If you're tiling a circular room with square tiles, how many do you need? What if your tiles are parallelograms? As the circle gets larger, is there a pattern? Mathematically, this can be framed as counting integer points in a large circle. We're interested in how this count changes as you change the shape of the tiles, and to understand this better, we started by counting the number of integer vector pairs within a ball of radius R so that the parallelogram they make has a fixed area (determinant). We created a Python program that would generate all of the primitive points out to radius R, which we then extended to count the number of vector pairs with a certain determinant k. We were able to compute the limiting density of this count, extending known results for the case of determinant 1. We are now studying other discrete sets, such as ones generated by objects from hyperbolic geometry, known as Hecke triangle groups. In the future, we plan to use our research for counting pairs of vectors in different spaces and generalize it for counts of k-tuple vectors. Come for floor tilings, stay for beautiful pictures!

With only a handful of species known to the general public, many of the 4000 bees endemic to the United States go unnoticed. As crucial pollinators, bees are incredibly important to the environment. There is a notable lack of data regarding bees in the United States, thus data collection about bee species diversity and abundance is prudent. Citizen science, or the use of volunteers in ecological studies, is a relatively new concept, introduced only a century ago by ornithological and astronomical sciences. The invention of digital single-lens reflex (DSLR) cameras makes it easy for novice photographers to take quality pictures, and they have become more accessible to citizen scientists in recent years. Our research introduces methods using visual observation and digital photography to study bee species diversity and abundance. It is impossible to identify most bee species without a microscope so we have taken a morphogroup approach, adapted from Xerces Society monitoring protocols, to gather data about bee species without needing an identification expert. We group bees into eight categories according to key characteristics visible to the naked eye, as similar-looking species are often closely related. Using digital cameras we implement two timed survey protocols, one tallying abundance and one “capturing” as many species on camera as possible. Two years of preliminary data has yielded information regarding the abundance, diversity, and phenology of bee groups on the UW Bothell/Cascadia College campus. Our surveys revealed the presence of all eight morphogroups on campus, 23% representing honey bees and 77% other bee species; bumble bee species (Bombus sp.) dominated in abundance representing 55% of all bees seen in 2018. Research has thus far informed improvements to monitoring protocols, identified bloom gaps on campus, and has resulted in the creation of UW digital photo archive documenting the findings of the research.

Bloodborne pathogens are wide spread and can pose risks to health care workers and vulnerable patient populations alike. Conventional diagnostic tests for bloodborne pathogens are costly and time intensive, so fast, affordable, and sensitive diagnostic methods are needed that can be performed under low-resource conditions by untrained personnel. Microfluidic self-digitization technology, developed in the Chiu Laboratory, provides the foundation for such low-cost diagnostics. As undergraduates, we work on the fabrication and optimization of cheap, robust devices used to load samples, as well as dPCR of samples required to detect diseases. As part of a larger research endeavor, we are developing a portable instrument that, in combination with our proprietary microfluidic chip technology, will expand rapid diagnostics to low-resource settings around the world. Expanding diagnostics for bloodborne pathogens will both help prevention as well as monitoring treatment of patients already infected.

As climate change progresses, increasing temperature is expected to have community and population-level consequences. The temperature-size rule predicts shifts in both community and population structure to favor smaller bodied organisms over larger bodied organisms as temperatures increases. We investigated this hypothesis in the context of rocky intertidal invertebrate communities on San Juan Island, and examined changes in community and population structure over the past 45 years. We replicated historical ecology studies on the abundance of six intertidal species; three of which body size data was also collected. These species included grazing and predatory molluscs (Katharina tunicata, Nucella lamellosa), anemones (Anthopleura elegantissima), and crabs (Hemigrapsus nudus, Pugettia gracilis, Pagurus sp). Additional abundance data was collected on smaller molluscs (limpets, littorine snails). Predicted decreases in body size were not apparent in populations of K. tunicata., N. lamellosa, and A. elegantissima. However, when comparing abundances, shifts in community structure towards smaller-bodied organisms were apparent. We observed an increased abundance of smaller-bodied gastropods and crustaceans (limpets and Pagurus sp), but a decrease in K. tunicata abundance, one of the largest grazers in this system. This research highlights the potential for a variety of community and population responses including both stability and variability in the face of climate change.

Natural armors, like those found on animals, provide inspiration for the development of next-generation engineered materials. In this study, the microstructure and composition of the cuticle of the terrestrial isopod armadillidium vulgare was evaluated. The cuticle, composed of layers of mineral particles and chitin fibers, is multifunctional. It protects the animal from impacts, predation, and maintains internal moisture, while facilitating sensing of the environment. Prior literature has focused on mineralized portions or anatomical features, while this work integrates composition data with chitin fiber structure and orientation. Cuticles of the armadillidium vulgare were evaluated by scanning electron microscopy (SEM) and Raman spectroscopy. Cuticle cross sections were prepared by freezing individual tergites in liquid nitrogen, then sectioned by fast-fracture. SEM was used to image cross sections and view chitin fibers and ply orientations over the cuticle thickness. This data was used to understand fiber layer morphology and orientation throughout the cuticle thickness. Raman was used to evaluate the spatial dependence of composition across the layers. Results showed that the cuticle is actually a nanolaminate consisting of plys of chitin fibrils, and with ply thickness that varies through the cuticle. The stacking arrangement of the plies appears to follow a Bouligand structure, which is characteristic for other natural composites. Results of Raman indicate that the plies are mineralized chitin, however, it is unclear if there are changes in composition through thickness. This work is ongoing. An understanding of the ply arrangement and mineral distribution in this armor can be used to pursue new strategies for material design where resistance to impact and puncture are needed. Applying design principles found in this isopod, more efficient and effective materials can be designed for applications such as light-weight armors or protective coatings.

Many plant species are known to undergo masting, where individuals within a species all produce a large amount of seed in a single year, with a sharp drop off in reproduction in the following years. Masting may be beneficial to plants by suppressing seed predator populations in low seed production years, allowing for seeds to escape predation in high seed production years. Masting is likely associated with climate, although this is poorly understood. In this study, I will use herbarium specimens to tackle questions related to masting in five plant species: Tsuga heterophylla, Pseudotsuga menziesii, Thuja plicata, Rubus spectabilis, and Rubus parviflorus. Specifically, I will note the number of cones, seeds or berries on herbarium specimens, as well as specimen size and the year it was collected. I will use these data to 1) examine patterns of masting in focal species as compared to field data from Mount Rainier. I hypothesize that herbarium specimens will show a similar pattern of masting as field data, with trees showing stronger patterns than shrubs. Next, I will use these data to 2) compare masting patterns to annual climate. I hypothesize that warm temperatures lead to masting the following year, and that masting has increased in frequency. Finally, I will 3) assess the relationship between masting and bird species that eat seeds and berries (e.g. grouse, jays), to determine whether masting influences population dynamics of higher trophic levels. I will do so by comparing masting patterns with bird count data from the Audobon Society. I hypothesize that population sizes of birds that rely on seeds and berries will be greater in mast years. This study provides additional information for how we might expect entire ecosystems to be affected by climate change, including resource distribution and population health.

Nowadays teamwork is becoming more and more important in business, and we may need to work together to fulfil projects’ goals. However, we still cannot find the exact reasons why some teams collapse. However, we think referent power and trust, in part, be likely culprits because it affects the way teams communicate to share knowledge about the project. Referent power is a form of reverence gained by a leader who has strong interpersonal relationship skills. Trust refers to a strong belief in the honesty and goodness of someone. Past research suggest that if the referent and trust are relatively low, it will affect the effectiveness and efficiency of communication or even no communication at all. Without communication, the team work will likely collapse. To test this contention without negatively affecting real-world teams, we used an agent-based modeling and simulation approach. Agent-based models are an impactful tool to study team phenomena because it can model the complex behaviors and probability of interactions and effects in a closed environment. So in our research, we analyzed the referent power and trust of a team by using agent-based models. We created a model for our research with two components “referent power and trust”. We 1) tested a mix of referent power and compared high referent power with low member trust, versus low referent power with high member trust to see what is the best scenario and 2) the results showed the scenarios when a team had higher effectiveness and efficiency in team work. Also, we share our NetLogo agent-based model code for other researchers to leverage.

The delivery of cell and drug-based chemotherapeutics to tumors have presented major challenges in effective cancer treatment. Opportunities to improve current small molecule drug delivery systems exist by increasing overall delivery specificity and decreasing harmful off-target effects. Towards this, we have recently developed a chemical framework for creating user-programmable hydrogels that undergo programmed degradation in response to multiple environmental cues following Boolean logic. Exploiting this methodology, user-specified combinations of environmental inputs (e.g., tumor-presented enzymes, reducing conditions) yield material breakdown and accompanying therapeutic release. To translate these materials for chemotherapeutic delivery in vivo, we established strategies to formulate these stimuli-sensitive materials into nanogels that circulate in the bloodstream before acting on the desired target site. We developed techniques to formulate gels on the 50-250 nanometers size scale, one which should enable circulation in the blood and uptake within tumors based on the enhanced permeability and retention effect. Different ultrasonication conditions allowed us to tune nanogel, size and dispersity. This system is scalable, translational, and simple to recreate. In the future, these materials can effectively hone and selectively deploy small molecule chemotherapeutics to tumors in patients.

Physical resilience is a measure of healthy aging, but current assessment methods are underdeveloped, relying on a frailty panel of subjective tests. A quantitative, reliable test for resilience using endpoints aligning with healthy aging is needed to provide a standardized measurement of resistance to age-associated decline. The chemotherapeutic drug cyclophosphamide (CYP) is an immunosuppressant used in clinical medicine for certain types of cancer and autoimmune diseases. Precursor neutrophils are especially sensitive to this chemical stressor, such that a single low-level dose stops cell cycling with a decrease in mature neutrophils, but neutrophils rebound as the drug is eliminated from the body. This study sought to characterize the neutrophil rebound response in aging mice as a biological sensor for how an organism might respond to physical stress with increasing age. One intraperitoneal dose of CYP at 100 mg/kg was given to groups of mice at various ages. White blood cell differential and total counts were enumerated to monitor cellular response over a 25-day period. A robust and predictable drop in neutrophils occurred across age groups five days after injection of CYP, followed by a robust increase in neutrophils beyond baseline measurements at day seven in an age-dependent manner. Strength of the neutrophil rebound had a moderate correlation with strength and stamina as measured by rod hanging time, a test that corresponds to hand grip assessment in humans as an indication of frailty. This observation suggests that the neutrophil rebound response to the chemical stressor CYP in young adult mice can predict resilience to aging in older adult mice. The clinical implications are that an in vitro bench test could be developed using CYP-induced rebound response of human precursor neutrophils to predict resistance to frailty and produce a more accurate assessment of resilient healthy aging in older adults.

Pre-hospital patient protocols standardize medical procedures for EMTs and increase the quality of care for patients. In Washington state, these protocols vary by county and often contain a section for psychiatric emergencies with variant restraint protocols. The immediate use of restraints in these situations can be seen as dismissive to the patient and can lead to agitation and violence. Verbal de-escalation can enhance provider-patient relations and decrease likelihood of restraints, seclusion, and hospital admissions.  The goal of this project was to explore restraint and de-escalation methods used in WA state county-level EMS pre-hospital patient protocols. To complete the project, a codebook was created with binary variables with definitions that determined whether the procedures listed in the protocol satisfied the definition. Using the codebook, each protocol was independently coded by 2 people and disagreements were reviewed by a third person. Finally, we created descriptive statistics from the restraint protocols and stratified based on rurality. Of the 39 counties in WA state, 77% of counties mentioned verbal de-escalation methods in their pre-hospital patient protocol. The de-escalation method with the highest proportion in all counties recommended reassuring the patient that the providers care for them (28%). The second highest proportion in all counties was to mention the use of de-escalation methods without explicit instructions (23%). Counties that do not have specific instructions and just mention the use of de-escalation methods can be confusing if the provider does not have de-escalation training or has not trained recently. These findings highlight the need for future research concerning the adherence of EMTs to these protocols and to see the what the outcomes are of the different protocols. More research can be done through contacting individual EMS agencies to see if internal protocols exist outside of county protocols.

c-fos is an immediate early gene that is commonly used to mark cell activation and can be identified using immunohistochemistry techniques. In this project, we optimized immunohistochemistry conditions for a phosphorylated c-fos antibody in brain tissue obtained from the rat lateral habenula (LHb). Male, Long-Evans rats (n=4) were injected with cocaine (10 mg/kg) or saline. Ninety minutes following intraperitoneal injections, rats were perfused with 4% paraformaldehyde. Brains were dissected and 40-micron slices across the rostro-caudal axis of the LHb were collected. We tested two dilutions of the rabbit phospho c-fos antibody (1:500 and 1:800), three blocking agents (normal goat serum, donkey serum and bovine serum albumin) and two secondary Alexa Fluor antibodies (goat anti-rabbit Alexa Fluor 488, and donkey anti-rabbit Alexa Fluor 488) to determine which combination allowed for the best visualization of c-fos in the LHb. Fluorescence microscopy analysis of the tissue sections is underway, and imaging is expected to show differences in brightness and visibility of c-fos with respect to the varying conditions. These results will identify the optimal conditions for more accurate identification and quantification of cell activation in the lateral habenula.

c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinases (MAPKs) family that are derived from three genes: Jnk1, Jnk2, and Jnk3. JNKs have been implicated in several cellular responses to homeostatic insults, including inflammation and apoptosis. We previously reported in a chronic epilepsy rat model significant elevated levels of phosphorylated JNKs (pJNKs), which indicate increased JNK activities. Additionally, we demonstrated that pharmacological manipulations of JNK proportionally affected seizure frequency. In this set of experiments, we attempted to identify which of the JNK isoforms (JNK1, JNK2, JNK3) contribute to the overall increased pJNK levels in our animal model of epilepsy. This would provide us insights as to the role(s) of JNKs in this disease. We measured the phosphorylation levels of the individual isoforms after pJNK enrichment from the CA1 hippocampal tissue of chronic epileptic rats and their age controls. The amount of protein was normalized by pJNK levels between experimental and control samples. We found a significant increase in activation levels of JNK2 in chronic epilepsy at 130 ± 9% (n=6, p=0.018) when compared to naïve, nonepileptic controls but insignificant changes in activation levels of JNK1 (97 ± 14%, n=5, p=0.83) and JNK3 (98 ± 17%, n=6, p=0.92). Previously, we had found in rats that JNK1 predominantly exists in the 46kDa size; JNK3 predominantly exists in the 54 kDa; and JNK2 exists in both sizes equally. We further analyze which of the JNK bands (46 kDa and 54 kDa or both) contribute to the elevated phosphorylated JNK levels. Given the previous pharmacological observation that JNK manipulation does influence seizure frequency in epilepsy, this investigation is imperative as it will allow us to narrow our focus to a specific JNK isoform to study further.

The receptor human protein kinase EphA2 has emerged as a therapeutic drug target for cancer and infectious diseases. However, the dearth of selective EphA2 inhibitors in the field is hampering EphA2-based research and therapies. Our lab recently developed a novel selective small molecule EphA2 inhibitor I268 using medicinal chemistry. Surprisingly, we observed the same inhibitory effects on cell proliferation paradoxically when inhibiting EphA2 with I268 or activating EphA2 with its agonist EphrinA1. We hypothesized that both I268 (inhibitor) and EphrinA1 (agonist) may lead to the same protein-protein interaction network of EphA2 that caused this paradoxical inhibition. We used a chemical proteomic method developed in our lab called “co-clickable precipitation (Co-CP)” to study the interaction network of EphA2 in colon cancer cell line HCT-116. We observed that the onset of inhibitory effect by I268 concurred with the association between EphA2 and a protein interactor called c-RAF, a key player in activating proliferation pathways in cancer cells containing Ras mutations such as HCT-116 cells. We are currently performing confirmatory experiments to characterize whether upon inhibition by I268 or activation by EphrinA1, EphA2 sequesters c-RAF in a form unable to transmit proliferative signals downstream. Our results may demonstrate a possible mechanism for inhibitory effects of EphA2 inhibition on cancer cell growth. These results validated EphA2 as a drug target and may provide novel ways for treating cancer cells by inhibiting cell proliferation pathways indirectly using EphA2 inhibitors.

This work replicates previous research used to criticize the use of principle component (PCA) and exploratory factor analysis (EFA). Factor analysis is often used when research is being conducted in a new area and there is limited background knowledge about what one might expect to find. It’s useful because of its ability to reduce the number of variables in a dataset by examining the correlations between items and identifying latent dimensions that account for measured item variance and covariation. A criticism of this method is that it detects patterns in random data where there is no underlying structure. We tested this criticism by replicating the methods in a 1981 paper by Robert Vierra and David Carlson. We generated random data in R Studio and looked at the variance accounted for by the factors identified by EFA and PCA. We also examined the Type I error rate in sample correlation matrices tested with Bartlett's test. Additionally, we have begun a literature review looking at the modern methods similar to exploratory factor analysis, including machine learning, in order to understand how these criticisms have been addressed by these modern approaches.

Pulmonary fibrosis is a disease marked by irreversible scarring and thickening of the lung tissue causing significant decline in lung function. Individuals afflicted will struggle to perform simple physical activities and often require mechanical assistance at some point in their lives. Currently, there are no permanent solutions for those with pulmonary fibrosis as most treatments only aim to slow down the progression of the disease. In these studies, we developed a novel therapeutic that could stop the progression through DNA modification of fibrotic gene targets using exosomes as a delivery vehicle. Additionally, regeneration of lung tissue is imperative for reinstating lung function and by using similar technologies we aim to target and overexpress critical regenerative genes. Using CRISPR Cas9 gene editing technology, we were able to knockdown key cytokine specific genes responsible for the development of fibrosis. We specifically targeted TGFß (Transforming Growth Factor ß) and Interleuken-6 (IL-6), both known to play a significant role in pro-inflammatory responses and fibrosis through exosome-mediated delivery mechanisms. CRISPR Cas9 vectors were designed to contain unique guide RNAs that could effectively target specific genes that the Cas9 complex could use to repress TGFß and IL-6. Cell lines were treated with the modified CRISPR Cas9 vectors and assessed for gene and protein expression. This study provides key insight into a novel therapeutic platform using a new delivery mechanism that mitigates and reduces fibrosis and promotes recovery of pulmonary function.

For years, scientists have been baffled by the imbalance between matter and antimatter in the universe. The properties of the neutrino, a subatomic chargeless particle, may ultimately help us explain this anomaly. Double beta decay takes place in an atomic nucleus, and it occurs when two neutrons spontaneously transform into two protons, emitting two electrons and two antineutrinos in the process. However, if the neutrino is its own antiparticle, the antineutrinos could annihilate each other, so that the two electrons are emitted with no balancing emission of antimatter. This is a postulated process known as “neutrinoless double beta decay.” The MAJORANA Demonstrator, a collection of germanium detectors, allows us to search for this creation of matter in a laboratory setting. However, there are naturally occurring background processes, such as gamma rays, which closely resemble that of the creation of matter. In order to accurately distinguish one process from the other, we must understand them extremely well. The Demonstrator relies heavily on the use of simulation software, called Geant4, in order to predict the occurrence of gamma ray backgrounds. Geant4 previously generated the gamma rays’ directions isotropically, but many sequences of gamma emission are emitted in correlated directions. We contributed new code that uses the computation of associated Legendre polynomials to correctly generate the gamma emission directions. This code used a recursive algorithm that was too slow for general use. A speedup was attempted using a cache, meaning it stored computations to avoid repetition. However, the cache was implemented inefficiently and incorrectly. Improved caching should speed up calculations. If it is insufficient, we will unwrap the recursive algorithm into a generative for-loop. For gamma emissions with extremely large angular momenta, the code may still be too slow. In that case, we will explore asymptotic formulae to speed up computations even further.

DNA polymerase (Pol) proofreading and mismatch repair (MMR) cooperatively guard against DNA replication errors and cancer. Defects in these activities produce “mutator” phenotypes characterized by elevated levels of base-substitutions and frameshifts. Haploid yeast with combined defects in these activities rapidly go extinct, in a process termed error-induced extinction (EEX). Organisms adapt by duplicating their genome without dividing (polyploidization) or by lowering their mutation rate via antimutator mutations. In evolution experiments with haploid mutators that display a synthetic-sick phenotype due to Pol ε proofreading and MMR defects we found that polyploids routinely beat out antimutator mutants. To investigate whether polyploids arise during the evolution of mutator diploid strains, we propagated diploids defective in Pol δ proofreading and base-base MMR, which have a mutation rate an order of magnitude below the diploid error threshold. Our mathematical modeling predicts that EEX mutants will overtake mutator cultures by 250 generations. We found only a single tetraploid in 89 independent cultures evolved for 300 generations. We then measured mutation rates of isolates from cultures that remained diploid and repeatedly found antimutator phenotypes. Whole genome sequencing of independent isolates from each culture revealed a single dominant antimutator clone that caused significant changes in mutation spectra. Thus, spontaneous antimutator alleles and polyploidization rescues haploid and diploid cells from EEX with markedly different efficiencies. Differences in the relative frequency of each escape mechanism may reflect the nature of the mutator alleles, the starting ploidy of the cells, or the magnitude of the initial mutation rate. Our findings in diploids suggest that mutator cancer cells near the edge of error-induced extinction may similarly be under selection for spontaneous antimutator mutations and polyploidization. 

The purpose of the UW-EXP research study is to understand the first-year UW student experience in a data-driven manner to create a cornerstone for institutional change. This study extends focus to investigating stressful life events of first-year students. Phase 1 data collection is complete, and we are in Phase 2 of the project. The UW-EXP research study is researching the overall well-being of first-year students. Can we detect stressful life events in students using passive technologies (mobile phone data, Fitbit and Social Media)? Which technology is a better indicator of a stressful life event or do they complement each other? Phase 1 data used a participant sample size of around 200 University of Washington first-year undergraduate students (around 50% pre-/engineers). Data consists of both active and passive collection methods: participants actively respond to Qualtrics surveys, with questions investigating stressors, and passively via our phone software and Fitbits. The stressors are defined as stressful events in the students' relationships, health, living situation, finances, and if they experienced discrimination, relationship violence, and assault. The following information collected from our phone software and Fitbits are number of steps, location, sleep, App usage, and when in conversation and with whom in generic terms (friend or relative). I have used the data collected in Phase 1 to answer my research questions. We identify critical data that can be analyzed to advance the science of behavioral health and trauma. This data is unique in providing a picture of student behavioral changes after stressful experiences. Additionally, this advancement in science can become a basis for institutional change for improving student wellness. As the first-year population is only a slice of the overall student experience, we hope to expand this study further by encompassing the graduate student experience and the full four-year college experience in addition to a larger sample size. We will help improve the lives and success of University of Washington students.

There has been significant concern regarding the global effects of climate change, especially on plant communities, which are influenced by individual plant species responses. Warming temperatures are having large repercussions for plant biodiversity, with rapidly changing environmental conditions causing shifts in species ranges and phenology. To better understand the implications of this phenomenon on plants, many studies have investigated responses of individual species to climate change. One approach that has grown in recent decades is the use of functional leaf traits, which are indicators of plant performance and reproductive capacity. Specific leaf area (SLA), the ratio of fresh leaf area to dry leaf mass, is one such trait that is attributed to plant growth and photosynthetic capacity, and found to be correlated with climate. We assessed the relationship between climate and SLA for Vaccinium ovalifoilum, a shrub that is prevalent in the montane ecosystems of Mt. Rainier. To accomplish this, we gathered SLA using samples from the Burke Museum’s herbarium collection whose distribution will be delineated across three elevational bands. Altitude was used as a proxy for climate due to variable environmental conditions found across elevation—namely temperature and precipitation—and the collection location of specimens allowed us to obtain temperature and snow accumulation data. With this information, we compared the variation between the three established elevational bands in which the specimens fell under. We hypothesized that as elevation increases, SLA will decrease, attributing this trend to a limited growing window to support high photosynthetic capacity as a result of later snowmelt and lower temperatures. Our results offered insight to understand how climate change may potentially affect plant functionality and guide future research to analyze how changes in individual species may influence community coexistence in the upcoming years.

Natural dermal armors are inspiring the development of advanced engineering materials and next generation flexible armors. Fish scales are an exemplary candidate and consist largely of laminated plies of unidirectional type I collagen fibrils. The mechanical properties of fish scales depend on the interpeptide bonds within the triple helix of the collagen fibrils. Adjusting the strength of these bonds to change the performance of the scales has applications to the design and functionality of bioinspired flexible armors. Here, elasmodine scales were exposed to polar solvents to adjust the extent of intermolecular bonding. Changes in the mechanical properties were evaluated in uniaxial tension and at two different strain rates. Results showed that the constitutive behavior was highly dependent on the intermolecular bonds. A significant increase was observed in elastic modulus (stiffness), strength and toughness as a result of increasing the extent of interpeptide bonding via solvents with low affinity for hydrogen bonding. A 300% increase was seen in the elastic modulus of scales soaked in acetone compared to HBSS at the highest strain rate. Furthermore, the importance of interfibril bonding was dependent on loading rate. Overall, results showed that the “protecto-flexibility” of fibrous armor materials can be improved by activating interfibril bonds and that this could spawn approaches for tuning armor performance.

We previously observed that turmeric could be used to manipulate gut motility in a microbiome-dependent-bile acid-mediated manner via the enteric nervous system (ENS). Bile acids comprise a class of metabolites synthesized by the host and modified by gut microbes. Bacterial bile salt hydrolases (BSH) are responsible for deconjugation, the first step in bile acid metabolism for generation of secondary bile acids. We hypothesized that homeostatic ENS signaling is dependent upon gut bacterial bile acid metabolism. To test this hypothesis, we colonized wild-type gnotobiotic mice with different defined consortia varying in BSH activity and subjected them to a two-week low-fat diet ± turmeric. As a control, one treatment group remained germ-free. mRNA isolated from small intestine and colon was subjected to gene expression profiling returning counts of 68 target genes, including ENS-specific genes, and 7 housekeeping controls. A machine learning algorithm was deployed to identify genes whose expressions were most impactful in discriminating between treatment groups. Glp2r and VIP were highly discriminatory with respect to BSH activity. Enteric neurons express Glp2r, activate the mTORC1 pathway in response to GLP-2, and modulate intestinal epithelial cell growth. We found that Glp2r expression was significantly greater in mice harboring consortia with BSH activity than in germ-free mice. GLP-2 induces VIP neurotransmission from enteric neurons. Indeed, VIP expression is correlated with Glp2r expression. These data suggest that gut bacterial bile acid metabolism regulates homeostatic ENS signaling, with implications for gut motility and colorectal carcinogenesis.

Inflammasomes are multimeric protein complexes involved in innate immune responses. Inflammasomes include a protein sensor, such as NLRP3 or pyrin, linked to the enzyme caspase-1 via the adaptor protein, ASC. Active caspase-1 is responsible for release of the cytokine interleukin (IL)-1ß and triggering inflammatory cell death. Inflammasomes are crucial in defense against pathogens. However, excess inflammasome activation is linked to diseases such as Alzheimer's, atherosclerosis, and other inflammatory conditions. Although inflammasomes are linked to several diseases, we do not fully understand how inflammasomes are activated. We are researching the role that potassium plays in inflammasome activation. To detect inflammasome activation, I measured released IL-1ß using enzyme-linked immunosorbent assays (ELISAs). I determined whether inflammasome activation requires potassium efflux by measuring IL-1ß released from cells stimulated in high extracellular potassium, which prevents potassium efflux. I found that IL-1ß release triggered by NLRP3 inflammasome activators is prevented when cells are stimulated in high extracellular potassium. However, IL-1ß release triggered by the pyrin inflammasome was not affected by high extracellular potassium. From these results, we conclude that the NLRP3 inflammasome is dependent on potassium efflux from the cell, whereas the pryin inflammasome is not. The implications of our research are two-fold. First, our findings argue against a long-standing hypothesis that high extracellular potassium blocks ASC binding. Both NLRP3 and pyrin need ASC, but our data show that only NLRP3 is affected by potassium concentration. This suggests that potassium affects NLRP3 activation at an unknown point. Second, understanding the role of potassium in regulating inflammasome activity provides a potential therapeutic target. There are drugs that regulate ion concentrations by controlling ion channel activity. Knowing whether an inflammasome pathway is potassium efflux dependent could be beneficial in limiting excess inflammasome activation that is linked to a variety of human diseases.

