The University of Washington CubeSat program needs a space environment simulator to test their satellite. This was done by simulating the magnetic field and light environments of low-earth orbit. We have accomplished this through the construction of a 3-axis Helmholtz cage and sun simulator. The cage is used as a testing rig for a small satellite's, DubSat-1, attitude determination and control systems. The cage is large enough to fit a 3U cubesat (30cmx10cmx10cm) inside and fully cancel Earth's magnetic field, in addition to generating an arbitrary magnetic field vector of the magnitude expected to be found in low-earth orbit. The sun simulator generates a sun vector over the entire sphere within 10 degrees of accuracy using a strip of LEDs. The LED strip is mounted on the cage and a motor is used to rotate the strip around the cage to change the angle of the light. The Helmholtz cage is controlled using feedback from a magnetometer placed inside the magnetic field of the cage. Based on the data from the magnetometer a control loop is used to control the current through the coils to adjust the magnetic field strength to its desired value.

Satellites require intelligent power distribution systems to manage the loads and sources onboard. The UW CubeSat team required a power distribution system to provide overcurrent protection, current monitoring, and current limiting ranging from 500 mA to 8 A for their satellite. Thus, we researched many different types of current limiting switches and current monitoring methods to find the most reasonable, power efficient, and effective way of designing a power distribution system that would be the best for the satellite. We examined many different solutions to this problem, including off-the-shelf chips, operational amplifier current limiting circuits, and metal-oxide semiconductor field-effect transistor (MOSFET) current limiting circuits. In the end, we found that a chip made by Texas Instruments was the best solution. We then researched and designed a printed circuit board that could be used to test the operation of the chip. After testing the chip, we found that it could limit current in the desired range as well as monitor the current and report it to a microcontroller. With this chip, the microcontroller is also able to turn electric subsystems on and off. This chip design could be used to construct the power distribution system for the cube satellite in years to come.

Influenza is a viral respiratory tract infection responsible for 15-60 million infections and more than 200,000 flu related hospitalizations in the United States annually. Diagnostics can help stop outbreaks and treat infections, however the current flu detection methods lack high sensitivity. The Yager Lab at the University of Washington is developing a simple diagnostic test to identify subtypes of the influenza virus. The assay targets the viral surface protein hemagglutinin (HA) and can specifically detect two strains of the influenza virus, the Solomon Islands strain of H1N1 and the Switzerland strain of H3N2. We have developed an automated device for this assay, supplying the reagents sequentially using a pinched valve and water soluble paper. I have tested and modified this device in order to enable hands free operation once the sample (a nasal swab) is inserted in the system. The paper based assay is fast, specific, and easy to use, allowing rapid diagnosis of influenza in any setting.

Gap Junctions (GJ) are specialized portions of cell membranes that connect adjacent cells and are critical for synchronous electrical signaling in the heart. Therefore, better understanding of GJs can lead to more effective treatment for individuals with arrhythmia. GJs are a collection of channels that pass small molecules and metabolites between cells. They are composed of connexin proteins, the most common being Connexin 43 (Cx43). Kinases phosphorylate Cx43 to control the assembly and disassembly of GJs. Specifically, Protein Kinase C (PKC) and Extracellular Signal-Regulated Kinase (ERK1/2) phosphorylate Cx43 during assembly and disassembly and are activated during cardiac ischemia. PKC phosphorylates amino acid S368 on Cx43, which changes channel permeability, while residues S279/282 can be phosphorylated by ERK1/2 which results in channel closure. Lastly, it is known that S262 is phosphorylated under conditions which lead to GJs disassembly. However, both PKC and ERK1/2 have been implicated as the S262 kinase, in part because typical reagents that activate PKC also result in activation of ERK1/2. My study was designed to clarify which of these kinases phosphorylates S262 by quantifying phosphorylation of Cx43 at S368, and S262 in the presence of PKC and ERK1/2 kinase activators and inhibitors. By examining S262 phosphorylation using different combinations of these reagents, we should be able to discern which kinase phosphorylates S262 or, at least, whether this event correlates with phosphorylation on S368. The data indicated that ERK1/2 is the kinase involved in S262 phosphorylation due to the greatly diminished phosphorylation activity observed when ERK1/2 inhibitors were introduced; and that the PKC activator, TPA, in reality plays a role in stimulating ERK1/2. I now want to find out which phosphorylation event is the most important aspect for gap junction disassembly between MAPK and PKC and perhaps if the two kinases work together.

