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

Females use many different sanitation products, including tampons and pads. However, it is speculated that some of these everyday products for some women may contain heavy metals and other chemicals. Lead is a heavy metal that presents a danger as can cause cancer and mental inefficiencies. Chromium is said to cause dermatitis and skin irritation. These metals are often thought of to be threatening in water and soil. However, man-made products such as tampons and sanitary napkins/pads can contain these heavy metals. Using standards from both metals and ICP technology, or an Inductively Coupled Plasma machine, analyzed the feminine hygiene products for chromium and lead. In my analysis of the hygiene products there does not seem to be a concerning amount of heavy metals, even with an increased mass analyzed. Very small amounts of chromium and lead were found in both pads and tampons. There appeared to be more lead than chromium in tampons, yet not a large amount to report stark differences.

Many research studies have shown that bilingual speakers tend to swear in their first language because it creates more emotional resonance than their second or third language. It is also found that swear words in people’s first language contain more emotional weight. Because of this, bilinguals can use second language swear words without violating first language norms. This research examines whether swearing in a second language helps bilingual speakers overcome sociocultural restrictions on swearing in their first language. Bilingual Taiwanese who are fluent in both Mandarin and English were recruited into the study. They were asked to read a combative conversation containing swear words in both Mandarin and English, and then, were interviewed about the emotional impact in reading and swearing in these two languages. In the interviews, participants reported having stronger emotional feelings in hearing first language swear words than second language; however, in the reading experiment, participants reported that their emotional response was similar for both prompts. The interview data confirmed the findings of previous quantitative analysis while adding rich detail about the decision of selecting different language swear words in various situations.

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

The purpose of this research is to generate immersive, natural-looking environments from geoengineering data that can be viewed on hardware-constrained virtual and mixed-reality platforms. Current mixed and virtual-reality technologies, such as the Microsoft HoloLens, are constrained by limited hardware resources. These constraints set limits on the realism and quality of immersion of virtual reality environments. Thus, no software previously existed to intelligently render immersive 3D terrain on hardware-constrained virtual and mixed reality devices. Through research in terrain generation and 3D rendering, the Viewpoint Generator application was created that can accurately and realistically render 3D environments. To render the terrain, 3D environments generated in Unity, a game rendering software, are pre-processed on a host machine. The 3D terrain is sliced into a grid containing individual slices. For each slice of terrain, a set of concentric panoramas and a skybox are generated. These static components require relatively little power to render. Performance was benchmarked through simulations in Unity and on-device with a Microsoft HoloLens, with a minimum frames-per-second (fps) requirement of 60 fps to prevent nausea induced by slow rendering. These components form the Viewpoint Generator application that can be deployed to hardware-constrained devices, which allows users to explore areas of a 3D environment not previously possible on hardware constrained devices. Applications for the Viewpoint Generator include uses in civil engineering and environmental restoration and visualization. Large-scale environmental projects too large to convey through static 3D renderings or by human imagination can now be explored in an accurate and realistic virtual environment. As the Viewpoint Generator is designed to operate on compact devices, it is easily deployed to remote environments. The Viewpoint Generator sets the foundation for development in procedural environment generation based on geoengineering data, allowing new visual insight into civil engineering and environmental restoration projects.

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

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

Patients undergoing ventricular assist device (VAD) therapy are by definition very sick and typically told that they have a high likelihood of dying in the next year without VAD therapy. Though most patients have improved functional status and quality of life, repeated hospitalizations and potentially catastrophic complications post implant can be burdensome. The research aims to assess for decisional regret or ambivalence about VAD implant. During a 3 year program all post VAD implant patients were referred to palliative care. Patients were administered a 5 question decision regret survey that correlates well with decision satisfaction and decisional conflict. On a scale of 0-100, scores >25 were considered to reflect significant ambivalence, and scores between 10-25 were defined as mild ambivalence. Patients were also administered the PHQ-9, a tool for depression inventory. The results found that 102/129 (79%) patients completed decision regret surveys. 64/102 (63%) scored 0, 7/102 (7%) scored >25, and 29/102 (28%) scored between 10-25. The average age of the patients scoring >25 was 44, compared to 55 of the whole cohort. Of those that expressed some degree of ambivalence (n=36), 11/36 (31%) met criteria for at least moderate depression, compared with 18% for the whole cohort. 26% of the total cohort had VADs as bridge to transplant (BTT), while 13/29 (44%) ambivalent patients and 4/7 (57%) very ambivalent patients were living with their VAD as BTT. A majority of patients do not regret their decision; however, a significant minority expressed mild ambivalence, and a small number expressed significant ambivalence. Ambivalence was more common in those who are younger, those with VAD as BTT, and in patients with depression. It is important to recognize pre-implant that patients have different decision-making needs, and that managing their expectations may be a key component of helping them to adjust post VAD.