Spanish is one of the fastest growing spoken languages in the United States. In 2017, the US census reported that about 41 million residents (13.4%) speak Spanish in the home. Since the number of Spanish speakers increases annually in the United States, it is important to research how Spanish heritage language (HL) learners and Spanish second language (L2) learners interact with each other. Many assume that speaking with a Spanish HL learner will improve the quality of the L2 learner’s Spanish. The goal of my project is to analyze the amount of English spoken between the two types of students and the opportunities for learning created during their interaction by correcting each other’s grammar or helping each other to use different vocabulary words. To this end, I have transcribed the Spanish conversations between two Spanish L2 students and formatted them using the F4/F5 transcription software. I am currently coding them using a qualitative data analysis program called Atlas.Ti, with the addition of three other transcriptions that had already been transcribed. I predict that our results will demonstrate that, while some learning will occur between the HL and L2 students, a majority of the learning will occur among Spanish L2 learners. Since the research is still ongoing, I predict that due to the greater level of comfort a Spanish L2 learner has while talking to a fellow Spanish L2 learner, they will work together to solve more language issues than with a Spanish HL learner. This research is important due to its implications for improving how Spanish is taught in schools across the United States.

Measurement in Psychology usually assumes continuous dimensions. Correspondingly, theoretical constructs are typically conceptualized as dimensions, meaning that observations differ in degree. However, theoretical constructs may also be categorical, i.e., rather than differing by degree, differing qualitatively. Different construct operationalization’s may lead to different scientific conclusions. Self esteem has long been of interest in Psychology. It has been conceptualized in various different ways, but always dimensional. Some self-esteem measures (for example, the The Rosenberg Self Esteem scale) seek to measure globally how much one values themselves as a person, without focusing on specific domains. On the other hand, other self-esteem measures (for example, the State self esteem scale) focus on perceived capabilities in specific domains, such as performance, social, and appearance. In the National Survey of Youth, participants rated themselves in specific areas, and then again as they would like to be. Using these data, we examined how different operationalization’s of self-esteem are associated with a few psychologically interesting outcomes such as drug use and crime. Specifically, we compared continuous and categorical representations of self-esteem. By examining the assumption of continuous latent variables, we shed light on how scientific conclusions differ based on construct operationalization.
 

Is the neutrino its own anti-particle? This is a question physicists do not have the answer to, but if a process known as neutrinoless double-beta decay were observed then it could be said with certainty that the neutrino is indeed its own anti-particle. In an attempt to search for said process, researchers at UW have joined forces with researchers from a number of other institutions to form the group known as LEGEND (Large Enriched Germanium Experiment for Neutrinoless Double-Beta Decay). The source used for this experiment that undergoes double beta-decay is Germanium-76. This source is also used for the detector itself. When a beta decay happens in the detector a pulse proportional to the energy of the electrons emitted is produced. If these electrons have all the energy available from the decay, then it will be known that no neutrinos are present. What makes things tricky is that if this process occurs it does so with a half-life greater than 10²⁶ years. Thus, counting rates for this process will be very low, and very low backgrounds will be needed to effectively carry out the experiment. Currently at UW we are working on developing pulse rejection techniques that will allow us to get rid of unwanted background events that our detectors measure. The project I'm presenting on involves aiming a collimated alpha source at our detector, and the goal is to develop techniques that allow us to reject pulses resulting from alpha decays. A number of other collimated sources are also aimed at the detector in order to study rejecting the pulses that they give rise to. In order to help design this experiment I am running simulations in the Geant4 based application “g4simple” to determine collimator dimensions and materials that will work best for different radiation sources.

Aging is characterized by the loss of physiological and cellular functionality, however the mechanisms that underlie this deterioration are still unclear. Emerging evidence indicates that aging is associated with increased cell-to-cell variation in gene expression within tissues: homologous cells within tissues start expressing the same gene at varying levels. The causes of this age-related variation of gene expression are not known. Here, we aim to investigate this age-related dissimilarity in gene expression using C. elegans as a model system. We hypothesize that increase of gene expression variation is an early event during aging that may therefore underlie subsequent deterioration of tissues functionality. By characterizing aging in C. elegans, we hope to provide further insight into the molecular characteristics of aging in humans, and possible points of intervention. Previously, we have found that young C. elegans animals exhibit nearly identical stoichiometry of independent genes expression with very little difference between individual animals of the same genetic background. Our initial results support the idea of increased cell-to-cell and animal-to-animal variation of gene expression with age in C. elegans. Here, I use quantitative microscopy to measure animal-to-animal and cell-to-cell variation of genes expression in middle aged C. elegans using fluorescently-tagged proteins and quantitative microscopy. In addition, I use the methodology of molecular cloning and transgenics developed by the Mendenhall lab to create new transgenic strains of C. elegans. These transgenic strains will be also be used for quantitative microscopic analysis. By examining existing strains and developing new ones, I will determine if increase of gene expression variation represents early event in the aging of C. elegans.

We present optical and infrared photometry of the T Tauri binary KH 15D acquired in the 2017/2018 observing season. The data were obtained from the A Novel Dual Imaging CAMera (ANDICAM) instrument on the 1.3m telescope operated by the Small Moderate Aperture Research Telescope System (SMARTS) at the Cerro-Tololo Inter-American Observatory (CTIO). KH 15D includes two young stars (A and B, with 0.72 and 0.74 solar masses, respectively) orbiting their common center of mass, surrounded by an inclined precessing circumbinary disk. The recent data reveals Star B gradually emerging from the trailing “fuzzy” edge of the disk and is now in a stage that is completely unocculted for the first time since the system’s discovery in 1995. We use time-series photometry, or light curves, to probe the composition of the disk, derive parameters of Star B, and demonstrate the overall mechanics of the system. Recent data also shows reddening during the egress of the last eclipse, proving that the trailing edge of the disk is transparent and consists of dust-sized particles. Additionally, the most recent data displays Star B at a brighter magnitude than ever than before, allowing us to calculate an apparent I magnitude of 14.079, which is 12% brighter than previously computed.

Freshwater is one of the most valuable resources in Washington State. In recent decades, water supply has been affected due to climate change and population growth. Understanding changes in water supply and demand is crucial for ensuring an abundance of water for residential, economic, and industrial needs. The proposed research analyzes changes in the streamflow regime of the Cedar and Tolt Rivers which provide drinking water for the greater Seattle area. The main goal is to calculate the water budgets for the Cedar and Tolt watersheds and estimate how the inputs and outputs to these budgets change over the 21st century. An existing ensemble of streamflow projections for the Cedar and Tolt Rivers are used to analyze changes in water supply. The mean streamflow for each month is compared between a 30-year control period (water years 1971-2000) and a 30-year future period (water years 2031-2060). For each of these periods, I determine “optimistic” and “pessimistic” scenarios for the streamflow. For the “drought” month the highest streamflow value is considered as “optimistic”, and the lowest as “pessimistic” since the goal is to assess potential shortages. I use existing monthly demand values provided by Seattle Public Utilities and create different future scenarios, based on the predictions of population and employment growth. Supply and demand values are compared to evaluate (1) the potential for water shortage and (2) water management and conservation methods to satisfy the unmet demand. One potential water management method is the construction of a new reservoir. The results of the research are aimed at helping to inform society and water managers about the potential changes in the water system. Based on this information, they might be able to introduce changes in their future plans to accommodate the predicted needs.

Gravity remains one of the biggest mysteries in physics. Since Einstein’s Theory of Relativity not many new discoveries have happened. There are several big questions remaining about it such as: Why is it so much weaker than the other forces of nature? Is it mediated by a particle like the other forces? Is there a unifying theory between gravity and quantum mechanics? Does gravity have a place in the standard model? There are many theories proposed today which attempt to answer these questions, such as the presence of extra dimensions in string theory. If the effects of these theories exist, they would be present at small length scales (less than 1 mm). In order to test these theories, our lab uses a torsion balance experiment at sub-millimeter lengths. Our torsion balance experiment consists of an attractor mass on a turn table, and a detector mass hanging from a thin wire. Each has wedges cut out of it in the same pattern. The test is run by operating the turntable and measures the gravitational torque experienced by the detector mass. My project is to use a code that simulates the gravitational torque in order to investigate its dependence on different orientations and geometrical aspects of the experiment, as well as to improve on future runs of the experiment. This experiment will shed light on previously unknown aspects of Gravity and hopefully provide new discoveries to the field of gravitational physics.

Age related immune decline may play a role in susceptibility to parasitic helminth infections among the octogenarian population. Few studies have examined very old age on susceptibility to helminth infections in people. Effects of age on host susceptibility using mouse models also has not been thoroughly studied for older ages of laboratory mice (Mus musculus), such as beyond about one year. In this study, the intestinal nematode (Heligmosomoides bakeri), a model for hookworm infection in humans, was used to infect 1-year-old and 2-year-old Swiss Webster mice. At 40 days post-infection, worms were removed and worm survival, sex ratio, ex vivo egg output, and worm length (as an index of growth) were measured and analyzed. I expect to find an increase in worm survival, ex-vivo egg output, and worm length in older mice. These results will be valuable because they will model the helminths capacity to proliferate in older hosts in comparison to younger hosts during a human hookworm infection.

The life history traits of parasites are dependent on the host environment and host immune response. Heligmosomoides bakeri, is an intestinal nematode parasite that infects laboratory mice (Mus musculus). This investigation used rapamycin, an immunosuppressant drug, in the diet of the host. Rapamycin is currently being considered as an over the counter drug to extend life expectancy. The purpose of this experiment was to determine if inhibition of the host immune system by rapamycin had a significant effect on parasite life history traits during a secondary infection. Mice were fed either a rapamycin diet or control diet for two months. Mice were then infected with H. bakeri, drug cleared of the primary infection, then re-infected with a secondary H. bakeri infection. Starting eight days after the secondary infection fecal egg counts were done daily to determine worm reproduction in vivo. Eighteen days after the secondary infection the mice were euthanized and worms were removed, sexed and counted. Length of ten female and ten male worms from each mouse was then measured, and 10 females were cultured to measure ex vivo reproduction. After 24 hours of incubation in culture media, egg output and worm motility (an index of viability) were measured. For this experiment I was involved in recording the worm length and was responsible for performing the ex vivo egg count and motility measurements. Results will provide important information about potential over-the-counter use of rapamycin in humans to slow biological aging and how changes in immune function may affect susceptibility to parasite infection.

The impacts of climate change may be partially ameliorated by positive interactions among species, such as native plants and arbuscular mycorrhizal fungi (AMF). Sea level rise caused by climate change will detrimentally impact saltmarsh habitats by increasing the elevation and rate of terrestrial salt water inundation, thereby reducing effective functionality of ecosystem services provided by saltmarshes. Facilitations, positive interactions between species, is key in determining which species can survive in habitats characterized by abiotic stress, such as salt marshes. Halophytic plants within salt marshes can benefit from symbiotic AMF facilitations through increased inundation tolerance, greater nutrient availability and uptake, and relief from saline stress. This study identifies the presence of AMF across multiple saltmarsh plant species and measures the impacts of AMF within Oregon’s Salmon River Estuary at Cascade Head. I have extracted samples from four monoculture plots within high and low marsh elevations across four species that grow at both elevations: jaumea (Jaumea carnosa), Lyngbye’s sedge (Carex lyngbyei), pickleweed (Salicornia virginica) and salt grass (Distichlis spicata). I have centrifuged soil samples from each plant, and calculated AMF spore abundance at each sample location. I am also processing soil samples for percent water in the sediment, soil salinity and pH. Additional samples from both elevations within polyculture plots have been collected to develop a secondary index to cross-reference my primary findings. Through this research, land managers developing sea-level rise mitigation plans will have data supporting which plants will be most resilient to sea level rise. Potential inoculation of host plants could protect estuaries from sea level rise and increased frequency and intensity of storm events in coastal regions. In identifying plants which host beneficial AMF, fungal inoculation of plants in marshes could help plants grow further into lower marsh elevations, increasing sediment accretion and decreasing erosion and soil degradation.

Hydrogen sulfide (H₂S) is a common cause of workplace injuries and deaths for industrial workers. In our project, we use Caenorhabditis elegans (C. elegans) as a model organism for investigating how cells behave under an environmental stressor and the long-lasting effects of that behavior. Previous work in our lab has shown that early exposure to low H₂S (50 ppm) enable C. elegans adults to survive a much higher subsequent exposure by forming a cellular memory known as a “bookmark.” Bookmarked animals survive at high H₂S (150 ppm), while animals without previous exposure do not. In a genetic screen, we identified various epigenetic factors that are involved in this process; however, it is still unclear when in the “life” of the bookmark and where in the animal these factors are required. The required bookmarking gene swsn-4 is part of the SWI/SNF complex, a group of proteins that regulate compaction of DNA and thus the accessibility of genes. We are interested in assessing the spatial requirements for swsn-4 by rescuing mutant animals that lack this chromatin-remodeling factor. For the first part of the project, we use Gateway recombination cloning technology to enable tissue-specific expression of swsn-4. In the next part of the project, we test whether introducing swsn-4 in specific tissues rescues bookmark retention. A recent study identified hif-1, a transcription factor, to be broadly needed to rescue animals exposed to both low and high H₂S, suggesting that the response is needed in most cells to ensure survival of the animal. Because swsn-4 is also present broadly in the body of C. elegans, we predict it will be needed in a similar way to hif-1. We hope that our investigation would lead us to discovering methods in which we can utilize the properties of H₂S as a chemical messenger to help patients.

Hybrid organic inorganic perovskites are a promising highly efficient photovoltaic material that can be solution processed at low temperatures enabling an inexpensive solution to rising renewable energy demands with high-volume, scalable manufacturing of solar cells. Small scale perovskite devices are successful using spin coating; however, this needs to translate to larger scale deposition systems such as roll-to-roll slot die printing. Understanding the crystallization and morphology dependence of these materials is essential to enabling slot-die coated perovskite films on scalable systems and transitioning this technology to the market. In order to model crystallization rates of printed layers, we used in-situ optical and photoluminescence microscopy during printing of perovksite films to determine crystal growth rates and evaluate perovskite conversion. Printing parameters were manipulated through variation of temperature, atmospheric conditions, ink recipes, and substrate surface energy generating a model to achieve desired grain size and morphology of the perovskite layer across an array of relevant potential perovskite photovoltaic device stacks. Following classical models, we determined the necessary parameters to translate these fundamentals to perovskite crystallization and grain growth. We further explored the conversion and degradation of the perovskite phases through the printing process, which plays a significant role in device performance, through in situ photoluminescence microscopy, as well as verification through X-ray diffraction. Verification of the observed grain sizes and morphology was also done through scanning electron microscopy, to ensure optical measurements and analysis were accurate. With efficiencies of perovskites approaching current industry standards of silicon, perovskites are increasingly becoming the clear answer to solar industry demands. This research is essential in enabling scalable methods with the potential to revolutionize the solar industry with large scale fully printable devices.

The US/Mexico border is a politically polarized and militarized place where each year millions of people cross, both with and without authorization. As a militarized border that divides two nations, the border region is prone to violence for at least two reasons: the presence of armed state officials on both sides of the border, and the presence of drug trafficking. National treaties such as the North American Free Trade Act (NAFTA), soon to be replaced by the United States–Mexico–Canada Agreement (USMCA), impact industries in Mexico. NAFTA removes trade tariffs, creating investment opportunities for the United States in their border countries (Canada and Mexico). These industries employ high numbers of womxn in their facilities. Notably, there is a history of high rates of murders and disappearances of womxn in border towns such as Tijuana, Ciudad Juárez, Mexicali, El Paso, etc. Many of these murders, disappearances, and other acts of violence are ignored or denied by local and state officials. My research explores the violence that is enacted through the actions and inactions of state and local officials. I will conduct an analysis of declassified documents, released by the United States and as well as related research publication. My research contributes to a larger conversation about how accountability can be upheld for the victims of violence who have been dismissed by state officials and the media. I also hope to contribute to a larger conversation about how to prevent such violence. Keeping a research focus on these questions also mitigates against the further violence of forgetting about the victims and their families.

Recent studies like the Coupled Model Intercomparison Project Phase 5, or CMIP5, have sought to quantify how atmospheric variables will change due to climate change. Even from decades of rigorous study, it is still uncertain how cloud feedbacks will respond to a warming climate. However, it is possible to try and minimize this uncertainty in part by examining deep convection, and specifically anvil clouds, in the Tropical Pacific. Anvil clouds are not simulated well by the current generation of climate models, but by studying both large scale motions and small scale, local cloud structural evolution, Professor Hartmann and I seek in this study to determine the underlying mechanisms of the differences in vertical cloud structure in the Eastern and Western Pacific. I use the ERA-Interim, CloudSat, Calipso, and CMIP5 datasets in addition to climate model output from the Community Earth System Model (CESM) to analyze differences in cloud structure and model output. This is important work, because reducing uncertainty in an era of global warming can mean better advanced warning systems and more informed, ethical policy decisions moving forward.

Plants take up silica over the course of their lives along with other essential nutrients in the water that they absorb through their roots. The silica becomes deposited within their tissues in the form of solid bodies (phytoliths); when the plant dies and decays, phytoliths are left in the soil where they can fossilize. Fossil phytoliths preserve the original morphology of plant cells and can be used to reconstruct past vegetation. The Middle Miocene Climatic Optimum (MMCO) was a major global warming event that happened ~17-14.5 million years ago. Researcher have reconstructed the climate of the Santa Cruz Formation in Patagonia, Argentina during this period as warmer and drier. While we would expect the vegetation to become more open (i.e., grassy or shrubby versus forested), phytoliths from the Santa Cruz Formation tell a different story; in particular grass phytoliths (and therefore grass abundance) seem to decrease through time, but also become smaller. Some researchers have noticed that grasses tend to respond to reduced water availability with reduced cell size. Therefore, we hypothesize that the reduced size of grass phytoliths observed in the Santa Cruz Formation during the MMCO is due to increasing aridity. However, because this trend of decreasing cell size was only noticed by qualitative observation, we need to quantitatively assess significant changes in cell size. To do so, I studied and imaged grass phytoliths under a microscope, categorized them by subfamily/tribe based on their shape, and measured their size. I then statistically compared the phytoliths from younger and older strata to test whether there was a change in size through time. This work will be used to predict how our current global warming event may impact plant life based on the trends of the past.

Methane reservoirs are commonly found throughout the world’s oceans and the release of methane from seafloor reservoirs is thought to make up 5 to 10% of the global atmospheric methane. In fact, the greatest deep-sea mass extinction in the last 97 Myr during the Paleocene-Eocene Thermal Maximum (PETM) may have been caused by methane release from seep sites along the upper continental slope margin. Recently, methane reservoirs along this margin have been gaining attention due to their potential to accelerate current global warming. Changes in seafloor pressure and temperature could destabilize these seafloor deposits and cause methane bubble plume release into the ocean. At SHR, an extensively studied active seep site located ~ 90 km offshore Oregon, discontinuity in methane plume release was observed, but still not well understood. Hence, using Acoustic Doppler Current Profiler (ADCP) and pressure data archived by the Ocean Observatories Initiative (OOI) Cabled Array, we are investigating the potential correlation between tides and the presence of methane plume at SHR. Our study detects methane plume structures based on the proxies of echo contrast caused by acoustic-bubble interaction. By analyzing the derived plume structures and their correlation with 226 tidal cycles, we expect a trend of plume release triggered by low tides. Our study provides the first high-temporal-resolution analysis on the methane plume release at SHR using OOI acoustic data. 

The physics behind wave-driven mixing of river and ocean waters and current-driven wave breaking are not well understood. The current body of work surrounding river-ocean interactions focuses on large rivers. However, small rivers, which are much more strongly influenced by waves, make up the majority of such systems, and contribute significantly to global riverine discharge. Examining the momentum balance of river flow in opposition to wave-driven forcing from the ocean is necessary to understand how waves influence the travel and mixing of river water. One way to measure this interaction is using instrumental drifting buoys that follow the path of the river water and take temporal measurements of water properties. These leave gaps in our knowledge, as such buoys do not provide a description of the entire system, only specific points. To fill in these gaps, Unmanned Aerial Vehicle (UAV) footage was used to understand broader wave-current interactions at the Quinault River mouth, a small river that feeds directly into the Pacific Ocean. The town of Taholah, WA, is on its banks, and faces challenges due to wave-driven flooding. The size of the surf zone, the nearshore region where waves break at high frequency, was mapped with UAV footage, and related back to local environmental conditions, such as tidal phase. At low water, the momentum from the river is maximized, and so is the cross-shore extent of the surf zone. This decreases salinity around the river mouth, as freshwater is trapped by the surf zone. At high tide, these conditions are reversed, and fresh water streams can be detected past the surf zone, suggesting the river water has escaped from this region of high turbulence. The conditions under which these escapes occur are to be understood by combining analyses of UAV footage with drifter and tidal data.

During embryonic development, a dormancy-like state known as diapause arises during the transition from pre to post implantation. This state of suspended development is a reproductive strategy which favors newborn survival in mammals during nutritional deprivation or stress. Studies from the Ruohola-Baker lab found potential candidate regulators of diapause by establishing an in-vitro diapause model using pluripotent mouse embryonic stem cells (mESC). One of the genes is Activating Transcription Factor 5 (ATF5) which encodes a protein capable of survival-mediated functions such as maintaining mitochondrial activity during stress, modulating cell differentiation, preventing apoptosis and regulating cancer pathway. ATF5 has been known to transcriptionally target mTOR, a mechanistic target of rapamycin. Energy stress in the form of starvation and pharmacological inhibition of mTOR has shown to induce diapause-like state in mESCs in vitro. Our hypothesis is that upregulation of ATF5 under energy stress will reestablish diapause-like state in naïve mouse embryonic stem cells in vitro. We will test our hypothesis by loss-of-function and overexpression experiments. We test if ATF5 gene knockout using CRISPR-Cas9 prevents the mutant lines from entering diapause-like state from energy stress. Using western blots, we will quantify phospho-mTOR levels and its downstream targets in the ATF5 KO lines and compare them with the wildtype lines. For the overexpression of ATF5, we will make rescue lines for the ATF5 KO cells. We predict that overexpressed ATF5 in rescue lines will enter diapause-like state, and have reduced mTOR and its downstream target signals compared to KO lines. Our discoveries of ATF5 function in diapause can be useful in understanding how early-staged cancer stem cells enter a diapause-like state or quiescent state which enables them to escape chemotherapy detection. We can potentially contribute to the development of therapies to target ATF5 mechanism so that these undetected cancer stem cells can be detected.

Synthetic CRISPR–Cas transcription factors enable the construction of complex gene expression programs, and chemically–inducible systems allow for precise, rheostatic-like control over the transcriptional dynamics. We have constructed a bio-orthogonal, chemically–inducible synthetic CRISPR regulatory system to activate and repress gene expression in yeast. By fusing chemically inducible transcriptional regulators to specific RNA binding proteins, we have expanded the tunability of this system of constructs. The RNA binding proteins are fused to half of one chemically-inducible system while the other half is fused to an effector. Upon addition of drug, the two halves come together to form the complete chemically-inducible system and either activate or repress the target gene. We use reporter gene assays to probe the dose-dependence, time-dependence, and reversibility of the systems. The use of multiple, orthogonal chemically-inducible systems and unique guide RNAs allows for more sophisticated, multi-gene programs that still maintain precise control of gene expression dynamics independently at different sites.

New techniques in genome mapping have revealed a high degree of 3D spatial organization in the nucleus. Long-range loops connect enhancers to their gene targets to regulate expression. In order to assess the mechanisms and functions behind 3D spatial organization of the nucleus we need a system that allows us to engineer DNA loops. We use programmable CRISPR-Cas DNA binding domains to target specific sites in the genome. The CRISPR-Cas complex is tethered to a targeting domain (TD) that can dimerize with another TD at a distant DNA locus. To promote interactions between TDs that are bound to DNA, we have designed an allosteric switch that assembles the TD only when the CRISPR-Cas complex has engaged its DNA target. To validate that our switch proteins can act as a DNA sensor we have developed a simple reporter assay; upon successful switch protein activation on DNA a transcription factor is recruited to drive expression of a fluorescent protein. Our results indicate that the protein switches activate when they are recruited to DNA, effectively acting as a sensor for DNA binding. Future steps include optimizing our reporter assay design and modifying the design for DNA looping.

Since 1982, with the introduction of insulin as the first recombinant protein therapeutic, peptide and protein drugs have grown to encompass 10% of the pharmaceutical market and are the fastest expanding class of drugs. Advantages of using peptides over small-molecule drugs include high potency, selectivity, and capability to be engineered for a diverse range of targets, most commonly to disrupt or facilitate key protein-protein interactions (PPIs) in the human body for a therapeutic effect. To search for strong binders for a therapeutic target, combinatorial peptide libraries of up to billions of different sequences are synthesized and screened against promising targets. Due to the enormous library size, screening for high affinity binders often requires multiple rounds of enrichment in order to isolate the most potent molecules, a laborious and potentially bottlenecking step in developing protein drugs. Current approaches that have strategies for enrichment, such as phage display and yeast surface display, are limited to screening a library of binders against one target instead of multiple targets (library-on-library). This proposal describes the development of a peptide binder screen utilizing a simple workflow of repeated mating and sporulation of genetically engineered S. cerevisiae, or baker’s yeast. This technology improves the throughput of established screening methods through a library-on-library format that efficiently isolates high-affinity peptide binding interactions.

Precise spatiotemporal control over biochemical cue presentation is necessary to mimic the complex, heterogenous environments found in biological systems. Achieving this level of control within engineered microenvironments would allow for the manipulation of cell growth and differentiation, which could be utilized in tissue engineering and drug delivery. To this end, we developed a method that utilizes fusion proteins made from a novel PhotoCleavable protein linker (PhoCl) and a protein of interest (POI). This method allows for spatiotemporal control of POI release from hydrogels in response to cytocompatible violet light (λ = 405). This system is flexible, as PhoCl can be conjugated to many different POIs, including fluorescent proteins, enzymes, and growth factors, and was found to not affect protein function. Additionally, PhoCl undergoes a green-to-red transition after photocleavage, allowing for real-time tracking and quantification of POI release. As PhoCl cleaves in response to visible light, which is less damaging to cell function and has a greater tissue penetration depth than the traditionally used UV light, PhoCl fusion proteins hold promise for use in vivo. To demonstrate the feasibility of this system, PhoCl fusion proteins were formed with several fluorescent proteins (e.g., mRuby, sfGFP, mCerulean). Conjugating these fusion proteins into gels and exposing them to patterned light produced spatiotemporal localized release of proteins with micron scale resolution, which was demonstrated through fluorescent imaging of the photopatterned gels. To support the potential in vivo applications of this system, PhoCl was also used in mammalian cell studies with epidermal growth factor (EGF). These studies showed the expected increased cell growth in response to photomediated EGF release. This illustrates the potential versatility of the PhoCl system in biological applications, thus supporting the relevance of this novel system to tissue engineering and drug delivery methods.

My guiding question is, “how we can I use storytelling to empower the stories and experiences of the black community in the Central Area that has been displaced through gentrification?” Redlining has historically made it harder for people of color to obtain a mortgage or buy a property in certain neighborhoods, and it continues to exist as gentrification. As tech companies like Microsoft, Amazon, and Airbnb among others continue to expand their presence in the greater Seattle area, rent prices have risen and property owners have started building expensive apartments and living complexes for the influx of workers with a high income. This expansion has displaced people of color who have historically lived in the Central Area. Currently there are stories of resilience in communities that have been displaced by gentrification, but they are often not showcased in the public eye and media outlets. I want to focus on the use of radical storytelling as an act of defiance against erasure, and the role of tech companies in the greater Seattle area in displacing communities. Inspired by the interactive digital stories like “Trump Wants a Border Wall. See What’s In Place Already” in the New York Times or “Microsoft By the Numbers” on Microsoft Story Labs, I hope to create a visual digital story about the ways and means of gentrification, and I hope this story can be used to educate the public about the mechanisms of gentrification and where communities move. By conducting interviews with activists and community members in the Central Area and highlighting their perspective through direct quotes and testimonials, I hope to educate everyone about the strength of communities in the Central Area. My goal is to create a project that focuses on empowering people of color and their stories through community activation.

