Increasing world energy consumption is accompanied by rising concerns over non-renewable fossil fuel usage and its detrimental environmental impacts. Even if we were to try to meet future energy demands exclusively with fossil fuels, we would fall woefully short of our projected global needs. Consequently, there are compelling economic and environmental reasons that support a dramatic shift in future energy utilization to renewable technologies. The development of solar-driven technologies to generate renewable, transportable, and storable fuel sources is desirable since these energy-dense fuels can be used in the future regardless of the time of day, season, or geographic location. My research explores the utilization of nanomaterials for solar energy-catalyzed biomass decomposition into fuels and valuable, small molecules with diverse industrial applications. Specifically, this work shows the potential of using nanomaterials of a variety of structures to convert lignin (which composes 20-30% of biomass) into more useful small molecule components. To do this, we strive to understand how to make a nanomaterial that both efficiently absorbs light and subsequently uses that absorbed energy to power lignin degradation. The ability of a nanomaterial to absorb light and catalyze this reaction depends on its electronic properties, which is dependent on both its elemental composition and morphology. Our work explores the various catalytic efficiencies of nanomaterial systems of different shapes and sizes.

The President’s Council of Advisors on Science and Technology estimates that the United States will need to increase its number of science, technology, engineering, and mathematics (STEM) professionals by 34% over the next decade. This guidance seems contrary to the rising trend of STEM majors becoming increasingly selective of the students they admit. Competitive degrees with additional requirements for entry (ARE) are a universally accepted phenomenon here at UW. However, there are no studies characterizing their prevalence in higher education or investigating their impact on students. We are conducting an exploratory study to characterize the number of STEM degrees across the U.S. with ARE and the factors departments use to admit students. First, we compiled institutional data for the research universities (R1) and Master’s-granting institutions with the highest number of undergraduates in each state. Next, we determined whether or not they offered STEM degrees with ARE. Programs were identified through a combination of data-mining institutional websites and contacting individuals at each institution. Finally, we administered an online survey to each department to gain deeper insight into the factors used in their admission decisions. We hypothesized that variables such as high school GPA, university GPA, and SAT score will commonly be used in the decisions to admit students. SAT scores and GPA often have significant socioeconomic and demographic biases. Consequently, if we confirm our hypothesis, this could raise questions about the equity of the admission processes for selective degrees. Preliminary results revealed over 250 degrees with ARE. Most of those degrees are offered in biology, health science, or engineering departments at R1 institutions. Ultimately, our results will inform a future study that will take a closer look at the impacts of degrees with ARE on STEM retention and diversity from a representative sample of institutions.

Foliar (leaf) diseases and epiphytes are a major hindrance to photosynthesis throughout the wet tropics. The research sought to determine whether there is a correlation between foliar epiphyte and disease coverage in a relatively small elevational gradient with a hypothesis that a decrease in elevation increases humidity levels which then correlates with greater coverage by foliar diseases and epiphytes on Geonoma oxycarpa. The study took place in three wet pre-montane secondary forest sites in southern Costa Rica, ranging in elevation from 1130.8 – 1188.7 m, and focused on G. oxycarpa due to its accessibility and observable patterns of foliar epiphytes coverage and disease. Three specimens of G. oxycarpa were chosen at each site, and initial estimates of the percentage of foliar epiphytes and diseases were done visually. Photos were then taken for analysis using ImageJ and a metric scale ruler was used to determine the total area of a leaf sample and the healthy portions of the leaf. Weather meters were used to determine the average relative humidity at each of the three sites. All the data was used in linear regression exploratory analysis using the open source software ‘R’. It was determined through clear, though not statistically significant, trends in the data that increased coverage of foliar diseases and epiphytes was related to a decrease in elevation and a corresponding increase in humidity, confirming our hypothesis. These findings indicate that native plants that can tolerate or better defend themselves against the onslaught of foliar epiphytes and diseases should be chosen for the more humid, lower microclimates. Because of this implication, this study is particularly important regarding the preservation of forest microclimates for indigenous cultures that depend upon native tropical plant life for a wide variety of ethnobotanical uses and crucial to the success of native plant restoration and preservation efforts.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi’s Sarcoma (KS), a highly vascularized tumor made up of cells of endothelial origin. KSHV establishes a predominantly latent infection in endothelial cells in culture and in the KS tumor. A previous study has shown that KSHV induction of the Warburg effect is required for the survival of latently infected endothelial cells. The Warburg effect, a common metabolic alteration in cancer cells, refers to an increase in glycolysis and a decrease in oxidative phosphorylation. The mechanism of Warburg induction by KSHV is currently unknown. I proposed to evaluate the role of endothelial cell specific hypoxia-induced factors (HIFs) on KSHV Warburg induction since HIFs have been implicated in Warburg induction in other types of cancer. I hypothesized that HIF2α mediates KSHV Warburg induction through expression of glycolytic genes. To test this, I constructed HIF2α knock-out cells using CRISPR/Cas9 gene editing. I then used RT-qPCR to measure glycolytic gene expression during KSHV infection of wild-type and HIF2α knock-out cells. I found that the transcript levels of certain glycolytic genes remained constant in KSHV-infected HIF2α knock-out cells as compared to KSHV-infected wild-type cells, showing that HIF2α is not responsible for glycolytic gene expression during KSHV infection. I am following up on the role of the KSHV latent gene KapA on the induction of glycolytic gene expression. KapA was previously found to interact with components of the Ras pathway. As the Ras pathway activates glycolytic genes through HIF1α, I hypothesize that exogenous expression of KapA will lead to increased glycolytic gene expression through increased expression of HIF1α. I will construct an endothelial cell line that overexpresses KapA using CRISPR/Cas9 system and then use RT-qPCR to evaluate glycolytic gene expression. These results will aid in the future efforts to develop antiviral drugs by targeting cellular metabolism.

