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

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

The endothelial to mesenchymal transition (EndMT) has been identified as a key part of organ development as well as many disease pathways. EndMT is characterized by endothelial cells, which make up the inner lining of blood and lymphatic vessels and are adhesive and non-migratory, gaining mesenchymal markers and invasive, migratory behaviors. This overall change in phenotype is normal in embryonic development where EndMT is linked to development of organs but has also been linked to numerous diseases in adults including cerebral malformations, Alport nephropathy, fibrosis, heart disease, and bronchopulmonary dysplasia. Whereas it appears that EndMT does not discriminate by organ, it does by sex. The diseases mentioned previously have a significantly higher incidence in males. To understand the role that sex plays on the EndMT pathway, human neonatal pulmonary cells with a gestational age of 18 to 19 weeks from three female and three male donors were routinely cultured and monitored for changes in phenotype. Using angiogenesis sprouting assays, western blot protein analysis and immunostaining, we collected quantifiable data on the reversibility of the EndMT process in each donor. We found that cells from male donors had lower plasticity, characterized as shifting between the two phenotypes, and generally existed in an endothelial state until pushed into a mesenchymal phenotype through a stressor. Female cells were more likely to shift between phenotypes regardless of conditions and exhibited more angiogenic potential, suggesting a heightened ability to transition between phenotypic states. Future experiments include placing cells in environments with differing stressors to mechanistically determine what drives EndMT processes and monitoring cells with time-lapse imaging to quantify the dynamics of the transition.

Fluctuating temperature patterns due to climate change could negatively impact the survival of species whose sex is determined by environmental cues rather than genetic factors. Species whose sex is determined by ecological cues or social interactions fall under the Environmental Sex Determination (ESD) category, while the Genetic Sex Determination (GSD) category includes species whose sex is determined by genotype at conception. There are numerous published examples of teleost fish species that rely on ecological factors for sex determination, but no studies have explored the extent to which the annual killifish Austrofundulus limnaeus, a species without sex chromosomes, relies on ESD. A. limnaeus inhabit ephemeral ponds in Venezuela, South America that experience extreme daily fluctuating temperatures. Climate change could affect this delicate lifecycle by disrupting seasonal temperature patterns that alter sex ratios or exposure to temperatures beyond their tolerance range. Therefore, it is essential to determine what impact temperature and social interaction may have on sex determination in developing A. limnaeus. To explore ESD, A. limnaeus larvae were exposed to constant temperatures of 20 and 30°C, and a daily fluctuating temperature regime from 20-30°C from hatching until the display of secondary sexual characteristics. Some fish were grown in isolation while others were grown in small groups. Fish grown in isolation at 30°C exhibited female-biased sex ratios with 64% developing as females whereas 69% in 20°C and 61% in fluctuating temperatures exhibited male-biased sex ratios. Ongoing experiments are being conducted to explore the possible interaction between temperature and social cues for determining the sex in this species. A. limnaeus has a relatively higher tolerance to fluctuating temperatures due to their ephemeral pond environment, and thus represents an opportunity to explore how even the most tolerant of species may respond to global climate change, especially in the context of ESD.

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

The nematode C. elegans is a common model organism for investigating cellular aging mechanisms and there is an extensive database of known genes which influence their lifespan. Protein kinases are regulatory enzymes which function in the cell as molecular switches. Here we use RNA interference to disrupt 11 classes of protein kinases to determine their role in 10 different C. elegans mutant backgrounds known to affect aging. With the included controls this involved over 120 individual lifespan experiments. In order to perform this many experiments we used a custom robotics platform called the Wormbot, a novel, high-throughput technique for measuring worm lifespans that our laboratory developed. We present here the results from 120 combinations of longevity mutants and inhibited protein kinases. The findings of this study may identify biochemical pathways or interactions which may play a role in the regulation of human aging or development of age related disease.