The Syrian crisis has become the defining humanitarian concern of the early 21st century. With over 400,000 dead and millions displaced, regional and international actors have been overwhelmed by the logistical and political strains of supporting Syrians. Because women and children comprise eighty percent of all refugees and Internally Displaced Persons across Syria, Lebanon, Jordan, Iraq and Turkey, this Task Force elected to focus on the specific needs of this vulnerable population. Our report employs an inter-sectoral approach to identify both short and long-term gaps in women and children's rights and representation, economic opportunities, and their access to healthcare and education in Syria and the four major refugee-hosting countries. Data was collected through a a wide array of sources ranging from government reports to academic articles, the diversity of which enabled the report to look beyond the symptomatic issues facing women and children towards some of the more systemic cultural, political and historical factors which have influenced the conflict and the place of women and children within it. Our report concludes with policy recommendations for short-term relief, as well as the eventual reconstruction of Syria. This report was prepared for the Jackson School's capstone course, and has been evaluated by Ryan Crocker, former U.S Ambassador to Syria, Kuwait, Lebanon, Afghanistan, Pakistan and Iraq.

Current trends in the healthcare industry are focused on providing precision medicine for more individualized patient care. Many researchers are actively designing specialized devices specific to the imaging and care of breasts. However, we are unaware of any comprehensive large-scale studies that analyze population breast sizes that provide design guidelines for such precision medicine machines. In order to fill this knowledge gap, we collected nearly 15,000 mammograms from the Seattle Cancer Care Alliance and built a database of the images and DICOM (Digital Imaging and Communications in Medicine standard) header information. Images which were selected to be analyzed were run through a MATLAB algorithm to extract length and width measurements of each individual breast in the General Electric mammography unit. Around 2,400 images with Cranio-Caudal view, without magnification, and without foreign objects, were run through the MATLAB algorithm. With the measurement results from this algorithm, we studied the statistical distribution of the breast size and shape from this population. The extent of the breasts from the chest wall into the scanner had a mean of 96.19 +/- 29.04 mm , while the width of the breast had a mean of 184.69 +/- 29.00 mm. With the database in place, the next phase of our research was dedicated to analyzing breast density among the population. Women with dense breasts are at higher risk of false negatives in cancer screening and many states require that clinicians notify a woman if she has dense breasts. Nevertheless, there exists the problem of intra-and interobserver variability for judgement of breast density. To address this, some researchers have proposed automated algorithms to quantify breast density. We used our existing database to analyze the variability within these automated algorithms.

Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine skin cancer, which primarily afflicts Caucasian and elderly patients. While MCC patients with intact immune systems have a 20-30% risk of recurrent MCC within 2 years of diagnosis, risk of recurrence or death is higher among immunosuppressed patients. However, it is unknown whether immunosuppression type affects progression-free and disease-specific survival. From a large data repository, we identified and retrospectively evaluated 95 MCC patients that were chronically immunosuppressed at the time of diagnosis and were treated with curative intent between 1988 and 2016. There were five types of chronic immunosuppression represented: chronic lymphocytic leukemia (CLL, 29% of immunosuppressed cases), solid organ transplant (SOT, 23%), autoimmune disorders (AD, 22%), other hematologic malignancies (OHM, 17%), and HIV/AIDS (9%). Progression free survival (PFS) and disease specific survival (DSS) were estimated by the Kaplan-Meier method. As part of curative-intent therapy, the majority of patients were treated with wide local excision (89%) followed by adjuvant radiation (82%). Immunosuppression was associated with poor outcomes among MCC patients: 72% of such MCC patients experienced recurrent disease at a median time of 7.4 months after diagnosis (range, 1-75 months). 45% of recurrences in immunosuppressed patients were distant, while 30% were regional and 25% local/in-transit. 2 yr-PFS and DSS by immunosuppression type was: CLL (PFS 20%/DSS 59%), SOT (0%/32%), AD (14%/52%), OHM (29%/43%), and HIV/AIDS (0%/0%). Immunosuppression is associated with a high risk of recurrence and mortality in MCC patients. While these results are preliminary, they suggest that the type of chronic immunosuppression may affect outcomes in MCC patients.