The Olympia oyster (Ostrea lurida), Washington's only native oyster, was brought to near extinction during the last century due to overharvesting and habitat destruction. Populations have failed to recover naturally leading to a need for active restoration. O. lurida provide ecosystem services including filtering water, providing habitat and food, and increasing the overall diversity of the ecosystem. In order to restore the Olympia oyster to healthy population numbers in Puget Sound, WA, spatial patterns and population connectivity of O. lurida veliger larvae need to be studied to provide efficient and targeted restoration for the species. It can be difficult to determine larval transport patterns due to high larval mortality, small size, difficult identification, and long potential dispersal distances. By following methods of Carson et al. 2010, using trace elemental fingerprinting, it is possible to trace veliger larvae back to predicted natal populations. This is achieved by comparing the chemical signatures of settlers, veligers and brooded larvae using analysis by Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS). During the 10-12 day brooding period, trace metals from larval bay of origin are embedded into the natal shell during early life, leaving a record of past locations in the shell. Analyzing samples collected at natural and restored sites of O. lurida in Puget Sound, WA allow us to compare site and spatial variability. These results will shed light on the dispersal and larval behavior of O. lurida which could inform restoration of native shellfish to create a network of self-sustaining sub-populations.

The North Atlantic right whale (Eubalaena glacialis, NARW) is among the most depleted species of whale existing today, with a current estimated population of only 450. Ship strikes and entanglement issues remain the predominant cause of mortality for this critically endangered species. Thus, understanding NARW distributions, and their overlap with human activities, are a top priority for management and conservation efforts. This study analyzed passive acoustic data collected within the migratory corridor from Cape Hatteras, NC to Brunswick, GA from Oct 2015 - June 2016 as part of a comprehensive seasonal distribution look at NARWs along the Western North Atlantic coast. Four lines of 5-8 Marine Autonomous Recording Units were deployed, stretching across the shelf, in the first deployment of a three year, continuous acoustic monitoring effort. These units were processed using the Low Frequency Detection and Classification System (LFDCS), and detections were screened for daily NARW presence. The results of this study show seasonal movements to and from their calving grounds while passing through Georgia, South Carolina, and North Carolina. Off the coast of Georgia and North Carolina, NARWs are detected primarily on inshore recorders from November to March, suggesting the species take paths closer to shore while migrating. However, in late April, the groups travelling north take a path further away from the Cape Hatteras, NC shore. NARWs were picked up predominantly on inshore recorders throughout all four lines of MARUs, further supporting that critical habitats exist close to the coast in southern US waters. This information provides a better understanding as to where NARWs are spatially and temporally located, which is essential in minimizing human impacts on the species.

I will be doing research alongside Dr. Ronnie Dunn, for the 50th Year Commemoration of the Stokes brothers. Carl Stokes was the first African American mayor of a major US city, and Brother Louis Stokes, was the first African-American congressman in the state of Ohio. The commemoration will focus on the contributions the Stokes brothers made within Greater Cleveland via a public policy perspective. This research will mainly focus on the category of Public Safety Sector, specifically on policing because local/national racial tensions then, mirror those of today. Since these racial tensions have not decreased, this means new policies and changes need to be implemented, thus I question why they have not been implemented. I will look at Mayor Stokes funding proposal titled: “Cleveland: NOW!” and other administrative documents from the early 1960’s. I will be analyzing the impact he had on public safety policies while in office, and use it as a model to study the current climate of public safety. I expect to find that public safety policies and police administration are still being ran how they were fifty years ago in spite of the evolving and ever-changing society. Collectively, this information will be used to help conduct a report with recommendations that policy makers can utilize for enhancing public safety.

For more than three decades, the innovation of cochlear implants (CI) has improved the lives of patients who have trouble hearing sounds. Using the technology of signal processing, the device can provide electric sound to patients at various frequencies. One thing cochlear implant patients have trouble with is the ability to perceive music. Cochlear implants can provide good speech performance but lack the ability to encode music in a better way. Our research is designed to provide the ability for patients to enjoy music through its three facets: rhythm, pitch, and timbre. The purpose of our study is to investigate pitch coding in cochlear implants which is important for music and speech and the quality of life for CI patients. The ultimate goal of our research is to improve sound-processing in deaf or hard-hearing patients all over the world. We will have determined how to deliver the best acoustic simulations of cochlear implants to study pitch coding and identify the ideal combination of carrier type frequency, channel and envelope that will deliver optimal music perception of any sound entering the cochlear implant. Features will be extracted from the simulated sounds to investigate spectral and temporal coding in cochlear implants. This will help in finding new ways of processing music and speech in cochlear implants for the benefit of its users.

