justice that we owe to each other, nonhuman animals, and nature.

Adverse Events (AEs) are detrimental medical incidents in a research participant that may or may not be related to the investigational agent. During clinical trials, physicians are required to document attribution of AEs with limited guidance regarding how to assign attribution. The objective of this study was to determine the accuracy of physician based attributions for AEs occurring in cystic fibrosis (CF) patients participating in blinded randomized controlled trials (RCTs). This retrospective study pooled clinical trial data from 4 CF RCTs; all participants who experienced ≥1 AE were included. A repeated measures logistic regression model using generalized estimating equations and adjusted for baseline demographic variables (gender, age, CF genotype), comorbidities, and lung function (forced expiratory volume in one second; FEV1). The primary predictor of interest was receipt of study drug versus placebo. The primary outcome of interest was physicians’ determination of the AE as related to study drug. Study patients were young with mild lung disease. Receipt of study drug was not significantly associated with the likelihood a physician would deem an AE to be related to study drug. However, we found significant associations between age, gender, and FEV1 and our outcome of interest– presence of an AE felt by a physician to be related to study drug. These findings suggest a biased assessment of physician attribution. Physician assessment of relatedness in RCT’s is a key requirement per the Food and Drug Administration (FDA). We found no significant association between AEs coded as related to study drug and actual study drug receipt. Interestingly, we found other associations implying an intrinsic bias in physician assessment based on age, gender and disease severity. Further research may clarify whether changes are needed to current FDA guidelines.

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.

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.

In Kenyan HIV-1 infected children, it is likely that exposure to household indoor air pollution (HAP) increases susceptibility to respiratory illness, cancer, growth failure, and compromised cognitive abilities. Kenyan families usually live in one-room houses and use wood or charcoal cook stoves indoors. Smoke from burning of wood and charcoal contains large quantities of polycyclic aromatic hydrocarbons (PAHs) which are among the many components of incomplete combustion. Many PAHs are carcinogens that undergo metabolism and are excreted in urine. In order to assess children’s exposure to wood and charcoal smoke, single spot urine samples were collected from 100 subjects: 50 recently diagnosed HIV-1 infected children, age-matched healthy HIV uninfected children, and their mothers. Urine samples were hydrolyzed by treatment with glucuronidase enzyme and cleaned up with solid phase extraction. High-performance liquid chromatography with fluorescence detection was used to analyze the samples for PAH metabolites. We anticipate that urinary hydroxyl PAH metabolite concentrations will be high in these cohorts of HIV-1 infected and healthy children reflecting the exposure to HAP by use of wood and charcoal for cooking. Findings of this research will help to understand the pathogenesis of cancer and respiratory diseases in HIV-1 infected children, healthy children, and their mothers.

This project focuses on adding unique telemetry and communication capability to one of the largest and fastest growing areas in the Washington State aerospace industry: Unmanned Aerial Systems (UAS). These systems have the potential to revolutionize a wide variety of commercial applications such as precision agriculture, disaster management, mapping, and ecological/biological monitoring and represents a $13.6 billion civilian market. One of the main challenges for UAS integration is to provide reliable and effective guarantees for safe operation that in turn requires high-performance and flexible control/navigation of the aircraft and reliable transmission modes from air to a ground control station or operator. For example, a typical commercially available UAV will have 3 distinct sensor-processing-radio chains, one each for onboard camera, one for data telemetry (navigation and strategic command) and one for primary command & control data. This effort fundamentally seeks to mature ongoing work at UW and will allow for greater communication bandwidth, enhanced flexibility, and levels of control between the UAS and GCS and achieve the required situational awareness as mandated for UAS operations. The outcome of this project will be a modular, encapsulated radio system that can be used to combine the myriad data links between a UAS and the GCS into a single, robust, and reconfigurable link. The link quality will be tested using a bladeRF software defined radio paired with a student built, Pixhawk based octocopter. Several flights tests will be conducted by students using custom built software which will measure the strength of the UAV based link in various environments. This project encapsulates the construction of the octocopter, the build of the software defined radio system, and flight testing. A publicly accessible link quality database will be populated with fight test data in order to encourage further development of software defined based communication links.