Not only a form of self expression, fashion is one of many markers used by society to assign class, race, gender and sexuality. These assignments are made based on a set of norms which dictate which people are, or are not, privileged. Because of its power to assign privileged traits to an individual, it is also able to create a certain norm for what certain identities are meant to look like. In queer spaces, these norms are often thought to be challenged. This study engages with queer and feminist theories to analyze the ways queer individuals interact with the world of fashion. It argues that fashion should be viewed not only as a way through which individuals show self-expression, and explore identity, but also a form of powerful discourse. In order to do this, I have conducted 15 filmed interviews of queer individuals int the city of Seattle. These interviews seek to provide a platform through which participants can express the complexities of identity formation within a liberal, capitalist system as it relates to fashion. Interviews are collaborative and semi-structured, allowing for participants to share what they find important to their person and communities. From these interviews, this research engages with how the intersections of queer identities with race, class, gender, and sexualities work to form identities through fashion. This research and knowledge is then produced in the form of a thesis paper as well as a video. I have chosen to represent this research in a video format as there is something inherently visual about engaging with identity formation and fashion. This research and its methods are important in exploring and displaying the ways complex and multifaceted intersectional identities interact with systems of power through expression and fashion.

Team leaders occasionally find themselves managing teams that have a member set on under-performing in the social group, or "social loafing." Many team leaders have difficulty managing this social loafer effectively. In our research, we focus on helping leaders identify social loafers on their team. Our goal is to create guidelines and strategies on how to work with social loafers after they have been identified. We use communication transcripts and performance data, gathered by social behavioral researchers, that conducted a hidden profile experimental simulation with rotating leadership and hidden social loafers amongst project members. This was done over time, as project requirements escalated. By applying recurrence quantification analysis, we gathered metrics on the development of communication patterns across time in terms of: being recurrent, deterministic, and chaotic. Statistical modeling was used with the intention of revealing the types of relationships between the ability to identify social loafers and project success. By providing a way to quantify the properties of dynamic systems, recurrence analysis offers group researchers a new approach for empirically studying group dynamics. Rather than presuming that such systems are linear, researchers can use recurrence analysis to assess the degree to which a system is stable, predictable, and complex. This work will provide value to researchers in demonstrating the application of the recurrence analysis method, and will help team leaders by exemplifying how they can manage tough situations where social loafers exist on a team.

This project deals with human robot-network collaboration for synchronization to desired reference velocities. A human interacts with the network of mobile robots by sending virtual source inputs to the leading robot. The information propagates through the network from each robot sensing its nearest neighbor. This research proposes a visual interface design to develop a real time, wireless communication channel between a human operator and the robot-network. The model developed introduces real time feedback from the human operator via a graphical interface of the relative positions of each robot in the network. The human operator’s ability to move the network cohesively with desired velocity trajectories require rapid information transfer, which is achieved using a delayed self-reinforcement (DSR) technique. We expect the human operator’s ability to move the network cohesively to improve with DSR hence enabling easier operation for the operator. The human-swarm interface designed has applications for semi-autonomous networks such as vehicle platoons. We can improve modern freight transportation safety and efficiency with a human remotely operating a robot-network of trucks.

Our research group performs one of the highest-precision tests of Einstein’s equivalence principle, perhaps the most fundamental property of gravitation, using a sensitive rotating torsion balance. Among the leading experimental challenges are temporal and spatial temperature variation. Notably, horizontal temperature gradients across the apparatus, if not properly characterized, can emulate an equivalence-principle violating signal. We have implemented thermal shielding and run tests to measure the thermal effects on our measurement. Past tests have shown a need for both absolute and differential temperature sensors with higher sensitivity. Hence, my research project focuses on investigating the effect of temperature gradient on our experiments by constructing a thermal monitoring system. I have designed, laid out, constructed, and tested sensitive bridge thermistor circuits that can function as both absolute and differential temperature sensors. Current tests of our prototypes have shown that temperature sensitivities reaching 10 micro-Kelvin in one second (10^-5K/Hz^0.5). We are scaling-up these sensors and plan to deploy them in this academic year. Successful completion of this project will yield improved understanding of the temperature gradients within our experimental apparatus, allowing us to test the equivalence principle with yet higher precision.

Isotropic scattering in various spatial dimensions is considered for arbitrary finite-range potentials using non-relativistic effective field theory. With periodic boundary conditions, compactifications from a box to a plane and to a wire, and from a plane to a wire, are considered by matching S-matrix elements. General relations among (all) effective-range parameters in the various dimensions are derived through a functional relationship, and the dependence of bound states on changing dimensionality are considered. This research was conducted by matching the effective range parameters in one dimension to the particle scattering within another dimension. This relationship ultimately leads to the immediate functional relationship of the effective range parameters in varying dimensions. Generally, it is found that compactification binds the two-body system, even if the uncompactified system is unbound. For instance, compactification from a box to a plane gives rise to a bound state with binding momentum given by ln(1/2(3 + √5)) in units of the inverse compactification length. This binding momentum is universal in the sense that it does not depend on the two-body interaction in the box. This research will in the future allow for the calculations of practical and important thermodynamic variables such as pressure, energy of Bose gases with varying lengths of a three dimensional box.

High purity germanium (HPGe) detectors are an important technology in several leading experimental searches for dark matter and neutrinoless double beta decay. Understanding the interaction of various types of radiation on the different surfaces of HPGe detectors is essential to developing methods to reject unwanted signals from radioactive background sources. I have taken a leading role in the construction and use of the Collimated Alphas, Gammas, and Electrons (CAGE) test stand at the University of Washington, whose goal is to evaluate the response of an HPGe detector to different types of radiation on its various surfaces. CAGE is a vacuum cryostat with an internal system of motors that move a radiation source while keeping the detector active. It requires the operation of a liquid nitrogen cryostat, vacuum pump, temperature sensors, and various radioactive sources, all of which must be integrated into a single data acquisition (DAQ) system. We are currently constructing this system, fabricating and installing parts, and are planning to take initial data with the HPGe detector in the summer. In this talk I will present the current status of the CAGE detector, as well as preliminary data from radiation signals in the detector.

Here at the University of Washington we are characterizing one ton of NaI[Tl] crystal scintillator detectors for use in the COHERENT project. NaI[Tl] scintillating crystals detectors work by producing photons from the kinetic energy of charged particles passing through the scintillating material. COHERENT aims to detect coherent elastic neutrino-nucleus scattering, a novel interaction between neutrinos and matter that was first observed less than two years ago. It employs a large scale of scintillator detectors in order to record these events at an appreciable scale. Our characterization campaign allows us to group crystals with similar outputs by voltage which will determine the setup of our detectors once at ORNL. During this characterization, the crystals exhibited behaviors that correlated with the ambient temperature of the lab. The temperature dependence was first noticed during voltage gain characterization tests taken at different times of the day in the uncontrolled temperature environment of our lab. We expect the gain of our crystals to fit to a curve function, which breaks down if data is taken at different times of the day. The goal of this study is to understand the impact of temperature dependencies on our characterization campaign, and in particular to derive a relationship between voltage gain and temperature. I will present the data gathered toward this goal, and also our larger body of data on the relationship between light yield, voltage gain, peak resolution, and waveform rise time, as well as the techniques used to re-characterize previous crystals gain curve based on the derived relationship from this study.

Latent class models are frequently used to classify observations into homogeneous albeit qualitatively different sub-groups. Typically, these models are unrestricted, meaning there is no a priori structure or restrictions on model estimates. This approach is similar to many cluster analysis approaches. The study examines modeling conclusions when an unrestricted model is applied to data generated from a highly restrictive population model through a simulation study. We expect that a researcher will conclude fewer classes than the true number specified in the population. Simulated data for this work is based on a substance use example identified in other research. The population was generated based on six classes, but with a within class error rate fixed to 5%. Preliminary analyses indicate that commonly employed model selection criteria indicate that models with fewer than six classes are preferable. This is important because most analysts using latent class models use unrestricted models with little apparent consideration to what realistic error rates may be. In research domains where smaller error rates may be plausible, restrictive models of the sort used to generate the data for this study may be worth considering.

 The United States’ participation in “othering” is nothing new. Government policies for centuries, including executive orders, have long targeted minority populations living in or seeking refuge within the U.S. These populations are often the scapegoats in politically turbulent times and are treated as such in order to not deal with greater issues. During World War II, Japanese Americans—including citizens and legal residents—were sent to internment camps not knowing when they would return home. This was the “solution” to war hysteria post-Pearl Harbor. Similarly, current immigrants from non-European populations are targeted by ICE and taken away from their families with no promise of return. Although the rationale is complex, this rhetoric often revolves around jobs and criminality despite any significant statistical back-up. My presentation seeks to compare these two situations: what is currently happening with Latin American and Southeast Asian immigrants in the U.S. to the incarceration of Japanese Americans during WWII. These families, whether nearly 80 years ago or today, have and are being systematically stripped of their livelihood and humanity for the sake of political scapegoating. Through intensive archival research—including collecting historical photographs, first-person accounts, and government propaganda of and against those incarcerated in the Japanese Internment Camps and current U.S. detention centers—, I examine the similarities in these families’ plights, the situations’ causes, and their inevitable long-term impacts. Through this analysis, I seek to interrogate the broader structure of U.S. immigration policies and our place in these events as global citizens. It is only through such analysis that we can began to understand the cyclical nature of rhetoric and trauma and have a chance to stop it.

Herpes simplex virus (HSV) infects mucosal epithelial cells and can cause recurrent, painful ulcers. HSV is not curable and establishes lifelong latency in host neuronal ganglia. Herpes affects more than 400 million people globally, with an increased risk of genital ulcer disease, HIV acquisition, and transmission of HSV-2 to partners or neonates. Persons with altered T cell immunity have been reported to have prolonged and more frequent lesions, suggesting that the adaptive immune responses are associated with HSV clearance. Additionally, there is evidence of innate cell and T lymphocyte recruitment to the infection site to help clear the lesion. While there is extensive evidence on the role of T cells in these lesion infiltrates, little is known about B cell activation and antibody response during HSV reactivation. The study of B cells and antibodies can be used in developing a HSV vaccine since all licensed vaccines induce robust antibody responses. Antibodies block virus entry and mediate antibody-dependent cell-mediated cytotoxicity (ADCC). That is why a better understanding of local antibody responses against HSV-2 is needed to develop a successful vaccine. My project examines whether HSV antibody-producing B cells are found in areas where HSV-2 reactivates and the potential role that these antibody-producing cells play in host clearance of the virus. To examine this question, ddPCR using B-cell-specific probes were used to identify different B-cell subtypes responding to the HSV infection. Results to date have shown high variability with few emergent patterns. In the future, additional samples will be examined using new protocols for better cDNA synthesis, and more positive and negative controls to increase the accuracy of the data.

The ability of an organism to dissociate environmental cues for either safe or threatening situations is key for survival. Generalized fear is an adaptation in which behavioral responses for threatening stimuli are produced to non-threatening cues. In mice, discriminative or generalized fear responses are modeled using a fear conditioning paradigm of two shock intensities, 0.3 mA or 0.5 mA. Mice trained at 0.3 mA intensities could discriminate between safe and threatening cues, yet mice trained at a 0.5 mA intensities displayed generalized fear behavioral responses. Previously, we had identified that dopaminergic neural activity was critical for the ability to discriminate between cues when shocked at 0.3 mA. Dopamine neurons express Kv7.3 potassium channel subunits, which modulate neural activity. I hypothesized that Kv7.3 subunits on dopamine neurons would be critical to threat discrimination and mice that had undergone mutagenesis of Kv7.3 would show generalized fear discrimination. My approach used transgenic mice expressing Cre-recombinase and viral-mediated gene delivery of cre-inducible CRISPR-Cas9 plasmids targeted for the specific mutagenesis of Kv7.3. Mice underwent a three day paradigm known as fear conditioning. Baseline freezing behavior was assessed by playing two tones, A and B. This was followed by fear conditioning trials where at the end of tone A, mice received a shock of either 0.3 mA or 0.5 mA (CS+) and at the end of tone B, mice received no shock (CS-). On the third day, mice underwent a probe trial to assess final ability to discriminate between threatening and non-threatening cues. These findings could provide insights into the mechanisms underlying neurological disorders and serve as a guide for future pharmacological interventions.

Over 350 million people are affected by major depression on a global scale according to the World Health Organization with an increasing economic impact of over $210 billion annually with a growth of 21.5% since 2005. The ability to switch behaviors or strategies when faced with chronic stress is an important determinant to the susceptibility and longevity of depression within people, also referred to as cognitive flexibility. The objective of this proposed study is to analyze the effects of the endogenous cannabinoid (ECB) system on cognitive flexibility through the lateral habenula. Cannula implant surgeries will be conducted on rats to input antagonist or agonist drugs into the brain. Behavioral tests will be conducted on rats to examine whether these manipulations will promote or deter cognitive flexibility.

Due to antibacterial resistance and the high recurrence of urinary tract infections (UTIs), studies have shifted to focus on anti-adhesive therapies as alternative to antibiotics. Often treated with antibiotics, UTIs are caused by uropathogenic Escherichia coli (UPEC). The bacterial adhesin, FimH, found on the terminal end of fimbria, hair like structures expressed on the perimeter of UPEC, is the main etiological factor of UTI prevalence and recurrence. FimH increases the virulence factor of E. coli by mediating the initial binding of the bacteria to glycosylated cells in the urinary tract. FimH has two domains. The lectin domain (LD) recognizes and binds the terminal mannose on glycosylated cells lining the urinary tract, whereas the pilin domain acts as an anchor to the fimbria. Previous studies have shown that α-methyl-mannose (αMM) competitively inhibits glycoproteins, such as horseradish peroxidase (HRP), from the FimH active site. We hypothesize that αMM can non-competitively inhibit HRP through a novel mechanism of inhibition. To determine the mechanism of inhibition of HRP in the presence of αMM, we are using Enzyme Linked Immunosorbent Assays to measure the dissociation of HRP in the presence and absence of αMM, after the FimH-HRP complex has formed. We expect to see an increase in the dissociation of HRP in the presence of αMM. HRP in this case, will act as a model to the glycosylated cells lining the urinary tract. This study aims to assist in the design of innovative anti-adhesive therapies that inhibit binding of FimH once bound to glycosylated cells lining the urinary tract.

Modern cells discriminate among the amino acids chosen to be included in protein synthesis: some, like leucine, serine, glycine, and alanine appear in most proteins across most cells, whereas other amino acids, like (g)-aminobutyric acid and aminoisobutyric acid do not appear. This raises the question of how selectivity among amino acids arose – does the selectivity rely on modern protein machinery or could it have arisen in the first protocells of the Early Earth? For example, could certain amino acids have, by some mechanism, associated more strongly with protocell membranes, increasing their chances of being integrated into the first peptide chains? To test this hypothesis, our group assembles rudimentary protocells from molecules that would have been present on the early-Earth: decanoic acid (a fatty acid), sodium mono-phosphate, salt, and water. Vesicles of the decanoic acid spontaneously form. We then add different amino acids to the solutions and measure their turbidity to determine whether each amino acid causes the number of lamellae in the vesicles to increase or decrease. Increased lamellarity correlates with a sturdier vesicle. If certain amino acids increase lamellarity of protocells, that could serve as a method of selection for certain amino acids rather than others. Our results are that particular amino acids (most notably serine, glycine, and alanine) do in fact increase the lamellarity of fatty acid vesicles significantly, whereas other, less common, amino acids do not. We are currently exploring the plausibility of a mechanism for this occurrence involving ease of rotation around the alpha carbon of the amino acids, and we are investigating other ways in which interactions between amino acids and fatty acid membranes might be manifested, for example by a shift in the solution’s critical vesicle concentration. Our results will fit into the overarching goal of understanding peptide formation and protocell stability in order to gain insight into the origins of life on Earth.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) have great potentials in biomedical research and can be used extensively in drug screening and heart simulations. To understand the cardiomyocytes, we need to perform functional analysis on these muscle cells. Therefore, we need a simple, controllable, yet biocompatible and high throughput tool to measure the cellular traction force. At the Sniadecki Lab, we are developing a new technique to measure the force generation of hiPSC-CM: dotted traction force microscopy platform. To create the platform, fluorescent proteins were first absorbed to a dotted polydimethylsiloxane (PDMS) negative and stamped onto a polyvinyl alcohol film. The film was then transferred to a soft PDMS substrate and subsequently dissolved using phosphate buffered saline solution while the patterned fluorescent proteins stained the substrate. Since the stiffness of the soft PDMS substrate is known, the force generation of the cardiomyocytes can be calculated in real time by optically tracking the deformation of the fluorescent dots. Currently, we are able to manufacture the platform with high fidelity and uniform alignment with a production time of less than 2 hours. Moreover, the cardiomyocytes can fully spread out to their in vivo state on the substrate which ensures the force measurement is valid and accurate. Potentially, this method is not limited to cardiomyocyte research and can be applied to study the interaction between force generation and cell performance of other cells. We are also exploring the possibility of automated manufacture and integration with 96-well to enable mass production.

Within the domains of graphic and video game design, there is often need for tools to quickly develop convincingly realistic models of plants. A common tool applied to this problem is L-systems, a kind of rewriting system that can be used to define rules for iteratively transforming plant models to increasingly fine detail. However, the connection between L-systems and the graphics they generate can sometimes be unintuitive. To enable more intuitive development of plant models, we propose a method for generating models of branching structures from simple specifications of a few given iterations of the model. Our approach involves encoding plant models as bracketed L-systems and applying SMT (Satisfiability Modulo Theory) solvers to solve a form of the inverse L-system problem. Iterations of growth in the form of simple vector graphics are compiled to formal constraints for an L-system that can indefinitely generate further growth iterations. The satisfactory system is then found using an SMT solver. This technique allows for branching structures to be conveniently developed by providing meaningful specifications.

Trigger-action platforms enable end-users to program their smart homes using simple conditional rules of the form: if condition then action. Although these rules are easy to program, subtleties in their interpretation can cause users to make errors that have consequences ranging from incorrect and undesired functionality to security and privacy violations. Based on prior work, we enumerate a set of nine error classes that users can make, and we empirically study the relationship between these classes and the interface design of eight commercially available trigger-action platforms. Particularly, we examine whether each interface prevents (e.g., via good design) or allows each class of error. Based on this analysis, we develop a framework to classify errors and extract insights that lay a foundation for the design of future trigger-action programming interfaces where certain classes of errors can be mitigated by technical means or by alerting the user to the possibility of an error. For instance, we identify that an analysis of a dataset of functionally-similar trigger-action rules could be used to predict whether certain types of error patterns are about to occur. We believe that this work is a first step towards trigger-action interface designs that significantly mitigate user error.

The ability to derive information through automated scanning of personal documents has significant economic and societal value, stemming from applications in surveillance and digital forensics, e-commerce, tailored advertising, recommender systems, human resource management, mental health care, and more. Giving applications access to one’s personal text messages and e-mails can easily lead to (un)intentional privacy violations. We have developed and implemented cryptographic protocols to scan personal documents in a privacy-preserving manner, using techniques from Machine Learning (ML) and Secure Multiparty Computation (SMC). In a typical scenario of interest for our research, there are two parties, nick-named Alice and Bob. Bob has a trained ML model that can automatically classify texts like e-mails, for instance inferring whether the author is depressed, suicidal, a terrorist threat, or whether the e-mail is a spam message. Our SMC based protocols allow for the classification of a personal text written by Alice with Bob’s ML model in such a way that Bob does not learn anything about Alice’s text (other than the class label resulting from the classification) and Alice does not learn anything about Bob’s model. We demo the cryptographic protocols in an application for privacy-preserving detection of hate speech against women and immigrants in text messages, built on top of the SMC framework Lynx developed at UW. In this use case, Bob has a boosted decision tree model that flags texts as hateful based on the occurrence of particular words. We show that Bob can label Alice’s texts as hateful or not without learning which words occur in Alice’s texts, and Alice does not learn which words are in Bob’s hate speech lexicon, no how these words are used in the classification process.

With the global refugee crisis, resettlement agencies do important work to insure successful refugee resettlement. Previous research suggests that some refugee families struggle with segmented assimilation, which is when a child assimilates faster than their parents, disassociating from their culture of origin, creating conflict with their parents. Building on this research, this qualitative ethnographic study investigated segmented assimilation concern among families through semi-structured interviewing. Refugee families and childcare providers for refugee children were interviewed to understand the needs of families in managing segmented assimilation and to gain insight from childcare providers serving those families. While it was hypothesized that families would be concerned about segmented assimilation, the recently arrived families in this study were not experiencing segmented assimilation. This is due to a lack of resources which maintains daily stresses of supporting themselves. The resettlement agency in the city where interviews were conducted solely provided housing and employment placement services, meaning that refugees had no resources for civic engagement, education, community building, and other necessary programs. While families are now physically safe, in terms of not being in a war zone or crowded refugee camp, their lives are still extremely strained, in part due to the lack of adjustment resources. Many families work long hours simply to have enough money to live in their apartment and feed their children. The lack of resources given to resettlement agencies, especially in the recent years under the Trump administration, have profound impacts on the lives of hundreds of refugees that are being resettled, and the thousands more that have not been allowed into the United States due to restrictions under the Trump administration. After refugees move to America, it takes time for them to establish their lives in their host country, and the lack of resources allocated to refugee resettlement impedes this process.

Dravet syndrome (DS) is a severe form of childhood epilepsy caused by a mutation in the SCN1A gene, which encodes the NaV1.1 voltage-gated Na+ channel. This channel is present in most GABAergic neurons, the main inhibitory neurons in the brain. Reduced activity of the channel in DS leads to loss of inhibitory activity in the brain; this, in turn, leads to seizures and developmental deficits. Through previous research using the mouse model of DS, the de la Iglesia lab has demonstrated that DS also affects circadian rhythms, which are the endogenous biological rhythms synchronized to the 24 hour day. These symptoms are likely caused by the loss of NaV1.1 in a sleep regulatory center called the suprachiasmatic nucleus (SCN), a set of cells which functions as the ‘master clock’ of the circadian system of mammals. However, the de la Iglesia lab found that selective deletion of the SCN1A gene from the SCN fails to replicate the abnormal circadian phenotype. We believe that these mutant mice are phenotypically normal either because there is a compensatory increase in the expression of another sodium channel, NaV1.3, or because the targeting strategy does not reach all cells within the SCN. To test the first hypothesis we employed in-situ hybridization to visualize the expression of the genes that code for NaV1.1 and NaV1.3 channels in either SCN-specific knock outs or their wild type littermates. My results will help explain the phenotype seen in the SCN-specific SCN1A mutants and determine whether developmental compensatory mechanisms are important in the SCN of DS mice.

Most organisms show a roughly 24-h cycle in their physiological and behavioral processes, called circadian rhythms, generated endogenously through the ~24h cyclic expression of genes known as clock genes. Clock gene expression oscillates in the master circadian clock of mammals – the suprachiasmatic nucleus (SCN) - and nearly every cell of the body. Typically, circadian clocks and the rhythms they sustain are ‘entrained’ by the 24-h light-dark (LD) cycle. Our lab has found that fear can also behave as an entraining factor. We observed that when mice or rats need to leave a safe nesting area to access a foraging area, they forage and feed during the dark phase of the LD cycle. If the foraging area is rendered dangerous with random uncued footshocks during the active dark phase, the animals’ foraging and feeding activity shifts to the light phase. My goal is to understand the neural circuits and molecular processes involved in fear entrainment. I have analyzed the expression of clock genes in animals exposed to nighttime fear and control animals exposed to daytime fear; this allowed me to assess the circadian rhythm of expression of clock genes of interest (Per1 and Bmal1) in the SCN and amygdala, and I found that the amygdala entrains to fear but the SCN does not. I have also performed trials with brain-specific-knockout mice and found that nocturnal fear entrainment requires an intact molecular clock. My current experiments use a more specific knockout strategy of viral injections into the brain to determine whether a functioning circadian oscillator in the basolateral amygdala (BLA) or the SCN is needed for nocturnal fear entrainment. These experiments serve to unmask the molecular mechanism of fear entrainment and could also help understand the mechanisms linking fear and anxiety disorders to problems with circadian rhythms and sleep.

Microglia, the immune cells of the central nervous system, are long lived. In mice, microglia have an average lifespan of 15.5 months. When microglia are experimentally depleted from the mouse brain, microglia populations quickly return to steady state levels. The mechanisms of this observed repopulation are unclear. More importantly, the mechanisms of microglia replenishment in the healthy brain are not well understood. The literature supports two competing hypotheses. One is that microglia proliferate simply by dividing. Another possibility is that pools of microglia progenitor cells within the central nervous system divide and differentiate into microglia. I hypothesize that microglia proliferate primarily through the differentiation of progenitor cells. Available data to date suggest CD133 as a potential marker for microglia progenitor cells. In order to study these putative progenitor populations, I used a genetic reporter mouse line in which administration of tamoxifen induces TdTomato expression specifically in CD133-expressing cells. TdTomato, a red fluorescent protein, allows these cells to be visualized under a fluorescence microscope. Importantly, all the progeny of these cells also express TdTomato, allowing us to determine whether CD133 cells generate new microglia over time. After tamoxifen treatment at the age of 10 weeks, mice were sacrificed at three and nine months of age. Brains were fixed, sectioned, and labeled with antibodies to a microglia specific protein and to TdTomato. Daughter microglia that differentiated from CD133-expressing cells express both markers. Using a fluorescence microscope, I identified several microglia daughter cells of CD133-expressing cells. This suggests that microglia populations replenish in the healthy brain at least in part through the division of CD133-expressing cells. We can apply this new knowledge about how new microglia are generated in the healthy mouse brain to further our understanding of how microglia population dynamics are affected in both health and disease.

Interferon-gamma Enzyme-Linked Immunosorbent Spot Assay (IFNγ ELISpot) is a laboratory technique that quantifies the number of cells producing interferon gamma (IFNγ) by utilizing antibodies that selectively bind to IFNγ molecules, resulting in spot formation corresponding to individual IFNγ-producing cells. Since cytotoxic T cells (CD8 T cells) and their helper Th1 cells (CD4 T cells) produce IFNγ to activate macrophages and inflammatory responses, quantifying IFNγ-producing cells allows for characterization of host immune responses. Our lab utilized a canine IFNγ ELISpot to test for immune responses against novel proteins expressed following gene therapy in dogs. Currently, we used a routinely given vaccine for canine flu, distemper, adenovirus, and parvovirus as a biological positive control. However, we tested our experimental proteins with peptides, making a whole-protein positive control, which required internal processing, flawed. Our goal was to optimize this ELISpot by identifying peptides from the vaccine that stimulates an IFNγ immune response in peripheral blood mononuclear cells (PBMCs) and splenocytes. Utilizing a commercially available canine IFNγ ELISpot, we stimulated PBMCs and splenocytes. These cells included lymphocytes (T cells) and macrophages, which acted as antigen presenting cells. We compared stimulation with the entire vaccine and various vaccine peptides in order to identify peptides that can be used as a biological positive control. These were compared to traditionally used mitogens that indiscriminately activated all lymphocytes. This optimization allows for greater confidence in the results obtained from our canine IFNγ ELISpot. The improved technique serves as a powerful tool to assist in preclinical trials of vaccine production and gene therapy. It is utilized in our lab to test for CD8 T cell-mediated immune responses against novel dystrophins following gene therapy in Duchenne muscular dystrophy dogs.

Development of technology, including artificial intelligence, has been controversial due to the potential negative effects. Many believe it is an auspicious industry and that technology will eventually replace the work of the human labor force. In this research project, our team analyzed the effects of technology in the labor market on the labor force within the next few decades. To accomplish our goal, we developed a model that can interpret the correlations between various factors. Specifically, this research analyzed data, including the employment rate, unemployment rate, number of occupation categories, and involvement rate of technology from varied industries. In addition, the Bureau of Labor statistics database was primarily used to regress the model. We expected to see a decrease in low-skilled occupations, such as cashier, in a short run. These types of occupations will be replace by IoT or artificial intelligence. High-skilled occupations, such as lawyer and surgeons, will still be demanded and irreplaceable in a short run, but would eventually be displaced in the long run. A shift would occur from low to high skilled occupations, due to factors such as social demand, working efficiency, and wage polarization. The employment rate goes down during the short run since more jobs are replaced by artificial intelligences and robotics. However, in a long run, more occupations will be created which will recuperate the fall in employment rate occurred during the short run. This research is very crucial and necessary because of the rapid growth of technology and its immense impact on the human labor force market. This research could ultimately provide information for governments and policy makers regarding potential growth sectors in the economy due to technological changes. Also, future generation can be more aware of this technological change, so that they can choose majors with accurately reflecting future employment trends.

