The release of chloride in response to the binding of the neurotransmitter GABA to the ligand-gated ion channel GABAAR mediates neurotransmission that plays critical roles during early development. Disturbed chloride homeostasis in early neuronal circuits results in imbalances in neuronal differentiation, cell growth, and synapse formation that are thought to trigger irreversible paths into neuropathological brain states associated with autism, epilepsy, and schizophrenia. However, the pathogenesis and fundamental physiological mechanisms underlying these disorders are unknown. To dissect these diseases, researchers must understand, in neurodevelopment, the specific role that chloride-mediated neurotransmission plays in triggering the imbalances of inhibitory and excitatory neuronal pathways observed in neuropathological states in adulthood. Zebrafish serve as a powerful model, as the fundamental mechanism of neurotransmission and the microarchitecture of neuronal circuits are preserved among vertebrate. Furthermore, the zebrafish central nervous system is accessible for experimental manipulation from early development. I aim to precisely control chloride-mediated neurotransmission during early development through the creation of versatile transgenic zebrafish lines in which GABA-producing (GABAergic) and glutamate-producing (glutamatergic) neuronal subtypes express the light-activated chloride channel iC++. We are currently designing DNA constructs encoding iC++ and UAS, a promoter element that binds to the yeast transcriptional factor Gal4, to be injected into embryonic wild-type zebrafish. Upon successful integration of these transgenes, Gal4 driver lines that express the transcription factor in specific neuronal subtypes such as GABAergic (Gal4:gad1b) and glutamatergic (Gal4:vglut2a) cells will be crossed with our UAS response line to achieve our desired expression. Ultimately, I will non-invasively activate iC++ in live animals through light stimulations to drive chloride signals with millisecond precision at any time throughout the embryonic and larval stages. This approach will serve as a high-throughput system, allowing us to collectively study a large number of conditions that trigger permanent neuropathological impairments.

Methodological advances have made it possible to generate predictions of brain activity using functional magnetic resonance imaging (fMRI) for cognitive architectures, such as Adaptive Control of Thought—Rational (ACT-R), thus expanding the range of model predictions and making it possible to distinguish between alternative models that produce otherwise identical behavioral patterns. However, for tasks associated with relatively brief response times, fMRI predictions are often not sufficient to compare alternative models. Presently, we outline a method based on effective connectivity, which significantly augments the amount of information that can be extracted from fMRI data to distinguish between models. We show the application of this method in the case of two competing ACT-R models of the Stroop task. Although the models make, predictably, identical behavioral and fMRI predictions, patterns of functional connectivity favor one model over the other. Finally, we show that the same data indicates, provided reasonable task constraints, that we may be able to automatically generate modifications to our models using measures of effective connectivity, further increasing the validity of our models and opening new avenues for model development. We see this an exciting opportunity for future research.

Inflammasomes are multimeric protein complexes involved in innate immune responses. Inflammasomes include a protein sensor, such as NLRP3 or pyrin, linked to the enzyme caspase-1 via the adaptor protein, ASC. Active caspase-1 is responsible for release of the cytokine interleukin (IL)-1ß and triggering inflammatory cell death. Inflammasomes are crucial in defense against pathogens. However, excess inflammasome activation is linked to diseases such as Alzheimer's, atherosclerosis, and other inflammatory conditions. Although inflammasomes are linked to several diseases, we do not fully understand how inflammasomes are activated. We are researching the role that potassium plays in inflammasome activation. To detect inflammasome activation, I measured released IL-1ß using enzyme-linked immunosorbent assays (ELISAs). I determined whether inflammasome activation requires potassium efflux by measuring IL-1ß released from cells stimulated in high extracellular potassium, which prevents potassium efflux. I found that IL-1ß release triggered by NLRP3 inflammasome activators is prevented when cells are stimulated in high extracellular potassium. However, IL-1ß release triggered by the pyrin inflammasome was not affected by high extracellular potassium. From these results, we conclude that the NLRP3 inflammasome is dependent on potassium efflux from the cell, whereas the pryin inflammasome is not. The implications of our research are two-fold. First, our findings argue against a long-standing hypothesis that high extracellular potassium blocks ASC binding. Both NLRP3 and pyrin need ASC, but our data show that only NLRP3 is affected by potassium concentration. This suggests that potassium affects NLRP3 activation at an unknown point. Second, understanding the role of potassium in regulating inflammasome activity provides a potential therapeutic target. There are drugs that regulate ion concentrations by controlling ion channel activity. Knowing whether an inflammasome pathway is potassium efflux dependent could be beneficial in limiting excess inflammasome activation that is linked to a variety of human diseases.