We are UW Smart USB Charger Team supervised by Plugable Technology and a UW EE faculty mentor. The main objective of this project is to design and implement a Smart USB charger that has five USB ports. It is called “smart” because the charger will be able to charge the higher priority phones at almost full rate before moving on to lower priority phones. With that function, this USB charger will fully charge several phones much faster than traditional USB chargers having only a 5V 3A power supply. However, there is no such five ports USB charger in the current electronic device market, so we see the important implications of solving this problem. What our team will have done by the day of our Symposium presentation is to implement the priority charge behavior module using C language and an Arduino Mega Board which has a microcontroller inside. The challenges of the task are that the actual charging behavior of different devices is often unpredictable or sometimes disappointing and the devices themselves decide how much current to draw and manufacturers have not settled on a single standard. Therefore, our module should be able to select the best charging mechanism for each of different devices while supporting priority charge. To do this, it requires us to test our module with as many different devices as possible and to collect data for future references. The other goal is to make the product as low-cost as possible: We will use lowest-cost microcontroller with sufficient I/Os to control all ports and lowest-cost external components for port power control. Finally, if more time is allowed, we will try to design and implement a PCB.

Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is the etiological agent of Kaposi’s Sarcoma (KS), a highly vascularized tumor predominantly made up of cells of endothelial origin. KSHV establishes a predominantly latent infection in endothelial cells in culture and in the KS tumor. KSHV latent infection alters cellular metabolism to improve the survival of the infected cells. These metabolic changes resemble a common alteration in cancer cells, termed the Warburg effect. This refers to an increase in glycolysis in the presence of oxygen and a decrease in oxidative phosphorylation. The mechanism of KSHV induction of the Warburg effect is currently unknown. Hypoxia-induced factors (HIFs) are likely candidates for KSHV induction of the Warburg effect as both HIF1α and 2α are induced by KSHV latent infection. To determine if HIF1α or 2α is required for the survival of KSHV infected endothelial cells, I constructed HIF1α or 2α knockouts using the CRISPR/Cas9 gene editing technique in a lentivirus vector. I expressed the endonuclease Cas9 and guide RNAs that lead Cas9 to cut at the HIF1α or 2α genomic sequences leading to genomic mutations in HIF1α and 2α respectively. To determine if HIF1α or 2α is required for the survival of latently infected endothelial cells, I infected the HIF1α and 2α knockout cells with KSHV and looked for cell death at 48 hours after infection. To evaluate if HIFs are required for KSHV induction of the Warburg effect, I will determine if one or both of the knockout cells produce less lactic acid and increase oxygen consumption at 48 hours post KSHV infection as compared to infection of wild type cells indicating the Warburg effect is not being induced by KSHV in the knockout cells. These results will aid in the future efforts to develop antiviral drugs by inhibiting viral latency.

The entirety of this research focuses on a cochlear implant test system. Cochlear is an implantable device which bypasses the middle and inner ear to directly stimulate the nerve system, in order to give patients hearing ability. Current implants are helpful for speech recognition, however, their performance decrease in recognizing pitch in music or speeches with background noises. This proposed test system has the hope to model after the conditions in which the cochlear implants is already implanted in a patient with cochlear impairment. The pitch test system is designed to simulate what a subject with a cochlear implant would hear. A set of sound through a series of filters programmed using MATLAB will be created to perform the simulations.Data will be collected from approximately 5 to 10 normal hearing subjects and 5 cochlear implant subjects. The testing system is intended to evaluate the subject's ability to recognize different controlled sound pitches. A standard deviation will be taken from the data collection versus the standard data to determine the validity of the result. As the outcome will determine the accuracy of the test system and for further construction of an improved system. The result from the tests for both groups will be graphed in MATLAB. The current pitch testing system has been tested on two normal hearing subjects. The subjects response to the test is consistent to what is expected in normal hearing subjects. Further studies will consist of more subjects, therefore, a larger data set will be accumulated allowing for more in depth data analysis and better understanding of cochlear implant patient’s pitch recognition.