The beta-amyloid (Aβ) peptide, implicated in Alzheimer’s Disease, forms toxic aggregates known as oligomers that cause brain degeneration. This aggregation process can be inhibited by designed peptides that have a novel structure, known as the alpha-sheet. This structure is similar to a beta-sheet except that the carbonyls all point in one direction and the amine groups in the other, generating a molecular dipole moment. The Daggett group hypothesizes that at some point, Aβ undergoes a conformational change that gives it this alpha-sheet character and the resulting molecular dipole causes Aβ monomers to be attracted to each other and form oligomers. There is also evidence to suggest from simulation that this structure occurs in many other amyloid-related diseases. As such, designed peptide inhibitors from the Daggett group all target this structure. One of the control designs, AP3, aggregates much more rapidly than Aβ does under acidic conditions. My project involves using AP3 to probe the reasons for Aβ toxicity as well as its aggregation mechanism – both of which are not understood. The residues involved in the turn sequence of AP3, which is hairpin peptide, are of particular interest due to their propensity to display β-sheet character. After synthesizing these “turn sequence” peptides, I found that the presence of these turn sequence peptides can increase the speed of AP3 aggregation, which is an analogue to the seeding of amyloids. I am characterizing these turn sequence peptides using circular dichroism to observe their secondary structure. In addition, I along with other undergraduates in the lab have optimized ELISA to quantify the interactions between our designed peptides and another peptide of interest. The behaviors of these turn sequence peptides as shown by characterization and various assays prove enlightening in understanding the pathology of amyloidogenic diseases.

Despite our society’s growing awareness that transgender people exist, transgender children remain an understudied group that falls victim to huge amount of misunderstanding in both the scientific community and the public sphere. While academic literature is decidedly mixed on how to best support children who express a cross-sex gender identity, a growing number of parents have begun to support their children in social transitioning, a process that allows the child to live as their identified gender in all social contexts. The objective of this study was to understand the impact of social transition on children’s mental health using structured interviews with the parents of socially transitioned transgender children. We focused on parent’s report of how transgender children’s mental health (child’s mood, anxiety symptoms, and externalizing behaviors) changes over time. We recruited 60 parents of transgender children whose child socially transitioned between the age of 3 and 12. To maximize representation and generalizability, we then stratified them into subgroups by age at social transition (3-5; 6-8; 9-12); gender (girls; boys); and race (white; non-white). The interview was structured around three moments in the child’s life: the first sign of gender non-conforming behaviors from the parent’s perspective, the parent’s first realization that the behaviors might signify something more deeply about the child’s stable identity, and the age at which the child socially transitioned. The interviewer asked the same set of questions regarding the child’s mental health condition at the first sign, at first realization, during the year before transitioning, and during the year after (if applicable). Two coders then coded the wellbeing of each child in each of the mental health domains at each time point. Findings of the study will have implications for the developments of research and clinical work as how to best support transgender children regarding their social transition.

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.

Duplex Sequencing (DS) is a Next-Generation Sequencing technology employing double strand molecular tagging to reduce technical errors.The high accuracy of DS enables the resolution of low frequency mutations in nuclear and mitochondrial DNA (mtDNA). We are interested in using DS to analyze mtDNA mutations in ulcerative colitis (UC), an inflammatory bowel disease that predisposes to colorectal cancer. Tumor progression in UC is related to mitochondrial dysfunction. By sequencing mtDNA mutations in colonic biopsies from UC patients, we aim to elucidate the role of these mutations in tumorigenesis. A critical limitation, however, is the high number of low frequency mtDNA mutations present in these biopsies and the lack of appropriate computational methods to process large amounts of information generated by DS. The goal of this study was to develop computational methods to enable the extraction of mtDNA mutation information from DS data. DS identifies the reads corresponding to each strand of DNA by grouping them by their unique tags, making single-strand consensus sequences (SSCSs), which are then paired to form duplex consensus sequences (DCS). SSCS reads remove technical errors, but contain errors caused by DNA damage. The first computational method that we developed compared sequencing information from SSCS and DCS reads to assess the level and type of DNA damage in each sample. Other computational methods consisted of a program to classify mutations according to their sequence context, and a script that calculates mutation types and frequencies inside and outside the D-loop region as well as in all mtDNA genes and allows comparison of these parameters in large batches of samples. These tools will enable the processing of mtDNA DS information in a fast and high-throughput way, leading to assessment of the range of different types of mtDNA mutations in the progression of ulcerative colitis and colorectal cancer.

Airborne and satellite remote-sensing surveys of Antarctica have revealed over 400 subglacial lakes beneath the ice sheet. These lakes are one component of a subglacial hydrology network that also includes drainage channels, swamps, and estuaries. The influence of this subglacial hydrology network on ice-sheet dynamics is unclear. Water under ice sheets can reduce friction at the ice-sheet base, and thereby increase ice velocity. Alternatively, partitioning of water into lakes or channels, can also limit the area lubricated by water, and thereby decrease ice velocity. Ice-penetrating radar can map the presence of subglacial water due to the differing electrical properties of an ice/water vs. ice/sediment (or rock) interface at the ice sheet bed. The radar surveys also map constant time (isochronal) layers within the ice sheet that are formed due to chemical (and thus also electrical) differences in the snow deposited at the ice-sheet surface, which is eventually compressed into ice. Burial by subsequent snowfall and ice flow deforms these layers, which were originally surface parallel. Thus mapping these layers reveals the internal stress state and flow history of the ice. Here we report on the internal architecture of the ice sheet in the vicinity of Subglacial Lake Whillans, West Antarctica, as imaged by ice-penetrating radar. In the 2010-2011 austral summer, 500 line kilometers of ice-penetrating radar data were collected over Subglacial Lake Whillans in support of subglacial lake access drilling. In radar profiles (2-dimensional cross sections through the ice), we trace isochronal, internal layers imaged by the radar. Compared to nearby grounded areas, layers in the ice over the lake are highly deformed, suggesting ice-flow disruption over the lake. This interpretation is consistent with the active nature of the lake, which fills and drains every few years, causing changes in friction at the ice-sheet base, and thus non-steady ice flow.