The World Health Organization reports that seasonal influenza (flu) epidemics result in 3 to 5 million cases of severe illness with upwards of 500,000 deaths. In the United States, 200,000 hospitalizations and 36,000 deaths occur annually with an economic burden of $87 billion. Hence, there is an urgent need to develop highly-sensitive, low-cost diagnostics for use at the point-of-care (POC) to facilitate immediate treatment at the early stages of infection. POC diagnostics can be implemented in paper-based, lateral flow assays to capture influenza nucleoproteins (NPs) with antibodies. Despite their utility, these assays suffer from moderately poor sensitivity. The highest sensitivity measured in a study evaluating six common colloidal gold colorimetric flu tests was 71%. To improve assay sensitivity, fluorescence detection has been demonstrated to detect analyte concentrations 1000-fold lower than with colloidal gold in lateral flow assays. My project aims to develop a fluorescence-based immunoassay for influenza A NPs to achieve improved sensitivities over those of colorimetric tests. The assay is carried out on lateral flow strips, making it readily translatable to point-of-care use. Upon capture of NP by immobilized antibodies, a biotinylated immunoglobulin G (IgG) binds to the NP at a different epitope. Subsequently, streptavidin-bound horseradish peroxidase (HRP) binds to the IgG. HRP oxidizes dihydrorhodamine 6G, a non-fluorescent substrate, into the fluorophore rhodamine 6G (R6G). Hence, an observable fluorescent signal is generated in the presence of NP. In preliminary experiments, R6G adsorbed to the membrane; R6G accumulates near HRP, increasing the signal-to-background ratio. An immunoassay for NP shows observable fluorescence signals at the capture and control regions for their respective positive and negative sample conditions. The remainder of the project aims to optimize reagent concentrations to further improve the assay’s sensitivity. Ultimately, the fluorescence-based assay developed would serve as a translatable platform for highly-sensitive detection of other viral analytes.

The leading cause of mortality in the developing world is infectious disease. Because such diseases are often treatable, the expanded use of accurate and rapid diagnostic devices plays an important role in reducing the burden of these illnesses. Point-of-care (POC) diagnostic devices can be inexpensive, which expands use in low resource settings, and rapid, which expands options for immediate patient care and treatment. The Yager lab designs low-cost, rapid and easy-to-use POC diagnostic devices based on paper microfluidics. These devices perform sample preparation, nucleic acid amplification and visual result detection in sequential, automated steps. As with lateral flow tests (LFTs), these devices have generally employed colorimetric detection, but this method increases test duration compared to fluorescence detection, which can give results in real time. Additionally, we hypothesize that fluorescence detection could provide a semi-quantitative evaluation of pathogen load via competitive thresholding. When a second DNA species is introduced, competition for reagents can generate different chemical sensitivities by varying the concentration of the secondary species. The competitive threshold, which we aim to calibrate by this method, is the point at which a known concentration of competitive targets amplifies in preference to the pathogenic target. We first performed amplification reactions in aqueous solutions with the number of copies of the pathogenic target and the competitor varied across five orders of magnitude. We visualized this calibration matrix as a surface plot, which we used to establish an accurate, order-of-magnitude competitive threshold that did not hinder pathogenic target amplification. Implementation of this method in paper-based amplification reactions will lead to shorter test times and, possibly, semi-quantitative detection in a new family of optics-enabled POC diagnostic devices.

