Alzheimer’s disease (AD) is a debilitating neurodegenerative disease that currently affects >5 million individuals. 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 Adult Changes in Thought (ACT) Study and the Religious Orders Study/Memory and Aging Project (ROS/MAP). These are prospective cohort studies of individuals who are dementia-free at enrollment. 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 domain with a substantial relative impairment; isolated substantial relative impairment in memory, in visuospatial functioning, in language, or in executive functioning, and multiple domains with substantial relative impairments. We evaluated risk factors for these six groups from self-report and/or diagnosis data on vascular conditions, including diabetes, stroke, hypertension, atrial fibrillation, cholesterol levels, and 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. The proportion of individuals in each subgroup differ between the two studies. Our results suggest possible differences in risk for specific cognitive domain deficits associated with stroke history, HDL cholesterol, and treated diabetes. Subsequent work will attempt to understand possible mechanisms behind these associations through literature review. These results support additional efforts to further understand cognitively-defined AD subgroups.

DNA replication is critical to cellular reproduction. In bacterial and eukaryotic cells alike, DNA replication occurs in immobile, discrete complexes (so-called “factories”) enriched for replication proteins. However, the size of these replication complexes is well below the optical diffraction limit (~250 nm), making them difficult to study using conventional microscopy. Using Escherichia coli as a model organism, the goal of my project is to create an ultrastructural model (50-nm resolution) of the bacterial replication complex. To this end, I have developed an expansion microscopy (ExM) protocol that is compatible with bacterial cells. In short, E. coli cells harboring fluorescent fusions to replication proteins are embedded in a swellable polymer gel and physically expanded by a factor of four. The expanded specimen is subsequently visualized at high-resolution using a conventional fluorescence microscope. Unlike more traditional super-resolution methods, ExM is compatible with simultaneous imaging of up to four replication proteins, which I expect will be critical in determining the overall organization of the replication complex. Although ExM is established for eukaryotic cells, I am the first to apply the method to visualize the interiors of bacterial cells. I expect that this project will not only contribute a new super-resolution-microscopy method for bacterial cells, but also reveal the structure of the replication complex with unprecedented detail, perhaps providing insight into the mechanism of the fundamental process of DNA replication. This, in-turn, could lead to new therapies for combating diseases associated with replication defects, including cancer.

Current studies have shown that changing climate is responsible for methane releases from the seafloor, making it important to understand how methane plumes can impact the ocean. Methane plumes impact the stratification and circulation within the water column which can influence primary productivity and seawater chemistry and potentially atmospheric greenhouse gas levels if the methane from the plume undergoes exchange with the atmosphere. Along the Washington margin there has been over 1772 individual bubble plumes located and identified in depths ranging from 40 meters to 1988 meters water depth. Compilation of archived CTD (conductivity, temperature, depth) data at active methane plume sites including Southern Hydrate Ridge, Grays Harbor, and Vancouver Island, can provide estimates of the plume fluids temperature and salinity. Using these estimates, the point where buoyancy reaches zero determines the possible depth of horizontal plume fluid intrusion into the water column and the entrainment coefficient to quantify the fluid that’s entrained by the plume can be calculated., I can determine the plumes’ ability to penetrate through sea water density interfaces such as the thermocline by calculating the Richardson number for the plumes. Using pre-established bubble models, I will also quantify sea water entrainment by the plumes. Through these methods, I will be able to determine how methane plumes impact water column stratification on the Washington Margin.

Merkel cell carcinoma (MCC) is a rare, aggressive, neuroendocrine skin malignancy that is typically radiosensitive. Standard-of-care for loco-regional MCC consists of a wide local excision and sentinel node biopsy, followed by post-operative radiotherapy (PORT) to a total dose of 50 Gy in 25 fractions over 5 weeks. This approach has been shown to reduce the incidence of loco-regional recurrence. In cancers characterized by a rapid growth pattern, delay in starting PORT after surgery is associated with increased loco-regional failures. However, the impact of delayed PORT on recurrence patterns in MCC has not been characterized. We evaluated 152 MCC patients from a prospective registry with the following criteria: diagnosed with loco-regional disease, received definitive surgical resection followed by PORT, treated at the University of Washington Medical Center (UWMC) within 6 months of initial diagnosis, and had at least 6 months of follow up data from the date of surgery. Statistical analyses were conducted in STATA 14.0 using univariate tests to assess associations between variables and survival outcomes. The group was dichotomized according to UWMC recommendations to initiate adjuvant RT no later than 6 weeks after date of definitive excision. Sixty-three patients had short delay to PORT, defined as 6 weeks or less, and 89 patients had a long delay of > 6 weeks (range: 12-419 days). There were no differences between the two groups in regards to age at diagnosis, stage, sex, tumor site, tumor size, and immunosuppression status. Among the short delay and long delay groups, no significant survival differences were observed: loco-regional recurrence-free survival (p=0.69), and MCC-specific survival (p=0.48). These preliminary results suggest that delay for up to 6 weeks does not impact loco-regional control or cancer-specific survival, and may allow for improved wound healing. Further analysis is underway to determine the upper limit of safe PORT delay.