With regards to most tissues, humans lack the ability to regenerate, instead scarring in response to injury. This often leads to poor patient outcomes, especially in the event of spinal cord damage. Xenopus tropicalis are capable of avoiding this scarring response as tadpoles but not as adults. They instead fully regenerate tail tissue and are thus an excellent model system for the investigation of how regenerative and non-regenerative organisms differ in their response to injury. Due to the complexity of this process, many transcription factors have been implicated to have a role in regeneration, though the precise roles of many such transcription factors remain unknown. Here, we focus on the transcription factor Hif1α, which is canonically involved in responses to hypoxia and oxidative stress. Using an Assay for Transposase-Accessible Chromatin (ATAC-Seq) we have found that over the course of regeneration, Hif1α response elements (HREs) increase in accessibility. To understand the role of Hif1α during regeneration, I have used echinomycin, a small molecule known to inhibit binding of Hif1α to HREs. Tadpoles treated with echinomycin fail to regenerate, indicating the necessity of Hif1α in regeneration. In order to determine the effects of Hif1α on gene expression, I have queried several genes known to be differentially expressed during regeneration through the use of quantitative polymerase chain reaction. I have shown that inhibition of Hif1α transcriptional activity via echinomycin significantly alters Wnt target gene expression, indicating that Hif1α regulates Wnt target genes. This provides an improved understanding of the regulatory processes that enable regeneration.

Reactive oxygen species (ROS) such as the superoxide anion (O₂-) and hydrogen peroxide (H₂O₂) are produced by enzymes such as NADPH oxidases (NOX), which have been identified in most cell types and tissues. Additionally, ROS is identified as a second messenger which regulates the activity of G-protein-coupled receptors. For example, ROS decreases the sensitivity of the kappa-opioid receptor (KOR), a signaling pathway in the brain involved in drug addiction. Thus, a fluorescent ROS sensor protein would allow us to monitor KOR signaling in animal models of drug addiction. However, the best performing ROS sensor called HyperRed is not suitable for in vivo studies due to the weak amplitude of fluorescent signal output. Therefore, the overall goal of the project is to increase the magnitude of fluorescent change of HyperRed, ease the detection of low H₂O₂ concentration, and decrease the time of fluorescent response after the addition of H₂O₂. First, using the structure-based engineering approach with the help of Dr Berndt, I construct a library of thousands of semi-randomized HyperRed variants, where mutations are only introduced to the amino-acid linkers between the main domains. Then I test the fluorescent output of all the variants in the library at high throughput utilizing a novel protein engineering platform. This approach allows me to rapidly identify the best performing ROS sensors and optimize their signal amplitude in several rounds of repeated mutagenesis mimicking an accelerated evolutionary process. The expected result is to detect one variant with the signal amplitude having at least a 100% change of fluorescence over baseline values which has been determined to be the minimum for applications in animal models. The improved sensor enables direct measurements of ROS signaling during drug-seeking behaviors and could potentially facilitate the development of novel therapeutic approaches.

Suspended sediment in the bottom boundary layer impacts both ecosystems and geomorphology. High concentrations of suspended sediment affect light attenuation, harming benthic plants, and sediment resuspension and transport can affect the distribution and size of sediment on the seafloor. The purpose of this project was to determine the variations and controls on suspended sediment in the Elwha River nearshore region and to find relationships between bed shear velocity and suspended sediment concentration. The 2011 Elwha River dam removal released a large pulse of sediment, giving us the opportunity to study a coastal environment with fine sediment deposits, and varying hydrodynamic conditions. Data collection measured near-bed turbidity and wave conditions, and sediment grab samples were collected to characterize bed conditions. Harmonic tidal analysis was used to predict tidal current velocity. Over the sampling period, on the east side of the river mouth, currents ranging from ~0 to 100 cm/s and wave heights up to 1.0 m were sufficient to resuspend sediment. Suspended sediment concentrations generally ranged from 1.5 to 25 mg/L. In Freshwater Bay, currents ranging from ~0 to 31 cm/s and wave heights up to 0.86 m were not sufficient to resuspend sediment. Instead, fine sediment settled out of the water column, resulting in near-bed sediment concentrations generally ranging from 1 mg/L to 101 mg/L. These findings show how variable the processes controlling sediment in suspension can be in a tidal environment with complex morphology.