Research has shown that positive outcome expectancy (i.e., anticipated positive consequences as a result of engaging in a behavior) and positive affective responses to exercise play an important role in predicting exercise behavior as well as future exercise intentions. Unlike most research in which the valence (i.e., positive - negative) of outcome expectancy and affective responses are typically assessed through survey items whose valence is predetermined, the current study used a spontaneous word association test (WAT) to examine the subjective meaning of exercise-related outcome expectancy and mood before exercise, and how they are related to exercise intentions and behaviors. One-hundred and eleven undergraduate and graduate students (74 females; 64 undergraduate students) performed an exercise-related WAT in which they were asked to write down the first word or a short phrase that comes to mind when they thought of (a) things that might happen from exercising and (b) their mood right before exercising. Subsequently, they were instructed to rate their subjective valence (i.e., positive-negative) to each of their responses to the WAT. They also completed a series of online self-report surveys that included assessments of exercise intentions and past exercise behavior. Path analyses will be conducted to test our hypotheses that the subjective valence of their mood right before exercising is expected to be directly associated with exercise behavior whereas the subjective valence of outcome expectancy is expected to be indirectly associated with exercise behavior through exercise intentions. This study examines the potential value and usefulness of a spontaneous word association procedure to more accurately measure affective evaluations of an individual’s feelings and thoughts toward exercise behavior.

Mosquitoes primarily navigate using their olfactory system, and can use this system to form “memories” that influence their choice in hosts. When a mosquito encounters an odor, information is sent through the antennal lobes in the brain to the mushroom bodies, which are structures responsible for learning and memory consolidation. This odor-learning pathway is mediated by neurotransmitters like dopamine and serotonin. Recent research has shown that the mosquito Culex quinquefasciatus has extremely low levels of dopamine in the antennal lobes compared to other species, and is unable to learn to avoid odors associated with a negative response. This led us to predict that dopamine is essential for aversive learning in mosquitoes. We hypothesized that Cx. quinquefasciatus differed from other mosquitoes in learning ability because they were previously tested in the light and they are the most nocturnal of the originally tested species. To test this hypothesis, we conditioned the mosquitoes in the absence of light in an aversive learning paradigm to measure how frequently they chose to avoid the conditioned odor. An inability to learn regardless of light condition would indicate that the role of dopamine as a neuromodulator in the antennal lobes evolved partly to allow diurnal mosquitoes to avoid defensive hosts. Next, specific neurotransmitters in the antennal lobe were mapped using confocal microscopy, revealing their concentrations which may explain behavioral differences from other mosquitoes. Most mosquito species show some plasticity in host selection, which can lead to the transmission of animal diseases, like West Nile Virus, to humans. Mosquitoes are the world’s deadliest disease vector, killing over 700,000 people globally each year, so understanding how and why this adaptation occurs can help us understand the framework that underlies the spread of mosquito borne diseases and bring us one step closer to solving this global issue.

Flow Cytometry is a quantitative data collection method which utilizes a laser and optics system to measure forward and side-scattering light from single cells in a heterogeneous solution, which when analyzed describe the structure and internal complexity of the cells in solution. Additionally, varying wavelengths of light emitted from the cytometer excite particular fluorescent dyes that can be conjugated to known antibodies, so that when cells contain the known antibody’s antigen, the dye color will be present. This allows for cell identification and/or protein expression to be determined and quantified within a heterogeneous mixture of cells. With the given technology, we optimized a flow cytometry panel for the use of analyzing immune responses to gene therapy treatments in canines. We stained extracellular and intracellular protein markers on canine peripheral blood mononuclear cells (PBMCs) with fluorescent dye conjugated antibodies thought to recognize canine antigens. This entailed staining the extracellular markers with conjugated antibodies, fixing and permeabilizing the cell, and doing the same to intracellular markers. Once this had been carried out, the cells were run through a flow cytometer to excite the dyes with varying wavelengths of light to highlight separate dye colors. Analysis of multicolored dye presence in cells post-excitement allowed for identification and quantification of cell types. We identified antibodies that recognize canine antigens and developed a multicolor panel identifying T helper cells, cytotoxic T cells, T regulatory cells, and B cells in canine samples. Once optimized, we used this panel to characterize immune responses in dogs following gene therapy. With a reliable canine cytometry panel, future canine immune responses, both broadly and in isolated muscular tissues, can be characterized.

The application of quantum dots (QDs) is increasing in optical manufacture (solar cells), biological and chemical devices (cancer research). Exposure to QDs can potentially lead to a variety of adverse health effects especially for children and pregnant women. The goal of this study is to determine the toxicity of CdSe/ZnS QDs on brain development of both sexes using in vitro techniques. To determine sex-specific response, we used human neural progenitor cells (hNPC) lines derived from male (NSC-H14) and female (hNP1). An in-vitro model was established using an adverse outcome pathway (AOP) framework. We plated cells in proliferation or differentiation media for treatment to assess the morphology and biochemistry response in cells. Two QDs, differing in their surface chemical properties: carboxyl functional group – ITK and polyethylene glycol (PEG) without reactive functional group (Qtracker) were assessed. Cadmium chloride (Cd) was also tested to determine whether the toxicity of QDs is associated with ion release. The NSC-H14 and hNP1 cells were exposed to QDs of concentration from 0, 2.5, 5, 10, 20 and 40 nM on days in vitro (DIV) 0. After 24 hours, we evaluated the number of cells remaining (cell viability) with an LDH assay at each dose treatment. The results showed that exposures to ITK QDs demonstrated significant dose-dependent decreases in viability of both cell lines during the proliferative stage. Male (NSC-H14) cells were more responsive than female (hNP1) cells to the cytotoxicity of ITK. We did not observe significant dose response for Qtracker and Cd at the same dose range as ITK, either in proliferation nor differentiation stages of both male and female cell lines. Our results indicate that the coating on quantum dots may play a significant role in the exposure and toxicity of the cells, suggesting potential strategies to reduce the adverse health effects of quantum dots.

Barcoding of physical objects with molecular tags holds an advantage over traditional paper or electronic barcodes in that they are discreet, durable, and difficult to falsify. Here, we developed a DNA tagging system that labels objects to verify their authenticity and trace their origin. We chose DNA as our tagging medium due to its information storage capacity and chemical stability, allowing us to generate a wide variety of unique barcode sequences that can be read by Oxford Nanopore’s MinION sequencing device. The MinION contains an array of thousands of nanopore sensors that are capable of sequencing single strands of DNA. The nanopore sequencing process creates distinct disruptions in the ionic current through the sensors that are indicative of the DNA sequence. However, the DNA basecalling software that processes the raw ionic current is computationally expensive, making it impractical when our goal is to quickly “scan” and identify a tagged sample. Because of this, we designed our barcode sequences to generate unique current patterns that are identified using a simple classification algorithm as opposed to arduous basecalling. So far, we have synthesized and classified a set of 96 barcodes that can be indiscriminately combined to create multi-bit tags. In a given tag, each bit is defined by the presence or absence of a particular barcode, and in practice, we have assembled and read up to 16-bits. We have also explored increasing bit capacity by independently varying barcode lengths, which adds another dimension to the barcode space. We also tested the durability of our barcodes by drying them onto filter paper and sequencing them 24 hours later, proving that our barcodes could survive in a dehydrated state. Future experiments will aim to lengthen this duration and expose the barcodes to different environments, in order to better simulate intended tagging conditions.

Cardiovascular disease (CVD) continues as the leading cause of death worldwide. The underlying cause of CVD is atherosclerosis, characterized by fatty plaques in the inner walls of the artery, and exacerbated by oxidative stress, inflammation and immune cells. The high-density lipoprotein (HDL)-associated enzyme paraoxonase-1 (PON1) plays a significant role in protecting against CVD. However, the mechanism of PON1’s protection is not well understood. Our laboratory and others have reported a progressive decrease of PON1 activity in several diseases. Our goal is to understand how PON1 prevents oxidative stress and its potential measurement as an early biomarker of risk of disease. Our hypothesis is that oxidative stress inhibits PON1 enzymatic activity, leading to progression of the atherosclerotic process in CVD. The objective of this research is to generate a mouse model to allow us to study the human enzyme PON1 in induced atherosclerosis in vivo.  Starting from two strains of knockout (KO) mice, we have generated the Pon1/apolipoprotein E (apoE) double KO mouse, known to be susceptible to atherosclerosis. We are now crossing this double KO mouse with Pon1 KO mice that express human PON1 (transgenic human PON1 mice, tgHuPON1). We collect ear punches for DNA extraction and blood from the saphenous vein for activity assays. We use polymerase chain reaction (PCR) genotyping methodology, which amplifies DNA, to identify mice that are KO for mouse Pon1 and apoE and that express human PON1. We run enzymatic assays to determine activity levels of the human PON1 in mice. Once we have created the tgHuPON1/apoE KO mouse, we will feed mice with an atherogenic diet, which will induce subclinical atherosclerosis. This knowledge will contribute to understanding the relationship between PON1 and CVD, and will likely generate a useful biomarker for risk of disease.      

Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous malignancy with a high propensity for recurrence and distant metastasis. Individuals with chronic immunosuppression have both a higher predilection towards developing MCC and tend to have a more aggressive disease course. Chronic lymphocytic leukemia (CLL) is among the most common types of immunosuppression associated with MCC. Immune-checkpoint inhibitors (such as anti-PD1 or anti PD-L1 therapies recently approved for cancer treatment) are associated with improved disease-specific survival and are often used to treat patients with progressive, metastatic MCC. However, the effectiveness and side-effect profile associated with treating metastatic MCC in CLL patients with immunotherapy is not well categorized. This study seeks to understand the risk-benefit profile of immunotherapy in this setting, and a possible combined role for radiation in the treatment of CLL patients. Data was abstracted from a Seattle-based prospective registry of 1,439 MCC patients in which 9 patients were identified to have had CLL and been treated with immunotherapy. Patients were assessed for side effects of immunotherapy, progression of disease and survival status. Six patients had side effects from immunotherapy with 5 of the patients having side effects that resulted in termination of treatment. The average number of doses of immunotherapy received before termination was 3. Eight patients had progressive disease after the initiation of immunotherapy, 4 of whom have died of progressive MCC. No patients have had a complete and ongoing response to immunotherapy. These findings suggest that despite the efficacy of immunotherapy in immune-competent MCC patients, this approach may be less effective in CLL patients than immunocompetent patients. In additional, the side effect profile seemed to be more frequent in CLL-MCC patients. Neutron radiation and combination therapy are discussed as potential treatment options. Further investigation into treatment options for MCC patients with immunosuppression, such as CLL, is needed.

Through mapping the complexities of spatial inequality, I examined the geographic differences in economic and socio-cultural inequality across the state of Indiana. I grew up in Indianapolis, Indiana in an upper-middle class neighborhood that undoubtedly limited my ability to see inequality. Examining inequality in my home state provided me with a deeper understanding of the gap between rich and poor within the state, how Indiana fits into the growing wealth gap in the United States, and how income inequality further impacts access to education, housing, and healthcare. I collected data from the US Census Bureau for Indiana and Indianapolis, as well as the three richest and poorest cities in the state, St. John, Zionsville, Carmel, Gary, East Chicago, and Muncie. Across the state and the seven cities studied, I measured educational attainment, employment status, health insurance coverage, housing costs, and income in the past 12 months. In Indiana, less than 25% of the population over 25 has a Bachelor’s degree. Rates of health insurance coverage vary greatly across the cities studied, but are fairly consistent across racial lines within cities. Income distribution varies drastically across the state, and by race across the seven cities in this study; however, single mothers, regardless of their race or ethnicity, are far more likely to be impoverished compared to the average in each city. In the three richest cities, populations are almost entirely white, while populations in the three poorest cities are almost entirely African American. Economic differences are not the only indicator of inequality in Indiana; socio-cultural differences also underlie many aspects of poverty and inequality. Mapping how inequality varies in terms of race, gender, and location provided a stronger sense of how geography affects the distribution of wealth and resources, and who is impacted the most.

Through the use of virtual and augmented reality technology, we want to record clinical interactions within the respective environments on acute stroke patients to help us design more acceptable gaming technologies for a wide range of ages and backgrounds in future studies. With the combined efforts of the computer science and electrical engineering team, we were able to produce two games; "Dolphin Days" for the augmented reality environment and "Apollo" for the virtual reality environment. "Apollo" will be available for play during the duration of the symposium. The purpose of exposing patients to these environments is to promote neuroplasticity in the recovery process. Neuroplasticity is when the brain can reform or grow new nerve endings to reconnect with the affected areas in order to accomplish a function. Clinically, our experience has been that patient motivation is key to obtaining good clinical outcomes. Stroke rehabilitation therapies can often be tedious and difficult to maintain interest in. These types of interventions, by incorporating the desired exercises into a motivating and game-like environment, could potentially address this issue. Additionally, these systems offer the potential advantage of not requiring the immediate presence of a skilled occupational and physical therapist. Cost and insurance limitations often mean patients get a limited number of skilled therapy sessions. Therapists skilled in the rehabilitation of stroke are often less available in rural regions and patients often struggle to travel to therapy appointments. All of these factors limit the patient’s overall access to skilled therapeutic interventions. Our results found the overall patient census enjoying the environments while also having them engaged with the technologies. We believe using these technologies would further motivate patients undergoing stroke therapy to continue participation and ultimately reach a state where the patient feels confident physically and mentally to resume their daily tasks pre-stroke.

Nanopore sequencing is a “third-generation” sequencing approach in which a constant electric voltage is applied across a nanoscale pore and the changes in the ionic current flow through the pore are measured as single molecules such as RNA or DNA pass through it. It is our goal to expand and adapt this sensing technology to enable single-molecule proteomics. Specifically, being able to characterize protein post-translational modifications at the single-molecule level is important for quantifying protein complexity and understanding how different protein mod-forms contribute to cellular processes such as differentiation and the progression of disease states like cancer. In this project, we modified a model protein to contain a protein kinase A phosphorylation motif with the purpose of demonstrating the ability to discriminate the modified protein from the unmodified with the Oxford Nanopore MinION, a high-throughput nanopore sequencing device. We hypothesize that the observed ionic current pattern will change upon phosphorylation and enable direct quantification of modified peptides. Ultimately, these analyses will inform us of the general ionic current signature that phosphorylated residues generate, which can then be added to our growing library of nanopore signal signatures that are informative of protein sequence and structure at the single-molecule level.

The University of Washington (UW) has a rich history around diversification and over the past 50 years anti-affirmative educational policies have reconfigured what “achieving diversity” looks like beyond admission statistics. Effective support is critical for the underrepresented minority (URM) student who gets into the UW and faces many disadvantages that make it difficult to succeed. The UW Educational Opportunity Program (EOP) Scholars Academy was created in 1968 to facilitate the metamorphosis (transformation) of low-income, first-generation URM students into independent and successful scholars. However, what is not publicly known is why the EOP Scholars Academy support program is effective and in what ways. This study explores how the EOP’s process of curating the student metamorphosis experience impacts 1) students’ academic outcomes and 2) their sense of belonging. The chosen field of perspective in this study is Organizational Communication (OC), looking at the interaction between parts of an organization (UW’s EOP and students) and the resulting behavior as seen through student actions. Some OC concepts used in exploring students’ responses to EOP affiliation include performance, social support, motivation, sense-making, diversity, and identity. Pre-existing data of two EOP-affiliated student-groups (~165 in EOP-1 and ~165 in EOP-3) include longitudinal grade point average, retention, major acceptance, and campus resource engagement metrics over the 2015-2016 school year. The data was statistically analyzed and compared across the two EOP-affiliated student groups, where EOP-3 consists of conditionally admitted Scholars Academy students and EOP-1 consists of non-conditionally admitted students. It is hoped these findings will assist in informing improved diversification policies, program changes, and future research on URM support programs within similar public selective universities.

Ovarian cancer is the deadliest gynecological malignancy: nearly 70% of patients are diagnosed at late stage and 70% relapse. Cytoreductive surgery and chemotherapy are the standard of care, yet fewer than 50% of patients live 5 years past diagnosis. Innovative therapies are desperately needed. Immunotherapy using T cells engineered to express a T cell receptor (TCR) targeting proteins overexpressed in tumors is a promising strategy that limits tumor growth and has minimal toxicity. Mesothelin (Msln) is overexpressed in over 75% of ovarian cancer patients and has been identified as a promising cancer antigen because it contributes to the malignancy and invasiveness. Our group has shown that targeting Msln with engineered T cells (TCRMsln) in the mouse ID8 ovarian tumor model, which recapitulates essential aspects of the human disease, can prolong survival of mice with advanced disease. However, our data also show that immunosuppressive features in the tumor cause T cells to become dysfunctional and/or die. Fas ligand (FasL) is a transmembrane death-inducing protein present in ovarian tumors, and may be used by tumor cells to escape the immune response by inducing cell death of beneficial lymphocytes expressing the Fas receptor (Fas). We hypothesized that we could overcome this T cell evasion mechanism using immunomodulatory fusion proteins (IFP). We generated IFPs that combine the Fas extracellular binding domain to a co-stimulatory domain, CD28 or 4-1BB, instead of the standard death domain, to trigger T cell activation. T cells expressing an IFP had enhanced proliferation and persistence in ID8 tumors relative T cells lacking an IFP. Further, ID8 mice treated with TCRMsln T cells expressing an IFP had prolonged survival compared to mice treated with the TCRMsln T cells alone. Many solid tumors overexpress FasL, thus our findings have the potential to enhance engineered T cell therapy against other malignancies.

Prostate cancer, the second most common cancer in American men, has a relatively low fatality rate – only around 2% of men will actually die of the disease. Prostate cancer has many treatment options. Each of these treatments comes with varying and oftentimes severe symptoms, so choosing a treatment plan is often based on patient preference. Since treatment is based heavily on patient’s values, it is important that patients are well informed about various treatments and related side-effects. This study aims to reveal how a patient’s socioeconomic background influences their decision making processes, with particular focus on how they receive information related to prostate cancer treatments. We review articles and studies from the past 10 years to determine how they address ethics in prostate cancer decision-making. The three framework topics used are: 1) how healthcare providers influence the decision-making process with their ethics and values, 2) how the socioeconomic and cultural factors of a patient informs their decision-making in prostate cancer treatment, and 3) how patients receive information and whether that information is reliable and accurate. In this literature review we find that patients are presented treatment options differently based on their education levels, race, and socioeconomic background, which has potential ethical implications. As this review progresses, we expect to organize these studies to inform how to best provide information to patients of varying cultural backgrounds. We aim to clarify future areas of study towards arriving at ethical consensus in decision-making in prostate cancer. Since decision-making in prostate cancer is a cost-benefit analysis, usually a trade-off between quantity and quality of life, studying how to best involve patients of varying backgrounds in the decision-making process is crucial to treating the disease.

Mitosis is an essential cellular process in which a cell divides to produce two genetically identical daughter cells. When this process becomes dysregulated cells divide uncontrollably leading to diseases such as cancer. Polo-like kinase 1 (Plk1) is a key enzyme that is necessary for coordinating numerous events during mitosis. When Plk1 becomes dysregulated or mislocalized, mitotic spindle assembly, protein organization, and mitotic timing impairments may occur. One of the many subcellular locations where Plk1 carries out essential mitotic functions is the kinetochore. The kinetochore is the interface between the chromosomes and the mitotic spindle and is critical for ensuring proper DNA to microtubule attachments early on in mitosis. Historically, the small-molecule inhibitor drug BI2536 has been used to inhibit the activity of Plk1 in order to study its role in regulating various mitotic processes. However, traditional inhibitor drugs turn off entire protein kinase populations, inhibiting the activity of Plk1 all throughout the cell, not just at the kinetochore. This can lead to unwanted side effects and limits our understanding of Plk1’s role at specific subcellular locations. To improve the specificity of BI2536 drug delivery, we utilized SNAP-tag, a tool in which a self-labeling enzyme can irreversibly react with substrates linked to a chloropyrimidine (CLP) functional group. By genetically manipulating human bone cancer (U2OS) cells, we expressed a kinetochore localizing SNAP. We also generated a BI2536 conjugated to a CLP group. By treating our genetically modified cells with CLP-BI2536, we can target Plk1 inhibiting drug to the kinetochore to study Plk1’s role at this specific location. Using super-resolution structured illumination microscopy (SIM), we demonstrate that we can effectively target fluorescently labeled CLP substrates to kinetochores in our cell line. In future work, we will target our CLP-BI2536 drugs to the kinetochore and investigate how local Plk1 inhibition affects mitotic timing.

Sleep deprivation has serious health consequence with disturbances in cognitive function and metabolism, and increased risk for age-related diseases such as Alzheimer’s disease. Intervention strategies are therefore of interest, especially in individuals unable to routinely obtain healthful sleep time hours. This study was designed to investigate the effects of the repurpose drug phenylbutyrate (PBA) on learning impairment in a mouse model of short-term sleep deprivation. Histone deacetylase (HDAC) is activated by neuronal stress resulting in decreased gene expression and under-acetylation of histones linked to decreased cognitive function. As an HDAC inhibitor, PBA would be expected to alleviate cognitive dysfunction associated with sleep deprivation. Further rationale for testing PBA was provided by showing the drug enhanced expression of acetylated histones in mouse and human neuronal cell cultures. Nine CB6F1 male mice, 17 months of age, were started on treatment with PBA in the drinking water at a concentration of 6 g/L for twelve weeks. Nine control mice were provided drinking water with diluent only. During the last week of treatment, mice were maintained in an awake state 4 hours during the day for 4 days using a non-stressful protocol. All mice were then immediately tested in a box maze learning paradigm consisting of four successive trials to find an escape hole. Control mice had variable escape times over the four-trial test, while mice treated with PBA showed a consistent decrease in escape times with each successive trial. This observation suggests that pretreatment with PBA can prevent learning impairment induced by short term sleep deprivation in mice. Since PBA is already clinically approved to treat urea cycle disorders in children, there are intriguing implications for repurposing this drug for experimental trials in individuals with primary or secondary sleep disorders.

Marine mammal strandings can serve as an indicator of marine ecosystem and mammal population health, providing an opportunity to learn about the life of individual animals prior to their deaths. Existing stranding networks are focused on response to opportunistic reports of stranded or beached marine mammals, which limits their usefulness in establishing baseline data. The Coastal Observation and Seabird Survey Team (COASST) engages volunteers along the western coast of the United States in collecting and reporting data for two effort-based programs, beached birds and marine debris. Looking to the future, COASST hopes to complement information gathered by marine mammal stranding networks by expanding their effort-based surveys to include marine mammals. COASST has already begun this process by asking volunteers to report sightings of marine mammals along with pictures of their beached bird or marine debris finds. These pictures can be used for species identification, and by looking at these photos in correspondence to other survey data such as region, beach, and survey date we can learn about species distribution along the western coast of the United States. We found at least 300 beached pinnipeds, through photo identification we were able to place them into at the least the families Otariidae or Phocidae. Using current literature on marine mammals, pinnipeds specifically, and their identifying characteristics, I aim to map pinniped species distribution and locate hotspots to ultimately test a pinniped species identification key that will be used by COASST volunteers. With this distribution and hotspot identification we will be able to idenify whether pinnipeds follow already established beaching patterns of beached birds or if they differ. These differentiations could lead to more information about the death of marine mammals and allow future research on the death of these animals in more focalized areas. 

Wound healing in the elderly can be a serious health issue because of a delay in tissue repair and compromised immune response. In these circumstances successful treatment can be challenging. New drugs and preclinical animal models are needed to develop alternative treatment strategies. This study was designed to investigate the effectiveness of a novel mitochondrial specific peptide, designated as SS20, in enhancing wound healing in an aging mouse model using a novel and relatively noninvasive ear punch wound. Ten C57BL/6 female mice 26 months of age were ear punched through and through in the central area of both ears using a 2 mm dermal biopsy punch. C57BL/6 mice were chosen due to them being well defined aging models both genetically and physiologically, allowing us to compare aging parameters in the same background. Five mice were randomly selected for daily topical treatment directly on the wound with SS20 at 3mM concentration in a volume of 10 ul. Five mice were treated daily with topical saline in the same manner. Two individuals separately and independently measured diameter of the wounds in two directions (because the wounds were more elliptical than circular) on days 1, 7 and 14 when treatment was stopped. Wound surface areas were then calculated and average change over the 2 weeks was determined between mice with SS20 and saline. A significant decrease in surface area was seen in the left ears of mice treated with SS20 but not with saline as measured by both individuals. Interestingly there was no difference in change in surface area in the right ears of mice treated with SS20 or saline over the 14-day period, confirmed by both individual measurements. These observations suggest that SS20 can prevent a delay in wound healing in very old mice by targeting mitochondrial activity. Additional studies are needed, not only for SS20 effects, but also to investigate utility of the ear punch in aging mice as a model of age-related wound healing.

Nearly 22 million HIV-positive people are receiving antiretroviral therapy in order to suppress their HIV infections. They need consistent viral load monitoring to track viral suppression and detect the possibility of viral rebound. Nucleic acid amplification tests (NAATs) are used to measure the viral load in a patient's blood. Traditional, laboratory-based NAATs require complex robotic systems to automate HIV RNA purification, amplification, and detection from blood. Since the majority of those living with HIV are located in low and middle income countries, there is a need for rapid viral load monitoring at the point of care (POC). We aim to provide accessible HIV viral load testing through low-cost, integrated POC NAAT devices. These proof-of-concept devices operate as a two-step assay to extract and detect nucliec acids in blood. An electrophoretic separation technique called isotachophoresis (ITP) separates HIV RNA from other components in a blood sample. An isothermal nucleic acid amplification assay amplifies the purified, concentrated nucleic acids in order to detect and quantify their presence. We present our development of a novel ITP system to remove potent contaminants from Proteinase K (PK) digested serum and extract highly pure nucleic acids automatically. Through computational modelling, a dual trailing electrolyte (TE) buffer system was designed to exploit the isoelectric point of PK for its removal, while simultaneously concentrating nucleic acids away from serum components. We demonstrate system control through comparison of experimental observations to model predictions by performing dual-TE ITP on pH paper. We also show that the dual-TE system improves upon previous limits of detection for DNA extraction and detection from complex samples. Our system processes 40 microliters of blood in 20 minutes using only simple buffers, a paper strip and an electric field - making it an ideal tool for use in a rapid NAAT for HIV viral load testing. 

Oral swab analysis (OSA) is a possible alternative sample type for tuberculosis diagnostics. It has been observed that tongue swabs contain greater amounts of Mycobacterium tuberculosis DNA than cheek swabs (p<0.0001) from tuberculosis patients. After determining that oral microbiota follows this same pattern, several factors including time-of-day swabbed and health status were analyzed to understand factors affecting the amount of bacteria on the tongue. This project aims to optimize the oral swab sampling methods in order to facilitate more sensitive diagnostic tests, using universal bacterial 16s rDNA as a proxy for Mtb DNA. Previously tested samples from South Africa were further analyzed to investigate amount of oral microbiota by day collected, HIV status, health status, and other demographic factors. To evaluate whether collecting multiple swabs per sample yielded more universal bacterial DNA, tongue swabs were taken from healthy volunteers in Seattle. Each subject provided a 1-swab sample and a 3-swab sample, which was then extracted and analyzed by a previously optimized universal bacterial PCR. Additionally, tongue scrapers are being assessed as an alternative to oral swabs. Swabs collected early in the morning had more bacterial DNA than swabs collected later (p<0.03). 3-swab samples yielded an average of 2-fold greater amounts of bacterial DNA than 1-swab samples. Bacterial biomass correlated with M. tuberculosis signal in most comparisons. Bacterial biomass may serve as a useful proxy when developing better oral swab sampling strategies for TB diagnosis.