How is star formation affected by its environment? How big is the effect of having differing metallicities during the star forming process? The goal of this research is to study star formation in low metallicity environments, which are well represented by dwarf galaxies. For this project, we used the Atacama Large Millimeter Array (ALMA) telescope, at an angular resolution of about 0.5 arcseconds (about 66 lys), to observe the 12CO (115 GHZ) distribution in the galaxy Henize 2-10 (He 2-10). He 2-10 is an irregular dwarf galaxy about 30 million lightyears (lys) away, about 1 kilolightyears (kly) across, and has a mass of about 10 billion solar masses. It has a high gas to dust ratio, is star forming, and has a low metallicity of about 12+log(O/H)~8.3. Additionally it contains a supermassive black hole candidate of about 1 billion solar masses, which could have significant dynamical interactions with the molecular gas. We created a zeroth moment map which we used to estimate several properties of the galaxy: including a molecular gas mass of about 38 million solar masses, an observable area of about 1850 square klys, and a molecular gas density of about 3.7x10^(-20) grams per cubic centimeter. We also created multiple channel maps which provide preliminary results that indicate the presence of at least 10 giant molecular clouds within He 2-10.  With what we have done so far, we will be able to compare our results to known giant molecular coulds in the Milky Way Galaxy, and begin to better understand the relationship, if any, between star formation and star forming environments. 

My research began with a discovery of manuscripts in the Royal BC Museum in Victoria, British Columbia. These manuscripts were written at Fort Nisqually during the 1840s. A series of journals and ledgers detailing the arrival of Americans into the Oregon Country in 1845 have been missing for over 150 years. They were taken to Victoria, BC in 1865 to provide evidence for the claims of the Hudson’s Bay Company and Puget Sound Agricultural Company in the joint commission hearings between Great Britain and the United States. Through letter books and other writings detailing events around the same time of the missing journals, I am piecing together some of this early 1840s history by working with the curators at the BC Archives. While the journals are yet to be found, my research at the Royal BC Museum has uncovered new leads to follow. The discovery of the journals will reveal farming practices employed by the fort and add detail to what is already known and would answer the unending questions regarding breeds of cattle and sheep arriving at Fort Nisqually, as well as specific cultivars of potatoes, grain, and other food stuffs grown at the fort. The discovery is not the last step in writing Fort Nisqually and the Puget Sound Agricultural Company history. After locating, transcribing and publishing the journals comes the true historiography. These journals hold the keys to a lifetime of research. By uncovering these stories, a box of knowledge will be opened which local historians will dig through for the rest of time. These manuscripts will be the keys to that box, and give future generations a chance to fully understand a history lost for 152 years.

Cas9 has long been used as an effective genome-editing tool for its ability to knock out genes with high efficiency; only in recent years has it been used to activate genes as well. Our current research aims to develop Cas9 as a gene activating tool in zebrafish, and to use that tool to target genes associated with rhabdomyosarcoma, a devastating pediatric cancer, in order to better understand the genetic factors and interactions leading to tumor growth and progression. When a dead form of Cas9 is used (dCas9), it will bind to the specified site without cutting/knocking out the gene. We have developed a gene-activating system using dead Cas9 fused with a transcriptional activator (dCas9-VPR), and are currently optimizing its use with Csy4 in order to activate multiple genes in a pathway or genetic network. We have seen low efficiency success so far by injecting zebrafish with DNA coding for Csy4, dCas9-VPR, and guide RNAs downstream of the ubiquitin promoter. Once the system is optimized, we hope to use it to test interactions between multiple genes simultaneously, which would allow us to determine how interactions between genes impact cell behavior.
This will allow us to gain a more wholistic understanding of how a genetic pathway works.