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

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

Human alpha defensins, a component of the innate immune system, are small cationic peptides that possess antiviral activity against non-enveloped viruses. The effect of defensins on human adenoviruses (HAdV) is serotype-dependent, infection by some serotypes is enhanced while for others it is neutralized. Enhanced infection correlates with increased cell binding; however, the mechanism of increased binding is unclear. One hypothesis is that defensins mediate receptor-independent binding. Inhibitor studies support this hypothesis, although formal proof is still needed. To test this hypothesis, we used CRISPR/Cas9 lentivirus to knockout the primary receptor, coxsackie adenovirus receptor(CAR), in A549 lung cells. In order to vet these cell lines, they were infected with different HAdV serotypes that use either CAR or an unrelated molecule, sialic acid, as their primary receptors. As expected, the sialic acid-utilizing but not the CAR-utilizing serotype was able to infect the CAR KO A549 cells. We have used these cell lines in combination with integrin co-receptor inhibitors to measure binding and infection of wildtype and mutant adenoviruses in the presence and absence of defensins. These experiments allowed us to determine the extent to which defensin-mediated attachment and entry is receptor-independent.
 

Mouse adenoviruses (MAdV), like human adenoviruses (HAdVs), have specific tissue tropisms. MAdV-1 infects macrophages and vascular endothelial cells, which can result in encephalitis, while MAdV-2 infects epithelial cells of the intestine but does not cause overt disease. The viral protein that determines MAdV tropism is unknown; however, for many viral families it is the viral attachment protein that is critical. For MAdVs, fiber is the viral attachment protein, and the receptors used by MAdV-1 and MAdV-2, although unknown, are distinct. To test whether MAdV receptor usage dictates tissue tropism, I constructed a MAdV-2 chimeric virus, replacing its fiber protein with that of MAdV-1 using a gene-editing recombination system. The chimera was used to infect a 3D culture model of the intestinal epithelium called “enteroids.” As expected, MAdV-1 does not replicate in enteroids and MAdV-2 does, consistent with their in vivo tropisms. Remarkably, the chimera replicated efficiently, indicating that the fiber protein is not the sole determinant of MAdV-2 intestinal tropism. Although fiber is not the main contributor to tropism, its interactions with host factors are still likely important for productive infection. A recent study identified N-acetylglucosamine (GlcNAc) as a specific ligand for MAdV-2 fiber. We have shown that GlcNAc is not the primary receptor for MAdV-2; however, binding to GlcNAc may aid in adhesion of MAdV-2 and penetration through the mouse intestinal mucus layer. To test this hypothesis, I mutated the GlcNAc interacting residues in MAdV-2 fiber to prevent GlcNAc binding. I am currently comparing the infectivity of this mutant virus to wild type MAdV-2 in both epithelial tumor cells and enteroids. Unlike tumor cell cultures, enteroids contain mucus-secreting goblet cells which will recreate the in vivo context more accurately. Together, these studies of MAdV may help us to understand why different HAdVs infect specific tissues.

Mouse adenoviruses (MAdVs) are non-enveloped double stranded DNA viruses. There are two different types of MAdVs with different tropisms or ability to infect particular cells or tissues. MAdV-1 infects macrophages and endothelial cells and causes encephalitis. MAdV-2 infects intestinal cells but causes no disease. Although, the MAdV fiber capsid proteins are important for attachment of the virus to host cells, it is not known if they are the major determinant of tissue tropism in the mouse. To address this question, I use recombination-mediated genetic engineering to make chimeric MAdVs, wherein I keep most of the genome of one strain but replace the fiber protein with that of the other strain. I then use transfection to introduce the DNA of the chimeric virus into a mouse cell line to allow the virus to replicate. I am currently designing and testing the proper chimeric fiber construct that will result in a replication-competent virus. Ultimately, I compare infection of the chimeric virus to that of the parent viruses in intestinal organoids, a tissue culture model that allows us to faithfully test tropism without the need for mouse studies. These experiments may reveal general principles of AdV tropism that will allow us to understand why different human AdVs cause disease in specific organs.