Mapping is a powerful tool to visualize space and data. However, working with historical data and maps is problematic as spaces change across time. Given this, the scope of this research is to create visual maps using GIS software that accurately represents the Baghdad-Basrah region of the Ottoman Empire in the late nineteenth century. Our key research question is: how can cholera data from primary sources in the nineteenth century be visualized through mapping? We gathered the data for this research from the diaries of Joseph Svoboda, a resident of the region who traveled frequently up and down the river system and recorded information about numbers of deaths and quarantines from cholera in the cities he passed. The method we used for mapping the data includes a comprehensive reading of the diaries currently being transcribed to assess which information is important for mapping purposes, including the location and time period of the event in question. We mapped the data using Geographic Information Systems (GIS), a software that is used to analyze and represent data through maps, to geocode locations and include cities and other features as they appeared in the nineteenth century. Anticipated results include an interactive choropleth and dot maps that are accurate to the specific time period and show deaths from cholera in specific locations over time. One key implication for further work would be a standardized way to map disease data from primary sources and to increase the number of individuals who use data visualization with historical data. Additionally, our mapped data can be used for other types of GIS analysis that may be of interest to those studying the history of cholera or historic epidemics.

Nearly 22 million HIV-positive people are receiving antiretroviral therapy in order to suppress their HIV infections. They need consistent viral load monitoring to track viral suppression and detect the possibility of viral rebound. Nucleic acid amplification tests (NAATs) are used to measure the viral load in a patient's blood. Traditional, laboratory-based NAATs require complex robotic systems to automate HIV RNA purification, amplification, and detection from blood. Since the majority of those living with HIV are located in low and middle income countries, there is a need for rapid viral load monitoring at the point of care (POC). We aim to provide accessible HIV viral load testing through low-cost, integrated POC NAAT devices. These proof-of-concept devices operate as a two-step assay to extract and detect nucliec acids in blood. An electrophoretic separation technique called isotachophoresis (ITP) separates HIV RNA from other components in a blood sample. An isothermal nucleic acid amplification assay amplifies the purified, concentrated nucleic acids in order to detect and quantify their presence. We present our development of a novel ITP system to remove potent contaminants from Proteinase K (PK) digested serum and extract highly pure nucleic acids automatically. Through computational modelling, a dual trailing electrolyte (TE) buffer system was designed to exploit the isoelectric point of PK for its removal, while simultaneously concentrating nucleic acids away from serum components. We demonstrate system control through comparison of experimental observations to model predictions by performing dual-TE ITP on pH paper. We also show that the dual-TE system improves upon previous limits of detection for DNA extraction and detection from complex samples. Our system processes 40 microliters of blood in 20 minutes using only simple buffers, a paper strip and an electric field - making it an ideal tool for use in a rapid NAAT for HIV viral load testing. 

The environment in which a molecule resides can drastically affect both its reactivity and its spectroscopic properties. These effects can be clearly seen in phenomena such as solvochromism or increased reactivity in a protein active site. The most accurate models that can capture these effects are purely quantum mechanical, but they also have a steep computational scaling cost that prohibits their use on large molecular systems. In order to circumvent this, less expensive models, such as molecular mechanics, can be used for the environment while maintaining the accurate model for the system of interest. In recent years, development of polarizable molecular mechanics has enhanced the accuracy of this technique for time-independent systems, but a gap still exists for time-dependent systems. In particular, the frequency dependence of polarizability has yet to be addressed. This research uses frequency dependent polarizabilities to parameterize a novel molecular mechanics scheme. Specifically, a machine learning model is trained to predict the best locations to place isotropic polarizabilities in order to recover the molecular, frequency-dependent polarizability.