The Nanoscribe is a high-resolution (500nm) Nano 3D printer that is new to the Washington Nanofabrication Facility. It was acquired through a National Science Foundation (NSF) Major Research Instrumentation (MRI) grant. The tool uses a technology called two-photon polymerization. This involves the use of photoresist, which is a light sensitive material, and an infrared laser that directs brief pulses of light through a microscope objective in different patterns to create 3D objects. The highly focused laser beam allows for dimensions of less than 500 nanometers in X and Y directions to be achieved. One major limitation of the tool is that unlike most photolithography equipment, it is not programmed to do basic alignment. Most fabrication applications require at least two layers of materials to be aligned. For example, when making electronic circuits, there is a top and bottom layer at a minimum, and a vertical layer in between, thereby requiring at least two alignments to the first layer. Our research is focused on understanding and testing test code to add pattern recognition alignment capabilities to the Nanoscribe instrument. For our test application, we applied test aligning microneedles to pre-patterned electrodes fabricated using conventional photolithography. We then 3D printed microneedles that are subsequently used to release drug therapies in test wells via an applied electrical potential (voltage). The alignment process that we developed can be used as a basic construct and capability for other applications being developed on the tool.

Study of the thermodynamic properties of pure water and aqueous salt solutions of NaCl and Na2SO4 were undertaken to determine the equations of state for ices VI and VII at pressures from 1 to 4 GPa and temperatures from room temperature to approximately 300 ℃. The analysis was performed on data along the melting curves of pure water and aqueous solutions of up to 4 mol/kg of Na2SO4 and NaCl as measured within the pressure chamber of a Diamond Anvil Cell (DAC) utilizing ruby fluorescence spectra as a pressure gauge. The data were fit using a local basis function representation, allowing the comparison of the melting surfaces for the two salt solutions over a range of concentrations. Equations estimating evolution of the liquid-ice VI-ice VII triple point with increasing salt concentration, and prediction of the eutectic were enabled from the fit, providing better constraints on structural and geodynamic modelling of icy worlds. Melting point depression with increasing concentration of salt and incorporation of the salt ions into the crystal lattice, and density inversion between the high-pressure ices and saline solutions suggest the possibility of deep oceans of alternating layers of water and ice. Quantification of the effects of aqueous salts on these ices will allow the development of realistic modelling of the hydrosphere of icy worlds and exoplanets. Such modelling can be used to test the possible existence of chemical composition and water/rock dynamics favorable to the existence of habitable zones in these extreme environments.

Speed of sound measurements in four geologically important aqueous solutions (sodium chloride, sodium sulfate, magnesium chloride, and magnesium sulfate) as a function of pressure (0.1 to 700 MPa), temperature (250 K to 360 K), and concentration allow determination of Gibbs Energy. Sound speeds, based on ultrasonic time-of-flight measurements in a temperature-controlled pressure vessel, are integrated as a function of pressure to get densities and heat capacities. Both of these are derivatives of Gibbs Energy, which can be used to find all thermodynamic properties of the solution at a given pressure and temperature. Aside from spending vast amounts of time in-lab to acquire ultrasonic data, my data processing programs have been helpful in assuring our data precision and accuracy throughout the research process. Since icy (ocean) worlds in our solar system (including Europa, Titan, and Enceladus) are believed to contain liquid water within the current range of measurements, this work is useful in constructing geochemical models for planetary evolution and guides astrobiology questions of whether habitable environments exist on these planets.

Accurate thermodynamic properties of aqueous solutions are typically described using arbitrary and custom combinations of global basis functions (GBFs) that are designed to fit specific data sets. This approach is revision-intolerant, since each set of GBFs is fixed to a particular data set, and an entirely new set of basis functions may be required when data sets are expanded. Reusable thermodynamic representations that can accommodate new data for an ever-widening range of conditions can be achieved using multivariate tensor B-splines, a series of smoothly-connecting local basis functions (LBFs) that each represents an arbitrarily narrow regime. However, the current implementation of LBFs for thermodynamic representations requires proprietary software, and widespread acceptance of this new paradigm may be contingent on the availability of easy-to-use, platform-agnostic tools that simplify the dissemination and utilization of such information. I am developing Jupyter notebooks that accept previously developed LBF-based equations of state covering a defined range of pressures, temperatures, and concentrations, and, within the specified regime, return graphical or numerical representations of thermodynamic properties including Gibbs energy and its derivatives including density, specific heat, isothermal and adiabatic bulk moduli, thermal expansivity, and chemical potential. These tools will eventually accommodate both those seeking a simple prediction of state variables and those who seek to expand, reproduce, or refine an existing LBF equation of state. Use by the latter group has the potential to strengthen a model over time by providing straightforward and open-source opportunities for collaborative evolution of thermodynamic representations, whereas the conventional methodology requires highly specialized skills that inhibit such improvement.