As the only bus that runs from the University District to Mount Baker across Seattle’s Eastside, the 48 provides a unique perspective on accessibility. Through a research paper, and map of the 48’s route I analysed the access to various forms of economic and social capital, such as grocery stores, schools, medical centers, and community centers. Access to forms of economic and social capital were analyzed through their relative proximity to the 48 bus route, or the walking distance from homes in the Central District. The purpose of my project was to start a conversation about the access provided by the 48 bus, and to create a map and record of what is in the quickly changing Central District, as well as Mount Baker and Capitol Hill neighborhoods relative to the 48. All to ask where can the 48 take you? With the Mount Baker Transit Center and University of Washington as end caps, all of the destinations in between mark different issues Seattle faces such as development of single family homes, food deserts, and access to community centers. Focused through the perspective provided by public transportation a new perspective is provided on these issues through the lens of public transportation.

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 small subunit of RNR, 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.

Neurodegenerative diseases, such as Alzheimer’s (AD), Parkinson's, and Huntington’s, affect millions of people. In AD, prior studies indicate the formation and accumulation of amyloid-beta proteins may play a crucial role in the pathology of the disease. The Herpes Simplex Virus (HSV-1) encodes an alkaline nuclease (UL12.5) known to cause degradation of the mitochondrial genome. HSV-1 infection has been previously associated with AD brain pathology. We hypothesize that UL12.5 activity in the brain may predispose an individual to amyloid-beta aggregation and AD neuropathology. Here, we controlledl the amyloid-beta protein aggregation using a degron attached UL12.5, which is induced by the plant hormone auxin through a molecular signaling pathway known as auxin-inducible degron. We have engineered an auxin UL12.5-degron construct in order to precisely control the temporal and cell type expression of UL12.5 in Caenorhabditis elegans (C.elegans). This construct was microinjected into the worms and by using auxin, we controleld the expression of UL12.5 and tested its effects on amyloid-beta and Huntington protein aggregation. Here, we have elucidated the relationship between HSV-1 infection, UL12.5 expression, and neurodegenerative disease which may form the basis of novel treatments.

Mycobacterium tuberculosis (Mtb) is a bacterium that can cause both latent Mtb infection (LTBI) and tuberculosis disease (TB). LTBI affects one quarter of the world’s population of those who are LTBI positive, 5-10% develop TB disease which left untreated can be fatal. Most individuals who are highly exposed to Mtb develop LTBI and show positive results for the tuberculin skin test (TST) or interferon-g release assay (IGRA). The Hawn lab has followed a cohort of Ugandans with heavy exposure to Mtb within the household but test negative by the TST and IGRA tests and define these individuals as Mtb 'resisters'. The presence of these Mtb ‘resister’ genes suggests a possible pathway for host directed therapies. Using transcriptomic profiling to compare the ex vivo Mtb response in LTBI and resister monocytes, several candidate resister genes were identified. We hypothesized that these candidate resister genes modulate antimicrobial functions through an effect on pro-inflammatory cytokine secretion which restricts Mtb growth. Individual ‘resister’ genes were inactivated by CRISPR/Cas9 gene editing in THP-1 cells and then differentiated into macrophage-like cells prior to various stimulations. Macrophage signaling pathways were stimulated with LPS (Toll-like Receptor 4 (TLR4)), PAM3 (TLR2/1) as well as with whole cell lysate from Mtb. I measured pro-inflammatory cytokine secretion in supernatants after 24h (IL-6, IL-1b, and TNF-a). We identified two genes, SETDB1 and NFIL3_03, that may differentially regulate pro-inflammatory cytokines compared to the control THP-1 cells. By uncovering the function of these genes, we hope to better understand how Mtb infections can result in variable immunopathogenesis.