Humans hear sound through two main pathways: air conduction (AC) and bone conduction (BC). The AC pathway is dominant under normal listening conditions, but conductive hearing loss (e.g. outer/middle ear diseases) impairs one’s ability to hear by AC. BC hearing aids can improve hearing under such conditions. To determine the presence of a conductive hearing loss, BC hearing thresholds must be compared to AC thresholds. For populations that cannot respond reliably to behavioral audiometric testing (e.g. infants, elderly, individuals with cognitive impairments), clinicians instead record sound-evoked brainstem activity through the auditory brainstem response (ABR) test. While AC ABR test results are clinically reliable, BC ABR test results suffer from electrical artefacts generated by the BC transducer and variable ABR waveform morphology. Therefore, determining the presence of a conductive hearing loss by comparing AC and BC ABRs is often unreliable. This study aimed to (1) reduce the severity of electrical artefacts present in the early BC ABR waveform (Wave I/II) by shielding bone-vibrating transducers (Radioear B81) with MuMETAL and (2) improve the quality of the ABR signal by using ‘chirp’ stimuli (ascending frequency sweep) to enhance the summating amplitude of auditory neural responses. Ongoing measurements leverage three transducer types in total (shielded and unshielded BC, earphone AC) and three stimulus types (chirps, constant-frequency tone bursts, and broadband “clicks”) to evaluate main and combinatorial effects of the transducer and stimulus on BC ABR data quality. Early indications show that shielding the BC transducer does not materially change its frequency response. Therefore, any observed changes in the BC ABR waveform can be attributed to electrical shielding and/or stimulus modifications per se and not to unintended changes in transducer acoustic output. Data will inform efforts to improve the clinical reliability and utility of BC ABR measures for detection and treatment of conductive hearing loss.

Electronic movement can be modeled using the Schrodinger equation. For many-electron systems, however, these equations are not algebraically solvable, which is why this research uses direct numerical integration of Schrodinger’s equation to model time dependent electronic characteristics of molecules. The choice of numerical integration scheme can drastically change the outcomes of the simulation – even causing qualitative changes in molecular behavior. First, an advanced numerical integration technique (4th order Magnus propagator) has been implemented in the ChronusQuantum software package. Next, the effects on molecular behavior of choosing this integration scheme over preexisting schemes are evaluated. The implemented integration algorithms can be used to model the elementary steps of chemical reactions, as well as analyzing the effects of light on electron movement. The research provides meaningful information about the impacts of different implementations of algorithms along with analyzing the simulated behavior of electrons.

Larval tadpoles of the frog Xenopus tropicalis exhibit a natural ability to regenerate multiple tissue types in response to injury. Unlike tadpoles, humans are incapable of regenerating a majority of their major organs and tissues following traumatic injury, often resulting in an irreversible loss of function of the affected tissues. While both non-regenerative and regenerative organisms undergo a period of wound healing in response to injury, the former then undergo scarring, whereas regenerative systems forgo scarring and ultimately regenerate the lost or damaged tissue. After tail amputation, reactivation of the cell cycle in the remaining tissue is required to promote cell proliferation in order to create the cells that will populate the regenerated tail. However, the molecular mechanisms that enable naturally occurring regeneration are not entirely understood. In order to better understand how wound healing promotes regeneration in tadpoles, I used immunofluorescent microscopy of Phospho-histone 3 (PH3) to assess the mitotic activity of Xenopus tropicalis tails during early regeneration. Over the first two hours post tail amputation, image analysis of PH3-positive cells shows that the amount and localization of mitotic activity varies greatly in the remaining tail tissue. Specifically, tissues adjacent to the amputation site transiently experience a dramatic decrease in mitotic activity beginning at 45 minutes post amputation (mpa), followed by the return of mitotic activity to these areas after 75mpa. I hypothesize that cell cycle inhibition during this 30 minute window is an important point of regulation during the regenerative process and may be a critical component of setting up a regenerative response to traumatic injury. Identifying mechanisms that enable regeneration will be critical for the development of clinical therapies that promote regeneration in humans

This research explores 19th century Middle Eastern and European quarantine practices through the personal diaries of Joseph Mattias Svoboda, written in Iraq from 1862 to 1908. Joseph Mattias Svoboda’s diaries document a detailed account of daily life and information in the Basra and Baghdad regions of modern day Iraq. Joseph himself was from a prominent European family and worked for an English steamship company, traveling across Iraq during a time when cholera was prevalent and occasionally reached epidemic proportions. I used Joseph Svoboda’s notes as a basis to piece together common quarantine practices and procedures utilized in the 19th century by the Ottoman Empire. Using digital transcriptions of the diaries, I used data-mining techniques to extract references to cholera outbreaks, quarantine procedures, and numbers of people afflicted by disease before and after quarantines were put in place. Then, using contemporary European newspapers, medical journals, and historical records, I compared the Ottoman procedures to European quarantine practices. I examined the efficacy of quarantine procedures in both regions depending on the size of each given cholera outbreak. Ultimately, these early forms of public health governance may have influenced global trade patterns. Future projects stemming from this research could look into the influence of European public health practices on Ottoman medical thought, or the comparative role of physicians in the Ottoman Empire and in 19th century Europe. Regardless, hopefully this research encourages others to explore beyond the Eurocentric narrative of developing public health in the 19th and 20th centuries.