Working memory is a cognitive skill that allows temporary storage and manipulation of information. It is an important skill as previous studies have shown relations between working memory and various cognitive abilities, such as reading comprehension and speech perception. So far, evidence have suggested that working memory can be enhanced with training. One of the training experiences that has been extensively researched is early music training. Most studies have shown that music training experience has a positive correlation with working memory while other studies has shown no difference in working memory capacity for musicians and non-musicians. For this study, our aim is to further delve into this controversial topic and seek more information and data to find out whether early music training could enhance working memory. We recruited a group of musicians (N=20) and non-musicians (N=20) and compared working memory capacities between the two groups with two tasks: Memory Updating and Operation Span, targeting slightly different aspects of working memory. We hypothesized that those who had early music training would have higher scores on both tasks. Data analysis has been completed and the initial results from the data shows that there is no significant difference in working memory capacity between musicians and non-musicians. Currently, further analysis of data is still ongoing to help understand the results we observed. We will be discussing the similarities and differences of the tasks compared to previously published studies and how our results contribute to this field. 

Prior research has demonstrated that there is a relationship between sleep and academic performance, yet few studies have assessed the role of stress on these variables. It was hypothesized that stress would act as a mediator between sleep and academic performance. Seventy-six undergraduate students made up the preliminary sample from a public university in Washington. Participants completed an online survey which asked demographic questions, including self-reported GPA. Additionally, students completed the Perceived Stress Scale and the Pittsburgh Sleep Quality Index. The preliminary regression analysis indicated a nonsignificant effect of sleep on GPA, R² = .03, F(1, 74) = 2.13, p = .15. The effect of stress on this relationship was also a nonsignificant effect, R² = .01, F(1, 73) = .84, p = .36. However, a power analysis indicated a minimum of 107 participants is necessary to reach beta of .95, alpha = .05, with an effect size of F² =.15, the number of predictors at 1 for level A and 2 for level B. Correlations show predicted, but nonsignificant, relationships between GPA and sleep (r = -.17, p = .07) and GPA and stress (r = .01, p = .45), with a moderate correlation between stress and sleep (r = .47, p < .01). The current sample is underpowered, making it difficult to confidently draw conclusions. Participants will continue to be recruited but if power is met and results continue to be nonsignificant, it is probable that this is due to GPA failing to have a normal distribution for each level of the independent variable. The lack of heterogeneity in GPA would then raise concern. If the necessary sample size is met and results are significant, this is likely due to poor sleep increasing student’s susceptibility to high levels of stress, which consequently leads to a decline in GPA. 

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.

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.

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.

Many court cases in the United States are resolved through plea bargaining. A plea bargain is an agreement between the prosecutor and the defense for a reduced sentence in exchange for a guilty plea from the defendant. This study hypothesized that a) strength of evidence, b) client acknowledgment of their substance use problem, and c) substance use rehabilitative history would predict attorney recommendations for taking a plea bargain. Participants were recruited through the Washington State Bar Association and included 491 active and retired attorneys. Each received an informed consent and one of eight counterbalanced vignettes. In this 2x2x2 between subject’s design, a potential client was described and the possibility of conviction was manipulated based on strength of evidence, defendant’s acknowledgement of a substance use problem, and prior history of substance abuse treatment. 91 participants had completed surveys and identified as Public Defender (51%), Private Defender (34%) and both (15%). This study found a significant interaction η2 = .08, F(1,85) = 8.12, p < .01 and three significant main effects that indicate defense attorneys consider all of the aforementioned variables when making recommendations that clients accept a plea of diversion to treatment. They placed greatest importance on clients’ rehabilitative histories η2 =.48, F(1,85) = 77.47, p = .00, followed by acknowledgements of substance use problems η2 = .26, F(1,85) = 30.24, p = .00, and then strength of evidence η2 = .19, F(1,85) = 19.52, p = .00. A possible limitation, though anonymous, the self-report nature may have been impacted by participant’s social desirability. This study had a strong counterbalance design, which eliminated the potentially confounding impact of order effects. Future research would benefit from investigating differences factors that may be significant to prosecutors offering a plea and moderating or mediating factors involved in this decision-making process.