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

 Many genetic mutations have little effect on phenotype. One hypothesis to explain this phenomenon is that strong genetic modifiers buffer genetic and environmental variation. Chromatin remodeling proteins are candidate genetic modifiers because they regulate the transcription of many genes, provide greater robustness to environmental changes, and cause pleiotropic genetic diseases when mutated in humans. To test the hypothesis that chromatin remodeling proteins buffer genetic variation, we will comprehensively assess how deletion of one of three chromatin remodeling proteins (Chd1, Hda1, and Cyc8) alters the phenotypic effect of variants of the transcription factor Ste12. Ste12 is a crucial component of the yeast mating pathway, as it activates the transcription of cell-type-specific genes needed for mating. A mutational scanning experiment of the STE12 gene done in the Fields lab yielded data on the effect on mating of thousands of mutations of STE12. These data provide a baseline for similar scans in strains with a chromatin remodeler deletion. First, we constructed double mutants by deleting each chromatin remodeler gene along with a STE12 deletion. We analyzed the growth rate of each double mutant. The chd1Δ ste12Δ and hda1Δ ste12Δ strains showed healthy growth, but the cyc8Δ ste12Δ strain grew poorly and was flocculent. We then assessed the mating ability of chd1Δ ste12Δ and hda1Δ ste12Δ strains containing the wild type STE12 gene, and found that they could mate sufficiently to continue with the deep mutational scan. We transformed both double mutant strains with a library of plasmids encoding the same Ste12 variants as previously studied, and will calculate a mating score of each variant in the two double deletion backgrounds. If the chromatin remodeler deletion alters the effect on mating of a large number of variants, this result would indicate the importance of chromatin remodeling in phenotypic robustness.

Progress in genomics research has led to increasing demands in scientific computing, especially in the field of DNA sequencing. As sequencing technologies have matured over the past decade, the rate of growth in data has outpaced the advancement of sequential processing devices such as CPU-based systems. Field-Programmable Gate Arrays (FPGAs) are reconfigurable computing devices that can be highly customized to suit specific applications, including genomic data processing. In contrast to CPU-based systems, FPGAs are parallel processing devices capable of carrying out computations more efficiently. Additionally, newly emerged memory architectures have greatly improved the random-access performance in genomics applications. One of these is the Hybrid Memory Cube (HMC), whose internal architecture incorporates fast serial communication links and efficient controllers that raise the memory bandwidth limit of the FPGA when compared to traditional DDR DRAM. In our research, we harness the high random-access rate of the HMC and the parallel computing ability of the FPGAs to establish a Bloom filter, an efficient data structure used to confirm membership in a group. The Bloom filter can be utilized to discard erroneous reads generated by normal sequencing techniques and can further relieve the need to account for false information in downstream systems. We constructed a filtering pipeline on the development board that houses a Xilinx Kintex FPGA and the HMC, using only one interface port and 2 GB storage of the HMC. The system is capable of processing 1 billion DNA segments with a maximum length of 64 base pairs. The computing ability of the FPGA along with the high random-access rate of the HMC are highly desirable when processing large amounts of genomic data.

Pacific cod (Gadus macrocephalus) is the target species of a large fishery in North America that ranges from Alaska to California. In a recent genetic sequencing study, very high divergence among Pacific cod from different geographic areas was observed in what appears to be the Zona pellucida sperm-binding protein 3 (ZP3) gene. In many other vertebrates, the ZP3 gene codes for proteins that are significant to reproduction, and this gene may be an important factor in the context of the species' life history and management. To increase resolution of how ZP3 varies across the Pacific cod range, this project investigates the entire coding sequence in Pacific cod from four geographic areas: Kodiak Island, Prince Williams Sound, Hecate Strait, and the Washington Coast using PCR and Sanger sequencing. Differences in amino acids and protein structure were identified. It appears that there are significant differences in the ZP3 gene between southern and northern populations of Pacific cod. Understanding these patterns is important for future studies of how differences in the ZP3 gene may affect reproduction and population connectivity.

The need for energy efficient chemical reactions is growing in response to the world’s energy crisis. Reactions must not only be high yielding but also require low energy input. Many redox processes of interest involve the transfer of electrons from the surfaces of electrodes and need an electrical input as driving force. The ability to promote redox reactions photochemically would greatly reduce the energy input needed. As of yet, interfacing electrochemical reactions with colloidal photosensitizers in order to promote them photochemically has not been done. This project experiments with CdSe quantum dots as the colloidal photosensitizer by absorbing visible light from an LED light source and transferring an electron in order to reduce ferrocenium to ferrocene as a model reaction. The goal of this project is display the ability of CdSe in the productive electron transfer promoted by visible light to ferrocenium. Knowledge of this model system can be applied to more complex reactions in the future and the work done here will help understand how to drive industrial chemical reactions via sunlight. 