Despite the intense coverage of the Syrian Civil War in US media, consensus about what started the Syrian Civil War is highly debated. The variety of explanations cited as leading to the Syrian Civil War range from: corruption, protests, sectarianism, climate change, and drought. My mixed methods research begins by exploring whether certain conflict explanations like the Arab Spring Protests are overrepresented in discourse whereas conflict explanations using climate change are underrepresented. A manifest content analysis of 134 American newspaper articles of causes for the conflict from the New York Times, Washington Post, and Wall Street Journal yielded that they did indeed favor Arab Spring protests but that the results varied by newspaper. The right-leaning Wall Street Journal failed to mention climate change in a statistically significant manner whereas the Washington Post repeatedly mentioned climate change as a major factor for the Syrian Civil War. Interpretations and analysis of the same conflict produced different ideas about what started the conflict. The second part of my research involved an inductive latent content analysis of these news sources to truly understand the perceptions that drove news organizations to report the way they did. Here I report two key findings: a better understanding of how causal factors for conflict are explained in American media and how media organizations justify and convey narratives of importance to them. Such research is important because climate change and conflict is an issue that is important to the stability of the globe. Understanding how and why it’s importance is overrepresented and underrepresented in news sources can help determine how popular geopolitics shapes perceptions and how to make discussions of climate change in the lens of conflict more acceptable.

Situated in the depths of a city’s music scene lies an underbelly of DIY (Do It Yourself) spaces. These spaces operate under the radar of the city’s commercial music scene found in for-profit venues, working to support experimental and small-scale artists who are often excluded from “legitimate” spaces. The DIY mentality developed from a desire to create venues that support musicians and audiences who refuse to adjust to the tastes of large music corporations. These communities host shows in spaces artists can afford, such as living rooms, basements, and warehouses. This project examines the creation and perpetuation of these underground music scenes, focusing on the development of a scene’s community and how identity is performed inside and outside of DIY spaces. This project explores how a city’s built environment (including its available housing, geographic layout, and ease of transport) impacts the way a scene functions and evolves. It teases out how these impacts are felt through observing how a scene’s social norms are performed at events in DIY spaces, examining the styles of music played within a scene, and other related factors. In order to determine how a scene's norms are specifically influenced by the spatial dynamics of a city, this paper examines the underground music scenes of Seattle and Philadelphia, a deliberate contrasting that shows how the differences and similarities in each scene can be attributed to both the cultural identity of the city as well as its unique urban environment. Whereas there are certain norms that arise out of the overarching DIY ideology (meaning they can be found in the scene of each city), there are disparate norms that are endemic to the each city’s scene. These latter norms prove a city’s built environment holds significant weight in the development of its underground scene. Ultimately, this data will provide evidence of the importance of these underground scenes within a city's arts community,advocating for their protection and support both locally and nationally. 

In the fabrication of integrated circuits and microscale devices a technique known as electroplating is often used to deposit thick layers of metals such as gold and copper. The electroplating process requires a conductive layer to facilitate electroplating, commonly referred to as a “seed” layer. In many cases, the seed material will not bond well with the base materials, necessitating the use of an intermediate “adhesion layer” to improve adhesion between the substrate material and the plated metal. In this project, Electron Beam Physical Vapor Deposition (EBPVD) was investigated as a means to deposit a 25nm titanium adhesion layer followed by a 300nm copper seed layer to promote the adhesion of electroplated copper to a silicon dioxide substrate. EBPVD is a process in which a collimated beam of high energy electrons is used to boil metal atoms off of a target and deposit them on a substrate. These systems operate in high vacuum to increase the mean free path of metal atoms ejected from the target surface, allowing for highly anisotropic deposition on the substrate. The stress states of EBPVD deposited seed layers and electroplated wafers were also observed as residual stress can adversely affect the performance of active circuitry that IPDs are bonded to. The deposition of this seed layer, followed by electroplating of copper, is but one step in the fabrication of prototype signal filtering devices for mobile applications. These micrometer scale inductors and capacitors, referred to as “Integrated Passive Devices,” are built directly atop silicon wafers, with the intent of eliminating the need for larger surface mounted signal filtering devices in applications where working volume is at a premium, such as in mobile phones and tablets. We hope our research will allow for production of higher quality prototypes and accelerate development of this emerging technology. 