Point-of-care diagnostics are needed to improve the detection of pathogens like the Zika virus. The Zika virus can be transmitted by mosquitoes, or by the exchange of blood or other bodily fluids. The implications of the virus in pregnant women and their children are still largely unknown. So monitoring, and if possible, preventing the spread of the disease is critical. Paper-based diagnostic assays enable complex assays in settings without laboratory assets. Today, sample preparation for molecular testing requires training and expensive machinery. The Yager lab recently created a fully automated multi-material paper-based system that can extract DNA from urine, but had not yet developed an automated system for blood targets. My initial work centered on adapting the paper-based system to enable diluted blood processing. Our updated design successfully flowed ~1.6 mL diluted blood sample through the paper in ~20 minutes. By passing large volumes through the system, low-concentration DNA can be extracted from the sample, removing assay inhibitors at the same time. This work continues the study of fundamentals of fluid movement through porous networks in the Yager lab, enabling a foundation for DNA sample preparation necessary for blood diagnostics. These advances will help detect the Zika virus in blood samples outside the laboratory, contributing to the monitoring and prevention of the disease.

My presentation focuses on research I conducted using immunohistochemistry to identify proteins in insulin stimulated pathways. My research contributed to the lab's ability to analyze p300 and CBP proteins for their use of acetylation. p300 and CBP often are talked about hand in hand as two proteins that function together to induce and carry out acetylation of other proteins. Generally, you can think of them as a messenger, carrying the information from one source to the next in a long line of messangers. Phosphorylation has long been seen as the primary driving force of downstream signaling. Our lab examined the importance and underestimation of acetylation through these two proteins. Initially, the lab used Western Blotting to look for protein presence, but the results we obtained were faint and difficult to analyze. I was assigned the task of trouble-shooting a protocol for the staining of these two proteins. I sliced tissue, plated it, stained it, and calculated the changes between knockout- and wild-type tissue. I was ultimately successful in identifying p300 but statistically unsuccessful in identifying CBP due to a technical error. My research showed a much more reliable way of identifying at least 1 of the proteins rather than Western Blotting techniques. This has a broader implication that there will possibly be more accurate or reliable research done on these p300 and CBP proteins, and acetylation as a primer for downstream pathway messaging done in the future.

Merkel cell carcinoma (MCC) is an aggressive, neuroendocrine skin cancer and one-third of patients develop metastatic disease. Specific sites and timing of distant metastases are largely un-described and, if characterized, could inform staging and surveillance protocols as well as optimize clinical  management. A cohort of 292 patients with metastatic MCC were prospectively followed and then grouped by the site(s) and timing of their initial distant metastases. Initial distant metastases were defined as the first distant metastatic lesions detected clinically and/or radiologically. Presentation of multiple lesions in a single organ site only counted as one initial distant metastasis for that organ site. MCC-specific survival was analyzed using a competing-risks regression, where 200 patients died of MCC. 292 patients had 420 distant metastatic sites, most commonly non-regional lymph nodes (40% of 292 patients), skin / body wall (26%), liver (22%), bone (20%), lung (9%), pancreas (8%), and brain (4%). Patients with only liver lesions (HR 2.1; p=0.02) or patients with lesions in multiple sites (HR 2.7; p<0.01) were over twice as likely to die of MCC than patients with distant metastases limited to the skin / body wall. Additionally, patients whose first distant metastasis occurred >3 years after initial diagnosis were twice as likely to survive MCC than patients whose MCC metastasized within two years of diagnosis (HR 0.5; p=0.02). Site of primary MCC is associated with a distinct pattern of initial distant metastasis, which may help inform surveillance practices. Survival outcomes vary among patients with metastatic MCC and prognosis may be more accurately estimated with stratification by timing and site(s) of initial distant metastasis. Accounting for these patient characteristics will help inform surveillance imaging intervals and define sub-staging for patients diagnosed with metastatic MCC.