The ion exchanger monosodium titanate (MST) has been proposed to therapeutically deliver calcium ions in dental situations that require calcium. However, previous results suggest that MST limits cell growth in vitro by mechanisms that remain obscure. It is critical to understand these mechanisms for any successful therapeutic use of MST. The current study tested the hypothesis that MST limits cell growth in vitro by binding to culture polystyrene, reducing attachments sites available to cells, thereby limiting cell culture growth. MST was added to culture wells (8 concentrations between 0 and 200 mg/L) either 24 h after addition of L929 fibroblasts/OSC2 osteoblasts or prior to cell addition. Cell cultures (n = 8/condition) were left in contact with the MST for 72 h, after which cell growth was assessed using Cell TiterBlue®. Control groups received no MST; statistical differences were detected using ANOVA with Tukey post-hoc tests (α = 0.05). When MST was added after plating of the cells, cell growth was inhibited in a dose-dependent fashion to a maximum of 40% (p < 0.05 compared to no-MST controls). Yet when MST was added prior to the cells, the maximal inhibition of growth was 80% vs. controls. (p < 0.05). The addition of MST before cells significantly decreased growth in the cultures (relative to conditions where cells were established first). Microscopic inspection of the culture wells revealed more attachment of the MST to the culture polystyrene than when cells were added first. These results and observations are consistent with our hypothesis that the MST attaches the polystyrene and reduces the ability of cells to attach and proliferate. Further testing with other cell types and additional cell densities is in progress.

The incorporation of trivalent ytterbium ion (Yb³⁺) impurities, or dopants, into nanoscale materials is of significant interest due to the unique optical properties of the Yb³⁺ ion. The electron configuration of Yb³⁺, 4f¹³, is associated with just one f-f electronic transition (²F_5/2↔²F_7/2) that is narrow and lattice-independent with an energy of ~1.27 eV. This makes luminescent ytterbium doped colloidal semiconductor nanocrystals, which are solution processable, ideal for use in silicon-based solar energy applications like luminescent solar concentrators. Doping colloidal nanocrystals with ytterbium ions, however, is not necessarily sufficient for creating a material with luminescent ytterbium. It is necessary for ytterbium to be sensitized by energy transfer from a visible light absorber in order to be emissive. Previous synthetic work indicates that the most promising route for sensitizing ytterbium is the direct doping of ytterbium ions into visibly absorbing host nanocrystals such as chalcogenide semiconductors. Incorporating ytterbium ions into chalcogenide semiconductor hosts, however, poses a significant synthetic challenge. Additionally, it is essential to optimize synthetic reaction conditions such as growth temperature, growth time, coordinating solvent used, precursor concentration in coordinating solvent, and ytterbium dopant concentration in order to produce a material with luminescent ytterbium. These synthetic variations are informed by physical characterization of the morphology, absorbance, and photoluminescence of previously synthesized ytterbium doped colloidal nanocrystal samples by transmission electron microscopy, absorbance spectroscopy, and photoluminescence spectroscopy, respectively. It is expected that current efforts to optimize these synthetic parameters and interface ytterbium doped colloidal nanocrystals with compatible ionic materials in a process known as ‘shelling’ will allow the reproducible creation of a novel nanoscale material with bright ytterbium luminescence at room temperature. Upcoming fundamental studies of these ytterbium doped colloidal nanocrystals will probe the dynamics of energy transfer by time-resolved photoluminescence spectroscopy and provide insight into the kinetics of ytterbium sensitization.

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

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

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

According to the World Health Organization, an estimated, 780 million people do not have access to clean water sources. Disasters are another potential cause of disruption to the supply of clean water. When the safety of a water source is unknown, a cost effective method to determine water safety is necessary to reduce unsafe water related disease and death. Prior synthetic biology research has been conducted utilizing transgenic yeast as a biosensor for heavy metal detection, such as using plasmids expressing Green Fluorescent Protein (GFP) which are promoted by heavy metal binding transcription factors. While able to detect copper ions in a dose dependent fashion, the relative instability of the plasmid, reporters which are difficult to detect in situ, and limited targeting to copper and silver ions make the efficacy of the designs impractical. Using modern methods of plasmid integration and the modulation of the copper sensitive pCUP1 promoter expression we sought to produce a stable and more sensitive biosensor for the detection of copper ions in water. We successfully integrated the genetic cassette pCUP1-yeGFP into the yeast genome and produced a viable biosensor for the detection of aqueous copper. We tested this yeast strain via a cytometer after exposure to copper of varying concentrations at varied time intervals. The results showed a dose response curve that positively correlated with copper concentration in a semi-linear fashion, which indicated the pCUP1-yeGFP transgene was a viable sensitive reporter for the detection of copper. Currently, variable constitutive promoters are being altered to increase copper detection sensitivity by modulation of the expression of the metal binding transcription factor Ace1. The team is currently exploring the effect of Ace1 upregulation on the sensitivity of copper sensing. After investigating sensitivity modulation, an exploration of practical field detectable reporters, such as violacein, will be undertaken.