Etching is one of the critical process steps in the fabrication of micro- and nano-scaled devices. Prior to etching, a pattern, usually defined in a temporary photo-sensitive polymer mask material called photoresist, is transferred onto a silicon wafer. One way to etch material, is through the use of plasma. Inductively Coupled Plasma (ICP), is distinct for its coil that shapes plasma and DC bias coupling for directionality. The machine has high etch rates and is used to etch oxide. Specifically for this project ICP-F (Fluorine) has been used. During etching, a photoresist reticulation problem has occurred. With this reticulation, features on the wafer are degraded, when reticulation is wide spread, the whole wafer must be stripped and restarted. This problem affects the speed and quality of production of wafers. In attempts to fix this problem, different variables such as the DC bias, chuck temperature, He backing, and RF power are monitored. Furthermore, the effects of varying pre-etch conditions, such as wafer bakes and different wait times, are examined to determine if there is a correlation between moisture in the photoresist film and resist reticulation or if the issues are predominately related to the etching system. Different inspection steps are made to ensure the quality of the etch, such as visual inspections and measurements of etch depths. These inspection steps concludes how much reticulation has occurred and what it has affected. ICP-F etching is just one step of the process in of fabrication of micrometer scale inductors and capacitors directly on the top of silicon wafers referred to as Integrated Passive Devices (IPD). This process decreases the amount of space used and increases the bandwidth of radio frequency (RF) analog filtering and the speed of digital information transfers. 

X-ray diffraction gratings are an array of equally spaced structures with defined width designed to diffract optical waves. These waves create a spectrum that allows for the characterization of the wavelength or angles created by each diffraction. X-ray diffraction is commonly used in x-ray crystallography, where x-rays are diffracted off crystal lattices and give information about its three-dimensional structure. The goal of this project is to develop a microfabrication process for high aspect ratio x-ray diffraction gratings. These devices will expand the x-ray diffraction capabilities of the University of Washington and the National Nanotechnology Coordinated Infrastructure (NNCI) at large. In developing this process, our main objectives are to attain smooth vertical sidewalls for the grating features, minimize gold waste in the plating process and to be highly repeatable. For our research, each fabrication step will be tested, troubleshot, and documented for future use. The microfabrication steps required in our process include photolithography, etching, grinding and polishing, wafer bonding, and electrochemistry metal plating. The main challenges of this project are producing a photoresist pattern thick enough to withstand deep etching without affecting the sidewall profile, attaining precise grinding thicknesses and overcoming wafer bonding complications. The work towards maturing each process through inspection and troubleshooting for fabricating high aspect ratio x-ray gratings will be discussed.

Deep Reactive Ion Etching (DRIE) is used for high aspect ratio etches in silicon wafers. The process is effective at achieving deep etches with high anisotropy and selectivity, however more process development is required such that side wall profile angle is selectable based on etch parameters. The goal of this study is to manipulate process variables (including pressure, etch time, and temperature) to allow the production of tapered, reentrant, or vertical side walls as needed. The SPTS-DRIE will be used to etch silicon targets with a repeating array of test patterns using a standard etch and deposition recipe. The standard etch recipe used will cycle between polymer deposition steps and plasma etching steps at low pressure using C4F8 and SF6 gases. Etch depths will be verified using a Bruker DekTak profilometer and sidewall angles will be measured using a Jeol scanning electron microscope. The parameters of the etch recipe will then be adjusted depending on the previous etch profile. Depending on the profile and aspect ratio of the etch, different parameters will be studied, including the process pressure, temperature, gas flow rates, and etch/deposition times. This process will be repeated until results within a reasonable margin of the target are reached. The goal will be to obtain an etch profile with sidewall angles of 90° ± 0.1° for use in industry and to determine parameters for reentrant and tapered profiles.

Oil-spills are a widespread hazardous environmental issue, often caused by mistakes when drilling or transporting crude oil. The effects are devastating to the ecosystem. There are very few ways to quickly clean up an oil spill efficiently, at low cost and without additional environmental damage, as often they are cleaned by using expensive chemical dispersants. We conducted research on an environmentally friendly and inexpensive solution to this problem by using pearl oyster mushroom mycelium. The pearl oyster mushroom has a lignin decomposing basidiomycotina that produces a set of extracellular ligninolytic enzymes that have bioremediation properties. Our hypothesis is that, oyster mushrooms can be used to clean oil spills. We cultivated the mushroom mycelium in straw on trays with different amounts of oil on them. We grew this in a greenhouse with controlled temperature and allowed 4-6 weeks for the mycelium to eat the oil. We are currently observing the results. After 6 weeks, we will filter out the remaining hydrocarbons with a solvent and determine if the mycelium reduced the amount of hydrocarbons or consumed them entirely. Our anticipated results are that the mushroom will clean up the oil in six weeks. For future work, we plan on conducting this same experiment but in a salt water environment to explore applications for mycoremediation in a simulated ocean environment.