Amongst the graffiti blanketing the cities of Athens and Berlin, depictions of children stand out as a solemn reminder of the dire nature of the current refugee crisis in Europe. Due to the large number of refugees passing through, or settling, in various European states, governments have reduced individuals to statics. This data-driven approach, focused on the number of people and the cost of providing basic resources, dominates the news and international discourse. Combating this statistical discussion, artists have created works that demonstrate the humanity of the crisis. Because paint and printers are easily accessible, street art portrays a broader public sentiment towards the crisis, countering the governmental narrative. This project examines graffiti in Athens and Berlin, and the insights it provides on the refugee crisis that cannot be derived from the official discourse. Using images of children, artists in both cities have manufactured an alternative narrative articulating the challenges facing the individuals involved in the crisis and highlighting their struggle in Europe. Because Athens and Berlin function as endpoints of the refugees’ journey, as many enter through Greece before traveling northwest to Germany, a study of the graffiti responding to the migration in these cities is of particular importance. After collecting hundreds of images in-situ during July of 2016, using geo-tagged web-based content and information from locals on hubs of graffiti, several themes emerged, the most prevalent being representations of children. The lack of scholarship on graffiti pertaining to this refugee crisis necessitated the use of art historical images of children in conjunction with current graffiti scholarship to create a methodological framework. Employing the iconographic, semiotic and socio-economic methodologies, I conclude that the works promote the humanization of the crisis and force recognition of the refugees as individuals, thereby opposing the governmental narrative.

Many scholars have constantly claimed that a shift from defined benefit plans to defined contribution plans has been the wrong course of action in policy and it has brought direr effect to the economy. Economists have argued that defined contribution plans are generating inefficiency pension income for employees in the private sectors, creating further cost burden for taxpayers and discouraging employees to stay at a job. While most observers are accepting the current situation by sticking to the traditional defined benefit plans, there are significant indicators in which the shift to defined contribution plans is inevitable. Economic shocks such as financial crisis, new health policy like Affordable Care Act and Congress abolishment of the Aid to Families with Dependent Children federal assistance program have changed the tides. They have brought a great deal of attention on how defined contribution pension plans can shape agents’ behavior in the labor market. Using datasets from the Survey of Income and Program Participation (SIPP) Synthetic Beta file and the National Quarterly Workforce Indicators, this paper examines the proposition that defined contribution plans motivate newly hired workers to stay at a job for a much longer period and gain higher weeks at pay without federal assistance programs taken place. This occurs particularly for male employees in public sectors. Regression analysis (OLS and logistic) demonstrates that workers enrolled in defined contribution pension plans have increasing personal level annual earnings taxed under FICA, higher probability in having employer- based health insurance and a larger family size in their individual households. Furthermore, this paper attempts to explain whether defined contribution plans have transitory or permeant effect on workers’ wellbeing during economic shocks using unit root test to determine whether there exists a stochastic trend.

Keywords: defined contribution pension plans, labor market, individual households, workers

Cultural festivals provide the opportunity to share and celebrate ideas, values, art, food, etc. from the heritage of other homelands. Aside from other smaller gatherings, these mass public festivities are typically held only once a year. With such a limited window for this type of cultural sharing, it is ideal that people of the represented community share fully their experiences of their culture and have the accommodating infrastructure to do so to increase cultural competency. Creating inclusionary spaces that allow cultural expression and celebration is termed cultural placemaking. After reviewing issues in Journal of Planning Education and Research from 2017-2010 I discovered that cultural placemaking was well intentioned but was not well reported or practiced. This is an issue when communities are projected to increase in density and in diversity. It is important for planners and city officials to know the significance of public spaces that allow for cultural expression in order to increase community development and cohesion as communities continue to grow. Through literature review, attending several festivals in Seattle, and interviews of students and festival participants, cultural festivals can provide a deeper understanding of culture instead of perpetuating stereotypes. The purpose of this project is to reintroduce the significance of cultural festivals to planners and city officials in the form of a white paper that also provides some suggestions to help local festival coordinators increase local participation, community support and create meaningful experiences for both visitors and people with cultural heritage.