It has been well documented in the literature that many children with autism spectrum disorder (ASD) display abnormalities in sensory processing, which is often displayed through hyper- or hyposensitivity to sensory stimuli. Auditory processing difficulties in particular are shown to be the most common. However, there is limited research into the association between abnormal auditory processing with other factors like language and cognitive ability. This study aims to further examine auditory processing difficulties in children with ASD by first looking at the auditory items on the Short Sensory Profile (SSP), a 38-item caregiver questionnaire measuring sensory reactivity. While the SSP is a measure often used in studies to quantify sensory behaviors and has commonly shown atypical behaviors within the Auditory Filtering domain in children with ASD, it should be noted that additional auditory items are also present within other SSP domains. This study examines all of the items on the SSP that have to do with auditory processing, and scores will be compared between two groups: children (nine to ten years old) with ASD and children with other developmental disorders within the same age range. This study also investigates the relationship between SSP auditory scores with receptive and expressive language scores from the Peabody Picture Vocabulary Test and the Expressive Vocabulary Test, as well as cognitive scores from the Differential Ability Scales. Data analysis is ongoing, and it is hoped that the results will help to increase conversation around this topic and the need for further research into the functional impact of sensory processing difficulties in children with ASD. 

Over the past few years’ silicon photonics has proven to be a viable bio-sensing platform for various diagnostic applications. The small size, label free detection and ability to be multiplexed with various assays make it a very attractive technology and potential replacement for many clinical tests. Silicon photonics has proven to be a viable technology for ABO blood typing. This technology has the potential to achieve a higher level of automation than the current clinical method along with a lower run time and lower costs. Two large limitations in clinical translation of this technology are reducing the time it takes to run the assays and the amount of processing that must be done before running them. Indirect blood typing using this technology currently requires centrifuging whole blood to isolate plasma or serum to use in the assay. This sample processing can take anywhere from 20-40 minutes and is not ideal for a clinical setting. This project seeks to increase silicon photonics clinical viability by designing an inline filtration system to isolate the plasma or serum from whole blood as it is running through the assay. This would mean that whole blood would not have to be centrifuged for indirect blood typing. Instead, the blood will be filtered in the assay and then travel through a paper network for reagent delivery recently developed in the Ratner Lab. Additionally, this assay will be developed for the commercial Genalyte silicon photonic test setup. This requires the development of a custom mount for the system along with determining positioning for the paper network. This research may help to replace the agglutination diagnostic test that has been in use for over 100 years and help save medical and monetary resources as well as patient lives.

In the US, someone needs blood every two seconds, making blood transfusion a frequently performed procedure in hospitals. Transfusion of incompatible blood due to clerical error in the clinical blood typing procedure can induce a multitude of life-threatening immune responses in the recipient’s body. The most prevalent typing procedures are based on the overly simplified characterization of surface antigens on an individual’s red blood cells (RBCs) into the commonly known ABO blood type system. However, studies have shown that the antigens embedded in the RBC membrane, comprising of glycolipids and glycoproteins, are structural determinants of variations in the blood type outside of the ABO system, elucidating the limitations of current typing methods. This project follows the hypothesis that gas-phase hydrogen/deuterium exchange (HDX) MS techniques can be employed to characterize and further elaborate the structure of these RBC antigen constituents. To test this hypothesis, genotypically and phenotypically defined donor blood samples are processed to obtain RBC ghosts, containing only the membrane components with the hemoglobin removed through an osmotic lysis procedure. Glycoprotein and lipid fractionation, followed by digestion are employed to isolate carbohydrate components in preparation for HDX-MS analysis, which provides insight into structural composition of the carbohydrate antigens. Through this study, we expect to develop a deeper understanding of the variations in the carbohydrate RBC antigens which dictates the blood type of individuals. This knowledge will be crucial in developing glycobiological sensors that can be functionalized onto silicon chips used by Ratner Lab, facilitating novel blood typing techniques.