Alzheimer’s Disease (AD) is a debilitating neurodegenerative disease that currently affects over 5 million Americans. We have previously demonstrated genetic and neuropathology-based variation across cognitively-defined AD subgroups. Here we sought to determine whether patterns of vascular risk factors differed across cognitively defined subgroups. We used data from the Seattle-based Adult Changes in Thought (ACT) prospective cohort study of individuals age 65+ and dementia-free at enrollment. ACT follows people at 2-year intervals to identify incident dementia and AD. We used cognitive data at the time of AD diagnosis to determine scores for memory, visuospatial abilities, language, and executive functioning. We used these scores to determine each individual’s average cognition and then domain-specific deficits below that individual average. We used these data to define six groups: no prominent domain, memory-, visuospatial-, language-, or executive functioning-prominent. We evaluated risk factors for these six groups from self-reported medical conditions including diabetes, stroke, and hypertension, and diagnosed atrial fibrillation or coronary artery disease. We used multinomial logistic regression models with the no prominent domain group as the reference. To account for multiple comparisons, we present tests of the null that each risk factor is unrelated to each subgroup, and an omnibus test of the association between each risk factor and any subgroup. Of 825 cases, nearly half had no prominent domain, a few had multiple prominent domains, and a single domain was prominent for the remainder. Memory-prominent domain was more common in people with diabetes and executive functioning-prominent was less common, although the omnibus test did not reach statistical significance (p=0.08). Despite low statistical power due to small group sizes, our results suggest possible differences in risk associated with diabetes. Subsequent work will refine these investigations using medications and evaluating glucose levels over time. These results support additional efforts to further understand cognitively-defined AD subgroups

Preliminary work has revealed genetic heterogeneity among Alzheimer’s disease (AD) subtypes defined by cognitive domain-specific impairments. There are currently no reports of risk factor associations for these AD subgroups. We used cognitive data from 825 Adult Changes in Thought (ACT) participants at the time they were identified with probable or possible AD to generate scores for memory, executive functioning/attention, language, and visuospatial ability. We determined individual mean scores across all domains, and identified specific impairments as >0.75 SD below each individual’s mean domain score. Depressive symptom severity was measured with the CES-D, and study staff collected self-reported data on traumatic brain injury (TBI) exposure. We used multinomial logistic regression with the “no prominent domain group” designated as the reference category. We determined risk ratios for depression and TBI for each subgroup vs. the no prominent domain subgroup, and tested significance of any heterogeneity with an omnibus test. We controlled for age, sex, APOE genotype, and years of education. Education's role as a risk factor varied across the five cognitively-defined AD subgroups (omnibus p=0.04). Education predicted development of memory prominent AD rather than AD with no prominent domain (risk ratio [RR] = 1.08 per year of education, 95% confidence interval (CI) 1.00, 1.16, p=0.05; omnibus p=0.04). Depression also varied across AD subgroups (omnibus p = 0.04). Higher CESD scores were associated with lower risk of developing memory prominent AD compared to no prominent domain AD (RR = 0.93 per point on the CESD, 95% CI 0.88, 0.98, p=0.01). We did not find differences in risk associated with TBI. Cognitively-defined AD subtypes show heterogeneity in their associations with depressive symptoms and education. It is possible that these risk factors are associated with biological differences across subgroups.

MSN2 is an environmental stress response transcription factor that is activated in yeast by multiple different stresses—e.g. glucose deprivation, heat, oxidative stress, misfolded proteins among many others—and in turn activates a collection of downstream transcriptional programs. Interestingly, while MSN2 becomes increasingly activated with age, deletion of MSN2 increases the replicative lifespan of the budding yeast. We show that in a glucose-rich environment, MSN2 misreads the environment and drives a pathological glucose starvation response that limits lifespan. Our experiments are facilitated by a novel budget microfluidic/microscopy method rather than the golden standard of microdissection. This innovative system analyzes data through time-lapse images to develop replicative lifespan curves rather than picking off daughter cells with a needle. Microfluidics drastically increases the throughput of experiments and allows for whole-lifespan monitoring of aging yeast cells. The system allows us to efficiently further the research of MSN2 and other transcription factors so that we can potentially translate this to other complex organisms that have similar types of transcription.