There is a current lack of art and outdoor environmental education within the public school system. Outdoor education refers to any topic that is taught outside, while environmental education concerns the natural environment and issues that pertain to the natural environment. Students are often taught about the problems that deal with our environment, though they are rarely taught to simply appreciate it. Art can be used as a tool to give students enjoyable hands on environmental learning in an outdoor setting. By using photography students are able to interact with their environment, show others how they see the world, and feel a deeper sense of appreciation towards nature. This project was inspired by the Literacy Through Photography program at Duke University and will address the question of how photography in an outdoor curriculum can increase a student’s environmental awareness within public schools. To begin answering this question I have compiled a comprehensive literature review and taught a pilot curriculum activity with a K/1 class at Leschi Elementary School that incorporates being outdoors, the environment, and photography. Preliminary literature review and observation results suggest that students enjoy interactive activities outside and that outdoor and environmental education is beneficial for student health, environmental awareness, and happiness. In the future, these findings could be studied further with more pilot programs in a variety of school locations, such as within intercity schools where nature is lacking.

Over the past ten years, esports, or competitive gaming, has come to the forefront of youth entertainment. In 2016, global awareness of esports surpassed 1 billion people. The largest segments of this population are college-age (18-25 years) and middle/high school-age (12-18 years). While significant activity is being undertaken by both universities and collegiate athletic conferences to develop varsity programs that structure the experience and growth of esports players, such efforts have not yet been made in high schools. My research clearly outlines the developmental impacts to students of varsity esports programs at the high school level. Based on the finding that such positive impacts scale directly with an administration’s support for the club, my paper provides recommendations on how administrators and teachers can support esports programs, even in the common case where esports is a foreign concept to staff. I draw on: 1) an extensive literature review synthesizing secondary research on the effects of participating in high school esports and ‘games in the classroom’ curricula, 2) programmatic recommendations based on analyses of similar extracurricular and youth development programs, and 3) observations and interviews with existing high school esports communities. The end goal of my research is to make high school esports a more productive developmental experience for high school students by de-stigmatizing and structuring participation. In addition, I highlight opportunities for further research into the mechanics and effects of high school esports programs.

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.

Organisms face a range of stressors over the course of their lifespans. As with most traits, we see significant variation in response to stress even between individuals within species. In our attempt to explain differences in stress responses between individuals, the Promislow lab studied the metabolome, the total profile of all small molecules in an organism, and found variation in stress response is correlated with variation in contents of the metabolome. Indeed, in a study of peroxide resistance in the fruit fly, Drosophila melanogaster, we found resistant flies tended to have increased galactose levels, compared to sensitive flies. This observation led to the novel hypothesis that flies fed supplemental galactose will have increased peroxide resistance compared to control flies. For this experiment, we chose flies among the most sensitive and resistant lines examined previously because the most sensitive flies, with low galactose levels, may show increased effects of supplemental galactose on lifespan. In this experiment, flies feed on food with varied galactose concentrations, including a negative control without galactose. We then place the flies into media with peroxide or water, the control. We record the number of flies that die in each condition multiple times a day, and the death rate provides a quantitative measure of peroxide resistance. By comparing the death rate of flies within a line kept in varying concentrations of galactose exposure, we can test the effect of galactose on resistance. If our hypothesis is correct, the flies receiving supplemental galactose should die at a slower rate than those without additional galactose. However, it is possible flies receiving supplemental galactose will die at faster rates, suggesting galactose makes flies more sensitive. Determining whether galactose makes flies more resistant or sensitive to peroxide may prove to be important in counteracting effects of oxidative stress in other organisms.

Development of antibody-based diagnostics, biomolecular sensors, and biomaterial with reactive surface requires the ability to control the adsorption of bioactive protein at a surface. All medical devices exposed in an in vivo biological environment are immediately coated with a layer of adsorbed protein that plays an important role in the body’s behaviors towards the devices and the effectiveness of the devices themselves. In this study, we use a multi-technique approach to characterize the effect of protein G B1 (6 kDa) orientation on multi-layer antibody binding. By conjugating a cysteine group at different positions along the protein’s surface, we can immobilize five variants of the protein (V21C, D35C, E42C, T11C, and WT) onto maleimide and bare gold surfaces. We have confirmed through X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry experiments that the proteins adsorb onto these surfaces. In addition, we introduced primary IgG and secondary F(ab’)2 antibodies to investigate if the protein’s orientation will affect the binding of the antibodies. The adsorbed mass are measured using quartz crystal microbalance with dissipation monitoring. We have demonstrated that the cysteine mutants orient better on maleimide surfaces and encourage more IgG binding. We will also study the effect of temperature on adsorption mass and extend the experiments to amine and carboxyl functionalized surfaces. Fully developed techniques to accurately characterize the orientation and quantify the amount of protein and secondary adsorption will significantly contribute to understanding the interactions of biomaterials with the biological environment.