Listeria monocytogenes is the causative agent of listeriosis, an infection which causes flu-like symptoms in most individuals after ingesting contaminated food, but can severely harm immunocompromised individuals, newborn children, and the fetus of an infected pregnant woman. L. monocytogenes is a facultative intracellular pathogen which escapes a phagocytic vacuole early in its life cycle. Once in the cytosol of the enterocyte, the master virulence regulator PrfA activates virulence factor production. However, what initiates PrfA and allows L. monocytogenes to sense that it is in the cytosolic environment is still not understood. Recent discoveries have indicated that the translation of at least one of the nine virulence genes, actA, requires several genes involved in bacterial redox regulation, including the protein YjbH. In the Bacillus subtilis model, a homologous YjbH interacts with the protein Spx, an important regulator of thiol-specific oxidative stress. Under normal conditions, YjbH binds to Spx and facilitates its degradation via the ClpXP protease. During disulfide stress, YjbH aggregates, freeing Spx to regulate oxidative stress response genes. Although the B. subtilis and L. monocytogenes YjbH are homologs, the function of the L. monocytogenes YjbH is uncharacterized. My research project seeks to extract and purify the protein. I first generated two strains of E. coli that express a tagged version of either the wild type YjbH or a mutant YjbH which lacks two cysteines that may be involved in its interaction with Spx. The second half of my project will focus on limiting the aggregation of recombinant YjbH. After successful purification of the proteins, these two strains will be a useful tool in characterizing the function of L. monocytogenes YjbH, and its role in the virulence pathway. Understanding the role of YjbH in the virulence pathway could ultimately lead to novel ways to inhibit the pathogenicity of L. monocytogenes.

The Olympia oyster (Ostrea lurida) is the only native oyster along the west coast of the U.S. and has been commercially extinct in Puget Sound since the 1940s. Olympia oysters are a beneficial part of the ecosystem by increasing intertidal species diversity through construction of oyster reefs that provide filtration, habitat, and food. Within the last two decades, they have been the subject of restoration efforts in Washington State. It is important to track the oyster in its larval planktonic stage to aid in restoration of the species because, during this stage, bivalves may travel vast distances of coastline as veliger larvae. To better understand larval transport, plankton samples were collected from two bays in Western Washington during the summer of 2015, Fidalgo Bay to the north, and Dyes Inlet in central Puget Sound. Plankton samples were collected weekly for 5 weeks during ebb and flood tides from two different depths in the water column, one meter from the surface and 0.25m from the bottom of the bay at both locations. O. lurida larval abundance in plankton samples were hand counted and identified using microscopy. The results indicate a high larval abundance in the ebb tide at Fidalgo bay between June 23 and June 30. In Dyes Inlet there is an abundance peak of larvae in the ebb and flood tide on July 9 and larvae were found in both surface and bottom samples at each site. Looking at the densities of oyster larvae in two bays and different tidal conditions can suggest where larvae are dispersing within their natal bays.

The Olympia oyster, Ostrea lurida, benefits the ecosystem for the health of marine environments. Olympia oysters provide natural filtration and restore complex nearshore habitats. Along the estuarine habitats of Washington State, native oyster populations have been declining. Invertebrates reproduce by producing thousands of larvae to ensure survival of a few. Most larvae will not survive due to natural causes such as predation or advection, or human causes such as poor water quality or ocean acidification. The larval stage is the most mobile form of development, and it determines whether they settle away from where they were reproduced or remain in the same area. Larval identification allows for the study of larval behavior and provides data for environmental organizations as to whether restored populations self-sustain or export new offpsring. In this study, we collected samples from Fidalgo Bay, WA from eight stations: five intertidal and three subtidal including three replicates of each sample. Larval samples have been quantified by microscopy and visual identification to describe spatial and temporal distribution of O. lurida larvae. Although the results are pending, data is being collected to find out if larvae will be found in intertidal sites near suitable substrate for adult settlement and adult populations. Results will also indicate if their narrow band as adults is due to their settling everywhere and then die when they get bigger, or if they settle only in their narrow band. If successful, restored populations of Ostrea lurida will attract fish, invertebrates and other marine organisms throughout Fidalgo Bay and therefore prevent these species from going extinct.