Paper fluidic devices expanded diagnostic medicine to applications beyond lab testing. A key example is the common pregnancy test, which relies on an antibody capture line and detection antibodies conjugated to a colorimetric label to provide a diagnostic readout. This detection method requires high concentrations of target proteins. However, by incorporating isothermal strand displacement amplification (iSDA) and antibody capture of proteins tagged with nucleic acids (rather than colorimetric labels) much smaller protein concentrations can be detected. The Yager Lab has shown that this method increases sensitivity by 10⁴ in comparison with conventional lateral flow methods. Incorporating iSDA into a multistep fluidic device poses the challenge of transporting, delivering, and holding solutions with accurate concentrations of iSDA reagents in an amplification region for thirty minutes. A specific difficulty is delivering the flow of rehydrated iSDA reagents and subsequently stopping fluid movement over the protein capture region. My initial work has been focused on designing and optimizing a device with stopped flow to create ideal amplification conditions evaluated with fluidic dye testing. By using fluorescein and ImageJ analysis to iterate device and membrane design, correct concentrations were delivered to the amplification region. To further the device automation, timers were incorporated so the only user step would be inputting the sample. The device then isolated the target protein, amplified the signal, and provided a diagnostic analysis for the user using a fluorescent probe. Such an advancement can further the reach of higher sensitivity protein diagnostic technologies to low resource settings and for in home testing.

Zika virus has become a major epidemic around the world due to the serious birth defects that it can cause; this has led to an increase in research regarding Zika and how to diagnose the disease. Diagnostic tests require a specific sample type to be tested for the presence of a virus, and because it has been shown that Zika viral levels are more consistent and reliable in whole blood compared to plasma or serum, whole blood is the more efficient sample type. Current strategies for diagnosing Zika fall short because there is limited data on how the virus presents itself in the body, and many of these strategies utilize expensive and time-consuming methods that are not feasible in low-resource settings where Zika is more prevalent. Therefore, there is a need for a low-cost and instrument-free process that can effectively purify Zika virus RNA from whole blood in order to allow for detection of the viral RNA. This project dealt with optimization of an existing lab protocol that required laboratory equipment and resulted in low efficiency. The aim was to develop a robust, high-efficiency, instrument-free RNA purification protocol for whole blood samples. The method of RNA purification consisted of a lysis and binding buffer, followed by RNA purification using silica beads embedded in paper to extract pure RNA from the sample. qRT-PCR was then used to quantify the resulting RNA. So far, the paper-based extraction method recovers approximately 20% of the initial target RNA; however, there are still some techniques that will be employed in the future to help improve this recovery rate. The goal of this project is to increase RNA purification efficiency and to provide an alternative mechanism to purify nucleic acids that can be used in a Zika virus diagnostic test.

Lateral flow tests are assays conducted on porous media, commonly referred to as paper, that can detect and quantify analytes in a sample. They have the potential to return results within minutes and can be used in low-resource or non-medical settings. Protein adsorption is critical in these tests because protein interaction with a media can affect the delivery of reagents or choice of blocking agents to prevent nonspecific binding. A computational model of the fluid and adsorption physics may improve the development of paper-based assays. Yet-to-be-published data from the Yager group evaluates protein adsorption based on the deposition and analysis of protein spots on paper. The computational model used is derived from Darcy’s law, commonly used to model fluid flow in porous media and to calculate volumetric flow rates. This model makes two main assumptions: the porous media is fully saturated, and there is zero outflow on the boundaries of the media. These assumptions are not applicable because the protein spot dries as a result of evaporation, creating a non-zero outflow at the surface of the paper. The saturation level in the paper will also vary therefore Darcy’s law is not an adequate representation of the paper. My project aims to develop a more accurate computational model of fluid and protein absorption dynamic by modeling protein spots on paper. I incorporated an evaporation component that will allow the use of Richards equation to model fluid flow, which applies to unsaturated conditions. This is coupled with a protein transport and adsorption component to achieve a complete model. Although literature on evaporation from sand or soil is abundant, these results cannot be directly applied to evaporation from paper. This model will aid the diagnostic community in better understanding their assays and ultimately help improve the sensitivity and specificity of their assays.