The focus of this research is on Spanish heritage language (HL) and second language (L2) learners. An HL learner is a student who comes from a house where Spanish is spoken, but still; needs Spanish classes to master certain aspects of his/her first language. Sometimes English is also spoken at home: e.g., one parent speaks Spanish and the other English, or the student speaks Spanish with his/her parents but English with his/her siblings. On the other hand, an L2 learner is a student who is learning Spanish in a classroom setting. His/her first language may be English or any language other than Spanish. Between these two groups of students, there are important differences when it comes to learning Spanish, for example, L2 students are more knowledgeable about Spanish grammar rules and academic vocabulary. On the other hand, HL students have more fluency and much more knowledge about the culture. The focus of this research is on how the interaction between an HL and an L2 learner in a classroom impacts learning for both students. My role in this research is to transcribe the audio-recorded conversations between two L2 students and to compare these conversations with those between the same L2 students and an HL learner. I will analyze how the students collaborate and how they work to find the solution to any challenge they face that is associated with vocabulary, grammar or orthography. I hypothesize that pairing an L2 and an HL student will generate more interaction since each student offers different sources of knowledge. Pairing an L2 student with an HL student creates a diversity of ideas that I expect to increase learning. This analysis will help us to better understand how to create a more efficient learning environment, for both HL and L2 students, within mixed classrooms.

Methods to differentiate stem cells into cardiomyocytes have been well established. However, a limitation for the successful application of these cells for research and medicine has been their fetal-like phenotype with respect to cell size, contractility, calcium handling, metabolism, and electrophysiology. We sought to increase the maturity of human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) through metabolic pathway regulation. We hypothesized that switching the main metabolic substrates from glucose to fatty acids, mimicking the switch from placental to breast milk nutrient consumption that occurs during development, will increase hPSC-CM maturation. RUES2 embryonic stem cells were differentiated into cardiomyocytes and then treated with base media with varying glucose and calcium concentrations and fatty acid supplementation. Using quantitative PCR to measure gene expression, we measured an inverse relationship between glucose levels and markers of cardiomyocyte maturation: increased expression of cardiac troponin I relative to skeletal troponin I isoforms (TNNI3:TNNI1), increased expression of metabolic (CPT1B, PPARGC1A) and electrical maturity markers (KCNJ2, RYR2). These maturation markers were not influenced with fatty acid supplementation but were enhanced upon the addition of thyroid hormone and dexamethasone. We further quantified nucleation and sarcomere spacing to assess structural features of maturation using confocal microscopy. To understand the mechanisms by which media nutrients and signaling molecules cause phenotypic changes we investigated the relationship between maturation and global epigenetic state. Western blot revealed increases in global acetylation levels, measured by histone H3 acetylation, linked to maturation signaling. These findings depict a direct relationship between glucose metabolism and the development of a mature phenotype in hPSC-CMs, mediated by epigenetic mechanisms.

Sleep problems are prominent in adolescence and most commonly include short sleep duration, poor sleep quality, and insomnia symptoms. These problems are even more pronounced in youth with headache, with research finding that poor sleep contributes to increased headaches and disability. It is important to screen for insomnia in order to accurately diagnose and treat this disorder. However, there is currently no brief, adolescent-focused measure of insomnia that would allow for a high level of accuracy in screening and diagnosis that is relevant for youth with headache. A recently developed screening tool, the Adolescent Insomnia Questionnaire (AIQ), appears to be a valid measure of insomnia in adolescents with chronic pain, diagnosed sleep problems, and in the general community. This study aims to extend validation of the AIQ to children and adolescents with headache from clinical and community settings. 169 adolescents (ages 12-18) were enrolled and completed the AIQ and the Adolescent Sleep Wake Scale (ASWS), a measure of sleep quality, to determine the validity of the AIQ as an insomnia screening tool for youth with headache. Reliability, or internal consistency, of the items in the questionnaire was tested by computing Cronbach’s Alpha. The correlation between the AIQ and ASWS was high (0.736), indicating high validity of the AIQ as an insomnia screening tool for youth with headache. Cronbach’s Alpha was 0.892, which indicates high internal consistency of the AIQ. Based on these results, the AIQ appears to be a valid and reliable tool in the screening and diagnosis of insomnia in adolescents with chronic headache. Further research should continue to focus on insomnia screening within specific adolescent populations at high risk for insomnia, and the development of effective treatments tailored to these populations.

A protein's function is dependent on its structure, which is made up of amino acids. Proline, (an amino acid) is known to cause the kinks and turns in protein structures. However, little is known about the influence of pH on the isomeric preference of proline-attached dipeptides. For this experiment, the isomeric preference of methionine-proline was measured in 10% solutions for pH levels of 7 and 11, the pH of the human body falls between the range of 6 and 9. At pH 11, NMR-90 spectra showed that the cis- isometric form was preferred at a rate of 14% more than the trans- isometric form, measured by the alpha hydrogen. At neutral pH of 7 trans- isomers are preferred 36% more than the cis- form measured by the alpha hydrogen and 73% more measured by the delta hydrogen. In conclusion, this experiment supports the hypothesis that proline-attached dipeptides’ isomerization is pH dependent and is more likely to be in cis- form when in high pH in comparison to a neutral pH. The purpose of this experiment is to determine if pH levels can change the structure of a protien, with further research exploring if the change of structure changes the function. This is important to determine if medications containing amino acids can have an optimal or range of pH.

Establish-Maintain-Restore (EMR) is a professional development training for teachers to strengthen their relationships with their students. Previous studies have shown that the strategies presented in the training have significantly improved academically engaged time and reduced disruptive behavior among elementary and middle school students. The proposed study will examine 9th grade high school student perceptions of the appropriateness and effectiveness of the EMR strategies to improve student-teacher relationships within the school context. Additionally, this study will analyze whether there is a significant difference in these perceptions between the genders of students. It is predicted that male students will tend to perceive the EMR strategies as less effective and appropriate in improving student-teacher relationships compared to their female peers. A focus group was held to present the EMR strategies to 9th grade students at a racially/ethnically diverse high school in the Pacific Northwest. After the strategies were presented, students were asked to provide ratings and comments regarding the appropriateness and effectiveness of each individual strategy. The students were asked about the following items for each of the EMR strategies: appropriateness for school context, appropriateness for both students and teachers, and effectiveness at improving relationships with high school students. Findings from this study will offer insight for improving student-teacher relationships with consideration for the students’ genders. Research has shown that strong student-teacher relationships can serve as a protective factor against high school dropout, and this study may help provide information about culturally responsive strategies to reduce the dropout rates among students of color and male students.

Neonatal mammalian heart tissues possess regenerative capabilities after injuries like myocardial infarctions that are mostly lost in adult mammalian tissues but conserved through adulthood in other vertebrates like zebrafish. Previous studies have shown that regeneration in ventricular cardiomyocytes (CM) occurs through de-differentiation and proliferation, but the underlying mechanisms that cause cardiomyocytes to enter the primed cell-cycle are unknown. Here we show that amino acid and metabolite levels in injured cardiomyocytes result in a primed state for regenerating cells. In chemically ablated zebrafish, it is shown that the amino acid profile activates the mTOR pathway to drive regeneration. Amino acid activation of mTOR is a result of high glutamine and leucine levels post-injury and in early heart regeneration in adult zebrafish, which is lost in adut mammals. Inhibition of the Wnt/β-catenin signaling pathway upstream of mTOR shows down regulation of mTORC1, showing that mTOR is necessary for CM proliferation in regenerating heart tissue. How Wnt signaling gets activated upon injury is unknown, and this study aims to understand the pathways upstream of Wnt signaling for activation. It is known that scarring needs to occur before regeneration occurs in heart tissue. This study also investigates why macrophages are essential for scar formation in ablated heart tissue and its underlying mechanisms. Further, single cell RNA sequencing one-week post injury is used to determine cell fates of the heart tissue. Cardiac cell types like CMs, endocardial and epicardial cells, and bulbus arteriosus (BA) cells were activated post-injury, with epicaridal cells promoting CM regeneration and BA cells activating signaling pathways during heart regeneration. This study demonstrates the signaling and metabolic pathways that activate cardiomyocyte regeneration in zebrafish hearts.

When bacteria experience high salinity environments, such as the brines of sea ice, they take up compatible solutes to protect against osmotic stress. Examples of compatible solutes include amino acids, betaines, and other organic molecules that accumulate in the cells, balancing the osmotic difference between cytosol and external environment without impacting intracellular functions. I am exploring the mutualism of sea-ice bacteria and diatoms (an algal source of compatible solutes for bacteria), and the effects of salinity shifts on this dynamic. I have determined specific growth rates for five strains of bacteria at different combinations of temperature (–3°C to 1°C) and salinity (17 – 55 ppt) that mimic sea-ice conditions. Four strains derive from a collection of Antarctic bacteria found growing mutualistically in diatom cultures: 1) strain Fc1, most closely related (by16S rRNA gene sequence analysis) to Marinobacter psychrophilus strain i20041; 2) Fc4, closest relative Pseudoalteromonas arctica strain A 37-1-2; 3) Nl1, closest relative Glaciecola pallidula strain DSM 14239; and 4) Tr1, closest relative Colwellia rossensis strain S51-W. The fifth was isolated from Arctic sediments but has since been found in sea ice: Colwelia psychrerythraea strain 34H These strains were tested in a defined medium, composed of glucose, vitamins, and a nitrogen source (GVaN), and a complex medium, Marine Broth 2216. Those subjected to higher salinities could be tested at subzero temperatures due to the lowering of freezing point by the salts. During the incubations optical density and cell counts were determined and used for calculations. After determining permissive growth conditions from calculated growth rates, strains will be selected for experiments using specific compatible solutes and/or diatom exudates.

The ability to respond to physical stress can be useful in gauging resilience to aging. Vaccine inoculation is an immune stress that triggers an easily quantifiable antibody response. This study was designed to identify antibody response patterns to a clinically relevant pneumococcal vaccine in mice, in order to show an association with resistance to age-related decline. Twenty C57BL/6 and twenty CB6F1 male mice in age groups of 8, 16, 24, and 32 months were inoculated with one subcutaneous dose of pneumococcal capsular antigen in adjuvant. Serum was collected 16 days later, diluted 1 to 625, and immunoglobulin G antibody levels were measured by an enzyme-linked immunosorbent assay (ELISA) using 410/490 optical density. All data points were standardized against serum from non-vaccinated mice. There was a clear differentiation between high and low antibody responders in both mouse strains, within the range of antibody levels following inoculation. Interestingly, the high antibody response pattern seen in mice at 8 months of age was also seen in a robust manner in mice at ages 16, 24, and 32 months, as opposed to their low-responding counterparts which actually showed decreasing antibody response patterns in an age-dependent manner. In general, C57BL/6 mice had a more vigorous antibody response than CB6F1 mice. These observations suggest that age impacts the effectiveness of a pneumococcal vaccine in conferring an adaptive immune response, and that certain individual mice can robustly respond regardless of age. This high response pattern can thus be very valuable in determining if a robust antibody response is a resilience factor capable of predicting resistance to age-related decline. The concept is clinically relevant since pneumococcal vaccine, for example Prevnar-13, is routinely used to protect elderly populations, and sera can be readily available to test robustness of antibody responses in relation to aging related parameters.     

In the last decade, a large amount of studies have been done using virtual environment (VE) because of the increased control over the sensory stimuli used and the detailed observation of the associated behavioral and sometimes neuronal responses. In this study, we placed honeybee in a VE and exposed them to visual stimulus in closed-loop. We showed that honeybees are able to fixate the stimulus regardless of the level of feedback fed to the sensors(e.g., the gain between the animal motion and the stimulus motion on the VE). Our next step is to investigate how different neuromodulators such as the octopamine and dopamine, may be important for fixation and adaptation to the different level of sensor feedback. Octopamine (OA) is key in modulates physiological process in invertebrates including honeybees. Recent studies have identified OA neurons that are critical for visual behaviors and that increase their activity during active behavioral state. To identify the effects on octopamine and dopamine (DA) on honeybee’s ability to fixate, we are injecting bees with OA, DA or their respective antagonists in a visual processing brain area. Potential results of this study will impact the fields of neuroethology, providing insights on the modularity of neural processes and the feedback between sensory and motor pathways.

This work describes an inexpensive and accurate gesture control implementation designed for an industrial setting. Sensing hand movements and being able to remotely operate devices without use of a tangible control can be useful, particularly in manufacturing applications where other methods of communication may not be available. One gesture recognition method is to use a camera or set of cameras to capture the motions of the user. However, this method imposes line-of-sight workspace constraints and is sensitive to environmental factors, such as consistent lighting conditions. My approach is to use an instrumented glove that detects the amount of bend in specific joints and sends those positions to a central processor that is programmed to recognize control gestures. Similar glove controllers are available but are either not well suited to an industrial setting because the sensors are vulnerable to metal dust and debris, or are not accurate enough to identify commands quickly and consistently. My version has custom sensors exactly fitted to this application and aims to have all sensors sealed and self contained to protect against contamination. This system is able to capture high resolution movement from the wearer and either save that data for machine training or send it immediately to be acted on. Going forward, onboard capabilities such as local gesture recognition will be added, as well as allowing the user to add custom gestures suited to their particular application.

Spatial variation in soil abiotic conditions at plant range limits may be important in determining how plant range limits will respond to climate change. One reason for this is that plants are known to change many characteristics of the soil around their roots in ways that influence the growth of other plants.The range limit of subalpine fir is expected to shift into subalpine meadows as the climate warms. Our goal for this project is to describe the abiotic soil qualities of a subalpine forest habitat and a subalpine meadow habitat on Mount Rainier to explore how these characteristics may affect the predicted range shift. We hypothesize that soils from subalpine meadows will have less organic matter and therefore less phosphorus, a lower carbon to nitrogen ratio, and less water holding capacity than soils from the subalpine forest. We will develop a protocol for measuring water availability and implement it on soil samples obtained from around the roots of subalpine fir trees in both the forest and meadow sites on Mount Rainier. We will use standardized tests to obtain the organic matter and phosphorus measurements. If our hypothesis of lower nutrient availability and water retention in meadow soils is supported, this may suggest that subalpine fir growing in the meadows have a greater dependency on fungal symbionts for obtaining soil resources. This would have important management implications given that suitable fungal symbionts are expected to be rare or absent at range limits.

As it stands today, there is a disconnect between the world of architecture and that of "art" - fine art, outsider art, and similar. This disconnect accounts for the loss of information on a grand scale, as each field continues to analyze itself from an insular perspective, rather than recognizing the interdisciplinary potential inherent in combining these studies. I am developing a foundation for understanding the built environment’s role in shaping society and the world, one that highlights the connective role architecture plays between all the various components of contemporary life. By examining various interdisciplinary examples such as political theory, art scholarship, outsider manifestos, etc., I am synthesizing an understanding of how architecture has historically been approached and interpreted in industrial society, while drafting a manifesto to propose a new methodology for approaching architecture in the contemporary age. In the future, analysis like this could have impact on political or social policy, as well as construction and development, if we utilize this more thorough, interdisciplinary analysis to better understand the implications of what we build and how we will experience it as both art and architecture in contemporary society.

Jacques-Louis David's 1789 The Lictors Bringing Brutus the Bodies of His Sons stages a harrowing scene from Roman history. Consul Brutus, upon discovery of his sons' treasonous plot to restore the monarchy, sanctions their deaths in defense of the Republic. Debuted in the 1789 Paris Salon and shortly after the fall of the Bastille, the political meaning of the colossal painting would have been explicit. In painting the return of the sons' bodies, David's composition reinforces the grave but necessary cost of revolution. Representing an event of classical antiquity as a moral exemplar, Brutus is firmly anchored in period conflict, evincing David's personal revolutionary values. This presentation is a survey of current art historic scholarship surrounding David’s Brutus as a dramatic tableau that synthesizes antiquarian interest and theatricality. The purpose is to understand the aesthetic, social and historic influences of the painting and its presentation as a cathartic, timeless exemplar of nationalistic virtue told through the myth of Brutus.

Organisms in the ocean depend ultimately on phytoplankton as it is the base of the marine food web. Through the use of satellite imaging, indicators of photosynthesis such as chlorophyll-A are used as an index of primary production in the ocean over large areas and variable time scales. Phytoplankton is dependent on large-scale ocean processes such as water temperature and water column mixing. Such estimates might provide insights in food availability for larger predators that eat plankton eating fish such as the Magellanic penguin. Located on the southeast coast of Argentina, Punta Tombo is among the largest breeding colonies for Magellanic penguins. The Boersma Lab at the University of Washington has studied the colony since 1982 and have determined reproductive success for 35 years. Although many factors influence the overall success of the colony, starvation of chicks is responsible for 40% of chick deaths. Through the use of spatial analysis tools, we have looked at chlorophyll-A patterns and seasonal water temperature variation around Punta Tombo and compared these two variables with reproductive success of Magellanic penguins. We expect a strong positive relationship between the two datasets: water temperature and chlorophyll-A. Further we expect that when values are higher closer to the colony, reproductive success of birds is higher, and parents forage closer to the colony.

Building upon our rat testicular o-culture system developed to improve evaluation of chemicals and identify their potential adverse health effects on humans, we have designed in vitro mouse system to expedite such assessments. Models of in vitro cell cultures are, however, limited in their approach typically using a monoculture of single cell types. Using a 3 dimensional organotypic approach can provide a framework for evaluating systematic interactions between cells with a goal to reduce the need for in vivo assessments. We utilized a novel organotypic, in vitro model of testicular development that mirrors the development shown by in vivo studies. We used isolated testicular tissue harvested from C57BL/6 male mice on post-natal day 9 and digested into a single cell suspension. Testicular co-cultures were maintained for up to 16 days in 24 well plates. Data collections occurred at days in vitro (DIV) 3, 7, and 16. These timepoints represent “windows of potential susceptibility” for testicular development. Plates were stained with antibodies providing markers corresponding to morphological features of specific cell type; six different primary antibodies were used as markers to visualize the change in cell populations; markers for Sertoli cells corresponded to Vimentin, Leydig cells with 3b-HSD, Germ cells to DAZL, SCP3 and c-kit, and proliferation with PCNA. We used an immunofluorescence dual staining technique with Scanning Laser Image Cytometery (iCys) to quantify the proportion of cell types and to determine changes in phenotypic distribution of the cell population. Our observations in vitro demonstrated concordance of changes in Sertoli cells, Leydig cells and Germ cells throughout testicular development with similar trends in vivo. Our observation of similarities in testicular development in our 3 D in vitro experiment and in vivo studies is important and supports the current trends in minimizing the need for in vivo studies for chemical toxicological.

Insects use feedback from multiple sensory modalities to control their motor output. Hawkmoths are crepuscular insects that fly in low light conditions, hovering over flowers as they pollinate and feed from them. They use their long and flexible mouthpart, the proboscis, to explore flower surfaces and feed from a tiny nectary opening in these flowers. We asked how moths combine visual and mechanosensory feedback to find the nectary opening in flowers. To test the effect of light level on their efficiency in locating the nectary we combined 3D printing technology to generate artificial flowers, with micro-sensing technology that allowed us to detect the proboscis tip inside the nectary, and computer vision techniques to track the motion of hovering moths at two different light levels; 0.1 lux (moonlight) and 50 lux (dawn/dusk). Using a combination of low light videography and machine vision, we quantified how floral exploration changed between visits, and between the different light levels. We also measured the length of time moths took to find the flower nectary each visit. We found that moths took less time to find the nectary at lower (moonlight) levels compared to higher (dawn/dusk) levels. Hawkmoths are typically active in low light conditions, hence the higher light levels might be adversely affecting flight control. Moreover, preliminary reconstruction of their flight paths suggests that moths hovered over the flower in tighter trajectories under lower light conditions, as compared to the higher light conditions. These results suggest a decreased control over their flight motor output at higher light levels, resulting in reduced hover feeding. These behavioral differences have led us to a series of questions looking at the physiological effects of the different stimuli.

Exposure to traumatic events during infancy can lead to adverse health effects later in life, stemming from an imbalance between the innate and acquired arms of the immune system. Trauma experienced at an early age, such as childhood abuse or environmental stress, is categorized as early life adversity. As the immune consequences of early life adversity are unclear, our study explores how early life adversity affects immunological development and parasite susceptibility in gelada monkeys (Theropithecus gelada). Geladas are an ideal species to study early life adversity due to the similarity of their immune response to that of humans and the fact that they may face adversity early in life. Male geladas compete for reproductive access to females, which often leads to attempted infanticide. Surviving juveniles experience trauma that may have similar physiological consequences to early life adversity in humans. Thus, we determine if there are differences in the immune development of the acquired immune system between geladas exposed to takeovers as infants and those who were not. Acquired immune system development is measured by the identification of gastrointestinal (GI) parasites species that commonly infect geladas, as these parasites trigger the adaptive immune response. We identify these GI parasites using high throughput methods to sequence a region of DNA with known variation across nematode species, allowing us to identify parasites at the level of genus. We expect to see greater GI parasite species diversity in geladas with underdeveloped acquired immune systems as we continue to identify GI parasite species in geladas that were exposed to early life adversity and those who were not.

Dynamic-contrast enhanced (DCE) MRI has a very high sensitivity for breast cancer detection. However, the high costs, long scan-times and safety issues associated with injecting gadolinium-based contrast agents prompt the need to explore non-invasive, non-contrast-based diffusion-weighted imaging (DWI) as a possible alternative. DWI reflects the microscopic cellular environment and at high sensitizations (b-values), DWI can highlight malignant breast tissues without the aid of gadolinium. Acquiring images at high b-values increases image distortions and lengthens scan times. By simulating these high b-value images, lesion conspicuity can be increased while minimizing scan time and maintaining image quality. The purpose of this study was to compare lesion conspicuity across b-values and between acquired (aDWI) and computed (cDWI) DWI. Twenty women with invasive breast cancer were enrolled to undergo a research DWI scan. aDWI was acquired at multiple b-values of b=0/100/800/1500/2500 s/mm². Apparent diffusion coefficient (ADC) maps were generated and cDWI images were then computed for b-values ranging from b=200-2500s/mm² using: S_b = S₁₀₀ e^-Δb*ADC. Lesion contrast-to-noise ratio (CNR) was calculated for both aDWI and cDWI at each b-value. CNR measures across b-values from cDWI and aDWI were compared by Wilcoxon signed-rank test. Lesion conspicuity, as measured by CNR, increased with increasing b-value, with no significant difference between aDWI and cDWI. Our findings show the maxium lesion conspicuity on DWI is achieved at b=1100-1500s/mm², which is higher than typical diagnostic breast DWI protocols. However, lesion conspicuity likely varies with breast density and other patient and tumor characteristics. Potential advantages of cDWI include shorter scan times and flexibility to retrospectively generate images at any b-value for optimal interpretation, warranting further exploration of the value of this technique for breast imaging.

Current methods of modeling tissues rely on two-dimensional (2D) cell cultures which fail to incorporate the three-dimensional (3D) aspect of native tissues in the body and therefore cannot mimic tissue-specific conditions in vivo. The inability to accurately mimic the native properties of tissues in vitro makes characterizing physiological cell behavior challenging and limits the applications of these models. As such, the lack of sufficient in vitro tissue models necessitates the need for a more advanced engineered tissue models that accurately recapitulates the native morphology and function of cells in vivo. Previously developed in vitro platforms that attempt to model human vasculature in vivo have been limited in their ability to imitate the circumferential architecture of smooth muscle cells which surround the veins and arteries. Through the production of a more advanced vascular tissue engineered platform that accurately recapitulates biomimetic conditions of native tissue, we could better study cardiovascular biology, disease modeling, and drug-response in a dish. We propose the fabrication of an architecturally-controlled multi-layered 3D smooth muscle cardiovascular model that mimics the tubular structure of blood vessels in vivo to study structure-function relationships. Utilizing a thermoresponsive nanopatterned film, we are able to direct cell alignment and layer sheets of cells to create a circumferentially aligned 3D smooth muscle tissue model that mimics the physiology of the vascular tunica media. Furthermore, we have designed a fibrin gel casting method to produce tubes with a hollow intraluminal space that has mechanical properties that are physiologically relevant to human tissue. We aim to determine how anisotropic alignment of smooth muscle affects vascular compliance. This model is highly versatile in nature and can be functionalized with a wide variety of cell types to accommodate different tubular tissue structure throughout the human body.

Bacteria inhabit a world filled with threats, including antagonism from other species throughout different environmental conditions. One mechanism microbes employ as protection from these hazards is the type VI secretion system (T6SS) - a system bacteria utilize to inject toxic proteins into neighboring cells, leading to cell death. The H1-T6SS of Pseudomonas aeruginosa comprises 7 pairs of toxins and cognate immunity proteins (which prevent self-intoxication). I hypothesized that each of these toxins could be maximally effective against specific kinds of competing bacteria, however the function of many effector proteins is unknown. My research focuses on characterizing the protein Tse7, encoded by the gene PA0099, and elucidating its potential role in species-specific antagonism. To begin, I identified its key functional amino acids and the gene encoding its immunity protein. I designed and created mutant strains with several genes adjacent to PA0099 deleted and co-cultured these with wild-type Pseudomonas aeruginosa to identify any mutants with a loss in competitive fitness. This lead to the discovery that the gene PA0100 encodes the cognate immunity protein. To determine key functional residues, I identified potential candidates using conserved motifs in the toxin’s amino acid sequence. By creating mutants of these residues and analyzing their change in competitive fitness compared to wild type, I recognized histidine 230 as the residue vital for Tse7 function. Going forward, I will attempt to determine whether Tse7 improves fitness of P. aeruginosa against any specific families of bacteria, indicating the toxin targets that particular bacteria. Almost one-third of Gram-negative bacteria have T6SSs. These systems largely dictate the ability of bacterial species to compete with one another, dramatically affecting bacterial community structure and the landscape of human infections. Therefore, this deepened understanding of its function could further our knowledge of and ability to manipulate bacterial interactions’ impact on environmental and human health.

In cystic fibrosis (CF), a genetic defect in the CFTR anion channel compromises host defenses and causes chronic lung infections with organisms like Staphylococcus aureus. Our lab has been studying the effects of combining ivacaftor, a CFTR modulator which increases CFTR channel activity, with a period of intensive antibiotic treatment. We found that S. aureus lung infections generally persisted despite this aggressive treatment. However, most subjects undergoing treatment were found to be infected by different S. aureus strains one year after treatment than were present before treatment was initiated. Understanding the dynamics of strain switching provide new knowledge about the natural history of chronic CF infections, help define the effects of CFTR modulators and antibiotics, and inform new approaches that might produce infection eradication. We hypothesize that (1) strain switching is most likely to occur during the period of combined ivacaftor and antibiotic treatment, as sputum bacterial burdens were lowest during combined treatment; and that (2) strain switching is rare in the absence of combined treatment. To test this, we used a new population-based multilocus sequence typing (PopMLST) method we developed to perform strain-level genotyping on S. aureus. PopMLST uses PCR amplification and next generation sequencing of housekeeping genes from bacterial isolate pools cultured from sputum. Sequencing determines the number and relative abundance of unique sequence types present, and the data can be used to infer the number of strains present. This analysis was performed on samples obtained before treatment, during treatment with ivacaftor alone, and during combined treatment. We also examined a cohort of subjects receiving usual care. These data improve understanding of strain dynamics during CF infections and suggest new strategies to eliminate infection.

Giardia lamblia, a microscopic flagellated parasite that causes giardiasis, is a highly divergent eukaryote in which conventional Golgi, endosomes, lysosomes, and mitochondria are absent. Similar to other parasites of medical importance, Giardia lamblia has two life cycle stages - proliferative trophozoite form and water-resistant, nonmotile, infectious cyst form. During encystation when Giardia trophozoites transform into infectious cysts, they secrete cyst wall proteins (CWP1-3) that are trafficked and processed in Encystation Specific Vesicles (ESVs). These vesicles are thought to be stage-induced Golgi in Giardia. Previous work in the lab has shown that the signaling activities of G. lamblia’s single Rho family GTPase, GIRac play an important role in regulating this encystation process. The aim is to characterize proteins in Giardia lamblia that potentially interact with GIRac, currently focusing on homologs of known players in membrane trafficking by examining their order of arrival using morphology of the ESVs based on CWP1 staining. Since this is subjective, there is a need for stage-specific molecular markers. In other eukaryotes, Rab GTPases have been established as markers of membrane identity and directionality of trafficking. Only two out of nine Giardia’s Rab GTPases have been localized and reportedly found at ESVs and based on published images, they appear to be recruited at different stages of ESV maturation. By tagging the N-terminus of all 9 Giardia Rab GTPases with fluorescent tags, we can screen them for their localization to ESVs and perform multi-color imaging to determine the order of arrival of these markers. Ultimately, this finding of stage-specific molecular markers could be a powerful tool to further suggests its potential as a novel target for drug development to treat giardiasis.