 Microbially Induced Calcite Precipitation (MICP), or bio-cementation, is a bio-mediated soil improvement process that can improve the engineering properties of granular soils through the precipitation of calcite on soil particle surfaces and contacts. In the urea hydrolysis driven process, soil microorganisms containing urease enzymes can catalyze a hydrolysis reaction that degrades urea, producing total ammonium, dissolved inorganic carbon, and hydroxide ions. In the presence of sufficient soluble calcium, the resulting carbonate production can supersaturate solutions with respect to calcite and initiate precipitation. The process has shown significant promise as an environmentally-conscious alternative to traditional ground improvement methods, which oftentimes rely on high mechanical energy and energy-intensive materials to improve soils. Despite many recent advances in MICP, environmental concerns regarding the fate of nitrogen by-products have remained largely unaddressed. In this study, soil column experiments were performed to examine the transport, removal, and transformation of nitrogen by-products following MICP. Columns were 7.6-cm in diameter, 17.8-cm long, and contained a poorly-graded sand material. Soil columns received identical treatment solution injections over 14 days targeting a post-treatment calcite content of 4% to 5% by mass. During treatments, non-destructive geophysical measurements were completed to monitor changes in soil stiffness and pore fluid compressibility resulting from carbonate degradation. Aqueous samples were obtained from all columns in time to examine changes in solution chemistry related to microbial urea hydrolysis, calcite precipitation, and ammonium sorption and transport. Following cementation treatments, columns received rinse injections of differing ionic strengths and pH values to investigate the effect of rinse solution chemistry on by-product removal. The results of this study will address critical knowledge gaps currently limiting practical application of MICP technology and will guide future efforts to model the transport and removal of ammonium by-products following bio-cementation.

Oysters support a multi-million-dollar aquaculture industry in Washington State and provide important ecosystem services in estuarine habitats. Sustaining aquaculture and restoring native oysters depends on oyster health. Food availability is a driver of oyster health that interacts with environmental effects of global climate change. These global changes may be altered by the effects of local species interactions like association with eelgrass. Measuring essential fatty acids, which are necessary for survival and physiological processes of oysters, can show the level of food availability to oysters in environments with and without eelgrass. We tested the hypothesis that eelgrass alters oyster health by growing oysters inside and outside of eelgrass at 5 sites in Washington state. We used fatty acid composition of tissue from Pacific and Olympia oysters to look at changes of assimilated food in relation to habitat and environment. We predict that eelgrass will slow water flow, resulting in a decrease in food availability and thus essential fatty acid concentration in oysters grown in eelgrass. Looking at essential fatty acids of oysters from different environments, specifically with or without eelgrass, provides a better understanding of environmental impacts on oyster health and contributes to a collaborative effort in Washington state focused on sustaining the valuable food production and ecosystem services of oysters in our ever-changing climate.

An Inductively Coupled Plasma fluorine (ICP-F) reactor is used to anisotropically etch silicon dioxide with octafluorocyclobutane (C4F8) and argon (Ar) gas. Microelectromechanical system (MEMS) and microelectronics applications require several micron wide features with high-resolution and precision features made on silicon wafers. The ICP-F is a fundamental of the pattern transfer process called a damascene process where a precision pattern is formed and then filled with metal and planarized to remove excess metal and leave the intended pattern. This step of the damascene process consists of lithographic resist patterning and subsequent plasma etching of the underlying material. In this work, C4F8 plasma with Ar additives are used to etch silicon dioxide to create patterns in the oxide that will later be used molds for copper electroplating. In this design, the ICP-F must have a consistent etch rate with high selectivity to achieve constant depth for the copper layer with high resolution patterns. Variables such as DC bias, Ar flow, and O2 flow were optimized to achieve a repeatable etch rate. Furthermore, wafer stress was measured at every step in the process as the varying stress of the IPD could adversely affect the electronic circuits that the IPDs are connected to. Compared to Discrete Surface Mount Devices (SMD), IPDs allow for high-density trench capacitors, Metal-Insulator-Metal Capacitors, and high-Quality inductors which are enabled by ICP-F technology. In addition, new radio frequency (RF) circuits for 5G applications are employing the use of Integrated Passive Devices (IPDs) for making RF filters.

Traditionally, drugs intended for use in the central nervous system (CNS) have lower success rates than drugs used to treat any other part of the body, with very little drug reaching target disease cells in the brain. This limited success is brought about by several physiological barriers, including the blood-brain barrier (BBB), the blood-cerebrospinal fluid barrier, and the highly dynamic and densely packed brain microenvironment. Nanoparticles, if designed properly, have been shown to bypass the BBB and diffuse into the brain extracellular space (ECS), making them a promising option as drug delivery vehicles for CNS diseases moving forward. However, evaluating a nanoparticle’s ability to overcome these barriers both in vivo and ex vivo remains difficult because the brain microenvironment is dynamic, heterogeneous, and variable from brain to brain. To combat this, we aim to develop a gel model of the brain microenvironment that can be used to assess the diffusive ability of nanoparticles in vitro. The model consists of a low concentration (0.4%) agarose gel loaded with proteins found within the brain ECS. To ensure the method used to construct the gels is both controllable and reproducible, characterization of the gels was carried out using a combination of rheological and multiple particle tracking experiments. Rheological experiments determine the gel’s bulk mechanical properties, while multiple particle tracking using probe nanoparticles characterized the gel’s pore size distribution. The insight gained from these characterization techniques allows us to make more informed predictions of how various nanoparticles will diffuse when loaded into these gels.