At the 2017 Undergraduate Research Symposium, I presented a low-cost, automated device for influenza detection that was being developed in the Yager lab. After creating, testing, and optimizing this device, we were left with many questions. While certain components of fluid flow in paper are understood, there is much more to learn. In this device alone, three different types of paper were used to run the two-dimensional assay. Current unanswered questions include the following: how do additives affect protein deposition, rehydration, and capture? How much of a dried protein on paper can be rehydrated by a passing solution, and which paper and solution properties affect this value? How do the viscosity and complexity of our patient samples affect the quality of our results? Our overall goal is to determine how to optimize our test line and generate a better understanding for optimization of future diagnostic devices. To make progress towards this goal, my project centers on the development and characterization of a laboratory technique to quantitatively study the effects of these parameters on protein interactions with membrane surfaces and other proteins bound to those membrane surfaces. Improving our understanding of these interactions in membranes will enable better diagnostic device optimization and enable illness detection with lower amounts of infected sample – allowing for earlier detection and better disease treatment. Through the development of this testing apparatus, I have begun to address these questions.

The reduction of drug craving and subsequent relapse is central to the recovery process for individuals with substance use disorders. One of the goals of this project is to improve pharmacological treatment interventions for substance use disorders by better understanding how the neurotransmitter dopamine is involved in relapse. We investigated the relationship between drug craving and dopamine release in the nucleus accumbens core (NAC), an area of the brain that plays an integral role in reward-based learning and addiction, in male rats that have been trained to consume cocaine or alcohol. We tested the effects of L-DOPA, a chemical precursor to dopamine and a treatment that could reduce drug consumption, on craving and relapse-like behavior. Recent evidence from our lab suggests that decreased dopamine release in the NAC correlates with increased cocaine consumption in rats, and that L-DOPA administration attenuates drug intake. Preliminary data from our current project shows a similar effect of L-DOPA on alcohol or cocaine relapse. Our characterization of dopamine release in the NAC during both periods of drug abuse and withdrawal provides new insights for future studies and our use of innovative electrode implants for monitoring dopamine neurotransmission will pilot the technology for other drug self-administration studies. This will help to determine whether L-DOPA should be considered for treatment of cocaine and alcohol addiction in humans.

Influenza is a prevalent infectious disease that claims the lives of 800,000 people globally. Current diagnostic methods include cell culturing, antibody staining, and nucleic acid amplification tests that take from 4 hours to 3 days. The need for rapid, cost-effective and accurate influenza detection tests can be met by lateral flow immunoassays (LFA), which are paper-based test strips similar to pregnancy tests. Most LFAs use colored particles such as gold nanoparticles to indicate the presence or absence of a biomarker by producing a line when the biomarker is present. However, influenza LFAs require over 10 fmol of influenza nucleoproteins to produce a readable signal, which is impractical for influenza because it is present at lower levels at earlier stages of infection. There is a need for a more sensitive influenza LFA that has an improved limit of detection. Here, we investigated how replacing gold nanoparticles with quantum dots, a fluorescent label, improves assay performance when imaged with a mobile phone. We optimized our LFA by varying the concentration and volume of the reagents needed to produce a visible signal. We excited 605 nm quantum dots with a UV LED and used an iPhone SE or Nexus 5X to image the LFAs. Results showed that quantum dot-labeled LFAs imaged with cell phones had a 10x lower-limit of detection than gold nanoparticle-labeled LFAs. Limits of detection with the mobile phones of 1.5-2.6 fmol were comparable to that on a lab-based fluorescence reader, a gel imager (limit of detection of 1.9 fmol). These results suggest that cell phone imaging and fluorescent labels can be combined to make cost-effective LFAs that can be used for rapid and efficient detection in point-of-care settings.

Mitosis is a process important for the regulated division of cells. In cancer cells mitosis is often dysregulated, which can cause cells to split uncontrollably. One important protein that plays a key role during mitosis is the protein kinase Polo-like Kinase 1 (plk1). The function of Plk1 is to regulate centrosome maturation, bipolar spindle assembly, and mitotic entry, making it a very important protein throughout mitosis. To understand how it functions during mitosis we can use drugs that turn off the activity of Plk1 to mimic what occurs when the protein is malfunctioning. Drugs like BI2536, a small-molecule inhibitor that turns off Plk1 activity in mitotic cells, have provided insight into how Plk1 functions; however, these drugs are not selective enough to inhibit protein activity at distinct locations in the cell. To address this problem, we use a technique called SNAP-tag to localize BI2536 to specific organelles within cells. Using genetic engineering approaches like cloning, virus generation and stable cell production, I helped make cells that express a SNAP protein linked to a PACT domain. PACT localizes to the centrosomes in dividing cells and allows us to bring SNAP protein only to that location. Our modified drug enters cells, binds to SNAP-PACT, and selectively inhibits Plk1 activity at the centrosome of cells. Through experiments I have run, we have shown that we are able to inhibit Plk1 activity in vitro and in vivo. Using immunofluorescences and super-resolution microscopy, I have shown that local targeting of CLP-BI2536 to the centrosomes causes a greater reduction in active Plk1 (p-plk1) than traditional global drug delivery. In the future, I hope to use this approach to investigate the activity of another mitotic protein, Aurora A, in order to further explore the role of mitotic kinases during the cell cycle. 