This is a decolonized narrative based on a three year body of collaborative work as a Native Student Advocate in community college and university student leadership programs. This paper will allow for Native American and Alaska Native students/faculty/staff (community members) to speak for themselves and to indigenize the way that Indigenous “research” is presented. The research question is more of a statement that has surfaced from the community, and so space is created for naming and documenting institutional racism, bias, and self-protective practices. Many Administrators, Department Chairs, Faculty, and Staff continue to cling to a one-sided narrative formulated by white cis-male society that has best suited the US federal and state governments and its institutions for centuries. Textbooks written, approved, and published by white cis-male faculty are allowed to dictate classroom conversations that are white-sided in perspective and perpetuate assaultive language on Indigenous lives and continue to create a dangerous environment for Native community members. This paper will speak to “decolonization” as a buzzword for work in diversity, equity and inclusion mission statements without disembedding settler logic so ingrained in higher education institutions. As a performative act of equity, Native community members are often called upon to speak for over 560+ tribes with different languages, cultural protocols, and cosmologies--only to be bullied or “corrected” when their truth does not suit the ideations of the institution. Decolonized writings and presentation styles by Indigenous scholars and authors will be invoked to support the body of work. It is my hope and utmost intention to create space to allow Indigenous voices to be heard by stating plainly our/their experiences without worry of dominant society conventions and opinions and to collaboratively claim self-determination on presentation of an Indigenized Research narrative that is respectful, relational, and responsible to the Indigenous community it engages.

Advanced oxidation treatment processes involve powerful and indiscriminate radical intermediates, including hydroxyl radicals (•OH) and sulfate radicals (SO4-•). Inefficiency in radical-driven treatment systems involves scavenging reactions where radicals react with non-target species in water and solids. Radical scavenging studies have been focused on soluble scavengers in the water and have not assessed radical scavenging by solids which are also present in oxidation treatment systems. The objective of this study was to quantify radical scavenging by solid surfaces. •OH were produced in iron (Fe)- and UV-activated hydrogen peroxide (Fe-AHP, UV-AHP) systems where the loss of rhodamine B (RhB) dye served as an indicator of •OH activity. The basis used to estimate the •OH surface scavenging rate constant (k≡S) were comparisons of treatment results between simple solids-free oxidation systems and more complex systems containing mineral solids. The solids-free system was based on Fe-AHP and UV-AHP reactions; the solids-amended systems were identical but contained different mineral species. Therefore, differences in the loss of RhB were attributed to •OH scavenging by the solid surfaces in the Fe-AHP and UV-AHP treatment systems. Alumina (Al₂O₃), silica (SiO₂), and montmorillonite (Al₂H₂O₁₂Si₄) (MMT) are solid minerals found in soil and aquifers. These minerals were used in this study to assess the solid surface scavenging rate constants. Preliminary results in the Fe-AHP system indicated that k≡S for silica (2.85×10⁶ 1/m²×s) was not statistically distinguishable from alumina (3.92×10⁶ 1/m²×s). k≡S values in the UV-AHP system for silica (4.50×10⁶ 1/m2×s) and alumina (7.45×106 1/m2×s) were greater than estimates in the Fe-AHP system and may be due to pH. k≡S,MMT (≤ 4.22×10⁵ 1/m²×s) was much less than k≡S for silica and alumina indicating k≡S is mineral-specific. A critical analysis suggests that radical scavenging by solid surfaces in aquifer systems is orders of magnitude greater than scavengers in the water.

Non-invasive prenatal testing is a recently approved form of genetic testing for pregnant women to screen for chromosomal problems such as Trisomy 21 (i.e., Down syndrome [DS]) without the risk of miscarriage. In the event that this test indicates DS, people may consider whether to continue pregnancy, and perceptions of DS may influence this decision. The present study examines whether positively and negatively framed information about DS influences perceptions of DS and the imagined decision to continue or terminate pregnancies when testing indicates a fetus has DS. It was hypothesized that participants who receive positively framed information would be more likely to imagine continuing pregnancy and have more positive perceptions of DS. Participants were given the same set of facts about DS, either framed positively (e.g., “50% have no heart problems”) or negatively (e.g., “50% have heart problems). The participants reported their perception of the syndrome, including personality traits, adult achievements, and level of learning disability. The participants were then asked whether their feelings toward a pregnancy might change if they were aware their child would have DS. Contrary to the hypothesis, there was no significant effect of framing on perceptions of DS or imagined decisions about pregnancy. However, perceptions of positive personality traits were significantly related to greater expected adult achievement (r=.33, p<.03) and lower expected level of learning disability (r=.39, p<.01). Although this study did not show framing effects, the framing manipulation studied was subtle. Given the possible implications for genetic counseling, more research—with a stronger manipulation—could be done to examine these effects.

Increasing energy demand coupled with over-reliance on fossil fuels and other non-renewable energy sources has created a need for alternative renewable energy sources. The sun is one of the most promising sources and photovoltaic cells are one way to capture solar energy. Halide perovskite thin-films have recently emerged as ideal materials for solar cells due to low fabrication costs, bandgap tunability, high extinction coefficients, and high carrier mobility. Moreover, they have demonstrated rapid gains in power conversion efficiencies from 3.8% to 23.7% in nine years. Halide perovskites have the molecular formula ABX3, where A and B are cations while X is a halide. Past research has shown that methylammonium(MA) lead triiodide, a commonly used perovskite, can be changed into formamidinium(FA) lead triiodide by exchanging the A-site cation in a formamidinium iodide solution. This highlights high ion mobility and interchangeability in perovskites. However, in perovskites with mixed-ion composition, high ion diffusion adversely affects the device performance due to ion segregation, but little is known about inter-diffusion of different ions in perovskites. Here, we investigate the inter-diffusion of A-site and X-site ions in halide perovskite films by creating a lateral heterojunction of the ions. We confirm the creation of the gradient using UV-Vis and steady-state photoluminescence (PL) measurements. We further confirm that there is no change in the film morphology and crystallinity as evidenced by SEM and XRD, respectively. With PL line scans across the lateral gradient, we image the inter-diffusion of the ions as a function of position and time. Using Fick’s Diffusion equations to fit the PL line scans, we determine the ion inter-diffusion coefficient and extract the activation energy using temperature dependent measurements. This study demonstrates a facile quantitative method of probing the ion inter-diffusion in halide perovskites and furthers understanding of mixed-ion perovskite compositions.

Unwanted Invaders. The “Others”. Deceitful Criminals. Victims of Injustice. These themes were not ripped from the headlines of newspapers describing immigrants today, but rather are found in newspapers from the 1880s and the 1940s to describe Asian-American immigrants to the United States during two separate times of crisis- Chinese Exclusion and Japanese Internment. This project explores how print media has constructed the accounts of immigrants by seeking the answer to the question: What rhetoric have newspapers used to portray Non-European immigrants to the United States? Articles from three time periods (1880s, 1940s, today) were selected and the rhetoric used within these sources were divided into four descriptive categories: Unwanted Invaders, The "Others," Deceitful Criminals, and Victims of Injustice. Quantifying the use of particular phrases within these categorical themes led to an analysis of the intersection between language and media, and how media repertoire has changed in regard to its identification and treatment of immigrants. Preliminary findings indicate that the description of immigrants by media sources has shifted over time, from "deceitful criminals" and "unwanted invaders," to "victims of injustice." This historical analysis of the common rhetoric used throughout these three separate times of crisis characterizes how media has continued to shape the lens that we view and have viewed immigrants through, which allows us to better understand and address the current state of the immigration crisis.

Crops are constantly exposed to pathogens and pests, but being sessile, they cannot physically move away from danger. In order to defend themselves, plants have mechanisms to recognize and respond to threats. One way that plants do this is through the Jasmonic acid pathway, which is specifically activated in response to the wounding of plant tissue. Surprisingly, there are many functionally similar Jasmonate-ZIM domain (JAZ) proteins involved in this response, and the reason for this redundancy is unknown. Studying this pathway in depth presents a challenge when using low-throughput methods. In order to better understand this complex system, we built a workflow, which will be integrated into an automated system using the Aquarium Laboratory Operating System, designed by the Klavins Lab. This will help us track data from high throughput experiments. By developing automated methods to track maintenance of cell cultures, production of protoplasts, and delivery of dCAS9 transcription regulators, we hope to produce sufficient data to better understand the JA-signaling pathway. We anticipate that these experiments will illustrate a relationship between the manipulated activity of JAZ gene regulators and subsequent immune response activity, and have potential implications in the agricultural and food industries, where pests and stress conditions are a significant concern.

The plant hormone Jasmonic acid (JA) interacts with JAZ proteins, proteins that play a crucial role in the regulation of signal cascades triggered by jasmonates, and controls the activity of MYCs, a family of genes that code for transcription factors. In the absence of JA, JAZ proteins bind to downstream MYCs and limit their activity. JA is produced in plant cells in response to tissue damage, and is crucial to plant fitness. The genome of model plant Arabidopsis thaliana encodes 13 JAZ and 5 MYC isoforms, but as of now, it is unclear why this apparent redundancy exists. Such redundancy is a common feature of plant genomes. In the case of JA-signaling, it may be that interaction strengths differ between JAZ and MYC isoforms and that this is important for system behavior. In A. thaliana, there are 65 pairwise combinations alone, which provides many opportunities for engineering incredibly fine-tuned and controlled gene expression. Our past work has been to clone and create a library of JAZ and MYC proteins. Our goal now is to create yeast mini-circuits, which are yeast cells that contain plasmids that carry certain selected JAZ and MYC proteins. After constructing these yeast mini-circuits, we will study the JA-response dynamics of these circuits to better understand the role of each of these proteins and study differences in strengths of interaction between the different proteins. We suspect that the absence of certain JAZ and MYC proteins will lead to different phenotypes in defense mechanisms. Additionally, by investigating this relatively familiar pathway through the construction of yeast mini-circuits, we are attempting to develop an efficient technique which can be used to characterize other unexplored plant systems.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s Sarcoma (KS), a highly vascularized tumor composed of cells of endothelial origin. KSHV, while possessing both lytic and latent replication programs, predominantly exists in the latent form during infection. While KSHV infects both blood (BECs) and lymphatic (LECs) endothelial cells, LECs are more susceptible to infection and express fewer antiviral genes during infection compared to BECs. Recent experiments have shown that LECs, but not BECs, have a defect in STING, a critical signaling protein that is activated during herpesvirus infections and results in the production of antiviral signaling molecules such as IFN-β. It remains unknown whether the defect in STING plays a direct role in increasing susceptibility to KSHV infection and if the defect impacts the ability of STING to suppress lytic reactivation in LECs. Accordingly, I propose to construct a constitutively active (CA)-STING and express it in LECs. Because CA-STING results in the continuous induction of IFN-β, I hypothesize that CA-STING-LECS will show decreased susceptibility to the establishment of latency by KSHV and have increased ability to suppress lytic reactivation compared to empty vector-expressing (EV)-LECs. First, I will infect EV-LECs and CA-STING-LECs with KSHV and measure the infection rates 48 hours post infection (hpi). I expect the number of infected cells in the CA-STING-LECs to be decreased relative to the EV-LECs. Next, I will infect EV-LECs and CA-STING-LECs with KSHV, and at 48 hpi, I will induce lytic reactivation in the two cell types and quantify the virus produced. If CA-STING suppresses lytic reactivation in LECs, I expect less virion production from CA-STING-LECs than from EV-LECs. The results from these experiments will further elucidate how KSHV exploits defects in innate-immunity to infect and transform host cells.

Labeling objects with DNA-based tags can provide a secure, difficult to fake identifier that is particularly useful for objects of high value or those that cannot be physically tagged. In this problem setup, a tag is a bit string, where each bit represents the presence or absence of a DNA strand containing a particular barcode. Our goal is to consistently and accurately identify the tag. These DNA barcodes were designed for use on a MinION nanopore sequencer, which outputs a time series signal corresponding to the DNA sequence. Ideally, each barcode should generate a dissimilar signal, which makes it easier to distinguish from other barcodes. We designed 96 barcodes that are signal orthogonal (i.e the signal output from the MinION was as dissimilar as possible), and detected them using signal processing algorithms. Using this system, I created an error analysis pipeline to ensure that we can identify tags both quickly and accurately. In order to optimize the time it takes to identify a tag, it was important to minimize the number of sequencing reads we needed to observe on the MinION, without sacrificing accuracy. I found that using a subset of the reads produced approximately the same error rate as a full run. Therefore, we can run the MinION for a shorter amount of time, and still identify tags at a similar error rate compared to a longer runtime.

Biological mineralization is the formation of minerals in hard tissues guided by proteins. Unique aspects of these minerals include the molecular control of hierarchical structure, intricate architectures, and multifunctional properties for inspiration in bionanotechnology and nanomedicine applications. Numerous biomineralization strategies have been developed in hard tissue regeneration therapies. However, there is currently no in-depth understanding of how proteins regulate the synthesis of these inorganics or the physiological formation of the minerals. The ability to control mineral formation for biomedical applications, therefore, is still limited to the use of a few mineral-directing proteins extracted from tissues. Biomineralization can also be controlled using short peptide domains derived from natural proteins known to have a regulatory role in mineralization. Our laboratory has designed peptides derived from amelogenin (ADPs), the key protein in tooth formation, using combinatorial selection and computational design, whose utility in rebuilding hydroxyapatite (HAp) mineral on tooth has been demonstrated in numerous case studies. The goal here is to understand the fundamental mechanisms of biomineralization guided by ADP5 and develop a methodology to form HAp with exclusive control of its growth kinetics and mineral crystallography. We designed mutants of ADP5 to investigate changes in mineralization kinetics, nucleation, and morphology. In the current study, we are establishing the conditions for ion-peptide interactions on the onset pH for mineral nucleation using calcium/phosphate and mutant ADPs. The goal is to gain insights into the correlation between sequence domains and biomineralization outcomes eventually facilitating greater control over the reaction and further optimize remineralization approach. The developed method has a high potential to develop non-invasive oral health care materials and methods by restoring mineral loss, the root cause of dental ailments and, eventually, help bring clinical and over-the-counter dental products into the market with preventive, restorative, therapeutic, and cosmetic characteristics. Sponsored by SoD Spencer Funds.

Anecdotal evidence suggests patients that undergo Targeted Muscle Reinnervation (TMR) surgery, an operation where nerves are implanted to muscle instead of cut and left between muscle, report less residual and phantom limb pain when compared to standard amputation patients. To our knowledge, this is the first study to investigate this claim. Based on the nature of the TMR procedure, it should facilitate higher pressure thresholds than a severed nerve from a standard amputation, resulting in less pain for the patient. The risk of neuromas, a collection of highly sensitive tissue that can develop on a damaged nerve, should also diminish. To evaluate the efficacy of TMR in relation to pain reduction, a populace upwards of 135 people (comprised of TMR amputees, standard amputees and a control cohort) will have their nerves stimulated using focused ultrasound, allowing the application of focal intense and transient pressure on the nerve and not on the surrounding tissue. In this study, diagnostic ultrasound imaging guided the application of intense focused ultrasound on intact and transected nerve endings in our test subjects. We increased the ultrasound intensity until we either elicited a sensation or reached the maximum output possible by our device. We anticipate TMR amputees to have a higher threshold for ultrasound-induced sensations than standard amputation test subjects, but a lower threshold than the control test subjects. Due to the limited availability of TMR amputees in the area, a definitive conclusion has not been made about the effect TMR has relative to the standard amputation. When analyzing results from standard amputees specifically, there are three subgroups that possess an intriguing feature that distinguish them from each other. This study can potentially influence the types of surgical techniques offered to future amputees if TMR operations do in fact reduce the amount of pain experienced by patients.

Quantitative PCR (qPCR) is a commonly-used method to analyze gene expression. However, carrying out a qPCR experiment is often daunting to a beginner such as an undergraduate student, as it involves a lot of advance planning, precise pipetting, and is susceptible to contamination. Our goal is to automate laboratory and data analysis protocols so that (1) experiments can be carried out more quickly and more efficiently; (2) data are carefully tracked and recorded; and (3) results are reproducible. Automation involves repeated testing, writing and integrating protocols onto Aquarium, the laboratory operating system developed by our lab. Since Aquarium can keep track of every data point that it is asked to collect, we can define metrics of protocol success (like product quality, time-to-completion and ease of execution) and compare them throughout the automation process to make sure that the final protocols satisfy the three criteria above. For the 2018-2019 school year, we are automating protocols to cultivate Arabidopsis thaliana and study the gene expression of its Jasmonate (JA) signaling pathway. Since the JA pathway in Arabidopsis thaliana involves many genes and has been well-studied by plant biologists, we reason that it would be an appropriate case-study for our automation project. As we apply our qPCR workflow to study the expression of these genes in response to application of exogenous JA, we expect to see results that are consistent between different sets of technical replicates performed by different student technicians, and results that are consistent with those in existing literature.

Single-cell gel electrophoresis, or a Comet Assay, can be used to assess DNA damage in cells as it liberates single-strand DNA breaks (SSB), alkali labile sites (ALS), and DNA cross-links, then uses microscopy to determine levels of chromosome damage. Various methods have been used to detect DNA damage; advantages of this assay include its capacity to detect low levels of DNA damage, the small number of cells required, low cost, and the ability to utilize various cell types. Our lab used lymphocytes withdrawn from elderly patients as we targeted quantifying the effects on an individual’s DNA due to aging, stress, and drugs. Lymphocytes were suspended in low-melting point agarose and placed on a slide pre-coated with regular agarose. Once cells were fixed on the slide, they were placed in lysis solution to remove membranes and liberate DNA. Lymphocytes then went through an alkaline treatment to unwind DNA super-coils. The assay has been tried under neutral conditions however, we used the alkaline method because it is known to increase reproducibility and specificity. The cells were then placed in an electrophoresis chamber at 5 V/cm for 30 minutes, the SSB’s, ALS’s, and DNA cross-link fragments traveled towards the anode, forming a comet like appearance behind the cell. The cells were then neutralized, stained with ethidium bromide, and the comets were visualized. Data can be quantified using software provided by Instem Technologies. A comparison of Comet values derived from varying cell densities as well as untreated control samples are also presented, confirming good reliability using this technique.

Symbiotic nitrogen fixation (SNF) in the interaction between the soil bacteria Sinorhizobium meliloti and legume plant Medicago sativa is carried out in specialized root organs called nodules. During nodule development, each symbiont must drastically alter their proteins, transcripts and metabolites in order to support nitrogen fixation. Moreover, bacteria within the nodules are under stress, including challenges by plant antimicrobial peptides, low pH, limited oxygen availability, and strongly reducing conditions, all of which challenge proteome integrity. S. meliloti stress adaptation, proteome remodeling and quality control are controlled in part by the large oligomeric protease complexes HslUV and ClpXP1. To improve understanding of the roles of S. meliloti HslUV and ClpXP1 in free-living conditions and in symbiosis with M. sativa, we generated ΔhslU, ΔhslV, ΔhslUV, and ΔclpP1 knockout mutants. Shoot dry weight of M. sativa plants inoculated with each deletion mutant was significantly reduced, suggesting a role in symbiosis. Further, slower free-living growth of ΔhslUV and ΔclpP1 suggest HslUV and ClpP1 were involved in adapting to heat stress, while ΔhslU and ΔclpP1 mutants were sensitive to kanamycin. All deletion mutants produced less exopolysaccharide and succinoglycan, as shown by replicate spot plating and Calcofluor binding. We also generated endogenous C-terminal eGFP fusions to HslU, HslV, ClpX and ClpP1 in S. meliloti. Using anti-eGFP antibodies, native coimmunoprecipitation experiments of proteins from free-living and nodule tissues were performed and analyzed by mass spectrometry. The results suggest HslUV and ClpXP were closely associated with ribosomal and proteome quality control proteins, and identified several novel putative protein-protein interactions.

Telomeres represent the structural ends of genomic chromosome molecules and are composed of hexamer repeats of the form [TTAGGG] which play a protective role against DNA damage and genetic information loss. The telomere length of normal diploid cells decreases over time and can be correlated with issues related to cancer and the aging process. Our preliminary work utilizes methods developed initially by Cawthon and O’Callaghan and is applied to DNA samples obtained from research studies involving the elderly as well as those with aneurisms. In contrast to standard measurements which use real-time PCR in order to calculate relative telomere lengths, our method uses absolute quantification through the use of standard curves generated with a synthetic 84-mer telomere (control gene) and another synthetic oligomer (36B4 ‘housekeeping gene’). Through the use of both synthetic standards in a typical RT-PCR experiment utilizing SybrGreen and specific primers for either telomere sequence or housekeeping gene, the true length of a diploid telomere can be calculated by extrapolation from standard curves. Telomere lengths obtained through this method have been compared to results derived from commercially available test kits which used relative RT-PCR as a quantification measure. Results obtained through the use of this technique can be incorporated in research studies encompassing cell apoptosis, processes involved with aging. This technique can also be used to ascertain the effects of environmental impacts involving DNA damage on plants, bacteria, and animal cells.

Often, when rodents are faced with a decision on a maze, they will pause to look back and forth between different paths. This behavior has been termed vicarious trial-and-error (VTE) and has been linked to the reactivation of hippocampal place field sequences as animals look back and forth. Previous research has shown that place field sequences represent a neural map of the paths the animals can potentially take as if they are pre-playing their future path. These reactivation events give merit to the idea that VTEs are reflective of a deliberative process. Additionally, studies show that when animals reach such decision points there is increased communication between the hippocampus (HPC) and medial prefrontal cortex (mPFC) as measured by increased correlates in local oscillatory power. We wanted to further study the mPFC’s role in initiating VTEs during a task that is HPC and mPFC dependent: the spatial delayed-alternation-task. This task was carried out on a plus maze; it required rats to alternate between reward arms every trial while the start arm was pseudo-randomly selected every trial. During this task we used a closed-loop system to optogenetically disrupt the mPFC upon detection of specific phases of HPC theta oscillations. Additionally, disruption was applied across three different task-epochs: delay, choice, and reward in order to determine under which epoch is the mPFC most important for VTE behavior. We hypothesized that mPFC disruption during the choice epoch would reveal the greatest change in the number of VTE’s as the mPFC is thought to initiate retrieval events when decisions are to be made. Because VTE’s are a unique and observable behavior that has clear neural activity correlates, we hope to use this study to better understand the relationship between the hippocampus and mPFC in learning and memory.

Chronic non-bacterial osteomyelitis (CNO), or chronic recurrent multifocal osteomyelitis (CRMO), is an auto-inflammatory bone condition that causes persistent bone pain, poor growth, and other complications. Physical exams, laboratory tests and radiographs are not sensitive in detecting the active disease. MRI is the gold standard assessment for diagnosis. Patient reported outcome (PRO) measurements have not been commonly used to determine the disease impact on the physical health of affected children. Pain score is not accurate in assessing disease activity due to the confounding effects from other associated conditions such as amplified pain syndrome. Consensus treatment plans (CTPs) were developed by the Childhood Arthritis and Rheumatology Research Alliance (CARRA). In this study, a prospective patient registry based on CTPs is used to compare the effects of different treatment plans. This study was approved by the Seattle Children’s Institutional Review Board (#1232). The center is the coordinating site of this multicenter observational study, Chronic nonbacterial Osteomyelitis International Registry (CHOIR). Inclusion criteria are: ≤21 years old at enrollment and CNO diagnosis. Consent and assents are obtained. Standard of care is provided to the subjects and no investigational intervention is performed. Detailed clinical information, laboratory, and imaging data along with patient/parent reported outcome measurements (PROMIS questionnaires) and childhood health assessment questionnaire (CHAQ) are collected at baseline and follow-up visits. We aim to enroll 2,000 subjects worldwide and follow up with subjects for at least 5 years. Our center has enrolled 74 subjects between June 2018-present and collected PROs at baseline visits. Data is being entered into REDCap database for further analysis. PROs collected throughout the course of the study will be correlated with imaging results and expectedly determine the effectiveness of commonly used medications for patients. The data will establish measurements for physicians to estimate disease burden and treatment responses in children with CNO.

During HIV infection, CD8+T-cells are crucial for the control of viral replication. Increased CD8+T-cell polyfunctionality, which is the ability for one cell to perform more than one function within the immune response, was associated with improved clinical outcomes, is now the focus of experimental curative therapies for HIV. One therapy that showed promise is a Conserved Elements DNA (CE) vaccine, which is designed to prime the immune system with essential regions of the virus, thereby eliciting polyfunctionality and circumventing escape mutants. A novel immunotherapeutic combinatorial approach was investigated for the ability to reduce or eliminate viral burden in Simian Human Immunodeficiency Virus (SHIV) infected macaques receiving antiretroviral drug therapy. We evaluated effects of this regimen on CD8+T-cell polyfunctionality and its correlation to viral control after analytical antiretroviral treatment interruption (ATI). Our combinatorial immunotherapy regimen uses the CE vaccine to target highly conserved viral sequences, latency reversal agent GS986, exhaustion reversal agent anti-PD-1, and CCR5 gene editing to delete the co-receptor for viral entry in CD4+T-cells. Analysis of CD8+T-cell polyfunctionality was performed using intracellular cytokine staining and flow cytometry to measure the frequency of CD8+T-cells secreting one or more of the following effector functions: TNFα, IFNγ, IL-2, and both CD107a and Granzyme B. Polyfunctionality induced by the combinatorial regimen was algorithmically quantified and compared to a control group receiving no interventions and a group receiving CE vaccine and CCR5 gene editing alone. Our analysis showed neither significant differences in polyfunctionality between treated and control groups, nor any changes in polyfunctionality within the subset of CD8 targeting conserved regions of the virus. However, we observed diverse viral control post-ATI among all animals, and additional experiments are in progress to determine if CD8 polyfunctionality played a role in improved viral control post-ATI.

Mental illness is a national concern; for rural residents, the availability of mental health services magnifies the problem. The 2017 National Survey on Drug Use and Health found close to 1.5 million non-urban residents had psychiatric illness or serious thoughts of suicide during 2017. Hypothesis: In Washington State, there is less access for outpatient mental health services in rural areas than in urban areas. The 2018 Washington State Directory of Certified Mental Health, Substance Use Disorder, and Problem & Pathological Gambling Services contains 503 entries of mental health facilities. Using the Office of Community Health Systems Series on Rural-Urban Disparities, facilities with available information were categorized as urban or rural. Statistics were calculated to characterize the availability of individual therapy, and crisis intervention information via public-facing websites for included clinics. The association between rural-urban status and the availability of outpatient psychotherapy and crisis intervention contact information was tested via the chi-squared test of independence. Approximately half (n=131, 50.6%) of clinics with available information regarding outpatient psychotherapy indicated that these services were available. Similarly, nearly half (n=120, 46.1%) of clinics with available information had crisis lifeline information presented via their public-facing website. There was no significant association between urban-rural status and availability of outpatient psychotherapy services (_X²=0.21, p=.647), nor between urban-rural status and publicly available crisis lifeline information on mental health clinic websites (_X²=0.80, p=.371). Washington State rural clinics are not significantly different from urban clinics with respect to saying they provide outpatient psychotherapy and crisis lifeline information. Limitations include methods relying on public reporting of services and limited scope, not allowing the study of other factors that might affect rural residents, such as distance traveled to obtain care. Rural counties in Washington State do not differ from urban counties with respect to the presence of clinics providing outpatient psychotherapy.