Oxidative stress arises from an imbalance between reactive oxygen species and an organism’s ability to avoid or repair the damage caused by these reactive molecules. The fruit fly, Drosophila melanogaster, demonstrates varying degrees of resistance to oxidative stress. To understand the genetic basis of this variation, our lab analyzed a collection of inbred, fully sequenced Drosophila lines, the Drosophila Genetic Reference Panel (DGRP), and tested the associations between resistance and each of more than two million single-nucleotide polymorphism (SNP) markers throughout the genome. We found a particular SNP at the second chromosome located within the gene u-shaped (ush), had the strongest statistical relationship with oxidaive stress resistance. We then hypothesized that this gene contributes to the degree of resistance in the flies. For my research, I am evaluating this hypothesis by comparing the peroxide resistance of two sets of genotypes to confirm if the SNP at 2L:531863 bp contributes to the trait. For this comparison, I used two groups of lines, one with the ‘resistant’ and the other with the ‘sensitive’ version of the SNP, and distributed mated females from each line into vials with or without peroxide added into the food. I counted the number of dead flies at regular intervals and used these data to calculate the mean lifespan for each line, where lifespan is a measure of peroxide resistance. If both groups show similar lifespans, it suggests that this particular SNP does not affect peroxide resistance and I may test other SNPs at nearby loci with a similar procedure. However, if the two groups show significant differences in resistance, this result would support the connection between the SNP and peroxide resistance. I would then go on to manipulate the expression of ush using RNA interference to test whether changing mRNA levels impacts peroxide resistance.

Imidacloprid (IMI), a neonicotinoid, 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 million ppb in artificial seawater (SW), but not killed and subsequently recover. Our objective was to determine if emamectin benzoate (EB) is a better alternative to IMI. EB, the active ingredient (a.i.) in Slice(R), is currently registered for use in marine waters for the control of sea lice on farmed salmon. We simulated a 6-hour tidal exposure of adult non-gravid female ghost shrimp to concentrations of EB (as the insecticide Proclaim(R)) ranging from 0.01 to 100 ppb a.i. within artificial seawater alone or sediment + seawater. No treatment-related mortality was observed within the 96-hour test, but overt effects were observed, particularly at 100 ppb in both seawater (abnormal body position) and sediment (lethargy, inability to burrow). Results suggest that the 6-hour exposure was not sufficient to cause mortality. In a subsequent 96-hour test, we will expose the shrimp to EB mixed directly into the sediment simulated tidal cycle. As the shrimp re-establish their burrows, we anticipate increased exposure to the chemical and mortality. EB is more effective as a pesticide when ingested and, in comparison to IMI, targets the primary neurophysiology of crustaceans including sea lice and burrowing shrimp.

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.

For our Jackson School of International Studies capstone class, Task Force, we created a strategic communications plan tied with a digital story to create a social media campaign. The goal of this campaign is to communicate Tribal Treaty Rights between tribal and non-tribal millennials in the Puget Sound area. The Treaty Rights target issues in the Puget Sound caused by climate change, including decreasing salmon populations and managing non-tribal fishing rights around the region. Most millennials are unaware of these rights, or these issues in general, despite living in the Puget Sound. Our research, primarily self-hosted surveys and focus groups, show a trend in millennials to be sensitive to cultural diversity and climate change acceptance, making them a prime demographic for a marketing campaign. After data collection, we created social media groups, including Facebook, Instagram, and a digital story for potential use in the Tulalip Tribes website. We worked closely with the Tulalip Tribes in Marysville, and visited their location, supervised by Tulalip Tribes' Public Affairs Coordinator Francesca Hillery. UW campus resources will be invaluable in our pursuits; we will contact the school newspaper "The Daily", as well as collaborate with various departments, such as the Intellectual House, to spread awareness and to set up relevant lectures. Millennials are apt to be interested in this topic, therefore, one of our main campaign goals is to create the knowledge in the first place. If successful, our social media campaign will grow towards increasing implementaion on campus and for the tribes themselves. Overall, this can lead to future, independent student involvement and efforts to make a change for themselves, for the tribes, and for all in the Salish Sea region. 

Although questionnaires are helpful for identifying employee retention targets in research contexts, such surveys do not benefit employers in small, real-world organizations due to the inevitability of gathering biased data (e.g., due to social desirability or lack of anonymity). The purpose of the current study was to test a tool employers could use to more accurately identify turnover risk and targets for individualized retention strategies. This tool focuses on employee engagement, which research has shown to be related to employee turnover (i.e. high engagement is related to lower employee turnover intentions). This tool enabled us to determine employee level of engagement and predict employee group identification (i.e. long-term retention vs turnover). We hypothesized that higher levels of engagement will predict employee long-term group identity, whereas, low levels of engagement will predict turnover group identity. We conducted a qualitative study using a between-subjects design to compare semistructured phone interviews with 25 community mental health clinicians (13 long-term, 12 turnover), in which we asked clinicians what “ideal changes” they would like to implement in their agency. Using the Q12 Engagement Measurement Model, derived from Maslow’s Hierarchy of Needs, we determined clinician engagement levels, which we then used to predict their group identification. We predict our analyses will reveal that clinicians who suggest more personal-level changes are more likely to be in the turnover group, while clinicians who suggest more altruistic-level changes are more likely to be in the long-term retention group. The literature indicates that employees with higher engagement levels are less likely to turnover; therefore, if we achieve our predicted results, future implications of this study could be that employers could use this tool in their annual review process to more accurately assess employee turnover risk and improve employee retention strategies.