Rhabdomyosarcoma (RMS) is an aggressive malignant pediatric cancer characterized by pathological skeletal muscle development. Due to a small accumulation of genetic mutations, RMS tumor progression is believed to be driven by epigenetic regulators, which modify chromatin structures to control gene expression. Through gene expression and knockout experiments, our lab identified components of the Switch/Sucrose non-fermentable (SWI/SNF) complex as being potentially involved in RMS growth. SWI/SNF is a family of chromatin remodeling complexes that may function in histone binding and chromatin organization to regulate gene expression. To identify any possible interactions and understand the role SWI/SNF genes in RMS, we developed a large-scale multiplex CRISPR/Cas9 genetic screening system that targets 50 epigenetic regulators, including all the SWI/SNF components. These genes are targeted individually and in every possible two-gene combination. CRISPR, a gene editing technology which uses guide RNA (gRNA) sequences to direct double-stranded cuts in DNA, allows us to knock out genes in cells to gain insight into their function. I have made the CRISPR targeting DNA constructs for the SWI/SNF genes to be included in the library of epigenetic regulators for the genetic screen. We are currently introducing the CRISPR library into human RMS cells to assess the effects of all possible combinations of single-gene and dual gene-knockouts on tumor cell growth. Using this large-scale multiplex genetic interaction screen, we hope to identify genes and gene combinations that are essential to the growth of RMS cells. If two genes have cooperative or redundant function in promoting RMS tumor growth, a dual-gene knockout is expected to result in a more significant reduction in cell growth compared to targeted disruption of either gene alone. The study will provide valuable insight into genetic interactions among key epigenetic regulators in RMS and potentially identify novel therapeutic targets for the treatment of RMS.

Rhabdomyosarcoma (RMS) is a devastating pediatric soft tissue sarcoma. The major RMS subtype, embryonal RMS (ERMS), is often driven by abnormal RAS activity. Activating mutations in the NRAS oncogene, for example, drive cell growth in many types of cancer, including RMS. Unfortunately, there are currently no drugs that can block NRAS activity. Recently developed CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 gene editing technology has become a powerful tool to disrupt gene function in a wide range of organisms and cell types. My research investigates the therapeutic potential of using CRISPR/Cas9 gene editing in RMS to target a cancer-causing mutation in the NRAS oncogene. To target the mutation in NRAS, I used a Gibson molecular cloning strategy to create a DNA construct expressing the SaCas9 coding sequence DNA and two CRISPR gRNAs. One of the gRNAs precisely matches the activating A183T NRAS mutation found in some ERMS tumors. Lentiviruses were produced from these constructs to deliver the gene editing NRAS A183T therapy to ERMS cancer cells. The efficiency for targeted disruption of the NRAS mutation by the CRISPR/Cas9 technology has been confirmed as comparable to general targeting of NRAS, and the mutations produced by this therapy have been profiled through next-generation sequencing to analyze targeting specificity in normal cells. If this therapy is effective in inhibiting RMS tumor growth, this mutation-specific CRISPR/Cas9 targeting strategy can be further developed into a treatment for cancer caused by this mutation. In contrast to conventional chemotherapy treatment, which kills both normal and cancer cells, this CRISPR/Cas9 gene editing therapy could be introduced with the aid of a suitable delivery system into the cancer cells of a patient carrying the NRAS A183T mutation. The treatment would then target and selectively kill NRAS mutant cancer cells, thereby achieving therapeutic results without causing significant side effects.

Viruses are nonliving entities and therefore lack their own metabolism, but they do have the ability to alter the host cell's metabolic pathways for their own gain. Kaposi's Sarcoma-associated herpesvirus (KSHV), a human herpesvirus, is the etiological agent of Kaposi's Sarcoma (KS), the most common tumor in AIDS patients worldwide. In KS tumors and cultured endothelial cells, KSHV is predominantly in the latent state, with only a small portion of viral genes and a microRNA cluster expressed, and no virion assembly. Our lab has shown that KSHV dramatically activates many host cell metabolic pathways during latent infection, including glycolysis. A global metabolomics screen suggested that glycolytic intermediates might be shuttled through the pentose phosphate pathway, (PPP) during KSHV infection. Our hypothesis is that the PPP is important for viral perseverance and the survival of human endothelial cells latently infected with KSHV. Infected endothelial cells were treated with the glucose-6-phosphate dehydrogenase (G6PD) inhibitor 6-aminonictotinamide (6-AN) at different concentrations and the amount of cell death was quantified. The inhibition of the PPP by 6-AN significantly increases cell death in KSHV infected TIME cells, but not in matched uninfected cells. KSHV microRNAs are expressed during latent infection and have been shown to be sufficient to induce glycolysis. Interestingly, a KSHV mutant, ∆miR, which lacks the microRNA cluster is less sensitive to inhibition of the PPP. This suggests that the microRNA cluster may be responsible for the reliance of KSHV infected endothelial cells on the PPP. The data suggest that inhibitors of the PPP provide novel therapeutic agents for KS tumors.