Microglia are innate immune cells in the CNS that exhibit a sustained pro-inflammatory response in the Alzheimer's disease (AD) brain. Sustained pro-inflammatory responses by microglia can promote excessive synaptic pruning and neuronal death, exacerbating neurodegeneration. MicroRNAs can regulate microglia inflammatory behaviors by modifying gene expression at the post-transcriptional level by suppressing expression of target genes. MiR-155 is a microRNA that targets suppressors of inflammation and is dysregulated in neurodegenerative disorders. Additionally, miR-155 deletion has been reported to be neuroprotective in several models of neural injury and degeneration. The impact of microglia specific miR-155 regulation on the neuroinflammatory response or behavioral outcomes of AD models has yet to be elucidated. We hypothesize that miR-155 deletion in microglia decreases neuroinflammatory response to AD, thus improving memory impairments typically observed in AD. We use a mouse model expressing a transgene of associated mutant forms of human amyloid precursor protein and presenilin 1 (APP/PS1). We crossed APP/PS1 mice with a tamoxifen-inducible Cre model or a constitutive Cre model to conditionally or constitutively delete miR-155 in microglia. We use open field chambers and T-maze to assess general behavior and spatial memory at 6, 9, and 12 months. When miR-155 was deleted specifically in microglia, no difference was seen in the spatial memory as measured through T-maze tests, compared to APP/PS1 mice. However, increased locomotor activity was seen in open field tests at 6 and 9 months. Similarly, when miR-155 was deleted in microglia and peripheral myeloid cells, there were no significant differences in spatial memory, though increases in locomotor activity at 6 and 9 months and potential decreases in anxiety at 6 months were also seen in open field. These results suggest that miR-155 may play a more complex role in the regulatory response of neuroinflammation during AD.

Studies show that disparities in healthcare access in ethnically diverse underserved communities are linked to healthcare barriers including cost of care and language differences. The purpose of the study was to evaluate the role of technology in improving healthcare access for ethnically diverse and underserved communities and thus helping to reduce healthcare disparities. We hypothesized that ethnically diverse patients’ ready access to mobile health technologies like smartphones and healthcare apps can improve patient care by promoting self-care management and improve doctor-patient communication. To test our hypothesis, we recruited N=20 healthcare providers from Washington, state. Healthcare providers comprising physicians, medical assistants, nurses and social workers participated in a 60-minute focus group session and were asked about the role of technology in increasing access to healthcare services for diverse ethnic communities. Respondents received a $75 gift certificate at the end of the study and responses were transcribed for later assessment. We evaluated transcripts by deriving codewords, codewords used more than two times were identified and recorded on three separate occasions, codewords with similar information were grouped into codeword clusters and finally, themes were derived based on a single idea from codeword clusters. From our analysis, we learned that healthcare providers see the importance of using mobile devices to increase healthcare access for ethnically diverse and underserved communities. They noted that mobile healthcare technologies can improve patient-provider relationships via enhanced communication. The implication of our finding is that mobile health technologies can improve patient-provider communication and help to eliminate barriers to healthcare access for ethnically diverse and underserved communities.

For women to have protection from unintended pregnancy and human immunodeficiency (HIV), current lead prevention options use oral antiretroviral drugs (ARV) for pre-exposure prophylaxis (oral PrEP) along with a form of contraception. Failure to adhere to these drug therapies will increase the risk of contracting HIV or pregnancy. We have proposed to integrate drug-eluting materials onto a copper-intrauterine device (IUD) that could provide both HIV prevention and contraception. We will evaluate two methods to formulate a matrix release drug delivery system. Injection molding is a method to inject material into a mold that can be used for constructing drug-eluting medical devices with low drug degradation. For our purpose, we injected a polymer and drug combination into a mold to construct a solid slab. Whereas, electrospinning is a method that uses electric force to formulate stable and high surface-to-volume ratio nanofibers with high drug encapsulation and porosity compared to the molded slab. Both delivery systems will be used to administer ARV drugs to the female genital tract for a year. We optimized the molded slab and electrospun nanofibers technique for maximum polymer-loading, and used 3-D printing and nanofiber wrapping technique as a process for slab integration and fiber integration onto the IUD respectively. The polymer and drug combinations for both electrospun nanofibers and molded slabs were chosen to have the maximum drug-loading and stable mechanical properties. Drug release was measured in vitro to predict daily release rates out to three years. The ideal matrix release drug delivery system method for the dual HIV prevention and conception IUD is determined based on the mechanical properties and drug release rate of the polymer and system combination. We also investigated the drug delivery systems for cytotoxicity to verify dosage safety.

Autophagy is a fundamental biological pathway that facilitates the degradation and recycling of intracellular components. While it is known to be activated in response to cellular stress, it may also have critical roles in developmental processes. To date, studies in mice have shown that deletion of core autophagy proteins impairs the production of many essential hematopoietic lineages, suggesting that autophagy is critical for blood cell differentiation. In particular red cell production is known to be dependent on autophagy however (1) when does autophagy occur? and (2) how is it regulated? In human erythropoiesis is unknown. Preliminary data from the Doulatov lab has identified a novel negative regulator of autophagy, ATG4A, in human erythropoiesis. The ATG4 family of proteins are cysteine proteases known to regulate LC3B a critical molecule which decorates the outside of the autophagosome. However, whether its expression is confined to erythropoiesis or broadly applies to other hematopoietic lineages is unknown. Therefore, I performed a bioinformatic analysis on a large RNA microarray dataset which profiles gene expression in 38 distinct cell populations in the 7 major hematopoietic lineages. Only ATG4A and ATG4B were detected in the dataset, and when mapped across the hematopoietic hierarchy had differing patterns of expression. In contrast to ATG4B the expression of autophagy protein ATG4A was selectively upregulated in the erythroid lineage. Previous studies have described a critical role BNIP3L(NIX) in erythropoiesis, therefore I compared the expression pattern of ATG4A to BNIP3L. ATG4A and BNIP3L had similar patterns of expression suggesting multiple autophagy proteins may be upregulated during erythroid differentiation. Taken together these data suggest that ATG4A ia a unique regulator in the human erythroid lineage.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multi-organ inflammation and damage, including skin and kidney. Ultraviolet B light (UVBL) is the only environmental factor known to precipitate both skin and kidney disease. How UVBL-triggered sterile inflammation in the skin influences kidney injury remains a pressing question for SLE patients, about 70% of whom suffer from sensitivity to UVBL. We previously demonstrated that neutrophils are the first immune cells to infiltrate the inflamed skin tissue after exposure to UVBL. To investigate the neutrophils’ role in UVBL-induced kidney injury, C57BL/6J mice were irradiated with a single dose of UVBL (500mJ/cm2). Cells in the bone marrow (BM), skin, blood, and kidney were characterized using flow cytometry. Gene expression of inflammatory mediators and adhesion molecules was evaluated using qPCR. Following acute exposure to UVBL, we observed a 10-fold increase in skin neutrophils, associated with a decline in neutrophils from the BM and a 5-fold increase in circulating neutrophils relative to baseline (no UVL). Relevant to SLE, neutrophils increased up to 10-fold in the kidney after skin UVBL injury (vs. no increase in monocytes/macrophages). Local skin response was characterized by rapid induction in inflammatory cytokines (IL1b, TNFa, IL6, IL33) and neutrophil chemoattractants (G-CSF, CXCL1, LIX) (day 1-2) that returned to baseline by day 6 after UV. Neutrophil infiltration into the kidney was accompanied by endothelial activation and inflammation: increased VCAM1, E-Selectin, IL1b, Ngal, and s1008/9 gene expression, markers of kidney injury in SLE, as well as by transient proteinuria. Neutrophils recruited to the kidney demonstrated two phenotypes: early activated CXCR2hi (day 1-2) and late aged CXCR4hi (day 2-6), which followed expression of CXCR4 ligand CXCL12, another marker of kidney injury. Together, our findings propose a novel neutrophil-dominated skin-kidney axis of pathogenesis and provide a model for UVBL-triggered disease UW flares in SLE.

From November 2015 through March 2016, the Olympic Mountains Experiment (OLYMPEX) was conducted on the Olympic Peninsula to study the evolution of wintertime clouds and precipitation in frontal systems passing over this coastal mountain range and to validate satellite-derived precipitation measurements from the U.S.-Japan Global Precipitation Measurement (GPM) mission. While most OLYMPEX research has focused on precipitation processes on the windward (usually southwest) side of the Olympic Mountains, this study uniquely examines the leeward (usually northeast) side of the mountains where there is climatological rain shadow (i.e. a minimum in precipitation relative to the windward side). The vertical structure of the frontal systems over the northern Olympic Mountains is examined using data from a radar managed by Environment and Climate Change Canada on Vancouver Island (EC-XBAND), including intensity inferred from radar reflectivity. Using environmental data from North American Regional Reanalysis on the windward side of the mountains, this study classifies the leeside radar data based on upstream large-scale conditions. The cloud and precipitation structure on the leeward and windward sides of the mountains are compared utilizing the Doppler on Wheels (DOW) radar in the Quinault River Valley, and the EC-XBAND radar. Cloud and precipitation particles measured by in situ aircraft over the windward and high terrain illuminate situations when particles are lofted over the mountains to the leeward side, reducing the rain shadow. These findings will inform local studies of snowpack and water supply in the Olympic Peninsula as many reservoirs there depend on precipitation that occurs on the leeward side. Outside of the Pacific Northwest, these findings can be applied to other midlatitude coastal mountain ranges on the west side of continents around the world.

Opsins are the light-sensitive proteins in photoreceptors that mediate vision. They are most commonly known to be expressed in rods and cones in the retina of the eye, but some are also expressed in various tissues throughout the body. Because previous research has shown that retinas from OPN3 knockout mice have altered circadian amplitudes, my goal is to further investigate the effects of OPN3 on circadian rhythms of various tissues. In order to determine which tissues actively express OPN3, rtPCR analysis will be done on a variety of Wild-Type (WT) mouse tissues. To determine how circadian amplitude and rhythmicity is affected by OPN3, tissues from both WT and OPN3 knockout mice with the Per2 Luciferase marker are cultured and have their circadian rhythms recorded and analyzed. These cultures are also used to determine the effect of OPN3 on ability to synchronize to light-dark cycles, and to begin investigating the mechanism through which OPN3 works. Preliminary results show that OPN3 knockout mice have significantly decreased amplitudes in tissues actively expressing OPN3, although the lack of OPN3 does not affect rhythmicity or the ability to synchronize to light-dark cycles. So far, I have seen that OPN3 is not acting through a diffusible substance, as other opsins have been shown to do. More investigation should be done in the future to determine the exact mechanism through which OPN3 works, as it will have important consequences for understanding the circadian system. 

Malaria affects close to half of the world’s population and kills nearly 3,000 children every day. Due to the rapid rise in drug-resistant parasites and insecticide-resistant mosquitoes, we need a vaccine now more than ever before. Malaria is caused when a mosquito infected with the parasite, Plasmodium, bites and injects thousands of sporozoites that infect the liver and replicate without causing symptoms for 7-10 days. After egressing out of the liver, the parasites infect red blood cells and can get taken up by another mosquito during a bite perpetuating disease transmission. In the blood stage, the population of parasites in circulation can reach the billions, symptoms occur, and disease complications can result in death. Our strategy is to develop a pre-erythrocytic malaria vaccine by creating a genetically attenuated parasite (GAP) strain. If infection could be stopped before the parasites break through into the proliferative blood stage, both the disease and transmission could be prevented. Our goal is to make a late-liver stage arresting Plasmodium falciparum GAP conferring a broad antigenic diversity. Based on late-liver stage transcriptomics, we identified genes that could be candidates for knockout. One of these genes is mei2 of which my team already generated a CRISPR/Cas9-mediated knockout. We conducted an analysis of invasion kinetics via a growth competition assay between wild-type and mei2- parasites to ensure the gene deletion did not reduce viability. We proceeded by exposing our human-liver chimeric mice to infectious mei2- sporozoites, tracked liver stage development, and looked for breakthrough into the blood stage as an indication of incomplete attenuation. We then built on the previously established role of mei2 as an RNA binding protein by studying the subcellular localization of Pf mei2 to better understand the effect it has on liver-stage development.

The human adaptive immune system is composed of B and T cell lymphocytes. Of the latter, these cells are further differentiated and identified by the presence of CD (cluster of differentiation) molecules. CD molecules are expressed on the surface of the cell and can range from receptors essential to the cell’s functionality to glycoproteins marking distinct stages in their maturation. For example, CD45RA is expressed on naïve T cells while the presence of CCR7 marks the change to effector memory cells. Flow cytometry can be used as a tool to aid in detecting multiple types of T cells in a peripheral blood sample both rapidly and accurately. The use of 10-color flow cytometry in the clinical laboratory can provide simultaneous detection of ten cell markers to help differentiate the populations of T cells in a patient. Seattle Children’s Hospital utilizes this technology to monitor immune status in patients with auto-immune disorders, immunodeficiencies and post transplantation. Extended T-cell immunophenotyping by flow cytometry is not a mainstream clinical test. Since only a few laboratories offer this service, samples are often received from sites outside the hospital. With distance comes the issue of peripheral blood stability, especially regarding the loss of CD markers among the T-cell population over time. This study will compare the stability of T cell subsets through peripheral blood samples collected in EDTA vs sodium heparin collection tubes through four days of daily T cell immunophenotyping testing, as well as to determine the last day of stability for each T cell subset. The results will help form the guidelines regarding specimen stability for transport and the availability of offering this test to both adult and pediatric patients across the globe and help aid the diagnosis and treatment of various diseases and conditions that these patients face.

PTEN is a tumor suppressing protein that carries out important cell functions such as inhibiting cell growth and promoting genomic stability. Somatic variants of PTEN can lead to cancer, and PTEN mutational status has shown to be an indicator of patient survival and prognosis. However, it is not clear whether cancer-associated PTEN variants affect cell growth, genome stability, or both. Here, we demonstrate that simple competition assays can quantitatively assess PTEN variants for their effect on these two important cellular functions. Cells expressing cancer-associated PTEN variants tagged to blue fluorescent protein are mixed with cells expressing wild-type (WT) PTEN tagged with a red fluorescent protein. The proportion of blue and red cells are analyzed over several days using flow cytometry. If the variant does not repress cell growth, variant (blue) cells will outcompete their WT (red) counterparts. To modify the competition assay for genome stability assessment, cells are treated with a PI3K inhibitor and the genotoxic chemotherapeutic temozolomide. These drugs isolate the genomic stability function of PTEN by removing its role in cell growth and causing genome instability, respectively. Here, cells harboring variants that cannot repair temozolomide-induced DNA lesions will be outcompeted by their WT counterparts. The assay generates a score that is based on the rate of change of variant populations relative to the WT population to quantitatively define the phenotype. Results can be interpreted to establish a relationship between a PTEN variant and its quantitative effect on the cell growth or genomic stability functionality of PTEN. Furthermore, the growth-based nature of these assays means that in future work they can be adapted to a pooled library format, allowing the simultaneous, quantitative assessment of thousands of PTEN variants. Data from both low-throughput and high-throughput experiments bring clarity to the relationship between specific PTEN functions and patient prognosis.

Diet influences mammalian physiology and has been linked to obesity and the development of carotid artery disease. Carotid artery disease (CAD) is a result of plaque buildup in arteries leading to the brain and other parts of the head, reducing blood flow and increasing the risk of stroke. Rates of obesity and carotid artery disease are higher in countries with Western diets (higher saturated fat and carbohydrate composition) compared to those with Mediterranean diets (higher produce and monounsaturated fat composition). Although there is evidence demonstrating the effect of diet on overall health and physiology, the mechanisms by which these changes arise could be better understood. These health consequences of diet are thought to be mediated by changes in gene expression caused by eating different diets. Understanding how diet influences gene regulation in different tissue types could give critical insight into our understanding of diet’s effect on physiology. To this end, I measured gene expression via RNA-Seq in 37 cynomolgus macaques (Macaca fascicularis) that were randomly assigned either a Western or Mediterranean diet. By analyzing gene expression in samples collected from two types of fat (subcutaneous and visceral) as well as two arterial tissue types (iliac and carotid), I aim to gain insight into the transcriptomic changes due to diet. Further, by understanding diet’s role in gene regulation, I aim to construct a clearer picture of the mechanisms through which western diet might heighten the risk of obesity and the development of CAD. I predict that the differentially expressed genes in fat tissues will be associated with obesity and that the differentially expressed genes in the arterial tissues will be associated with plaque build up and CAD. This study will illuminate how diet mediates gene regulation in a variety of tissues, providing a mechanistic link to diet-related diseases such as obesity and CAD.

Physiological and behavioral rhythms are controlled in mammals by a central circadian clock located in suprachiasmatic nuclei of the hypothalamus (SCN). This master clock has outputs to other organs and tissues crucial to keeping the organism properly synchronized. The SCN clock is synchronized by environmental cues, most importantly is the light-dark cycle (LD). Fearful stimuli (i.e. presence of predators) can also present cyclic variations. The de la Iglesia lab has recently shown that timed fearful stimuli during the night can switch the locomotor activity rhythms of mice to the light phase, overriding their natural nocturnal behavior. Interestingly, while the expression of the so-called “clock genes” (which sustain the circadian rhythms at the molecular level) remains unchanged in the SCN, it displays a complete inversion in the amygdala, the brain region that encodes fear. Currently, we aim to determine the pattern of expression of clock-genes in peripheral organs of mice subjected to cycling fear stimuli. Using qPCR, we will assess RNA expression of the clock genes bmal1, per1, and per2 in the adrenal gland, kidney, and liver to determine whether entrainment of activity by cyclic fear also impacts peripheral clocks at the molecular level. We hypothesize that the pattern of expression the clock genes in the liver and kidneys will be modified in mice subjected to nocturnal fear, due to altered feeding and drinking patterns. However, the adrenal gland is difficult to make predictions about the pattern of expression of the clock genes given the fact that preliminary data from our lab showed that cortisol shows two peaks in mice displaying diurnal activity after nocturnal cyclic fear exposure compared to the single peak displayed by nocturnally active mice. Thus, it is unclear if we will observe an inversion peak, much like the amygdala, or a peak similar to the LD cycle.

Hydrogen is a non-polluting, sustainable energy alternative to fossil fuels. One method of generating hydrogen is by splitting water with sunlight and a semiconductor photocatalyst. In this study, determining the most viable semiconductor in terms of efficiency and safety for the environment is critical for making hydrogen energy available for commercial usage. When the catalyst absorbs UV light, water molecules in contact with the activated surface are split. Hydrogen is produced more rapidly when the photocatalytic crystals have a high surface area to volume ratio. Therefore, greater efficiency can be attained by reducing the size of the photocatalyst crystals. Nanometer-sized crystals can be synthesized within nanopores, thereby avoiding loss of surface area by agglomeration. Particular attention has been focused on placing the most efficient and cost-effective semiconductor inside a matrix of cellulose fibers and introducing doping, quantum dots, and other engineering strategies to improve the suitability of the photocatalyst. Selecting the most fitting photocatalyst candidate for large scale mass production of hydrogen is imperative for providing a viable source of renewable energy.

The study of the behavior of multiple agents, specifically human and machine, in a dynamic environment is challenging due to the unpredictable individual behaviors. Humans will naturally formulate beliefs about the machine’s behavior, which would directly affect their future decisions. Our research aims to develop a framework for the study of human-machine dynamic interactions. With the imperfect information humans and machines have about each other and their environment, a game-theoretic approach was done to study the natural model of their interactions. We derive theoretical models for steady-state (i.e. equilibrium) and transient (i.e. learning) behaviors of humans interacting with other agents (humans and machines). We also design experiments to validate our theory. A haptic testbench, in the shape of a robotic arm, is used as a dynamic simulation platform for studying the trajectories of the human/machine interaction, allowing us to study both theoretically and experimentally. The robotic arm has a position control system that supports a wide variety of human/machine experiments. The user is provided with visual and haptic feedback, which allows for experiments to be designed to study the sensorimotor learning processes. The robotic arm is built using direct-drive brushless motors, force sensors, an open-source ODrive motor controller, and an arm lever. The motor firmware is designed in C/C++, and integrated with a user-interface in Python. With the wide variety of potential applications, we hope our research will give insights into the different natures of human motion and be a fundamental platform for technological breakthroughs in the medical field.

Coevolution is a process where two or more species reciprocally affect each other's evolution over time. The traits of one species evolves in response to the other. This process can lead to the diversification of organisms with unique adaptive traits. Previous experiments tested whether coevolution occurred during the 2000 generations of evolution between the bacteria Desulfovibrio vulgaris and archaea Methanococcus maripaludis by pairing populations from 1000 generations with mutualist partners from their evolutionary past or future. Results suggested patterns of coevolution, but hypothesizing that those patterns occurred by accident, we tested whether or not patterns can occur that look like coevolution with microbes that could not possibly coevolve. We conducted a timeshifts control experiment using freezer stocks of D. vulgaris and M. maripaludis that evolved alone for 2000 generations and created pairings of the mutualists with five different generational pairings. They then were paired with an ancestral control group and a modern test group from the 1000th generation. Growth rates were calculated for each coculture. Graphs of the effects the 1000th generation of M. maripaludis and D. vulgaris had on coculture growth rate showed that M1000 had similar growth rates irrespective of its partners evolution while D1000 results were variable. An ANOVA test showed the mean log ratios statistically indistinguishable across all timepoints, indicating that the null hypothesis should be accepted. This result suggests that both partners have the same effects on fitness no matter how long they evolved. However, due to variations in results between D. vulgaris and M. maripaludis it is still unclear whether these populations have the ability to create patterns of coevolution due to their adaptation to their abiotic environment.

The American crow (Corvus brachyrhynchos) emits a variety of vocalizations, but the meaning of these calls is not well understood. To better understand crow vocal communication, we tested whether the playback of call sequences with accelerated, decelerated, or randomized pauses, or bouts of silence between calls, caused different behavioral responses in wild crows. Previous research has shown that the pattern of silence between call syllables may code important information in this species. We manufactured crow vocal sequences using Audacity audio editing software to produce variations of the same overall sequence with differing pause rates. We then played them to crows on diurnal foraging areas in Western Washington and measured the latency to move towards the playback speaker, closest approach, and the number of crows responding. After 20 trials we detected non-significant trends suggesting an effect of pause rate. We are currently increasing our sample size in an attempt to resolve these trends.

The Yerkes-Dodson law suggests that performance is optimal when physiological arousal is moderate. Prior research has shown that arousal induced by listening to music will affect task performance on a cognitive task. The goal of this study was to test whether different music tempos would affect arousal levels and performance on a cognitive task. We recruited 16 UW undergraduates and exposed them to different tempos of the same song while performing a math test. We tested their accuracy, completion time, and recorded their heart rate. Results suggest that accuracy was higher under moderate music tempo than slow but there was no difference between moderate and fast. Interestingly enough there was no difference in completion time or physiological arousal. Although we failed to establish an effect of arousal and completion time the fact that participants did better on accuracy when music tempo was moderate is in line with what we predicted. Music tempo could still be influential to performance and future research can be done to understand these effects by operationalizing performance differently. Future directions about ways to improve the study will be discussed especially whether music/music tempo affect people subjective arousal and physical arousal differently.

Molecular interactions that occur between surface components of pathogens, termed adhesins, and complementary receptors presented on host cells are critical for establishment of infection. The adhesion-receptor binding event may trigger diverse signaling cascades in the host cell that result in activation of innate host defenses or the subversion of cellular processes facilitating bacterial colonization or invasion. Hence, microbial adhesins are important targets for the development of antimicrobial therapeutics and vaccines. One of the interests of the Sokurenko Lab at UW is understanding the structure and function of the ubiquitous Escherichia coli adhesin, FimH, and its potential as a vaccine candidate against urinary tract infections. FimH is expressed on top of filamentous hair-like appendages called type 1 fimbriae that confer bacterial binding to glycoproteins with terminally exposed mannose. In the laboratory, we use a variety of assays to study FimH-mediated bacterial adhesion and test different mannose-containing compounds and glycoproteins for optimal inhibition of FimH-mediated interactions. We also elicited a great number of FimH-specific monoclonal antibodies that are tested for their potency to inhibit binding properties of the FimH, and to understand the structural mechanisms of antibody-mediated inhibition. By generating and analyzing various FimH mutants, we seek to elucidate how the function of FimH protein can be efficiently regulated. The ultimate goal of these experiments is to gain knowledge on the optimal structure (conformation) of the FimH protein that can be used as an antigen for efficient induction of protective antibodies during immunization.

Manu National Park in southeastern Peru is an incredibly bio-diverse national reserve at the meeting point of the Tropical Andes and the Amazon lowland rain forest, and an important location for tropical ecology research. Cocha Cashu Biological Station is one of few research locations in the Neotropical rain forest in which mammal communities remain almost completely undisturbed by human impact, including hunting and road construction. As a result, mammal population densities and compositions accurately reflect their natural environmental state. Among these mammals are fifteen known species of primates, and as major seed dispersers they play a critical role in the stability of the ecosystem. The aim of this study was to learn more about the behaviors of eight primate species within the Cocha Cashu trail system, during September of the dry season, using quantitative and qualitative observational methods. We used the focal sampling methods to quantify the proportions of time spent on daily activities for each species, and created activity budgets from this data to compare time spent on each activity. We also collected data on troop size, social interactions, habitat, and location throughout the day in order to compare and contrast the different groups. We were correct in our prediction that most species spent the largest proportion of time moving and feeding, but varied in other activity categories such as curiosity and aggressiveness. We found that there was significant overlap in the habitats and resources used by different species, but there were notable differences in foraging behaviors, troop size, canopy level, and the time of day designated to different activities.

The Wrangel Island State Nature Reserve (WISNR) serves as a vital refuge for the Alaska-Chukotka (AC) population of polar bears (Ursus maritimus) during the ice-free season. In September 2017, a total of 181 polar bears were observed near a bowhead whale (Balaena mysticetus) carcass on the island. This gathering is the largest aggregation of polar bears ever recorded for the AC population. This study sorted, labeled, and processed photographs of the polar bear aggregation taken by a professional photographer from a boat a day before initial ground-based observations were made. Our objective was to use the photographs to evaluate characteristics of the polar bear aggregation including animal sex, age, reproductive composition (e.g., adult females that have first-year or second-year cubs), and body condition (i.e., fatness). To do this, we selected representative subsets of photos, categorized them by time and location, and labeled individual bears across multiple photographs. The resulting set of processed photographs was evaluated by multiple polar bear experts, and the results were statistically analyzed. Based on knowledge of polar bear social systems and an initial review of the photographs, we hypothesized that both feeding activity and the locations of bears in the vicinity of the carcass will be structured by sex, age, reproductive status, and time of day. This study provides a unique opportunity to collect information on a large number of polar bears and document behavioral interactions. The resulting information will help address key conservation challenges for the AC polar bear population, including the effects of sea-ice loss due to climate warming, increased industrial activity, and identifying a sustainable rate for subsistence harvest.

Control of the cell-cycle is a topic of fundamental importance in cell biology. We are using the unicellular eukaryotic green alga Chlamydomonas reinhardtii to better understand how DNA damage affects transition of these cells in and out of the mitotic cell cycle. We find that UV treatment of cultures of C. reinhardtii gametes (in Go) delays re-entry of these cells into the mitotic cell cycle upon addition of nitrogen to cultures of gametes, a standard treatment which releases these cells to re-enter mitosis. Using a C. reinhardtii mutant which is unable to carry out photorepair of UV-induced DNA damage, we are characterizing the degree to which the kinetics of re-entry into mitosis depends on this very important repair system; we are thus beginning the 'parsing out' of the relative importance of the various DNA repair systems in controlling the transition from Go, a non-dividing stage, to G1,  the start of DNA replication and cell division. This will potentially provide insight into other downstream effects of UV exposure, such as mutagenesis. Our work is of a very basic nature, but with application to understanding e.g. dynamics of natural populations of microalgae in a changing global environment.

The use of mindfulness for emotion regulation has led to its adoption as a strategy to manage emotion and mood. Studies on eating behavior have utilized mindfulness exercises for those with eating disorders and Type 2 diabetes and found that mindfulness improves healthy eating habits. This study extends prior work by examining a mindfulness-based eating intervention among college students. Participants were randomly assigned to a weekly 30-minute mindfulness group or relaxation control group for six weeks. Mindfulness exercises ranged from meditations on nonjudgmental self-acceptance to advanced mindfulness procedures utilizing common snack foods. These exercises encouraged the use of mindfulness strategies to help regulate emotional eating, mood, and eating strategies in daily life. Mindful eating participants reported significant decreases in depressive symptoms compared to controls. However, there was no significant differences between mindful eating, anxiety symptoms, and emotional eating in response to stress over time in mindful practicing participants and compared to controls. Results suggest that a mindful eating intervention for a non-clinical sample helps maintain mindful awareness of dietary behavior and decrease depressive symptoms, but has little effect on emotional eating and mindfulness.