Endothelial cells (ECs) are cells that line our blood vessel walls. They differ throughout the circulatory system. For example, high pressure arteries typically have tighter endothelial junctions/gaps than the leaky, low pressure veins. ECs are known to sense fluid shear stress and hydrostatic pressure. These cues play a large part in embryonic development of the vasculature and induce short term responses in adults, such as dilation and constriction of arterioles, but it is unknown if adult ECs adapt to them in the long term. This matters for many medical procedures, such as venous graft bypass surgery, where the saphenous vein is implanted to bypass a blocked artery. These grafts are often lifesaving successes, but they sometimes re-occlude. Venous and arterial endothelium are very distinct, and it is important to know if the venous ECs in the graft can adapt to their new arterial environment. If not, that could explain the pathological re-occlusion. Here, we measure adaptation by changes in gene expression from a venous to an arterial phenotype. We designed a novel in vitro pump system to expose a flow chamber with endothelial cells to controlled flow and pressure. HUVECs (human umbilical vein endothelial cells) were then cultured and seeded into the chamber. Finally, a quantitative real time polymerase chain reaction (qRT-PCR) will measure gene expression at various time points in the venous (20 cmH₂O, low flow) and arterial (60 cmH₂O, high flow) cases. We analyze the markers EphrinB4 (EphB4) (higher in veins), EphB2 (arteries), Cx40 (veins) and endomucin (arteries). We present preliminary gene expression data from the 24-hour time point. We hypothesized that HUVECs will respond to arterial conditions with increased arterial gene expression, while retaining their venous state in venous conditions. Supporting or falsifying this will illuminate endothelial cell response to changing conditions in the body.

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.

Targeted therapies designed to inhibit hyperactive oncogenic signaling have demonstrated some encouraging clinical responses. However, many of these responses are not durable as tumors may develop new mutations to “work around” pathway inhibition leading to drug resistance. Technologies that enable more efficient discovery of “suites” of drugs that inhibit multiple pathway nodes are needed. The SABRE (Splice Acceptor Brilliant Reporter) platform was developed to provide an unbiased method to screen large compound libraries and dramatically improve the speed and efficiency for which novel targeted therapeutics can be identified. SABRE is built on the premise that oncogenic changes in gene transcription can be harnessed as powerful reporters of pathway activation status. SABRE utilizes gene trap technology coupled with a drug selection process to isolate cells that generate a robust “off to on” luciferase signal in response to pathway inhibition. Via massively parallel comparative analysis, multiple traps let nature provide the best reporter for further analysis and drug discovery. We employed the SABRE technology to identify insertion sites specific to the MAPK (mitogen-activated protein kinase) pathway, an oncogenic pathway that is implicated in a plethora of cancers. Experiments were performed using the human melanoma cell line A375, containing BRAFV600E mutation. SABRE lentiviral transduced A375 cells were treated with trametinib, an inhibitor of MAPK pathway, and clones were isolated that emitted a positive luciferase signal upon drug treatment. To determine if these reporters were MAPK pathway specific, the platform was used to screen a 6000+ compound library. Results from the screen found that 70% (28/40) of the top drug hits were known to directly modulate the MAPK pathway revealing the power of the SABRE gene trap technology to generate a panel of reporters specific to an unlimited number of cancer signaling pathways. SABRE holds the promise for discovering untapped drug therapies for cancer.

Many infectious diseases are caused by bacteria growing in biofilms, a group of cells that stick together and adhere to surfaces, such as the human lung or implanted medical devices. Biofilms affect millions of people worldwide each year, often contributing to death. When a biofilm is successfully established, bacteria are up to 1,000-times more resistant to antimicrobial agents and extremely resistant to clearance by the immune system. These molecular fortifications make bacterial infections difficult to treat, with the possibility of relapse weeks or months after the use of antibiotics. Many infectious diseases caused by biofilm-forming bacteria disproportionately affect the elderly and immunocompromised, like Legionella and Bordetella, which cause pulmonary infections. My research focuses on preventing biofilm formation, and forcing bacteria that have already formed a biofilm to disperse. Molecular engineering was used to design a protein to activate LapG, a protein effector responsible for balancing whether the bacteria are free floating versus anchored. When LapG is active, bacteria shed their adhesive proteins anchoring them to surfaces, and become free floating. I am trying to develop a protein-based drug to fight biofilm-based infections by permanently switching LapG to the “on position.” Because this drug will be made of protein, there will be no toxic breakdown products, and the highly specific binding inherent to protein-based drugs will minimize the risk of side effects. Furthermore, protein-based drugs are genetically encodable, allowing us to genetically modify self-replicating probiotics, such as yogurt cultures or baker’s yeast, to produce this medicine. This will empower low cost drug manufacturing and distribution because local communities in developing countries can produce these therapeutics using readily available cooking equipment and ingredients, and administer them without fear of overdose or toxicity. The goal is to test the LapG binder in animal models of chronic infection, eventually moving to human clinical trials.