2-deoxy-ATP (dATP) is a nucleotide used in DNA synthesis and its presence has been seen to improve the magnitude and rate of contractions in heart muscle cells. However, levels of dATP are naturally low in mature cells. As an attempt to develop a novel treatment for heart failure, methods to increase the expression of ribonucleotide reductase (RNR), a key enzyme in the production of dATP are being investigated. RNR is regulated by ubiquitin-proteasome degradation of the Rrm2 subunit. We constructed a variant version in which two regions were changed to prevent ubiquitination. This new variant should lead to higher levels of RNR in cardiomyocytes, which also indirectly increases levels of dATP. Our preliminary results show a successful increase in levels of both RNR protein and dATP in cultured neonatal rat cardiomyocytes. Although levels of RNR and dATP were increased, the level present in our cultured samples are much higher than expected for adult rat and mice cells. Therefore, we are currently testing this RNR variant in vitro in cultured adult rat cells, as well as in vivo in aged adult mice. These models are more representative of a therapeutic use. Preliminary results have been promising toward identifying a more effective method of increasing dATP levels for improving cardiac function.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s Sarcoma (KS), a highly vascularized tumor composed of cells of endothelial origin. While KSHV infects both blood (BECs) and lymphatic (LECs) endothelial cells, LECs are more susceptible to infection and express fewer antiviral genes during infection compared to BECs. Recent experiments have shown that LECs, but not BECs, have a defect in STING-mediated signaling, a critical antiviral pathway that is activated during herpesvirus infections. STING induces expression of the antiviral signaling molecule interferon and pro-inflammatory cytokines through the transcription factors IRF3 and NF-kB, respectively. While STING is unable to signal through IRF3 in LECs, it is unknown if NF-kB activation is similarly impaired. Moreover, it is unknown if the defect contributes to the increased susceptibility to KSHV infection in LECs. Accordingly, I hypothesize that in LECs, NF-kB signaling via STING will be impaired and that the STING defect increases susceptibility to infection relative to BECs. To determine if STING activates NF-kB, I will stimulate STING in BECs and LECs and measure expression of NF-kB-activated genes. I expect NF-kB-activated genes to be expressed in BECs but not in LECs, indicating that STING is unable to signal through NF-kB in LECs. To determine if the STING defect renders LECs more susceptible to KSHV infection, I will use CRISPR-Cas9 to knock out STING in BECs. I will then use KSHV to infect the knock-out BECs as well as wild-type BECs and LECs and compare infection rates. I expect the infection rate in the knockouts to be increased relative to wild-type BECs and similar to the infection rate in LECs. The results from these experiments will further elucidate how KSHV exploits defects in innate-immunity to infect and transform host cells.

Researchers often deal with massive longitudinal data sets. Currently-available tools visualize large amounts of longitudinal data but are very rigid or require strict formatting of the data to process it and translate it into visuals. We created an online longitudinal data visualization tool that can be customized by its user in nearly every way. We aimed to visualize trends in sequentially-applied anti-cancer therapies in a cohort of patients from the Fred Hutchinson Cancer Research Center who received cell transplantation for leukemia, but later relapsed. The researchers wanted to visualize multiple disease statuses in parallel to the treatment-and-response trajectories. My objective was to make this tool as customizable as possible, so that it could be used by other researchers with different longitudinal data. I first created a prototype using JavaFX that allowed the user to construct customizable swimmer plots. My design allowed the user to customize visuals such as the colors, shapes, and scale of events in the swimmer plot. To further improve the dynamic features and efficiency of this tool, I approached the developers of Oncoscape, an open source JavaScript software that allows for data visualization and exploratory data analysis, to refine and incorporate my tool into the Oncoscape suite. The current iteration of our tool provides unparalleled flexibility to accommodate any longitudinal data set. It allows researchers to select subsets of subjects based on multiple shared characteristics, allow simultaneous visualization of two or more sequences per subject, and allow researchers to zoom in to a granular level of detail for each subject. This tool will provide doctors a means to quickly study which treatments were administered in which order to similar, historical patients, and their associated outcomes. Thus, by disseminating this tool, we will help doctors make better therapeutic decisions during key moments in an individual patient’s treatment course.