The Svoboda Diaries Project works with personal diaries written at the turn of the 19th century, capturing over 40 years of the life, politics, and landscape of Ottoman Iraq. Written through the unique lens of a British steamship purser with a rich family history and connections in the area, these texts provide a unique insight into a locale on which there exists minimal literature for this time period. Undergraduate interns transcribe these diaries and develop open-source tools to make the texts available in a variety of formats. We are currently redesigning our website to better serve the needs of its various users, and our main question is: How do we realize the needs of prospective users when creating a digital platform for viewing historical manuscripts? Our current website is not sufficient in meeting the needs of the project’s diverse users: historians/researchers, contributors, and the general public. Therefore we intend to create an engaging and interactive user interface. At its core, the Svoboda Diaries comprise a personal narrative. We also intend to infuse a storytelling approach to present these unique documents in a larger political and historical context, and allow the user to explore them in different ways. We utilize a variety of user-centered research and design methods, such as conducting user interviews with domain experts and other interested individuals, creating prototypes, and conducting pilot usability sessions to refine the website.We anticipate that our website redesign will allow users greater access to explore the diaries. In addition, the redesign will draw attention to the most important aspect of the website: the diaries themselves, and the fascinating and valuable accounts within them.

In humans, limb amputation and recovery post-amputation is characterized by inflammation and scarring that lead to poor clinical outcomes. In contrast, amphibians such as the frog Xenopus tropicalis are capable of healing scarlessly and can fully regenerate previously amputated appendages. Successful limb regeneration depends on precisely choreographed expression of genes, directed in part by the deposition and removal of epigenetic markers. The broad aim of this research is to identify the spatiotemporal dynamics of epigenetic modifications and how they play a role in regulating gene expression during regeneration. It is known that histone deacetylases (HDACs) and H3K27-specific methyltransferase EZH2 enzymes limit chromatin accessibility and are necessary for regeneration to occur properly. However, the precise mechanisms and genomic targets of these enzymes remain unknown. We hypothesize that inhibiting these enzymes will leave chromatin in a constitutively accessible state, disrupting the gene expression required for successful regeneration. I am utilizing the drugs Trichostatin A (TSA) and DZNep to inhibit HDACs and EZH2 respectively at differing sequential time points throughout tail regeneration. In addition to characterizing the morphological outcome of regenerating tails that have been treated with these drugs at varying intervals post-amputation, I  also use immunofluorescence to identify the targeted location relative to the injury site and tissue types as they are affected across time. For humans and other mammals with limited regenerative capability, studying these epigenetic changes and their impact on Xenopus tropicalis tadpole tail regeneration is especially significant: it has the potential to determine how changes in gene regulation may enable and facilitate a broader capacity for limb regeneration by informing future therapeutic possibilities.

Joseph Mathia Svoboda, a European residing in Baghdad and working as a steamship officer, kept a detailed daily diary from 1861 to 1908, in which he recorded his trips on the Tigris, family events, and his medical and financial concerns, as well as each day’s weather. Working with the UW Svoboda Diaries Project, part of the Newbook Digital Texts Collective, I transcribe these diaries and help develop resources for their analysis and the promotion of digital humanities. Digital humanities work changes not only the literal, physical forms of texts but their literary forms as well, and digitizing a text such as a diary and opening it to public scholarship presents new questions about how we think about private writings. Literary scholarship on diaries addresses the unique psychological and philosophical aspects of keeping a diary and attempts to analyze its own position in relation to its object of study, as what is personal to a diarist is placed in plain view. Svoboda himself wrote in a largely impersonal, factual style, with little personal commentary, for instance, but scholars mine social and political sentiments from his choice of language or attention. In this project, I will consider the Svoboda diaries (focusing on three diaries written between 1897 and 1899) as textual objects and describe the transformations they have undergone since their writing, with a special focus on the transformation of the private nature of a diary. What do the diaries become as we study them? While digital humanities seeks faithfulness to the original contents of a text, drastic transformation of forms such as “making the private public” naturally opens those same contents to new uses, and understanding these changes is crucial to understanding the full extent of digital humanities scholarship.