Bees and flowers trade nectar for reproduction. Rhinos provide food to birds in exchange for removal of parasites. Mutually beneficial interactions like these pervade the natural world, but their impact on the process of evolution is not well understood. Here, we test whether each species in a microbial mutualism repeatedly adapts to the changes in their partners, or coevolves. In our model system 22 communities of the Methanococcus maripaludis (archaea) and Desulfovibrio vulgaris (sulfate reducing bacteria) evolved for over 3000 generations in the laboratory. To test whether coevolution occurred, we separated the D. vulgaris and the M. maripuludis in the mutualistic community at 1000 generations. We then paired these 1000 generation D. vulgaris populations with M. maripaludis at different evolutionary time points and measured the growth rate and yield of each population. If M. maripuludis coevolved with D. vulgaris then we would expect the 1000 generation D. vulgaris to perform better with future partners (0-1000 generations) than past partners (1000-2000 generations) because mutualists are expected to have positive effects on each other's fitness. A preliminary experiment with 6 communities indicated the opposite, that D. vulgaris grew faster with past generations compared to future generations. The yield of communities was similar across pairings. These results could indicate D. vulgaris has coevolved with M. maripaludis, and that there is an underlying antagonism between these species that has not been recognized. We present the results of a more extensive experiment, testing more mutualistic communities at more time points, and looking for patterns of coevolution in both species.  
 

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.

The forefront of modern medical research involves decoding the biological pathways that make disease transmission and perpetuation possible. Protein-based site-specific affinity reagents could allow us to antagonize harmful molecular interactions employed by pathogens, disrupt their toxic effects, and develop cutting-edge treatments. Consequently, the ability to express and purify rationally-designed proteins is the key to assessing their structure and function. To increase solubility and stability during recombinant expression, a genetic fusion to a small-ubiquitin-like-modifier (SUMO) is commonly used. The highly-specific SUMO protease is then used to cleave the SUMO fusion, leaving a completely intact and unmodified protein-of-interest. The current protease used originates from Saccharomyces cerevisiae. Known as Ulp1, the instability of this enzyme due to thermosensitivity and solvent-exposed hydrophobicity presents considerable limitations and can result in incomplete digestion of SUMO-tagged constructs. We present two approaches to mitigate these complications. First, we have discovered a homologous protease with a conserved catalytic site in the thermophilic eukaryote Chaetomium thermophilium. Second, using the Baker Lab’s pioneering computational design program, Rosetta, we have exchanged the hydrophobic surface residues of Ulp1 with hydrophilic side chains with the aim of improving stability in aqueous solution. We have characterized the kinetic function of these protease variants by monitoring digest efficacy through gel-based assays. Effectively, the improved proteases can equip the scientific community with a powerful new tool to facilitate protein-based drug design research.

Small Ubiquitin-like Modifier, SUMO for short, is a protein involved in cellular regulation which can also be used as a biochemistry tool to express difficult-to-express proteins in the lab. I am investigating several SUMO proteases in order to learn more about the regulatory pathways it is involved in. We are focused on the traditionally used protease from Saccharomyces cerevisiae, a thermophilic version from Chaetomium thermophilium, as well as several redesigns of a homologous protein from the Saccharomyces SUMO protease to improve solubility by mutating solvent-exposed hydrophobic amino acids to polar amino acids using a computational protein design program called Rosetta. I am using thermal melting techniques to find melting points and gel comparison techniques to analyze the efficiency of each protease. Understanding of the kinetics and properties of these proteases and comparing them to each other will yield a greater understanding of how the system works, which versions are more efficient for in vitro use, and how the system can be manipulated in vivo.The goal of my research is to create a set of biochemical tools associated with SUMO proteins to be used in cancer research, as the SUMO system is known to be dysregulated in cancer. Understanding of the role SUMO plays in cancer will provide context for the development of a new generation of therapeutics, and understanding of the SUMO system as a whole will improve a current biochemical technique.

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.

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.