This work is an investigation into the multifacited world of fairy tales. Through history, fairy tales and fables have been collected from story tellers, edited, and presented to the world in print. Through this process, tales with dark or racy subjects are painstakingly polished, added on to, and moralized. Many were warped and nearly completely overwritten to fit within the views and biases of the collectors and editors. Hundreds of years down the line and through the Disney lens, fairytales are mostly known by their bright, cheery exteriors, with little of their original content to be found. Through the use of stage design and illustration, I worked to create an environment that would display this dichotomy. Once installed, the work creates a bright fairy tale forest for the audience to enter. When they turn to exit, they are faced with the dark underside of the fairy tale world. 

 A method of negative-pulse partial modulation for comprehensive two-dimensional gas chromatography (GC×GC) using a pulse flow is studied. The capability of a pulse flow valve modulator has been shown to enhance many aspects of GC performance such as chemical selectivity and reduced analysis time, allowing complex GC×GC analysis in a shorter time scale. In our study, we explored a particular configuration of a pulse flow valve that periodically (specified by modulation period, P_M) disrupts the carrier gas flow to achieve partial modulation. This disruption of flow creates a "plug" of higher concentration on top of the primary GC signal, which is further separated in a secondary column. The size of the plug determined by pulse width, P_W, has a direct relationship to chemical selectivity and sensitivity. For our experiment, we determined the ideal P_W to be around 8 ms. Therefore, a 20-component mixture was evaluated using P_W of 8 ms and P_M of 100 ms, producing an average peak width, ²W_b, of 12 ms and approximated ideal peak capacity, ²n_c, of 8 on the second dimension. All 20 compounds were separated in a 12 second separation window. This powerful partial modulation method was achieved by the unique concept of the flow disruption with short modulation period, demonstrating high peak capacity and improved selectivity between compounds and chemical sensitivity. Due to the significant increase in GC performance, this form of partial modulation using a pulse flow valve allows efficient and faster GC×GC analysis that can be implemented in a variety of GC×GC applications.

Natural proteins often assemble into various complex geometric structures based on their interactions with each other. These structures can hold and transport "cargo" as well as display antigens, making them extremely useful in vaccine design. The King Lab at the University of Washington uses the way these proteins assemble to develop computational models that help them design novel self-assembling protein cages, or nanoparticles. These nanoparticles are then used to develop vaccines or treatments for diseases. Components of the designed protein cage can be modified and expressed individually before being assembled together into the nanoparticle. I am working on stabilizing one of these protein cages known as T33DN2, so it can be used towards creating a vaccine. T33DN2 is a tetrahedral cage comprised of two trimeric proteins known as T33DN2A and T33DN2B. When expressed individually through E.coli, DN2A is produced in a soluble form while DN2B is produced in a mostly insoluble form. T33DN2 is currently an unstable cage, as only the A component is expressed solubly. Soluble proteins are generally more stable and thus easier to work with than their insoluble counterparts. To increase the solubility of DN2B, I have been making mutations to specific amino acids in the DNA that produces this protein, as well as expressing and purifying this component to determine its stability.

Nearly a century ago, Lothrop Stoddard’s The Rising Tide of Color (1920) articulated his growing concern that the scourge of racial diversity exacerbated by immigration was particularly acute within the United States. This scourge not only threatened to transform America’s national character in terms of religion, laws, and customs, but also served as a profoundly destructive force to white labor and standards of living. Trump’s zero-tolerance immigration policies and the rationale behind them parallel not only Stoddard’s concerns about the threat immigrants pose to the United States, but also Stoddard’s solution of shoring up the “inner dikes” against undesirable immigrants. This study compares the immigration policies of Presidents Bush, Obama, and Trump to explore issues of border control, detention methods, arrest rates, and use of media in shaping public perception of immigrants. It also utilizes statistical data to assess the economic impact of legal and illegal immigration within the United States under the three presidential administrations listed. Findings suggest that the claims made by the Trump administration about the threat immigration poses not only contradict statistical data, but also perpetuate negative racialized stereotypes about immigrants. By drawing on the basic premise of critical whiteness theory--that race as a social construction is an important feature in American society and includes racialized practices of essentializing, colorblindness, and microaggression—this study provides an example of how Trump’s zero tolerance policies on immigration utilize early twentieth-century narratives of race and racialized differences to institutionally normalize discrimination and prejudice as natural features of American society as opposed to social challenges within it.

Cerebral palsy is a congenital disorder which impacts movement, muscle tone, and cognitive ability. This disorder affects 2-3 people per 1,000 births annually. My lab is currently developing a technology to assess motor learning. We’re working on quantifying motor planning deficits in cerebral palsy to aid in targeted therapy. However, the small test population combined with the nature of cerebral palsy means it can be difficult to bring these subjects into the lab to verify our technology. I developed a “simulation” of the controller we use to interface with our technology that can be downloaded onto android devices and can connect to a subject’s bluetooth enabled computer wirelessly. This allows subjects to test our technology remotely in a setting which best suits their needs. Upon testing this virtual controller against the currently used controller, the virtual controller offered a lower mean-squared error to the normal controller, and was proved a viable option for remote testing. This development allows us to expand our testing pool to those who may not be able to physically come to the laboratory for testing, and can be expanded to future developments which require unique controllers.

The #MeToo movement has sparked a national conversation about what constitutes sexual consent, and a series of debates have erupted over the actions of famous men like Brett Kavanaugh, Louis C.K., and Aziz Ansari. Popular hits like Robin Thicke’s song “Blurred Lines” have also come under fire for promoting sexual violence by celebrating confusion around sexual consent. At this moment, when norms and expectations around sexual interactions seem to be changing quickly, it is especially important that we have comprehensive and systematic data on how people define sexual consent and interpret ambiguous sexual experiences. Existing studies have shown characteristics of an individual matter when it comes how they conceptualize appropriateness. However, we still do not know to what extent these characteristics matter. This study addresses this gap by answering the question: How do people of varying groups define appropriate sexual behaviors and boundaries? A Q-Method study of over 200 people will reveal how individuals of different characteristics sort scenarios of various sexual interactions into categories ranging from “most appropriate” to “most inappropriate.” Q method is similar to a rank-order techinque and allows for representation or reconstruction of perspectives concerning any given topic of interest. In addition to providing insight about how different people make sense of sexual behaviors, this study shows how Q-sorts can provide a more detailed look at sexual norms than the survey and vignette studies currently used in the field.

Enteric diseases, or diseases of the Gastrointestinal (GI) tract, remain one of the most prevalent killers of children in sub-Saharan Africa. The most practical way to prevent such diseases is through vaccination, but antigens for enteric diseases need to be delivered directly to the GI tract to be most efficient, making vaccination difficult. Recent studies by the von Adrian group at Harvard University have found that both T and B cells are reprogrammed to home to the GI tract when they encounter retinoic acid, a metabolite of vitamin A. The King Lab at the University of Washington is working to develop a novel vaccine candidate using recently developed self-assembling protein nanoparticles, that can simultaneously package all-trans retinoic acid (ATRA) and multivalently display enteric antigens. Previous work has suggested that two cystine mutations to Cellular Retinoic Acid Binding Protein I (CRABP-I) create a disulfide bond as a result of the conformational change that CRABP-I undergoes when it binds ATRA. This disulfide bond would essentially lock ATRA into CRABP-I, reducing its dissociation constant in vivo and maintaining the gut-homing properties of the nanoparticle post-injection. In order to assess the efficacy of these cysteine mutations, I expressed two versions of CRABP-I, the wildtype protein with no cysteine residues, and a version with no cystine residues except for the two that create the disulfide bond. After establishing that these new CRABP-I mutants folded into the approximate shape of wildtype CRABP-I via circular dichroism, I designed and tested new assays that measured free thiol concentrations of each protein after binding ATRA, as well as free ATRA concentration overtime. This data will help us determine whether these two cystine mutations make a significant difference in the ATRA binding quality of CRABP-I, which could improve the immune response generated by our vaccine candidate.

Cancer is increasingly a global health issue. Access to radiation therapy, the primary method of cancer treatment, is limited in low- and middle-income countries due to difficulty translating technologies in resource-limited settings. We are developing a new generation of cost effective IMRT (intensity modulated radiotherapy) devices for low- and middle-income countries. The current IMRT is performed using linear accelerators with multi-leaf collimators (MLC) which is very expensive and challenging to maintain in low- and middle-income countries. As a team, we are replacing the current technology with an alternative one using physical compensators which have lower commitments in terms of cost, overhead, and complexity. During my time working in the Imaging Lab, I have implemented all of the custom scripting protocols in a Python script (IronPython Interpreter) which is connected to the treatment planning system (RayStation - RaySearch Laboratories). Treatment planning systems are at the heart of radiation therapy systems and the key to improved patient outcomes. My script has enabled the creation of a compensator map and contour from the fluence data of the beamset. To test validity and correctness of the equations derived - I created a treatment plan based on the CT image for a head and neck cancer patient. The preliminary results of my script on this treatment plan indicate that compensators with ray lines divergent with the radiation source can be constructed for use in the treatment planning system. My contribution satisfies a key requirement of the project. A prototype of the compensator will be developed in commercial and partner sites in India, validating the script with a large number of patients.

Supraspinatus tendon tears are a type of rotator cuff tear, accounting for 15% of overhead workplace musculoskeletal injuries. These tears disproportionately affect blue-collar workers and cost millions in healthcare every year, but there is still relatively little known about the appropriate work-rest cycles to prevent the risk of occurrence during work. Directly measuring the rotator cuff in vivo is difficult because the supraspinatus is covered by the bursa sac, the acromion, and the deltoid, making its material properties hard to accurately record. This presents a need for a material that can model an in vivo shoulder tendon. There are many options of what materials can be used: organic and in vitro models are the most common, with relatively new inorganic models being designed. However, none of these models fulfill all modeling needs; overlap between all models is needed to get an idea of how an in vivo tendon accumulates damage. Organic models can provide tissue repair and degradation rates and these can be projected for a human supraspinatus. From in vitro studies stress-strain curves and maximum load can be recorded, and from inorganic models tear propagation can be observed. This work compiles research on candidates for tendon proxy materials by cross-referencing a variety of papers in tendon literature to find the foundational papers. Then builds off those with other works by the foundational authors or other highly regarded works that cite those foundational papers. From the collection of these papers, the shortcomings of current tendon modeling can accurately be seen, showing what research is needed to better model in vivo tendons. For instance, to confirm the hypothesized projection from organic models, psychophysical testing that isolates the supraspinatus needs to be conducted. Better modeling of tendons will allow for better prediction of appropriate work-rest cycles that may slow tendon fatigue damage.

For many globular proteins, the sequence and native structure are known. However, less is understood about how a string of amino acids folds into a functional protein. Experimental study of folding presents challenges due to the transience and variability of folding/unfolding transition states and intermediates. Alternatively, computational study of unfolding can provide significant insight into folding. Here, molecular dynamics simulations have been used to study the unfolding pathways of the SH3 domain structural family and to investigate the factors that determine the path and outcome. To separate folding determinants from amino acid sequence, 17 SH3 proteins were chosen with an average sequence identity of only 27%. Six unfolding simulations were performed for each protein, and the unfolding transition state ensemble was identified by locating the large, rapid conformational changes that signal the start of unfolding. Contact analysis was used to characterize the structure of the transition states ensembles. Two general pathways at the transition state were identified, distinguished based on the specific β-sheet structure lost at the transition state. In the first, more populated pathway contacts in the β-sheet containing the N- and C- terminal β-strands were lost while the second pathway was defined by structure loss in the other β-sheet. Though many of the investigated proteins went through both pathways in different simulations, most showed a clear bias towards one pathway. This work demonstrates that similar protein structures can fold through different pathways. The bias of many SH3 proteins towards one folding pathway also suggests the presence of some elements of primary structure that direct folding. Further investigation of the SH3 domain may yield ‘rules’ that determine the structure and folding pathway of the domain, and these rules may inform the study of other, similar proteins.

Manufacturing workers are often subjected to many rigorous and repetitive shoulder and arm motions, usually leading to shoulder injuries. Assessing the likelihood of an injury before it occurs and adjusting practices accordingly can keep the individual from the severe pain that shoulder injuries can cause. This research focuses on creating such a predictive model to warn individuals before they sustain an injury. I created a mathmatical model to assess critical positions that would cause the most stress in the shoulder joint. A Kinect sensor locates the arm joints in space and my Matlab code calculates the expected reaction forces in the shoulder. My current results focus on single, static positions defined by common industry working positions. Future work will focus on dynamic positions and comparing the results from the mathematical model with biological indicators to determine if this predictive model is indicative of injury.

A national organization named 2-1-1 provides assistance to populations with basic needs, mainly informing and answering questions, and researching resources covering 50 states. Not only is the demand for information growing, but also the complexity of calls is increasing. For example, a person may need information on shelters, medical issues, legal assistance, debt management and other services. Consequently, calls are taking longer, and with limited resources, wait times are increasing and even critical calls are sometimes abandoned. Assigning more staff to a call center is not realistic under limited budgets. Hence, an efficient call center system must be designed. Our research seeks to address the efficiency of the WA 2-1-1 call center system, in Washington State. We hypothesize that the application of call prioritization and skill-based routing can reduce the holding-call time, and eliminate call abandonment for those with urgent needs. We will identify classifications of calls, and evaluate how automation can be used to navigate or direct callers to a correct resource and expedite the whole process. Appropriate classification of calls and prioritization may not only decrease the waiting time but also help training operators with an appropriate skill set. We will apply the concept of operations research to forecast seasonal demand using available data within Washington State, and will estimate the number and allocation of resources the organization should provide. The anticipated result of the research is a call center design that will decrease the number of abandoned calls, provide an immediate response to an urgent call, decrease waiting time, and assign calls to available operators to exploit the limited resources. The WA 2-1-1 call center is important to provide assistance to efficiently serve populations in need. Increasing the efficiency of call centers with limited resources will enable WA 2-1-1 to reach more people in need.

Prion diseases occur from the misfolding of the Prion Protein cellular form (PrP^C) under low pH conditions to the infectious scrapie species (PrP^Sc), which can aggregate further into insoluble fibrils. Previous studies have demonstrated that along with other amyloid oligomers, the prion scrapie oligomers cause neurotoxicity by disrupting the membrane, increasing its permeability and affecting calcium ion influx; however, the molecular mechanism for this effect is unknown. Molecular Dynamics simulations were performed to gain insight into the molecular mechanism of PrP^Sc-induced misfolding of PrP^C and oligomer toxicity in a membrane environment. The system was composed of the hexameric bovine PrP^Sc spiral model oligomer and the di-glycosylated human PrP^C attached to a POPC membrane via a glycophosphatidylinositol (GPI) anchor. Prior unpublished membrane simulations of this system have suggested that PrP^Sc induced PrP^C conformational changes as well as significant membrane disruption from oligomer-binding. Here we confirm and build upon these earlier studies demonstrating the reproducibility and robustness of oligomer binding affinity by varying the proximity of the oligomer to the membrane, providing key insight into infectious scrapie propagation and PrP^Sc cellular toxicity.

The HIV Vaccine Trials Network (HVTN) has flow cytometry labs at both the Fred Hutchinson Cancer Research Center (FHCRC) and the Cape Town HVTN Immunology Lab (CHIL) which both perform testing on HIV vaccine clinical trial samples. There can be a significant level of variability in the performance of flow cytometers, potentially leading to inconsistent data. The large degree of intra- and inter-instrument variability necessitates a quality assurance (QA) protocol be followed to standardize new flow cytometers to the instruments currently in use in both HVTN laboratories to ensure accurate, consistent results between instruments. Using a modified version of Perfetto et al.’s protocol which utilized sets of standardized calibration beads as well as singly stained CD4 T-cells, we successfully optimized, calibrated, standardized, and qualified a new BD FACSymphonyTM (Becton-Dickinson, San Jose, CA) flow cytometer for future use at CHIL. The successful completion of this QA protocol ensures that this instrument will be able to collect accurate and reproducible data for HIV vaccine clinical trials. It is iimportant to guarantee the quality of the data collected during these clinical trials so that the effectiveness of candidiate HIV vaccines can be properly assessed.

Headache is common in childhood and can be associated with disability and distress. Family history of chronic pain may increase risk for pain and disability in childhood, although prior research has primarily focused on non-headache pain populations. We aimed to characterize the prevalence of headache, abdominal pain, and musculoskeletal pain in first- and second-degree relatives of youth with headache and determine whether family and parental pain history is associated with children’s headache experiences. We hypothesized that a positive family pain history would be common, and that greater extent of family and parental pain history would be associated with greater headache frequency, intensity, and disability. We enrolled 239 youth (Mage = 14.7, 66.5% female) with headache and their parents. Parents reported whether they and 16 of their first- and second-degree relatives had a history of migraine, tension-type headache, abdominal pain, or musculoskeletal pain. Youth completed a 28-day headache diary to assess headache frequency and pain intensity and completed the PedMIDAS, a measure that assesses headache-related disability. As expected, a family pain history was common: 65.7% of youth had a relative with migraine, 39.3% had a relative with tension-type headache, 44.8% had a relative with abdominal pain, and 66.9% had a relative with musculoskeletal pain. Children’s headache frequency was positively associated with the number of first- and second- degree relatives with a history of tension-type headache (r=.14, p=.04) and musculoskeletal pain (r=.14, p=.04), but not migraine or abdominal pain. We found that youth had greater disability when both parents had a history of headache compared to only one parent with a positive history, F(2,222)=3.27, p=.04. No associations were detected between family history and youth’s pain intensity. Our findings indicate children’s adaptation to headache may be influenced by their family. Further research is needed to understand potential mechanisms of intergenerational transmission of headache.

Dementia and learning impairment are associated with increasing age, and age-related diseases such as Alzheimer’s disease. There is evidence to suggest age-related cognitive decline is also associated with increased metabolic stress and disturbances in insulin signaling. Diets high in animal fat and table sugar are metabolically stressful, especially with increasing age, and place greater demands on mitochondrial bioenergetic machinery. This study was designed to show that a metabolically stressful diet would increase the severity of cognitive dysfunction in aging mice. Ten C57BL/6 male mice were fed a high fat and sugar (HFS) diet starting at 21 months of age. The diet consisted of 15% protein (casein), 58% fat (lard), and 27% carbohydrates (sucrose), as well as a vitamin and mineral mix. Nine mice were fed regular rodent chow (RC) diet, consisting of 24.5% protein (balanced amino acid mix), 13.1% fat (soy bean oil), 62.4% carbohydrates (28.2% starch and 3.25% sucrose), and a vitamin and mineral mix, for the same time period. Mice were weighed weekly and assessed for body fat using quantitative magnetic resonance imaging. After 7 weeks, mice were tested for learning impairment using a box maze paradigm. Mice on the HFS diet gained an average of 9.62 grams of body weight, with an average 18.7% increase in fat mass compared to mice on the RC diet. Mice on the RC diet showed moderate levels of learning impairment. Surprisingly, mice on the HFS diet showed very little learning impairment, suggesting that some component in the HFS diet had a sparing effect on cognitive decline. This observation may be unique to mice, and certainly requires additional studies, but still raises an intriguing implication for how clinicians and dieticians might consider alternative intervention strategies for cognitively impaired individuals.

Pain is a major contributor to disability, and is more prevalent among low income groups. Rehabilitation care can be an effective treatment, although access and improvement may be associated with social factors. We examined the association of social factors (English proficiency and income) with rehabilitation use, improvement in rehabilitation, and meeting treatment goals among older adults in bothersome pain. We hypothesized: 1) participants with lower English proficiency will experience less improvement and achieve treatment goals less often than participants who are proficient in English and 2) participants with lower incomes will experience less improvement and achieve their treatment goals less often than higher income participants. This is a secondary analysis of the National Health and Aging Trends Study (NHATS), a cohort study representative of Medicare beneficiaries. Participants are interviewed yearly with a self-reported health and socioeconomic survey. We included community dwelling participants who indicated being bothered by pain in the last month. Participants reported how well they understood or spoke English. “Not well” or “not at all” was considered not proficient. Income was self-reported as total household income. Outcomes reported are rehabilitation use for pain related reasons in the next year, improvement with rehabilitation, and meeting treatment goals. Descriptive analysis suggests participants with lower English proficiency had higher rates of rehabilitation improvement. Rehabilitation users have a median income about $6,000 higher than non-users, however there is no difference in income between improvement groups or those that do or do not meet rehabilitation goals. We found lower income is associated with lower rehabilitation use but not improvement among participants in bothersome pain. These results indicate improvement is consistent among rehabilitation users however, access is limited for lower income participants. More work is required to determine how barriers can be eliminated to improve access to rehabilitation care for people in pain.

Roborun is a simulation game that utilizes crowdsourcing to identify legged movement patterns for land-based robotic movement. By using a game controller, keyboard, or by providing a set of instructions to execute in order (e.g. “rotate front leg X degrees”, “move back leg forward Y meters”, etc.), players can control joint torques and leg movements in order to navigate a virtual two-legged robot through several 2D obstacle courses that contain varied terrain and movable boxes. The game can be played through a web browser on either a computer or smartphone, making the game accessible to players across multiple platforms. In future development, we intend to implement a scoring system based on efficiency and speed of course solutions and will replicate the best scoring solutions from players on a commercially-available robot in our testing laboratory. It is our hope that their solutions will help develop reliable robotic movement algorithms and shed some insight into the dynamics of land-based movement.

Mindfulness involves being present, aware, and non-judgmental. The present study investigates the efficacy of a brief weekly mindfulness intervention within the college classroom as a means of increasing psychological well-being among students. We hypothesized that mindfulness would have emotional, cognitive, and social benefits, and that students who participated more frequently would benefit more. Across six 10-week classes, N=800 university psychology students participated in a five-minute weekly mindfulness activity. Students completed measures of emotional, cognitive, and social functioning in both the first and last weeks as well as some questions specific to the mindfulness activities. We found strong support for our hypotheses for emotional and cognitive benefits. Emotional benefits included increased happiness, resilience, and significantly lower anxiety. Cognitive benefits included engagement in the activities, engagement in the class, being more focused in class, and a positive impact on grades. Students who participated more frequently and to a greater degree saw more positive effects from the intervention. We conclude that a brief mindfulness intervention may be an effective way to improve wellness on the university campus.

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition associated with deficits in social interaction and the presence of restricted patterns of behavior. ASD is clinically and phenotypically heterogeneous. The most recent update to the diagnostic definitions for mental disorders (DSM-5) provides additional flexibility for capturing the diverse array of phenotypes shown by diagnosed individuals. Despite categorized behavioral symptoms across core and comorbid dimensions of ASD, the neural mechanisms are unknown. Studies have shown atypical neuroanatomy and functional connectivity of the brains of individuals with ASD, suggesting a neural etiology. Here, we focus our attention on the superior temporal sulcus (STS), a region of the brain demonstrated to play a role in processing communication, social information, and theory of mind. Our research question evaluates whether neural activity in STS is affected by behavioral phenotype in children with ASD. We speculate that neural activity between individuals with ASD varies significantly more than typically developing (TD) children, who show more clustered, similar outcomes. Functional near infrared spectroscopy (fNIRS) distinguishes concentrations of oxygenated (HbO) and deoxygenated (HbR) hemoglobin in regions of cortical vasculature, signifying neurovascular coupling. This study uses HbO and HbR measured as subjects watch the events of a social scene on a monitor and analyzes ASD individuals’ deviance from average TD activity. Eye-tracking, clinical assessments, and parent questionnaires are considered to extrapolate possible correlations between ASD individuals’ variation in neural outcome and the behavioral phenotype expressed. We expect that individuals showing higher degree of neural variance from TD activity will show more severe autism behavioral phenotype. It is possible that in the search for neural mechanisms of ASD, considering behavioral factors signifying atypical variance could reveal significant differences. This exploratory analysis aims to examine the wide range of possible influential factors on neural heterogeneity within the social brain of individuals with ASD.

Assessments of tropical insect diversity largely lacks known groups from which appropriate interpretations can be made, and ultimately undervalues the taxa of some regions. The ecology of Diptera within the tropics constitutes largely on the roles of nutrient breakdown, recycling, and pollination; as numerous Orchidaceae rely on pseudocopulatory mechanisms for effective pollination. Our study was conducted to explore and investigate the nocturnal diversity of the Order Diptera, with a special emphasis on Mosquitoes (Family: Culicidae), across multiple habitat types. The study site is in Cocha Cashu biological station along the Manu River in the Manu National Park, Peru. Sampling was conducted in early September during the dry season where we sampled several areas of varying vegetation communities, canopy coverage, and disturbance: river-side sample, lake-side sample, second-growth forest, and high-altitude cloud forest. We hypothesized that the highest diversity of morphospecies would occur in closed canopy areas with closer proximity to water bodies. Insects were collected using traps baited with a visual lure and soiled socks. Samples were filtered of other insect orders and individual dipterans were categorized using visual differences and similarities to create morphospecies groupings. Due to numerous variables, primarily trap efficacy and testing, our sample size was limited and as a result, our data could be expanded upon for further collection and analysis. The proceeding decreases in samples across a time-scale is largely due to bait methods. Overall, our hypothesis was supported by results indicating a higher species richness and diversity at sites in dense canopy coverage in proximity to a larger water body. Calculated Shannon diversity indices of 1.574 at the river closed canopy sampling site compared to 0.689 in the open canopy river site. These results may reflect habitat affinities of Dipterans in the tropics. Future testing will allow for increased understanding of Diptera communities in the tropics, taxa, and environmental health using certain species as indicators.

There is a critical lack of knowledge regarding the effects of human immunodeficiency virus (HIV) and zika virus (ZIKV) coinfection in respect to ZIKV pathogenesis, vertical transmission and current vaccine strategies. ZIKV has received global attention because of its clinical complications including congenital malformations during vertical transmission and Guillain-Barré syndrome, a neurocognitive disorder in adults. Recently, ZIKV outbreaks have occurred in tropical and subtropical regions endemic with HIV, therefore it is imperative to understand the impact HIV-ZIKV coinfection may have when moving forward with ZIKV vaccine design. Furthermore, in humans and non-human primates (NHPs) frequencies of blood monocytes increase during HIV and simian immunodeficiency virus (SIV) infection and monocytes are early targets of ZIKV infection. Therefore, we hypothesize that cells from HIV infected individuals have the capacity to harbor increased ZIKV replication and could lead to enhanced ZIKV viremia and pathogenesis. For this study, we use a NHP model to determine the impact of acute SIV infection on ZIKV pathogenesis. We determine ZIKV cellular targets in the blood and preliminary results show an increase in NHP blood monocyte within the first six weeks of SIV infection. We evaluate whether SIV infection increases the susceptibility of cells to ZIKV infection by isolating cells from SIV- and SIV+ animals, infecting them in vitro with ZIKV, and assessing viral replication by plaque assay. Our preliminary findings suggest that in vitro ZIKV replication may increase in cells from SIV+ NHP blood when compared to SIV- blood. Future studies will look at the impact of SIV infection on in vivo ZIKV replication and whether ZIKV replication is enhanced in cells from HIV-infected individuals.

The North Creek Wetlands Restoration on the University of Washington Bothell campus is host to a communal crow roost, with upwards of 16000 crows in the autumn and winter and significantly fewer during the spring and early summer. Crows congregate in larger and larger groups, called pre-roost aggregations, as they approach their roost each night. The function of these aggregations is not yet clear. We set up a night vision video camera and four audio recorders to observe the social behavior of crows on a pre-roost aggregation on the roof of Discovery Hall, adjacent to the aforementioned communal roosting site. The average duration of stay at this pre-roost aggregation was 24 minutes. We assessed video footage using a mix of scan and ad libitum sampling with a focus on aggressive interactions. We defined aggressive interactions between crows as approaching, pecking, and tail and wing pulling. I compared the frequency of these interactions for crows in the breeding and non-breeding seasons. Preliminary analyses suggest a significant increase in tail pulling during breeding (2.9 +/- 0.8) as compared non-breeding periods (0.3 +/- 0.3), but no differences in approaching, pecking, and wing pulling between these life history stages. I am currently increasing my sample size to better discern any differences, and am beginning to investigate social dynamics in pre-roost aggregations using social network analyses.