Restoration of threatened marine invertebrate species necessitates information on cryptic life history stages, including the distribution and abundance of the crucial larval stage. The larval stage is the most mobile stage of development, and information on their location and migratory tendencies gives insight into whether or not a restored population will be able to persist after initial aid from humans. Traditionally, larval samples have been quantified through visual identification methods that rely on knowledge of shell morphology. More recently there has been a shift to using molecular methods including DNA extraction and quantitative polymerase chain reaction (qPCR) to quantify and identify larvae. QPCR is a time efficient and cost effective way to identify species by specific DNA code, which is copied thousands of times and quantified using fluorescent markers. The results, when placed against known standard samples, can describe the abundance of specific larvae in environmental samples. Limited studies have successfully used qPCR with environmental samples due to potential inhibitors within the sample that can influence amplification. In this study, two methods of DNA extraction were tested on native Olympia oysters for qPCR using environmental samples from Puget Sound. Of the two methods, one was a more expensive DNA extraction and isolation kit while the other was a cheaper, modified DNA extraction method using proteinase-K that did not include DNA isolation. Results indicate issues with inhibition in the proteinase-K modified method that could potentially be due to high sediment collected in the samples. The DNA extraction and isolation kit was proven to work, but at a cost 3 times the amount of the modified proteinase-K method. A fast, accurate method of processing environmental samples such as qPCR is a crucial step for successful restoration efforts of marine invertebrates, but cost must also be taken into account moving forward.

Müller glia (MG) are the predominant glia of the neural retina. In fish and birds, retinal injury causes these glial cells to de-differentiate into retinal progenitor cells (RPCs), and these RPCs repair the damaged tissue by generating new neurons. This process does not happen in mammals, and so retinal damage is permanent. microRNAs (miRNAs, miRs) are small RNA molecules that regulate gene expression. Several miRNAs are expressed in the RPCs, but not in the Müller glia, and we call these RPC-miRs. This study focused on if artificially expressing the RPC-miRs would stimulate mammalian MG to regenerate new neurons in postnatal mouse retinal explants. Two leading questions were explored: Will the Müller glia treated with RPC miRNAs regenerate new neurons? What is the best time window to convert Müller glia into retinal progenitors with high efficiency? Retinas from postnatal (P) 4 up to P12 wild type or transgenic reporter mice were dissected from surrounding eye tissue and electroporated to introduce RPC artificial miRNAs (such as miR-15a, miR-17, miR-19, and miR-20a mimics) or control miRNA mimics, along with a reporter mRNA.  Explants were cultured for 3/4 or 6/7 days ex vitro (DEV), fixed, cross sectioned, stained (immunofluorescent labeling) and analyzed using confocal microscopy. The proliferation marker EdU was added to track cell proliferation. Our data showed that RPC-miRs led to an increased proliferative response in the Müller glia, indicating that the cells were adopting an RPC-like state. Moreover, the age of the explants was an important factor; the areas that were successfully electroporated became smaller in older explants and therefore fewer Müller glia can be targeted in older tissue. Our data shows that miRNAs from RPCs can promote Müller glia to adopt some RPC-like characteristics and therefore could be used to stimulate regeneration in the retina.

Duplex ultrasound scanning with both color Doppler and Doppler spectral waveforms is relied upon for diagnosis of vascular disease and selection of patients for further evaluation and treatment. In most duplex ultrasound applications, classification of disease severity is based primarily on alterations in blood flow velocities. Our research group in the Applied Physics Laboratory is collaborating on the development of a Doppler Ultrasound Simulator for training and assessment of scanning skills. The ultrasound simulator allows learners to repeat examinations as many times as necessary to acquire and improve skills. With the addition of clinical cases covering a range of disease severity, this duplex ultrasound simulator will be a useful tool for training health-care providers in vascular ultrasound applications and for assessing their skills in an objective and quantitative manner. The Doppler Ultrasound Simulator extracts virtual blood flow measurements from computational flow models, with real-time scanning linked to a physical mannequin. The Simulator reproduces the key components of a clinical Doppler ultrasound examination including steering the Doppler beam, selecting the size and location of the Doppler sample volume, specifying the Doppler angle relative to the vessel axis, and adjusting the pulse repetition frequency (scale control) and baseline to avoid aliasing. My research role in this larger-scale project is to use a data processing program, MATLAB, for image reconstruction, processing and analysis. MATALB is used for the creation of 3D image volumes, which are used as the foundation for vessel reconstruction as 3D computer models. MATLAB is also a useful tool for correction of image motion artifacts. My effort in this project involves applying image correcting algorithms to create 3D volumes with enhanced vessel alignment and minimized interference from real-time shifting of 2D ultrasound images.