Hosted through the Earth and Space Sciences department, HuskySat-1 is a 3U (30cm x 10cm x 10cm) satellite manifested for launch into low earth orbit in late 2018 or early 2019. HuskySat-1 is primarily an undergraduate research effort, but includes graduate students and industry partnerships. It spans many departments including Earth and Space Sciences, Electrical Engineering, Mechanical Engineering, Aerospace Engineering, Physics, Mathematics, and many others. The mission goals are to establish a space presence for the University of Washington, and to test novel hardware fabricated here at the University of Washington. Of particular interest are the Pulsed Plasma Thruster (PPT), a plasma propulsion device designed and fabricated entirely at the University of Washington in the Advanced Propulsion Labratory, and the K-band communication system, a high-frequency, high-gain antenna capable of transmitting data at megabits per second from orbit to Earth. The satellite also includes several other subsystems including power, attitude control, structures, computers and data handling, and low-gain communication. The mission strives to provide experience in real-life engineering to the entire team, and we achieve this by attempting to build as much of each of these subsystems from scratch as possible. Not only does this provide the most possible experience, but we are also able to grow our mission scope and customize our requirements without the excessive budget attached with many pre-made satellite components. We plan to present on the current and planned state of the mission, what we have learned in the process of designing and building this mission, and our next plans for further space exploration at the University of Washington.

Cell divisions require proper replication and distribution of genetic materials between mother and daughter cells, and mistakes made during the process may result in aneuploidy (abnormal number of chromosomes in a cell) and possible carcinogenesis. Thus, checkpoints exist along the cell cycle to ensure that cellular errors are corrected. Likewise, during the mitotic processes of budding yeast, Saccaromyces cerevisiae, numerous checkpoint mechanisms evolved to prevent catastrophic genomic missegregations. By observing the replicative life span of aging yeasts fluorescently tagged with histone 2B through single cell imaging, we identified a new mechanism that is needed in aging cells for correcting nuclear missegregation. This Retrograde Transport Nuclear (RETRN) pathway fixes genomic missegregation by delaying the incorrect mitotic division and returning the genetic material from the daughter to the mother cell. Following the correction, mother cells could continue to divide and produce healthy daughter cells. In our research, we generated new strains of mutant yeasts that underwent incorrect mitotic divisions to further observe the activation of the RETRN mechanism. Each mutant strain had different non-essential genes relevant to the cell cycle deleted. We confirmed the genetic makeup of each mutant strain through replica plating and PCR. Then, we imaged mother cells from verified mutant strains and observed the budding events throughout their lifespans. This allowed us to see specific points along the lifespan where mitotic divisions occurred, and whether the deletions affected the RETRN pathway. We have speculated that this mechanism is a result of cellular damage due to increasing genomic instability in aging cells, and thus is rarely observed in young, healthy cells. The RETRN pathway could be part of many new pathways needed when cells age and become genomically unstable. Since mammalian cells also become genomically unstable with age, similar mechanisms may be necessary for age-associated genomic instability in multicellular eukaryotes.

Brain-Computer Interface (BCI) research is a rapidly growing area of development in biomedicine. As this neurotechnology continues to be developed it is important to address the following question: what is the most appropriate way to conceptualize BCIs from the ethical point of view? This is important to consider because how we understand the device will shape how it is developed and used in ethical discussions. Potential end-users and ethicists while beginning to investigate BCIs have identified nuanced issues specific to BCIs in the areas of privacy, security, identity and intimacy. To understand these issues physicians, scientists, ethicists and patients are conceptualizing BCIs through analogies drawn from both the medical and consumer realm. This has been shown through a comprehensive lit review and analysis of coded focus group discussions with a spinal cord injury group, a non-disabled group and a mixed group. Analogies to medical and consumer technologies, such as the cardiac pacemaker, wheelchair and iPhone, were most commonly drawn from. Although descriptive, these analogies do not fully encompass the nuanced issues presented by BCIs. Drawing on analogies that do capture these nuances could change the direction of our research, miscommunicate risks to patients or change the way we fundamentally understand brains and computers.